Growth Of hESCs Research Articles

Investigations into the Metabolism of Two-Dimensional Colony and Suspended Microcarrier Cultures of Human Embryonic Stem Cells in Serum-Free Media

Metabolic studies of human embryonic stem cells (hESCs) can provide important information for stem cell bioprocessing. To this end, we have examined growth and metabolism of hESCs in both traditional 2-dimensional (2D) colony cultures and 3-dimensional microcarrier cultures using a conditioned medium and 3 serum-free media. The 2D colony cultures plateaued at cell densities of 1.1-1.5 × 10⁶ cells/mL at day 6 due to surface limitation. Microcarrier cultures achieved 1.5-2 × 10⁶ cells/mL on days 8-10 before reaching a plateau; this growth arrest was not due to surface limitation, but probably due to metabolic limitations. Metabolic analysis of the cultures showed that amino acids (including glutamine) and glucose are in excess and are not limiting cell growth; on the other hand, the high levels of waste products (25 mM lactate and 0.8 mM ammonium) and low pH (6.6) obtained at the last stages of cell propagation could be the causes for growth arrest. hESCs cultured in media supplemented with lactate (up to 28 mM) showed reduced cell growth, whereas ammonium (up to 5 mM) had no effect. Lactate and, to a lesser extent, ammonia affected pluripotency as reflected by the decreasing population of cells expressing pluripotent marker TRA-1-60. Feeding hESC cultures with low concentrations of glucose resulted in lower lactate levels (∼10%) and a higher pH level of 6.7, which leads to a 40% increase in cell density. We conclude that the high lactate levels and the low pH during the last stages of high-density hESC culture may limit cell growth and affect pluripotency. To overcome this limitation, a controlled feed of low levels of glucose and online control of pH can be used.

Comparison of the Toxicity of Smoke from Conventional and Harm Reduction Cigarettes Using Human Embryonic Stem Cells

This study evaluated the hypothesis that smoke from harm reduction cigarettes impedes attachment and proliferation of H9 human embryonic stem cells (hESCs). Smoke from three harm reduction brands was compared with smoke from a conventional brand. Doses of smoke were measured in puff equivalents (PE) (1 PE = the amount of smoke in one puff that dissolves in 1 ml of medium). Cytotoxic doses were determined using morphological criteria and trypan blue staining, and apoptosis was confirmed using Magic Red staining. Attachment and proliferation of hESC were followed at a noncytotoxic dose in time-lapse videos collected using BioStation technology. Data were mined from videos either manually or using video bioinformatics subroutines developed with CL-Quant software. Mainstream (MS) and sidestream (SS) smoke from conventional and harm reduction cigarettes induced apoptosis in hESC colonies at 1 PE. At 0.1 PE (noncytotoxic), SS smoke from all brands inhibited attachment of hESC colonies to Matrigel with the strongest inhibition occurring in harm reduction brands. At 0.1 PE, SS smoke, but not MS smoke, from all brands inhibited hESC growth, and two harm reduction brands were more potent than the conventional brand. In general, hESC appeared more sensitive to smoke than their mouse ESC counterparts. Although harm reduction cigarettes are often marketed as safer than conventional brands, our assays show that SS smoke from harm reduction cigarettes was at least as potent or in some cases more potent than smoke from a conventional brand and that SS smoke was more inhibitory than MS smoke in all assays.

Autologous Feeder Cells from Embryoid Body Outgrowth Support the Long-Term Growth of Human Embryonic Stem Cells More Effectively than Those from Direct Differentiation

Autologous feeder cells have been developed by various methods to minimize the presence of xenogenic entities in human embryonic stem cell (hESC) cultures. However, there was no systematic comparison of supportive effects of the feeder cells on hESC growth, nor comparison to the supportive effects of various feeder-free culture systems and standard mouse feeder cells. In this study, we aimed to compare the supportive abilities of autologous feeders derived either directly from H9 hESCs (H9 dF) or from outgrowth of embryoid body predifferentiated in suspension from H9 hESCs (H9 ebF). Mouse feeder system and matrigel-mTeSR1 feeder-free system were used as controls. H9 ebF was found to secrete more basic fibroblast growth factor in the conditioned medium than H9 dF did. The undifferentiated state of H9 hESCs was sustained more stably on H9 ebF than on H9 dF, and the differentiation potential of H9 hESCs on H9 ebF was higher than on H9 dF. We concluded that H9 ebF was an optimal autologous feeder to maintain the long-term undifferentiated state of hESCs in our current culture system. This study helps to standardize the autologous culture of hESCs. It also suggests a more definite direction for future development of xeno-free culture system for hESCs.

