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.
- MeSH
- aktivinové receptory typu I chemie metabolismus MeSH
- aktiviny metabolismus MeSH
- benzamidy farmakologie MeSH
- buněčná diferenciace * účinky léků MeSH
- buněčný rodokmen účinky léků MeSH
- dioxoly farmakologie MeSH
- embryonální kmenové buňky cytologie účinky léků enzymologie MeSH
- endoderm cytologie účinky léků metabolismus MeSH
- extracelulárním signálem regulované MAP kinasy metabolismus MeSH
- fosforylace účinky léků MeSH
- lidé MeSH
- MAP kinasový signální systém účinky léků MeSH
- mezoderm cytologie účinky léků metabolismus MeSH
- molekulární sekvence - údaje MeSH
- myši MeSH
- neurony cytologie účinky léků metabolismus MeSH
- pluripotentní kmenové buňky cytologie účinky léků metabolismus MeSH
- protein Smad2 metabolismus MeSH
- sekvence aminokyselin MeSH
- signální transdukce * účinky léků MeSH
- tyrosinfosfatasa nereceptorového typu 1 metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- analýza kolonii tvořících jednotek MeSH
- biologické markery metabolismus MeSH
- buněčné kultury metody MeSH
- buněčné linie MeSH
- indukované pluripotentní kmenové buňky cytologie metabolismus MeSH
- lidé MeSH
- myši MeSH
- pluripotentní kmenové buňky cytologie metabolismus MeSH
- podkladové buňky cytologie MeSH
- proliferace buněk MeSH
- teratom patologie MeSH
- viabilita buněk MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- buněčné linie MeSH
- DNA-lyasa (apurinová nebo apyrimidinová) genetika metabolismus MeSH
- dvouřetězcové zlomy DNA MeSH
- embryonální kmenové buňky enzymologie metabolismus MeSH
- imunohistochemie MeSH
- karyotypizace MeSH
- lidé MeSH
- oprava DNA genetika fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- akrylamidy chemie MeSH
- buněčné linie MeSH
- embryonální kmenové buňky cytologie MeSH
- hydrogely chemie MeSH
- lidé MeSH
- myši MeSH
- oligopeptidy chemie MeSH
- polymery chemie MeSH
- proliferace buněk MeSH
- tkáňové inženýrství metody MeSH
- tkáňové podpůrné struktury MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- apoptóza účinky léků MeSH
- buněčná diferenciace účinky léků MeSH
- buněčné kultury metody MeSH
- buněčný cyklus účinky léků MeSH
- embryonální kmenové buňky cytologie MeSH
- fibroblasty účinky léků MeSH
- fixní kombinace léků MeSH
- indukované pluripotentní kmenové buňky MeSH
- kolagen MeSH
- kultivační média bez séra farmakologie MeSH
- kultivační média speciální farmakologie MeSH
- kultivační média MeSH
- laminin MeSH
- lidé MeSH
- pluripotentní kmenové buňky cytologie MeSH
- proliferace buněk účinky léků MeSH
- proteoglykany MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
