Academic literature on the topic 'Human Umbilical Cord Matrix-Derived Mesenchymal Cells'

Tissue engineered vascular grafts with long term patency are in great need in the clinics. An accessible source of human smooth muscle cell (SMC) is important for constructing functional vascular grafts. Human mesenchymal stem cells from the umbilical cord (UCMSCs) exhibit multi-lineage differentiation abilities, including the potential to differentiate towards vascular lineages such as SMCs. MicroRNAs (miRNAs) are short non-coding regulatory RNAs. They widely participate in regulation of stem cell differentiation and may play an important role in SMC differentiation. Understanding how to generate SMCs from UCMSCs as well as its underlying mechanism might greatly contribute to our knowledge of manufacturing functional vascular grafts. We hypothesise that vascular grafts could be generated with SMCs differentiated from human UCMSCs, and further explore the role of miRNAs in the differentiation process. We utilised transforming growth factor β 1 (TGFβ1) to stimulate the UCMSCs differentiation towards SMCs. A panel of SMC markers including αSMA, SM22, Calponin and SMMHC were highly upregulated both at the gene expression and the protein level at day 5 of TGFβ1 treatment. Micro-RNA (miR) array analysis showed that miR-503 was increased at early time points (6 h and 24 h) after TGFβ1 treatment, which was confirmed by TaqMan microRNA assay. We further demonstrated that miR-503 mimics promoted SMC differentiation both at the gene expression and the protein level and miR-503 inhibitors downregulated SMC markers at the protein level. Smad7, which is a negative regulator of TGFβ1-related signalling pathways, was identified to be a direct target of miR-503 by luciferase reporter experiments. The expression level of miR-503 was Smad4-dependent as shown by the Smad4 knockdown experiments. Also, Smad4 was demonstrated to be enriched at the promoter region of miR-503 as shown by Chromatin immunoprecipitation experiments. In addition to miR-503, miR-222-5p was also downregulated in the differentiation process. The gain-of-function study with the treatment of miR-222-5p mimics significantly inhibited the induction of SMC markers Calponin and αSMA both at the gene expression and protein level during differentiation. αSMA was confirmed to be a direct target of miR-222-5p. Moreover, ROCK2, which could mediate SMC differentiation through RhoA/ROCK pathway, was downregulated by miR-222-5p mimics both at the gene expression and protein level. The 3’-UTR segment of ROCK2 was identified to be a direct target of miR-222-5p. Finally, SMCs differentiated from UCMSCs exhibited the ability to migrate into decellularised mouse aorta grafts. Seeding of the cells onto the decellularised scaffold gave rise to vascular graft with smooth muscle layer that is comparable to its analog of the native vessel. In conclusion, we demonstrated the potential of using hUCMSCs-derived SMCs to generate vascular grafts which are in critical need in the clinics.

Master of Science<br>Department of Anatomy and Physiology<br>Mark L. Weiss<br>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.

