During embryogenesis, the Activin/Nodal pathway promotes the mesendodermal lineage and inhibits neural fate. The molecular mechanisms underlying this role of the Activin/Nodal pathway are not clear. In this study, we report a role for protein tyrosine phosphatase 1B (PTP1B) in Activin-mediated early fate decisions during ESC differentiation and show that PTP1B acts as an effector of the Activin pathway to specify mesendodermal or neural fate. We found that the Activin/ALK4 pathway directly recruits PTP1B and stimulates its release from the endoplasmic reticulum through ALK4-mediated cleavage. Subsequently, PTP1B suppresses p-ERK1/2 signaling to inhibit neural specification and promote mesendodermal commitment. These findings suggest that a noncanonical Activin signaling pathway functions in lineage specification of mouse and human embryonic stem cells.

The inevitable accumulation of chromosomal abnormalities in human embryonic stem cells (hESCs) during in vitro expansion represents a considerable obstacle for cell replacement therapies. To determine the source of chromosomal abnormalities, we examined hESCs maintained in culture for over 55 months for defects in telomere maintenance and DNA repair. Although prolonged culture affected neither telomerase activity nor nonhomologous end joining, the efficiency of base excision repair (BER) was significantly decreased and correlated with reduced expression of apurinic/apyrimidinic endonuclease 1 (APE1), the major nuclease required for BER. Interestingly, the expression of other BER enzymes was unchanged. Addition of human recombinant APE1 protein to nuclear extracts from late passage hESCs increased BER efficiency to the level typical of early passage hESCs. The link between BER and double-strand breaks (DSB) was demonstrated by decreased DSB release after downregulation of APE1 in early passage hESCs via siRNA. Correspondingly lower APE1 level in late passage hESC resulted in slower and less intensive but long lasting DSB release upon ionizing radiation (IR). Downregulation of APE1 in early passage hESCs also led to approximately 30% decrease in γ-H2AX signaling following IR, similar to that in late passage hESCs. We suggest that downregulation of APE1 significantly contributes to the failure of BER during long-term culture of hESCs, and further that BER failure is one of the factors affecting the genomic instability of hESCs by altering BER-dependent DSB release and cell cycle/checkpoint signaling.

The generation of human pluripotent stem cells (hPSCs) of sufficient quantity and quality remains a major challenge for biomedical application. Here we present an efficient feeder-free, high-density monolayer system in which hPSCs become SSEA-3-high and gradually more viable than their feeder-dependent counterparts without changes attributed to culture adaptation. As a consequence, monolayer hPSCs possess advantages over their counterparts in embryoid body development, teratoma formation, freezing as a single-cell suspension, and colony-forming efficiency. Importantly, this monolayer culture system is reversible, preserving the competence of hPSCs to gradually reacquire features of colony growth, if necessary. Therefore, the monolayer culture system is highly suitable for long-term, large-scale propagation of hPSCs, which is necessary in drug development and pluripotent stem cell-based therapies.

Basic fibroblast growth factor (FGF-2) promotes self-renewal, survival, and adhesion of human ESCs. The diverse functions of FGF-2 may be attributed to its interactions with various fibroblast growth factor receptors (FGFRs), which in turn lead to different signalling cascades. Our study explores the role of FGFR1 in the human ESC adhesion, signalling and proliferation. FGFRs are tyrosine kinase receptors involved in a broad range of developmental processes, but their functions or expression often is dysregulated in developmental malignancies and cancers. Our data showed that hESCs express all types of FGFRs (1, 2, 3 and 4), to which FGFR1 is the most abundant. Intriguingly, the phosphorylation induced by FGF-2 treatment in FGFR1 is the least amongst the FGFRs. Our results suggest that the tepid reaction of FGFR1 to FGF-2 in phosphorylation implicates the action of FGFR1 in non-canonical FGF pathway. We also observed that FGFR1 localises mainly in the cell membranes where the cell-to-cell contacts occur. We also found that FGFR1 co-stains with ZO-1 protein, which is involved in tight junction assembly. Interestingly, in lower density cultures, we observed that FGFR1 and ZO-1 co-localise in nucleus in addition to the location in the cell membrane. Our data suggest that FGFR1 may play a role in cell adhesion and contact inhibition. Herein, we propose a potential novel role of FGFR1 in the microenvironment of human ESC interactions.

