Diverse hematopoietic potentials of five human embryonic stem cell lines

Despite a growing body of literature concerning the hematopoietic differentiation of human embryonic stem cells (hESCs), the full hematopoietic potential of the majority of existing hESC lines remains unknown. In this study, the hematopoietic response of five NIH-approved hESC lines (H1, hSF6, BG01, BG02, and BG03) was compared. Our data show that despite expressing similar hESC markers under self-renewing conditions and initiating mesodermal differentiation under spontaneous differentiation conditions, marked differences in subsequent hematopoietic differentiation potential among these lines existed. A high degree of hematopoietic differentiation was attained only by H1 and BG02, whereas this process appeared to be abortive in nature for hSF6, BG01, and BG03. This difference in hematopoietic differentiation predisposition was readily apparent during spontaneous differentiation, and further augmented under hematopoietic-inducing conditions. This predisposition appeared to be intrinsic to the specific hESC line and independent of passage number or gender karyotype. Interestingly, H1 and BG02 displayed remarkable similarities in their kinetics of hematopoietic marker expression, hematopoietic colony formation, erythroid differentiation, and globin expression, suggesting that a similar, predetermined differentiation sequence is followed. The identification of intrinsic and extrinsic factors governing the hematopoietic differentiation potential of hESCs will be of great importance for the putative clinical utility of hESC lines.     

CD marker expression profiles of human embryonic stem cells and their neural derivatives,determined using flow-cytometric analysis,reveal a novel CD marker for exclusion of pluripotent stem cells

Human embryonic stem cells (hESCs) are pluripotent cells that can differentiate into neural cell lineages. These neural populations are usually heterogeneous and can contain undifferentiated pluripotent cells that are capable of producing teratomas in cell grafts. The characterization of surface protein profiles of hESCs and their neural derivatives is important to determine the specific markers that can be used to exclude undifferentiated cells from neural populations. In this study, we analyzed the cluster of differentiation (CD) marker expression profiles of seven undifferentiated hESC lines using flow-cytometric analysis and compared their profiles to those of neural derivatives. Stem cell and progenitor marker CD133 and epithelial adhesion molecule marker CD326 were more highly expressed in undifferentiated hESCs, whereas neural marker CD56 (NCAM) and neural precursor marker (chemokine receptor) CD184 were more highly expressed in hESC-derived neural cells. CD326 expression levels were consistently higher in all nondifferentiated hESC lines than in neural cell derivatives. In addition, CD326-positive hESCs produced teratomas in SCID mouse testes, whereas CD362-negative neural populations did not. Thus, CD326 may be useful as a novel marker of undifferentiated hESCs to exclude undifferentiated hESCs from differentiated neural cell populations prior to transplantation.     

Intrinsic properties and external factors determine the differentiation bias of human embryonic stem cell lines

A major goal of human embryonic stem cell (hESC) research is to regulate differentiation through external means to generate specific cell types with high purity for regenerative medicine applications. Although all hESC lines express pluripotency‐associated genes, their differentiation ability to various lineages differs considerably. We have compared spontaneous differentiation propensity of the two hESC lines, RelicellhES1 and BG01. Spontaneous differentiation of hESC lines grown in different media conditions was followed by differentiation using two methods. Kinetic data generated by real‐time gene expression studies for differentiated cell types were analyzed, and confirmed at protein levels. Both cell lines showed upregulation of genes associated with the 3 germ layers, although stark contrast was evident in the magnitude of upregulation of lineage specific genes. A distinct difference was also found in the rate at which the pluripoteny factors, Oct‐4 and Nanog, were downregulated during differentiation. Once differentiation was initiated, both Oct‐4 and Nanog gene expression was drastically reduced in RelicellhES1, whereas a gradual decrease was observed in BG01. A clear trend is seen in RelicellhES1 to differentiate into neuroectodermal and mesenchymal lineages, whereas BG01 cells are more prone to mesoderm and endoderm development. In addition, suspension versus plated methods of cell culture significantly influenced the outcome of differentiation of certain types of cells. Results obtained by spontaneous differentiation of hESCs were also amplified by induced differentiation. Thus, differential rates of downregulation of pluripotency markers along with culture conditions seem to play an important role in determining the developmental bias of human ES cell lines.     

