Dissertations / Theses: 'Wharton’s jelly'

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Seshareddy, Kiran Babu. "Human Wharton’s jelly cells-isolation and characterization in different growth conditions." Thesis, Kansas State University, 2008. http://hdl.handle.net/2097/1054.

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Master of Science
Department of Anatomy and Physiology
Mark L. Weiss
Wharton's jelly is a non-controversial source of mesenchymal stromal cells. Isolation of the cells is non-invasive and painless. The cells have been shown to have a wide array of therapeutic applications. They have improved symptoms when transplanted in a variety of animal disease models, can be used in tissue engineering applications to grow living tissue ex vivo for transplantation, and can be used as drug delivery vehicles in cancer therapy. The cells have also been shown to be non-immunogenic and immune suppressive. This thesis focuses on optimizing isolation protocols, culture protocols, cryopreservation, and characterization of cells in different growth conditions. Results from the experiments indicate that isolation of cells by enzyme digestion yields cells consistently, a freezing mixture containing 90% FBS and 10% DMSO confers maximum viability, and the expression of mesenchymal stromal cell consensus markers does not change with passage and cryopreservation. The results of the experiments also show that cells grow at a higher rate in 5% oxygen culture conditions compared to 21% oxygen culture conditions, serum does not have an effect on growth of the cells, serum and oxygen do not have effects on the expression of mesenchymal stromal cell consensus markers and the cells are stable without nuclear abnormalities when grown in 5% oxygen and serum free conditions for six passages after first establishing in serum conditions.

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Morton, Jodi Mirissa. "Effects of intrauterine growth restriction on Wharton’s jelly cells and preweaning traits in pigs." Diss., Kansas State University, 2018. http://hdl.handle.net/2097/38757.

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Doctor of Philosophy
Department of Animal Sciences and Industry
Duane L. Davis
Intrauterine growth restriction (IUGR) affects all mammals. In the swine industry IUGR pigs result from intrauterine crowding. Prenatal programming in IUGR pigs has substantial effects on myogenesis and adipogenesis. Prenatal programming due to IUGR is also a problem in humans and long-term effects on adipogenesis are well established for small for gestational age (SGA) babies. Mesenchymal stem cells (MSCs) are the precursors for adipocytes. The umbilical cord contains a population of MSCs in Wharton’s jelly (WJ) and they can be harvested postnatally without ethical issues. Therefore, WJMSCs are proposed as models for studying prenatal programming of adipogenesis. We selected genes from studies of adipogenesis in humans and other species and examined their expression in pig WJ. We assigned pigs within litter as High, Medium, or Low birth weight and evaluated these categories for expression of Cox1, Cox2, EGR1, PPARɣ1, PPARɣ2, and Pref1. Differences due to size classification within litter were limited but there were correlations between weaning weight and delta cycle threshold (ΔCt) for EGR1 (r = 0.28; P < 0.009), PPARɣ1 (r = 0.29; P < 0.007), and PPARɣ2 (r = 0.30; P < 0.005). This may be consistent with the reports for SGA babies where EGR1 is upregulated by prenatal growth restriction. To gain insight into when during pregnancy IUGR affects WJ cells we collected umbilical cords at d 60 and d 95. In d 60 umbilical cords, small fetuses had increased (P = 0.06) Cox1 gene expression. We tested the ability of d 60 WJ cells to undergo adipogenic differentiation using standard protocols and a cycling protocol that exposed the cells to adipogenic differentiation conditions interposed with a rest phase with high insulin. It has been reported that the cycling protocol revealed increased glucose uptake in WJ cells from human SGA babies. We found that d 60 WJ cells did not show adipogenic differentiation in any of the protocols tested however glucose uptake correlated negatively with birth weight at Cycle 0 (P < 0.02; r = 0.61). In summary, pig WJ cells reveal some effects of IUGR but they appear to differ from the relationship demonstrated reported for human SGA babies. A new finding was that at midgestation pig WJ cells do not appear to be competent to complete adipogenesis. We also studied nursing managements to improve outcomes for IUGR pigs. Colostrum intake may be a problem, particularly for light weight pigs and those born later during farrowing. Split suckling is the removal of some pigs to allow others unrestricted nursing access. We temporarily removed the six heaviest pigs and this treatment increased gain and weight by d 7 of age. Colostrum intake was highest for the high birth weight pigs. When we temporarily removed the first half of the litter, colostrum intake was increased for the second half of litter born and the difference in immunocrit was reduced between the two litter halves.

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Reeds, Kimberly. "In vitro effects of canine Wharton’s jelly mesenchymal stromal cells and nanoparticles on canine osteosarcoma D17 cell viability." Thesis, Kansas State University, 2011. http://hdl.handle.net/2097/11990.

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Master of Science
Department of Clinical Sciences
Mary Lynn Higginbotham
Objectives – To isolate and maintain canine Wharton’s jelly mesenchymal stromal cells (WJMSCs) in culture, to determine the effects of micellar nanoparticles containing doxorubicin (DOX) on WJMSCs and canine osteosarcoma (OSA) D17 cell viability, and to determine the effects of conditioned media from WJMSCs loaded with micellar nanoparticles containing DOX on OSA D17 cell viability. Sample Population – Canine WJMSCs containing various concentrations of DOX micelles and canine OSA D17 cells. Procedures – WJMSCs were isolated from canine umbilical cords. Micellar nanoparticles containing DOX were prepared and added to culture plates containing canine OSA D17 cells to determine micelle effects on cell growth and viability. Conditioned media from culture plates containing canine WJMSCs incubated with various DOX micelle concentrations was added to OSA D17 cells for conditioned media experiments. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to assess OSA D17 cell viability. A trypan blue stain was also utilized to perform cell counts to determine the effect of the DOX micelles on stromal cell growth. Results – WJMSCs were successfully isolated and maintained in culture. Micellar nanoparticles containing DOX decreased OSA D17 cell viability. OSA D17 cell viability was also decreased following incubation with conditioned media from canine WJMSCs loaded with micellar nanoparticles containing DOX. Significant decreases with the conditioned media of canine WJMSCs loaded with 10μM micelles occurred at 48 hours (p < 0.005) and at 72 and 96 hours (p < 0.0001). Significant decreases were also observed with the 1 μM DOX micelles at 72 hours (p < 0.005) and 96 hours (p < 0.0001). WJMSC numbers decreased in a dose dependent manner following incubation with DOX micelles. Changes in WJMSC number was not caused by increased cell death as all variables produced similar percentages of dead cells. Conclusions – Canine WJMSCs were successfully isolated and maintained in culture. Stromal cells containing DOX micellar nanoparticles induced OSA D17 cell cytotoxicity while inducing an anti-proliferative, rather than cytotoxic effect, on the WJMSC. These data support future in vivo experiments utilizing canine WJMSCs and micellar nanoparticles.

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Sankaramaddi, Jeevan Reddy [Verfasser]. "Evaluation of human umbilical cord Wharton’s Jelly Cells as a potential cell source for cardiovascular tissue engineering / Jeevan Reddy Sankaramaddi." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2017. http://d-nb.info/1148425780/34.

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Ferreira, Daniela Filipa Alves. "Influence of substrates composition on immunomodulation by MSCs." Master's thesis, Universidade de Aveiro, 2016. http://hdl.handle.net/10773/16048.

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Mestrado em Biomedicina Molecular
Mesenchymal stem cells (MSCs) are non-hematopoietic multipotent stem cells capable to self-renew and differentiate along different cell lineages. MSCs can be found in adult tissues and extra embryonic tissues like the umbilical cord matrix/Wharton’s Jelly (WJ). The latter constitute a good source of MSCs, being more naïve and having a higher proliferative potential than MSCs from adult tissues like the bone marrow, turning them more appealing for clinical use. It is clear that MSCs modulate both innate and adaptive immune responses and its immunodulatory effects are wide, extending to T cells and dendritic cells, being therapeutically useful for treatment of immune system disorders. Mechanotransduction is by definition the mechanism by which cells transform mechanical signals translating that information into biochemical and morphological changes. Here, we hypothesize that by culturing WJ-MSCs on distinct substrates with different stiffness and biochemical composition, may influence the immunomodulatory capacity of the cells. Here, we showed that WJ-MSCs cultured on distinct PDMS substrates presented different secretory profiles from cells cultured on regular tissue culture polystyrene plates (TCP), showing higher secretion of several cytokines analysed. Moreover, it was also shown that WJ-MSCs cultured on PDMS substrates seems to possess higher immunomodulatory capabilities and to differentially regulate the functional compartments of T cells when compared to MSCs maintained on TCP. Taken together, our results suggest that elements of mechanotransduction seem to be influencing the immunomodulatory ability of MSCs, as well as their secretory profile. Thus, future strategies will be further explored to better understand these observation and to envisage new in vitro culture conditions for MSCs aiming at distinct therapeutic approaches, namely for immune-mediated disorders.
As células estaminais mesenquimais (MSCs) são células não-hematopoéticas, multipotentes, capazes de se auto-renovar e de diferenciar em diferentes tipos celulares. As MSCs estão presentes em tecidos mesenquimais e de tecidos extra embrionários, tais como a matriz do cordão umbilical/Wharton’s Jelly(WJ). Estes últimos constituem uma boa fonte de de MSCs, sendo estas mais naive e tendo um maior potencial de proliferação do que as MSCs obtidas de tecidos adultos, como a medula óssea, tornando as MSCs da matriz do cordão umbilical/Wharton’s Jelly sejam mais apelativas para uso clínico. As MSCs possuem a capacidade de modularem tanto o sistema imune inato como o adquirido e os seus efeitos são vastos, afectando todas as células do sistema imune. Esta capacidade é bastante vantajosa para o uso terapêutico destas células em doenças do sistema imunitário. A mecanotransducção é por definição o mecanismos pelo qual as células convertem estímulos mecânicos em uma resposta bioquímica e com mudanças na sua morfologia. Apartir destas observações colocámos a hipotese de que mantendo MSCs in vitro em diferentes substratos poderia influencia a sua capacidade imunomoduladora. Com este trabalho, demonstrámos que ao plaquear MSCs em diferentes substratos de PDMS, estas mostram uma tendência para secretar quantidades diferentes de vários factores soluveis analisados, relativamente a MSCs mantidas em cultura em plataformas convencionais (placas de cultura de células - TCP). Para além disto, foi também observado que MSCs plaqueadas em substratos de PDMS aparentavam possuir uma maior capacidade imunomoduladora quando comparadas com MSCs mantidas em condições convencionais. Em conjunto todos os resultados obtidos sugerem que elementos relacionados com a mecanotransdução parecem influenciar a capacidade imunomoduladora de MSCs e a sua secreção de factores solúveis. Deste modo, estudos futuros poderão elucidar os mecanismos responsáveis por estas observações, de modo a permitir que se possa constitutuir melhores estratégias de cultura de MSCs para futuro uso terapêutico dirigido a doenças do sistema imunitário.

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Laroye, Caroline. "Le cordon ombilical : une source alternative de cellules souches/stromales mésenchymateuses dans le traitement du choc septique ?" Thesis, Université de Lorraine, 2017. http://www.theses.fr/2017LORR0279.

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Le choc septique est actuellement la dixième cause de mortalité à travers le monde à égalité avec les infarctus du myocarde. Sa physiopathologie extrêmement complexe, entrelaçant un état pro-inflammatoire et anti-inflammatoire, rend caduque l’action des thérapeutiques conventionnelles. En ce sens, les recherches s’orientent vers les thérapeutiques innovantes et notamment les cellules souches/stromales mésenchymateuses (CSM). En effet, les études murines ont mis en évidence que les CSM étaient en mesure, notamment par leurs actions paracrines, d’améliorer la survie, la défaillance d’organes mais également la bactériémie de souris soumises à un choc septique. Cependant, les propriétés des CSM varient en fonction du tissu dont elles sont issues et particulièrement selon qu’elles proviennent de tissus fœtaux (cordon ombilical, placenta, liquide amniotique) ou adultes (moelle osseuse, tissu adipeux...). Ainsi, notre premier objectif a été de comparer, dans un modèle murin de choc septique, l’action des CSM issues de la moelle osseuse (MO) à celle des CSM issues de la gelée de Wharton (GW) du cordon ombilical. Cette étude murine a permis de mettre en évidence une action quelque peu différente, entre les CSM-GW et les CSM-MO, sur la physiopathologie du choc septique sans que pour autant, l’une des deux sources de CSM, ne se dégage significativement de l’autre en termes d’efficacité. Cependant, en raison de leur importante capacité de prolifération et de l’accessibilité du tissu source, les CSM-GW apparaissent comme étant nettement plus avantageuses que les CSM-MO. En conséquence, notre deuxième objectif a été d’évaluer l’action des CSM-GW dans un modèle porcin de péritonite afin de se rapprocher un peu plus près de la clinique humaine. Cette étude, menée en double aveugle et en présence continue d’un médecin réanimateur expérimenté, a permis de mettre en évidence que les CSM-GW, produites en grade clinique et utilisées juste après décongélation, étaient en mesure d’améliorer la survie, les paramètres hémodynamiques ainsi que les défaillances d’organes, selon un mécanisme d’action différent de celui rapporté par les études murines
Septic shock, equal to the myocardial infraction, is currently the tenth cause of death in the world. The pathophysiological complexity of this syndrome, with a simultaneous pro and anti- inflammatory state, results in the failure of conventional treatments. In this sense, research is focusing on innovative therapeutics agent, including mesenchymal stem cells (MSC). Indeed, murine studies of septic shock showed that MSC improve organ injuries, bacteremia and survival by notably a paracrine mechanism. However, MSC properties vary according to the source tissue, especially if they are derived from a fetal tissue (Wharton’s jelly (WJ), placenta amniotic fluid) or an adult tissue (bone marrow (BM), adipose tissue...). Our first objective was to compare, in a septic shock murine model, the effect of BM-MSC with that of WJ-MSC. Although some differences were observed, the same efficiency was demonstrated between these two sources. However, WJ-MSC present large advantages in comparison to BM-MSC due to their important proliferation capacities and potential quantities of umbilical cord donation. Consequently, our second objective was to investigate the effect of WJ-MSC administration in a relevant pig model of peritonitis in order to better mimic a clinical approach in humans. This study, conducted in double-blind and in presence of an experimented intensivist, showed that WJ-MSC produced in clinical grade and used immediately after thawing, improve survival, hemodynamic parameters and organ injuries by another action than that described in murine studies

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Reppel, Loïc. "Potentialité des cellules stromales de la gelée de Wharton en ingénierie du cartillage." Thesis, Université de Lorraine, 2014. http://www.theses.fr/2014LORR0164/document.

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Les cellules stromales/souches mésenchymateuses de la gelée de Wharton humaines (CSM-WJ) représentent une source abondante et intéressante de cellules souches pour des applications en ingénierie cellulaire et tissulaire. Leur origine fœtale leur confère des caractéristiques spécifiques par rapport aux cellules stromales/souches mésenchymateuses isolées à partir de moelle osseuse humaine (CSM-MO). Tout d'abord, le but de ce travail est d'optimiser les conditions de culture des CSM-GW pour leur utilisation clinique ultérieure. Nous nous concentrons sur l'influence de la concentration en oxygène lors de l'expansion en monocouche de P1 à P7 sur plusieurs paramètres permettant de caractériser les CSM. Les résultats obtenus sont comparés à ceux obtenus avec les CSM-MO. Notre travail a montré des différences entre les deux sources cellulaires en termes de prolifération et de différenciation adipocytaire. D’après nos résultats, l'hypoxie, au cours de l'expansion, est un paramètre important à prendre en compte en ce qui concerne la prolifération et le potentiel de différenciation chondrocytaire. L'influence des facteurs obstétricaux sur les caractéristiques des CSM-GW est également explorée. Cette étude se situant également dans le cadre de l’ingénierie tissulaire du cartilage, la seconde phase du projet consiste à induire la différenciation des cellules en chondrocytes en ensemençant ces dernières dans un biomatériau à base d’alginate et d’acide hyaluronique, et sur une cinétique de 28 jours. Les résultats obtenus sont comparés à ceux obtenus avec les CSM-MO. Après 4 semaines de culture, les CSM-GW sont capables de s'adapter à leur environnement et d’exprimer des gènes et des protéines matriciels spécifiques du cartilage tels que le collagène de type 2, qui se trouve plus exprimé après différenciation à partir des CSM-GW qu’à partir de CSM-MO
Mesenchymal Stromal/Stem Cells from human Wharton’s jelly (WJ-MSC) are an abundant and interesting source of stem cells for applications in cell and tissue engineering. Their fetal origin confers specific characteristics compared to Mesenchymal Stromal/Stem Cells isolated from human bone marrow (BM-MSC). First, the aim of this work is to optimize WJ-MSC culture conditions for their subsequent clinical use. We focus on the influence of oxygen concentration during monolayer expansion on several parameters to characterize MSC. The results are compared to those obtained with BM-MSC. Our work distinguishes WJ-MSC from BM-MSC in terms of proliferation and adipogenic differentiation. Considering our results, hypoxia during cell expansion is an important parameter to take into account regarding proliferation potential but also chondrogenic differentiation potential. The influence of obstetric factors on WJ-MSC characteristics is also explored. In cartilage tissue engineering context, the second phase of the project is to induce cell differentiation into chondrocytes by seeding them in Alginate/Hyaluronic Acid hydrogel scaffold, and during 28 days. The results obtained are compared to those obtained with BM-MSC. After 4 weeks of culture, WJ-MSC are able to adapt to their environment and express specific cartilage-Related genes and matrix proteins such as type 2 collagen, which is found more expressed after differentiation fromWJ-MSC, than from BM-MSC

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Yu, Hao. "Évaluation des caractéristiques des hydrogels d’alginate supplémentés en acide hyaluronique ou en hydroxyapatite lors de la différenciation des cellules souches mésenchymateuses issues de la gelée de Wharton." Thesis, Université de Lorraine, 2017. http://www.theses.fr/2017LORR0087/document.

