Hemangioblasts or blood islands only arise in early development thereby the sources to obtain these bi-potential cells are limited. While previous studies have isolated both lineages in vitro through the hemangioblast, derivation efficiency was rather low due to cellular damage attributed by enzyme usage and fluorescent activated cell sorting (FACS). This study focused on avoiding the use of damaging factors in the derivation of endothelial cells (ECs). Single cell H9-human embryonic stem cells (hESCs) were obtained by using a mild dissociation protocol then human embryoid body (hEB) formation was performed under hemangioblast differentiation conditions. The hEBs were subjected to a two-stage cytokine treatment procedure. Subsequent culture of the adhesive cells in day 4 hEBs gave arise to a seemingly pure population of ECs. The hESC-derived ECs were characterized by identifying signature endothelial gene and protein markers as well as testing for in vitro functionality. Furthermore, in vivo functionality was also confirmed by transplanting the cells in hindlimb ischemic murine models. We demonstrate that the genetic change required for EC derivation precedes blast colony formation. Furthermore, cell damage was prevented by abating enzyme usage and FACS, resulting in a high yield of ECs upon adhesion. Under this method, confluent cultures of ECs were obtainable 4 days after hEB formation which is significantly faster than previous protocols.
Adhesives ; Animals ; Embryoid Bodies ; Embryonic Stem Cells ; Endothelial Cells* ; Hemangioblasts ; Hindlimb ; Human Embryonic Stem Cells* ; Humans* ; In Vitro Techniques ; Islands ; Methods

Embryonic stem (ES) cells have potential for use in evaluation of developmental toxicity because they are generated in large numbers and differentiate into three germ layers following formation of embryoid bodies (EBs). In earlier study, embryonic stem cell test (EST) was established for assessment of the embryotoxic potential of compounds. Using EBs indicating the onset of differentiation of mouse ES cells, many toxicologists have refined the developmental toxicity of a variety of compounds. However, due to some limitation of the EST method resulting from species-specific differences between humans and mouse, it is an incomplete approach. In this regard, we examined the effects of several developmental toxic chemicals on formation of EBs using human ES cells. Although human ES cells are fastidious in culture and differentiation, we concluded that the relevancy of our experimental method is more accurate than that of EST using mouse ES cells. These types of studies could extend our understanding of how human ES cells could be used for monitoring developmental toxicity and its relevance in relation to its differentiation progress. In addition, this concept will be used as a model system for screening for developmental toxicity of various chemicals. This article might update new information about the usage of embryonic stem cells in the context of their possible ability in the toxicological fields.
Animals ; Embryoid Bodies ; Embryonic Stem Cells* ; Germ Layers ; Humans* ; Mass Screening ; Mice

OBJECTIVE: The aim of this study was to investigate whether embryonic stem (ES) cells can be established from isolated blastomeres of mouse embryos. METHODS: Blastomeres were separated from mouse (C57Bl/6J) 2- or 4-cell embryos. Isolated blastomeres or whole 4-cell embryos were co-cultured with mitosis-arrested STO feeder cells in DMEM supplemented with recombinant murine leukemia inhibitory factor and ES-qualified fetal bovine serum. After the tentative ES cell lines were maintained from isolated blastomeres or whole embryos, some of them were frozen and the others were sub-cultured continually. Characteristics of tentative ES cell lines as were evaluated for specific gene expressions with immunocytochemistry and RT-PCR. RESULTS: One ES cell line (3.0%) was established from isolated blastomere of 2-cell embryo and one cell line (4.0%) from isolated two blastomeres of 4-cell embryo. And five cell lines (16.7%) were established from whole 4-cell embryos. Both cell lines from isolated blastomere and whole embryo expressed mouse ES cells specific markers such as SSEA-1, Oct-4 and alkaline phosphatase. Marker genes of three germ layers were expressed from embryoid bodies of both cell lines. CONCLUSION: This study suggests that mouse ES cells could be established from isolated blastomeres, although the efficiency is lower than whole embryos. This animal model could be applied to establishment of autologous human ES cells from biopsied blastomeres of preimplantation embryos in human IVF-ET program.
Alkaline Phosphatase ; Animals ; Antigens, CD15 ; Blastocyst* ; Blastomeres* ; Cell Line ; Embryoid Bodies ; Embryonic Stem Cells* ; Embryonic Structures ; Feeder Cells ; Gene Expression ; Germ Layers ; Humans ; Immunohistochemistry ; Leukemia Inhibitory Factor ; Mice* ; Models, Animal