Mesenchymal stem cells have been recently investigated for their potential use in regenerative medicine. It has been suggested that there may be a stem cell population within both umbilical cord matrix and amniotic fluid. However, little knowledge exists about the characteristics of these progenitor cells within these sources in the equine species. This study wanted to investigate an alternative and non-invasive stem cell source for the equine tissue engineering and to learn more about the properties of these cells for future cell banking. Moreover, population of adult stem cells were recently identified in human and lab animal tendons, but no detailed investigations have been made in the equine species. The aim of the study was to compare in vitro the stemness features of horse progenitor cells derived from bone marrow (BM-MSCs), amniotic fluid (AF-MSCs), umbilical cord matrix (EUC-MSCs) and tendon derived progenitor cells (TSPCs). This work defines a protocol for extraction, isolation, expansion and characterization of mesenchymal stem cells from equine bone marrow, amniotic fluid, umbilical cord matrix (Wharton’s jelly) and tendon. Their localization into the tissues from which they were extracted, was reported. During the cell culture, cell expansion, CFU-F assay, doubling time, plasticity and immunophenotype were analyzed. Furthermore, a specific cell labeling was realized that could be used for non-invasive magnetic resonance cell tracking through endosomal incorporation of superparamagnetic iron oxide particles through the in vitro evaluation of the efficiency of this labeling method. In the next future, this technique should facilitate translation of the approach into clinical trials, in particular to track cells in vivo after transplantation and to follow their homing, viability and repair potential during time. The mesenchymal stem cells were grown on control medium, such as DMEM with the addition of basic FGF. Our results pointed out that these cells performed similarly in terms of CFU-F formation and growth kinetic. The immunocytochemical and RT-PCR analysis of MSCs isolated from all tissues showed the presence of antigens such as CD44, CD105, CD29, Oct-4, c-Myc, SSEA4 and HLA-ABC, whereas they were negative for CD34 and HLA-DR. These cells, differentiated into osteogenic, adipogenic, chondrogenic and tenogenic lineages confirming the nature of mesenchymal stem cells. These findings suggest that AF-MSCs appeared to be a readily obtainable and high proliferative cell line that may represent a good model system for stem cell biology. EUC-MSCs need to be further investigated regarding their particular behavior in vitro represented by spheroid formation. Equine TSPCs have high clonogenic properties and proliferating potential, they express stem cell markers and have the capability to be multipotent as well as BM-MSCs. These findings suggest that TSPCs may represent a good model for stem cell biology and could be useful for future tendon regenerative medicine investigations. These data could be useful for optimization of horse’s mesenchymal stem cell isolation and expansion protocol that could be used in the experimental clinic application for tissue regeneration because to their biological properties. The MSCs isolated from equine extra-embryonic tissues and tendons, showed characteristics of stem cells and therefore can be regarded as good candidates to be used in regenerative medicine, with special reference to orthopaedic diseases of horses. Furthermore, future investigations in vivo are useful to evaluate the efficacy of cell labeling with contrast agents for magnetic resonance with particular attention to the muscle-skeletal tissues.

碩士<br>南臺科技大學<br>生物科技系<br>104<br>Spinal cord injury (SCI) is spinal cord and nerves damage by trauma, resulting in motor, sensory and excretory dysfunctions. There are more than 65,000 patients with spinal cord injury in Taiwan nowadays. SCI not only seriously affect victims' personal and family lives, but also cost a lot of resources from community. Current treatments for SCI include medications, medical procedures, functional rehabilitation, gene therapy and other modalities. Unfortunately, those treatments have their limits and there is no real functional recovery experience. Therefore, to find an effective treatment of spinal cord injury is indeed imperative. In recent years, the use of stem cell therapy for spinal cord injury has been widely discussed, is considered to be a viable treatment option. Previous studies have pointed out that mesenchymal stem cell transplantation in the treatment of cells for the treatment of a new model. Human umbilical cord derived mesenchymal stem cells (hUCMSCs), one such candidate with high potential, are a fetus-derived stem cell source collected from discarded tissue (Wharton's jelly) after birth. Compared with human bone marrow derived mesenchymal stem cells, hUCMSCs have the advantages of abundant supply, painless collection, no donor site morbidity, and faster and longer self-renewal in vitro. From other researchers studies, hUCMSCs improved hindlimb