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Dans le domaine de l'ingénierie du cartilage, les hydrogels à base d'alginate (Alg) et de cellules souches mésenchymateuses (CSM) sont utilisés comme biomatériaux pouvant être utilisés pour combler des lésions cartilagineuses plus ou moins profondes. Cependant, pour reproduire l’organisation zonale du cartilage, des biomatériaux multiphasiques sont nécessaires. Afin de guider la différenciation des CSM dans les différentes strates du biomatériau, sans apports de facteurs de croissance, des composants naturels du cartilage (acide hyaluronique, HA) ou de la matrice osseuse (hydroxyapatite, Hap) peuvent être ajoutés à l’alginate. L’objectif de ce travail de thèse consiste à analyser l’impact de la composition de biomatériaux à base d’alginate enrichi soit en HA soit en Hap sur le comportement des CSM. La première partie de notre travail à consister à évaluer le comportement des CSM issues de la gelée de Wharton dans ces hydrogels. Nos résultats mettent en évidence que les hydrogels d’Alg/Hap possèdent non seulement de meilleures propriétés mécaniques que les hydrogels Alg/HA et favorisent la viabilité des CSM ainsi que leur différenciation par rapport aux CSM ensemencées dans un hydrogel d’Alg/HA. La méthode de stérilisation du biomatériau représente une étape incontournable, dont on doit impérativement évaluer les multiples effets, en particulier pour ce qui touche au comportement des cellules, mais aussi au maintien de l’intégrité des propriétés physicochimiques de l'hydrogel. Ainsi, dans une seconde partie du travail, nous avons montré que le traitement de stérilisation par autoclave induisait un effet négatif sur les caractéristiques initiales de l'hydrogel à base d'alginate. Il ressort également de cette investigation sur les modes de stérilisation, que la stérilisation des hydrogels avec des UV est plus efficace et permet de préserver au mieux les propriétés spécifiques de l'hydrogel, notamment de l’Alg/HA. Enfin, dans une troisième partie de notre travail, nous avons évalué l’évolution des propriétés mécaniques au cours de la différenciation et l’impact de celles-ci sur la différenciation des CSM ainsi que sur leurs propriétés immunomodulatrices. À partir de ces résultats, nous avons montré que les caractéristiques physico-chimiques des hydrogels d’Alg/ha et Alg/hap influençaient non seulement le potentiel de différenciation des CSM-GW mais également la sécrétion des facteurs solubles impliqués dans l’immunomodulation. Ces propriétés physico-chimiques étant influencées dès le procédé de stérilisation, il est alors conseillé de les prendre en compte dans toutes les étapes de l’ingénierie tissulaire
In the field of cartilage engineering, alginate (Alg)-based hydrogels and mesenchymal stem cells (MSC) are widely used as raw biomaterials and stem cells which can be used to fill cartilage lesions of varying depth. However, to reproduce the zonal organization of articular cartilage, a graft multilayer is necessary. In order to guide the differentiation of MSCs in different strata of the biomaterials, without input of growth factors, natural cartilage components (hyaluronic acid, HA) or bone matrix (hydroxyapatite, Hap) can be added into the alginate. The aim of this work is to analyze the impact of the composition of alginate enriched either in HA or in Hap on the behavior of MSCs. The first part of our work is to evaluate the behavior of WJ-MSCs into these hydrogels. Our results have shown that Alg/ Hap hydrogels not only possess better mechanical properties than Alg/HA hydrogels, but also promote the viability of MSCs and their differentiation from MSC seeded into the Alg/HA hydrogel. The sterilization method of biomaterial is an essential step, the multiple effects of which must be evaluated, in particular as regards the behavior of the cells, but also to maintain the integrity of the physicochemical properties of hydrogel. Thus, in a second part of this work, we showed that the autoclave sterilization treatment induced a negative effect on the initial characteristics of alginate hydrogel. It is also apparent from this investigation of the sterilization modes that the sterilization of hydrogels with UV is more efficient and makes it possible to preserve the specific properties of the hydrogel as best as possible, in particular Alg/HA. Finally, in a third part of our work, we also evaluated the evolution of the mechanical properties during the differentiation and the impact of these on the differentiation of MSCs and their immunomodulatory properties. From these results, we have shown that the physico-chemical characteristics of Alg / ha and Alg/hap hydrogels influence not only the differentiation potential of WJ-MSC but also the secretion of soluble factors involved in immunomodulation. Since these physicochemical properties are influenced by the sterilization process, it is advisable to take them into account in all stages of tissue engineering

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Seshareddy, Kiran Babu. "Human Wharton's jelly cells-isolation and characterization in different growth conditions." Manhattan, Kan. : Kansas State University, 2008. http://hdl.handle.net/2097/1054.

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Badraiq, Heba Ghazi O. "Effects of maternal body weight on Wharton's Jelly mesenchymal stromal cells (pilot study)." Thesis, King's College London (University of London), 2017. https://kclpure.kcl.ac.uk/portal/en/theses/effects-of-maternal-body-weight-on-whartons-jelly-mesenchymal-stromal-cells-pilot-study(dac6be9c-1f9d-4c00-88dc-2a73ec4489b4).html.

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To investigate whether the maternal metabolic environment affects the DNA methylation of mesenchymal stromal/stem cells (MSCs) from umbilical cord (UC) Wharton’s Jelly (WJ), potentially rendering them unsuitable for clinical use in multiple recipients, a pilot study was conducted on fourteen UCs obtained post partum from healthy non-obese (BMI=19-25; n=7) and obese (BMI≥30; n=7) donors receiving elective Caesarean sections. The time of first WJ-MSCs outgrowth from UC explants was similar in samples from obese and non-obese donors. However, the cells from non-obese donors proliferated faster after 34 hours of culture than cells from obese donors. Differentiation into adipogenic, osteogenic and chondrogenic lineages was similar between obese and non-obese donor samples as demonstrated by tissue-specific staining and RT-PCR for lineage markers. However, WJ-MSCs from obese donors exhibited stronger immunosuppressive activity than those from non-obese donors. Genome-wide DNA methylation of triple-positive (CD73+CD90+CD105+) WJ-MSCs sorted from the first passage of a mixed population of cells was assessed. Samples from the obese and non-obese donors clustered separately, and 5,767 of the analysed CpG sites (1%) exhibited different methylation. Sixty-seven genes were observed with at least one CpG site with a methylation difference ≥0.2 in four or more obese donors. These 67 genes were further refined based on a list of polymorphic CpG sites and segmental duplications. In 18 of the 67 genes with a different CpG methylation pattern, the CpG sites were in non-polymorphic regions. However, two genes (DCAF6 and ZNF714) resided in segmentally duplicated regions. To determine whether methylation differences altered gene expression, the samples were analysed using a HumanHT-12 Expression BeadChip array and, of the 18 genes, only PNPLA7 was significantly affected at the mRNA level, which was confirmed independently by RT-PCR and Western blotting. Although the number of analysed donors was limited, the data suggest that an abnormal metabolic environment related to excessive body weight might alter the properties of WJ-MSCs used for cellular therapy.

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Morton, Jodi Mirissa. "Effects of porcine jelly matrix (JMX) on gene expression of porcine umbilical cord (PUC) stem cells." Thesis, Kansas State University, 2014. http://hdl.handle.net/2097/17684.

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Master of Science
Department of Animal Sciences and Industry
Duane L. Davis
Culturing stem cells is usually done on tissue-culture treated plastic. Over time the cells change their gene expression and start to differentiate. Porcine umbilical cord (PUC) stem cells express the embryonic transcription factors Oct4, Nanog and Sox2 and changes in their expression may be useful for to evaluating culture-induced changes in the cells. We developed an extract of porcine Wharton’s jelly matrix (JMX) that may provide some characteristics of the stem cell niche located in the umbilical cord. Our extract used whole cords and enzyme digestion to simplify preparation of the product. We compare cells cultured on plastic to those grown on thin and thick gels of JMX in four experiments. In Exp 1a and b, growing PUCs on a thick JMX coating for 3(1a) or 4(1b) d increased the number of cells at the end of culture (P < 0.05) with minimal effects on gene expression. In Exp 2 we compared PUCs grown on thin and thick layered JMX with added collagen (+C) and to control cells. The JMX layers caused the cells to adopt a small, round shape and to form clumps or colonies during culture. No differences (P > 0.10) were seen between thin10 +C and control wells for viable and total cell counts but thick layered +C resulted in decreased numbers of viable cells compared to thin + C (P < 0.10) and control wells (P < 0.05). In a follow up experiment (Exp. 3) growing the PUCs mixed within, rather than plating on top of, a thick layer of JMX + C caused marked morphological changes with dense 3-dimensional structures formed. Exp 4 compared JMX allowed to gel for 10 (Thin10 +C) or 60 (Thin60 +C) min before the non-gelled fraction was removed. There were no effects on cell numbers at the end of culture (P > 0.10) but Sox2 expression was increased in Thin60 +C compared to controls on plastic (P < 0.05) and Thin10 +C (P < 0.10). In summary, JMX extracts change cell morphology and in some formats increased cell proliferation and may increase Sox2 expression. Further investigation is needed to fully understand the effects of JMX on PUCs.

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Ueda, Minoru, Fumitaka Kikkawa, Hideharu Hibi, Akira Iwase, Sachiko Takikawa, Akihito Yamamoto, and Ryutaro Shohara. "Mesenchymal stromal cells of human umbilical cord Wharton's jelly accelerate wound healing by paracrine mechanisms." Thesis, Informa healthcare, 2012. http://hdl.handle.net/2237/18891.

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Liu, Xing. "A contribution to the selection of suitable cells, scaffold and biomechanical environment for ligament tissue engineering." Thesis, Université de Lorraine, 2019. http://www.theses.fr/2019LORR0058/document.

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L'ingénierie tissulaire du ligament constitue une approche prometteuse pour réparer ou remplacer un ligament endommagé. Les trois piliers essentiels de l'ingénierie tissulaire ligamentaire sont la matrice de support (aussi appelée scaffold), la source cellulaire, ainsi que l'apport de stimulations biomécaniques/biochimiques : ces trois piliers ont été partiellement étudiés par le passé dans le but de s’orienter vers une régénération ligamentaire. Dans la présente étude, le polymère synthétique poly (L-lactide-co-ε-caprolactone) (PLCL) et la soie ont été proposés et comparés comme de potentiels candidats pour la constitution d’une matrice de support. Une série de matrices tressées multicouches à base de PLCL et de soie, ainsi qu'un nouveau composite soie/PLCL ont été développés et comparés. Les caractérisations physico-chimiques et biologiques ont démontré que le PLCL et la soie constituent des candidats pertinents, tant sur les plans mécaniques que biologiques, pour la constitution d’une matrice de support. De plus, nous avons montré que le composite soie/PLCL offrait des propriétés mécaniques et une biocompatibilité accrue par rapport aux autres matrice testées, et constituait probablement le candidat le plus approprié pour l'ingénierie tissulaire du ligament. Les cellules souches mésenchymateuses (CSM) de la gelée de Wharton (WJ-MSCs) ainsi que les cellules souches mésenchymateuses de la moelle osseuse (BM-MSCs) ont été évaluées et comparées en tant que sources cellulaires potentielles pour la régénération ligamentaire. Les caractéristiques biologiques de ces cellules incluent l’adhésion cellulaire, la prolifération, la migration et la synthèse de matrice extracellulaire. Ces deux types de cellules ont montré une bonne biocompatibilité dans leurs interactions avec les matrices de support en PLCL et en soie. Aucune différence significative n'a été observée entre les WJ-MSCs et les BM-MSCs. Enfin, l'effet de la stimulation biomécanique sur la différentiation des CSM en tissu ligamentaire a été évalué par le biais d’un bioréacteur de traction-torsion. Bien que peu de cellules aient été détectées la matrice après 7 jours de stimulation, des CSM de forme allongée le long des fibres ont été détectées, ce qui permet de penser qu'il est possible de promouvoir la différenciation des biosubstituts matrice-cellules grâce à la stimulation mécanique en bioréacteur. En conclusion, cette étude démontre le potentiel prometteur de l’association de cellules souches mésenchymateuses issues de la gelée de Wharton ou de la moelle osseuse avec une matrice de support composite soie/PLCL pour la régénération ligamentaire dans le futur
Ligament tissue engineering offers a potential approach to recover or replace injured ligament. The three essential elements that have been investigated towards ligament regeneration consist in a suitable scaffold, an adapted cell source, and the supply of biomechanical/biochemical stimulations. In the current study, synthetic polymer poly (L-lactide-co-ε-caprolactone) (PLCL) and silk have been evaluated as suitable candidates to constitute an adapted scaffold. A series of multilayer braided scaffolds based on PLCL and silk, as well as an original silk/PLCL composite scaffold, have been developed and compared. The conducted physicochemical and biological characterizations have demonstrated that both PLCL and silk constitute adapted candidate material to form ligament scaffolds from the mechanical and biological points of view. Moreover, it has been observed that silk/PLCL composite scaffold resulted in adequate mechanical properties and biocompatibility, and therefore could constitute suitable candidate scaffolds for ligament tissue engineering. Both Wharton’s Jelly mesenchymal stem cells (WJ-MSCs) and Bone marrow mesenchymal stem cells (BM-MSCs) have been evaluated to be cell source for ligament regeneration. MSCs behaviors including cell attachment, proliferation, migration and extracellular matrix synthesis have been investigated. In the present study, both MSCS showed a good biocompatibility to interact with PLCL and silk scaffolds. No significant differences have been detected between WJ-MSCs and BM-MSCs. Finally, the effect of biomechanical stimulation on MSCs differentiation towards ligament tissue has been carried out with a tension-torsion bioreactor. Although few cells were detected on scaffold after 7 days of stimulation, MSCs were observed to exhibit an elongated shape along the longitudinal direction of fibers, which may indicate that an adapted mechanical stimulation could promote MSC-scaffold constructs differentiation towards ligamentous tissue. As a conclusion, this study demonstrates the potential of WJ-MSCs and BM-MSCs combined with a new silk/PLCL composite scaffold towards ligament regeneration

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14

Walker, Kristen Elizabeth. "Effects of isolation methods on proliferation and GD2 expression by porcine umbilical cords stem cells." Thesis, Kansas State University, 2011. http://hdl.handle.net/2097/7067.

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Master of Science
Department of Animal Sciences and Industry
Duane L. Davis
Cell isolation method may have effects on the characteristics of the cells isolated from porcine umbilical cords. As stem cells age or approach senescence, it is hypothesized that their properties change. We expect that isolation method and age of cells will have effects on the phenotype of porcine umbilical cord (PUC) cells during in vitro expansion. We investigated the effects of three isolation methods on PUC population doublings, ability to produce colony forming units (CFU), and amount of ganglioside GD2 (GD2) expression over eleven passages. Isolation methods were explant (Exp) in which the Wharton's Jelly was removed from cords, minced and plated, enzyme digest (Dig), and stomacher assisted enzyme digestion (Stom). Cell isolates were analyzed for GD2 expression, CFU, and population doublings at early (3), middle (7), and late (11) passage. The Exp method produced greater (P<0.05) population doublings and more (P<0.05) CFU at passage 7. Explant isolates also were numerically more likely to survive to passage 11 (9/9 isolates vs 5/9 for Dig and 7/9 for Stom). In contrast, the percent cells expressing GD2 was greater (P<0.05) for Stom isolates than Exp isolates at passage 11. There were no trends for increased passage number to decreased population doubling, CFU formation, or percent GD2 positive cells. In summary, our results indicate that the Exp isolation method produced the greatest number of population doublings over 11 passages and there were minimal effects of isolation method on CFU and GD2 expression. Although Exp may be more difficult to scale up to isolate all of the PUCs in a cord, it provided greater in vitro expansion than the enzyme methods in our experiment and may provide the most cells for biotechnological and biomedical applications.

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15

McElreavey, Kenneth David. "An investigation of the colony-stimulating activity (CSA) produced by the Wharton's jelly portion of human umbilical cord." Thesis, Queen's University Belfast, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.317456.

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16

Lopez, Rodriguez Yelica Virginia. "Immunosuppressive properties of Wharton's jelly derived mesenchymal stromal cells in the treatment of graft versus host disease in rat model." Diss., Kansas State University, 2013. http://hdl.handle.net/2097/16331.

