Academic literature on the topic 'Aggregated stem cells'
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Journal articles on the topic "Aggregated stem cells"
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Allen, Leah M., John Matyas, Mark Ungrin, David A. Hart, and Arindom Sen. "Serum-Free Culture of Human Mesenchymal Stem Cell Aggregates in Suspension Bioreactors for Tissue Engineering Applications." Stem Cells International 2019 (November 7, 2019): 1–18. http://dx.doi.org/10.1155/2019/4607461.
Full textAbstract:
Mesenchymal stem cells (MSCs) have the capacity to differentiate towards bone, fat, and cartilage lineages. The most widely used culture and differentiation protocols for MSCs are currently limited by their use of serum-containing media and small-scale static culture vessels. Suspension bioreactors have multiple advantages over static culture vessels (e.g., scalability, control, and mechanical forces). This study sought to compare the formation and culture of 3D aggregates of human synovial fluid MSCs within suspension bioreactors and static microwell plates. It also sought to elucidate the benefits of these techniques in terms of productivity, cell number, and ability to generate aggregates containing extracellular matrix deposition. MSCs in serum-free medium were either (1) inoculated as single cells into suspension bioreactors, (2) aggregated using static microwell plates prior to being inoculated in the bioreactor environment, or (3) aggregated using microwell plates and kept in the static environment. Preformed aggregates that were size-controlled at inoculation had a greater tendency to form large, irregular super aggregates after a few days of suspension culture. The single MSCs inoculated into suspension bioreactors formed a more uniform population of smaller aggregates after a definite culture period of 8 days. Both techniques showed initial deposition of extracellular matrix within the aggregates. When the relationship between aggregate size and ECM deposition was investigated in static culture, midsized aggregates (100-300 cells/aggregate) were found to most consistently maximize sGAG and collagen productivity. Thus, this study presents a 3D tissue culture method, which avoids the clinical drawbacks of serum-containing medium that can easily be scaled for tissue culture applications.
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Lee, D. K., C. H. Park, Y. I. Jeong, et al. "24 IMPROVEMENT OF PORCINE CLONED EMBRYONIC STEM-LIKE CELLS DERIVATION BY ZONA-MEDIATED EMBRYO AGGREGATION." Reproduction, Fertility and Development 26, no. 1 (2014): 126. http://dx.doi.org/10.1071/rdv26n1ab24.
Full textAbstract:
Porcine embryonic stem cells (ESC) have become an important model for therapeutic cloning using embryonic stem cells derived by somatic cell nuclear transfer (SCNT). However, embryo quality and blastocyst formation have been major limitations for derivation of cloned embryonic stem-like cells. In this study, we tried to overcome these problems by treating with histone deacetylase inhibitors (HDACi) and aggregating porcine embryos. A porcine embryonic fibroblast (PEF) cell line was used as the source of donor cells injected into the enucleated oocytes. First, to confirm the effect of HDACi in cloned embryo quality, cloned embryos were treated with Scriptaid (histone deacetylase inhibitor). The Scriptaid-treated blastocysts (n = 26) showed significantly increased total cell number (29.50 ± 2.10; P < 0.05) than nontreated blastocysts (n = 21; 22.29 ± 1.50). Then, the cloned embryo quality and blastocyst formation were analyzed in aggregates. Three zona-free reconstructed 4-cell stage SCNT embryos were injected into empty zonae from hatched parthenogenic blastocysts. The blastocyst formation and total cell number of cloned blastocysts was significantly elevated for all the aggregates (76.3% and 83.18 ± 8.33 cells/blastocyst) compared with nonaggregated (31.0%, and 27.11 ± 1.67 cells/blastocyst; P < 0.05). Finally, aggregated blastocysts were cultured on a feeder layer to examine the efficiency of porcine embryonic stem-like cells derivation. Aggregated blastocyst showed higher primary colony formation percentage than nonaggregated cloned blastocysts (20.0 ± 12.3% v. 2.2 ± 1.35%, respectively; P < 0.05). In conclusion, the aggregation of pig SCNT embryos at the 4-cell stage could be a useful technique for improving the development rate and quality of cloned pig blastocyst and derivation efficiency of cloned embryonic stem-like cells.
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Kim, J. Y., S. J. Uhm, K. S. Chung, and H. T. Lee. "188FATE OF CHIMERIC EMBRYONIC STEM CELLS RECONSTRUCTED WITH PARTHENOGENETIC MOUSE EMBRYOS." Reproduction, Fertility and Development 16, no. 2 (2004): 216. http://dx.doi.org/10.1071/rdv16n1ab188.
Full textAbstract:
Mouse parthenogenetic embryos have been known to be unable to develop to term and are absorbed from Day 11. However, Nagy et al. (1990 Development 110, 815–821) reported that the aggregated tetraploid with embryonic stem (ES) cell developed to term and survived after birth. Thus, our study investigated the developmental capacity of the aggregated ES cells with mouse parthenogenetic embryos. Oocytes obtained from superovulated female mice (BCF1) were treated with 7% ethanol and 5μgmL−1 cytochalasin B for the production of pathenotes and co-cultured with sperm (1×106mL−1) for production of fertilized embryos. The reporter vector (pNeoEGFP) was introduced into ES cells (129S4/SvJae) by electroporation. At the 8-cell stage, pathenotes or fertilized embryos, from which the zona pellucida was removed, were co-cultured with ES cells for 4 h. The aggregated parthenotes or fertilized embryos with 5∼10 ES cells were cultured to the blastocyst stage, and transferred into the uteri of 2.5-day post-coitum pseudopregnant recipients. In experiment I, 144 parthenogenetic blastocysts were transferred into the uterine horns of 9 pseudopregnant recipients, and 5 recipients became pregnant. At Day 9, all fetuses were observed visible in uteri of pregnant fonder mice. At Days 10–11, many fetuses were observed in the progress of absorption in uteri of pregnant fonder mice, but a few fetuses were still alive. However, pathenogenetic fetuses were not detected alive beyond 11 days. In experiment II, the 171 aggregated fertilized embryos with ES cells were transferred (15–20 blastocysts/recipient) into 10 recipients and successfully produced 5 offspring from a recipient. We found that three newborn were chimeric mice derived from ES cells. In experiment III, the 209 aggregated parthenotes with ES cells failed to produce offspring, but inserted pNeoEGFP gene in ES cells was detected in the parthenogenetic 1 of 7 fetuses at 15-days of post-gestation by polymerase chain reactions. Therefore, this result suggests that the parthenotes show restricted development to fetus stage, but the aggregated parthenotes with ES cells might extend their developmental capacity. In the future, we will characterize the mechanism of this unusual phenomenon to understand the role of ES cells during development of chimeric parthenotes with ES cells.
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Wu, Fred D., Michael R. Jagir, Thomas T. Luu, and Jerry S. Powell. "Cellular Aggregates Induced from Monolayer Cultures of Human Pancreatic Cells Correct Hyperglycemia in Diabetic Mice and Demonstrate Physiological Production of Human C-Peptide." Blood 104, no. 11 (2004): 4184. http://dx.doi.org/10.1182/blood.v104.11.4184.4184.
Full textAbstract:
Abstract Diabetes is due to loss of appropriate insulin production by pancreatic islet cells, resulting in hyperglycemia and significant morbidity. We have developed cell cultures of pancreas-derived precursor cells that can be maintained for more than 20 population doublings in culture, growing as monolayers of undifferentiated cells. These monolayer cells can then be induced to differentiate into cellular aggregates that resemble islets when grown on different extracellular matrix components, including matrigel, or poly-d-lysine. The induced cellular aggregates, but not monolayer cells, correct hyperglycemia in diabetic (streptozotocin) SCID (severe combined immunodeficiency) mice after implantation under the kidney capsule. This animal model can be used to follow the process of differentiation from undifferentiated precursor cell to fully functional insulin producing beta cells, and to identify key proteins and genes involved in differentiation of pancreatic islet cells. Here we report that in the mice, after implantation of cellular aggregates, human c-peptide concentrations are controlled physiologically appropriately in response to glucose tolerance testing (challenged with 2 grams of glucose injected intraperitoneally) in the treated diabetic SCID mice (n = 6) as well as in normal mice that received implanted cellular aggregates (n = 6). Human c peptide is not detected in any of the control animals, nor in animals implanted with monolayer cells. Comparative RNA microarray data analyses, using U133 arrays (Affymetrix), were compared between the monolayer cells in culture and the cellular aggregates 48 hours after induction with poly-d-lysine. We consider of particular interest the up-regulation of BMP, CXCR4, and HGF in the cell aggregates, and the down-regulation of VCAM-1. We believe that these genes are necessary for either aggregation or for physiologically functional insulin secretion. When examined for greater than two-fold gene expression differences between monolayer and aggregated cells, 424 gene sequences were upregulated and 690 genes down regulated. In addition, RAGE display analysis for tyrosine kinases also showed that the kinases Erb-B2 and PDGFR-B were upregulated at 48 hours in the aggregating process. In contrast, DDR2, a collagen I receptor tyrosine kinase, was expressed equally in both monolayer and aggregated cells. In summary, the results suggest: 1) that a population of pancreatic stem cells can be isolated and cultured in vitro, 2) that these cells can be induced to form functional islet cells that correct hyperglycemia in diabetic mice, 3) that human c-peptide is physiologically regulated in aggregate implanted mice in response to glucose challenge, and 4) HGF, along with other genes of potential interest, is upregulated in the process of differentiation from monolayer to aggregated cell phenotype.