Human Embryonic Stem Cells (hESCs) Cultured Under Distinctive Feeder-Free Culture Conditions Display Global Gene Expression Patterns Similar to hESCs from Feeder-Dependent Culture Conditions

Human embryonic stem cell (hESC)-based assay systems and genetically modified hESCs are very useful tools for screening drugs that regulate stemness and differentiation and for studying the molecular mechanisms involved in hESC fate determination. For these types of studies, feeder cell-dependent cultures of hESCs are often problematic because the physiology of the feeder cells is perturbed by the drug treatments or genetic modifications, which potentially obscures research outcomes. In this study, we evaluated three commonly used feeder-free culture conditions to determine whether they supported the undifferentiated growth of hESCs and to determine whether the hESCs grown in these conditions displayed gene expression patterns that were similar to the expression patterns of feeder cell-dependent hESCs. Our results demonstrate that hESCs grown in the three feeder-free conditions expressed undifferentiation marker genes as strongly as hESCs that were grown in the feeder-dependent cultures. Furthermore, genome-wide gene expression profiles indicated that the gene expression patterns of hESCs that were grown under feeder-free or feeder-dependent culture conditions were highly similar. These results indicate that the feeder-free culture conditions support the undifferentiated growth of hESCs as effectively as the feeder-dependent culture conditions. Therefore, feeder-free culture conditions are potentially suitable for drug screening and for the genetic manipulation of hESCs in basic research.

Human Fetal Trophonema Matrix and Uterine Endometrium Support Better Human Embryonic Stem Cell Growth and Neural Differentiation than Mouse Embryonic Fibroblasts

The concerns over xenogeneic pathogens and immunogenic molecules derived from mouse embryonic fibroblasts (MEFs) trigger the development of human-derived feeder layers for human embryonic stem cell (hESC) maintenance. It is essential to evaluate the capability of these human feeder layers to retain the stemness and pluripotency of hESCs. In the present study, two Chinese hESC lines, SDU-hESCm-1 and SDU-hESCm-2, were continuously cultured on human adult uterine endometrial cells (hUEC), human fetal trophonema matrix cells (hFTMC), and MEFs for at least two month (up to 10 passages). A side-by-side comparison of the abilities to support: (1) self-renewal of the hESCs, (2) expression of undifferentiated markers, and (3) neural differentiation, was made between the human and mouse feeder layers. We demonstrated that the hESCs maintained on hUEC and hFTMC exhibited significantly higher growth rates and generated higher levels of DNA content than those on MEFs. Under neural differentiation-promoting conditions, greater neural differentiation was found in the hESCs maintained on human than on mouse feeder layers. These results suggest that human feeder layers derived from hUECs and hFTMCs are more efficient in supporting a long-term growth and neural differentiation of hESCs than MEFs.

