Jeong Tae Do

Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule compounds.

Somatic cells can be induced into pluripotent stem cells (iPSCs) with a combination of four transcription factors, Oct4/Sox2/Klf4/c-Myc or Oct4/Sox2/Nanog/LIN28. This provides an enabling platform to obtain patient-specific cells for various therapeutic and research applications. However, several problems remain for this approach to be therapeutically relevant due to drawbacks associated with efficiency and viral genome integration. Recently, it was shown that neural progenitor cells (NPCs) transduced with Oct4/Klf4 can be reprogrammed into iPSCs. However, NPCs express Sox2 endogenously, possibly facilitating reprogramming in the absence of exogenous Sox2. In this study, we identified a small-molecule combination, BIX-01294 and BayK8644, that enables reprogramming of Oct4/Klf4-transduced mouse embryonic fibroblasts, which do not endogenously express the factors essential for reprogramming. This study demonstrates that small molecules identified through a phenotypic screen can compensate for viral transduction of critical factors, such as Sox2, and improve reprogramming efficiency.

Self-renewal of embryonic stem cells by a small molecule

A cell-based screen of chemical libraries was carried out to identify small molecules that control the self-renewal of ES cells. A previously uncharacterized heterocycle, SC1, was discovered that allows one to propagate murine ES cells in an undifferentiated, pluripotent state under chemically defined conditions in the absence of feeder cells, serum, and leukemia inhibitory factor. Long-term SC1-expanded murine ES cells can be differentiated into cells of the three primary germ layers in vitro and also can generate chimeric mice and contribute to the germ line in vivo. Biochemical and cellular experiments suggest that SC1 works through dual inhibition of RasGAP and ERK1. Molecules of this kind may not only facilitate practical applications of stem cells in research and therapy, but also provide previously undescribed insights into the complex biology of stem cells.

Nuclei of Embryonic Stem Cells Reprogram Somatic Cells

The restricted potential of a differentiated cell can be reverted back to a pluripotent state by cell fusion; totipotency can even be regained after somatic cell nuclear transfer. To identify factors involved in resetting the genetic program of a differentiated cell, we fused embryonic stem cells (ESCs) with neurosphere cells (NSCs). The fusion activated Oct4, a gene essential for pluripotency, in NSCs. To further identify whether cytoplasmic or nuclear factors are responsible for its reactivation, we fused either karyoplasts or cytoplasts of ESCs with NSCs. Our results show that ESC karyoplasts could induce Oct4 expression in the somatic genome, but cytoplasts lacked this ability. In addition, mitomycin C–treated ESCs, although incapable of DNA replication and cell division, could reprogram 5‐azacytidine–treated NSCs. We therefore conclude that the Oct4 reprogramming capacity resides in the ESC karyoplast and that gene reactivation is independent of DNA replication and cell division.

Conversion of mouse epiblast stem cells to an earlier pluripotency state by small molecules.

Epiblast stem cells (EpiSCs) are pluripotent cells derived from post-implantation late epiblasts in vitro. EpiSCs are incapable of contributing to chimerism, indicating that EpiSCs are less pluripotent and represent a later developmental pluripotency state compared with inner cell mass stage murine embryonic stem cells (mESCs). Using a chemical approach, we found that blockage of the TGFβ pathway or inhibition of histone demethylase LSD1 with small molecule inhibitors induced dramatic morphological changes in EpiSCs toward mESC phenotypes with simultaneous activation of inner cell mass-specific gene expression. However, full conversion of EpiSCs to the mESC-like state with chimerism competence could be readily generated only with the combination of LSD1, ALK5, MEK, FGFR, and GSK3 inhibitors. Our results demonstrate that appropriate synergy of epigenetic and signaling modulations could convert cells at the later developmental pluripotency state to the earlier mESC-like pluripotency state, providing new insights into pluripotency regulation.

Neuronal Properties, In Vivo Effects, and Pathology of a Huntington's Disease Patient‐Derived Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) generated from somatic cells of patients can be used to model different human diseases. They may also serve as sources of transplantable cells that can be used in novel cell therapies. Here, we analyzed neuronal properties of an iPSC line derived from a patient with a juvenile form of Huntingtons disease (HD) carrying 72 CAG repeats (HD‐iPSC). Although its initial neural inducing activity was lower than that of human embryonic stem cells, we found that HD‐iPSC can give rise to GABAergic striatal neurons, the neuronal cell type that is most susceptible to degeneration in HD. We then transplanted HD‐iPSC‐derived neural precursors into a rat model of HD with a unilateral excitotoxic striatal lesion and observed a significant behavioral recovery in the grafted rats. Interestingly, during our in vitro culture and when the grafts were examined at 12 weeks after transplantation, no aggregate formation was detected. However, when the culture was treated with a proteasome inhibitor (MG132) or when the cells engrafted into neonatal brains were analyzed at 33 weeks, there were clear signs of HD pathology. Taken together, these results indicate that, although HD‐iPSC carrying 72 CAG repeats can form GABAergic neurons and give rise to functional effects in vivo, without showing an overt HD phenotype, it is highly susceptible to proteasome inhibition and develops HD pathology at later stages of transplantation. These unique features of HD‐iPSC will serve as useful tools to study HD pathology and develop novel therapeutics. Stem Cells2012;30:2054–2062

Pluripotential Reprogramming of the Somatic Genome in Hybrid Cells Occurs with the First Cell Cycle