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Nishina, Hironobu, Chisa Katou-Ichikawa, Mizuki Kuramochi, Takeshi Izawa, Mitsuru Kuwamura, and Jyoji Yamate. "Participation of Somatic Stem Cells, Recognized by a Unique A3 Antibody, in Mucosal Epithelial Regeneration in Dextran Sulfate Sodium (DSS)–Induced Rat Colonic Lesions." Toxicologic Pathology 48, no. 4 (2020): 560–69. http://dx.doi.org/10.1177/0192623320906817.
Full textAbstract:
A3, generated as a monoclonal antibody against rat malignant fibrous histiocytoma cells, recognizes somatic stem cells in rats. We analyzed the distribution of A3-positive cells in dextran sulfate sodium (DSS)–induced colonic lesions consisting of regenerating mucosa and fibrosis. Male 6-week-old F344 rats were administered 5% DSS in drinking water for 5 to 7 days, and lesions at recovery stage were also examined. In untreated control adult colons, A3-positive cells are localized around the crypts where stem cell niche is formed. Histopathologically, in colons of DSS-administered rats, mucosal atrophy, inflammatory cell infiltration, and fibrosis were observed in the lamina propria; thereafter, mucosal epithelia were desquamated, and crypts were decreased gradually with decrease in surrounding A3-positive cells. At the early recovery stage, crypts showed regeneration with reappearance of A3-positive cells. Interestingly, A3-positive cells aggregated in desquamated mucosa surface of fibrosis. Aggregated A3-positive cells coexpressed with vimentin, Thy-1, and partly CK19 but did not react simultaneously with α-SMA. Likely, aggregated A3-positive cells may be rescue cells with nature of both mesenchymal and epithelial cells to maintain self-renewal after injury in the colon. A3 antibody would become a useful tool to investigate the participation of stem cells in rat colonic lesions.
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Lupperger, Valerio, Carsten Marr, and Prisca Chapouton. "Reoccurring neural stem cell divisions in the adult zebrafish telencephalon are sufficient for the emergence of aggregated spatiotemporal patterns." PLOS Biology 18, no. 12 (2020): e3000708. http://dx.doi.org/10.1371/journal.pbio.3000708.
Full textAbstract:
Regulation of quiescence and cell cycle entry is pivotal for the maintenance of stem cell populations. Regulatory mechanisms, however, are poorly understood. In particular, it is unclear how the activity of single stem cells is coordinated within the population or if cells divide in a purely random fashion. We addressed this issue by analyzing division events in an adult neural stem cell (NSC) population of the zebrafish telencephalon. Spatial statistics and mathematical modeling of over 80,000 NSCs in 36 brain hemispheres revealed weakly aggregated, nonrandom division patterns in space and time. Analyzing divisions at 2 time points allowed us to infer cell cycle and S-phase lengths computationally. Interestingly, we observed rapid cell cycle reentries in roughly 15% of newly born NSCs. In agent-based simulations of NSC populations, this redividing activity sufficed to induce aggregated spatiotemporal division patterns that matched the ones observed experimentally. In contrast, omitting redivisions leads to a random spatiotemporal distribution of dividing cells. Spatiotemporal aggregation of dividing stem cells can thus emerge solely from the cells’ history.
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Zhao, Xiaozhi, Xuefeng Qiu, Yanting Zhang, Shiwei Zhang, Xiaoping Gu, and Hongqian Guo. "Three-Dimensional Aggregates Enhance the Therapeutic Effects of Adipose Mesenchymal Stem Cells for Ischemia-Reperfusion Induced Kidney Injury in Rats." Stem Cells International 2016 (2016): 1–11. http://dx.doi.org/10.1155/2016/9062638.
Full textAbstract:
It has been shown that administration of adipose derived mesenchymal stem cells (AdMSCs) enhanced structural and functional recovery of renal ischemia-reperfusion (IR) injury. Low engraftment of stem cells, however, limits the therapeutic effects of AdMSCs. The present study was designed to enhance the therapeutic effects of AdMSCs by delivering AdMSCs in a three-dimensional (3D) aggregates form. Microwell was used to produce 3D AdMSCs aggregates. In vitro data indicated that AdMSCs in 3D aggregates were less susceptible to oxidative and hypoxia stress induced by 200 μM peroxide and hypoxia/reoxygenation, respectively, compared with those cultured in two-dimensional (2D) monolayer. Furthermore, AdMSCs in 3D aggregates secreted more proangiogenic factors than those cultured in 2D monolayer. 2D AdMSCs or 3D AdMSCs aggregates were injected into renal cortex immediately after induction of renal IR injury. In vivo data revealed that 3D aggregates enhanced the effects of AdMSCs in recovering function and structure after renal IR injury. Improved grafted AdMSCs were observed in kidney injected with 3D aggregates compared with AdMSCs cultured in 2D monolayer. Our results demonstrated that 3D AdMSCs aggregated produced by microwell enhanced the retention and therapeutic effects of AdMSCs for renal IR injury.
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Yoon, S. W., C. H. Park, S. G. Lee, et al. "225 ANTI-APOPTOTIC EFFECT OF AGGREGATION ON PREIMPLANTATION DEVELOPMENT OF BOVINE IN VITRO-FERTILIZED EMBRYOS." Reproduction, Fertility and Development 21, no. 1 (2009): 210. http://dx.doi.org/10.1071/rdv21n1ab225.
Full textAbstract:
Apoptosis occurs during embryonic development, and is related to early embryonic loss. It is important to produce high-quality blastocysts in vitro for research on the establishment of embryonic stem (ES) cells and transgenic animal production. Therefore, our objectives were to compare the anti-apoptotic effect of bovine aggregate v. nonaggregate IVF embryos and to determine whether aggregation could improve the quality of bovine embryos. The cumulus–oocyte complexes were matured for 20–22 h, and the oocytes were fertilized with cryo-preserved bovine sperm using the swim-up method. After removal of the zona pellucida (ZP), three 4-cell-stage embryos (3X) were aggregated by co-culture in an aggregation hole that was made by an aggregation needle on the culture dish. Embryos were cultured either singularly (1X, ZP removed) or in aggregates of three (3X), and IVF intact embryos served as a control. Five days after aggregation, the developmental rate was observed. The numbers of total cells and apoptotic cells were determined by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assay using blastocyst-stage embryos. Moreover, the mRNA expression pattern related to apoptosis and embryo quality was verified by real-time PCR of the aggregated (3X) and nonaggregated (1X) embryos (at least 3 embryos). The percentage of blastocysts was higher in the 3X aggregated embryos (41.3%) compared with that of the 1X ZP-free embryos (24.3%), whereas there was no significant difference in the 1X embryos and the intact controls (24.3 and 25.8%, respectively; P < 0.05). The total cell number of blastocysts also increased approximately threefold (P < 0.05) in 3X aggregated embryos compared with that of 1X controls. In contrast, the percentage of TUNEL-positive cells, an indication of apoptotic cells, was decreased by approximately threefold in 3X aggregated embryos when compared with that of 1X embryos (7.7 and 2.6%, respectively). The mRNA levels for the Oct-4, NANOG, and bcl-2 genes were higher (P < 0.05) and for the Bax gene were lower in the 3X aggregated embryos than for those of the 1X controls. Therefore, our results indicated that aggregation of bovine IVF embryos at a 4-cell stage could promote the quality and suppress the apoptosis of bovine pre-implantation-stage embryos produced in vitro. Further studies are required to investigate the quality of the aggregated embryos in terms of increasing the establishment rate of ES cell lines by seeding on the feeder layer and raising the efficiency of embryo transfer. This work was supported by the BioGreen 21 Program (#20070401034031, #20080401034031), Rural Development Administration, Republic of Korea (HK).