Progressive accumulation of epigenetic heterogeneity during human ES cell culture

Human embryonic stem cells (hESCs) can be maintained in culture over a large number of passages while maintaining apparently normal colony morphology. However, recent reports describe variability in epigenetic states in comparisons among different human ES cell lines. These epigenetic differences include changes in CpG methylation, expression of imprinted genes, and the status of X chromosome inactivation (XCI). We report here that the status of XCI in the female hESC line H9 (WA09) is hypervariable. We find that XIST expression can differ between individual culture isolates of H9. In addition, we find that XIST expression status can vary even between different colonies present within the same H9 culture, effectively rendering the culture mosaic. H9 cultures that lack XIST expression, but have cytological evidence of completed XCI, can also exhibit altered response to BMP4, a growth factor known to induce differentiation of hESCs to a trophectodermal lineage. In the same cultures we find biallelic expression of X-linked genes suggesting that these lines consist of mixtures of cells that retain inactivation of one of two X chromosomes following random choice. Prolonged culture of the XIST-negative isolates to high passage numbers did not result in changes in global epiproteomic signatures, demonstrating rather stable levels of post-translational nucleosome modifications within the culture-adapted hESC lines. The results show that epigenetic variants arise within human ES cell cultures after cell line derivation. In addition, the results indicate that apparently normal cultures of hESCs may contain mixtures of cells with differing epigenetic states. Assays of epigenetic integrity are warranted as quality control measures for the culture of hESCs.     

Similar pattern in cardiac differentiation of human embryonic stem cell lines, BG01V and ReliCellhES1, under low serum concentration supplemented with bone morphogenetic protein-2

Human embryonic stem cells (hESCs) can differentiate into cardiomyocytes, but the efficiency of this process is highly variable. So, developing generic differentiation protocols and their empirical testing on a range of independently derived hESC lines pose a daunting challenge due to considerable diversity in culture methods practiced between lines. Maintenance of BG01V and ReliCellhES1 has routinely been on mouse embryonic fibroblast (MEF) feeder layers using manual passaging. We assessed cardiac differentiation from both the cell lines via embryoid body (EB) formation. Subsequent culture in low fetal bovine serum (5%)-containing medium produced spontaneously contracting EBs, in the presence of bone morphogenetic protein-2 (BMP-2; 25 ng/ml). Derived cardiomyocytes expressed cardiac genes and proteins and responded to functional assays. Further, the activation of the Smad signaling machinery evoked by BMP-2 has been confirmed through inhibitor studies. Therefore, in our hands, the same differentiation conditions functioned in two independently derived hESC lines. Similar studies in other lines may facilitate development of universal protocols. The present data may also provide valuable insights for testing of other factors that might promote cardiomyocyte differentiation in low-serum formulations.     

Derivation and characterization of human embryonic stem cell lines from the Chinese population

Human embryonic stem cells (hESCs) can self-renew indefinitely and differentiate into all cell types in the human body. Therefore, they are valuable in regenerative medicine, human developmental biology and drug discovery. A number of hESC lines have been derived from the Chinese population,but limited of them are available for research purposes. Here we report the derivation and characterization of two hESC lines derived from human blastocysts of Chinese origin. These hESCs express alkaline phosphatase and hESC-specific markers, including Oct4, Nanog, SSEA-3, SSEA-4,TRA-1-60 and TRA-1-81. They also have high levels of telomerase activity and normal karyotypes. These cells can form embryoid body in vitro and can be differentiated into all three germ layers in vivo by teratoma formation. The newly established hESCs will be distributed for research purposes.The availability of hESC lines from the Chinese population will facilitate studies on the differences in hESCs from different ethnic groups.     

Common culture conditions for maintenance and cardiomyocyte differentiation of the human embryonic stem cell lines, BG01 and HUES-7

Development of generic differentiation protocols that function in a range of independently-derived human embryonic stem cell (hESC) lines remains challenging due to considerable diversity in culture methods practiced between lines. Maintenance of BG01 and HUES-7 has routinely been on mouse embryonic fibroblast (MEF) feeder layers using manual- and trypsin-passaging, respectively. We adapted both lines to trypsin-passaging on feeders or on Matrigel in feeder-free conditions and assessed proliferation and cardiac differentiation. On feeders, undifferentiated proliferation of BG01 and HUES-7 was supported by all three media tested (BG-SK, HUES-C and HUES-nL), although incidence of karyotypic instability increased in both lines in BG-SK. On Matrigel, KSR-containing conditioned medium (CM) promoted undifferentiated cell proliferation, while differentiation occurred in CM containing Plasmanate or ES-screened Fetal Bovine Serum (FBS) and in unconditioned medium containing 100 ng/ml bFGF. Matrigel cultures were advantageous for transfection but detrimental to embryoid body (EB) formation. However, transfer of hESCs from Matrigel back to feeders and culturing to confluence was found to rescue EB formation. EBs formed efficiently when hESCs on feeders were treated with collagenase, harvested by scraping and then cultured in suspension in CM. Subsequent culture in FBS-containing medium produced spontaneously contracting EBs, for which the mean beat rate was 37.2 +/- 2.3 and 41.1 +/- 3.1 beats/min for BG01-EBs and HUES-7-EBs, respectively. Derived cardiomyocytes expressed cardiac genes and responded to pharmacological stimulation. Therefore the same culture and differentiation conditions functioned in two independently-derived hESC lines. Similar studies in other lines may facilitate development of universal protocols.     