motor dysfunction of spinal cord injury rats. Professor Chen Sheng- Hsien and other scholars from the past research that human umbilical cord mesenchymal stem cells improves limb motor dysfunction after SCI in rats. From other researchers studies, hUCMSCs improved hindlimb motor dysfunction of spinal cord injury rats. A laminectomy with removal of the vertebral peduncle, was performed at T8 or T9 on male Sprague-Dawley rats anesthetized with zoletil (25 mg/kg, i.p.) and atropine (0.1 mL, s.c.) The jaws of a calibrated aneurysm clip with a closing pressure of 55 g were placed between the dorsal and ventral surface of the spinal cord and left in place for 1 min. All SD rats are randomly assigned to five groups: (1) N=6; sham control; (2) N=6; after SCI, 1 cc saline iv immediately and 7 days after SCI, 10μl culture medium transplanted into epicenter of injury; (3) N=6; after SCI, hUCMSCs (2.5×105) in 1 cc saline iv immediately and 7 days after SCI, 10μl culture medium transplanted into epicenter of injury; (4) N=6; after SCI, 1 cc saline iv immediately and 7 days after SCI, hUCMSCs (2.5×105) in 10μl culture medium transplanted into epicenter of injury; (5) N=6; after SCI, hUCMSCs (2.5×105) in 1 cc saline iv immediately and 7 days after SCI, hUCMSCs (2.5×105) in 10μl culture medium transplanted into epicenter of injury; (6) N=6; after SCI, hUCMSCs (2.5×105) in 1 cc saline iv immediately and 7 days after SCI, hUCMSCs (2.5×105) in 10μl culture medium transplanted into epicenter of injury. All rats except for group 6 received cyclosporine A (10 mg/kg, IP) daily from day 5 after SCI till the completion of the experiments. Herein, The Basso, Beattie, and Bresnahan (BBB) locomotor rating scale was used to measure functional recovery of the rats on the first, second and third week after SCI. After spinal cord injury, in addition to causing animal hindlimb paralysis and dysfunctional behavior, which also occurs after spinal cord injury apoptosis (cell apoptosis) case, it is also more after spinal cord injury, apoptosis in spinal cord tissue and Caspase- 3's performance. Experiment utilizing TUNEL stain (The terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay) and immunofluorescence assay of caspase-3 was measured to do.Spinal cord injury can also cause nerve cells to shrink and sheath (demyelination) and other direct injury, spinal cord injury will start from restoration, during which there will be a lot of scar tissue filler (glial scar formation) lesions, interference neuraxis sudden regeneration (axonal regeneration) thereby affecting nerve conduction. GFAP stain from the results that, given the continuous implantation of spinal cord injury between human umbilical cord mesenchymal stem cell therapy and Sham, saline + CM group and Saline + hUC-MSCs compared to some significant improvement in the injured spinal cord tissue glial scar formation . Therefore, we investigate therapeutic effect of human umbilical cord derived mesenchymal stem cells sequential transplantation after experimental SCI.

Dissertação de mestrado Biologia Celular e Molecular, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra.<br>Está descrito que as células mesenquimais estaminais (MSCs) são extremamente reactivas à modulação por mecanotransdução (Chen, 2008; Eyckmans et al., 2011; Moore et al., 2010), nomeadamente através da expressão tipica de genes especificos da linhagem de certos tecidos quando cultivados in vitro em substratos com propriedades mecanicas similares ás dos mesmos. Nomeadamente, as MScs quando cultivadas em substratos com rigídez semelhante a dos tecidos neuronais (1-10 kPa) expressam genes neuronais (Engler et al., 2006)Também tem sido discrito que estas células parecem reter algum tipo de memoria relacionada com a rigídez dos subtratos em que estiveram cultivadas (Tse et al., 2011). Normalmente as MSCs são isoladas e cultivadas em placas de cultura de poliestireno e so depois de vários passagens transferidas para substratos apropriados para determinar a sua pasticidade em termos de expressão de marcadores de linhagem celular especifica , como já descrito nos casos de “compromisso” dos tipos osteogénico, miogénico e neurogénico (Engler et al., 2006). No entanto, as MSCs podem reter alguma “memoria” do contacto anterior com o poliestireno de rigidez extremamente alta quando comparada a de tecidos humanos, possivelmente diminuindo o potencial em termos de diferenciação (em termos de compromisso com as diferentes linhagens celulares). É do nosso interesse perceber quais serão os efeitos de isolar as MSCs directamente em substratos com rigidez similar a dos tecidos neuronais em termos do seu potencial para expressar marcadores neuronais. Propomos então isolar e cultivar MSCs da matriz do cordão umbilical humano (hUCM) directamente em substratos mais moles, nomeadamente, hidrogéis semelhantes em rigidez ao tecido neuronal (1 a 10 kPa). Como control parte da matriz do cordão umbilical de cada amostra irá ser usado para isolar MSCs usando o protocolo base em placas de cultura de tecidos de poliestireno (TCPs) (Secco et al., 2008) sendo depois transferidas