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Lee, Dong-Kyung, Chi-Hun Park, Kwang-Hwan Choi, et al. "Aggregation of cloned embryos in empty zona pellucida improves derivation efficiency of pig ES-like cells." Zygote 24, no. 6 (2016): 909–17. http://dx.doi.org/10.1017/s0967199416000241.
Full textAbstract:
SummaryThe development of embryonic stem cells (ESCs) from large animal species has become an important model for therapeutic cloning using ESCs derived by somatic cell nuclear transfer (SCNT). However, poor embryo quality and blastocyst formation have been major limitations for derivation of cloned ESCs (ntESCs). In this study, we have tried to overcome these problems by treating these cells with histone deacetylase inhibitors (HDACi) and aggregating porcine embryos. First, cloned embryos were treated with Scriptaid to confirm the effect of HDACi on cloned embryo quality. The Scriptaid-treated blastocysts showed significantly higher total cell numbers (29.50 ± 2.10) than non-treated blastocysts (22.29 ± 1.50, P < 0.05). Next, cloned embryo quality and blastocyst formation were analyzed in aggregates. Three zona-free, reconstructed, four-cell-stage SCNT embryos were injected into the empty zona of hatched parthenogenetic (PA) blastocysts. Blastocyst formation and total cell number of cloned blastocysts increased significantly for all aggregates (76.4% and 83.18 ± 8.33) compared with non-aggregates (25.5% and 27.11 ± 1.67, P < 0.05). Finally, aggregated blastocysts were cultured on a feeder layer to examine the efficiency of porcine ES-like cell derivation. Aggregated blastocysts showed a higher primary colony formation rate than non-aggregated cloned blastocysts (17.6 ± 12.3% vs. 2.2 ± 1.35%, respectively, P < 0.05). In addition, derived ES-like cells showed typical characters of ESCs. In conclusion, the aggregation of porcine SCNT embryos at the four-cell stage could be a useful technique for improving the development rate and quality of porcine-cloned blastocysts and the derivation efficiency of porcine ntESCs.
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Layer, Paul G., Andr??e Rothermel, and Elmar Willbold. "From stem cells towards neural layers: a lesson from re-aggregated embryonic retinal cells." Neuroreport 12, no. 7 (2001): A39—A46. http://dx.doi.org/10.1097/00001756-200105250-00001.
Full textDissertations / Theses on the topic "Aggregated stem cells"
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Sullivan, Denise D. "Incorporation of bio-inspired microparticles within embryonnic stem cell aggregates for directed differentiation." Thesis, Georgia Institute of Technology, 2015. http://hdl.handle.net/1853/54909.
Full textAbstract:
Embryonic stem cells (ESCs) are a unique cell population that can differentiate into all three embryonic germ layers (endoderm, mesoderm, and ectoderm), rendering them an invaluable cell source for studying the molecular mechanisms of embryogenesis. Signaling molecules that direct tissue patterning during embryonic development are secreted by ESC aggregates, known as embryoid bodies (EBs). As many of these signaling proteins interact with the extracellular matrix (ECM), manipulation of the ESC extracellular environment provides a means to direct differentiation. ECM components, such as glycosaminoglycans (GAGs), play crucial roles in cell signaling and regulation of morphogen gradients during early development through binding and concentration of secreted growth factors. Thus, engineered biomaterials fabricated from highly sulfated GAGs, such as heparin, provide matrices for manipulation and efficient capture of ESC morphogens via reversible electrostatic and affinity interactions. Ultimately, biomaterials designed to efficiently capture and retain morphogenic factors offer an attractive platform to enhance the differentiation of ESCs toward defined cell types. The overall objective of this work was to examine the ability of microparticles synthesized from both synthetic and naturally-derived materials to enhance the local presentation of morphogens to direct ESC differentiation. The overall hypothesis was that microparticles that mimic the ECM can modulate ESC differentiation through sequestration of endogenous morphogens present within the EB microenvironment.
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Bratt-Leal, Andrés Miguel. "Biomaterial integration within 3D stem cell aggregates for directed differentiation." Diss., Georgia Institute of Technology, 2011. http://hdl.handle.net/1853/45934.
Full textAbstract:
The derivation of embryonic stem cells (ESCs) has created an invaluable resource for scientific study and discovery. Further improvement in differentiation protocols is necessary to generate the large number of cells needed for clinical relevance. The goal of this work was to develop a method to incorporate biomaterial microparticles (MPs) within stem cell aggregates and to evaluate their use for local control of the cellular microenvironment for directed differentiation.
The effects of unloaded MPs on ESC differentiation were first determined by controlled incorporation of poly(lactic-co-glycolic acid) (PLGA), agarose and gelatin MPs. Embryoid body (EB) formation, cell viability, and gross morphology were not affected by the presence of the MPs. Further analysis of gene expression and patterns of phenotypic marker expression revealed alterations in the differentiation profile in response to material incorporation. The ability of MPs to direct ESC differentiation was investigated by incorporation of growth factor loaded MPs within EBs. MPs were loaded with bone morphogenetic protein-4 (BMP-4). BMP-4 loaded MPs incorporated within EBs induced mesoderm gene expression while inhibiting expression of an ectoderm marker compared to untreated EBs.
Finally, magnetic MPs (magMPs) were incorporated within EBs to induce magnetic sensitivity. The responsiveness of EBs to applied magnetic fields was controlled by the number of magMPs incorporated within the aggregates. Magnetic guidance was then used to control the precise location of single EBs or populations of EBs for bioreactor culture and for construction of heterogeneous cell constructs. Overall, the results indicated that PSC differentiation within spheroids is sensitive to various types of biomaterials. Incorporation of MPs within EBs can be used to direct ESC differentiation by control of the cellular environment from microscale interactions, by delivery of soluble factors, to macroscale interactions, by control of EB position in static and suspension cultures.
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Nguyen, Anh H. "Proteolytically degradable microparticles for engineering the extracellular microenvironment of pluripotent stem cell aggregates." Diss., Georgia Institute of Technology, 2014. http://hdl.handle.net/1853/54845.
Full textAbstract:
During embryo development, extracellular matrix (ECM) remodeling by matrix metalloproteinases (MMPs) and promotes downstream cell specifications. Pluripotent stem cell (PSC) aggregates can recapitulate various aspects of embryogenesis in vitro, and incorporation of biomaterial microparticles also provides an ideal platform to study cell-biomaterial interactions. Stem cell interactions with ECM-based biomaterials can impact tissue remodeling and differentiation propensity via modulation of MMP activity. This work investigated the MMP activity and subsequent mesenchymal differentiation of embryonic stem cell (ESC) aggregates with incorporated gelatin methacrylate (GMA) MPs with either low (20%) or high (90%) cross-linking densities, corresponding to faster or slower degradation rate, respectively. GMA MP incorporation increased total MMP and MMP-2 levels within 3D ESC aggregates in a substrate-dependent manner. GMA MP-incorporated aggregates also expressed higher levels of epithelial-to-mesenchymal transition markers and displayed enhanced mesenchymal morphogenesis than aggregates without MPs, and the MP-mediated effects were completely abrogated with MMP inhibitor treatment. This work predicts that control of proteolytic responses via introducing ECM-based MPs may offer a novel avenue to engineer the ECM microenvironment to modulate stem cell differentiation.
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McAlister, William. "Novel size separation techniques for aggregates of embryonic stem cells using the Stokes equation." Thesis, University of Nottingham, 2016. http://eprints.nottingham.ac.uk/38754/.