Optimization of Culture Conditions to Support Undifferentiated Growth of Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) are usually maintained in an undifferentiated state by coculture with mitomycin C-treated mouse embryonic fibroblasts (MEFs) as feeder cells in the presence of animal sera such as fetal bovine serum (FBS). Here, we use primary human amnion epithelial cells (hAECs) as feeder cells and human umbilical cord blood serum (CBS) as a replacement for FBS to support undifferentiated growth of hESCs. The 5 approximately 10-fold higher expression levels of ES cell markers including FGF, Oct-4, Nanog, Sox-2, Rex, and TERT were found in hESCs grown on hAECs compared with that on MEFs as measured by quantitative real-time polymerase chain reaction (PCR). By immunofluorescence, the expresisons of Oct-4 and Nanog is also higher in cells grown on hAECs than those on MEFs. Importantly, the ES cells grown on hAECs exhibit normal karyotypes on passage 25, thus ruling out the possibility of transformation. Using flow cytometry analysis, we show that both the ES cells grown on hAECs and MEFs have the same cell cycle distribution pattern. Further, hESCs cultured on hAECs for at least 20 passages could differentiate into three germ layers via teratoma formation. In addition, chromatin immunoprecipitation assay revealed that histone H3 is highly acetylated, and H3 lysine (K) 4 is hypermethylated at the Nanog locus and the Oct-4 locus in hESCs grown on hAECs. Conversely, hESCs grown on MEFs show histone deacetylation and H3-K4 demethylation. Taken together, these results suggest that hAECs supplemented with 10% CBS are suitable for hESC culture, and that this method may prove to be valuable for use in future regenerative therapies.

Defined and Serum-Free Media Support Undifferentiated Human Embryonic Stem Cell Growth

Four commercially available serum-free and defined culture media tested on 2 human embryonic stem cell (hESC) lines were all found to support undifferentiated growth for >10 continuous passages. For hESC cultured with defined StemPro and mTeSR1 media, the cells were maintained feeder-free on culture dishes coated with extracellular matrices (ECMs) with no requirement of feeder-conditioned media (CM). For xeno-free serum replacer (XSR), HEScGRO, and KnockOut media, mitotically inactivated human foreskin feeders (hFFs) were required for hESC growth. Under the different media conditions, cells continued to exhibit alkaline phosphatase activity and expressed undifferentiated hESC markers Oct-4, stage-specific embryonic antigens 4 (SSEA-4), and Tra-1-60. In addition, hESC maintained the expression of podocalyxin-like protein-1 (PODXL), an antigen recently reported in another study to be present in undifferentiated hESC. The cytotoxic antibody mAb 84 binds via PODXL expressed on hESC surface and kills >90% of hESC within 45 min of incubation. When these cells were spontaneously differentiated to form embryoid bodies, derivatives representing the 3 germ layers were obtained. Injection of hESC into animal models resulted in teratomas and the formation of tissue types indicative of ectodermal, endodermal, and mesodermal lineages were observed. Our data also suggested that StemPro and mTeSR1 media were more optimal for hESC proliferation compared to cells grown on CM because the growth rate of hESC increased by 30%-40%, higher split ratio was thus required for weekly passaging. This is advantageous for the large-scale cultivation of hESC required in clinical applications.

Notch and hedgehog signaling cooperate to maintain self-renewal of human embryonic stem cells exposed to low oxygen concentration.

Expansion and maintenance of human embryonic stem cells (hESCs) in undifferentiated state is influenced by complex signals in the microenvironment, including those contingent upon oxygen availability. Responses mediated by Notch and Hedgehog (Hh) have essential role in the growth and maintenance of hESCs, therefore this study examined their effect on the self-renewal of hESCs exposed to low oxygen. Using potent antagonists γ-secretase inhibitor and cyclopamine, we inhibited Notch and Hh pathways, respectively, in the CLS1 hESC line expanded continuously in a hypoxic atmosphere of 5% oxygen. Immunohistochemical staining and protein assays revealed loss of Oct4 and gain of stage-specific embryonic antigen 1 (SSEA1) markers in the inhibited cells. Semiquantitative real-time RT-PCR, and bromodeoxyuridine and thymidine incorporation assays demonstrated low Oct4 and Nanog mRNA expression, and decreased DNA synthesis, respectively, resulting from the block of each of the pathways. The loss increased significantly with co-inhibition of both pathways. Importantly, Notch and Hh downstream targets, including Hes1, Hey1, GIi1, and Ptc1, were surprisingly suppressed not only by the pathway-specific but also the unrelated inhibitor. These findings demonstrate complementary effect of Notch and Hh signaling in hypoxia enhanced maintenance of hESCs.