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Doctor of Philosophy
Department of Anatomy and Physiology
Mark L. Weiss
Graft Versus Host Disease (GVHD) is the major complication following hematopoietic stem cell transplantation. GVHD is activated by immunocompetent T cells presented in the donor grafted tissue. Due to the increased use of bone marrow transplantation to treat diverse malignancies, the incidence of GVHD has shown a notable increase. Depending of the degree of immunological mismatch between donor and host, 50-70% of patients develop GVHD after allogeneic Bone Marrow Transplantation (BMT). Once GVHD develops, mortality reaches up to 50% in humans. Several studies using Mesenchymal Stromal Cells (MSCs) to prevent and treat GVHD have produced controversial results. It is thought that distinct MSCs sources used in those studies might be an important factor that produces different outcomes. For cellular therapy, the most attractive characteristics of MSCs are their reduced immunogenic potential, and their abilities to modulate immune responses. This dissertation addressed the hypothesis that Wharton’s jelly cells (WJCs) would prevent the pathology and death associated with GVHD after BMT. To accomplish this, I created a clinically relevant model of GVHD by transplanting allogeneic bone marrow across minor histocompatibility antigen (HA) barriers in the rat. To enhance alloreactive T-cell stimulation, bone marrow (BM) was co-administered with a fraction of CD8[superscript]+ cells magnetically selected from spleen to induce GVHD. Bone marrow tissue was isolated from a donor rat Fischer 344 (F344, RT1lv) and transplanted into lethally irradiated (10 Gray) Lewis rat (LEW, RT1l). Once GVHD was induced, MSCs derived from umbilical cord WJCs were either co-transplanted at day 0 with bone marrow, or given on day 2 post-BMT intravenously. The prophylactic potential of WJCs in an in vivo GVHD model was assessed as survival time, clinical symptomatology occurrence, and histopathology injuries in target tissues. Results indicate that while co-administration of WJCs with hematopoietic cells on day 0 failed to alleviate GVHD associated symptomatology and mortality. WJCs administered on day 2 post-induction ameliorated GVHD-associated symptomatology, improved engraftment and survival.

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17

Ebrahim, Neven. "Cellular and molecular mechanisms underlying extravasation of human Wharton's jelly mesenchymal stem cells across fetal and adult endothelial cell monolayers." Thesis, University of Nottingham, 2016. http://eprints.nottingham.ac.uk/33246/.

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The Wharton’s Jelly (WJ) of human umbilical cord (HU) contains multipotent stem cells (WJ-MSC) which express mesenchymal markers but not hematopoietic markers. WJ-MSC are increasingly being tested for use in stem cell therapy, with intravenous delivery being the preferred route. Fetal stem cells from embryonic germ layers are present in maternal blood and can home to damaged maternal tissues. This study investigates how WJ-MSC cross the fetal and adult endothelial barriers; including the cellular and molecular mechanisms employed. WJ-MSC were isolated from HU (n=27) which were taken from normal term pregnancies after elective Caesarean section. Flow cytometry and immunofluorescence were used to check presence/absenc of mesenchymal versus haematopoietic markers. Cells were induced to become adipocytes, chondrocytes and osteocytes by using specific induction medium. Isolated WJ-MSC were added after labelling with PKH26 to confluent monolayers of isolated human umbilical vein endothelial cells (HUVEC) or commercially bought human uterine microvascular endothelial cells (HUtMEC) at a 1:5 ratio. Cell-cell interactions were monitored with real time microscopy for 24 to 40h. Fluorescence and confocal scanning microscopy, after vascular endothelial (VE) cadherin immunocytochemistry were used for detailed analysis of VE-cadherin junctional occupancy and spatio-temporal location of stem cells. Tyrosine phosphorylation status of VE-cadherin, whether at Tyr685 or Tyr731, at different time points were investigated by immunoblotting whilst levels of vascular endothelial growth factor (VEGF) in the conditional media (CM) were measured by ELISA. Three different isolates were tested, with 3 experimental repeats for all expermints. Statistical analyses were performed with ANOVA (One or Two way). Cells (>95%) from each passage were positive for the mesenchymal markers CD 29, CD 105, CD 90, CD 73 and CD 44. <2% cells showed positivity to the haematopoietic markers CD 34, HLA-DR, CD 14, CD 19 and CD 45. WJ-MSC differentiated into adipocytes, osteocytes and chondrocytes. WJ-MSC displayed exploratory behaviour for a minimum of 30 min on HUtMEC or 60 min on HUVEC with interrogation of paracellular openings before crossing rather than replacing endothelial cells. By 2h, half were found at sub-endothelial positions, with a majority reaching this within 16-22h. There was accompanying loss of junctional VE-cadherin (64.9 + 3.7 %; p<0.001 in HUVEC; 63 + 4.6%; p< 0.001 in HUtMEC) in the endothelial monolayers followed by a return at 16h and increased continuity by 22h (p<0.01 in HUVEC; p<0.001 in HUtMEC). Junctional disruptions were found close to overlying or migrating WJ-MSC. Confocal microscopy confirmed paracellular extravasation. VE-cadherin protein levels matched controls in the early hours (0-2h) and increased after 22h co-culture in both fetal and uterine endothelium. VE-cadherin showed a 2-fold increase in phosphorylation at Tyr685 from 30 min to 2h. P-Tyr731 remained unchanged, similar to untreated endothelial layers, then decreased at 2h and 22h. VEGF levels in WJ-MSC – HUtMEC co-culture supernatants was highest at 2h (88 + 3 pg/ml) and decreased by 22h, reaching negligible levels by 48h. Anti-VEGF blocked Tyr685 phosphorylation but did not affect the decrease in P-Tyr731; this was accompanied by a 25% decrease in transmigration of cells in the first two hours and a 43% decrease in total by 22h. in WJ-MSC – HUtMEC co-cultures. However, in HUVEC-WJ-MSC co-cultures, no VEGF were detected and anti-VEGF did not block Tyr685 phosphorylation and Tyr731 de-phosphorylation. WJ-MSC from term umbilical cords can be easily isolated and expanded in culture. They retain mesenchymal stem cell properties for the passages tested (up to P5) making them a valuable model for studies into mechanisms underlying extravasation. The data obtained suggest that WJ-MSC can influence expression of VE-cadherin, with perturbation during transmigration followed by upregulation and repair once the adlumenal side is reached. There was a similarity in the cellular and molecular mechanisms employed by WJ-MSC in their paracelluar migration across fetal and uterine endothelium, although VEGF may not be the key player in HUVEC interactions. For both endothelial types, WJ-MSC appear to induce phosphorylation events linked with paracellular permeability and de-phosphorylation events normally associated with leukocyte extravasation. The data from the uterine endothelial investigations suggests that fetal stem cells are able to influence paracellular junctional dynamics and strengthens the growing hypothesis that they may also play a role in re-modelling the uterine circulation for fetal advantage. The extra-embryonic WJ-MSC holds the promise of use in restoring junctional maturity and vascular repair in future therapeutic applications.

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18

Campos, Loreta Lemes. "Isolamento, cultivo, caracterização e criopreservação de células tronco mesenquimais derivadas de membrana amniótica, geléia de Wharton e sangue do cordão umbilical de fetos bovinos /." Botucatu, 2014. http://hdl.handle.net/11449/123860.

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Orientador: Fernanda da Cruz Landim
Coorientador: Bruna De Vita
Banca: Leandro Maia
Banca: Cláudia Barbosa Fernandes
Resumo: As células tronco mesenquimais (CTMs) estão presentes na maioria dos tecidos adultos e possuem grande capacidade de multiplicação, o que as tornam muito atrativas para a terapia celular, tanto na medicina humana como na medicina veterinária . Quando cultivadas in vitro são capazes de se auto renovar e dar origem a novos tipos celulares. Atualmente há uma grande tendência para a utilização de CTMs obtidas de tecidos fetais, como a membrana amniótica (MA), matriz extravascular do cordão umbilical (CU) e sangue do cordão umbilical (SCU), podendo ser obtidas no momento do parto por uma técnica não invasiva. Dessa forma, o objetivo deste estudo foi isolar, caracterizar, diferenciar e criopreservar CTMs obtidas de MA, CU e SCU de fetos bovinos colhidos no momento do parto e em abatedouro. As células foram recuperadas por meio de digestão enzimática tecidual, realizada com solução de colagenase 0,1%. Foram colhidas amostras de MA e CU no momento do parto (n=6) e de MA e CU no terço inicial de gestação (n=6), bem como de SCU no terço médio de gestação em abatedouro (n=6), as quais foram submetidas às análises morfológica, imunocitoquímica, imunofenotípica por citometria de fluxo e diferenciações in vitro nas linhagens osteogênica, adipogênica e condrogênica e criopreservação. Além disso, foram criopreservadas e analisadas por citometria de fluxo para observação da viabilidade celular. Todas as amostras dos diferentes períodos gestacionais demonstraram adesão ao plástico e morfologia fibroblastóide. No ensaio imunocitoquímico todas as amostras foram imunomarcadas para CD44, NANOG e Oct-4, com ausência de marcação para MHC II. Na análise imunofenotipica por citometria de fluxo, todas as amostras apresentaram marcação para CD44, ausência de marcação para CD34 e baixa expressão de MHC II. Apresentaram também capacidade de diferenciação in vitro nas três linhagens mesodermais e quando analisadas ...
Abstract: Stem cells are undifferentiated cells with a high proliferation potential. These cells can be characterized by their in vivo ability to self-renew and to differentiate into specialized cell lines. The most used stem cell type, in both human and veterinary field, is the mesenchymal stem cells (MSC). Nowadays, there is a great tendency to use stem cells derived from fetal tissues, such as amniotic membrane (AM), extravascular matrix of umbilical cord (EMUC) and umbilical cord blood (UCB), which can be obtained non-invasively at delivery time. Thus, the aim of this study was to isolate, characterize and differentiate. MSC derived from bovine fetal annexes cow dairy neonates after assisted delivery and of fetus obtained in slaughterhouse. Cells were isolated by enzymatic digestion of the tissue fragments with 0,1% collagenase solution. Six samples of AM and EMUC at delivery time, six samples of AM and EMUC at first third of pregnancy and six samples of UCB at secondary third of pregnancy were subjected to morphology evaluation, immunocytochemistry, flow cytometry and in vitro osteogenic, adipogenic and chondrogenic differentiation. Moreover, sample were cryopresrved and evaluated for viability by flow citometri. All samples showed adherence to plastic and fibroblast-like morphology. Immunocytochemistry revealed expression of CD 44, NANOG and OCT-4 and lack of expression of MHC II in MSC from all samples. Flow cytometry demonstrated that cells from all samples expressed CD 44, lacked CD 34 expression and showed low expression of MHC II. They were also capable of trilineage mesenchymal differentiation and showed 80-90% viability after cryopreservation. According to the results, AM, EMUC and UCB, either obtained at delivery time or from slaughterhouse, are a painless and non-invasive source of MSC and can be used for stem cell bank creation
Mestre

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19

Campos, Loreta Lemes [UNESP]. "Isolamento, cultivo, caracterização e criopreservação de células tronco mesenquimais derivadas de membrana amniótica, geléia de Wharton e sangue do cordão umbilical de fetos bovinos." Universidade Estadual Paulista (UNESP), 2014. http://hdl.handle.net/11449/123860.

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Made available in DSpace on 2015-06-17T19:34:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-11-10. Added 1 bitstream(s) on 2015-06-18T12:47:21Z : No. of bitstreams: 1 000831319.pdf: 1267209 bytes, checksum: c23ca8ee7c298aed36bdf625fb1e71a9 (MD5)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
As células tronco mesenquimais (CTMs) estão presentes na maioria dos tecidos adultos e possuem grande capacidade de multiplicação, o que as tornam muito atrativas para a terapia celular, tanto na medicina humana como na medicina veterinária . Quando cultivadas in vitro são capazes de se auto renovar e dar origem a novos tipos celulares. Atualmente há uma grande tendência para a utilização de CTMs obtidas de tecidos fetais, como a membrana amniótica (MA), matriz extravascular do cordão umbilical (CU) e sangue do cordão umbilical (SCU), podendo ser obtidas no momento do parto por uma técnica não invasiva. Dessa forma, o objetivo deste estudo foi isolar, caracterizar, diferenciar e criopreservar CTMs obtidas de MA, CU e SCU de fetos bovinos colhidos no momento do parto e em abatedouro. As células foram recuperadas por meio de digestão enzimática tecidual, realizada com solução de colagenase 0,1%. Foram colhidas amostras de MA e CU no momento do parto (n=6) e de MA e CU no terço inicial de gestação (n=6), bem como de SCU no terço médio de gestação em abatedouro (n=6), as quais foram submetidas às análises morfológica, imunocitoquímica, imunofenotípica por citometria de fluxo e diferenciações in vitro nas linhagens osteogênica, adipogênica e condrogênica e criopreservação. Além disso, foram criopreservadas e analisadas por citometria de fluxo para observação da viabilidade celular. Todas as amostras dos diferentes períodos gestacionais demonstraram adesão ao plástico e morfologia fibroblastóide. No ensaio imunocitoquímico todas as amostras foram imunomarcadas para CD44, NANOG e Oct-4, com ausência de marcação para MHC II. Na análise imunofenotipica por citometria de fluxo, todas as amostras apresentaram marcação para CD44, ausência de marcação para CD34 e baixa expressão de MHC II. Apresentaram também capacidade de diferenciação in vitro nas três linhagens mesodermais e quando analisadas ...
Stem cells are undifferentiated cells with a high proliferation potential. These cells can be characterized by their in vivo ability to self-renew and to differentiate into specialized cell lines. The most used stem cell type, in both human and veterinary field, is the mesenchymal stem cells (MSC). Nowadays, there is a great tendency to use stem cells derived from fetal tissues, such as amniotic membrane (AM), extravascular matrix of umbilical cord (EMUC) and umbilical cord blood (UCB), which can be obtained non-invasively at delivery time. Thus, the aim of this study was to isolate, characterize and differentiate. MSC derived from bovine fetal annexes cow dairy neonates after assisted delivery and of fetus obtained in slaughterhouse. Cells were isolated by enzymatic digestion of the tissue fragments with 0,1% collagenase solution. Six samples of AM and EMUC at delivery time, six samples of AM and EMUC at first third of pregnancy and six samples of UCB at secondary third of pregnancy were subjected to morphology evaluation, immunocytochemistry, flow cytometry and in vitro osteogenic, adipogenic and chondrogenic differentiation. Moreover, sample were cryopresrved and evaluated for viability by flow citometri. All samples showed adherence to plastic and fibroblast-like morphology. Immunocytochemistry revealed expression of CD 44, NANOG and OCT-4 and lack of expression of MHC II in MSC from all samples. Flow cytometry demonstrated that cells from all samples expressed CD 44, lacked CD 34 expression and showed low expression of MHC II. They were also capable of trilineage mesenchymal differentiation and showed 80-90% viability after cryopreservation. According to the results, AM, EMUC and UCB, either obtained at delivery time or from slaughterhouse, are a painless and non-invasive source of MSC and can be used for stem cell bank creation

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20

Karadas, Ozge. "Collagen Scaffolds With In Situ Grown Calcium Phosphate For Osteogenic Differentiation Of Wharton." Master's thesis, METU, 2011. http://etd.lib.metu.edu.tr/upload/12612975/index.pdf.

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COLLAGEN IN SITU GROWN CALCIUM PHOSPHATE SCAFFOLDS FOR OSTEOGENIC DIFFERENTIATION OF WHARTON&rsquo
S JELLY AND MENSTRUAL BLOOD STEM CELLS Karadas, Ö
zge M.Sc., Department of Biotechnology Supervisor : Prof. Dr. Vasif Hasirci Co-Supervisor: Assoc. Prof. Dr. Gamze Torun Kö
se February 2011, 91 pages The importance of developing new techniques for the treatment of bone and joint diseases is increasing continuosly together with the increase of human population and the average life span. Especially bone fractures as a result of osteoporosis are often seen in humans older than 50 years old. The expenses of bone and joint disease operations are very high and the duration of recovery is long. Because of these reasons World Health Organization, The United Nations and 37 countries announced that the years 2000-2010 is the Bone and Joint Decade. Tissue engineering is an alternative approach to clinically applied methods. In this study collagen scaffolds crosslinked with genipin, to improve the stability of foams in culture media, were prepared by lyophilization. To mimic the natural bone structure calcium phosphate mineral phase in the foam was formed by wet chemical precipitation. Collagen concentration (0.75% and 1%, w/v), freezing temperature (-20 oC and -80 oC) of the collagen solution before lyophilization and immersion duration (2x4 h and 2x48 h) of the foams in calcium and phosphate solutions for wet chemical precipitation were changed as process v parameters of foam production. Pore size distribution and porosity analysis as well as compression test were performed for characterization of the scaffolds. The foam with 1% w/v collagen concentration, frozen at -20 oC before lyophilization and immersed for 2x4 h in calcium and phosphate solution was chosen for in vitro cell culture studies. The defined foam had 70% porosity and pore sizes varying between 50 and 200 &mu
m. The elastic modulus and compressive strength of the foam was calculated as 127.1 kPa and 234.5 kPa, respectively. Stem cells isolated from Wharton&rsquo
s jelly (WJ) and menstrual blood (MB) were seeded to foams to compare their osteogenic differentiation. Both cells are isolated from discarded tissues and used in this study as an alternative to the commonly used cells which are isolated by invasive techniques such as bone marrow stem cells. Cells were seeded to collagen foams with and without calcium phosphate (CaP). It was observed that WJ cells proliferated during 21 days on collagen foams without CaP, but MB cell number decreased after day 14. Collagen foams with CaP supported the alkaline phosphate (ALP) activity compared to tissue culture polystyrene (TCPS) and foams without CaP. Contrarily lower cell numbers achieved on CaP containing collagen foams, possibly because of the calcium and phosphate concentration changes in the medium and as the result of osteogenic differentiation. ALP activity of both cell types increased almost 10 times and specific ALP activity (activity per cell) increased 40 times and 150 times for WJ and MB cells, respectively on the CaP containing foams compared to TCPS. Therefore, in this study it was shown that in situ CaP formed collagen foams induce osteogenic differentiation of WJ and MB cells, and these cells isolated from discarded tissues can be used as alternative cell sources in bone tissue engineering applications.

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21

Mechiche, Alami Saad. "Substrats phospho-calciques pour la régénération osseuse." Thesis, Reims, 2016. http://www.theses.fr/2016REIMS003.