PURPOSE: Human embryonic stem (ES) cell is pluripotent cell derived from a group of cells called the inner cell mass and has the ability to reproduce itself for long periods and give rise to types of cells that develop from the three germ layers. Due to its pluripotency, ES cell holds the promise of being able to replace cells that are damaged or destroyed by many devastating diseases. However, the potential for the recipient of an ES cell transplant to reject this cell as foreign is very high. Thus, it is essential to determine whether human ES cells express MHC antigens. The purpose of this study is to characterize the stem cell properties of our cell line (SNUhES1) and the expression profile of MHC antigens on the surface of these cells and their differentiated derivatives, embryoid bodies (EBs). METHODS: The ES cells were grown on STO fibroblast in DMEM-F12. The EBs were grown in the same medium with exception that it lacked LIF and bFGF. The expression of self-renewal-associated genes and three germ layer cell-specific genes in ES cells and EBs were measured by RT-PCR at varying time point of incubation (1, 7, 14 and 28 day). The expression of MHC molecules were measured by RT-PCR and FACS analysis. RESULTS: The SNUhES1 cells expressed all self-renewal- associated genes (Fgf4, FoxD3, Oct4, Sox2 and TERT) we tested. During the differentiation three germ layer cell-specific genes in EBs were expressed as following order: ecto-, meso- and endodermal cell-specific genes. MHC class I proteins (HLA-ABC and beta2m) on the surfaces of ES cells and EBs were expressed in very low levels. MHC class II proteins (HLA-DP, -DQ and -DR) and HLA-G were not expressed on the surface of these cells. However, the expression of MHC class II proteins were detected in 1% more or less cells of 28-day-old EBs which were hardly detected in the population of 1-day-old EBs. CONCLUSION: These data imply that SNUhES1 cells and EBs have stem cell properties. Although they express very low MHC antigens, further investigation determining whether the MHC expression in the ES cells and EBs may alter under inflammatory condition which can be occurred in damaged tissue or through surgical process.
Cell Line ; Embryoid Bodies* ; Embryonic Stem Cells* ; Endoderm ; Fibroblasts ; Germ Layers ; HLA-G Antigens ; Humans* ; Stem Cells ; Transplants

Human Embryonic Stem Cells ; Humans ; Safety

Human embryonic stem cell (hESC) culture system has been changing culture conditions from conventional to xeno-free for therapeutic cell applications, and N-glycolylneuraminic acid (Neu5Gc) could be a useful indicator of xenogeneic contaminations in hESCs because human cells can no longer produce it genetically. We set up the humanized culture condition using commercially available humanized materials and two different adaptation methods: sequential or direct. SNUhES4 and H1 hESC lines, previously established in conventional culture conditions, were maintained using the humanized culture condition and were examined for the presence of Neu5Gc. The hESCs showed the same morphology and character as those of the conventional culture condition. Moreover, they were negative for Neu5Gc within two passages without loss of pluripotency. This study suggested that this method can effectively cleanse previously established hESC lines, bringing them one step closer to being clinical-grade hESCs.
Human Embryonic Stem Cells* ; Humans* ; Methods

Previously, the majority of human embryonic stem cells and human induced pluripotent stem cells have been derived on feeder layers and chemically undefined medium. Those media components related to feeder cells, or animal products, often greatly affect the consistency of the cell culture. There are clear advantages of a defined, xeno-free, and feeder-free culture system for human pluripotent stem cells (hPSCs) cultures, since consistency in the formulations prevents lot-to-lot variability. Eliminating all non-human components reduces health risks for downstream applications, and those environments reduce potential immunological reactions from stem cells. Therefore, development of feeder-free hPSCs culture systems has been an important focus of hPSCs research. Recently, researchers have established a variety of culture systems in a defined combination, xeno-free matrix and medium that supports the growth and differentiation of hPSCs. Here we described detailed hPSCs culture methods under feeder-free and chemically defined conditions using vitronetin and TeSR-E8 medium including supplement bioactive lysophospholipid for promoting hPSCs proliferation and maintaining stemness.
Animals ; Cell Culture Techniques ; Embryonic Stem Cells ; Extracellular Matrix ; Feeder Cells ; Human Embryonic Stem Cells ; Humans ; Induced Pluripotent Stem Cells ; Pluripotent Stem Cells ; Stem Cells