para hidrogéis similares após algumas passagens em poliestireno (P1-P5), para verificar se a cultura prolongada em poliestireno rigo é um factor de restrição na sua capacidade de expressar marcadores neuronais após a cultura em plastico. Para promover a adesão das MSCs aos hidrogéis para a isolação e cultura, estes vão ser covalentemente funcionalizados com colagénio (Engler et al., 2006) e em alguns casos fibronectina. vi Conseguimos optimizar um novo protocolo para isolar MSCs humanas do cordão umbilical, permitindo-nos obter uma população de MSCs humanas indiferenciadas mais homogenea quando comparado com o protocolo de isolação em TCPs. Os hidrogéis de poliacrilamida (PA) usados para a isolação já são utilizados comumente em experiencias de mecanotransdução, mas tanto esta formulação dos hydrogéis como a isolação das hUCM-MSCs em hidrogeis, nunca foi feito antes à luz do nosso conhecimento. Podemos concluir que a FN juntamente com a rigidez do substrato tem um papel importante na proliferação inicial das MSCs humanas quando cultivadas em substratos moles, nomeadamente a 10kPa (Figura 17). Os resultados preliminares (Figuras 18, 19 e tabela III) mostram o que parece ser uma população de MSCs humanas mais indeferenciada e mais homogeneas quando isoladas e cultivadas nos hidrogéis de PA. Finalmente, parece-nos que certos marcadores neuronais (B-III tubulin, Nestin, O4 e GFAP) estão mais expressos nas células já em diferenciação cultivadas nos hidrogéis moles do que nas cultivadas e em diferenciação no plástico (TCP), isto para as células expandidas durante 5 passagens no plástico (TCPs). Em relação as MSCs humana isoladas exclusivamente nos hidrogéis de PA as diferenças entre estas e as MSCs isoladas no plastico não são muito evidentes, mas parece que o O4 está mais expresso nas células isoladas em hidrogéis moles de PA.<br>It is described that Mesenchymal Stem Cells (MSCs) are extremely responsive to modulation by mecanotransduction (Chen, 2008; Eyckmans et al., 2011; Moore et al., 2010), namely by expressing typical lineage-specific genes when cultured in vitro on substrates with mechanical properties similar to those of the target tissues. Namely, MSCs express neural genes when cultured on substrates compliant with neural tissues (1-10 kPa) (Engler et al., 2006). It has also been described that these cells seem to retain some memory related to the stiffness of the substrates in which they were previously cultured on (Tse et al., 2011). Typically, MSCs are isolated and cultured on polystyrene culture dishes (Tse et al., 2011) and eventually transferred onto compliant substrates after several passages to assess their plasticity in terms of lineage-specific expression markers, as reported in case of osteogenic-, myogenic- or neural-like commitment (Engler et al., 2006). Nevertheless, MSCs might retain memory (Tse et al., 2011) from the extremely high stiffness of polystyrene, possibly restraining their full potential in terms of lineage commitment. It is of interest to understand what would be the effect of isolating MSCs directly on substrates with stiffness similar to that of neural tissues in terms of their potential to express neural markers. We propose to isolate and culture human umbilical cord matrix MSCs directly on softer substrates, namely hydrogels compliant with neural tissue (1 to 10KPa). As a control, part of the umbilical cord matrix of every sample will be used to isolate MSCs using normal tissue-culture polystyrene plates (the typical isolation and culture protocol) (Secco et al., 2008) and then transferred onto similar hydrogels after several passages on polystyrene (P1-P5), to address if prolonged culture on hard polystyrene is restraining their capacity to express neural markers later on. To promote the attachment of MSCs onto the hydrogels for isolation and culture, these will be covalently functionalized with collagen (Engler et al., 2006) and Fibronectin. We optimized a new hMSCs isolation protocol for MSCs from UCM, allowing us to obtain naive hMSCs with a more homogenous population when compared to the isolation in TCPs. The PA hydrogels used for the isolation are commonly used in mechanotransduction experiments, but neither this specific formulation neither the iv isolation of hUCM-MSCs was ever done before in PA hydrogels to the best of our knowledge. We can conclude that FN together with substrate stiffness have an important role in the initial proliferation impulse of hMSCs when cultured on soft substrates, namely at 10kPa (Figure 17). Preliminary results (Figure 18, 19 and Table III) show what appears to be a more naive and more homogenous population of hMSCs isolated and cultured on the PA hydrogels. Finally, it seems that neural markers (B-III tubulin, Nestin, O4 and GFAP) are more expressed in differentiating hMSCs plated on soft hydrogels than on plastic for hMSCs expanded for 5 passages on plastic. In terms of hMSCs isolated exclusively on PA hydrogels, the differences between these and hMSCs isolated on plastic were very evident, but O4 seems to be more expressed in cells isolated on soft PA hydrogels.