Full textAbstract:
Embryoid body formation is a commonly used procedure in embryonic stem cell differentiation as it recapitulates the early stages of embryogenesis and in doing so induces the formation of the three germ layers. Despite being a commonly used step in the differentiation of embryonic stem cells there is still a raft of inconsistencies in this process. As a result, heterogeneity exists in the cells produced in terms of their number and differentiation status; an issue which must be overcome in order to realise the full potential of embryonic stem cells for regenerative medicine. The work produced here is focussed on the issue of size heterogeneity during embryoid body formation. First of all, a closer look at the inherent size-dependent characteristics of embryoid bodies was explored over a 120-hour formation period. This showed that the mean diameter and span of embryoid bodies continued to increase throughout the duration of the 120 hours and provided insight into the population dynamics over this formation period. A non-scalable mesh separation technique was produced to further explore the inherent characteristics of embryoid bodies. This was shown to be successful at collecting size fractions of < 100µm, 100-200µm and > 200µm. Immunohistochemistry was used to show size-dependent differences in embryoid body differentiation by showing differential surface expression of Gata-4, Brachyury and Nestin in different sized populations. Meanwhile cell counts and a growth rate assay were performed which showed further size-dependent differences in the ability of each fraction to produce large numbers of cells. This showed that EBs within the 100-200µm fraction contained the largest numbers of cells and were the most proliferative fraction. However, they were also the only fraction to not express proteins from all three germ layers to any level. Due to the clear size-dependent differences in these size fractions perfusion technique was developed to separate embryoid bodies according to their size in a glass column. This depended on the relationship between particle diameter and terminal falling velocity. Key experimental factors that impacted on the efficacy of this technique were shown to be settled bed height, flow rate, sampling height and flow rate. As a result, two artificial neural networks were developed using a model system of Spehadex beads to show how varying these factors impacted the mean diameter and span of collected particles at a constant settled bed height. These experiments showed that this technique was limited to collecting particles from the lower reaches of the stock for both Sephadex beads and embryoid bodies. This was because the most successful factor for increasing mean diameter, flow rate, also induced the greatest increase in span. Therefore, collecting larger sized fractions reduced their discrete nature to such an extent that they were no longer distinct from one another. This suggested that this technique had potential, but was limited by the flow characteristics caused by increasing flow rate. As a result, the separation technique was adapted to overcome this by inverting the column and removing the impact of flow rate; instead allowing gravity alone to separate the particles using a top-loading method. This method was shown to be substantially more successful at collecting larger particles in discrete fractions, however, there were issues with collecting the smaller fractions during a 15-minute separation. Adaptation of this technique allowed all three size fractions to be collected effectively. Finally, a growth rate assay was performed to determine if the embryoid bodies could survive the conditions encountered in this technique and continue to grow post-separation. The cells collected from the column were able to continue growing when dissociated and grown in two-dimensional culture. However, their growth rate was lower than that in the unseparated control and it is not known whether this was because the technique is destructive. This research has therefore identified a cheap, effective, tuneable, novel size-separation technique for embryoid bodies that can collect multiple fractions in a single run with high throughput.
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Garvin, Joshua (Joshua J. ). "Scalable production of cellular aggregates for the differentiation of embryonic stem cells into cardiac muscle." Thesis, Massachusetts Institute of Technology, 2008. http://hdl.handle.net/1721.1/45793.
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Thesis (S.B.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2008.<br>Includes bibliographical references (leaves 36-37).<br>Embryonic stem (ES) cells have the potential to treat many diseases, such as heart disease, diabetes, and Parkinson's disease. However, large numbers of desired differentiated or progenitor cells must be generated from ES cells for many regenerative medicine applications to be successful. Current methods of culture used in the laboratory either cannot be scaled-up to produce sufficiently large numbers of cells or do not consistently produce aggregates of uniform size. In this study, novel methods for aggregating and encapsulating embryonic stem cells were investigated. Latex microspheres and the [beta]TC3 cell line were used in place of ES cells during the development of the methods. Microspheres and cells were encapsulated in an alginate solution coated with poly-L-lysine using an established drip method and a novel fluorinated oil "floating drop" method. Results from these experiments demonstrate that both methods can be used for encapsulating and growing cells. However aggregation, an important aspect for the directed differentiation of ES cells, only occurred using the "floating drop" method, and this method was used to encapsulate a predetermined number of cells in capsules of a specified size. The "floating drop" method has the advantage that culture media can be changed during cell culture to increase the duration of experiments without transferring the aggregates to culture flasks and can potentially be scaled up to produce large numbers of encapsulations.<br>by Joshua Garvin.<br>S.B.
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Baillie-Johnson, Peter. "The generation of a candidate axial precursor in three dimensional aggregates of mouse embryonic stem cells." Thesis, University of Cambridge, 2017. https://www.repository.cam.ac.uk/handle/1810/267818.
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Textbook accounts of vertebrate embryonic development have been based largely upon experiments on amphibian embryos, which have shown that the tissues of the trunk and tail are organised from distinct precursors that existed during gastrulation. In the mouse and chick, however, retrospective clonal analyses and transplantation experiments have demonstrated that the amniote body instead arises progressively from a population of axial precursors that are common to both the neural and mesodermal tissues of the trunk and tail. For this reason, they are known as neuro-mesodermal progenitors (NMps). Detailed studies of NMps have been precluded by their lack of a unique gene expression profile and the technical difficulties associated with isolating them from the embryo. Mouse embryonic stem cells (ESCs) provide the possibility of instead deriving them in vitro. ESCs have been used to model developmental processes, partly through large cellular aggregates known as embryoid bodies. These structures do not, however, resemble the axial organisation of the embryo and they develop in a disordered manner. This thesis presents a novel culture system of small, three-dimensional aggregates of ESCs (gastruloids) that can recreate the events of early post-implantation development, including axial elongation. Gastruloids are the first ESC-based model for axial elongation morphogenesis; this body of work characterises their development and identifies a candidate population of NMps within their elongating tissues. Additionally, this work establishes a xenotransplantation assay for testing the functional properties of in vitro-derived NMp populations in the chicken embryo and applies it to NMps from gastruloid cultures. The results of this assay show that gastruloids are a credible source of NMps in vitro and therefore offer a new experimental means to interrogate their properties. The use of gastruloids to recreate embryonic development has implications for basic research as a synthetic system and for the therapeutic derivation of other embryonic progenitors through bioengineering.
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Terrasso, Ana Paula Barreto. "Development of novel human cellular models for neurotoxicity studies." Master's thesis, Faculdade de Ciências e Tecnologia, 2012. http://hdl.handle.net/10362/8488.
Full textAbstract:
Dissertation to obtain master degree in
Genética Molecular e Biomedicina<br>Information currently available on neurotoxicity of chemicals is scarce and there are a growing number of new compounds to be tested. Therefore, new strategies are necessary to identify neurotoxic agents with speed, reliability and respect for animal welfare.
The limited availability of primary human brain cells means that there is a need for human cell lines that reliably model human neurons and astrocytes. Despite the advances in stem cell research, numerous challenges must be overcome before this technology can be widespread used, such as low differentiation efficiency.
Human pluripotent embryocarcinoma NTera2/cloneD1 (NT2) cell line is an alternative cell source from which neurons and astrocytes can be derived in vitro.
The aim of this work was to develop scalable and reproducible novel human cellular models using NT2 cells as source of differentiated neural phenotypes.
A 2D culture system for astrocytic differentiation was implemented. After 4 weeks of differentiation with retinoic acid followed by 5 weeks maturation with mitotic inhibitors, astrocytes obtained expressed vimentin, GFAP, S100- and GLT-1 as characterized by immunodetection and qRT-PCR.
Then, a 3D culture approach was adopted, using stirred suspension culture systems, in which cell-cell and cell-extracellular matrix interactions occur, mimicking better the in vivo situation. NT2 cells, inoculated as single cells, spontaneously aggregated without compromising their pluripotency. Optimization of stirring rate allowed control of aggregate size along time. After 3 weeks of RA treatment and 2 weeks of maturation, neurons expressing βIII-tubulin, MAPs and synaptophysin and astrocytes expressing vimentin, GFAP, S100- and GLT-1 were detected, as characterized by immunodetection and qRT-PCR. Furthermore, astrocytes presented a 2.5-fold higher yield than that observed in 2D culture systems.
Results showed that NT2 differentiated cells are promising models for neurotoxicity testing. Furthermore, the 3D culture systems developed herein can contribute to increase the relevance of these studies, recapitulating human neuron-astrocyte interactions in a 3D cellular context.<br>Fundação para a Ciência e Tecnologia - PTDC/EEB-BIO/112786/2009
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Dang, Phuong Ngoc. "READILY IMPLANTABLE HIGH DENSITY STEM CELL SYSTEMS WITH CONTROLLED GROWTH FACTOR PRESENTATION FROM BIOACTIVE MICROPARTICLES FOR BONE REGENERATION VIA ENDOCHONDRAL OSSIFICATION." Case Western Reserve University School of Graduate Studies / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=case1421864780.