FGF2 secreting human fibroblast feeder cells: a novel culture system for human embryonic stem cells

Human embryonic stem cell (hESC) lines are traditionally derived and maintained on mouse embryonic fibroblasts (MEF) which are xenogeneic and enter senescence rapidly. In view of the clinical implications of hESCs, the use of human fibroblast as feeders has been suggested as a plausible alternative. However, use of fibroblast cells from varying sources leads to culture variations along with the need to add FGF2 in cultures to sustain ES cell pluripotency. In this study we report the derivation of FGF2 expressing germ layer derived fibroblast cells (GLDF) from hESC lines. These feeders could support the pluripotency, karyotypes and proliferation of hESCs with or without FGF2 in prolonged cultures as efficiently as that on MEF. GLDF cells were derived from embryoid bodies and characterized for expression of fibroblast markers by RT-PCR, Immunofluorescence and by flow cytometry for CD marker expression. The expression and secretion of FGF2 was confirmed by RT-PCR, Western blot, and ELISA. The hESC lines cultured on MEF and GLDF were analyzed for various stemness markers. These feeder cells with fibroblast cells like properties maintained the properties of hESCs in prolonged culture over 30 passages. Proliferation and pluripotency of hESCs on GLDF was comparable to that on mouse feeders. Further we discovered that these GLDF cells could secrete FGF2 and maintained pluripotency of hESC cultures even in the absence of supplemental FGF2. To our knowledge, this is the first study reporting a novel hESC culture system which does not warrant FGF2 supplementation, thereby reducing the cost of hESC cultures.     

BMP4 promotes formation of primitive vascular networks in human embryonic stem cell-derived embryoid bodies

The vasculature develops primarily through two processes, vasculogenesis and angiogenesis. Although much work has been published on angiogenesis, less is known of the mechanisms regulating the de novo formation of the vasculature commonly called vasculogenesis. Human embryonic stem cells (hESC) have the capability to produce all of the cells of the body and have been used as in vitro models to study the molecular signals controlling differentiation and vessel assembly. One such regulatory molecule is bone morphogenetic protein-4 (BMP4), which is required for mesoderm formation and vascular/hematopoietic specification in several species. However, hESC grown in feeder-free conditions and treated with BMP4 differentiate into a cellular phenotype highly expressing a trophoblast gene profile. Therefore, it is unclear what role, if any, BMP4 plays in regulating vascular development in hESC. Here we show in two National Institutes of Health-registered hESC lines (BG02 and WA09) cultured on a 3D substrate of Matrigel in endothelial cell growth medium-2 that the addition of BMP4 (100 ng/ml) for 3 days significantly increases the formation and outgrowth of a network of cells reminiscent of capillary-like structures formed by mature endothelial cells (P<0.05). Analysis of the expression of 45 genes by quantitative real time-polymerase chain reaction on a low-density array of the entire culture indicates a rapid and significant downregulation of pluripotent and most ectodermal markers with a general upregulation of endoderm, mesoderm, and endothelial markers. Of the genes assayed, BMPR2 and RUNX1 were differentially affected by exposure to BMP4 in both cell lines. Immunocytochemistry indicates the morphological structures formed were negative for the mature endothelial markers CD31 and CD146 as well as the neural marker SOX2, yet positive for the early vascular markers of endothelium (KDR, NESTIN) and smooth muscle cells (alpha-smooth muscle actin [alpha SMA]). Together, these data suggest BMP4 can enhance the formation and outgrowth of an immature vascular system.     

