•Bexarotene was identified as a hit to convert myoblasts into brown-like adipocytes•Bexarotene induces brown reprogramming via RXR•PRDM16 is required for bexarotene/RXR-induced browning•Oral bexarotene treatment enhances BAT mass and function in miceBrown adipose tissue (BAT) has attracted considerable research interest because of its therapeutic potential to treat obesity and associated metabolic diseases. Augmentation of brown fat mass and/or its function may represent an attractive strategy to enhance energy expenditure. Using high-throughput phenotypic screening to induce brown adipocyte reprogramming in committed myoblasts, we identified a retinoid X receptor (RXR) agonist, bexarotene (Bex), that efficiently converted myoblasts into brown adipocyte-like cells. Bex-treated mice exhibited enlarged BAT mass, enhanced BAT function, and a modest browning effect in subcutaneous white adipose tissue (WAT). Expression analysis showed that Bex initiated several “browning” pathways at an early stage during brown adipocyte reprogramming. Our findings suggest RXRs as new master regulators that control brown and beige fat development and activation, unlike the common adipogenic regulator PPARγ. Moreover, we demonstrated that selective RXR activation may potentially offer a therapeutic approach to manipulate brown/beige fat function in vivo.Download high-res image (244KB)Download full-size image

In vitro cell models are invaluable tools for studying diseases and discovering drugs. Human induced pluripotent stem cells, particularly derived from patients, are an advantageous resource for generating ample supplies of cells to create unique platforms that model disease. This manuscript will review recent developments in modeling a variety of diseases (including their cellular phenotypes) with induced pluripotent stem cells derived from patients. It will also describe how researchers have exploited these models to validate drugs as potential therapeutics for these devastating diseases.

