Sulfated disaccharide protects membrane and DNA damages from arginine-rich dipeptide repeats in ALS.

Hexanucleotide repeat expansion in C9ORF72 (C9) is the most prevalent mutation among amyotrophic lateral sclerosis (ALS) patients. The patients carry over ~30 to hundreds or thousands of repeats translated to dipeptide repeats (DPRs) where poly-glycine-arginine (GR) and poly-proline-arginine (PR) are most toxic. The structure-function relationship is still unknown. Here, we examined the minimal neurotoxic repeat number of poly-GR and found that extension of the repeat number led to a loose helical structure disrupting plasma and nuclear membrane. Poly-GR/PR bound to nucleotides and interfered with transcription. We screened and identified a sulfated disaccharide that bound to poly-GR/PR and rescued poly-GR/PR-induced toxicity in neuroblastoma and C9-ALS-iPSC-derived motor neurons. The compound rescued the shortened life span and defective locomotion in poly-GR/PR expressing Drosophila model and improved motor behavior in poly-GR-injected mouse model. Overall, our results reveal structural and toxicity mechanisms for poly-GR/PR and facilitate therapeutic development for C9-ALS.

Using human Pompe disease-induced pluripotent stem cell-derived neural cells to identify compounds with therapeutic potential.

Pompe disease (OMIM # 232300) is a glycogen storage disease caused by autosomal recessive mutations of the gene encoding alpha-1,4-glucosidase (GAA; EC 3.2.1.20). Despite the relatively effective employment of enzyme replacement therapy, some critical medical issues still exist in patients with this disease, including the persistence of abnormalities in the central nervous system (CNS), probably because of the inability of the recombinant GAA to pass through the blood-brain barrier. To address this issue, identification of more therapeutic agents that target the CNS of patients with Pompe disease may be required. In this study, we derived neuronal cells from Pompe disease-induced pluripotent stem cells (Pom-iPSCs) and proved that they are able to recapitulate the hallmark cellular and biochemical phenotypes of Pompe disease. Using the Pom-iPSC-derived neurons as an in vitro drug-testing model, we then identified three compounds, ebselen, wortmannin and PX-866, with therapeutic potential to alleviate Pompe disease-associated pathological phenotypes in the neurons derived from Pom-iPSCs. We confirmed that all three compounds were able to enhance the GAA activity in the Pom-iPSC-derived neurons. Moreover, they were able to enhance the GAA activity in several important internal organs of GAA-deficient mice when co-injected with recombinant human GAA, and we found that intraperitoneal injection of ebselen was able to promote the GAA activity of the GAA-heterozygous mouse brain. Our results prove the usefulness of Pom-iPSC-derived neuronal populations for identifying new compounds with therapeutic potential.

Modeling spinocerebellar ataxias 2 and 3 with iPSCs reveals a role for glutamate in disease pathology.

Spinocerebellar ataxias 2 and 3 (SCA2 and SCA3) are dominantly inherited neurodegenerative diseases caused by expansion of polyglutamine-encoding CAG repeats in the affected genes. The etiology of these disorders is known to involve widespread loss of neuronal cells in the cerebellum, however, the mechanisms that contribute to cell death are still elusive. Here we established SCA2 and SCA3 induced pluripotent stem cells (iPSCs) and demonstrated that SCA-associated pathological features can be recapitulated in SCA-iPSC-derived neurons. Importantly, our results also revealed that glutamate stimulation promotes the development of disease-related phenotypes in SCA-iPSC-derived neurons, including altered composition of glutamatergic receptors, destabilized intracellular calcium, and eventual cell death. Furthermore, anti-glutamate drugs and calcium stabilizer treatment protected the SCA-iPSC-derived neurons and reduced cell death. Collectively, our study demonstrates that the SCA-iPSC-derived neurons can recapitulate SCA-associated pathological features, providing a valuable tool to explore SCA pathogenic mechanisms and screen drugs to identify potential SCA therapeutics.

Subcellular Proteome Landscape of Human Embryonic Stem Cells Revealed Missing Membrane Proteins.