博士<br>國立交通大學<br>生物科技系所<br>94<br>Abstract During human entire lifespan, from fetus to adult, mesenchymal stem cells (MSCs) proliferate and differentiate into mesenchyme-lineage tissues. Bone marrow and umbilical cord blood are reported to be the main sources of MSCs and have been proposed for possible clinical applications. This study evaluates the tendency in bone marrow-derived MSCs (bmMSCs) and cord blood-derived MSCs (cbMSCs) by in vitro induction and quantification of their characteristics. Results indicated that cbMSCs had a significantly stronger osteogenic potential but less capacity in adipogenic differentiation than bmMSCs. Leptin, an important regulator of mesenchymal differentiation, also acted in bmMSCs and cbMSCs. In both types of MSCs, leptin was found to support osteogenesis, and inhibited adipogenesis. However, Cbfa1 mRNA expression in bmMSCs and cbMSCs was affected at different degree by leptin during osteogenesis. In contrast, leptin reduced PPARγ2 mRNA expression at same level during adipogenesis in both types of MSCs. These results demonstrate the diverse capacity of MSCs from bone marrow and cord blood and suggest that they be used differently in experimental and therapeutic studies. We also isolated the clonogenic MSCs from cord blood by limiting dilution method. These cells exhibited two different morphologic phenotypes, including flattened fibroblastic clones (majority) and spindle-shaped fibroblastic clones (minority). Both types of MSCs shared similar cell surface markers except CD90 and had similar osteogenic and chondrogenic potentials. However, the spindle-shaped clones possessed the positive CD90 expression and a greater tendency in adipogenesis than the flattened clones. The high number of flattened MSCs might actually be linked to the less sensitivity of cbMSCs in adipogenic differentiation. Amniotic fluid and amniotic membrane were also the good sources of mesenchymal stem cells. We successfully isolated MSCs from second-trimester amniotic fluid (AFMSCs) and term amniotic membrane (AMMSCs). AFMSCs and AMMSCs were very similar with MSCs from other sources in the phenotypic morphology, marker profiles (positive for SH2, SH3, SH4, CD29, CD44, CD90 and HLA-I; negative for CD26, CD31, CD34, CD45 and HLA-II). All AFMSCs and AMMSCs samples have mesenchymal-lineage differentiated potentials to differentiate into osteoblasts and adipocytes. The telomerase activity was not detected in AFMSCs, cbMSCs and bmMSCs, and the telomere length of AFMSCs was longer than cbMSCs and bmMSCs. AFMSCs could express embryonic regulators, Oct-4 and Nanog, but they could not express the embryonic markers SSEA-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. AMMSCs could be induced into neuronal cells, which expressed neuronal markers β-tubulin III, tyrosine hydroxylase and NeuN, by insulin and isobutyl-methylxanthine.