BACKGROUND: Embryonic stem cells (ESCs) can be expanded infinitely in vitro and have the potential to differentiate into hematopoietic stem cells (HSCs); thus, they are considered a useful source of cells for HSC production. Although several technical in vitro methods for engineering HSCs from pluripotent stem cells have been developed, clinical application of HSCs engineered from pluripotent stem cells is restricted because of the possibility of xenogeneic contamination resulting from the use of murine materials. METHODS: Human ESCs (CHA-hES15) were cultured on growth factor-reduced Matrigel-coated dishes in the mTeSR1 serum-free medium. When the cells were 70% confluent, we initiated HSC differentiation by three methods involving (1) knockout serum replacement (KSR), cytokines, TGFb1, EPO, and FLT3L; (2) KSR, cytokines, and bFGF; or (3) cytokines and bFGF. RESULTS: Among the three differentiation methods, the minimal number of cytokines without KSR resulted in the greatest production of HSCs. The optimized method resulted in a higher proportion of CD34⁺CD43⁺ hematopoietic progenitor cells (HPCs) and CD34⁺CD45⁺ HPCs compared to the other methods. In addition, the HSCs showed the potential to differentiate into multiple lineages of hematopoietic cells in vitro. CONCLUSION: In this study, we optimized a two-step, serum-free, animal protein-free, KSR-free, feeder-free, chemically defined monolayer culture method for generation of HSCs and hematopoietic stem and progenitor cells (HSPCs) from human ESCs.
Animals ; Cytokines ; Embryonic Stem Cells ; Hematopoietic Stem Cells* ; Human Embryonic Stem Cells* ; Humans* ; In Vitro Techniques ; Methods* ; Pluripotent Stem Cells ; Stem Cells

The study aims to investigate the effects of cardiac fibroblast (CF) paracrine factors on murine embryonic stem cells (ESCs). Conditioned mediums from either neonatal cardiac fibroblasts (ConM-NCF) or adult cardiac fibroblasts (ConM-ACF) were diluted by 1:50 and 1:5, respectively, to investigate whether these conditioned mediums impact murine ESCs distinctly with RT-real time PCR techniques, cell proliferation essay, ELISA and by counting percentage of beating embryoid bodies (EBs) during ESCs differentiation. The data showed that the paracrine ability of CFs changed dramatically during development, in which interleukin 6 (IL6) increased with maturation. ConM-NCF 1:50 and ConM-NCF 1:5 had opposite effects on the pluripotent markers, although they both reduced mouse ESC proliferation. ConM-ACF 1:50 promoted ESCs pluripotent markers and proliferation, while ConM-ACF 1:5 exerted negative effects. All CF-derived conditioned mediums inhibited cardiac differentiation, but with distinguishable features: ConM-NCF 1:50 slightly decreased the early cardiac differentiation without altering the maturation tendency or cardiac specific markers in EBs at differentiation of day 17; ConM-ACF 1:50 had more significant inhibitory effects on early cardiac differentiation than ConM-NCF 1:50 and impeded cardiac maturation with upregulation of cardiac specific markers. In addition, IL6 neutralization antibody attenuated positive effect of ConM-ACF 1:50 on ESCs proliferation, but had no effects on ConM-NCF 1:50. Long-term IL6 neutralization reduced the percentage of beating EBs at early developmental stage, but did not alter the late cardiac differentiation. Taken together, both the quality and quantity of factors and cytokines secreted by CFs are critical for the ESC fate. IL6 could be a favorable cytokine for ESC pluripotency and the early cardiac differentiation.
Animals ; Embryonic Stem Cells ; Fibroblasts ; Heart ; Mice ; Mouse Embryonic Stem Cells ; Paracrine Communication

Researches on manipulating pluripotent stem cells derived from blastocysts or primordial germ cells (PGCs) have a great advantage for developing innovative technologies in various fields of life science including medicine, pharmaceutics, and biotechnology. Since their first isolation in the mouse embryos(1), stem cells or stem cell-like colonies have been continuously established in the mouse of different strains(21), cattle(2, 3), pig(4, 5), rabbit(6, 7), and human(9). However, full-term development originated from established pluripotent cells, which is an absolute criterion for proving cell pluripotency and differentiation, has only been reported in the mouse(22). Due to technical difficulties, no further progress has been made in the establishment of animal embryonic stem (ES) cell line. Alternatively, the use of embryonic germ (EG) cells was selected to establish an animal stem cell line. EG cells also have pluripotent characteristics, which were proven by morphological assay, intracellular alkaline phosphatase activity, and reactions with cell surface-specific markers. The finding of Labosky et al.(23) on germline chimera development after transfer to embryos clearly proved the pluripotency of EG cells and their similar characteristics with ES cells. Avian transgenesis has an unlimited value in biotechnology industry, since its applicability as a bioreactor has proven to be greater than that of mammalian species(24). In the chicken, EG cells can be extensively utilized instead of ES cells for efficiently inducing transgenesis mediated by germline transmission. Recently, PGCs collected from the embryonic gonad were suggested to be useful in establishing avian stem cells. Technical feasibility and applicability of gonadal PGCs (gPGCs) to germline chimera production were also confirmed(25) and a gPGC culture system to establish EG cells was subsequently developed(15).
Alkaline Phosphatase ; Animals* ; Biological Science Disciplines ; Bioreactors ; Biotechnology ; Blastocyst ; Cell Line ; Chickens ; Chimera ; Embryonic Germ Cells ; Embryonic Stem Cells ; Embryonic Structures ; Gene Transfer Techniques ; Germ Cells ; Gonads ; Mice ; Pluripotent Stem Cells ; Stem Cell Research* ; Stem Cells*