Human embryonic stem cells (hESCs) have the capacity for self-renewal and multilineage differentiation, which are of clinical importance for regeneration medicine. Despite the significant progress of hESC study, the complete hESC proteome atlas, especially the surface protein composition, awaits delineation. According to the latest release of neXtProt database (January 17, 2018; 19 658 PE1, 2, 3, and 4 human proteins), membrane proteins present the major category (1047; 48%) among all 2186 missing proteins (MPs). We conducted a deep subcellular proteomics analysis of hESCs to identify the nuclear, cytoplasmic, and membrane proteins in hESCs and to mine missing membrane proteins in the very early cell status. To our knowledge, our study achieved the largest data set with confident identification of 11 970 unique proteins (1% false discovery rate at peptide, protein, and PSM levels), including the most-comprehensive description of 6 138 annotated membrane proteins in hESCs. Following the HPP guideline, we identified 26 gold (neXtProt PE2, 3, and 4 MPs) and 87 silver (potential MP candidates with a single unique peptide detected) MPs, of which 69 were membrane proteins, and the expression of 21 gold MPs was further verified either by multiple reaction monitoring mass spectrometry or by matching synthetic peptides in the Peptide Atlas database. Functional analysis of the MPs revealed their potential roles in the pluripotency-related pathways and the lineage- and tissue-specific differentiation processes. Our proteome map of hESCs may provide a rich resource not only for the identification of MPs in the human proteome but also for the investigation on self-renewal and differentiation of hESC. All mass spectrometry data were deposited in ProteomeXchange via jPOST with identifier PXD009840.

Trans-spliced long non-coding RNA: an emerging regulator of pluripotency.

With dual capacities for unlimited self-renewal and pluripotent differentiation, pluripotent stem cells (PSCs) give rise to many cell types in our body and PSC culture systems provide an unparalleled opportunity to study early human development and disease. Accumulating evidence indicates that the molecular mechanisms underlying pluripotency maintenance in PSCs involve many factors. Among these regulators, recent studies have shown that long non-coding RNAs (lncRNAs) can affect the pluripotency circuitry by cooperating with master pluripotency-associated factors. Additionally, trans-spliced RNAs, which are generated by combining two or more pre-mRNA transcripts to produce a chimeric RNA, have been identified as regulators of various biological processes, including human pluripotency. In this review, we summarize and discuss current knowledge about the roles of lncRNAs, including trans-spliced lncRNAs, in controlling pluripotency.

Combining membrane proteomics and computational three-way pathway analysis revealed signalling pathways preferentially regulated in human iPSCs and human ESCs.

Owing to the clinical potential of human induced pluripotent stem cells (hiPSCs) in regenerative medicine, a thorough examination of the similarities and differences between hiPSCs and human embryonic stem cells (hESCs) has become indispensable. Moreover, as the important roles of membrane proteins in biological signalling, functional analyses of membrane proteome are therefore promising. In this study, a pathway analysis by the bioinformatics tool GSEA was first performed to identify significant pathways associated with the three comparative membrane proteomics experiments: hiPSCs versus precursor human foreskin fibroblasts (HFF), hESCs versus precursor HFF, and hiPSCs versus hESCs. A following three-way pathway comparison was conducted to identify the differentially regulated pathways that may contribute to the differences between hiPSCs and hESCs. Our results revealed that pathways related to oxidative phosphorylation and focal adhesion may undergo incomplete regulations during the reprogramming process. This hypothesis was supported by another public proteomics dataset to a certain degree. The identified pathways and their core enriched proteins could serve as the starting point to explore the possible ways to make hiPSCs closer to hESCs.

The circular RNA circBIRC6 participates in the molecular circuitry controlling human pluripotency.