We previously developed a novel paradigm of cell activation and signaling-directed (CASD) lineage conversion for direct reprogramming of fibroblasts into cardiac, neural and endothelial precursor cells. This method is based on the transient overexpression of iPSC factors (cell activation, CA) in conjunction with lineage-specific soluble signals (signaling directed, SD). Such a strategy was used to generate human induced neural stem cells (hiNSCs) with 4 to 6 pluripotency factors, including OCT4, SOX2, KLF4 and microRNAs, from cells in human urine1 and fibroblasts2. Using a different strategy, SOX2 was found to induce the generation of hiNSCs, although only from fetal fibroblasts with low efficiency and by a tedious process3.Our long-term goal is to develop simpler and safer reprogramming methods for cell-based applications and, ultimately, to apply this reprogramming strategy pharmacologically in vivo for tissue regeneration. Thus, we are developing a strategy to identify and combine small molecules to replace genetic factors. To this end, we report here a proof-of-concept study that a cocktail of only small molecules could replace 3 of the 4 reprogramming factors under the CASD lineage-reprogramming paradigm to enable OCT4-only iNSC reprogramming of human neonatal and adult fibroblasts.First, we introduced OCT4 and SOX2 (OS) or OCT4 alone into human neonatal fibroblasts (CRL-2097) that lacked neural or pluripotency marker expression (Supplementary information, Figure S1). After 4-5 weeks under reprogramming conditions containing A83-01 (a TGFβ inhibitor) and CHIR99021 (a GSK3β inhibitor), which were similar to human primitive NSC (hNSC) cultures4 (Figure 1A and Supplementary information, Data S1), CRL-2097 transduced with either OS or OCT4 generated colonies (average 10-16 and 1-2 colonies from 6 × 104 CRL-2097 transduced with OS and OCT4, respectively) that were morphologically distinct from background cells and homogeneously expressed hNSC marker PAX6 (Figure 1B and 1C). However, adult dermal fibroblasts (AHDF) transduced with OCT4 alone failed to generate hNSC colonies under the same condition. Through chemical screenings under basal conditions containing A83-01, CHIR99021 and sodium butyrate (NaB, an HDAC inhibitor)5, we found that a combination of lysophosphatidic acid (LPA, a phospolipid derivative), rolipram (a PDE4 inhibitor), and SP600125 (a JNK inhibitor) facilitated the reprogramming of AHDF transduced with OCT4 alone. Thereafter, we formulated a chemical cocktail, containing 0.5 μM A83-01, 3 μM CHIR99021, 0.2 mM NaB, 2 μM LPA, 2 μM rolipram, and 2 μM SP600125, which combined with the ectopic expression of OCT4 could convert AHDF into hiNSC colonies that homogeneously expressed PAX6 (average 6 colonies from 2 × 105AHDF) (Figure 1D and 1E). Interestingly, ectopic expression of SOX2 alone under these conditions failed to generate hiNSC colonies (Figure 1E). After isolation and expansion, the reprogrammed hiNSC colonies continued to homogeneously express PAX6, PLZF and OTX2, supporting their hNSC identity4 (Figure 1C and 1F). We designated these reprogrammed cells as ONE (OCT4 only-induced neuro-epithelium). These hiNSCs expressed the proliferative marker Ki67 and showed growth rate comparable to human embryonic stem cell-derived NSCs (control hNSCs) (Supplementary information, Figure S2). We expanded and maintained these hiNSCs stably for more than 5 months. Additionally, we established hiNSC lines by using an episomal system expressing OCT4, SOX2, KLF4, and p53 shRNA6 in combination with the chemical cocktail from both neonatal (average 20-25 colonies from 4 × 105 CRL-2097) and adult fibroblasts (average 8-10 colonies from 4 × 105 AHDF) around 4-5 weeks after electroporation (Supplementary information, Figure S3), confirming that our chemical cocktail efficiently facilitates hiNSC reprogramming.Flow cytometry analysis highlighted the close similarity of hiNSCs to control hNSCs (Supplementary information, Figure S4A). Quantitative RT-PCR revealed that hiNSCs expressed PAX6, NESTIN and SOX1 at levels comparable to control hNSCs4 (Supplementary information, Figure S4B). Exogenous OCT4 was silenced and endogenous OCT4 expression was not observed in most established hiNSC lines (Supplementary information, Figure S5A and S5B). Notably, vector integration was not apparent in these episomal vector-driven hiNSCs (Supplementary information, Figure S6). The global gene-expression profile of hiNSCs closely resembled that of control hNSCs (Pearson correlation value: 0.96) and distinctly diverged from human fibroblasts (Pearson correlation value: 0.76) (Figure 1G). Collectively, these results suggest that our hiNSCs are comparable to control hNSCs.When we examined gene expression changes during hiNSC reprogramming, we found that PAX6 expression became upregulated from day 7 (Supplementary information, Figure S7A). Most importantly, endogenous OCT4 (Supplementary information, Figure S7B) and the pluripotency marker TRA-1-60 (Supplementary information, Figure S7C) were undetectable during the entire process. At epigenetic level, the PAX6 and SOX1 promoters had repressive H3K27me3 marks in the starting fibroblasts (Supplementary information, Figure S8). However, by day 24, the H3K27me3 marks were considerably reduced at these loci, which then showed active H3K4me3 marks similar to control hNSCs. In contrast, the OCT4 promoter was persistently marked by H3K27me3 throughout the conversion process (Supplementary information, Figure S8), suggesting that the epigenetic status of directly reprogrammed hiNSCs are comparable to that of control hNSCs. Therefore, these results indicate a rapid direct conversion to hiNSCs in our reprogramming paradigm.After 4 weeks of spontaneous or directed neural differentiation (Figure 1H and Supplementary information, Figure S9), these hiNSCs developed into cells expressing the early neuronal marker doublecortin (DCX) (Supplementary information, Figure S9), and subsequently the mature neuronal markers neuronal nuclear antigen (NeuN) and microtubule-associated protein-2 (MAP2) (Figure 1H). These hiNSCs could also develop into glutamatergic, GABAergic and dopaminergic neurons (Figure 1H), as well as peripheral neurons. We observed the synaptic protein Synapsin1 expression in these differentiated neurons (Figure 1H and Supplementary information, Figure S9). Moreover, hiNSCs could be differentiated into astrocytes and oligodendrocytes (Figure 1H). These results indicate that these hiNSCs are multipotent.To evaluate the functional properties of the hiNSC-derived neurons, we performed patch-clamp electrophysiological recordings. Under voltage clamp, the majority of 6-week-differentiated cells (n = 8/10) displayed fast sodium currents (Figure 1Ii), and under current clamp, we recorded spontaneous and evoked action potentials (Figure 1Iii and Iiii). Whole-cell recordings revealed a subset of cells (3/10) displaying N-methyl-D-aspartate (NMDA)-type glutamate receptor currents after NMDA application (Figure Iiv), and a majority (17/29) exhibited an increase in intracellular calcium in response to NMDA, which was sensitive to the specific NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (Supplementary information, Figure S10). We also observed spontaneous excitatory post-synaptic currents in these cultures (Figure 1Iv), indicating the formation of functional synapses. These data suggest that hiNSCs can be differentiated into functional neurons.Finally, we transplanted EGFP-labeled hiNSCs (~1 × 105) into the lateral ventricle of neonatal mice. Two weeks after transplantation, we found that most EGFP-expressing hiNSCs migrated and integrated into several areas of the mouse brain, such as the lateral periventricular region, subventricular zone and subcallosal zone (Supplementary information, Figure S11A). A few transplanted cells also migrated into the nearby cerebral cortex and olfactory bulb (Supplementary information, Figure S11B). After 4 weeks, the transplanted hiNSCs formed cell clusters expressing neuronal lineage markers, such as Tuj1, DCX and NeuN (Figure 1J), and a subset of cell clusters contained GFAP-expressing astrocytes (Figure 1J). Importantly, these cell clusters were not labeled by a 2-h pulse of bromodeoxyuridine, and we could not find any sign of tumor formation (data not shown). These results show that the transplanted hiNSCs engraft well in neonatal mouse brains and retain their potential to give rise to neurons and glias in vivo.In summary, we developed a novel chemical cocktail that enables the generation of expandable hiNSCs from human fibroblasts transduced with OCT4 alone. We found that SOX2 overexpression combined with the chemical cocktail treatment3 was not sufficient to reprogram adult fibroblasts, suggesting SOX2-mediated hiNSC reprogramming may follow a different reprogramming trajectory from our OCT4-mediated hiNSC reprogramming. These results further highlight the unique ability of the OCT4/CASD strategy and chemical cocktail in hiNSC reprogramming when considering the difficulty of reprogramming adult fibroblasts7. In the OCT4/CASD reprogramming paradigm8, environmental cues were found to be critical for committing cell fates1,9,10. Thus, the novel chemical cocktail we developed in this study can facilitate future investigations into the mechanistic basis of CASD reprogramming. Finally, we also anticipate that discovery of more small molecules and fine-tuning their combinations following the logic and strategy described here may increase the efficiency of hiNSC reprogramming and kinetics of this transition, and ultimately enable hiNSC reprogramming with only small molecules.JK, H-SK and YSC were supported by the MEST/Stem Cell Research Program [2010-0020272], Basic Science Research Program (2012R1A1A2043433), KRCF National Agenda Program, and KRIBB Research Initiative Program. SZ was supported by Gladstone CIRM Scholars Program. RA, MT, JDZ, SS-B, EM, SRM, and SAL were supported by P01 ES016738, P01 HD29587, and P30 NS076411. WS and HJK were supported by Basic Science Research Program (2012M3A9C6049933) through Korea NRF. TL, YZ and SD were supported by funding from California Institute for Regenerative Medicine, NICHD, NHLBI, NEI, NIMH/NIH, Prostate Cancer Foundation, and the Gladstone Institute. We thank Jianwei Che at Genomics Institute of the Novartis Research Foundation for analyzing the microarray data and Gary Howard at the Gladstone Institute for editing this manuscript.(Supplementary information is linked to the online version of the paper on the Cell Research website.)

