scholarly journals Establishment of a feeder and serum-free culture system for human embryonic stem cells

Zygote ◽  
2020 ◽  
Vol 28 (3) ◽  
pp. 175-182
Author(s):  
LiYun Wang ◽  
RuiNa Zhang ◽  
RongHua Ma ◽  
GongXue Jia ◽  
ShengYan Jian ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Culture System ◽  
Embryonic Stem ◽  
Hesc Line ◽  
Self Renewal ◽  
Serum Free ◽  
Genes Encoding

SummaryStem cells are an immortal cell population capable of self-renewal; they are essential for human development and ageing and are a major focus of research in regenerative medicine. Despite considerable progress in differentiation of stem cells in vitro, culture conditions require further optimization to maximize the potential for multicellular differentiation during expansion. The aim of this study was to develop a feeder-free, serum-free culture method for human embryonic stem cells (hESCs), to establish optimal conditions for hESC proliferation, and to determine the biological characteristics of the resulting hESCs. The H9 hESC line was cultured using a homemade serum-free, feeder-free culture system, and growth was observed. The expression of pluripotency proteins (OCT4, NANOG, SOX2, LIN28, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) in hESCs was determined by immunofluorescence and western blotting. The mRNA expression levels of genes encoding nestin, brachyury and α-fetoprotein in differentiated H9 cells were determined by RT-PCR. The newly developed culture system resulted in classical hESC colonies that were round or elliptical in shape, with clear and neat boundaries. The expression of pluripotency proteins was increased, and the genes encoding nestin, brachyury, and α-fetoprotein were expressed in H9 cells, suggesting that the cells maintained in vitro differentiation capacity. Our culture system containing a unique set of components, with animal-derived substances, maintained the self-renewal potential and pluripotency of H9 cells for eight passages. Further optimization of this system may expand the clinical application of hESCs.

Generation of T Cells from Human Embryonic Stem Cells.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1527-1527
Author(s):  
Frank Timmermans ◽  
Imke Velghe ◽  
Lieve Van Walleghem ◽  
Magda De Smedt ◽  
Stefanie Van Coppernolle ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Early Stage ◽  
Embryonic Stem ◽  
Hesc Line ◽  
Double Positive

Abstract Background: Human embryonic stem cells (hESC) are derived from early stage blastocysts and are characterized by the ability to both self-renew and to generate differentiated functional cell types. One of the major challenges in the field of hESC research, is to set up a culture system that drives hESC down a particular lineage fate. To date, studies reporting hematopoietic development have not provided evidence on the differentiation capacity of hESC into T lineage cells in vitro. Material and Methods: hESC line H1 (National Institutes of Health [NIH] code: WA01), Wisconson, Madison, USA) was used (Passage 30–60) in all experiments. The hESC line was kept in an undifferentiated state on MEFs as previously described. OP9 cells and OP9 cells that express high levels of the Notch ligand Delta-like 1 (OP9-DLL1, a gift from J. C. Zuniga-Pflücker, University of Toronto, Canada) were cultured as previously described in MEM-α with 20 % FCS. Results: Our data show that T cells can be generated in vitro from hESC in a robust and highly reproducible manner using the sequential exposure of hESC to the murine OP9 cell line and OP9-DLL1. On OP9 stromal layers, a CD34highCD43dim hematopoietic precursor population is generated that is confined to vascular-like structures, reminiscent of blood islands that emerge during in vivo embryonic development. This precursor population becomes T lineage committed when exposed to OP9-DLL1 monolayers, passing sequentially through a CD34+CD7+ phenotype, a CD4+CD8+ double positive intermediate stage and eventually differentiates into a mature T cells. Polyclonal T cells are generated, cell receptor (TCR) alpha-beta and TCRgamma-delta which are functional based on proliferative capacity and production of cytokines after TCR crosslinking. Conclusion: We show that mature and functional T cells can be generated from hESC using well defined in vitro conditions. This protocol in combination with the recently described induced pluripotent cells may find clinical applicability in tumor immunology.

Generation of Megakaryocytes from Human Embryonic Stem Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1265-1265
Author(s):  
Deborah L. French ◽  
Marketa Jirouskova ◽  
Marion Kennedy ◽  
Barry S. Coller ◽  
Gordon Keller
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Embryoid Body ◽  
Embryonic Stem ◽  
Pcr Analysis ◽  
Serum Free ◽  
Colony Assays ◽  
Low Levels

Abstract The ability to generate multiple cell types from human embryonic stem cells (hESC) in culture offers an unprecedented opportunity to produce an unlimited supply of cells for research and clinical purposes. Megakaryocytes comprise only 0.02–0.05% of the nucleated cell population in the bone marrow, thus making them a difficult cell to isolate and study. The ES cell technology provides a resource for generating megakaryocyte progenitors for in vitro and in vivo analyses. For this approach to be successful, reproducible differentiation schemes that generate sufficient numbers of cells are necessary. With an embryoid body (EB)-based protocol, in serum-free media we are able to generate hematopoietic populations with megakaryocyte potential from two different hESC lines; H1 and HES2. Using CD41 as an early marker of definitive hematopoiesis, we found that 4 to 12% of the day 11–12 EB population expressed high levels of the marker (CD41Hi) whereas 10 to 20% expressed low levels of the marker (CD41Lo). Cells from the CD41Hi but not the CD41Lo population expressed the megakaryocyte marker GPIb. To further analyze these populations, both were isolated from day 12 EBs by cell sorting and cultured in media containing TPO and SCF. Following several days in culture, the CD41Hi population expressed increasing levels of GPIb, as determined by flow cytometry and real time PCR. PF4 was detected in the sorted CD41Hi population and levels increased 10-fold following culture. These cells also adhered to fibrinogen and collagen and stained positive for von Willebrand factor (vWF) and P-selectin. Following activation by PAR1, the cells bound soluble fibrinogen and expressed P-selectin on their surface. Few to no progenitors were detected in the CD41Hi fraction by colony assays suggesting that this population contained cells that were already committed to the megakaryocyte lineage. Incubation of the CD41Lo population of cells in the presence of TPO resulted in 70–80% of the cells expressing increased levels of CD41 and GPIb. Immunostaining revealed vWF and P-selectin expression following adhesion of the cells to fibrinogen and collagen. Both fibrinogen binding and P-selectin surface expression were detected following activation with PAR1. Quantitative PCR analysis demonstrated low levels of PF4 message in the sorted population with an increase of 3-fold following incubation in the presence of TPO. Progenitor cells were detected in colony assays suggesting that this population of cells contained megakaryocyte-like progenitors. Taken together, these finding demonstrate the efficient and reproducible generation of megakaryocytes from hESC using a serum-free EB protocol.