Megakaryocyte Production From Feeder Cell-Free Cultures of Human Embryonic Stem Cells (hESC).

Abstract 2528Poster Board II-505 Introduction:As the understanding of hESC biology and technology improves, one long-term goal is to utilize expanded numbers of undifferentiated hESC to produce clinically relevant numbers of blood cells in vitro to reduce the need for donors for red blood cell and platelet transfusions. At one end of the production system is the need to create systems for efficient culture of very large numbers of hESC. Methods have been developed using various cytokine combinations to expand end-stage hematopoietic cells from CD34+ cells from bone marrow, peripheral blood, or umbilical cord blood, although little work has been done on megakaryocytopoiesis. Previous systems for bridging the differentiation gap from undifferentiated hESC to mesoderm to hematopoietic progenitor cells have required xenogeneic or in some cases allogeneic supportive cells ± serum at various stages of culture. We have developed a culture system that utilizes feeder cell-free matrix for undifferentiated hESC growth, followed by embryoid body formation, with subsequent shedding of individual hematopoietic progenitor cells that expand and mature to platelet-shedding megakaryocytes. Methods:After extensive evaluations of culture conditions, we arrived at the following optimized conditions. Culture dishes coated with extracellular matrix residual of chemically-digested human foreskin fibroblasts (HFF1 cells from ATCC) support expansion of undifferentiated hESC for more than 30 passages in the presence of DMEM/F12 medium with serum replacement and basic fibroblast growth factor (bFGF). Clusters of cells (∼2 × 105 cells per 2 ml in 6 well plates) from these hESC cultures were transferred to ultra-low attachment culture dishes to form embryoid bodies (EB) in Stemline II medium with (1) basic fibroblast growth factor (bFGF) + vascular endothelial growth factor (VEGF) + bone morphogenic protein (BMP)4 for 2 days; (2) then Stemline II medium with VEGF + BMP4 + thrombopoietin (TPO) + stem cell factor (SCF) + flt3 ligand (FL) for 2 days; (3) then a defined medium with VEGF + BMP4 + TPO + SCF + FL + interleukin (IL)3 + IL6 + IL11 + granulocyte-macrophage colony-stimulating factor (GM-CSF) for 10 days; (4) then cells were transferred to single cell suspension in defined medium with GM-CSF + SCF + FL + TPO + IL6 + Heparin for up to 3 additional weeks. Markers of all stages of differentiation from hESC through megakaryocytes were analyzed by immunofluorescence throughout the course of the cultures. Results:Expression of hESC markers Oct4 and Sox2 was decreased by EB day 7 and was undetectable by EB day 10, although SSEA4 expression persisted until EB day 14. The early mesoderm markers brachyury and Mixl1 were barely detectable until EB day 14. Somewhat surprisingly, hemangioblast/hematopoietic progenitor markers, CD34, GATA1, and CD31, were readily detectable in nearly 100% of cells several days earlier than brachyury and Mixl1, by EB day 7. By EB day 14, there was approximately a 10-fold expansion of cell numbers from the beginning of the EB cultures, with large numbers of single almost exclusively mononuclear cells routinely being found in suspension outside of the EBs. Virtually 100% of these cells were strongly CD34+/GATA1+, a majority were brachyury+ and Mixl1+, and the majority of free floating cells, but not cells within the day 14 EBs were mildly positive for the megakaryocytic lineage markers CD41, CD42a, and CD42b, with 5–10% of these cells outside of the EBs being very strongly positive for these megakaryocytic lineage markers. Differentiation toward megakaryocytes progressed from 7 to 14 days after the EB day 14 suspension cells were transferred to new feeder-free, non-adherent culture dishes. Nearly 100% of cells still were CD34+/GATA1+ (decreased intensity compared to EB Day 14), and >80% of cells became CD41+/CD42a+/CD42b+/CD61+/vWF+/PF4+, and many were shedding platelet-like particles by 14 days after transfer from EB cultures. Conclusions:We have developed a multi-stage culture system that, in the presence of appropriate growth factors, supports specific differentiation from hESC to EB to mature megakaryocytes, avoiding any need for xenogeneic or allogeneic feeder cells or serum. Disclosures:No relevant conflicts of interest to declare.