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L’ingénierie du tissu osseux est un domaine qui représente un enjeu majeur dans le cadre de la médecine régénératrice. Trois composants sont généralement décrits dans le cadre de l’ingénierie tissulaire : un biomatériau pour pallier le volume de tissu défectueux, une source de cellules progénitrices qui seront responsables de la synthèse des composants tissulaires ainsi que des facteurs de croissance ou des signaux issus des propriétés physico-chimiques du biomatériau afin de guider la prolifération et la différenciation cellulaire. Le but de cette étude a été de synthétiser des substrats phospho-calciques à l’aide de la technique de pulvérisation simultanée d’espèces réactives et de caractériser les différentes propriétés physico-chimiques des substrats obtenus. Nous avons pu démontrer la possibilité d’inclure des molécules organiques (chitosane et acide hyaluronique) à la phase minérale avec cette technique. Nous avons aussi montré la possibilité de faire varier des propriétés telles la rugosité (entre 300 et 700 nm), l’élasticité (entre 2 à 6 GPa), la composition chimique (phosphate octacalcique ou phosphate dicalcique dihydraté) et la bioactivité (précipitation des phosphates de calcium à la surface des substrats) avec la technique de pulvérisation. Par ailleurs, des cellules souches issues de la gelée de Wharton de cordons ombilicaux humains ont été isolées, puis caractérisées sur le plan génique et protéique. Ces cellules étant candidates pour l’utilisation en ingénierie tissulaire osseuse, nous nous sommes intéressés à plusieurs types de marqueurs dont les marqueurs mésenchymateux et les cytokines immuno-modulatrices.La dernière partie de cette thèse a concerné l’association des cellules souches isolées à partir de la gelée de Wharton aux substrats phospho-calciques obtenus à l’aide de la technique de pulvérisation. Nous avons pu démontrer que les cellules adhéraient sur ces substrats et s’organisaient en structures nodulaires au sein desquelles a été observée une couche de cellules sécrétrices entourant des fibres de collagène, des formations cristallines faîtes de phosphates de calcium et des cellules dont la morphologie rappelait celle des ostéocytes. Des variations dans l’expression de marqueurs ostéoblastiques ont aussi été observées, et ce en l’absence de facteurs solubles ostéogéniques dans le milieu de culture. En conclusion, les substrats phospho-calciques obtenus avec la technique de pulvérisation sont capables d’induire la différenciation de cellules souches issues du cordon ombilical en ostéoblastes. Ce modèle se révèle être prometteur pour la mise en place de thérapies en vue de la régénération du tissu osseux
Bone tissue engineering is a major issue within regenerative medicine. There are three main components in the field of tissue engineering: a scaffold providing a structure for tissue development, a source of stem cells for tissue formation and growth factors or physical stimuli from the biomaterial to direct growth and differentiation of cells. The purpose of this study was to synthesize calcium phosphate substrates by simultaneous spraying of interacting species and to carry out the physico-chemical characterization of the built substrates. We showed that the spraying technique allows the inclusion of organic molecules such as chitosan and hyaluronic acid. The spraying technique allows several physio-chemical characteristics to be varied, rugosity (300 – 700 nm), elasticity (2 – 6 GPa), chemical composition (octacalcium phosphate or dicalcium phosphate dehydrate), but also studied the bioactivity of the substrates (calcium phosphate from the culture medium precipitates at thesurface of the substrates). In another hand, our aim was to isolate stem cells from human umbilical cords’ Wharton’s Jelly and to carry out their genic and proteic characterization by focusing on mesenchymal markers and immunomodulating cytokines, knowing that these cells are candidates for a use in bone regeneration therapy.The last purpose of our study was to evaluate the potential of Wharton’s jelly stem cells to adhere and proliferate onto the sprayed substrates, and also the formation of nodules. The ultrastructural analysis of nodules formed by Wharton’s jelly stem cells showed a layer of secretory cells surrounding collagen fibers, calcium phosphate crystals and cells with a similar morphology to that of osteocytes. Osteoblastic markers appeared to be regulated in cells cultured without osteogenic supplements. To conclude, sprayed calcium phosphate substrates seem to induce osteoblastic differentiation of Wharton’s jelly stem cells through the substrate’s physico-chemical properties. Our model appears as promising for further bone regenerative therapies

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Paladino, Fernanda Vieira. "Potencial imunomodulador de células-tronco mesenquimais humanas de geleia de Wharton submetidas à senescência replicativa." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/5/5146/tde-10112017-122109/.

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INTRODUÇÃO: Células-tronco mesenquimais da geleia de Wharton (GW-CTM) exibem a capacidade de modular a resposta das células T e geralmente esses efeitos imunomoduladores são anti-inflamatórios. Devido ao seu potencial imunossupressor, as CTM emergiram recentemente como uma ferramenta promissora para terapia celular. No entanto, essas células têm uma vida útil limitada in vitro, com redução progressiva da sua capacidade de autorrenovação e parada irreversível do ciclo celular. O resultado deste processo é a perda da funcionalidade das CTM, o que limita a sua utilização para fins terapêuticos. Pouco se sabe sobre a variabilidade individual das amostras de GW-CTM e como isso poderia afetar sua expansão in vitro, seu potencial imunomodulador e o processo de envelhecimento. Neste estudo, avaliamos o perfil de citocinas imunomoduladoras e a capacidade das GW-CTM inibir a proliferação de células T durante o processo de senescência replicativa para determinar se a resposta esperada é afetada. MÉTODOS: GW-CTM foram cultivadas até a senescência replicativa ser atingida; as amostras foram coletadas numa fase inicial (passagem 5), numa etapa intermediária (passagem 15) e na senescência replicativa (passagem geralmente entre 20 e 25) para análise do perfil basal de moléculas imunomoduladoras. GW-CTM foram cocultivadas com amostras de duas células mononucleares de sangue periférico (PBMC), obtidas de doadores de plaquetas saudáveis. PBMC foi estimulado com fitohemaglutinina (PHA) durante 72 horas e cocultivado com três amostras de GW-CTM diferentes para medir a supressão da proliferação das células T. Os experimentos foram realizados utilizando as passagens P5 e P10. As análises foram feitas por PCR em tempo real, western blot e citometria de fluxo. RESULTADOS: Nossos resultados mostram que a expressão gênica e a secreção de moléculas imunomoduladoras variam entre amostras de GW-CTM, sem padrão específico. Em cocultura, todas as GW-CTM foram capazes de inibir a proliferação de células T CD3+ ativadas por mitogéno, embora em diferentes graus. E cada PBMC respondeu à cocultura com um nível diferente de inibição. Além disso, o perfil imunomodulador de todas as GW-CTM foi essencialmente mantido, mesmo após várias passagens. CONCLUSÃO: Nossos dados indicam que cada GW-CTM exibe um comportamento único, diferindo nos padrões de expressão e secreção de citocinas e capacidade imunomoduladora. Essa variabilidade intrínseca entre amostras pode influenciar a eficácia de GW-CTM quando utilizadas em terapia celular
INTRODUCTION: Wharton\'s jelly mesenchymal stem cells (WJ-MSC) exhibit the ability to modulate T cell responses and these immunomodulatory effects are usually anti-inflammatory. Due to their immunosuppressive potential, MSC have recently emerged as a promising tool for cell therapy. However, MSC have a limited lifespan in vitro, with a progressive reduction in their capacity for selfrenewal leading to irreversible arrest of cell division. The result of this process is the loss of stem cell functionality, which limits its use for therapeutic purposes. Information on the variability of individual cell samples impacting upon in vitro expansion, immunomodulatory potential, and aging processes is still lacking. In this study, we evaluated the immunomodulatory cytokine profile and capacity to inhibit T cell proliferation of WJ-MSC progressing to replicative senescence to determine if the expected response is affected. METHODS: WJ-MSC were cultured until replicative senescence was reached and the samples were collected at an early stage (passage 5), at an intermediate stage (passage 15), and in replicative senescence (passage generally between 20 and 25) to analyze the basal profile of immunomodulatory molecules. WJ-MSC were co-cultured with samples from the same two peripheral blood mononuclear cells (PBMC), obtained from healthy platelet donors. PBMC were stimulated with phytohemagglutinin (PHA) for 72 hours and tested against 3 different WJ-MSC to measure suppression of T cell proliferation. The experiments were performed using passages P5 and P10. Analyses were done by real-time PCR, western blot, and flow cytometry. RESULTS: Our results show that gene expression and secretion of immunomodulatory molecules varied among WJ-MSC samples with no specific pattern discernible. In co-culture all WJ-MSC were capable of inhibiting mitogen-activated CD3+ T cell proliferation, although to different extents and each PBMC responded with its unique level of inhibition. In addition, the immunomodulatory profile of each WJ-MSC sample was essentially maintained even after several passages. CONCLUSION: Our results indicate that each WJMSC displays a unique behavior, differing in patterns of cytokine mRNA expression and immunomodulatory capacity. The intrinsic variability between samples may influence the effectiveness of WJ-MSC when employed therapeutically

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Millan, Rivero Jose Eduardo. "= Estudio del efecto terapéutico de las células mesenquimales de la gelatina de Wharton del cordón umbilical humano en un modelo Murino de cicatrización de heridas = Study of the therapeutic effect of human Wharton's jelly mesenchymal stem cells in an experimental Murine model of wound healing." Doctoral thesis, Universidad de Murcia, 2016. http://hdl.handle.net/10803/371747.

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Introducción: La piel es el órgano más extenso del cuerpo humano. Las heridas cutáneas son producto de la ruptura de la integridad de la piel. Las células mesenquimales (MSCs) están emergiendo como fuertes candidatas para ser usadas como terapia celular en el tratamiento de heridas crónicas, dado su enorme potencial para mejorar la reparación y regeneración de los tejidos posterior a una lesión. Constructos biosintéticos fabricado con la fibroína de la seda celularizados con MSCs de diferentes fuentes han demostrado ser eficaces en la curación de heridas experimentales de la piel. Objetivos: investigar el comportamiento de las células mesenquimales de la gelatina de Wharton del cordón umbilical humano (hWj-MSCs) tanto in vitro como in vivo tras ser sembradas en un constructo electrohilado compuesto de fibroína de la seda y posteriormente implantado en ratones inmunocompetentes. Determinar si las hWj-MSCs aplicadas mediante constructos de fibroína de seda en heridas creadas en el dorso de ratones SKH1 contribuyen a mejorar la cicatrización de la lesión. Metodología: inicialmente hemos puesto a punto el protocolo para el aislamiento y caracterización de las hWj-MSCs. Posteriormente, estas MSCs en combinación con un constructo fabricado a partir de la fibroína de la seda fueron utilizados para probar sus propiedades terapéuticas en un modelo murino experimental de heridas. Resultados: Las hWj-MSCs mostraron una morfología fibroblástica, y presentaron en su superficie la expresión de marcadores mesenquimales que fueron positivos para CD90, CD105, CD73, y sin presencia detectable de marcadores de células hematopoyéticas. Las hWj-MSCs mostraron una capacidad limitada para diferenciarse hacia adipocitos, condrocitos, y osteoblastos. Las hWj-MSCs inhibieron de forma significativa la proliferación de linfocitos T tras ser estimulados con esferas anti-CD3/CD28, así como con células dendríticas maduras alogénicas. Las hWj-MSCs disminuyeron la producción in vitro de la citoquina pro-inflamatoria IFN- por los linfocitos T activados. Este efecto inmunosupresor también estuvo mediado por la producción de los factores anti-inflamatorios TGF-, IDO, y PGE2. Los experimentos con cultivos mixtos de linfocitos en presencia de diferentes inhibidores específicos de la biosíntesis o la señalización de estos factores anti-inflamatorios tales como SB-431542, indometacina (IDM), o 1-metil-triptófano (1-MT) respectivamente, recuperaron casi en su totalidad el nivel de proliferación de las células T estimuladas con células dendríticas maduras. El tratamiento de las heridas de la piel en ratones SKH1 demostró que la combinación de hWj-MSCs y el constructo de fibroína de la seda mejoró significativamente la capacidad de cicatrización de heridas. Conclusiones: Las hWj-MSCs son poco inmunogénicas, no expresan moléculas coestimuladoras, y expresan citoquinas que pueden modular la función inmune. La combinación de hWj-MSCs con los constructos de fibroína de seda contribuyó a la generación de un tejido de granulación de alta calidad, y escasa formación de tejido cicatrizal. La gelatina de Wharton del cordón umbilical humano puede ser una fuente adecuada para la obtención de MSCs para ser utilizadas en la cicatrización de heridas debido a sus diversas ventajas y junto con los beneficios sinérgicos de un constructo pudieran conformar una combinación ideal como apósitos para heridas crónicas de lenta y difícil curación.
Introduction: the skin is the largest organ in the human body. Cutaneous wounds are the result of disrupted skin integrity. Mesenchymal stem cells (MSCs) are emerging as a promising candidate for cell-based therapy for the treatment of chronic wounds because of their enormous potential for enhancing tissue repair and regeneration following injury. Silk fibroin cellularized with MSCs from different sources has been shown to be effective in repairing experimental wounds of the skin. Objectives: to investigate the behavior of Wharton’s jelly mesenchymal stem cells (Wj-MSCs) both in vitro and in vivo when cultivated on electrospun silk fibroin scaffolds and implanted in immunocompetent mice. To determine whether Wj-MSCs delivered in silk fibroin scaffolds into skin defects in SKH1 mice would contribute to dermal wound healing. Methodology: we have first standardized the protocol for the isolation and characterization of Wj-MSCs. Further, these MSCs along with the combination of silk fibroin scaffolds were used to test their wound healing properties by creating skin wounds in an experimental murine model. Results: Wj-MSCs exhibited a fibroblastic morphology and displayed MSC surface markers positive for CD90, CD105, CD73 with no detectable presence of hematopoietic cells markers. Wj-MSCs were limited on their ability to differentiate into adipocytes, chondrocytes, and osteoblasts. Wj-MSCs suppressed T cell proliferation after stimulation with anti-CD3/CD28 coated beads and allogeneic mDCs. Wj-MSCs downmodulated the in vitro production of the pro-inflammatory cytokine IFN- by activated T lymphocytes. This immunosuppressive effect was also mediated by the production of the anti-inflammatory cytokines TGF-, IDO, and PGE2. MLCs experiments in the presence of different specific inhibitors of the biosynthesis or signaling of these anti-inflammatory factors such as SB-431542, indomethacin (IDM) or 1-methyl-tryptophan (1-MT), respectively, recovered almost entirely the proliferation rate of mDCs-stimulated T cells. Treatment of skin injury of SKH1 mice model demonstrated that combination of Wj-MSCs and silk fibroin scaffold exhibited significantly better wound-healing capabilities. Conclusions: Wj-MSCs have reduced immunogenicity, did not express costimulatory molecules, and express cytokines that may modulate immune function. Wharton's jelly MSCs combined with silk fibroin scaffolds in the wound bed contributed to the generation of a high-quality, well-vascularized granulation tissue, enhanced re-epithelialization of the wound, and attenuated the formation of fibrotic scar tissue. Wharton's jelly from the human umbilical cord may be an adequate source for obtaining MSCs for wound healing given its several advantages and together with the synergistic benefits of a nanoscaffold they make ideal combinations as wound dressings for slow healing and hard-to-heal chronic wounds.

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Margossian, Talar. "Caractérisation des cellules souches mésenchymateuses du sang placentaire et de la gelée de Wharton." Thesis, Université de Lorraine, 2013. http://www.theses.fr/2013LORR0030/document.

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Les cellules souches suscitent de grands espoirs pour la thérapie cellulaire et l'ingénierie tissulaire. Les CSM du tissus foetaux (sang placentaire et gelée de Wharton du cordon ombilical), à l'origine d'épiblaste embryonnaire, sont considérées comme plus primitives que les CSM provenant de sources adultes. Les conditions de culture ayant un impact sur le comportement des cellules, dans notre étude, nous avons exploré l'effet de la concentration de l'oxygène sur l'expansion, l'immunophénotypage et la différenciation de ces cellules. L'objectif de ce travail est d'identifier la méthode optimale d'isolation des CSM issues de tissus foetaux. Compte tenu du faible taux de succès dans l'isolement des CSM extraites du sang placentaire, nous nous sommes dirigés vers les CSM-GW. Nous y avons déterminé in situ, les marqueurs spécifiques exprimés dans la gelée de Wharton et à la périphérie. Des études sur la morphologie, la cinétique de croissance, et sur l'expression phénotypique des marqueurs de surface, des CSM-GW, ont été effectuées sur une longue durée (7 passages) à différentes conditions de culture. Nous avons montré que la GW est composée d'une abondante matrice extracellulaire riche en collagènes et glycosaminoglycannes et que les cellules possèdent un phénotype variable selon leur localisation dans la gelée. Ce tissu est capable de fournir une quantité importante de CSM (6,7x105 Cs/cm de cordon) qui gardent une morphologie constante. Enfin, quel que soit le passage, la concentration de l'oxygène ne semble pas avoir d'effet sur le phénotype des cellules. En revanche, une faible teneur en oxygène durant l'expansion semble diminuer le temps de doublement des cellules, favoriser la chondrogénèse et inhiber la différenciation ostéogénique. Enfin, quelles que soient les conditions de culture, la différenciation adipogénique des CSM-GW semble difficile à obtenir
Stem cells are the hopes for cell therapy and tissue engineering. MSCs from fetal tissue (umbilical cord blood and WJ), which are a source of embryonic epiblast grow relatively faster comparing to other adult sources. The culture condition can affect cell behavior. In our study, we explored the effect of oxygen concentration on the expansion, immunophenotyping, and differentiation of these cells. The aim of this work is to identify the optimal method for isolation of MSCs derived from fetal tissue. Given the low rate of success in the isolation of MSCs from cord blood, we headed to WJ-MSCs. We have determined in siu, the specific markers expressed in the WJ and in the perivascular region. Studies on the morphology growth kinetics, and phenotypic expression of surface makers of MSCs isolated from WJ were made over a long period (7 passages) in different culture conditions. We have shown that WJ is composed of an abundant extracellular matrix rich in collagen and glycasominoglycans and have variable phenotype depending from their localization in the jelly. This tissue is able to provide a large amount of MSCs (6.7x105 Cs/cm of cord) that maintain a constant morphology. Finally, regardless of the passage, the oxygen concentration does not effect on the phenotype of the cells. In contrast, a low oxygen concentration during expansion appears to decrease the doubling time of MSCs, promote chondrogenesis and inhibit osteogenic differentiation. Finally, whatever the culture conditions, adipogenic differentiation of WJ-MSC seems difficult to obtain

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Carvalho, Ana Carolina Bazan de. "Expressão da resistência à múltiplas drogas em células-tronco mesenquimais do cordão umbilical humano." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/5/5167/tde-08112013-094537/.