Accumulating evidence indicates that circular RNAs (circRNAs) are abundant in the human transcriptome. However, their involvement in biological processes, including pluripotency, remains mostly undescribed. We identified a subset of circRNAs that are enriched in undifferentiated human embryonic stem cells (hESCs) and demonstrated that two, circBIRC6 and circCORO1C, are functionally associated with the pluripotent state. Mechanistically, we found that circBIRC6 is enriched in the AGO2 complex and directly interacts with microRNAs, miR-34a, and miR-145, which are known to modulate target genes that maintain pluripotency. Correspondingly, circBIRC6 attenuates the downregulation of these target genes and suppresses hESC differentiation. We further identified hESC-enriched splicing factors (SFs) and demonstrated that circBIRC6 biogenesis in hESCs is promoted by the SF ESRP1, whose expression is controlled by the core pluripotency-associated factors, OCT4 and NANOG. Collectively, our data suggest that circRNA serves as a microRNA "sponge" to regulate the molecular circuitry, which modulates human pluripotency and differentiation.

Delayed epidural transplantation of human induced pluripotent stem cell-derived neural progenitors enhances functional recovery after stroke.

Induced pluripotent stem cell-derived neural progenitor cells (iPSC-NPCs) are a promising source of tailor-made cell therapy for neurological diseases. However, major obstacles to clinical use still exist. To circumvent complications related to intracerebral administration, we implanted human iPSC-NPCs epidurally over the peri-infarct cortex 7 days after permanent middle cerebral artery occlusion in adult rats. Compared to controls, cell-treated rats showed significant improvements in paretic forelimb usage and grip strength from 10 days post-transplantation (dpt) onwards, as well as reductions in lesion volumes, inflammatory infiltration and astrogliosis at 21 dpt. Few iPSC-NPCs migrated into rat peri-infarct cortices and exhibited poor survival in tissue. To examine the paracrine therapeutic mechanisms of epidural iPSC-NPC grafts, we used transmembrane co-cultures of human iPSC-NPCs with rat cortical cells subjected to oxygen-glucose deprivation. Compared to other human stem cells, iPSC-NPCs were superior at promoting neuronal survival and outgrowth, and mitigating astrogliosis. Using comparative whole-genome microarrays and cytokine neutralization, we identified a neurorestorative secretome from iPSC-NPCs, and neutralizing enriched cytokines abolished neuroprotective effects in co-cultures. This proof-of-concept study demonstrates a relatively safe, yet effective epidural route for delivering human iPSC-NPCs, which acts predominately through discrete paracrine effects to promote functional recovery after stroke.

Direct Conversion of Human Fibroblasts into Neural Progenitors Using Transcription Factors Enriched in Human ESC-Derived Neural Progenitors.

Early human embryonic stem cell (hESC)-derived neural populations consist of various embryonic neural progenitors (ENPs) with broad neural developmental propensity. Here, we sought to directly convert human somatic cells into ENP-like phenotypes using hESC-ENP-enriched neural transcription factors (TFs). We demonstrated that induced ENP could be efficiently converted from human fibroblasts using two TF combinations. The iENPs exhibit cellular and molecular characteristics resembling hESC-ENPs and can give rise to astrocytes, oligodendrocytes, and functional neuronal subtypes of the central and peripheral nervous system. Nevertheless, our analyses further revealed that these two iENP populations differ in terms of their proliferation ability and neuronal propensity. Finally, we demonstrated that the iENPs can be induced from fibroblasts from patients with Huntington's disease and Alzheimer's disease, and the diseased iENPs and their neuronal derivatives recapitulated the hallmark pathological features of the diseases. Collectively, our results point toward a promising strategy for generating iENPs from somatic cells for disease modeling and future clinical intervention.

Elucidating the role of the A2A adenosine receptor in neurodegeneration using neurons derived from Huntington's disease iPSCs.

