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.     

Efficient iPS cell production with the MyoD transactivation domain in serum-free culture

A major difficulty of producing induced pluripotent stem cells (iPSCs) has been the low efficiency of reprogramming differentiated cells into pluripotent cells. We previously showed that 5% of mouse embryonic fibroblasts (MEFs) were reprogrammed into iPSCs when they were transduced with a fusion gene composed of Oct4 and the transactivation domain of MyoD (called M(3)O), along with Sox2, Klf4 and c-Myc (SKM). In addition, M(3)O facilitated chromatin remodeling of pluripotency genes in the majority of transduced MEFs, including cells that did not become iPSCs. These observations suggested the possibility that more than 5% of cells had acquired the ability to become iPSCs given more favorable culture conditions. Here, we raised the efficiency of making mouse iPSCs with M(3)O-SKM to 26% by culturing transduced cells at low density in serum-free culture medium. In contrast, the efficiency increased from 0.1% to only 2% with the combination of wild-type Oct4 and SKM (OSKM) under the same culture condition. For human iPSCs, M(3)O-SKM achieved 7% efficiency under a similar serum-free culture condition, in comparison to 1% efficiency with OSKM. This study highlights the power of combining the transactivation domain of MyoD with a favorable culture environment.     

Regulation of Induced Pluripotent Stem (iPS) Cell Induction by Wnt/β-Catenin Signaling

Wnt signaling has been implicated in promoting somatic cell reprogramming. However, its molecular mechanisms remain unknown. Here we report that Wnt/β-catenin enhances iPSCs induction at the early stage of reprogramming. The augmented reprogramming induced by β-catenin is not due to increased total cell population or activation of c-Myc. In addition, β-catenin interacts with reprogramming factors Klf4, Oct4, and Sox2, further enhancing expression of pluripotency circuitry genes. These studies reveal novel mechanisms underlying the regulation of reprogramming somatic cells to pluripotency by Wnt/β-catenin signaling.     

Derivation of iPSCs in stirred suspension bioreactors

Induced pluripotent stem cells (iPSCs) are typically derived in adherent culture. Here we report fast and efficient derivation of mouse iPSCs in stirred suspension bioreactors, with and without the use of c-Myc. Suspension-reprogrammed cells expressed pluripotency markers, showed multilineage differentiation in vitro and in vivo, and contributed to the germline in chimeric mice. Suspension reprogramming has the potential to accelerate and standardize iPSC research.     

Live cell imaging of X chromosome reactivation during somatic cell reprogramming

Generation of induced pluripotent stem cells (iPSCs) with naive pluripotency is important for their applications in regenerative medicine. In female iPSCs, acquisition of naive pluripotency is coupled to X chromosome reactivation (XCR) during somatic cell reprogramming, and live cell monitoring of XCR is potentially useful for analyzing how iPSCs acquire naive pluripotency. Here we generated female mouse embryonic stem cells (ESCs) that carry the enhanced green fluorescent protein (EGFP) and humanized Kusabira-Orange (hKO) genes inserted into an intergenic site near either the Syap1 or Taf1 gene on both X chromosomes. The ESC clones, which initially expressed both EGFP and hKO, inactivated one of the fluorescent protein genes upon differentiation, indicating that the EGFP and hKO genes are subject to X chromosome inactivation (XCI). When the derived somatic cells carrying the EGFP gene on the inactive X chromosome (Xi) were reprogrammed into iPSCs, the EGFP gene on the Xi was reactivated when pluripotency marker genes were induced. Thus, the fluorescent protein genes inserted into an intergenic locus on both X chromosomes enable live cell monitoring of XCI during ESC differentiation and XCR during reprogramming. This is the first study that succeeded live cell imaging of XCR during reprogramming.     

Generation of disease-specific induced pluripotent stem cells from patients with rheumatoid arthritis and osteoarthritis

Introduction

Since the concept of reprogramming mature somatic cells to generate induced pluripotent stem cells (iPSCs) was demonstrated in 2006, iPSCs have become a potential substitute for embryonic stem cells (ESCs) given their pluripotency and “stemness” characteristics, which resemble those of ESCs. We investigated to reprogram fibroblast-like synoviocytes (FLSs) from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) to generate iPSCs using a 4-in-1 lentiviral vector system.