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INTRODUÇÃO: As células-tronco mesenquimais (CTM) são células adultas multipotentes capazes de se diferenciarem em várias linhagens celulares. Estudos com CTM derivadas do cordão umbilical humano (CUh), mais especificamente da Geleia de Wharton (GW), tem demonstrado um grande potencial para a terapia de reposição celular e regeneração tecidual. O papel dos genes de resistência a múltiplas (MDR) drogas, tais como ABCB1 e LRP é bem conhecido nos processos de regulação, fisiologia e defesa. Entretanto, nenhum estudo demonstrou ainda a presença desses genes nas CTM da GW do CUh. Assim, o objetivo do presente estudo foi analisar a expressão de Pg-p e LRP em CTM derivadas da GW. MATERIAIS E MÉTODOS: CTMs da GW isoladas de CUh (n = 20) foram caracterizadas quanto ao seu estado indiferenciado por: citometria de fluxo; expressão de genes Oct-4 e Nanog por RT-PCR e capacidade de diferenciação adipogênica e osteogênica in vitro. Foi analisado ainda a expressão dos genes ABCB1 e LRP por RT-PCR em tempo real de células indiferenciadas. A resistência a doxorrubicina (DOX) foi determinada através do ensaio de MTT nessas mesmas amostras. RESULTADOS: As CTM da GW do CUh apresentaram positividade para marcadores de superfície presentes nas CTM, tais como, CD29, CD44, CD90 e CD105. Os genes de indiferenciação celular Oct-4 e Nanog foram expressos em todas as amostras, que também foram capazes de se diferenciar em adipócitos e osteócitos, comprovando assim que essas células são CTM. Com relação à Pg-p, o gene ABCB1 não amplificou em 18 amostras sendo que somente 2 apresentaram baixa expressão gênica. O gene LRP amplificou de forma intensa em todas as amostras analisadas. CONCLUSÃO: Concluímos que as CTM derivadas da GW do CUh apresentam um elevado nível de expressão de LRP, sugerindo que este gene pode estar envolvido no processo de regulação das células-tronco mesenquimais, bem como na fisiologia e defesa celular
INTRODUCTION: Mesenchymal stem cells (MSCs) are multipotent adult cells that can differentiate into various cell lineages. Studies with MSC derived from human umbilical cord (hUC), more specifically from Wharton jelly (WJ), have shown great potential for cellular replacement therapy and tissue regeneration. The role of multidrug resistance genes, such as ABCB1 and LRP is well known in the regulation, physiology and cellular defense. However, the presence of these genes in the MSC of the WJ from hUC was not demonstrated yet. The aim of this study was to analyze the expression of Pg-p and LRP in MSCs derived from WJ. MATERIALS AND METHODS: The MSC from WJ were isolated from hUC (n = 20) and were characterized by: flow cytometry; Oct-4 and Nanog gene expression by RT-PCR and adipogenic and osteogenic in vitro differentiation capability of these cells. It was also analyzed gene expression of ABCB1 and LRP by real time RT-PCR of undifferentiated cells. Doxorubicin (DOX) resistance was determined by MTT assay in all the samples. RESULTS: MSC WJ from hUC was positive for MSC surface markers, such as CD29, CD44, CD90 and CD105. The undifferentiated cell genes Oct-4 and Nanog were expressed in all samples, which were also capable to differentiate into adipocytes and osteocytes, proving that these cells are MSC. Concerning to Pgp, the ABCB1 gene, 18 samples didn\'t show any amplification product (no expression) while 2 showed little gene expression. The LRP gene amplified intensely in all samples. CONCLUSION: We conclude that MSC derived from the WJ hUC have a high level of LRP expression, suggesting that this gene may be involved in the regulation of mesenchymal stem cells as well as in physiology and cellular defense

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Barbieri, Cristiane. "Medidas ultra-sonograficas da secção transversal do cordão umbilical e de seus vasos, segundo idade gestacional, em gestações de baixo risco." [s.n.], 2007. http://repositorio.unicamp.br/jspui/handle/REPOSIP/310048.

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Orientador: Jose Guilherme Cecatti
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
Made available in DSpace on 2018-08-08T20:46:57Z (GMT). No. of bitstreams: 1 Barbieri_Cristiane_M.pdf: 3185188 bytes, checksum: b566f66bd024181e78245a8dd5211fe7 (MD5) Previous issue date: 2007
Resumo: Introdução: mais recentemente, demonstrou-se que o diâmetro do cordão umbilical pode se modificar nos casos de diabetes mellitus, pré-eclâmpsia, restrição de crescimento intra-uterino e baixo peso ao nascimento, podendo talvez ser utilizado como um marcador para detecção precoce destas condições. Objetivo: obter intervalos de referência das medidas ultra-sonográficas da área do cordão umbilical, dos diâmetros de seus vasos e da área da superfície da Geléia de Wharton da secção transversa do cordão umbilical em função da idade gestacional em gestações de baixo risco, entre 12 e 40 semanas, avaliar a variabilidade inter- e intra-observador destas medidas e investigar sua correlação com o peso fetal estimado. Método: foram avaliadas 2310 gestantes no período entre junho de 2005 e dezembro de 2006, seguindo critérios de inclusão e exclusão pré-estabelecidos. Em uma sub-amostra destas gestantes foi avaliada a variabilidade inter- e intraobservador, estimando-se o coeficiente de correlação de Spearman, o coeficiente de correlação intra-classe e o alfa de Crombach. Para cada idade gestacional, foi avaliado um número mínimo de 59 casos, calculando-se a média e seu respectivo desvio-padrão e os percentis 10, 50 e 90 de cada uma das medidas. Para a análise estatística foram utilizados os testes t de Student, Anova e Wilcoxon para amostras independentes. Os intervalos de referência foram estimados por regressão polinomial de terceiro grau. Foi avaliado também o desempenho da área da secção transversa do cordão umbilical, do diâmetro do cordão umbilical e da área de geléia de Wharton do cordão umbilical em predizer alterações do peso fetal estimado (PFE) nestas gestações, estimando-se sua sensibilidade, especificidade, valor preditivo positivo e negativo. Resultados: Foram obtidas elevadas correlação, reprodutibilidade e confiabilidade na avaliação da variabilidade inter e intra-observador das medidas do cordão umbilical e de seus vasos. Os intervalos de referência apresentaram valores crescentes até cerca de 32 semanas e depois estabilizaram-se. As medidas avaliadas apresentaram baixa sensibilidade para predição de alterações do PFE. Portanto, elas não devem ser utilizadas para rastreamento com esta finalidade. Conclusões: os valores normais padronizados para essas medidas em todas as idades gestacionais, para gestações de baixo risco na população de referência, foram determinados, havendo a necessidade de que sejam posteriormente validados como preditores de situações perinatais adversas
Abstract: Introduction: recently it has been demonstrated that the diameter of the umbilical cord may be modifyed in the case of diabetes, preeclampsia, intrauterine growth restriction and low birthweight. In this way it could perhaps be used as a marker for early detection of these conditions. Objective: to obtain reference ranges for the ultrasonographic measurements of the umbilical cord area, the diameters of its vessels and the area of the Wharton Jelly surface from a cross sectional plan of the umbilical cord according to gestational age in low risk pregnancies between 12 and 40 weeks; to evaluate the inter- and intra-observer variability of these measurements; and to investigate their correlation with the estimated fetal weight. Method: a total of 2310 pregnant women were evaluated in the period from June 2005 and December 2006, following previously stablished inclusion and exclusion criteria. Inter and intra-observer variability were evaluated in a sub sample of these pregnant women, with the estimation of Spearman correlation coefficient, the intraclass correlation coefficient and alfa of Crombach. For each gestational age a minimum number of 59 cases were evaluated. For statistical analysis mean and standard deviation and the percentiles 10, 50 and 90 for each one of the measurements were estimated. Student t, Anova and Wilcoxon tests for independent samples were used. The reference ranges were estimated by third degree polynomial regression. The performance of the area of the transverse section of umbilical cord, its diameter and the area of the Wharton Jelly in predicting deviations of the estimated fetal weight (EFW) was also estimated among these pregnancies, with their sensitivity, specificity, positive and negative predictive values. Results: High correlation, reproductibility and feasibility were obtained when evaluating the inter- and intra-observer variability of the measurements of umbilical cord and its vessels. The reference intervals presented increasing values up to around 32 weeks, and afterwards they estabilized. The measurements evaluated showed very low sensitivity for predicting deviations of the EFW. Therefore they should not be used for screening with this purpose. Conclusions: the normal standardized values for these measurements in all gestational ages, for low risk pregnancies in the reference population, were determined. There is still the need of them being validated as predictors of adverse perinatal conditions
Mestrado
Tocoginecologia
Mestre em Tocoginecologia

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Capcha, José Manuel Cóndor. "Células-tronco mesenquimais derivados da geleia de Wharton na injúria cardiopulmonar e neuroimunomodulação sistêmica na sepse." Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/5/5160/tde-11092018-132813/.

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A sepse causa uma alta taxa de mortalidade no mundo. A fisiopatologia da doença envolve uma rede complexa de mediadores inflamatórios que promovem a lesão de diversos tecidos, além de diversas alterações hemodinâmicas e disfunção do sistema nervoso autonômico (SNA). Assim sabe-se que o sistema nervoso cumpre um papel importante no controle da inflamação sistêmica mediante a via colinérgica anti-inflamatória (VCA) através do receptor nicotínico de acetilcolina alfa7 (alfa7nAChR). O uso das células-tronco mesenquimais (CTM) tem mostrado efeitos benéficos em diversos ensaios clínicos de doenças inflamatórias. Neste contexto, as células-tronco mesenquimais derivadas da geleia de Wharton do cordão umbilical (CTM-GW) tornam-se promissórias, uma vez que essas células são reconhecidas pela regulação da resposta imunológica, reparação neural, efeito anti-apoptose, assim como a melhora da sobrevida na sepse, em modelos experimentais. Nossa hipótese foi de que as CTM-GW poderiam cumprir um papel neuroimunomodulador através da VCA e atenuar a disfunção de múltiplos órgãos em um modelo animal de sepse de ligadura e punção do ceco (LPC). Inicialmente células da matriz do cordão umbilical foram isoladas e caracterizadas de acordo com o consenso internacional vigente. Ratos Wistar machos adultos foram subdivididos em grupos: 1) sham (operação simulada); 2) LPC; 3) LPC+CTM-GW (injetado 106 CTM-GW via intraperitoneal, i.p. 6 h após LPC) e 4) LPC+MLA+CTM-GW (MLA: Metillicaconitine, antagonista do alfa7nAChR, i.p., 5:30 h após LPC e 106 CTM-GW 6h após). Às 24 horas após LPC, foram avaliadas a função cardiovascular, hemodinâmica assim como os outros parâmetros. Interessantemente, o tratamento com CTM-GW na sepse atenuou a disfunção diastólica e protegeu a sensibilidade baroreflexa. Além disso, as CTM-GW estimularam a atividade autonômica, simpática e parassimpática no coração. Observamos que o tratamento celular induziu uma regulação da expressão do receptor alfa7nAChR e TLR4 no baço e no coração, assim como a redução da relação p-STAT3TYR705 e STAT3 total no baço. Outros efeitos importantes e adicionais foram a diminuição da infiltração de leucócitos e a regulação das citocinas pró-inflamatórias pelas células. O bloqueio da VCA usando MLA confirmou que o receptor alfa7nAChR pode ser um provável alvo, chave da ação das CTM entre vários outros mecanismos envolvidos na resposta imune. Finalmente, as CTM-GW conseguiram reduzir a apoptose no pulmão e no baço independentemente da VAC reforçando o conceito de que as células-tronco tem efeitos diversos além da imuno-regulação. Em conclusão, as CTM-GW na sepse foram capazes de atenuar a lesão cardiopulmonar assim como modular a atividade autonômica, reduzindo a inflamação sistêmica, pelo menos em parte, através da via colinérgica anti-inflamatória. Indubitavelmente todos estes efeitos anteriormente descritos e em associação se demonstraram fundamentais no mecanismo de reparo e proteção tecidual em resposta a sepse. Mais estudos pré-clínicos e futuros testes clínicos precisam ser realizados para maior compreensão destes mecanismos bem como uma possível validação terapêutica
Sepsis induces organ dysfunction due to overexpression of the inflammatory host response, involving cardiorespiratory and autonomic dysregulation, thus increasing the associated morbidity and mortality. The cholinergic anti-inflammatory pathway (CAP) is mediated by nervous system through alpha7 nicotinic acetylcholine receptor (alpha7nAChR). This receptor has an important role in systemic inflammation control. Wharton\'s jelly-derived mesenchymal stem cells (WJ-MSCs) are known to express genes and secreted factors related to neurological and immunological protection, as well as to improve survival in experimental sepsis. We hypothesized that WJ-MSCs play a modulatory role through the CAP and attenuate sepsis-induced organ injury in a cecal ligation and puncture (CLP) model. Rats were randomly divided into 4 groups: 1) Control (sham-operated); 2) submitted to CLP without treatment; 3) submitted to CLP and treated with 106 WJ-MSCs 6 h later and 4) CLP+MLA+WJ-MSC group (MLA: Methyllycaconitine, alpha7nAChR antagonist). All experiments were performed 24 h post-surgery. Echocardiographic parameters and heart rate variability were assessed. Importantly, treatment with WJ-MSCs attenuated diastolic heart failure and recovered barorreflex sensitivity. Moreover, WJ-MSCs injection increased cardiac sympathetic and cardiovagal activity. In cardiac and splenic tissue, WJ-MSC treatment downregulated TLR4 and alpha7nAChR expression, as well as it reduced p-STAT3/Total STAT3 ratio in the spleen. In addition, WJ-MSC reduced leukocyte infiltration and pro-inflammatory cytokines, which only were abolished by MLA treatment. Finally, WJ-MSC treatment diminished apoptosis in lung and spleen tissue. Together these findings suggest that treatment with WJ-MSCs appears to protect against sepsis-induced organ injury reducing systemic inflammation, at least in part, through cholinergic anti-inflammatory pathway

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Kenar, Halime. "3d Patterned Cardiac Tissue Construct Formation Using Biodegradable Materials." Phd thesis, METU, 2008. http://etd.lib.metu.edu.tr/upload/3/12610315/index.pdf.

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The heart does not regenerate new functional tissue when myocardium dies following coronary artery occlusion, or is defective. Ventricular restoration involves excising the infarct and replacing it with a cardiac patch to restore the heart to a more efficient condition. The goal of this study was to design and develop a myocardial patch to replace myocardial infarctions. A basic design was developed that is composed of 3D microfibrous mats that house mesenchymal stem cells (MSCs) from umbilical cord matrix (Wharton&rsquo
s Jelly) aligned parallel to each other, and biodegradable macroporous tubings to supply growth media into the structure. Poly(glycerol sebacate) (PGS) prepolimer was synthesized and blended with P(L-D,L)LA and/or PHBV, to produce aligned microfiber (dia 1.16 - 1.37 &
#956
m) mats and macroporous tubings. Hydrophilicity and softness of the polymer blends were found to be improved as a result of PGS introduction. The Wharton&rsquo
s Jelly (WJ) MSCs were characterized by determination of their cell surface antigens with flow cytometry and by differentiating them into cells of mesodermal lineage (osteoblasts, adipocytes, chondrocytes). Cardiomyogenic differentiation potential of WJ MSCs in presence of differentiation factors was studied with RT-PCR and immunocytochemistry. WJ MSCs expressed cardiomyogenic transcription factors even in their undifferentiated state. Expression of a ventricular sarcomeric protein was observed upon differentiation. The electrospun, aligned microfibrous mats of PHBV-P(L-D,L)LA-PGS blends allowed penetration of WJ MSCs and improved cell proliferation. To obtain the 3D myocardial graft, the WJ MSCs were seeded on the mats, which were then wrapped around macroporous tubings. The 3D construct (4 mm x 3.5 cm x 2 mm) was incubated in a bioreactor and maintained the uniform distribution of aligned cells for 2 weeks. The positive effect of nutrient flow within the 3D structure was significant. This study represents an important step towards obtaining a thick, autologous myocardial patch, with structure similar to native tissue and capability to grow, for ventricular restoration.

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Capcha, José Manuel Cóndor. "Avaliação de células-tronco mesenquimais do cordão umbilical humano em lesão de órgãos e disfunção endotelial na sepse." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/5/5148/tde-14092015-102858/.