Huntington's disease (HD) is an autosomal-dominant degenerative disease caused by a cytosine-adenine-guanine trinucleotide expansion in the Huntingtin (htt) gene. The most vulnerable brain areas to mutant HTT-evoked toxicity are the striatum and cortex. In spite of the extensive efforts that have been devoted to the characterization of HD pathogenesis, no disease-modifying therapy for HD is currently available. The A2A adenosine receptor (A2AR) is widely distributed in the brain, with the highest level observed in the striatum. We previously reported that stimulation of the A2AR triggers an anti-apoptotic effect in a rat neuron-like cell line (PC12). Using a transgenic mouse model (R6/2) of HD, we demonstrated that A2AR-selective agonists effectively ameliorate several major symptoms of HD. In the present study, we show that human iPSCs can be successfully induced to differentiate into DARPP32-positive, GABAergic neurons which express the A2AR in a similar manner to striatal medium spiny neurons. When compared with those derived from control subjects (CON-iPSCs), these HD-iPSC-derived neurons exhibited a higher DNA damage response, based on the observed expression of γH2AX and elevated oxidative stress. This is a critical observation, because oxidative damage and abnormal DNA damage/repair have been reported in HD patients. Most importantly, stimulation of the A2AR using selective agonists reduced DNA damage and oxidative stress-induced apoptosis in HD-iPSC-derived neurons through a cAMP/PKA-dependent pathway. These findings support our hypothesis that human neurons derived from diseased iPSCs might serve as an important platform to investigate the beneficial effects and underlying mechanisms of A2AR drugs.

Granulosa cell-derived induced pluripotent stem cells exhibit pro-trophoblastic differentiation potential.

INTRODUCTION: Human induced pluripotent stem cells (hiPSCs) have been derived from various somatic cell types. Granulosa cells, a group of cells which surround oocytes and are obtained from the (normally discarded) retrieved egg follicles of women undergoing infertility treatment, are a possible cell source for induced pluripotent stem cell (iPSC) generation. Here, we explored the possibility of using human granulosa cells as a donor cell type for iPSC reprogramming, and compared granulosa cell-derived iPSCs (iGRAs) with those derived from other cell sources, to determine the potential ability of iGRA differentiation. METHODS: Granulosa cells were collected from egg follicles retrieved from women undergoing infertility treatment. After short-term culture, the granulosa cells derived from different patients were mixed in culture, and infected with retroviruses encoding reprogramming factors. The resulting iPSC clones were selected and subjected to microsatellite DNA analysis to determine their parental origin. IGRAs were subjected to RT-PCR, immunofluorescence staining, and in vitro and in vivo differentiation assays to further establish their pluripotent characteristics. RESULTS: Microsatellite DNA analysis was used to demonstrate that hiPSCs with different parental origins can be simultaneously reprogrammed by retroviral transfection of a mixed human granulosa cell population obtained from multiple individuals. The iGRAs resemble human embryonic stem cells (hESCs) in many respects, including morphological traits, growth requirements, gene and marker expression profiles, and in vitro and in vivo developmental propensities. We also demonstrate that the iGRAs express low levels of NLRP2, and differentiating iGRAs possess a biased differentiation potential toward the trophoblastic lineage. Although NLRP2 knockdown in hESCs promotes trophoblastic differentiation of differentiating hESCs, it does not result in exit from pluripotency. These results imply that NLRP2 may play a role in regulating the trophoblastic differentiation of human pluripotent stem cells. CONCLUSIONS: These findings provide a means of generating iPSCs from multiple granulosa cell populations with different parental origins. The ability to generate iPSCs from granulosa cells not only enables modeling of infertility-associated disease, but also provides a means of identifying potential clinical interventions through iPSC-based drug screening.

Ectopic DNMT3L triggers assembly of a repressive complex for retroviral silencing in somatic cells.