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White, Douglas. "Analyzing multicellular interactions: A hybrid computational and biological pattern recognition approach." Diss., Georgia Institute of Technology, 2015. http://hdl.handle.net/1853/54876.
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Pluripotent embryonic stem cells (ESCs) can differentiate into all somatic cell types, making them a useful platform for studying a variety of cellular phenomenon. Furthermore, ESCs can be induced to form aggregates called embryoid bodies (EBs) which recapitulate the dynamics of development and morphogenesis. However, many different factors such as gradients of soluble morphogens, direct cell-to-cell signaling, and cell-matrix interactions have all been implicated in directing ESC differentiation. Though the effects of individual factors have often been investigated independently, the inherent difficulty in assaying combinatorial effects has made it difficult to ascertain the concerted effects of different environmental parameters, particularly due to the spatial and temporal dynamics associated with such cues. Dynamic computational models of ESC differentiation can provide powerful insight into how different cues function in combination both spatially and temporally. By combining particle based diffusion models, cellular agent based approaches, and physical models of morphogenesis, a multi-scale, rules-based modeling framework can provide insight into how each component contributes to differentiation. I propose to investigate the complex regulatory cues which govern complex morphogenic behavior in 3D ESC systems via a computational rules based modeling approach. The objective of this study is to examine how spatial patterns of differentiation by ESCs arise as a function of the microenvironment. The central hypothesis is that spatial control of soluble morphogens and cell-cell signaling will allow enhanced control over the patterns and efficiency of stem cell differentiation in embryoid bodies.
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Lefebvre, Omar Cynthia. "Défauts intrinsèques de motoneurones spinaux dérivés de cellules souches pluripotentes induites issues d’individus atteints de différentes formes de Sclérose Latérale Amyotrophique." Thesis, Sorbonne université, 2018. http://www.theses.fr/2018SORUS507.
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La Sclérose Latérale Amyotrophique (SLA) est une maladie neurodégénérative caractérisée par la mort des motoneurones (MNs). Malgré plusieurs hypothèses pouvant expliquer les mécanismes à l’origine de leur mort sélective, l’hétérogénéité de la SLA rend difficile la compréhension des causes exactes de la dégénérescence. Dans ce contexte, les cellules souches pluripotentes induites humaines (iPSC) permettent l’étude des formes familiales de la maladie comme des formes sporadiques. Contrairement à la majorité des travaux publiés à ce jour qui étudient des iPSC de patients porteurs de mutation dans un seul gène de SLA, mon projet a eu pour objectif de comparer plusieurs formes de SLA dans un même contexte expérimental. A partir d’iPSC de patients présentant différentes formes génétiques de SLA (C9ORF72, SOD1, TARDBP), nous avons obtenu des cultures pures de MNs humains. Alors que nous n’avons pas observé de mort des MNs mutants après plusieurs semaines, des études fonctionnelles d’électrophysiologie ont montré une altération tardive de l’excitabilité des MNs en fonction des patients. De façon plus précoce, nous avons observé la présence d’agrégats protéiques communs ou spécifiques aux différentes formes de SLA, avec certaines accumulations localisées au niveau du segment proximal de l’axone, une région importante pour la maintenance de l’identité axonale et le déclenchement des potentiels d’action. Des altérations physiques ou moléculaires ont été mises en évidence au niveau de ce segment dans les MNs mutants, suggérant qu’une perturbation du segment proximal de l’axone pourrait être un évènement très précoce altérant ainsi l’intégrité et la fonctionnalité des MNs de patients<br>Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder characterized by motor neurons death (MNs). Despite several hypothesis trying to explain this selective loss, the exact reasons of MNs degeneration remain unidentified mainly due to the disease heterogeneity. In this respect, the use of human induced pluripotent stem cells (iPSC) are opening up opportunities to model not only familial but also sporadic forms of ALS. In comparison to previously published studies, which focus only on one type of ALS mutation, my thesis had the objective to compare in a same experimental context multiple forms of ALS in order to distinguish similarities and discrepancies inherited by the mutation. Using iPSC obtained from genetic forms of ALS patients (C9ORF72, SOD1, TARDBP) as well as control subjects, we generated pure cultures of human MNs. While ALS MNs were not sensitive to death after few weeks of culture, electrophysiological functional studies revealed a patient-dependent late alteration in MNs excitability. Early defects were also reported, with observations of generic and mutation-specific protein aggregates. Interestingly, some accumulations were localized at the axonal initial segment (AIS) region, which is important for maintaining axonal identity and crucial for action potentials’ initiation. Physical and/or molecular alterations were reported at the AIS in ALS MNs, suggesting that AIS perturbation could be an early event in MN degeneration by disruption of ALS patients’ MNs integrity and functionality
Books on the topic "Aggregated stem cells"
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Kastritis, Efstathios, and Meletios A. Dimopoulos. The patient with myeloma. Edited by Giuseppe Remuzzi. Oxford University Press, 2018. http://dx.doi.org/10.1093/med/9780199592548.003.0153_update_001.
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Renal impairment is a common feature of multiple myeloma and often the presenting symptom of a patient with symptomatic myeloma. ‘Myeloma kidney’ results from the excess of immunoglobulin light chains which form aggregates and casts that result in tubular obstruction; however, light chains may cause renal damage with a variety of mechanisms, which often may coexist in the same patient. The presence of significant renal dysfunction in a patient with myeloma is associated with a risk for significant complications, including early death. Myeloma kidney is usually associated with high tumour burden and high rates of paraprotein production.Patients with renal impairment should be managed immediately with appropriate antimyeloma therapy and supported vigorously. New drugs such as bortezomib are probably the most effective therapies for patients with renal dysfunction and may improve renal function in a significant proportion of patients with myeloma-related renal impairment, especially with dexamethasone at high doses. Other drugs such as thalidomide or lenalidomide may also be helpful in certain patients. Direct removal of the toxic free light chains may improve outcomes in some patients, but randomized studies are still ongoing. The role of plasmapheresis has not been established.Autologous stem cell transplantation, with appropriate dose adjustments for high-dose melphalan should be offered in eligible patients, even those on dialysis, although this procedure may be associated with a higher risk of toxicity in patients with more severe renal dysfunction. Renal transplantation may be an option for selected patients who have responded well to therapy.
Book chapters on the topic "Aggregated stem cells"
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Kondoh, Hisato, and Mai Fujii. "Definitive Endoderm from EpiSC Aggregates in Matrigel." In Epiblast Stem Cells. Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2281-0_15.
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Sui, Bing-Dong, Bin Zhu, Cheng-Hu Hu, Pan Zhao, and Yan Jin. "Reconstruction of Regenerative Stem Cell Niche by Cell Aggregate Engineering." In Stem Cell Niche. Springer New York, 2018. http://dx.doi.org/10.1007/7651_2018_186.
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Abu-Hakmeh, Ahmad E., and Leo Q. Wan. "High-Throughput Cell Aggregate Culture for Stem Cell Chondrogenesis." In Methods in Molecular Biology. Springer New York, 2014. http://dx.doi.org/10.1007/7651_2014_75.
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Miyatake, Yukiko, and Masanori Kasahara. "Anchorage-Dependent Multicellular Aggregate Formation Induces CD44 High Cancer Stem Cell-Like Phenotypes in Adult T Cell Leukemia/Lymphoma Cells." In Inflammation and Immunity in Cancer. Springer Japan, 2015. http://dx.doi.org/10.1007/978-4-431-55327-4_6.
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Ashok, Preeti, Yongjia Fan, Mahboubeh R. Rostami, and Emmanuel S. Tzanakakis. "Aggregate and Microcarrier Cultures of Human Pluripotent Stem Cells in Stirred-Suspension Systems." In Methods in Molecular Biology. Springer New York, 2015. http://dx.doi.org/10.1007/7651_2015_312.
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Schulz, J. C., F. K. Groeber, A. F. J. Beier, et al. "Methods for Encapsulation and Storage of Human Stem Cells in Three Dimensional Alginate Aggregates." In IFMBE Proceedings. Springer Berlin Heidelberg, 2009. http://dx.doi.org/10.1007/978-3-642-03900-3_64.