Generation of Red Blood Cells From Human Embryonic Stem Cells (ES) with Increased Efficacy

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4334-4334
Author(s):  
Anand S Srivastava ◽  
Babak Esmaeli Azad ◽  
Rosalia De Necochea Campion ◽  
Ewa Carrier
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Red Blood Cells ◽  
Human Embryonic Stem Cells ◽  
Blood Cells ◽  
Culture System ◽  
Embryonic Stem ◽  
Employment Equity ◽  
Equity Ownership

Abstract Abstract 4334 It is estimated that for every unit of donated blood, two units are required in North America. The current rate of blood donation is stagnant while the need increases by 6–8% annually. In order to overcome this difficulty, we have developed an improved method to generate red blood cells from human embryonic stem cells (H9) with increased efficacy. In addition to xeno-free conditions and standard cytokine cocktail used for hematopoietic differentiation of human embryonic stem cells (Carrier et al. J Transl Med. 2009; Vol 7: 27), we have introduced a new method of improved growth and differentiation of human ES cells with hypoxia-induced mesenchymal stem cells, obtained from allogeneic adult bone marrow donors. This technique increased efficacy of red blood cell production by 5–25 fold. We have developed a bioscaffold–> microsphere-based culture system with highly porous surface allowing culturing of a very large number of embryonic stem cells per one culture condition. This culture system avoids shear forces and damage to the cells, and facilitates removal and recycling of the microspheres. The in vitro obtained human ES-derived red blood cells are enucleated and do not produce tumors (efficacy of enucleation is 65–95%). The laser-based system is utilized to eliminate nucleated cells from the culture. The problem with hES-derived red blood cells is that they are produced in small numbers and process is very costly. We are developing a 3-phase bioreactor with computerized programming, which will increase every step of the differentiation process and allow recycling of feeder cells and cytokines. In this system we will utilize iron-loaded microspheres coated with hypoxia-processed mesenchymal stem cells as a main culture unit. The in vitro generated human ES-derived red blood cells upscaled in a bioreactor will be used for the off-shelf production of red blood cells for clinical use. Disclosures: Srivastava: Giostar: Employment, Equity Ownership. Azad:Dnamicroarray, Inc.: Employment, Equity Ownership. Carrier:Giostar: Consultancy; Samaritan Pharmaceuticals: Consultancy; Entest Biomedical: Consultancy, Equity Ownership; America Stem Cells: Consultancy, Equity Ownership; Millenium: Speakers Bureau; NovaRx: Employment, Equity Ownership.

scholarly journals Effects of Ionizing Radiation on Self-Renewal and Pluripotency of Human Embryonic Stem Cells

2010 ◽  
Vol 70 (13) ◽  
pp. 5539-5548 ◽  
Author(s):  
Kitchener D. Wilson ◽  
Ning Sun ◽  
Mei Huang ◽  
Wendy Y. Zhang ◽  
Andrew S. Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Ionizing Radiation ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Self Renewal

scholarly journals Rapid Differentiation of Human Embryonic Stem Cells into Testosterone-Producing Leydig Cell-Like Cells In vitro

2021 ◽  
Author(s):  
Eun-Young Shin ◽  
Seah Park ◽  
Won Yun Choi ◽  
Dong Ryul Lee
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Male Hypogonadism ◽  
Testosterone Levels ◽  
Sequence Of Events ◽  
Short Period ◽  
Molecular Compounds

Abstract Background: Leydig cells (LCs) are testicular somatic cells that are the major producers of testosterone in males. Testosterone is essential for male physiology and reproduction. Reduced testosterone levels lead to hypogonadism and are associated with diverse pathologies, such as neuronal dysfunction, cardiovascular disease, and metabolic syndrome. LC transplantation is a promising therapy for hypogonadism; however, the number of LCs in the testis is very rare and they do not proliferate in vitro. Therefore, there is a need for an alternative source of LCs. Methods: To develop a safer, simple, and rapid strategy to generate human LC-like cells (LLCs) from stem cells, we first performed preliminary tests under different conditions for the induction of LLCs from human CD34/CD73 double positive-testis-derived stem cells (HTSCs). Based on the embryological sequence of events, we suggested a 3-step strategy for the differentiation of human ESCs into LLCs. We generated the mesendoderm in the first stage and intermediate mesoderm (IM) in the second stage and optimized the conditions for differentiation of IM into LLCs by comparing the secreted testosterone levels of each group. Results: HTSCs and human embryonic stem cells can be directly differentiated into LLCs by defined molecular compounds within a short period. Human ESC-derived LLCs can secrete testosterone and express steroidogenic markers. Conclusion: We developed a rapid and efficient protocol for the production of LLCs from stem cells using defined molecular compounds. These findings provide a new therapeutic cell source for male hypogonadism.

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