Regenerative medicine for heart failure seeks to replace lost cardiomyocytes. Chemical approaches for producing ample supplies of cells, such as pluripotent stem cells and cardiomyocytes, hold promise as practical means to achieve safe, facile cell-based therapy for cardiac repair and regenerative medicine. In this review, we describe recent advances in the application of small molecules to improve the generation and maintenance of pluripotent stem cells. We also describe new directions in heart repair and regeneration in which chemical approaches may find their application.

Understanding the mechanisms by which compounds discovered using cell-based phenotypic screening strategies might exert their effects would be highly augmented by new approaches exploring their potential interactions with the genome. For example, altered androgen receptor (AR) transcriptional programs, including castration resistance and subsequent chromosomal translocations, play key roles in prostate cancer pathological progression, making the quest for identification of new therapeutic agents and an understanding of their actions a continued priority. Here we report an approach that has permitted us to uncover the sites and mechanisms of action of a drug, referred to as “SD70,” initially identified by phenotypic screening for inhibitors of ligand and genotoxic stress-induced translocations in prostate cancer cells. Based on synthesis of a derivatized form of SD70 that permits its application for a ChIP-sequencing–like approach, referred to as “Chem-seq,” we were next able to efficiently map the genome-wide binding locations of this small molecule, revealing that it largely colocalized with AR on regulatory enhancers. Based on these observations, we performed the appropriate global analyses to ascertain that SD70 inhibits the androgen-dependent AR program, and prostate cancer cell growth, acting, at least in part, by functionally inhibiting the Jumonji domain-containing demethylase, KDM4C. Global location of candidate drugs represents a powerful strategy for new drug development by mapping genome-wide location of small molecules, a powerful adjunct to contemporary drug development strategies.

Stem cells, including both pluripotent stem cells and multipotent somatic stem cells, hold great potential for interrogating the mechanisms of tissue development, homeostasis and pathology, and for treating numerous devastating diseases. Establishment of in vitro platforms to faithfully maintain and precisely manipulate stem cell fates is essential to understand the basic mechanisms of stem cell biology, and to translate stem cells into regenerative medicine. Chemical approaches have recently provided a number of small molecules that can be used to control cell self-renewal, lineage differentiation, reprogramming and regeneration. These chemical modulators have been proven to be versatile tools for probing stem cell biology and manipulating cell fates toward desired outcomes. Ultimately, this strategy is promising to be a new frontier for drug development aimed at endogenous stem cell modulation.