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Bauwens, Celine L., and Mark D. Ungrin. "Scalable Cardiac Differentiation of Human Pluripotent Stem Cells as Microwell-Generated, Size Controlled Three-Dimensional Aggregates." In Methods in Molecular Biology. Springer New York, 2014. http://dx.doi.org/10.1007/978-1-4939-1047-2_2.
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Li, Quan, Guangyan Qi, and Xiuzhi Susan Sun. "Advanced Hydrogel for Physiological 3D Colonies of Pluripotent Stem Cells." In Advances in Pluripotent Stem Cells [Working Title]. IntechOpen, 2023. http://dx.doi.org/10.5772/intechopen.112656.
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Human induced pluripotent stem cells (hiPSCs) demonstrated great potential in basic research, disease modeling, drug development, cell therapeutics, and regenerative medicine, as various distinct somatic cell types such as hepatocytes can be derived from hiPSCs. However, highly efficient hiPSC to somatic cell differentiation has not yet been achieved because of various challenging problems, one of which is less-optimal culture methods for hiPSC expansion. Conventionally, hiPSCs have been cultured as monolayers on flat surfaces, usually resulting in unstable genetic integrity, reduced pluripotency, and spontaneous differentiation after numerous passages. Recently, three-dimensional (3D) spheroids of hiPSCs have shown potential for somatic cell differentiations. However, these hiPSC spheroids are generated using 2D-cultured cells in either nonadherent U-bottom 96-well plates or agarose microarray molding plates, in which single hiPSCs are forced to aggregate into spheroids. These “aggregation molding” methods are neither typically suited for large-scale hiPSC manufacturing nor for tissue engineering. In addition, the aggregated hiPSC spheroids present limited functions compared to physiologically formed hiPSC 3D colonies. In this chapter, advanced 3D cell culture technologies will be reviewed, and comprehensive discussions and future development will be provided and suggested.
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Papaioannou, Virginia, and Randall Johnson. "Production of chimeras by blastocyst and morula injection of targeted ES cells." In Gene Targeting. Oxford University Press, 1999. http://dx.doi.org/10.1093/oso/9780199637928.003.0008.
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The ability of mammalian embryos to incorporate foreign cells and develop as chimeras has been exploited for a variety of purposes including the elucidation of cell lineages, the investigation of cell potential, the perpetuation of mutations produced in embryonic stem (ES) cells by gene targeting, and the subsequent analysis of these mutations. The extent of contribution of the foreign cells depends on their developmental synchrony with the host embryo and their mitotic and developmental potential, which may be severely restricted if the cells bear mutations. If the goal in making chimeras is the transmission of a mutation produced by gene targeting to the next generation, the mutant ES cells must have the capacity to undergo meiosis and gametogenesis. Cells from two different mammalian embryos were first combined experimentally to produce a composite animal, dubbed a chimera, nearly four decades ago. Pairs of cleaving, pre-implantation embryos were mechanically associated in vitro until they aggregated together to make single large morulae; these in turn resulted in chimeric offspring (1). Genetic markers were used to distinguish the contributions of the two embryos in these animals. Since then, various methods for making chimeras have been explored to address different types of questions (2). In 1972 it was reported that highly asynchronous embryonic cells, which had been cultured in vitro, could contribute to chimeras upon re-introduction into pre-implantation embryos (3). Not long afterward, several groups working with teratocarcinomas, tumours derived from germ cells of the gonad, discovered that stem cells from these tumours, known as embryonal carcinoma cells, could contribute to an embryo if introduced into pre-implantation stages (4-6). It appeared that the undifferentiated stem cells of the tumour had enough features in common with early embryonic cells that they could respond to the embryonic environment, differentiating in a normal manner, even after long periods in vitro. Their embryonic potential was limited, however, and many teratocarcinoma cell lines made only meagre contributions to the developing fetus or even produced tumours in chimeras (7). Either their derivation from tumours or their extended sojourn in vitro rendered these cells so dissimilar from early embryonic cells that they rarely, if ever, had full embryonic potential.
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Tarasova, Yelena S., Daniel R. Riordon, Kirill V. Tarasov, and Kenneth R. Boheler. "In vitro differentiation of mouse ES cells into muscle cells." In Embryonic Stem Cells. Oxford University PressOxford, 2006. http://dx.doi.org/10.1093/oso/9780198550006.003.0006.
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Abstract Following the derivation of ES cell lines from mouse embryos (1, 2), Wobus et al. 1 and Doetschman et al. (4) observed that mouse ES (mES) cells when grown in suspension culture spontaneously form three-dimensional cell aggregates, termed embryoid bodies (EBs), which generate multiple differentiated cell types. These aggregates were later shown to give rise to, in vitro, cell lineages including those typical of striated and non-striated muscle. This chapter provides current techniques for the production of muscle derivatives from mES cells via EBs, and for their functional analysis. In vertebrates, striated cardiac and skeletal muscle cells are derived from mesoderm. Commitment of mesodermal cells to the cardiogenic lineage is established during and shortly after gastrulation, at which time cardiogenic cells are organized in bilateral epithelial sheets that are part of the visceral mesoderm lining the developing coelomic cavity (5). Skeletal muscle forms in the vertebrate limb from progenitor cells that originate in somites; these cells delaminate from dermomyotome, and migrate into the limb bud where they proliferate, express myogenic determination factors, and differentiate into skeletal muscle (6). Non-striated smooth-muscle cells (SMC) originate both from neuroectoderm (neural crest), which contains cells committed to the SMC lineage, and from mesodermal sources (endothelium, epicardium) that have undergone limited differentiation (7).
Conference papers on the topic "Aggregated stem cells"
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Kondo, Masaki, Hideki Kamiya, Tetsuji Okawa, et al. "3D-aggregated dermal stem cells with partial-pluripotency." In 2012 International Symposium on Micro-NanoMechatronics and Human Science (MHS). IEEE, 2012. http://dx.doi.org/10.1109/mhs.2012.6492401.
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Isu, Giuseppe, Diana Massai, Giulia Cerino, et al. "A Novel Perfusion Bioreactor for 3D Cell Culture in Microgravity Conditions." In ASME 2013 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2013. http://dx.doi.org/10.1115/sbc2013-14502.
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Cell suspension culture methods based on the generation of microgravity environment are widely used in regenerative medicine for (1) the production of native-like three-dimensional (3D) cell aggregates and engineered tissues [1,2,3], for (2) low cost scalable cell expansion and long-term cell viability maintenance [4,5], and for (3) guiding differentiation of stem cells (SCs) [6]. The generation of a microgravity environment for 3D cell cultures, mimicking the native environment, promotes spatial freedom, cell growth, cell-cell interaction and improves mass transfer and cell exposure to nutrients. Nowadays, microgravity cell cultures are obtained by using stirred or rotating bioreactors, but both devices suffer from limitations: stirring bioreactors generate non-physiological shear stresses, which could damage cultured cells, interfere with SC pluripotency, and limit reproducibility of the culture process; rotating bioreactors are expensive devices due to the complex technological solutions adopted for obtaining rotation [5].
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Wan, Chen-rei, Seok Chung, Ryo Sudo, and Roger D. Kamm. "Induction of Cardiomyocyte Differentiation From Mouse Embryonic Stem Cells in a Confined Microfluidic Environment." In ASME 2009 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2009. http://dx.doi.org/10.1115/sbc2009-203995.
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Embryonic stem cell derived cardiomyocytes are deemed an attractive treatment option for myocardial infarction. Their clinical efficacy, however, has not been unequivocally demonstrated. There is a need for better understanding and characterization of the cardiogenesis process. A microfluidic platform in vitro is used to dissect and better understand the differentiation process. Through this study, we find that while embryoid bodies (EBs) flatten out in a well plate system, differentiated EBs self-assemble into complex 3D structures. The beating regions of EBs are also different. Most beating areas are observed in a ring pattern on 2D well plates around the center, self-assembled beating large 3D aggregates are found in microfluidic devices. Furthermore, inspired by the natural mechanical environment of the heart, we applied uniaxial cyclic mechanical stretch to EBs. Results suggest that prolonged mechanical stimulation acts as a negative regulator of cardiogenesis. From this study, we conclude that the culture environments can influence differentiation of embryonic stem cells into cardiomycytes, and that the use of microfluidic systems can provide new insights into the differentiation process.
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Sperling, Patrick, Rainer Horstkotte, Jan Sommer, et al. "Challenges in the Production of Fuel Cells for Aviation." In ASME Turbo Expo 2024: Turbomachinery Technical Conference and Exposition. American Society of Mechanical Engineers, 2024. http://dx.doi.org/10.1115/gt2024-125888.