Naturally Immortalised Mouse Embryonic Fibroblast Lines Support Human Embryonic Stem Cell Growth

Human embryonic stem cell (hESC) growth is dependent on various factors released by feeder cells. Some of them have already been elucidated, although much research is still needed to understand the biology of stem cell maintenance in culture. Traditionally, primary mouse embryonic fibroblasts (PMEFs) have been used as feeder layers, and both murine and human fibroblast cell lines have been shown to support pluripotency and self-renewal of hESC. Here we report the derivation of three new mouse embryonic fibroblast cell lines, MEFLU-M, MEFLU-T, and MEFLU-TB, with different properties regarding growth and support for undifferentiated hESCs. MEFLU-TB is able to support continuous growth of the newly derived Man-1, as well as H1, HUES-1, HUES-7, HUES-8, and HUES-9 human embryonic stem cell lines. After more than 50 passages and doublings, MEFLU-TB feeders compare to early passage primary mouse embryonic fibroblasts in their ability to support undifferentiated hESC growth. Our results contradict a previous paradigm that PMEFs tend to lose their capacity to support proliferation of hESCs with increasing passages, and show that the MEFLU-TB mouse embryonic fibroblast cell line and its conditioned medium have the potential to support the maintenance of hESC lines. Also, our results clearly show that spontaneous immortalization of primary fibroblasts can be achieved in culture without any chemical addition or genetic modification.

Phosphorylation Dynamics during Early Differentiation of Human Embryonic Stem Cells

Pluripotent stem cells self-renew indefinitely and possess characteristic protein-protein networks that remodel during differentiation. How this occurs is poorly understood. Using quantitative mass spectrometry, we analyzed the (phospho)proteome of human embryonic stem cells (hESCs) during differentiation induced by bone morphogenetic protein (BMP) and removal of hESC growth factors. Of 5222 proteins identified, 1399 were phosphorylated on 3067 residues. Approximately 50% of these phosphosites were regulated within 1 hr of differentiation induction, revealing a complex interplay of phosphorylation networks spanning different signaling pathways and kinase activities. Among the phosphorylated proteins was the pluripotency-associated protein SOX2, which was SUMOylated as a result of phosphorylation. Using the data to predict kinase-substrate relationships, we reconstructed the hESC kinome; CDK1/2 emerged as central in controlling self-renewal and lineage specification. The findings provide new insights into how hESCs exit the pluripotent state and present the hESC (phospho)proteome resource as a complement to existing pluripotency network databases.

Identification of the Critical Extracellular Matrix Proteins that Promote Human Embryonic Stem Cell Assembly

Human embryonic stem cells (hESC) exist as large colonies containing tightly adherent, undifferentiated cells. Disaggregation of hESC as single cells significantly affects their survival and differentiation, suggesting that adhesion mechanisms are critical for the assembly and maintenance of hESC colonies. The goal of these studies was to determine the key extracellular matrix (ECM) components that regulate assembly and growth of hESC. Our studies demonstrate that undifferentiated hESC express a specific subtype of laminin (laminin-511) and nidogen-1. The addition of a purified protein complex comprised of human laminin-511 and nidogen-1 to single-cell suspensions of hESC is sufficient to restore hESC assembly in the absence of murine embryonic fibroblasts or exogenous chemicals. The mechanism of hESC aggregation is through binding of the alpha6beta1 integrin receptor highly expressed in the membranes of undifferentiated hESC; aggregation can be inhibited by an antibody against alpha6 and almost completely blocked by an antibody against the beta1 subunit. Reassembly of defined numbers of purified hESC with the laminin-nidogen complex allows consistent production of uniform embryoid bodies (EBs) ("LN-EBs") that differentiate into endodermal, ectodermal, and mesodermal derivatives, and are highly efficient in generating hematoendothelial progenitors. These data reveal for the first time the crucial role of the ECM proteins laminin-511 and nidogen-1 in hESC assembly, and provide a novel practical tool to investigate hESC differentiation in a xenogen-free microenvironment.