The fusion of pluripotent embryonic cells with somatic cells results in reprogramming of the somatic cell genome. Oct4‐green fluorescent protein (GFP) transgenes that do not contain the proximal enhancer (PE) region are widely used to visualize reprogramming of the somatic to the pluripotent cell state. The temporal onset of Oct4‐GFP activation has been found to occur 40–48 hours postfusion. We asked whether activation of the transgene actually reflects activation of the endogenous Oct4 gene. In the current study, we show that activation of an Oct4‐GFP transgene that contains the PE region occurs within 22 hours of fusion. In addition, demethylation of the Oct4‐GFP transgene and that of the endogenous Oct4 and Nanog genes was found to occur within 24 hours of fusion. As this timing corresponds with the timing of cell cycle completion in embryonic stem cells and fusion hybrids (∼22 hours), we postulate that pluripotential reprogramming of the somatic cell genome begins during the first cell cycle after the fusion of a somatic cell with a pluripotent cell and has been completed by day 2 postfusion.

Regulatory circuits underlying pluripotency and reprogramming

The ability of pluripotent stem cells to differentiate into all cell types of an organism has received widespread attention in basic and clinical research and holds tremendous potential for pharmacologic and medical applications. In this review, we provide an overview of the factors and pathways involved in pluripotency and discuss a possible mechanism underlying genetic reprogramming using defined transcription factors. We specifically address the association between core transcription factors (e.g. Oct4, Sox2 and Nanog) and the cellular machinery (e.g. chromatin remodeling complex, DNA methylation, microRNA and X chromosome inactivation), which has an important role in cell fate determination.

Quantitative proteomic analysis of induced pluripotent stem cells derived from a human Huntington's disease patient.

HD (Huntingtons disease) is a devastating neurodegenerative genetic disorder caused by abnormal expansion of CAG repeats in the HTT (huntingtin) gene. We have recently established two iPSC (induced pluripotent stem cell) lines derived from a HD patient carrying 72 CAG repeats (HD-iPSC). In order to understand the proteomic profiles of HD-iPSCs, we have performed comparative proteomic analysis among normal hESCs (human embryonic stem cells; H9), iPSCs (551-8) and HD-iPSCs at undifferentiated stages, and identified 26 up- and down-regulated proteins. Interestingly, these differentially expressed proteins are known to be involved in different biological processes, such as oxidative stress, programmed cell death and cellular oxygen-associated proteins. Among them, we found that oxidative stress-related proteins, such as SOD1 (superoxide dismutase 1) and Prx (peroxiredoxin) families are particularly affected in HD-iPSCs, implying that HD-iPSCs are highly susceptible to oxidative stress. We also found that BTF3 (basic transcription factor 3) is up-regulated in HD-iPSCs, which leads to the induction of ATM (ataxia telangiectasia mutated), followed by activation of the p53-mediated apoptotic pathway. In addition, we observed that the expression of cytoskeleton-associated proteins was significantly reduced in HD-iPSCs, implying that neuronal differentiation was also affected. Taken together, these results demonstrate that HD-iPSCs can provide a unique cellular disease model system to understand the pathogenesis and neurodegeneration mechanisms in HD, and the identified proteins from the present study may serve as potential targets for developing future HD therapeutics.

Reprogramming somatic gene activity by fusion with pluripotent cells.

Fertilized eggs and early blastomeres, that have the potential to develop to fetuses when placed into a uterus, are totipotent. Those cells in the embryo, that can give rise to all cell types of an organism, but not to an organism itself, are pluripotent. Embryonic stem (ES), embryonic carcinoma (EC), and embryonic germ (EG) cells are powerful in vitro artifacts derived from different embryonic stages and are pluripotent. Totipotent and pluripotent cells have the potential to greatly benefit biological research and medicine. One powerful feature is that the genetic program of somatic cells can be converted into that of totipotent or pluripotent cells, as shown by nuclear transfer or cell fusion experiments. During reprogramming by cell fusion various features of pluripotent cells are acquired. These include the typical morphology of the respective pluripotent fusion partner, a specific epigenetic state, a specific gene profile, inactivation of tissue-specific genes expressed in the somatic fusion partner, and the developmental as well as differentiation potential of pluripotent cells. In this review, we will discuss what is known about the reprogramming process mediated by cell fusion and the potential use of fusion-induced reprogramming for therapeutic applications.

Erasure of Cellular Memory by Fusion with Pluripotent Cells

Pluripotent cells have been suggested as a prime source to reprogram somatic cells. We used F9 EC cells as a pluripotent partner to reprogram neurosphere cells (NSCs) because they exhibit a nonneural differentiation potential in the presence of retinoic acid. F9‐NSC hybrid cells displayed various features of reprogramming, such as reactivation of pluripotency genes, inactivation of tissue‐specific genes, and reactivation of the inactive X chromosome. As the hybrid cells undergo differentiation, the pluripotency markers Oct4 and Nanog were downregulated. Whereas neural marker genes were not upregulated, endodermal and mesodermal markers were, suggesting that NSCs lose memory of their neural origin and preferentially differentiate to the lineages corresponding to the F9 program. After fusion, the methylation status in the Xist region was similar to that of F9 EC cells. However, upon differentiation, the Xist region failed to resume the methylation patterns of differentiated cells, suggesting that the Xist in F9‐NSC hybrids does not easily acquire a differentiated state.