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A sepse é uma doença relacionada como a presença de infeção junto a uma resposta inflamatória sistêmica; sua fisiopatologia envolve uma rede complexa de citocinas e mediadores inflamatórios que causam a injúria de diversos tecidos. Na atualidade são muitas as tentativas para diminuir a mortalidade, porém até agora, não existe uma estratégia específica para tratar a doença. As células-tronco mesenquimais da geleia de Wharton do cordão umbilical (CTM-GW) são conhecidas por expressar genes e fatores envolvidos na angiogênese e imunomodulação. Nós usamos o modelo de ligadura e punção do ceco (LPC) para analisar o papel da CTM-GW em disfunção orgânica relacionada à sepse. Foi utilizada a citometria de fluxo para avaliar o fenótipo das células isoladas. Dividimos ratos Wistar em grupos: sham (operação simulada); LPC; e LPC + CTM (106 CTM-GW i.p., 6 horas após LPC). Às 24 h pós-LPC, foram avaliadas a função renal, hepática e outras variáveis do estudo. As CTM-GW foram negativas para CD3, CD34, CD45 e HLA-DR, enquanto eles foram positivos para CD73, CD90 e CD105. O tratamento com CTM na sepse reduziu a mortalidade, melhorou a filtração glomerular (aferido pelo clearance de inulina), função tubular, reduziu a lesão hepática e demostrou uma ação anti-inflamatória. O tratamento também apresentou um efeito anti-apoptótico e protetor do tecido renal e do endotélio, mediante a regulação da expressão de VEGF, AQP2 e eNOS. Em conclusão as CTM-GW diminuem a injúria renal e hepática, portanto, pode desempenhar um papel protetor na sepse
Sepsis is a disease related to the presence of infection with a systemic inflammatory response. The pathophysiology involves complex cytokine and inflammatory mediator networks that cause injury to various tissues. Currently, there are many attempts to reduce mortality, but so far, there is no specific strategy for treating the disease. Human umbilical cord Wharton\'s jelly-derived mesenchymal stem cells (hWJ-MSCs) are known to express genes and factors involved in angiogenesis and immunomodulation. We used a cecal ligation and puncture (CLP) model to analyze the role of hWJ-MSCs in sepsis-related organ dysfunction. We used flow cytometry to evaluate hWJ-MSC phenotypes. We divided Wistar rats into groups: sham (sham-operated); CLP; and CLP+MSC (106 WJ-MSCs, i.p., 6 h after CLP). At 24 h post-CLP, we evaluated renal function, liver and other variables. hWJ-MSCs were negative for CD3, CD34, CD45 and HLA-DR, whereas they were positive for CD73, CD90 and CD105. In sepsis, treatment with MSC reduced mortality, improved glomerular filtration rate (measured by inulin clearance), tubular function, reduced liver damage and decreased the inflammatory markers. The treatment also showed an anti-apoptotic effect and protected the renal tissue and endothelium by up-regulation the expression of VEGF, AQP2 and eNOS. In conclusion, hWJ-MSCs decrease renal and hepatic injury, therefore, may play a protective role in sepsis

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30

Raicevic, Gordana. "Influence of microbial products and inflammation on the function of mesenchymal stromal cells isolated from different sources." Doctoral thesis, Universite Libre de Bruxelles, 2011. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/209790.

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Mesenchymal stromal cells (MSC) are adherent, clonogenic, fibroblast-like cells endowing with unique multipotent differentiation potential and immunosuppressive properties. They are considered as promising candidates for regenerative medicine and immunotherapy.

MSC can be isolated from different tissue sources including bone marrow (BM), adipose tissue (AT) and Wharton’s Jelly (WJ). Although fulfilling the ISCT criteria required to be recognized as MSC, MSC from these different sources could disclose some differences taking into account their different anatomical origin and ontogeny as well.

In the present work, we investigated the influence of MSC source on their immunosuppressive as well as differentiation properties. We further extended our study to the role of the microenvironment (infection and inflammation) on these features.

We show that BM-MSC express Toll-like receptors (TLR) from TLR1 to TLR6. In an inflammatory environment, TLR2, 3 and 4 are significantly upregulated. By upregulating TLR3 and TLR4 transcription, inflammation increases BM-MSC responsiveness to LPS (TLR4 ligand) and poly(I:C) (TLR3 ligand) leading to a pro-inflammatory shift of their cytokine profile. The effect of TLR ligation on BM-MSC osteogenic potential is donor dependent. Inflammation as well as stimulation with LPS and poly(I:C) result in a decrease of BM-MSC immunosuppressive capabilities.

We further observed that BM-, AT- and WJ-MSC do not have the same pattern of TLR expression and consequently do not respond the same way to bacterial or viral infection. WJ-MSC do not express TLR4 and although TLR3 is present at the protein level it is not functional as its ligation do not trigger cytokine expression. Inflammation modulates this TLR pattern expression by upregulating TLR3 in all three MSC types and TLR4 only in BM-MSC. TLR ligation increases the production of inflammatory cytokines in BM- and AT- but not in WJ-MSC and augments anti-inflammatory cytokines in AT-MSC. Although inflammation increases in all MSC types the secretion of inflammatory cytokines, additional TLR triggering does not further affect WJ-MSC. The immunosuppressive potential of WJ-MSC on mixed leucocytes reaction (MLR) is not affected either by inflammation or by TLR triggering.

On the differentiation side, WJ-MSC has the lower potential to differentiate into osteoblast as compared to BM- and AT-MSC, as revealed by alkaline-phosphatase (ALP) activity and by measuring extracellular Ca2+ deposits. However, inflammation is able to strongly increase the osteogenic differentiation of WJ-MSC as calcification and ALP activity appears as early as at day 7. However this latter enzymatic activity remains much lower than that disclosed by BM-MSC. TLR3 or TLR4 triggering does not affect the osteogenesis of WJ-MSC while it increases it in AT- and also, although to lesser extent, in BM-MSC.

Our work establishes that the source from which MSC is derived is of major importance for the design of MSC based immunointervention. WJ-MSC appear to be the most attractive cell type when an immunosuppressive action is required in an inflammatory or infectious context. Although WJ-MSC are poorly osteogenic, a complete osteogenic differentiation can be obtained under inflammatory conditions. Taking into account their easy accessibility as well as their huge proliferative potential, these data open an avenue for using these cells in regenerative medicine particularly in clinical settings where chronic inflammation or infection have to be considered.

Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished

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31

Sekiya, Elíseo Joji. "Avaliação do efeito de células-tronco mesenquimais humanas de várias origens na atividade de linhagem de células tumorais HepG-2." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/5/5167/tde-09022015-142850/.

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INTRODUÇÃO O câncer é uma das principais causas de morte no mundo, sendo responsável por cerca de 8 milhões de mortes por ano, segundo dados da OMS. As mortes por câncer são provocadas por tumores que se originam em órgãos como pulmão, fígado, estômago, intestino, mama e esôfago. As células-tronco mesenquimais (CTM) foram identificadas em vários órgãos e estudos da sua interação com células tumorais têm apresentado resultados indicando ação inibitória sobre alguns tipos de tumores. Para explorar essa questão foram analisados os efeitos sobre células tumorais de carcinoma hepatocelular humano (HepG-2) do meio condicionado (MC) obtido do cultivo de CTM isoladas de tecido adiposo (TA), líquido amniótico (LA) e geleia de Wharton (GW). MÉTODOS Os MCs foram coletados após 24 horas de incubação das CTM sub-confluentes com ?-MEM contendo 20% de soro fetal bovino (SFB). Os MCs foram centrifugados e passados através de filtros de 0,22 ?M e armazenadas a -20 °C. O MC da própria célula HepG-2 foi utilizado como controle. Os efeitos dos MCs sobre a proliferação de células HepG-2 foram testados por ensaio MTT, em várias concentrações após 24 h de incubação. O ciclo celular de células HepG-2 tratadas com MC a 25%, 50% ou 75% foi analisado por citometria de fluxo (coloração IP) utilizando o software Modfit LT. A expressão dos genes bcl-2, bcl-6, CCND1 foi analisada por RT-PCR. A proliferação celular foi avaliada pela expressão das proteínas survivina, Bcl-2, PCNA e Ki-67 e pela quantificação de mitocôndrias com corante MitoTracker, assim como pelo potencial de membrana mitocondrial por corante JC-1 Mitoscreen utilizando equipamento de high content analysis. RESULTADOS Os meios condicionados de células-tronco de tecido adiposo (MC-TA) não alteraram a proliferação de células tumorais HepG-2 e os meios condicionados de células-tronco de líquido amniótico (MC-LA) e de geleia de Wharton (MC-GW) provocam aumento da proliferação, confirmada pela contagem de células com núcleos corados com Hoescht 33342. A análise de alterações do ciclo celular demonstrou que a exposição de células HepG-2 aos meios MC-LA diminuem as células na fase G0/G1 e aumentam na fase G2/M do ciclo celular. A expressão dos genes CCND1, Bcl2 e Bcl6 que estão relacionados à proliferação e morte celular não apresentaram alteração. A quantificação das proteínas PCNA e survivina não apresentou alteração sob efeito dos MC, porém a comparação direta entre células tratadas com MC-LA e MC-TA indicou a tendência das células tratadas com MC-LA proliferarem. O percentual de células HepG-2 expressando a proteína Ki-67 foi significativamente menor em relação ao controle quando tratadas com MC-TA, não apresentando diferenças quando tratadas com MC-LA e MC-GW. A contagem de mitocôndrias evidenciou aumento de mitocôndrias nas células HepG-2 tratadas com MC-LA e efeito não significativo dos tratamentos com MC-TA e MC-GW. A diferença do potencial de membrana mitocondrial por JC-1 apresentou aumento da polarização nas células HepG-2 cultivadas com MC-TA. CONCLUSÃO Os resultados confirmam que as células-tronco mesenquimais diferem de acordo com a sua origem tecidual em sua ação na proliferação das células HepG-2. Mais estudos são necessários para estabelecer a causa destas ações, que não parecem ser devido a mediadores mais comuns na proliferação e morte celular
INTRODUCTION Cancer is a leading cause of death worldwide, accounting for about 8 million deaths per year, according to WHO data. Cancer deaths are caused by tumors that originate in organs such as lung, liver, stomach, bowel, breast and esophagus. The mesenchymal stem cells (MSCs) have been identified in many organs and studies of their interaction with tumor cells have shown results indicating an inhibitory effect on some types of tumors. To explore this question the effects of the conditioned medium (CM) obtained from mesenchymal stem cell isolated from adipose tissue (AT), amniotic fluid (AF) and Wharton jelly (WJ) on tumor cells of human hepatocellular carcinoma (HepG-2) were analyzed. METHODS The MSC CM was collected after 24 hours incubation of sub confluent MSC with ?-MEM containing 20% fetal bovine serum (FBS). The MSC CM were centrifuged and passed through 0.22 ?M filter and stored at -20° C. The CM of HepG-2 cell itself was used as control. The effects of contrast media on proliferation of HepG-2 cells were tested by MTT assay at various concentrations after 24 h of incubation. The cell cycle HepG-2 cells treated with CM at 25%, 50% or 75% was analyzed by flow cytometry (PI staining) using Modfit software LT. The expression of the genes Bcl-2, Bcl-6, CCND1 was analyzed by RT-PCR. Cell proliferation was assessed by the expression of survivin, Bcl-2, Ki-67 and PCNA proteins, and the quantization of mitochondrial by MitoTracker dye, as well as the mitochondrial membrane potential by JC-1 Mitoscreen dye using high content analysis equipment. RESULTS The conditioned media of mesenchymal stem cells (MSC) from adipose tissue (AT-CM) did not alter the proliferation of tumor HepG-2 cells and conditioned media of MSC cells from amniotic fluid (AF-CM) and Wharton jelly (WJ-CM) caused increased proliferation, confirmed by counting cells with nuclei stained with Hoechst 33342. The cell cycle analysis showed that exposure of HepG-2 cells to AF-CM means decrease the cells in G0 / G1 cell cycle phase and increase in phase G2 / M. The expression of Bcl2, Bcl6 and CCND1 genes that are related to proliferation and cell death did not change. The quantization of PCNA and survivin protein did not change under the effect of conditioned media, but a direct comparison between cells treated with AF-CM and AT-CM indicated the tendency of cells treated with AF-CM proliferate. The percentage of HepG-2 cells expressing the protein Ki-67 was significantly lower than the control when treated with AT-CM and no differences when treated with AF-CM and WJ-CM. The counting of mitochondria showed increased mitochondria in HepG-2 cells treated with AF-CM and no significant effect of treatment with WJ-CM and AT-CM. The difference in mitochondrial membrane potential by JC-1 showed an increase in polarization in HepG-2 cells cultured with AT-CM. CONCLUSION The results confirm that mesenchymal stem cells differ according to their tissue origin in its action on the proliferation of HepG-2 cells. More studies are needed to establish the cause of these actions, which seem to be not related to the most common mediators in cell proliferation and death

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Beroud, Jacqueline. "Différenciation de cellules mésenchymateuses périnatales vers un phénotype musculaire lisse : base de la construction d'un feuillet vasculaire." Thesis, Université de Lorraine, 2015. http://www.theses.fr/2015LORR0344/document.

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Les pathologies vasculaires représentent aujourd’hui l’une des principales causes de mortalité mondiale et leur nombre ne cesse d’augmenter. Les greffons autologues (disponibilité faible) et les prothèses synthétiques inadaptées pour des vaisseaux de diamètre inférieur à 6 mm ne répondent pas à la demande et il existe aujourd’hui, un réel besoin en substitut vasculaire pour les petits vaisseaux. Ainsi, le concept de l’ingénierie vasculaire semble très prometteur. Cette approche est fondée sur l’utilisation de matrices « scaffold » associées à une composante cellulaire pour construire, dans des conditions environnementales adaptées, un vaisseau qui réponde et réagisse aux contraintes physiologiques. Dans cet objectif, la fonctionnalisation d’une media vasculaire constituée de cellules musculaires lisses (CML) est prérequise. Aux CML matures qui ne sont pas de bons candidats (perte de leur phénotype contractile lors de la culture), nous avons identifié les cellules souches mésenchymateuses (CSM) de la gelée de Wharton (tissu conjonctif du cordon ombilical) comme source cellulaire majeure. Leur facilité de récupération, leur présence en grand nombre, leur faible immunogénicité et leur capacité de prolifération et différenciation en font d’excellents candidats en ingénierie tissulaire. Dans ce travail nous avons déterminé les conditions favorables à l’obtention d’un phénotype CML fonctionnelles et montré l’impact de différents paramètres environnementaux (apport en oxygène, facteurs de croissance, teneur en sérum…) sur le comportement des CSM de la gelée de Wharton. Nous avons pu montrer que 1) ces cellules étaient capables de se différencier en cellules au phénotype contractile comparable à celui des CML matures. 2) L’utilisation des films multicouches de polyéléctrolytes (FMP) en tant que support d’adhérence cellulaire a montré que les CSM de la gelée de Wharton avaient un comportement spécifique selon la charge de surface conduisant vers une cultures tridimensionnelle inadaptée sur (PAH-PSS)3 PAH et en monocouche sur films (PAH-PSS)4, 3) Ces cellules pouvaient être cultivées sur des hydrogels d’alginate fonctionnalisés par les FMP pour fournir un feuillet cellulaire susceptible de recréer une media vasculaire
Vascular diseases represent today one of the leading causes of global mortality and the number is increasing. Autologous transplants (limited availability) and synthetic prostheses unsuitable for vessels with a diameter less than 6 mm are not sufficient and there is now a real need of vascular substitute for small vessels. Thus, the concept of vascular engineering seems very promising. This approach is based on the use of "scaffold" associated with a cellular component to build in suitable environmental conditions, a vessel that reacts with the physiological constraints. To this aim, the functionalization of an incorporated media vascular smooth muscle cells (SMC) is a prerequisite. Insteag of using Mature CML which are not good candidates (loss of contractile phenotype in culture), we identified mesenchymal stem cells (MSCs) from Wharton's jelly (connective tissue of the umbilical cord) as a major cellular source. Their easiness of recovery, their presence in large numbers, their low immunogenicity, their proliferation and differentiation capacity make them excellent candidates for tissue engineering. In this work we determined the conditions for obtaining a functional CML phenotype and showed the impact of different environmental parameters (oxygen level, growth factors, serum content ...) on the behavior of CSM jelly Wharton. We have shown that: 1) these cells were able to differentiate into cells in contractile phenotype comparable to that of mature SMC. 2) The use of multilayer films of polyelectrolytes as cell adhesion support has shown that MSCs from the Wharton jelly had a specific behavior according to surface charge leading to an inappropriate three-dimensional cultures (PAHPSS)3-PAH and monolayer films on (PAH-PSS)4, 3) These cells could be grown on functionalized alginate hydrogels to provide a cellular sheet which may recreate a vascular media

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33

Fernandes, Douglas Bandeira. "Valores de referência para área de secção transversa do cordão e vasos umbilicais aferidos pela ultrassonografia em gestações gemelares dicoriônicas." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/5/5139/tde-13082014-144551/.