Multipotent mesenchymal stem cells of desquamated endometrium: Isolation,characterization, and application as a feeder layer for maintenance of human embryonic stem cells

In this study, we characterize new multipotent human mesenchymal stem cell lines (MSCs) derived from desquamated (shedding) endometrium of menstrual blood. The isolated endometrial MSC (eMSC) is an adhesive to plastic heterogeneous population composed mainly of endometrial glandular and stromal cells. The established cell lines meet the criteria of the International Society for Cellular Therapy for defining multipotent human MSCs of any origin. The eMSCs have positive expression of CD13, CD29, CD44, CD73, CD90, and CD105 markers and lack hematopoietic cell surface antigens CD19, CD34, CD45, CD117, CD130, and HLA-DR (class II). Multipotency of the established eMSCs is confirmed by their ability to differentiate into other mesodermal lineages, such as osteocytes and adipocytes. In addition, the isolated eMSCs partially (over 50%) express the pluripotency marker SSEA-4. However, they do not express Oct-4. Immunofluorescent analysis of the derived cells revealed the expression of the neural precursor markers nestin and β-III-tubulin. This suggests a neural predisposition of the established eMSCs. These cells are characterized by a high proliferation rate (doubling time 22–23 h) and a high colony-forming efficiency (about 60%). In vitro, the eMSCs undergo more than 45 population doublings without karyotypic abnormalities. We demonstrate that mitotically inactivated eMSCs are perfect feeder cells for maintenance of human embryonic stem cell lines (hESCs) C612 and C910. The eMSCs, being a feeder culture, sustain the hESC pluripotent status that verified by expression of Oct-4, alkaline phosphatase and SSEA-4 markers. The hESCs cocultured with the eMSCs retain their morphology and proliferative rate for more than 40 passages and exhibit the capability for spontaneous differentiation into embryoid bodies comprising three embryonic germ layers. Thus, an easy and noninvasive isolation of the eMSCs from menstrual blood, their multipotency and high proliferative activity in vitro without karyotypic abnormalities demonstrate the potential of use of these stem cells in regenerative medicine. Using the derived eMSCs as the feeder culture eliminates the risks associated with animal cells while transferring hESCs to clinical setting.     

Spa-1 regulates the maintenance and differentiation of human embryonic stem cells

Human embryonic stem cells (hESCs) are pluripotent, whereby they can proliferate endlessly and differentiate into many different cell types. At the molecular level, little is known of the mechanisms underlying their capability for self-renewal and differentiation. In the present study, we established two new hESC lines (AMC-hES1 and AMC-hES2) and demonstrated the existence of a regulator that may be a key molecule in hESC dynamics. Spa-1 is a principal Ras-proximate 1 (Rap1) GTPase-activating protein in hematopoietic progenitor cells that regulates Rap1-related signal transduction and is expressed restrictively in human adult tissues (bone marrow, thymus, and spleen). To investigate its functions in hESCs, we examined spa-1 expression profiles during hESC differentiation and used RNA interference (RNAi) to downregulate spa-1 in these cells. Our results show that Spa-1 is expressed in undifferentiated hESCs and is downregulated during hESC differentiation. In addition, the process of passing from the mode of self-renewal to that of differentiation in hESCs was regulated by spa-1 via Rap1/Raf/mitogen-activated protein kinase kinase/extracellular signal-related kinase signaling. An RNAi expression vector against spa-1 (pSUPER.retro.puro) was transfected into hESCs, which were seen to differentiate into three germ layers in spite of being in the undifferentiated condition. Based on our findings, therefore, it appears that spa-1 may be involved in hESC dynamics, and our results provide fundamental information regarding the self-renewal and differentiation of hESCs.     

Differing lectin binding profiles among human embryonic stem cells and derivatives aid in the isolation of neural progenitor cells

Human embryonic stem cells (hESCs) and their differentiated progeny allow for investigation of important changes/events during normal embryonic development. Currently most of the research is focused on proteinacous changes occurring as a result of differentiation of stem cells and little is known about changes in cell surface glycosylation patterns. Identification of cell lineage specific glycans can help in understanding their role in maintenance, proliferation and differentiation. Furthermore, these glycans can serve as markers for isolation of homogenous populations of cells. Using a panel of eight biotinylated lectins, the glycan expression of hESCs, hESCs-derived human neural progenitors (hNP) cells, and hESCs-derived mesenchymal progenitor (hMP) cells was investigated. Our goal was to identify glycans that are unique for hNP cells and use the corresponding lectins for cell isolation. Flow cytometry and immunocytochemistry were used to determine expression and localization of glycans, respectively, in each cell type. These results show that the glycan expression changes upon differentiation of hESCs and is different for neural and mesenchymal lineage. For example, binding of PHA-L lectin is low in hESCs (14±4.4%) but significantly higher in differentiated hNP cells (99±0.4%) and hMP cells (90±3%). Three lectins: VVA, DBA and LTL have low binding in hESCs and hMP cells, but significantly higher binding in hNP cells. Finally, VVA lectin binding was used to isolate hNP cells from a mixed population of hESCs, hNP cells and hMP cells. This is the first report that compares glycan expression across these human stem cell lineages and identifies significant differences. Also, this is the first study that uses VVA lectin for isolation for human neural progenitor cells.     