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Abstract The influence of increasing resource scarcity and climate change has a significant impact on aviation. Therefore, the European Commission has set ambitious goals with a 75 % reduction in CO2 and a 90 % reduction in NOX as part of the Flightpath 2050 vision. Achieving these goals requires the use of disruptive technologies in aviation, especially in propulsion. In addition to the use of Sustainable Aviation Fuel (SAF), the use of hydrogen is a promising alternative. There is the possibility of using a fuel cell which acts as an energy converter and powers the primary electric drives, offering the potential to fly emission-free, especially for short- and medium-range flights. However, successful integration of the fuel cell into the aircraft’s powertrain requires a significantly increased gravimetric power density compared to the status quo. Furthermore, the fuel cell and the entire manufacturing process chain must fulfill the stringent safety requirements of aviation. Therefore, the bipolar plate and, respectively, its production process are key elements for the fuel cell. This paper describes how to establish a basis for the production of the bipolar plate in accordance with aviation requirements. For this purpose, the process chain is explained step by step and for each process step different technology alternatives and corresponding challenges are identified. Finally, an aggregated view of the holistic process chain is given.
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Ravichandran, Dharneedar, and Kenan Song. "One-Step 3D Printed Layers Along With xy-in Plane Directions for Enhanced Multifunctional Nanocomposites." In ASME 2022 17th International Manufacturing Science and Engineering Conference. American Society of Mechanical Engineers, 2022. http://dx.doi.org/10.1115/msec2022-85056.
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Abstract Composite and hybrid materials displaying layered structures have broad applications in structural composites, fire retardant barriers, tissue scaffolds, and microelectronics. Inspired by biosystems, in this study, we explore the invention of a new 3D printing principle that can produce layered structures similar to those in trees, overcoming the bottleneck in additive manufacturing to include multi-materials. We use polyvinyl alcohol (PVA) and carbon nanotubes (CNTs) as material examples for producing alternating layers. With the unique 3D printing platform, Multiphase Direct Ink Writing (MDIW), the optimized dispersion quality and rheology behaviors allow the number of layers within an individual printing line to change between 4 and 512 layers. The mechanical tests consistently increased young’s modulus and ultimate tensile strength with decreased layer thickness and dispersion quality. The best-performed composites have 128 layers in one printing line, beyond which the dispersion of CNTs deteriorated due to aggregates. Due to the thin layer thickness, the improved composite mechanics relate to the closely packed CNTs and their alignment. Moreover, we will also demonstrate this MDIW printing with different polymers (e.g., thermoplastic urethane and polylactic acid) and nanoparticles (e.g., iron oxide, carbon fibers) for mechanical enhancement and intelligent behaviors. This research demonstrated one new 3D printing method, MDIW, that can fabricate multilayered composites containing well-managed content in each layer. Our advanced manufacturing method is compatible with other materials and has potential use in batteries, supercapacitors, solar cells, regenerative medicine, and energetic systems requiring layered structures.
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Narzary, Diganta, David Stasenko, and Nikhil Rao. "Thrust Force Measurements in an Axial Steam Turbine Test Rig: Effect of Disk Balance Holes." In ASME Turbo Expo 2021: Turbomachinery Technical Conference and Exposition. American Society of Mechanical Engineers, 2021. http://dx.doi.org/10.1115/gt2021-59242.
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Abstract A full-size, full-speed, axial flow steam turbine test rig capable of measuring turbine thrust, and static pressures in the rotor-stator disk cavity was built and commissioned. The test rig was operated in a single-stage configuration for the test results first reported in Stasenko et al. [1], and now in this paper. The stage has stationary axial face seals radially inward of the airfoils, near the rotor disk rim. The face seals divide the rotor-stator cavity into inner and outer circumferential cavities, both of which were instrumented with static pressure probes on the stator radial wall. Axial thrust was measured with load cells in every thrust bearing pad. The test rig was operated over a range of three nominal stage pressure ratios (designated as LPR, MPR, and HPR), five nominal stage velocity ratios (0.25–0.6), and five admission fractions (0.38–0.88). This latest group of tests was conducted without rotor disk balance holes, which were mechanically plugged, and will be compared to the original block of tests with disk balance holes opened. In the upstream disk cavity, the two disk balance hole configurations shared many similar pressure characteristics: nearly uniform pressures in the inner cavity, circumferential pressure distributions in the outer cavity that corresponded with the direction of axial thrust, and radial pressure distributions in the outer cavity that were a direct function of rotor speed. General trends of thrust coefficients with the disk holes plugged were correlated to stage pressure ratio, stage velocity ratio, admission fraction, and leakage mass flow rate. Those trends were consistent with the first block of tests with open disk balance holes, although there was an offset toward more operating conditions with negative aggregate thrust coefficients. This suggests that the rotating disk induces a low-pressure gradient in the inner (upstream) cavity, and the opened disk balance holes tend to equalize the inner cavity static pressure toward the higher static pressure on the exit side of the disk. Additionally, thrust coefficients tended to become less negative (or more positive) with stage pressure ratio and with velocity ratio, but tended to become more negative with admission fraction. Significant thrust coefficient reductions were realized with the open disk balance hole configuration, and were determined to be consistently speed-dependent.
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Lai, W. M., D. D. Sun, G. A. Ateshian, X. E. Guo, and V. C. Mow. "Effects of Inhomogeneous Fixed Charge Density on the Electrical Signals for Chondrocytes in Cartilage." In ASME 2000 International Mechanical Engineering Congress and Exposition. American Society of Mechanical Engineers, 2000. http://dx.doi.org/10.1115/imece2000-1931.
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Abstract An important step toward understanding the signal transduction mechanisms that modulate cellular activities is the accurate prediction of the mechanical and electro-chemical environment of the cells in well-defined experimental configurations. One such configuration is the steady permeation experiment (e.g., bioreactors) in the open circuit condition. Using our triphasic theory, we have calculated the strain, velocity and the electric potential fields inside a layer of charged articular cartilage, through which a uni-univalent salt (e.g., NaCl) solution permeates under a constant pressure difference across the layer. The fluid flow through the tissue gives rise to an electrical potential difference across the tissue. This potential difference is the well-known “streaming potential” that is measured by Ag/AgCl electrodes placed across the tissue on the outside. Our results show that inside the tissue, in addition to the streaming potential caused by fluid convection, there is also a “diffusion potential” caused by cation and anion concentration gradients that are induced by the gradient of fixed charge density (FCD) inside the tissue. The gradient of FCD may be intrinsic, i.e., the tissue has an inhomogeneous FCD distribution, or it may also be caused by a non-uniform compaction of the solid matrix as is the case in steady permeation where the drag force exerted by the permeating fluid onto the solid matrix causes a compressive strain field inside the tissue. In this experimental configuration, the diffusion potential would compete against the streaming potential. The magnitude and the polarity of the electric field depend, amongst other material parameters, on the compressive stiffness of the tissue. For softer tissue (e.g., aggregate modulus &lt;0.54 MPa for a set of realistic material and testing parameters), the diffusion potential dominates over the streaming potential and vice versa for stiffer tissue. For articular cartilage what the cells see in situ is the combined electrical effect of intrinsic and deformation induced inhomogeneity of FCD. The present results provide not only quantitative information, but also new insight into an important problem in biotechnology. These results also demonstrate that for proper interpretation of the mechano-electrochemical signal transduction mechanisms that is needed for modulating cellular biosynthetic activities, one must not ignore the important effects of diffusion potential.
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Mattson, C. J., and W. D. Estry. "EFFECTS OF 12-O-TETRADECANOYL PH0RB0L-13-ACETATE (TPA) ON PLATELET ADHESION-INDUCED SHAPE CHANGE." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643552.