UNLABELLED: Mammalian genomes are replete with retrotransposable elements, including endogenous retroviruses. DNA methyltransferase 3-like (DNMT3L) is an epigenetic regulator expressed in prospermatogonia, growing oocytes, and embryonic stem (ES) cells. Here, we demonstrate that DNMT3L enhances the interaction of repressive epigenetic modifiers, including histone deacetylase 1 (HDAC1), SET domain, bifurcated 1 (SETDB1), DNA methyltransferase 3A (DNMT3A), and tripartite motif-containing protein 28 (TRIM28; also known as TIF1ß and KAP1) in ES cells and orchestrates retroviral silencing activity with TRIM28 through mechanisms including, but not limited to, de novo DNA methylation. Ectopic expression of DNMT3L in somatic cells causes methylation-independent retroviral silencing activity by recruitment of the TRIM28/HDAC1/SETDB1/DNMT3A/DNMT3L complex to newly integrated Moloney murine leukemia virus (Mo-MuLV) proviral DNA. Concurrent with this recruitment, we also observed the accumulation of histone H3 lysine 9 trimethylation (H3K9me3) and heterochromatin protein 1 gamma (HP1γ), as well as reduced H3K9 and H3K27 acetylation at Mo-MuLV proviral sequences. Ectopic expression of DNMT3L in late-passage mouse embryonic fibroblasts (MEFs) recruited cytoplasmically localized HDAC1 to the nucleus. The formation of this epigenetic modifying complex requires interaction of DNMT3L with DNMT3A as well as with histone H3. In fetal testes at embryonic day 17.5, endogenous DNMT3L also enhanced the binding among TRIM28, DNMT3A, SETDB1, and HDAC1. We propose that DNMT3L may be involved in initiating a cascade of repressive epigenetic modifications by assisting in the preparation of a chromatin context that further attracts DNMT3A-DNMT3L binding and installs longer-term DNA methylation marks at newly integrated retroviruses. IMPORTANCE: Almost half of the mammalian genome is composed of endogenous retroviruses and other retrotransposable elements that threaten genomic integrity. These elements are usually subject to epigenetic silencing. We discovered that two epigenetic regulators that lack enzymatic activity, DNA methyltransferase 3-like (DNMT3L) and tripartite motif-containing protein 28 (TRIM28), collaborate with each other to impose retroviral silencing. In addition to modulating de novo DNA methylation, we found that by interacting with TRIM28, DNMT3L can attract various enzymes to form a DNMT3L-induced repressive complex to remove active marks and add repressive marks to histone proteins. Collectively, these results reveal a novel and pivotal function of DNMT3L in shaping the chromatin modifications necessary for retroviral and retrotransposon silencing.

Suppression of the SOX2 neural effector gene by PRDM1 promotes human germ cell fate in embryonic stem cells.

The mechanisms of transcriptional regulation underlying human primordial germ cell (PGC) differentiation are largely unknown. The transcriptional repressor Prdm1/Blimp-1 is known to play a critical role in controlling germ cell specification in mice. Here, we show that PRDM1 is expressed in developing human gonads and contributes to the determination of germline versus neural fate in early development. We show that knockdown of PRDM1 in human embryonic stem cells (hESCs) impairs germline potential and upregulates neural genes. Conversely, ectopic expression of PRDM1 in hESCs promotes the generation of cells that exhibit phenotypic and transcriptomic features of early PGCs. Furthermore, PRDM1 suppresses transcription of SOX2. Overexpression of SOX2 in hESCs under conditions favoring germline differentiation skews cell fate from the germline to the neural lineage. Collectively, our results demonstrate that PRDM1 serves as a molecular switch to modulate the divergence of neural or germline fates through repression of SOX2 during human development.

Integrative transcriptome sequencing identifies trans-splicing events with important roles in human embryonic stem cell pluripotency.

Trans-splicing is a post-transcriptional event that joins exons from separate pre-mRNAs. Detection of trans-splicing is usually severely hampered by experimental artifacts and genetic rearrangements. Here, we develop a new computational pipeline, TSscan, which integrates different types of high-throughput long-/short-read transcriptome sequencing of different human embryonic stem cell (hESC) lines to effectively minimize false positives while detecting trans-splicing. Combining TSscan screening with multiple experimental validation steps revealed that most chimeric RNA products were platform-dependent experimental artifacts of RNA sequencing. We successfully identified and confirmed four trans-spliced RNAs, including the first reported trans-spliced large intergenic noncoding RNA ("tsRMST"). We showed that these trans-spliced RNAs were all highly expressed in human pluripotent stem cells and differentially expressed during hESC differentiation. Our results further indicated that tsRMST can contribute to pluripotency maintenance of hESCs by suppressing lineage-specific gene expression through the recruitment of NANOG and the PRC2 complex factor, SUZ12. Taken together, our findings provide important insights into the role of trans-splicing in pluripotency maintenance of hESCs and help to facilitate future studies into trans-splicing, opening up this important but understudied class of post-transcriptional events for comprehensive characterization.