The need for a robust, invariable, and cost-effective culture of human embryonic stem cells (hESCs) resulted in several large-scale systems. However, the novelty was often hampered by changes in hESC phenotype, genomic instability, or difficult manipulation. Here we report a comprehensive analysis of pluripotency features by hESCs, which have been propagated for over 100 passages in monolayer (ML) culture. The ML system is based on single-cell dissociation and plating of cells in high densities on a matrix-coated surface, so they become fully confluent within 72 hours. This results in homogenization of the cell population and efficient expansion with 1:20 ratio every 3 days. While the in vitro and in vivo differentiation capacity remained to be the same, successful development of a teratoma required 5-times lower input of hESCs from the ML system then of their feeder-dependent counterparts. This indicates (a) different portion of teratoma initiating cells or (b) different survival rate early after injection. As the presence of SSEA-5- and Oct-4- positive cells was comparable in both culture systems after differentiation, teratoma development from ML hESCs was not driven by an increased pool of differentiation-resistant cells. Also, we found none of the recently described adaptation-related proteins (Bcl-xL, Bcl-2 and survivin) to be overexpressed in ML hESCs. In two viability assays, ML hESCs showed superior survival and participation to assays than colony-dependent culture. Finally, we found that the reverted feeder-dependent culture displayed normal phenotype of the colony growth, gradually developed back the dependence on Y27632 in viability assays, and lowered teratoma efficiency towards the ground state represented by feeder-dependent hESC colonies at the beginning. In addition to the stable karyotype, the reversibility rather points to an unselective process of adaptation and challenges the hypothesis of clonal expansion in ML system. In addition to that, a simple automation and compatibility with xeno-free conditions make the ML system highly suitable for production of vast numbers hESCs necessary in cell-based therapies.

During embryogenesis, the Activin/Nodal pathway plays a crucial role in lineage decision. In vivo studies showed that different concentrations of Activin elicit distinct responses from Xenopus animal caps, ultimately producing a range of mesodermal fates. Nodal-/- mice fail to form both the mesoderm and the definitive endoderm, but show precocious neural differentiation, suggesting that the Activin/Nodal pathway promotes mesodermal lineage and inhibits neural fate decision. This is corroborated by a series of studies in vitro. Blockage of Activin signalling has been shown to promote neural fate in human ES cells and in mouse ES cells (our unpublished data). What molecular mechanism underlies the dual role of the Activn/Nodal pathway is not clear. We have discovered that protein tyrosine phosphatase 1B (Ptp1B) acts as a novel partner of the Activin/Alk4 pathway to select between mesodermal or neural fates. We employed human and mouse embryonic stem (ES) cell-differentiation model to investigate the function of the Activin/Alk4 pathway in the early fate decision. We found that the treatment of Activin at the different stages of ES cell differentiation exhibit diverse influences in cell-type derivatives. Inhibition of the Activin pathway at different time windows also shows a stage-dependent fate adaptation. A downstream factor of this pathway, Ptp1B, has been identified to interact with Alk4, which in turn governs Activin-directed fate decision in combination with p-Smad2/3 signalling.

Poly(N,N-diethylacrylamide) (PDEAAm) hydrogel scaffolds were prepared by radical copolymerization of N,N-diethylacrylamide (DEAAm), N,N'-methylenebisacrylamide and methacrylic acid in the presence of (NH₄)₂SO₄ or NaCl. The hydrogels were characterized by low-vacuum scanning electron microscopy in the water-swollen state, water and cyclohexane regain, and by mercury porosimetry. The pentapeptide, YIGSR-NH₂, was immobilized on the hydrogel. Human embryonic stem cells (hESCs) were cultured with the hydrogels to test their biocompatibility. The results suggest that the PDEAAm hydrogel scaffolds are nontoxic and support hESC attachment and proliferation, and that interconnected pores of the scaffolds are important for hESC cultivation. Immobilization of YIGSR-NH₂ pentapeptide on the PDEAAm surface improved both adhesion and growth of hESCs compared with the unmodified hydrogel. The YIGSR-NH₂-modified PDEAAm hydrogels may be a useful tool for tissue-engineering purposes.

For human embryonic stem cells (ESC) to be used in cell replacement therapies, they must be grown under good manufacturing conditions in a chemically defined medium that lacks animal proteins. This study examined the ability of a newly designed medium containing the plant-derived serum replacement VegetaCell and other reagents of human origin to support undifferentiated growth and pluripotency of human ESC. This medium was tested in several culture systems, using human fibroblasts as a feeder layer or Matrigel in a feeder-free culture. Even under the most stringent feeder-free conditions without conditioned medium, human ESC exhibited an undifferentiated morphology, expressed markers of undifferentiated cells, demonstrated high alkaline phosphatase activity and multilineage differentiation and retained a normal karyotype. Compared with human ESC grown in standard culture conditions, human ESC maintained in humanized VegetaCell medium show longer cell cycles and decreased cell death. The availability of an animal protein-free medium supplemented with the low-cost VegetaCell reagent expands the repertoire of media for culturing human ESC as well as induced pluripotent stem cells for drug testing and cell replacement therapy.

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