Kidney-Specific Microscaffolds and Kidney-Derived Serum-Free Conditioned Media Support In Vitro Expansion, Differentiation, and Organization of Human Embryonic Stem Cells

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.

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

Zygote ◽

10.1017/s0967199419000625 ◽

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.

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Faculty Opinions recommendation of INS(GFP/w) human embryonic stem cells facilitate isolation of in vitro derived insulin-producing cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.13984975.15442093 ◽

2012 ◽

Author(s):

Gordon Weir

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Embryonic Stem ◽

Insulin Producing Cells

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Faculty Opinions recommendation of CORTECON: a temporal transcriptome analysis of in vitro human cerebral cortex development from human embryonic stem cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718478232.793498745 ◽

2014 ◽

Author(s):

Vijay Tiwari ◽

Johannes Jung

Keyword(s):

Stem Cells ◽

Cerebral Cortex ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Transcriptome Analysis ◽

Embryonic Stem ◽

Human Cerebral Cortex ◽

Cerebral Cortex Development

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

Tissue Engineering and Regenerative Medicine ◽

10.1007/s13770-021-00359-8 ◽

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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Nanofiber-Based in Vitro System for High Myogenic Differentiation of Human Embryonic Stem Cells

Biomacromolecules ◽

10.1021/bm4009843 ◽

2013 ◽

Vol 14 (12) ◽

pp. 4207-4216 ◽

Cited By ~ 29

Author(s):

Matthew Leung ◽

Ashleigh Cooper ◽

Soumen Jana ◽

Ching-Ting Tsao ◽

Timothy A. Petrie ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Myogenic Differentiation ◽

Embryonic Stem ◽

In Vitro System

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Smooth-Muscle-Like Cells Derived from Human Embryonic Stem Cells Support and Augment Cord-Like Structures In Vitro

Stem Cell Reviews and Reports ◽

10.1007/s12015-010-9144-3 ◽

2010 ◽

Vol 6 (2) ◽

pp. 237-247 ◽

Cited By ~ 49

Author(s):

Elaine Vo ◽

Donny Hanjaya-Putra ◽

Yuanting Zha ◽

Sravanti Kusuma ◽

Sharon Gerecht

Keyword(s):

Stem Cells ◽

Smooth Muscle ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Embryonic Stem

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Correlation of mesodermal and pluripotency gene expression during in vitro differentiation of human embryonic stem cells

Fertility and Sterility ◽

10.1016/j.fertnstert.2013.07.487 ◽

2013 ◽

Vol 100 (3) ◽

pp. S457

Author(s):

S.-Y. Ku ◽

Y.Y. Kim ◽

H. Kim ◽

S.H. Kim ◽

Y.M. Choi ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Embryonic Stem ◽

In Vitro Differentiation ◽

Pluripotency Gene

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Monocytic cells derived from human embryonic stem cells and fetal liver share common differentiation pathways and homeostatic functions

Blood ◽

10.1182/blood-2010-07-295246 ◽

2011 ◽

Vol 117 (11) ◽

pp. 3065-3075 ◽

Cited By ~ 36

Author(s):

Olena Klimchenko ◽

Antonio Di Stefano ◽

Birgit Geoerger ◽

Sofiane Hamidi ◽

Paule Opolon ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Fetal Liver ◽

Critical Role ◽

Embryonic Stem ◽

Blood Monocytes ◽

Monocytic Cell ◽

Monocytic Cells

Abstract The early emergence of macrophages and their large pattern of tissue distribution during development suggest that they may play a critical role in the initial steps of embryogenesis. In the present study, we show that monocytic cells derived from human embryonic stem cells (hESCs) and from fetal liver follow a differentiation pathway different to that of adult cells, leading to specific functions. Embryonic and fetal monocytic cells differentiated from a CD14lowCD16− precursor to form CD14highCD16+ cells without producing the CD14highCD16− cell population that predominates in adult peripheral blood. Both demonstrated an enhanced expression of genes encoding tissue-degrading enzymes, chemokines, and scavenger receptors, as was previously reported for M2 macrophages. Compared with adult blood monocytes, embryonic and fetal monocytic cells secreted high amounts of proteins acting on tissue remodeling and angiogenesis, and most of them expressed the Tie2 receptor. Furthermore, they promoted vascular remodeling in xenotransplanted human tumors. These findings suggest that the regulation of human fetal and embryonic monocytic cell differentiation leads to the generation of cells endowed mainly with anti-inflammatory and remodeling functions. Trophic and immunosuppressive functions of M2-polarized macrophages link fetus and tumor development, and hESCs offer a valuable experimental model for in vitro studies of mechanisms sustaining these processes.

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