Statins inhibit the growth of variant human embryonic stem cells and cancer cells in vitro but not normal human embryonic stem cells

Statins inhibit proliferation of various human cancer cell lines in vitro. As human embryonic stem cells (hESCs) possess neoplastic-like properties we have evaluated the role of various statins on karyotypically normal hESCs (HES3 and BG01), abnormal hESCs (BG01V) and breast adenocarcinoma cells (MCF-7) to evaluate whether the mode of action of the statins was via a stemness pathway. All cell lines were treated with simvastatin, pravastatin, lovastatin and mevastatin (1 micromol x L(-1) to 20 micromol x L(-1)) up to 7 days and their effects on cell proliferation, cell cycle, apoptosis and pluripotency studied. All four statins did not inhibit HES3 and BG01 proliferation, but BG01V and MCF-7 were inhibited by simvastatin, lovastatin and mevastatin. These inhibitory effects were reversed by the endogenous isoprenoids, farnesylpyrophosphate and geranylgeranylpyrophosphate. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling and cell cycle assay confirmed apoptosis in BG01V and MCF-7. Stem cell surface markers [stage-specific embryonic antigen-4, tumour rejection antigen-1-81, octamer-4 (OCT-4)] were expressed in HES3 and BG01, but not in BG01V cells, even after prolonged treatment with simvastatin. In BG01V and MCF-7, the pro-apoptotic Bcl-2-associated X protein genes were up-regulated, while the antiapoptotic BCL2 and SURVIVIN genes were down-regulated. Expression of the stemness-related genes namely, the growth differentiation factor-3, NANOG and OCT-4 was decreased in BG01V compared with BG01 and HES3. Normal hESCs were resistant to prolonged exposure to statins over a range of doses, compared with BG01V and MCF-7, probably because of genetic and behavioural differences. The statins not only have anti-cancer properties but can suppress abnormal hESCs thus promoting growth of normal hESCs in vitro.

Analysis of Human Embryonic Stem Cell Behavior in Control and Experimental Conditions Using Time-Lapse Video Microscopy Endpoints.

Since their first derivation in 1998, researchers have used human embryonic stem cells (hESC) for various purposes. However, the current methods for understanding hESC behavior have been problematic, and better technology is needed for efficient evaluation of hESC behavior in vitro. The purpose of this study was to evaluate and use time-lapse video microscopy (TLVM) to analyze hESC dynamics in vitro using various experimental conditions. The endpoints that were evaluated by observing cellular dynamics included pre-attachment "dancing", attachment/spreading, proliferation, and cell death (apoptosis or necrosis). For all experiments, WiCell H9 hESCs were cultured on Matrigel or gelatin-coated Petri dishes, and images were recorded with a Nikon BioStation IM at 1 or 2 minutes intervals for 3 hours using different experimental conditions. Quantification of the endpoints was done by different individuals counting multiple frames from each experiment. Different individuals also evaluated the same data repeatedly, and the coefficients of variation for both an individual's multiple counts as well as counts by different individuals were low (<1.0%), demonstrating the reliability of the method. One phenomenon observed in the control was pre-attachment "dancing" or rapid movements of paddle-like protrusions on cell surfaces in control culture environments. Similar behavior was observed in prostate stromal cells and mouse embryonic fibroblasts, but the movements were less vivacious and were short-lived compared to hESC. A number of protocols have been used to passage hESCs. Here, we utilized TLVM to compare the attachment/spreading ability of hESCS on Matrigel versus gelatin coated dishes after trypsin or Accutase treatment. The time lapse data showed that hESC survived best when removed from plates using trypsinization followed by subsequent plating on Matrigel-coated dishes. Changes in cellular dynamics also enabled analysis of apoptosis and necrosis. The surface of apoptotic cells underwent rapid blebbing, while necrotic cells became swollen. The toxicity of cigarette smoke and alcohol on hESC survival and growth was evaluated with TLVM endpoints. Treatment of hESC with sidestream (SS) smoke (0.1PE, PE=puff equivalent = smoke in 1 puff that dissolves in 1 ml of solution) from traditional (Marlboro Red) and harm reduction (Advance Lights, Marlboro Lights, and Quest) cigarettes decreased the percentage of cell attachment to Matrigel when compared to untreated controls. hESCs treated with 0.5% alcohol were undergoing apoptosis by 2 hours of treatment, and attached colonies treated with 0.1% alcohol (a level found in the blood of human drinkers) also showed apoptotic blebbing. hESC treated with 0.1% alcohol also had activated caspases that were demonstrated using a poly-caspase FLICA inhibitor. In summary, we have demonstrated that TLVM is a valuable and effective method to evaluate hESC behavior allowing close observation and detailed analysis of cellular dynamics under various experimental conditions. (poster)