The advent of powerful genomics technologies has uncovered many fundamental aspects of biology, including the mechanisms of cancer; however, it has not been appropriately matched by the development of global approaches to discover new medicines against human diseases. Here we describe a unique high-throughput screening strategy by high-throughput sequencing, referred to as HTS2, to meet this challenge. This technology enables large-scale and quantitative analysis of gene matrices associated with specific disease phenotypes, therefore allowing screening for small molecules that can specifically intervene with disease-linked gene-expression events. By initially applying this multitarget strategy to the pressing problem of hormone-refractory prostate cancer, which tends to be accelerated by the current antiandrogen therapy, we identify Peruvoside, a cardiac glycoside, which can potently inhibit both androgen-sensitive and -resistant prostate cancer cells without triggering severe cytotoxicity. We further show that, despite transcriptional reprogramming in prostate cancer cells at different disease stages, the compound can effectively block androgen receptor-dependent gene expression by inducing rapid androgen receptor degradation via the proteasome pathway. These findings establish a genomics-based phenotypic screening approach capable of quickly connecting pathways of phenotypic response to the molecular mechanism of drug action, thus offering a unique pathway-centric strategy for drug discovery.

Human embryonic stem cells (hESCs) hold enormous promise for regenerative medicine. Typically, hESC-based applications would require their in vitro differentiation into a desirable homogenous cell population. A major challenge of the current hESC differentiation paradigm is the inability to effectively capture and, in the long-term, stably expand primitive lineage-specific stem/precursor cells that retain broad differentiation potential and, more importantly, developmental stage-specific differentiation propensity. Here, we report synergistic inhibition of glycogen synthase kinase 3 (GSK3), transforming growth factor β (TGF-β), and Notch signaling pathways by small molecules can efficiently convert monolayer cultured hESCs into homogenous primitive neuroepithelium within 1 wk under chemically defined condition. These primitive neuroepithelia can stably self-renew in the presence of leukemia inhibitory factor, GSK3 inhibitor (CHIR99021), and TGF-β receptor inhibitor (SB431542); retain high neurogenic potential and responsiveness to instructive neural patterning cues toward midbrain and hindbrain neuronal subtypes; and exhibit in vivo integration. Our work uniformly captures and maintains primitive neural stem cells from hESCs.

The simple yet powerful technique of induced pluripotency may eventually supply a wide range of differentiated cells for cell therapy and drug development. However, making the appropriate cells via induced pluripotent stem cells (iPSCs) requires reprogramming of somatic cells and subsequent redifferentiation. Given how arduous and lengthy this process can be, we sought to determine whether it might be possible to convert somatic cells into lineage-specific stem/progenitor cells of another germ layer in one step, bypassing the intermediate pluripotent stage. Here we show that transient induction of the four reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) can efficiently transdifferentiate fibroblasts into functional neural stem/progenitor cells (NPCs) with appropriate signaling inputs. Compared with induced neurons (or iN cells, which are directly converted from fibroblasts), transdifferentiated NPCs have the distinct advantage of being expandable in vitro and retaining the ability to give rise to multiple neuronal subtypes and glial cells. Our results provide a unique paradigm for iPSC-factor–based reprogramming by demonstrating that it can be readily modified to serve as a general platform for transdifferentiation.

Recently we have witnessed an array of studies on direct reprogramming that describe induced inter conversion of mature cell types from higher organisms including human. While these studies reveal an unexpected level of plasticity of differentiated somatic cells, they also provide unprecedented opportunities to develop regenerative therapies for many debilitating disorders and model these ‘diseases-in-a-dish’ for studying their pathophysiology. Here we review the current state of the art in direct lineage reprogramming to neural cells, and discuss the challenges that need to be addressed toward achieving the full potential of this exciting new technology.Highlights► Somatic cell-specific or pluripotent cell-specific factors can directly reprogram fibroblasts to neural cells. ► The hiNs and hiNSCs may offer a faster and robust alternative to iPSCs for generating patient-specific neural cells. ► We review here the state-of-the art and the challenges that remain in this exciting field.

The discovery that somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by the expression of a few transcription factors has attracted enormous interest in biomedical research and the field of regenerative medicine. iPSCs nearly identically resemble embryonic stem cells (ESCs) and can give rise to all cell types in the body, and thus have opened new opportunities for personalized regenerative medicine and new ways of modeling human diseases. Although some studies have raised concerns about genomic stability and epigenetic memory in the resulting cells, better understanding and control of the reprogramming process should enable enhanced efficiency and higher fidelity in reprogramming. Therefore, small molecules regulating reprogramming mechanisms are valuable tools to probe the process of reprogramming and harness cell fate transitions for various applications.