LHX2 regulates the neural differentiation of human embryonic stem cells via transcriptional modulation of PAX6 and CER1.

The LIM homeobox 2 transcription factor Lhx2 is known to control crucial aspects of neural development in various species. However, its function in human neural development is still elusive. Here, we demonstrate that LHX2 plays a critical role in human neural differentiation, using human embryonic stem cells (hESCs) as a model. In hESC-derived neural progenitors (hESC-NPs), LHX2 was found to be expressed before PAX6, and co-expressed with early neural markers. Conditional ectopic expression of LHX2 promoted neural differentiation, whereas disruption of LHX2 expression in hESCs significantly impaired neural differentiation. Furthermore, we have demonstrated that LHX2 regulates neural differentiation at two levels: first, it promotes expression of PAX6 by binding to its active enhancers, and second, it attenuates BMP and WNT signaling by promoting expression of the BMP and WNT antagonist Cerberus 1 gene (CER1), to inhibit non-neural differentiation. These findings indicate that LHX2 regulates the transcription of downstream intrinsic and extrinsic molecules that are essential for early neural differentiation in human.

Induced pluripotent stem cell technology for disease modeling and drug screening with emphasis on lysosomal storage diseases.

The recent derivation of disease-specific induced pluripotent stem cells (iPSCs) from somatic cells of patients with familial and sporadic forms of diseases and the demonstration of their ability to give rise to disease-relevant cell types provide an excellent opportunity to gain further insights into the mechanisms responsible for the pathophysiology of these diseases and develop novel therapeutic drugs. Here, we review the recent advances in iPSC technology for modeling of various lysosomal storage diseases (LSDs) and discuss possible strategies through which LSD-iPSCs can be exploited to identify novel drugs and improve future clinical treatment of LSDs.

Meiotic competent human germ cell-like cells derived from human embryonic stem cells induced by BMP4/WNT3A signaling and OCT4/EpCAM (epithelial cell adhesion molecule) selection.

The establishment of an effective germ cell selection/enrichment platform from in vitro differentiating human embryonic stem cells (hESCs) is crucial for studying the molecular and signaling processes governing human germ cell specification and development. In this study, we developed a germ cell-enriching system that enables us to identify signaling factors involved in germ cell-fate induction from differentiating hESCs in vitro. First, we demonstrated that selection through an OCT4-EGFP reporter system can successfully increase the percentage of meiotic-competent, germ cell-like cells from spontaneously differentiating hESCs. Furthermore, we showed that the pluripotency associated surface marker, epithelial cell adhesion molecule (EpCAM), is also expressed in human fetal gonads and can be used as an effective selection marker for germ cell enrichment from differentiating hESCs. Combining OCT4 and EpCAM selection can further enrich the meiotic-competent germ cell-like cell population. Also, with the percentage of OCT4(+)/EpCAM(+) cells as readout, we demonstrated the synergistic effect of BMP4/pSMAD1/5/8 and WNT3A/ß-CATENIN in promoting hESCs toward the germline fate. Combining BMP4/WNT3A induction and OCT4/EpCAM selection can significantly increase the putative germ cell population with meiotic competency. Co-transplantation of these cells with dissociated mouse neonatal ovary cells into SCID mice resulted in a homogenous germ cell cluster formation in vivo. The stepwise platform established in this study provides a useful tool to elucidate the molecular mechanisms of human germ cell development, which has implications not only for human fertility research but regenerative medicine in general.

Human Pompe disease-induced pluripotent stem cells for pathogenesis modeling, drug testing and disease marker identification.