Methods

A 4-in-1 lentiviral vector containing Oct4, Sox2, Klf4, and c-Myc was transduced into RA and OA FLSs isolated from the synovia of two RA patients and two OA patients. Immunohistochemical staining and real-time PCR studies were performed to demonstrate the pluripotency of iPSCs. Chromosomal abnormalities were determined based on the karyotype. SCID-beige mice were injected with iPSCs and sacrificed to test for teratoma formation.

Results

After 14 days of transduction using the 4-in-1 lentiviral vector, RA FLSs and OA FLSs were transformed into spherical shapes that resembled embryonic stem cell colonies. Colonies were picked and cultivated on matrigel plates to produce iPSC lines. Real-time PCR of RA and OA iPSCs detected positive markers of pluripotency. Immunohistochemical staining tests with Nanog, Oct4, Sox2, Tra-1-80, Tra-1-60, and SSEA-4 were also positive. Teratomas that comprised three compartments of ectoderm, mesoderm, and endoderm were formed at the injection sites of iPSCs. Established iPSCs were shown to be compatible by karyotyping. Finally, we confirmed that the patient-derived iPSCs were able to differentiate into osteoblast, which was shown by an osteoimage mineralization assay.

Conclusion

FLSs derived from RA and OA could be cell resources for iPSC reprogramming. Disease- and patient-specific iPSCs have the potential to be applied in clinical settings as source materials for molecular diagnosis and regenerative therapy.     

Reprogramming factor stoichiometry influences the epigenetic state and biological properties of induced pluripotent stem cells

We compared two genetically highly defined transgenic systems to identify parameters affecting reprogramming of somatic cells to a pluripotent state. Our results demonstrate that the level and stoichiometry of reprogramming factors during the reprogramming process strongly influence the resulting pluripotency of iPS cells. High expression of Oct4 and Klf4 combined with lower expression of c-Myc and Sox2 produced iPS cells that efficiently generated "all-iPSC mice" by tetraploid (4n) complementation, maintained normal imprinting at the Dlk1-Dio3 locus, and did not create mice with tumors. Loss of imprinting (LOI) at the Dlk1-Dio3 locus did not strictly correlate with reduced pluripotency though the efficiency of generating "all-iPSC mice" was diminished. Our data indicate that stoichiometry of reprogramming factors can influence epigenetic and biological properties of iPS cells. This concept complicates efforts to define a "generic" epigenetic state of iPSCs and ESCs and should be considered when comparing different iPS and ES cell lines.     

Application of induced pluripotency in cancer studies

As soon as induced pluripotent stem cells (iPSCs) reprogramming of somatic cells were developed, the discovery attracted the attention of scientists, offering new perspectives for personalized medicine and providing a powerful platform for drug testing. The technology was almost immediately applied to cancer studies. As presented in this review, direct reprogramming of cancer cells with enforced expression of pluripotency factors have several basic purposes, all of which aim to explain the complex nature of cancer development and progression, therapy-resistance and relapse, and ultimately lead to the development of novel anti-cancer therapies. Here, we briefly present recent advances in reprogramming methodologies as well as commonalities between cell reprogramming and carcinogenesis and discuss recent outcomes from the implementation of induced pluripotency into cancer research.     

诱导多潜能干细胞(iPSCs)的研究与应用进展

诱导多潜能干细胞(induced pluripotent stem cells,iPSCs)是体细胞在外源因子作用下,经直接细胞核程序重整而重新获得多潜能的干细胞.iPSCs在疾病的模型建立与机理研究、细胞治疗、药物的发现与评价等方面有着巨大的潜在应用价值.在过去几年中,科学家们致力于改进体细胞重编程技术并取得许多突破.然而,为实现其在临床上的应用,必须克服体细胞重编程效率低和iPSCs成瘤风险两大挑战,而且重编程机制有待进一步阐明.结合iPSCs最新研究成果,评述了有关领域国内外研究进展,重点讨论当前存在问题,并展望未来研究方向.