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Background: The Taiwan Human Disease iPSC Service Consortium was established to accelerate Taiwan's growing stem cell research initiatives and provide a platform for researchers interested in utilizing induced pluripotent stem cell (iPSC) technology. The consortium has generated and characterized 83 iPSC lines: 11 normal and 72 disease iPSC lines covering 21 different diseases, several of which are of high incidence in Taiwan. Whether there are any reprogramming-induced recurrent copy number variant (CNV) hotspots in iPSCs is still largely unknown., Methods: We performed genome-wide copy number variant screening of 83 Han Taiwanese iPSC lines and compared them with 1093 control subjects using an Affymetrix genome-wide human SNP array., Results: In the iPSCs, we identified ten specific CNV loci and seven "polymorphic" CNV regions that are associated with the reprogramming process. Additionally, we established several differentiation protocols for our iPSC lines. We demonstrated that our iPSC-derived cardiomyocytes respond to pharmacological agents and were successfully engrafted into the mouse myocardium demonstrating their potential application in cell therapy., Conclusions: The CNV hotspots induced by cell reprogramming have successfully been identified in the current study. This finding may be used as a reference index for evaluating iPSC quality for future clinical applications. Our aim was to establish a national iPSC resource center generating iPSCs, made available to researchers, to benefit the stem cell community in Taiwan and throughout the world.

Mesenchymal stem cells (MSCs) are multipotent precursors that can undergo multilineage differentiation, including osteogenesis and adipogenesis, which are two mutually exclusive events. Previously, we demonstrated that enhancer of zeste homolog 2 (EZH2), the catalytic component of the Polycomb-repressive complex 2, mediates epigenetic silencing of histone deacetylase 9c (HDAC9c) in adipocytes but not in osteoblasts and that HDAC9c accelerates osteogenesis while attenuating adipogenesis of MSCs through inactivation of peroxisome proliferator-activated receptor gamma 2 activity. Importantly, disrupting the balance between adipogenesis and osteogenesis can lead to age-associated bone loss (osteoporosis) and obesity. Here, we investigated the relationship between age, and osteogenic and adipogenic differentiation potential of MSCs by comparing EZH2 and HDAC9c expression in osteoblasts and adipocytes of both human and mice origins to determine whether the EZH2-HDAC9c axis regulates age-associated osteoporosis and obesity. Our findings indicated that a decline in HDAC9c expression over time was accompanied by increased EZH2 expression and suggested that a therapeutic intervention for age-associated osteoporosis and obesity may be feasible by targeting the EZH2-HDAC9c axis. Stem Cells 2016;34:2183-2193., (© 2016 AlphaMed Press.)

Establishing an efficient differentiation procedure is prerequisite for the cell transplantation of pluripotent stem cells. Activating fibroblast growth factor (FGF) signals and inhibiting the activin/nodal pathway are both conserved principles to direct the neural induction (NI) of developing embryos and human embryonic stem cells (hESCs). Wnt signal and OCT4 expression are critical for the hESC pluripotency; however, their roles in cell differentiation are largely unclear. We demonstrate that in the presence of FGF2 and activin inhibitor SB431542, applying a small-molecule Wnt agonist, BIO, efficiently and rapidly steers the NI of all our tested hESCs. A human induced pluripotent stem cell (iPSC), which is refractory for efficient neural conversion by FGF2, effectively differentiated to SOX1(+) cells after the BIO/SB431542/FGF2 treatment. In addition, BIO promoted cell survival and transiently sustained OCT4 expression at the early NI stage with FGF2 and SB431542. Interestingly, at the late NI stage, the OCT4 level rapidly declined in the treated hESCs and consequently initiated the formation of neural rosettes with forebrain neuron characteristics. This study illustrates the distinct effects of Wnt activation on maintaining pluripotency and committing neural lineages at the early and late NI stages of hESCs and iPSCs, respectively.

A short G1 phase is a characteristic feature of the cell cycle structure of pluripotent cells, and is reestablished during Yamanaka factor-mediated pluripotent reprogramming. How cell cycle control is adjusted to meet the requirements of pluripotent cell fate commitment during reprogramming is less well understood. Elevated levels of cyclin D1 were initially found to impair pluripotency maintenance. The current work further identified Cyclin D1 to be capable of transcriptionally upregulating Pax6, which promoted reprogramming cells to commit to a neural progenitor fate rather than a pluripotent cell fate. These findings explain the importance of reestablishment of G1-phase restriction in pluripotent reprogramming., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

In regular culture conditions with leukemia inhibitory factor (LIF), the majority of mouse embryonic stem cells (mESCs) are maintained in a self-renewal stage; very few mESCs have differentiated morphology. When LIF is withdrawn, mESCs tend to differentiate; this differentiation process can be enhanced by the introduction of exogenous fibroblast growth factor (FGF). Here, we show that even in the presence of exogenous FGF1, mESCs can maintain self-renewal and expression of pluripotency markers in the presence of LIF. To elucidate the mechanism in which LIF dominates over the FGF1, extracellular signal-regulated kinase 1/2 (Erk1/2) signaling of mESCs cultured in a medium containing FGF1 or LIF/FGF1 was examined. The results demonstrate that Erk1/2 was activated by FGF1 in the absence of LIF; however, the FGF1-induced Erk1/2 phosphorylation was suppressed when LIF was introduced. Moreover, FGF1-Erk1/2 downregulation was inhibited by a signal transducer and activator of the transcription 3 (Stat3) inhibitor WP1066, suggesting that LIF-induced Stat3 activation plays an important role in the FGF1-Erk1/2 inhibition in mESCs. We further demonstrate that the binding affinity of phospho-Erk1/2 and Sprouty2 was increased via Stat3 activation. Binding of phospho-Erk1/2 and Sprouty2 blocks the activation of Erk1/2 signaling, thus inhibiting the downstream differentiation process in mESCs. Our findings demonstrate, for the first time, that LIF-induced Stat3 phosphorylation plays an important role in promoting the binding of phospho-Erk1/2 and Sprouty2, and thus inhibiting FGF-induced differentiation.

The level of Gs alpha activity has been shown to modulate the rate of adipogenesis in mouse embryo fibroblast 3T3-L1 cells (H.-Y. Wang, D. C. Watkins, and C. C. Malbon. Nature Lond. 358: 334-337, 1992). For the current work the role of Gi alpha 2, a G protein mediator of inhibitory control of adenylyl cyclase, in regulating terminal differentiation of these cells was explored by stable transfection of fibroblasts expressing wild-type and a constitutively active mutant of Gi alpha 2 (Q205L). Under the influence of the cytomegalovirus promoter, the expression vector yielded a 1.7-fold (Q205L mutant Gi alpha 2) and 2.2-fold (wild-type Gi alpha 2) increase in steady-state levels of these G protein alpha-subunits. Elevation of Gi alpha 2 expression or expression of constitutively active Gi alpha 2 (Q205L) promoted lipid accumulation in these clones, the hallmark of terminal differentiation of 3T3-L1 fibroblasts to adipocytes. Increasing Gi alpha 2 activity promotes adipogenic conversion, as was previously observed by decreasing Gs alpha either by inducers of differentiation or by oligodeoxynucleotides antisense to Gs alpha. Thus Gs alpha and Gi alpha 2 are shown to be counterregulatory with respect to promoting differentiation of 3T3-L1 mouse embryo fibroblasts to adipocytes in the absence of exogenously added inducers of differentiation. This is the first report demonstrating the induction of terminal differentiation of cells by the overexpression of a G protein alpha-subunit, further implicating G proteins as regulators of complex biological responses such as adipogenesis.