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Adherent platelets undergo changes in shape which involve conversion of the resting discoid platelet to a sphered or dentritic/pseudopodial form followed by cytoplasmic spreading to produce a fully spread cell with centralized organelles. Platelets from donors who have taken 10 gr aspirin (ASA) 4 hr prior to venipuncture show adequate pseudopod formation but defective spreading. Addition of 0.5 uM ADP is sufficient to reconstitute normal spreading suggesting that shape change is a two step process and that formation of spread platelets may require release of dense granule ADP. Since activation of platelets in suspension by ADP is associated with degradation of phosphoinositides and phosphorylation of a 40 KD protein, the potential role of this pathway in the formation of spread platelets was examined. Citrated platelets formed large aggregates when stimulated by TPA interfering with evaluation of adhesion and contact-activated shape change. When EGTA treated platelets were stimulated with TPA, aggregation was blocked but adhesion and spreading were unaffected by calcium chelation in the presence of magnesium. Therefore ASA-inhibited, EGTA-treated platelets were stimulated with 100 ng/ml TPA to activate protein kinase C, and platelet morphology was evaluated by whole mount transmission electron microscopy. TPA stimulated platelets underwent normal shape change to the fully spread stage despite endoperoxide inhibition by ASA. This data supports a role for ADP-trigqered phosphoinositide degradation in the spreading phase of adhesion induced shape change.
Reports on the topic "Aggregated stem cells"
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Gafny, Ron, A. L. N. Rao, and Edna Tanne. Etiology of the Rugose Wood Disease of Grapevine and Molecular Study of the Associated Trichoviruses. United States Department of Agriculture, 2000. http://dx.doi.org/10.32747/2000.7575269.bard.
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Rugose wood is a complex disease of grapevines, characterized by modification of the woody cylinder of affected vines. The control of rugose wood is based on the production of healthy propagation material. Detection of rugose wood in grapevines is difficult and expensive: budwood from tested plants is grafted onto sensitive Vitis indicators and the appearance of symptoms is monitored for 3 years. The etiology of rugose wood is complex and has not yet been elucidated. Several elongated clostero-like viruses are consistently found in affected vines; one of them, grapevine virus A (GVA), is closely associated with Kober stem grooving, a component of the rugose wood complex. GVA has a single-stranded RNA genome of 7349 nucleotides, excluding a polyA tail at the 3' terminus. The GVA genome includes five open reading frames (ORFs 1-5). ORF 4, which encodes for the coat protein of GVA, is the only ORF for which the function was determined experimentally. The original objectives of this research were: 1- To produce antisera to the structural and non-structural proteins of GVA and GVB and to use these antibodies to establish an effective detection method. 2- Develop full length infectious cDNA clones of GVA and GVB. 3- Study the roll of GVA and GVB in the etiology of the grapevine rugose wood disease. 4- Determine the function of Trichovirus (now called Vitivirus) encoded genes in the virus life cycle. Each of the ORFs 2, 3, 4 and 5 genes of GVA were cloned and expressed in E. coli and used to produce antisera. Both the CP (ORF 4) and the putative MP (ORF 3) were detected with their corresponding antisera in-GVA infected N. benthamiana and grapevine. The MP was first detected at an early stage of the infection, 6-12 h after inoculation, and the CP 2-3 days after inoculation. The MP could be detected in GVA-infected grapevines that tested negative for CP, both with CP antiserum and with a commercially available ELISA kit. Antisera to ORF 2 and 5 encoded proteins could react with the recombinant proteins but failed to detect both proteins in GVA infected plants. A full-length cDNA clone of grapevine virus A (GVA) was constructed downstream from the bacteriophage T7 RNA polymerase promoter. Capped in vitro transcribed RNA was infectious in N. benthamiana and N. clevelandii plants. Symptoms induced by the RNA transcripts or by the parental virus were indistinguishable. The infectivity of the in vitro-transcribed RNA was confirmed by serological detection of the virus coat and movement proteins and by observation of virions by electron microscopy. The full-length clone was modified to include a gus reporter gene and gus activity was detected in inoculated and systemic leaves of infected plants. Studies of GVA mutants suggests that the coat protein (ORF 4) is essential for cell to cell movement, the putative movement protein (ORF 3) indeed functions as a movement protein and that ORF 2 is not required for virus replication, cell to cell or systemic movement. Attempts to infect grapevines by in-vitro transcripts, by inoculation of cDNA construct in which the virus is derived by the CaMV 35S promoter or by approach grafting with infected N. benthamiana, have so far failed. Studies of the subcellular distribution of GFP fusion with each of ORF 2, 3 and 4 encoded protein showed that the CP fusion protein accumulated as a soluble cytoplasmatic protein. The ORF 2 fusion protein accumulated in cytoplasmatic aggregates. The MP-GFP fusion protein accumulated in a large number of small aggregates in the cytoplasm and could not move from cell to cell. However, in conditions that allowed movement of the fusion protein from cell to cell (expression by a PVX vector or in young immature leaves) the protein did not form cytoplasmatic aggregates but accumulated in the plasmodesmata.
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Manulis-Sasson, Shulamit, Christine D. Smart, Isaac Barash, Laura Chalupowicz, Guido Sessa, and Thomas J. Burr. Clavibacter michiganensis subsp. michiganensis-tomato interactions: expression and function of virulence factors, plant defense responses and pathogen movement. United States Department of Agriculture, 2015. http://dx.doi.org/10.32747/2015.7594405.bard.
Full textAbstract:
Clavibactermichiganensissubsp. michiganensis(Cmm), the causal agent of bacterial wilt and canker of tomato, is the most destructive bacterial disease of tomato causing substantial economic losses in Israel, the U.S.A. and worldwide. The goal of the project was to unravel the molecular strategies that allow Cmm, a Gram-positive bacterium, to develop a successful infection in tomato. The genome of Cmm contains numerous genes encoding for extracellular serine proteases and cell wall degrading enzymes. The first objective was to elucidate the role of secreted serine proteases in Cmm virulence. Mutants of nine genes encoding serine proteases of 3 different families were tested for their ability to induce wilting, when tomato stems were puncture-inoculated, as compared to blisters formation on leaves, when plants were spray-inoculated. All the mutants showed reduction in wilting and blister formation as compared to the wild type. The chpCmutant displayed the highest reduction, implicating its major role in symptom development. Five mutants of cell wall degrading enzymes and additional genes (i.e. perforin and sortase) caused wilting but were impaired in their ability to form blisters on leaves. These results suggest that Cmm differentially expressed virulence genes according to the site of penetration. Furthermore, we isolated and characterized two Cmmtranscriptional activators, Vatr1 and Vatr2 that regulate the expression of virulence factors, membrane and secreted proteins. The second objective was to determine the effect of bacterial virulence genes on movement of Cmm in tomato plants and identify the routes by which the pathogen contaminates seeds. Using a GFP-labeledCmm we could demonstrate that Cmm extensively colonizes the lumen of xylem vessels and preferentially attaches to spiral secondary wall thickening of the protoxylem and formed biofilm-like structures composed of large bacterial aggregates. Our findings suggest that virulence factors located on the chp/tomAPAI or the plasmids are required for effective movement of the pathogen in tomato and for the formation of cellular aggregates. We constructed a transposon plasmid that can be stably integrated into Cmm chromosome and express GFP, in order to follow movement to the seeds. Field strains from New York that were stably transformed with this construct, could not only access seeds systemically through the xylem, but also externally through tomato fruit lesions, which harbored high intra-and intercellular populations. Active movement and expansion of bacteria into the fruit mesocarp and nearby xylem vessels followed, once the fruit began to ripen. These results highlight the ability of Cmm to invade tomato fruit and seed through multiple entry routes. The third objective was to assess correlation between disease severity and expression levels of Cmm virulence genes and tomato defense genes. The effect of plant age on expression of tomato defense related proteins during Cmm infection was analyzed by qRT-PCR. Five genes out of eleven showed high induction at early stages of infection of plants with 19/20 leaves compared to young plants bearing 7/8 leaves. Previous results showed that Cmm virulence genes were expressed at early stages of infection in young plants compared to older plants. Results of this study suggest that Cmm virulence genes may suppress expression of tomato defense-related genes in young plants allowing effective disease development. The possibility that chpCis involved in suppression of tomato defense genes is currently under investigation by measuring the transcript level of several PR proteins, detected previously in our proteomics study. The fourth objective was to define genome location and stability of virulence genes in Cmm strains. New York isolates were compared to Israeli, Serbian, and NCPPB382 strains. The plasmid profiles of New York isolates were diverse and differed from both Israeli and Serbian strains. PCR analysis indicated that the presence of putative pathogenicity genes varied between isolates and highlighted the ephemeral nature of pathogenicity genes in field populations of Cmm. Results of this project significantly contributed to the understanding of Cmm virulence, its movement within tomato xylem or externally into the seeds, the role of serine proteases in disease development and initiated research on global regulation of Cmm virulence. These results form a basis for developing new strategies to combat wilt and canker disease of tomato.