一种新的分化心肌细胞筛选策略：稳定表达αMHC-Blar-2A-EGFP-Rex-mCherry-2A-neo融合基因的人胚胎干细胞系的建立及其向心肌细胞定向分化的鉴定

心肌梗死是威胁人类健康的重要疾病之一,胚胎干细胞来源的心肌细胞移植是目前治疗心肌梗死的研究热点. 但是由于受到分化的心肌细胞纯度的影响,限制了心肌分化的机理研究以及临床应用. 本实验构建含有心肌特异性启动子α MHC启动的灭瘟素（Blasticidin,简称Blar）抗性基因及增强绿色荧光蛋白（EGFP）的慢病毒表达载体αMHC-Blar-2A-EGFP-Rex-mCherry-2A-neo. 应用慢病毒转染技术将慢病毒转染到人胚胎干细胞（hESCs）,胚胎干细胞特异性启动子Rex启动mCherry和neo抗性蛋白的表达. 经过G418药物筛选,建立G418和mCherry阳性的细胞系. 通过PCR及流式细胞术,进一步鉴定稳定转染的hESCs细胞系;核型分析表明,该细胞系在建立过程中仍保持细胞核型的稳定. 在诱导稳定转染的hESCs向心肌细胞分化的实验中,分化的心肌细胞表达心肌细胞特异的肌钙蛋白(cTnT),同时具有EGFP和Blasticidin药物筛选的双标记.本实验建立的这一细胞系可用于心肌细胞的纯化,为深入研究心肌发生发育的关键调控机制及临床应用奠定基础.     

Identification and characterisation of the early differentiating cells in neural differentiation of human embryonic stem cells

One of the challenges in studying early differentiation of human embryonic stem cells (hESCs) is being able to discriminate the initial differentiated cells from the original pluripotent stem cells and their committed progenies. It remains unclear how a pluripotent stem cell becomes a lineage-specific cell type during early development, and how, or if, pluripotent genes, such as Oct4 and Sox2, play a role in this transition. Here, by studying the dynamic changes in the expression of embryonic surface antigens, we identified the sequential loss of Tra-1-81 and SSEA4 during hESC neural differentiation and isolated a transient Tra-1-81(-)/SSEA4(+) (TR-/S4+) cell population in the early stage of neural differentiation. These cells are distinct from both undifferentiated hESCs and their committed neural progenitor cells (NPCs) in their gene expression profiles and response to extracellular signalling; they co-express both the pluripotent gene Oct4 and the neural marker Pax6. Furthermore, these TR-/S4+ cells are able to produce cells of both neural and non-neural lineages, depending on their environmental cues. Our results demonstrate that expression of the pluripotent factor Oct4 is progressively downregulated and is accompanied by the gradual upregulation of neural genes, whereas the pluripotent factor Sox2 is consistently expressed at high levels, indicating that these pluripotent factors may play different roles in the regulation of neural differentiation. The identification of TR-S4+ cells provides a cell model for further elucidation of the molecular mechanisms underlying hESC neural differentiation.     

Self-renewal and differentiation capabilities are variable between human embryonic stem cell lines I3, I6 and BG01V

Background  

A unique and essential property of embryonic stem cells is the ability to self-renew and differentiate into multiple cell lineages. However, the possible differences in proliferation and differentiation capabilities among independently-derived human embryonic stem cells (hESCs) are not well known because of insufficient characterization. To address this question, a side-by-side comparison of 1) the ability to maintain an undifferentiated state and to self-renew under standard conditions; 2) the ability to spontaneously differentiate into three primary embryonic germ lineages in differentiating embryoid bodies; and 3) the responses to directed neural differentiation was made between three NIH registered hES cell lines I3 (TE03), I6 (TE06) and BG01V. Lines I3 and I6 possess normal XX and a normal XY karyotype while BG01V is a variant cell line with an abnormal karyotype derived from the karyotypically normal cell line BG01.     

Cells derived from human umbilical cord blood support the long-term growth of undifferentiated human embryonic stem cells

Various types of human cells have been tested as feeder cells for the undifferentiated growth of human embryonic stem cells (hESCs) in vitro. We report here the successful culture of two hESC lines (H1 and H9) on human umbilical cord blood (UCB)-derived fibroblast-like cells. These cells permit the long-term continuous growth of undifferentiated and pluripotent hESCs. The cultured hESCs had normal karyotypes, expressed OCT-4, SSEA-4, TRA-1-60, and TRA-1-81, formed cystic embryonic body in vitro and teratomas in vivo after injected into immunodeficient mice. The wide availability of clinical-grade human UCB makes it a promising source of support cells for the growth of hESC for use in cell therapies.