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OBJETIVO: Determinar os valores de referência, e examinar a correlação da área de secção transversa do cordão umbilical, e de seus componentes, com a idade gestacional (IG), em gestações gemelares. Examinar a correlação da área de secção transversa do cordão umbilical com o peso fetal estimado (PFE). MATERIAIS E MÉTODOS: Estudo prospectivo longitudinal envolvendo gestações gemelares dicoriônicas, não complicadas. Medidas ultrassonográficas das áreas de secção transversa do cordão umbilical (ASTCU), da veia (AVU) e artérias umbilicais (AAU), e da geleia de Wharton (AGW) foram obtidas em plano adjacente, próximo ao abdômen fetal, a cada três semanas. A correlação entre os parâmetros avaliados e a idade gestacional foi investigada por meio de modelo de regressão polinomial hierárquica, levando-se em consideração a variância segundo a idade gestacional, entre medidas obtidas em fetos da mesma gestação e, entre diferentes gestações. Para cada parâmetro estudado, foram calculados os valores correspondentes aos percentis 5, 10, 50, 90 e 95, para cada semana gestacional. RESULTADOS: Foram realizadas 334 avaliações ultrassonográficas em 44 gestações gemelares, entre 18 e 33 semanas (média: 3,8 ± 0,7 exames/gestação; intervalo médio entre exames: 3,3 ± 0,9 semanas). Os valores log-transformados de todos os parâmetros avaliados apresentaram correlação significativa (p<0,001) com a idade gestacional: Log(ASTCU) = - 2,287498 + 0,149298 x IG - 0,002302 x IG2, desvio-padrão DP = 0,113, R2 = 0,65; Log (AVU) = - 2,721487 + 0,119853 x IG - 0,001507 x IG2, DP=0,165, R2 = 0,58; Log (AAU) = - 4,223546 + 0,195454 x IG - 0,003080 x IG2, DP=0,163, R2 = 0,57; Log (AGW) = - 2,511648 + 0,157737 x IG - 0,002564 x IG2, DP=0,123, R2 = 0,55. A área de secção transversa do cordão umbilical apresentou correlação significativa com o peso fetal estimado (Log (ASTCU) = -1,602447 + 0,554502 x Log (PFE), R2 = 0,65, p < 0,001). CONCLUSÃO: Em gestações gemelares dicoriônicas, a área de secção transversa do cordão umbilical, e de seus componentes, mostram correlação positiva e significativa com a idade gestacional. A área de secção transversa do cordão umbilical também correlaciona-se significativamente com o peso fetal estimado
OBJECTIVE: To determine reference values, and examine the correlation between the cross-sectional area of the umbilical cord, and its components, with gestational age (GA) in twin pregnancies. To examine the correlation between the cross-sectional area of the umbilical cord with the estimated fetal weight (EFW). MATERIALS AND METHODS: A prospective longitudinal study involving uncomplicated dichorionic twin pregnancies. Sonographic measurements of the cross-sectional areas of the umbilical cord (UCCSA), umbilical vein (UVA) and arteries (UAA) and Wharton\'s jelly (WGA) were obtained in a plane adjacent to the fetal abdomen, every three weeks. The correlation between these parameters and gestational age was examined with hierarchical polynomial regression analysis. This modeling took into account the variance according to gestational age, fetuses within the same pregnancy and changes across different pregnancies. For each parameter, 5th, 10th, 50th, 90th and 95th centiles were calculated for each gestational week. RESULTS: 334 ultrasound scans were performed in 44 twin pregnancies, between 18 and 33 weeks (mean: 3.8 ± 0.7 scans/pregnancy, mean interval between scans: 3.3 ± 0.9 weeks). All umbilical cord log-transformed values showed a significant correlation (p < 0.001) with gestational age: Log (UCCSA) = - 2.287498 + 0.149298 x GA - 0.002302 x IG2, SD = standard deviation 0.113, R2 = 0.65, Log (UVA) = - 2.721487 + 0.119853 x GA - 0.001507 x IG2, SD = 0.165, R2 = 0.58, Log (UAA) = - 4.223546 + IG x 0.195454 - 0.003080 x IG2, SD = 0.163, R2 = 0.57, Log (WGA) = - 2.511648 + 0.157737 x GA - 0.002564 x IG2 , SD = 0.123, R2 = 0.55. The cross-sectional area of the umbilical cord also correlated significantly with the estimated fetal weight (Log (UCCSA) = -1.602447 + 0.554502 x Log (EFW), R2 = 0.65, p < 0.001). CONCLUSION: In dichorionic twin pregnancies, the cross-sectional areas of the umbilical cord, and its components, show a positive and significant correlation with gestational age. The cross-sectional area of the umbilical cord also has correlates significantly with the estimated fetal weight

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Lim, Yah-Teem, and 林雅婷. "Applications of Wharton’s Jelly Cell Therapy on Light-induced Retinopathy in Rat Model." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/83003198406870009549.

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碩士
輔仁大學
基礎醫學研究所碩士班
97
The death of photoreceptor is the final event that leads to several degenerative diseases of retina such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD). AMD and RP comprise a large group, about 17% of retinal diseases that often lead to blindness. At present, there are no effective vision-restoring treatments for such disorders. Some treatments including gene therapy and growth factor injections have been developed to delay or prevent the loss of retinal neurons. A cell-based therapy is the only treatment for retinal regeneration by re-supplying the damaged or faulty cells with neural progenitor cells or stem cells. Stem cells, as a potent resource for treating several degenerative diseases including retinal disorders. There are two types of stem cells found in umbilical cord, including Wharton’s Jelly Cells (WJCs), which is a noncontroversial, inexhaustible source for transplant therapies. In addition to several genetically engineered and transgenic animal models of RP and AMD, there are several studies mimicking these diseases by exposure to bright and luminous environment. The purpose of this study is to establish a light-induced retinopathy model and to investigate the fate of WJCs transplanted into rat retina. Firstly, SD rats were exposed for 7 days to bright, cyclic, luminous environment of 7000-10,000 lux. Immediately after that, WJCs were transplanted subretinally into the eyes of light-induced retinal damage rats. The rescue effect of WJCs was examined functionally by measuring the electroretinogram (ERG) before the light exposure and each week after the light exposure. Retinas were harvested for immunohistochemistry and histology analysis by measuring the thickness of the outer nuclear layer (ONL) at 2, 4 and 6 weeks after cell transplantation. Photoreceptor cell death was also detected by TUNEL assays. The data showed 7 days exposure of light causing significant reductions in a-wave and b-wave in ERG. In the histological study, the thickness of the ONL also decreased after the light exposure. The TUNEL staining shows that a higher number of TUNEL-positive cells on ONL in rats exposed for bright light compared to unexposed rats. Exposure to bright light caused severe retinal damage, thus light-induced retinopathy animal model mimicking AMD was established. After WJCs transplantation, the amplitude of a-wave and b-wave in WJCs-treated eyes are larger than that measured from non-treated eyes and control eyes. The ONL of treated retina was thicker in the inferior and superior hemisphere than non-treated eyes. After 3 weeks of injection, part of the grafted cells migrated as single scattered cells into INL, ONL and GCL. The grafted WJCs were differentiated into retinal cells including retinal pigmented epithelium cells, amacrine cells & horizontal cells which were determined by retinal immunohistochemistry. The results demonstrate that transplantation of WJCs improving both physiological function and morphology of retina. This study provides a potential therapeutic method in rescuing light-induced retinopathy.

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Hsu, Hsin-Chih, and 許馨之. "Trans-differentiation of human mesenchymal stem cells in Wharton’s jelly into insulin-producing cells." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/35760938452030078274.

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碩士
國立陽明大學
解剖暨細胞生物學研究所
95
Diabetes mellitus (DM) is a chronic metabolic disorder. Due to insulin insufficient, the body fails to effectively utilize or control postprandial blood glucose levels. It is characterized by persistent hyperglycemia. Recently, the number of the diabetic patients raise gradually. In spite of unlethal, the complication are ten leading cause of death, like apoplexy、Coronary heart disease…etc. At present, the therapies for diabetes patients are limited in using drugs or injecting insulin directly. Even using the latest technique, islet transplantation, allow patients have ability to produce insulin, the chock point is limited by the scarcity of donors. These days, many groups invested in regenerative medicine which is related to use stem cells producing injured tissues. Many studies show that the stem cells from different sources under different treatments have potential to differentiate into β cells；also have potency to cure diabetic symptom after transplantation to mice. Accordingly, in this study, we investigate whether mesenchymal stem cells in Wharton’s jelly could differentiate into insulin producing cells under 1% dimethylsulfoxide. By using Western blotting, increase expression of insulin and C-peptide were found in cultured cells under defined conditions after 12 days. At immunocytochemistry, we also can observed insulin、C-peptide and glucagon expression after treatment. mRNA of pancreatic related transcription factors Isl-1、Pdx-1、insulin and glucose transporter Π(GLUT2) also expression via RT-PCR. From Ultrastructure, we discovered β cells characteristics：abundant rough endoplasmic reticulum (rER)、secretary granules and microvilli…etc. In summary, we suggested that mesenchymal stem cells in Wharton’s jelly have potential in differentiating into insulin producing cells. It maybe a new source for islet transplantation and probably provide better therapies for diabetic patients.

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Pan, Chia-Hsin, and 潘佳欣. "TGF- β2 enhances cardiomyogenic differentiation of human mesenchymal cells from Wharton’s Jelly in vitro." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/64213307604779815883.

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碩士
國立陽明大學
解剖暨細胞生物學研究所
94
Myocardial infarction is one of the leading cause of public mortality. Nowadays, stem cell transplantation shows great promise as a treatment of dysfunctional heart failure. Mesenchymal stem cells (MSCs) derived from human bone marrow underwent cardiomyogenic differentiation after 5-azacytidine exposures in vitro; however, treatment with 5-azacytidine remains question because of its cytotoxicity. In addition, mouse embryonic stem cells treated with transforming growth factor β2 (TGF-β2) exhibited spontaneous beating and expressed cardiac-specific proteins. In this study, we have found that after exposure to TGF-β2, MSCs obtained from Wharton’s jelly of human umbilical cord have the capacity to differentiate into cardiomyocytes. After exposure to TGF-β2, the expression of myogenic transcription factors, myogenin and MyoD1, was increased at the first 6 days. The expression of C-troponin I and connexin 43 was also increased in the first 6 days. These results suggested that TGF-β2 may play a role in the early stage of cardiomyogenic and myogenic differentiation. It is possible that other factors may be involved in this process. Additional factors may be necessary to fully commit these cells to cardiomyocytes.

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37

Gouveia, Analuce Canha. "Osteogenic Potential of Human Wharton’s Jelly Stromal Cells Cultured on Hierarchical Fibrous-Based Scaffold." Master's thesis, 2010. http://hdl.handle.net/10348/2174.

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Dissertação de Mestrado em Genética Molecular Comparativa e Tecnológica
A Engenharia de Tecidos é um novo conceito que surgiu como uma estratégia alternativa às terapias actuais aplicadas nas desordens musculoesqueléticas. Esta estratégia difere da transplantação de órgãos pela regeneração do tecido do próprio paciente, evitando assim a rejeição imunológica e a fraca biocompatibilidade. Na estratégia de engenharia de tecidos desenvolvida pelo Grupo de Investigação 3Bs, o scaffold desempenha um papel crítico. A arquitectura do scaffold é uma característica importante pois permite modular a resposta biológica ao respectivo scaffold. Apesar da completa interconectividade que caracteriza os scaffolds produzidos por prototipagem rápida (RP), a baixa eficiência de seeding continua a ser uma limitação para aplicações de engenharia de tecidos. Uma solução possivel para esta limitação é a integração de nanofibras nas estruturas de prototipagem. Os scaffolds hierarquicos (6 RP+ 5NFM) são obtidos pela combinação de microfibras e nanofibras, produzidas respectivamente por prototipagem rápida e electrospinning. Este estudo demonstrou que estes scaffold à base de SPCL são favoráveis para as estratégias de engenharia de tecidos. As imagens de Microscopia Eletrónica de Varrimento e Hematoxilina-Eosina provam que a integração das nanofibras nos scaffold RP melhora a eficiência de seeding. Estas imagens também demonstraram que hWJSCs aderiram preferencialmente às malhas de nanofibras. Além disso, as células foram capazes de se multiplicar e colonizar as regiões internas dos scaffolds. Este facto indica que a porosidade e interconectividade dos scaffolds produzidos são suficientes para a infiltração de células. A quantificação de DNA e os ensaios de viabilidade (MTS) também corroboram a hipótese destes scaffolds serem uma válida alternativa aos scaffolds de prototipagem rápida. O potencial osteogénico das construções celulares formadas pelos scaffolds 6 RP+NFM e hWJSCs foi comprovado pelos níveis mais elevados de ALP, o que indica uma diferenciação precoce das hWJSCs. No espectro de EDS é possivel detectar a presença de iões cálcio e fósforo o que confirma a minerilização destas construções celulares. A diferenciação osteogénica também foi confirmada pela análise de PCR em Tempo Real. Após 3 semanas de cultura, é possivel a detecção de sobreexpressão de genes associados à diferenciação osteogénica tais como RUNX2, SP7, BGLAP, SPP1 e IBSP. Finalmente, dadas às inúmeras vantagens das hWJSCs e o potencial osteogénico das mesmas quando colocadas em scaffolds 6RP e em scaffolds 6RP+5NFM, esta fonte de células estaminais adultas é uma alternativa promissora para as estratégias de engenharia de tecido ósseo.
Tissue engineering is a new concept emerged as an alternative approach to tissue and organ reconstruction. It differs from organ transplantation by regenerating patient’s own tissue and organs avoiding the biocompatibility and low biofunctionality problems as well as severe immune rejection; which are the main problems of organ transplantation. In tissue engineering approach developed in 3Bs Research Group, the scaffold performs a critical role. The architecture of the tissue engineered scaffold is an important factor to take into consideration that can modulate biological response and the clinical success of the scaffold. Despite the periodical and completely interconnected pore network that characterizes rapid prototyped (RP) scaffolds, cell seeding efficiency still remains a critical factor for optimal tissue engineering applications. Hierarchical fibrous scaffolds, obtained by the combination of RP micro- and electrospun nano-motifs, have been considered a solution to overcome this drawback. This study demonstrated that hierarchical starch-based fibrous scaffolds are favorable for tissue engineering strategies and represent a solution to overcome the cell seeding limitation of rapid prototyped scaffolds. SEM micrographs and HE images prove that the integration of nanofiber meshes into 3D RP scaffolds improved the seeding performance, as they functioned as a cell entrapment system. SEM micrographs demonstrated that hWJSCs adhered preferentially to the nanofiber meshes. Moreover, cells were able to proliferate and colonize the inner regions of the scaffolds, highlighting that the porosity and interconnectivity of the developed scaffolds allow better cell infiltration and ingrowth than the traditional RP scaffolds. Our hypothesis that integration of nanofiber meshes enhances the cell seeding efficiency and provides a better environment for cell growth is also corroborated by the results of MTS viability assay and DNA quantification. Besides the chondrogenic potential of this scaffolds have already been proved, this work also demonstrates their osteogenic potential. Higher levels of ALP expression in WJSCs RP+NFM constructs were detected the second week, reflecting the cells early osteogenic differentiation stage. Moreover EDS spectra showed the presence of calcium and phosphorous elements at the surface of the cell cultured scaffolds, confirming the mineralization. Ultimately osteogenic differentiation of hWJSCs onto RP and RP+NFM scaffolds was demonstrated by Real Time Quantitative-PCR. After 3 weeks of culture, hWJSCs showed upregulation of genes linked to osteogenic differentiation such as transcription factors RUNX2 and SP7, and the matrix proteins BGLAP, SPP1, IBSP. Furthermore, a significantly higher fold change of these genes was detected on hWJSCs RP+NFM constructs, when compared to hWJSCs RP constructs. Concomitantly, given the numerous advantages of adult stem cells as cell source and their successful osteogenesis in 3D polymeric structures, hWJSCs may be a promising alternative for bone tissue engineering strategies.

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38

Yen, Ju-Ching, and 嚴如慶. "Elucidation of the mechanism underlying fluvastatin-enhanced osteogenic differentiation of Wharton’s jelly mesenchymal stem cells." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/66174746355603043966.

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碩士
國立陽明大學
生物藥學研究所
95
Wharton’s jelly mesenchymal stem cells (WJ-MSC) derived from umbilical cord connective tissue can differentiate into cells of neurogenic, adipogenic, osteogenic and myogenic lineages. Inhibitors of 3-hydroxy-2-methylglutaryl coenzyme A (HMG-CoA) reductase, statins, have been described as the most effective class of drugs to reduce serum cholesterol. Previous report suggested that simvastatin enhances osteoblastic differentiation and inhibits adipocytic differentiation of bone marrow stromal cells (BMSCs). On the other hand, we have shown a neuroglial differentiation of human bone marrow-derived MSCs induced by lovastatin and fluvastatin. To evaluate the effects of statin on osteogenic differentiation of WJ-MSCs, we treated them with fluvastatin alone or in the presence of induction medium. The mRNA level of osteocalcin and the activity of alkaline phosphatase in WJ-MSCs were significantly increased when they were cultured in osteogenic induction medium plus fluvastatin. Furthermore, osteoblast-like morphological change induced by osteogenic induction medium plus fluvastatin could be inhibited by the addition of geranylgeranyl pyrophosphate (GGPP). To find membrane proteins that participate in modulating osteogenic differentiation of these cells, two-dimensional electrophoresis was subsequently used to analyze the membrane proteins prepared from WJ-MSCs incubated in osteogenic medium supplemented without or with 1 mM fluvastatin and 1 mM fluvastatin plus GGPP. Five proteins whose membrane distribution was reduced by fluvastatin but was restored by the addition of GGPP were found. After mass spectrometry tropomyosin 4, calnexin and reticulocalbin 3 were identified. Taken together, our results suggest that fluvastatin enhances osteogenic medium-induced osteoblastic differentiation of human WJ-MSCs which might be attributed to a decreased membrane distribution of tropomyosin 4, calnexin and reticulocalbin 3.