Pompe disease is caused by autosomal recessive mutations in the acid alpha-glucosidase (GAA) gene, which encodes GAA. Although enzyme replacement therapy has recently improved patient survival greatly, the results in skeletal muscles and for advanced disease are still not satisfactory. Here, we report the derivation of Pompe disease-induced pluripotent stem cells (PomD-iPSCs) from two patients with different GAA mutations and their potential for pathogenesis modeling, drug testing and disease marker identification. PomD-iPSCs maintained pluripotent features and had low GAA activity and high glycogen content. Cardiomyocyte-like cells (CMLCs) differentiated from PomD-iPSCs recapitulated the hallmark Pompe disease pathophysiological phenotypes, including high levels of glycogen and multiple ultrastructural aberrances. Drug rescue assessment showed that exposure of PomD-iPSC-derived CMLCs to recombinant human GAA reversed the major pathologic phenotypes. Furthermore, l-carnitine treatment reduced defective cellular respiration in the diseased cells. By comparative transcriptome analysis, we identified glycogen metabolism, lysosome and mitochondria-related marker genes whose expression robustly correlated with the therapeutic effect of drug treatment in PomD-iPSC-derived CMLCs. Collectively, these results demonstrate that PomD-iPSCs are a promising in vitro disease model for the development of novel therapeutic strategies for Pompe disease.

Epithelial cell adhesion molecule (EpCAM) complex proteins promote transcription factor-mediated pluripotency reprogramming.

Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein that is highly expressed in embryonic stem cells (ESCs) and its role in maintenance of pluripotency has been suggested previously. In epithelial cancer cells, activation of the EpCAM surface-to-nucleus signaling transduction pathway involves a number of membrane proteins. However, their role in somatic cell reprogramming is still unknown. Here we demonstrate that EpCAM and its associated protein, Cldn7, play a critical role in reprogramming. Quantitative RT-PCR analysis of Oct4, Sox2, Klf4, and c-Myc (OSKM) infected mouse embryonic fibroblasts (MEFs) indicated that EpCAM and Cldn7 were up-regulated during reprogramming. Analysis of numbers of alkaline phosphatase- and Nanog-positive clones, and the expression level of pluripotency-related genes demonstrated that inhibition of either EpCAM or Cldn7 expression resulted in impairment in reprogramming efficiency, whereas overexpression of EpCAM, EpCAM plus Cldn7, or EpCAM intercellular domain (EpICD) significantly enhanced reprogramming efficiency in MEFs. Furthermore, overexpression of EpCAM or EpICD significantly repressed the expression of p53 and p21 in the reprogramming MEFs, and both EpCAM and EpICD activated the promoter activity of Oct4. These observations suggest that EpCAM signaling may enhance reprogramming through up-regulation of Oct4 and possible suppression of the p53-p21 pathway. In vitro and in vivo characterization indicated that the EpCAM-reprogrammed iPSCs exhibited similar molecular and functional features to the mouse ESCs. In summary, our studies provide additional insight into the molecular mechanisms of reprogramming and suggest a more effective means of induced pluripotent stem cell generation.

Surface marker epithelial cell adhesion molecule and E-cadherin facilitate the identification and selection of induced pluripotent stem cells.

The derivation of induced pluripotent stem cells (iPSCs) requires not only efficient reprogramming methods, but also reliable markers for identification and purification of iPSCs. Here, we demonstrate that surface markers, epithelial cells adhesion molecule (EpCAM) and epithelial cadherin (E-cadherin) can be used for efficient identification and/or isolation of reprogrammed mouse iPSCs. By viral transduction of Oct4, Sox2, Klf4 and n- or c-Myc into mouse embryonic fibroblasts, we observed that the conventional mouse embryonic stem cell (mESC) markers, alkaline phosphatase (AP) and stage-specific embryonic antigen 1 (SSEA1), were expressed in incompletely reprogrammed cells that did not express all the exogenous reprogramming factors or failed to acquire pluripotent status even though exogenous reprogramming factors were expressed. EpCAM and E-cadherin, however, remained inactivated in these cells. Expression of EpCAM and E-cadherin correlated with the activation of Nanog and endogenous Oct4, and was only seen in the successfully reprogrammed iPSCs. Furthermore, purification of EpCAM-expressing cells at late reprogramming stage by FACS enriched the Nanog-expressing cell population suggesting the feasibility of selecting successful reprogrammed mouse iPSCs by EpCAM expression. We have thus identified new surface markers that can efficiently identify successfully reprogrammed iPSCs and provide an effective means for iPSC isolation.