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1.
Nat Biomed Eng ; 2(3): 173-182, 2018 03.
Article in English | MEDLINE | ID: mdl-31015717

ABSTRACT

The large-scale and cost-effective production of quality-controlled human pluripotent stem cells (hPSCs) for use in cell therapy and drug discovery would ideally require a chemically defined xenobiotic-free culture system. Towards the development of such a system, costs associated with the use of recombinant proteins as supplements in basal culture media need to be reduced. Here, we describe a growth-factor-free culture medium that uses just three chemical compounds and a lower number of recombinant proteins than used in commercially available media. We show that the culture medium supports the long-term propagation of hPSCs, as confirmed by karyotype, the expression of pluripotency markers and the capacity to differentiate into cell types derived from the three embryonic germ layers. hPSCs growing in the medium were less dependent on glycolytic pathways than cells grown in medium containing growth factors. Moreover, the medium supported the generation of induced pluripotent stem cells derived from either human dermal fibroblasts or peripheral blood mononuclear cells. Our findings should facilitate the ongoing development of a completely xeno-free, chemically defined, synthetic culture system for hPSCs.


Subject(s)
Cell Culture Techniques/methods , Pluripotent Stem Cells , Cell Differentiation , Cells, Cultured , Culture Media/chemistry , Culture Media/metabolism , Fibroblasts/cytology , Fibroblasts/physiology , Humans , Intercellular Signaling Peptides and Proteins , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/physiology , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/physiology
2.
Curr Mol Pharmacol ; 9(3): 272-279, 2016.
Article in English | MEDLINE | ID: mdl-26008738

ABSTRACT

Despite their high degree of identity and even higher homology, the two Kat3 transcriptional coactivators, CBP and p300, have distinct functions, particularly within the Wnt/ß-catenin signaling cascade. ICG-001, by directly binding to CBP but not p300, inhibits CBP/ß-catenin transcription and has served as an invaluable chemical genomic tool to dissect the Wnt signaling cascade and the divergent roles of these two coactivators. However, to date no direct antagonist of the p300/ß-catenin interaction has been reported. We now report the identification and validation of the first highly specific, direct p300/ß-catenin antagonists, YH249/250 and their ability to maintain pluripotency in ESC.


Subject(s)
E1A-Associated p300 Protein/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Stem Cells/metabolism , beta Catenin/antagonists & inhibitors , Animals , CREB-Binding Protein/metabolism , Cell Differentiation/drug effects , Cell Line , Drug Evaluation, Preclinical , E1A-Associated p300 Protein/metabolism , HEK293 Cells , Human Embryonic Stem Cells/drug effects , Human Embryonic Stem Cells/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Mice , Protein Binding/drug effects , Satellite Cells, Skeletal Muscle/drug effects , Satellite Cells, Skeletal Muscle/metabolism , Small Molecule Libraries/chemistry , Stem Cells/drug effects , beta Catenin/metabolism
3.
Stem Cells Transl Med ; 1(1): 18-28, 2012 Jan.
Article in English | MEDLINE | ID: mdl-23197636

ABSTRACT

An optimal culture system for human pluripotent stem cells should be fully defined and free of animal components. To date, most xeno-free culture systems require human feeder cells and/or highly complicated culture media that contain activators of the fibroblast growth factor (FGF) and transforming growth factor-ß (TGFß) signaling pathways, and none provide for replacement of FGF/TGFß ligands with chemical compounds. The Wnt/ß-catenin signaling pathway plays an important role in mouse embryonic stem cells in leukemia inhibitory factor-independent culture; however, the role of Wnt/ß-catenin signaling in human pluripotent stem cell is still poorly understood and controversial because of the dual role of Wnts in proliferation and differentiation. Building on our previous investigations of small molecules modulating Wnt/ß-catenin signaling in mouse embryonic stem cells, we identified a compound, ID-8, that could support Wnt-induced human embryonic stem cell proliferation and survival without differentiation. Dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) is the target of the small molecule ID-8. Its role in human pluripotent cell renewal was confirmed by DYRK knockdown in human embryonic stem cells. Using Wnt and the DYRK inhibitor ID-8, we have developed a novel and simple chemically defined xeno-free culture system that allows for long-term expansion of human pluripotent stem cells without FGF or TGFß activation. These culture conditions do not include xenobiotic supplements, serum, serum replacement, or albumin. Using this culture system, we have shown that several human pluripotent cell lines maintained pluripotency (>20 passages) and a normal karyotype and still retained the ability to differentiate into derivatives of all three germ layers. This Wnt-dependent culture system should provide a platform for complete replacement of growth factors with chemical compounds.