A Complex Role for FGF-2 in Self-Renewal, Survival, and Adhesion of Human Embryonic Stem Cells

The transcription program that is responsible for the pluripotency of human ESCs (hESCs) is believed to be comaintained by exogenous fibroblast growth factor-2 (FGF-2), which activates FGF receptors (FGFRs) and stimulates the mitogen-activated protein kinase (MAPK) pathway. However, the same pathway is stimulated by insulin receptors, insulin-like growth factor 1 receptors, and epidermal growth factor receptors. This mechanism is further complicated by intracrine FGF signals. Thus, the molecular mechanisms by which FGF-2 promotes the undifferentiated growth of hESCs are unclear. Here we show that, in undifferentiated hESCs, exogenous FGF-2 stimulated the expression of stem cell genes while suppressing cell death and apoptosis genes. Inhibition of autocrine FGF signaling caused upregulation of differentiation-related genes and downregulation of stem cell genes. Thus, exogenous FGF-2 reinforced the pluripotency maintenance program of intracrine FGF-2 signaling. Consistent with this hypothesis, expression of endogenous FGF-2 decreased during hESC differentiation and FGF-2 knockdown-induced hESC differentiation. In addition, FGF-2 signaling via FGFR2 activated MAPK kinase/extracellular signal-regulated kinase and AKT kinases, protected hESC from stress-induced cell death, and increased hESC adhesion and cloning efficiency. This stimulation of self-renewal, cell survival, and adhesion by exogenous and endogenous FGF-2 may synergize to maintain the undifferentiated growth of hESCs. Stem Cells 2009;27:1847–1857

Analysis of the Factors that Limit the Ability of Feeder Cells to Maintain the Undifferentiated State of Human Embryonic Stem Cells

Human embryonic stem cell (hESC) culture is routinely performed using inactivated mouse embryonic fibroblasts (MEFs) as a feeder cell layer (FL). Although these cells maintain pluripotency of hESCs, the molecular basis for this is unknown. Objectives of this study were to determine whether timing between MEF inactivation and their use as a FL influenced hESC growth and differentiation, and to begin defining the mechanism(s) involved. hESCs were plated on MEFs prepared 1 (MEF-1), 4 (MEF-4), and 7 (MEF-7) days earlier. hESC colony morphology and Oct3/4 expression levels were evaluated to determine the influence of different FLs. Significant enhancement of hESC growth (self-renewal) was observed on MEF-1 compared with MEF-4 and/or MEF-7. Conditioned media (CM) collected from MEF-1 supported significantly better hESC growth in a FL-free system compared to MEF-7 CM. Effects of MEFs on hESC growth were not caused by differences in cell density or viability, although indications of apoptosis were observed in MEF-7. Scanning electron microscopy demonstrated that MEF-7 were morphologically distinct from MEF-1 and MEF-4. Microarray analysis identified 19 genes related to apoptosis with significantly different levels of expression between MEF-1 and MEF-7. Several differentially expressed RNAs had gene ontology classifications associated with extracellular matrix (ECM) structural constituents and growth factors. Because members of Wnt signaling pathway were identified in the array analysis, we examined the ability of the Wnt1 CM and secreted frizzled-related proteins to affect hESC growth and differentiation. The addition of Wnt1 CM to both MEF-1 and MEF-7 significantly increased the number of undifferentiated colonies, while the addition of Sfrps promoted differentiation. Together, these results suggest that microenvironment, ECM, and soluble factors expressed by MEF-1 are significantly better at maintaining self-renewal and pluripotency of hESCs. Our findings have important implications in the optimization of hESC culture when MEFs are used as FL or CM is used in FL-free culture.