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39

Huang, Chun-Wei, and 黃峻暐. "Xenograft of Human Umbilical Mesenchymal Stem Cells from Wharton’s Jelly Reverses Peritoneal Fibrosis in Rats." Thesis, 2018. http://ndltd.ncl.edu.tw/handle/726vmk.

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40

Chu, Pei-Chun, and 朱培君. "TGF-β2-treated Mesenchymal Stem Cells from Wharton’s Jelly Improve Cardiac Function in Myocardial Infarction Rats." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/01449684888430960303.

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碩士
國立陽明大學
解剖學及細胞生物學研究所
99
Myocardial infarction (MI) is one of the leading causes of death worldwide with an increasing morbidity and mortality of heart failure after MI. Recent studies showed that mesenchymal stem cells (MSCs) transplantation signiﬁcantly ameliorated the dysfunctional heart failure. In this study, we investigate the improvement in the left ventricular function and fibrosis of the myocardial infarct rat which induced by left anterior descending artery ligation that transplantation of undifferentiated, and TGF-β2 induced human umbilical cord mesenchymal stem cells from Wharton’s jelly. We make sure the induction of left anterior descending artery ligation by examining the electrocardiogram, echocardiography, and the ability of c-Troponin I in the serum. The observation of electrocardiogram, echocardiogram, Masson’s Trichrome staining, and immunohistochemistry in different time spot to assess the repair of myocardial infarct rat. We find that transplantation of undifferentiated and TGF-β2 induced human umbilical cord mesenchymal stem cells from Wharton’s jelly improve the left ventricular systolic function, reduce the fibrotic area of heart, and make better the survival rate.

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41

Tan, Joo-Shin, and 陳裕信. "Hepatocyte-like Cells derived from Wharton’s Jelly in the treatment of CCl4-induced Liver Injury Rats." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/17937740563425522043.

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碩士
國立陽明大學
解剖暨細胞生物學研究所
97
Aim：To study the capacity of mesenchymal stem cells from the Wharton’s jelly differentiating into hepatocyte-like cells and to observe the effect of portal vein transplantation of hepatocyte-like cells in the treatment of CCl4-induced acute liver failure rats. Methods：Mesenchymal stem cells were isolated from Wharton’s jelly of the human umbilical cord and induced to differentiate into hepatocyte-like cells under differentiating conditions (combine of condition medium and growth factor). Differentiated cells were evaluated with immunocytochemistry, RT-PCR, ammonia test and glycogen storage test. After that hepatocyte-like cells were transplanted into acute liver failure rats via portal vein. Blood biochemical analysis and body weight were measured, then Human nuclei and human albumin were detected in the liver by immunohistochemistry. Results：Hepatocyte-like cells were formed after induction. The hepatocyte-like cells expressed hepatocyte related genes and proteins on day 6 and morphology change on day12. The hepatocyte-like cells also have ammonia clearance and glycogen storage functions. After transplantation, hepatocyte-like cells could lower the AST, ALT and TBil of acute liver failure rats. Conclusion：Mesenchymal stem cells derived from the Wharton’s jelly of the human umbilical cord could be differentiated into hepatocyte-like cells in vitro. Transplantation of hepatocyte-like cells into portal vein could alleviate the liver injury of acute liver failure rats.

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42

Li, Po-Yu, and 李柏諭. "Study of transplantation of insulin-producing cells of mesenchymal stem cells from Wharton’s jelly into diabetic animals." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/05143298750380552095.

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碩士
國立陽明大學
解剖學及細胞生物學研究所
98
Aim：To study mesenchymal stem cells (MSCs) from the Wharton’s jelly differentiated into insulin-producing cells and to observe the effect of portal vein transplantation of insulin-producing cells via port-A in treatment with pancreatectomized-induced diabetic pigs. Methods：Mesenchymal stem cells (MSCs) were isolated from Wharton’s jelly of the human umbilical cord and induced into insulin-producing cells under differentiating medium (SFM-A, SFM-B, SFM-C).Undifferentiated MSCs and insulin-producing cells were transplanted into diabetic pigs via port-A to portal vein. Blood glucose levels, body weight and human C-peptide levels in serum were measured in diabetic pigs. Results：Mesenchymal stem cells (MSCs) were isolated from Wharton’s jelly and shaped like spindle. And MSCs were induced into insulin-producing cells in ten days. To transplant mesenchymal stem cells from Wharton’s jelly via portal vein in diabetic pigs could prolong life expectancy than those of control. To transplant insulin-producing cells via portal vein in diabetic pigs could lower blood glucose levels. Conclusion：Mesenchymal stem cells derived from the Wharton’s jelly of the human umbilical cord could differentiated into insulin-producing cells in vitro. Transplanted mesenchymal stem cells (MSCs) could prolong the life expectancy of diabetic pigs compared with the control. Transplant insulin-producing cells could lower the blood glucose levels of diabetic pigs compared with the control.

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43

Lin, Yun-Li, and 林昀勵. "Application of collagen scaffolds for differentiation of human Wharton’s jelly mesenchymal stem cells into cardiac progenitor cells." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/q3r549.

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碩士
國立陽明大學
解剖學及細胞生物學研究所
102
Myocardial infarction (MI) is the leading cause of morbidity and mortality in the world. Tissue engineering and stem cell therapy have emerged as potential therapies for MI. The aim of this study is to develop a material as the carriers which can allow mesenchymal stem cells to proliferate and differentiate into cardiac progenitor cells. The three components for tissue engineering are scaffolds, cells and signals. In this study, human Wharton’s jelly mesenchymal stem cells (hWJ-MSCs) were treated with transforming growth factor 2 (TGF-2) and cultured on type I collagen scaffolds which were cross-linked with different concentration of cross-linkers. Rheometer results showed that two different concentration of cross-linkers resulted in different Young’s moduli of scaffolds: 15 and 31 kPa. Young’s modulus indicates the stiffness of elastic materials. The higher modulus of scaffolds means they are much stiffer. The alamarBlue results showed that stiffer scaffolds allow a better viability of hWJ-MSCs. Moreover, hWJ-MSCs differentiated into cardiac progenitor cells when cultured in higher stiffness scaffolds, as confirmed by the expression of cardiac markers at both the gene and protein expression levels. Scanning electron microscope and laser confocal microscope revealed that hWJ-MSCs could grow inside the scaffolds and have a good interaction with collagen scaffolds. Taken together, these results indicated that the developed collagen scaffolds can be carriers which can allow hWJ-MSCs to proliferate and differentiate into cardiac progenitor cells. These scaffolds hold great potential for delivering hWJ-MSCs into an infracted heart.

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44

Hsieh, Jui-Yu, and 謝睿瑜. "Comparative Transcriptome Analysis of Mesenchymal Stem Cells Derived from Bone Marrow and Wharton’s Jelly of Umbilical Cord." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/36849809033388474398.

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博士
國立陽明大學
微生物及免疫學研究所
101
Mesenchymal stem cells (MSCs) are promising tools for the treatment of diseases because of their differentiating abilities and their ability to promote endogenous angiogenesis and neurogenesis via a variety of secreted factors. MSCs found in the Wharton’s jelly of the human umbilical cord are easily obtained and are capable of transplantation without rejection. Here, we aim to understand the functional comparison of WJ-MSCs with MSC from bone marrow (BM-MSCs) and to identify molecular regulation involved in MSC phenotypes. First, the gene expression patterns and genetic networks of BM-MSCs and WJ-MSCs were compared. BM-MSCs expressed more genes related to skeletal development, while WJ-MSCs express more growth-related genes. WJ-MSCs are more primitive since they share more common genes with embryonic stem cells. Drylab results could be verified by wetlab experiments, in which BM-MSCs were more efficient in osteogenic and adipogenic differentiation, while WJ-MSCs proliferated faster. Second, we compared the secretomes of BM-MSCs and WJ-MSCs, and found that WJ-MSCs express more secreted factors related to angiogenesis and neurogenesis. Functional assays showed that WJ-MSCs induced better neural differentiation and neural cell migration. WJ-MSCs had superior neuropeotective effects on primary cortical cells injured by oxygen-glucose deprivation culture. WJ-MSC secreted factors also induced better microvasculature formation and cell migration on co-cultured endothelial cells. Third, since the migration of transplanted MSCs to injured sites is also a critical property of engraftment, our aim was to identify critical miRNAs controlling MSCs proliferation and migration. WJ-MSCs showed poorer motility and better proliferation ability compared to BM-MSCs. Small RNA sequencing revealed that miR-146a-5p is significantly overexpressed in WJ-MSCs with high abundance. Knockdown of miR-146a-5p in WJ-MSCs inhibited their proliferation yet enhanced their migration. CXCL12, together with SIKE1, which is an IKKε suppressor, are direct targets of miR-146a-5p in MSCs. miR-146a-5p is also downstream of CXCL12, and a negative feedback loop is therefore formed in MSCs. Our study provides the basis for the development of cell-based therapy and the transcriptomic results also reveals of follow-up mechanistic studies related to MSC biology.

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45

Feng, Jui-Hsun, and 馮睿勛. "The Potential Application of the Human Umbilical Cord Mesenchymal Stem Cells from Wharton’s Jelly for Skin Wound Healing." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/63084814835085357859.

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碩士
國立陽明大學
解剖學及細胞生物學研究所
98
Skin is to serve as a protective barrier against foreign invaders. Burns and chronic skin ulcers even some severe occupational injuries can badly lose this innate immunity and leads to serious results. Cutaneous wound healing is a complex process involving inflammation, epithelialization, granulation tissue formation, neovascularization, wound contraction and extracellular-matrix (ECM) reorganization. The aim of this study, we investigated the effects of transplantated human umbilical mesenchymal cells in Wharton's jelly (HUMSCs) into full thickness wounded male Sprague-Dawley rats, HUMSCs have been shown possess stem cell properties and it’s also a good source to application in cell therapy. HUMSCs is the easily available and process, furthermore, it can be applied as a treatment without ethical consideration and immunological suppression in our animal models. Our preliminary data reveal that transplantation of HUMSCs is beneficial to skin wound healing, after transplantation of HUMSCs into the wounded rat, the wound healing process began more efficient and well construction. The underlying mechanism will be elucidated.

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46

LI, TUNG-YUEH, and 李東岳. "Stem Cell Therapy for Diabetes TreatmentEstablishment of Transduction and Cell Tracking Methods in Wharton’s Jelly Mesenchymal Stem Cell." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/y3yvc3.

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碩士
國防醫學院
生物及解剖學研究所
104
Diabetes mellitus (DM) threatens people health all over the world. Type 1 diabetes mellitus (T1D), caused by autoimmune attacking islet cells, affects 5-10% of total DM. Insulin injection is the predominant treating of T1D that cannot prevent islet cells loosing and poor immunosuppression. The recent progress in regenerative medicine, especially stem cell therapy, has suggested several novel and potential cures for T1D. According to our previous study, Wharton’s jelly mesenchymal stem cells (WJ-MSCs) can reduce hyperglycemia in nonobesity diabetic (NOD) mice. The function of WJ-MSCs is probably due to immune-modulatory and they may become insulin producing cells (IPCs) in vivo. However, blood sugar does not return to normal rang in NOD mice. In this study, we induced WJ-MSCs into IPCs by transduction of pancreatic and duodenal homeobox 1 (PDX1) in these cells. To reveal the mechanism of WJ-MSCs homing, we introduce luciferase and GFP by lentivirus and use IVIS Spectrum to track IPCs in vivo. In addition, we establish a method of stimulating MSCs to IPCs differentiation and tracking these cells in vivo. Furthermore, we plan to introduce more potential genes like PAX4, NKX6.1 to drive WJ-MSC to IPCs more effectively. In conclusion, the expansion and subsequent differentiation of stem cells, appears to have considerable potential to overcome the shortage of donor organs. Cellular replacement therapy may offer the best approach to achieve physiologic glucose control in diabetic patients.

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47

Lin, Meng-Feng, and 林孟鋒. "Characterization of Decoy Receptor 3-Transduced Adult Human Pancreatic Endocrine Precursor Cells and Wharton’s Jelly Mesenchymal Stem Cells." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/25663968703174680812.

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碩士
國防醫學院
生物及解剖學研究所
97
Type I diabetes Mellitus (IDDM) is a major T-cell mediated autoimmune disease that affect children and adolescence. Patients are so far depending on injected or pumped insulin for life while insulin doesn’t cure such disease, and bad blood sugar control will suffer the patients from several chronic complications. Although there are several therapies such as pancreas transplantation, islet transplantation, regeneration therapy or gene therapy trying to cure IDDM, researchers are still looking for ways to avoid these transplanted cells being attacked by autoimmune T-cells. Members of the tumor necrosis factor (TNF) family have been shown to play important roles in T-cell differentiation, activation, apoptosis and co-stimulation. Decoy Receptor 3 (DCR3), belongs to tumor necrosis factor receptor super family (TNFR), is a decoy receptor for Fas ligand (FasL), a member of TNF family. DCR3 binds to FasL and suppresses immune function of T-cells. Thus, our lab tried to transduce DCR3 gene into pancreatic endocrine precursor cells by lentivector expression system. The DCR3-transduced human pancreatic precursor cells were proliferated in conditioned culture medium. The expression of DCR3 in neoislets was analyzed. The immune modulatory function of DCR3 was examined through TNF-competing test. In conclusion, we have successfully constructed DCR3-GFP lenti-viral expression system, and DCR3-lentivirus transduction doesn’t affect cell morphology or function. We demonstrated that DCR3 decreases FasL-induced apoptosis rate as though pancreatic precursor cells would not apoptosis under the stimulation of FasL apoptosis may due to the immunoregulation of mesenchymal stem cells.

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48

Cheng, Jung-Tzu, and 鄭蓉慈. "Establish Type 1 Diabetes Osteoporosis Animal Model and Explore the Therapeutic Potential of Wharton’s Jelly Mesenchyaml Stem Cells Transplantation." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/01907162227489438359.

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碩士
國防醫學院
生物及解剖學研究所
102
Osteoporosis (OP) and diabetes are both systemic metabolic diseases. More and more studies have shown that type 1 diabetes mellitus (T1DM) have a negative effect on the bone. The mechanism for T1DM leading to low bone mass has not been clarified yet. However most studies suggest that impaired bone formation has been proposed as a major factor. Current T1DM therapy such as continuous subcutaneous insulin infusion and transplantation of pancreas or islet cells cannot avoid the complications. Renal failure may occur in advanced diabetic patients and increase the difficulty of treatment. The major current treatment of OP is inhibition of bone resorption. Increasing bone formation and find the most appropriate treatment for OP with T1DM is an urgent issue. There have been many studies using cell therapy in treatment of OP or T1DM. Because of mesenchymal stem cells (MSCs) from the umbilical cord can be easily isolated and expanded in culture, these cells may prove to be a useful new source of cells for cellular therapies for OP. In this study, we established T1DM OP rodent models first, and then transplanted Wharton’s jelly MSCs (WJ-MSCs) to spontaneous T1DM mice and streptozotocin(STZ)-induced T1DM rats and investigate therapeutic potential. The results show a significant decline in blood glucose in spontaneous T1DM mice. In micro computed tomography 3D analysis data, there is a tendency to improve bone quality. And we also found that systemic infusions of WJ-MSCs promote an osteogenic response in bone formation of osteoporotic in each type of T1DM animal model. By this study, we consider that WJ-MSCs have potential as a source of cell therapy on T1DM and OP.

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49

Cheng, Chih-Min, and 鄭智珉. "Study of the Immunomodulatory effect of Exosome Derived by Human Wharton’s Jelly Mesenchymal Stem Cell in Type1 Diabetic Rat." Thesis, 2019. http://ndltd.ncl.edu.tw/handle/2ph7my.

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50

Shen, Wen-Sheng, and 沈文聖. "Transplantation of insulin producing cells derived from mesenchymal stem cells of the Wharton’s jelly for control STZ-induced diabetic rat." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/94782293656288077589.

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碩士
國立陽明大學
解剖暨細胞生物學研究所
96
Aim：To study the capacity of mesenchymal stem cells from the Wharton’s jelly differentiating into insulin-producing cells and to observe the effect of portal vein transplantation of insulin-producing cells via port-A in the treatment of streptozotocin-induced diabetic rat. Methods：Mesenchymal stem cells were isolated from Wharton’s jelly of the human umbilical cord and induced to differentiate into insulin-producing cells under differentiating conditions (combine of activin A, nicotinamide). Differentiated cells was evaluated with immunocytochemistry, real-time PCR, RT-PCR. C-peptide release and glucose challenge was tested with ELISA. Then insulin-producing cells were transplanted into diabetic rats via port-A into portal vein. Blood glucose levels and body weight were measured and human C-peptide concentration in the serums was monitored with ELISA. Human nuclei and C-peptide were detected in the liver by immunohistochemistry. Results：Mesenchymal stem cells from Wharton’s jelly were spindle and round adherent monolayers. Insulin-producing cells were formed after induction. The insulin-producing cells expressed C-peptide and pancreatic β-cell development related genes. The insulin-producing cells secreted C-peptide that was increased 2-fold than low glucose group after glucose challenge. After transplantation, insulin-producing cells could locate in the liver expressing C-peptide and human nuclei and lower the glucose levels of diabetic rats during day 2 to day 28. Conclusion：Mesenchymal stem cells derived from the Wharton’s jelly of the human umbilical cord could be differentiated into insulin-producing cells in vitro. Transplantation of insulin-producing cells via port-A into portal vein could alleviate the hyperglycemia of diabetic rats.

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