Subject(s)
Embryonic Stem Cells/drug effects , Induced Pluripotent Stem Cells/drug effects , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/antagonists & inhibitors , Wnt Signaling Pathway/drug effects , Animals , Cell Culture Techniques , Cell Differentiation/drug effects , Cell Line , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Embryonic Stem Cells/enzymology , Gene Knockdown Techniques , Humans , Induced Pluripotent Stem Cells/enzymology , Karyotyping , Mice , Mice, SCID , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , RNA Interference , Time Factors , Wnt3A Protein/metabolism , Dyrk Kinases
4.
Stem Cell Rev Rep ; 7(4): 836-46, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21603945

ABSTRACT

Embryonic stem cells and induced pluripotent stem (iPS) cells are characterized by their ability to self-renew and to generate differentiated cells of all three germ layers. This potential makes them an attractive source to address question of developmental and also for use in clinical regenerative medicine. Although the culture conditions to maintain pluripotency and reprogramming technologies have been established, the underlying molecular mechanisms are incompletely understood. Accumulating evidence indicates that the Wnt/ß-catenin signaling pathway plays a pivotal role in the maintenance of pluripotency as well as in the process of somatic cell reprogramming. Reciprocally, Wnt/ß-catenin signaling also plays a critical role in the lineage decision/commitment process. These dramatically different outcomes upon activation of the Wnt signaling cascade has fueled enormous controversy concerning the role of Wnt signaling in the maintenance of potency and induction of differentiation in stem cells. Here, we discuss and explore the divergent roles of the Wnt signaling pathways based on findings from our lab. Accumulated results from our lab indicate the usage of a critical switching mechanism that regulates the divergent Wnt/catenin transcriptional programs associated with either maintenance of potency or initiation of differentiation.


Subject(s)
Embryonic Stem Cells/cytology , Wnt Signaling Pathway , beta Catenin/metabolism , Animals , Cell Differentiation , Cell Lineage , Embryonic Stem Cells/metabolism , Humans , Mice , Protein Interaction Domains and Motifs , TCF Transcription Factors/genetics , TCF Transcription Factors/metabolism , Transcription, Genetic , Wnt Proteins/genetics , Wnt Proteins/metabolism , beta Catenin/genetics
5.
Genes Cells ; 15(5): 455-70, 2010 May.
Article in English | MEDLINE | ID: mdl-20384793

ABSTRACT

Human embryonic stem cell (ESC) pluripotency is thought to be regulated by several key transcription factors including OCT4, NANOG, and SOX2. Although the functions of OCT4 and NANOG in human ESCs are well defined, that of SOX2 has not been fully characterized. To investigate the role of SOX2, we modulated the level of SOX2 expression in human ESCs. Reduction of SOX2 expression in human ESCs induced trophectodermal and partial endodermal differentiation. Interestingly, CDX2, a typical trophectoderm-associated gene, was not up-regulated. In contrast, using the Tet-on gene inducible system, SOX2 over-expression in human ESCs induced trophectoderm differentiation accompanied by increased CDX2 expression. Additionally, SOX2 over-expression resulted in an increase in CGalpha-positive cells, which marks later stage trophectoderm development, rather than placental lactogen-positive cells. Thus, over-expression as well as repression of SOX2 expression in human ESCs resulted in their differentiation into the trophectoderm lineage. Our data show that SOX2 plays an important role in the maintenance of pluripotency of human ESCs and possibly, trophoblast development.


Subject(s)
Embryonic Stem Cells/physiology , Pluripotent Stem Cells/physiology , SOXB1 Transcription Factors/metabolism , Animals , Cell Differentiation/physiology , Embryonic Stem Cells/cytology , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Mice , Nanog Homeobox Protein , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , Pluripotent Stem Cells/cytology , RNA Interference , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , SOXB1 Transcription Factors/genetics , Trophoblasts/cytology , Trophoblasts/metabolism
6.
Genes Cells ; 11(9): 1115-23, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16923129

ABSTRACT

Nanog is a homeodomain transcription factor that is expressed specifically in undifferentiated embryonic stem (ES) cells and has been shown to be essential in the maintenance of pluripotency in mouse ES cells. To examine the function of NANOG in primate ES cells, we generated transgenic monkey ES cell lines expressing three- to seven-fold higher levels of NANOG protein compared to wild-type ES cells. These NANOG over-expressing cell lines retained their undifferentiated state in the absence of a feeder layer, as shown by expression of undifferentiated ES cell markers such as alkaline phosphatase (ALP) and OCT-4. We also demonstrated that in vitro differentiation of transgenic cell lines was mostly restricted to the ectodermal lineage, as examined by reverse transcriptase-polymerase chain reaction (RT-PCR). Knockdown experiments using NANOG small interfering (si) RNA resulted in induction of differentiation markers such as AFP, GATA4 and GATA6 for the endoderm and CDX2 for the trophectoderm. These results suggest that NANOG plays a crucial role in maintaining the pluripotent state of primate ES cells.


Subject(s)
DNA-Binding Proteins/metabolism , Embryo, Mammalian/cytology , Homeodomain Proteins/metabolism , Primates/metabolism , Stem Cells/cytology , Animals , Cell Differentiation , Cells, Cultured , Culture Media, Conditioned , DNA-Binding Proteins/genetics , Gene Expression Regulation , Homeodomain Proteins/genetics , Humans , Mice , Nanog Homeobox Protein , RNA, Messenger/genetics , RNA, Messenger/metabolism
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