Manipulating the microenvironment for vascular differentiation and regeneration

Microvasculature differentiation and regeneration is guided by both biochemical and biophysical cues. Human embryonic stem cells (hESCs) and adult endothelial progenitor cells (hEPCs), which are capabile to differentiate and form blood vessels de novo, can be used as a model system to elucidate vasculogenic and angiogenic mechanisms and explore central issues in vascular regeneration. Our goal is to understand the molecular mechanisms underlying human vascular differentiation and regeneration. To achieve this we exploit microengineering approaches to manipulate, study, and direct adult and embryonic stem cell differentiation. As an example, we found that the extracellular matrix polysaccharide hyaluronic acid (HA) is a developmentally relevant material for the growth of hESCs. Human ESCs encapsulated in HA hydrogels maintained undifferentiated and induced to initiate vasculogenesis by supplementation with VEGF. In continues studies, utilizing mechanically tunable HA hydrogels, we show that VEGF and matrix stiffness regulate angiogenesis from hEPCs. For the binding interactions between cells and surfaces, it has become increasingly evident that cells are influenced also by spatial domains, structural compositions, and mechanical forces at the microscale and nanoscale. We discovered that micro‐nanotopography of a substrate can be used to control vascular assembly and tubulogenesis.

Adapting collagen/CNT matrix in directing hESC differentiation

The lineage selection in human embryonic stem cell (hESC) differentiation relies on both the growth factors and small molecules in the media and the physical characteristics of the micro-environment. In this work, we utilized various materials, including the collagen–carbon nanotube (collagen/CNT) composite material, as cell culture matrices to examine the impact of matrix properties on hESC differentiation. Our AFM analysis indicated that the collagen/CNT formed rigid fibril bundles, which polarized the growth and differentiation of hESCs, resulting in more than 90% of the cells to the ectodermal lineage in Day 3 in the media commonly used for spontaneous differentiation. We also observed the differentiated cells followed the coarse alignment of the collagen/CNT matrix. The research not only revealed the responsiveness of hESCs to matrix properties, but also provided a simple yet efficient way to direct the hESC differentiation, and imposed the potential of forming neural-cell based bio-devices for further applications.

Laminin isoforms in human embryonic stem cells: synthesis, receptor usage and growth support

To reveal the functional intrinsic niche of human embryonic stem cells (hESC) we examined the production of basement membrane (BM) proteins and the presence of their receptors in feeder-free cell culture conditions. In addition, we investigated binding of hESCs to purified human BM proteins and identified the receptors mediating these contacts. Also, we tested whether purified human laminin (Lm) isoforms have a role in hESC self-renewal and growth in short-term cultures. The results show that hESCs synthesize Lm alpha(1) and Lm alpha(5) chains together with Lm beta(1) and gamma(1) chains suggesting the production of Lms-111 and -511 into the culture medium and deposits on cells. hESCs contain functionally important integrin (Int) subunits, Int beta(1), alpha(3), alpha(6), alpha(5), beta(5) and alpha(V), as well as the Lm alpha(5) receptor, Lutheran (Lu) glycoprotein and its truncated form, basal cell adhesion molecule (B-CAM). In cell adhesion experiments, Int beta(1) was crucial for adhesion to most of the purified human BM proteins. Lu/B-CAM mediated adhesion to Lm-511 together with Int alpha(3)beta(1), and was essential for the adhesion of hESCs to embryonic feeder cells. Adhesion to Lm-411 was mediated by Int alpha(6)beta(1). Lm-511 supported hESC growth in defined medium equally well as Matrigel. These results provide consequential information of the biological role of BM in hESCs, warranting further investigation of BM biology of human pluripotent stem cells.

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.