Abstract
Human ES cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are usually generated and maintained on living feeder cells like mouse embryonic fibroblasts or on a cell-free substrate like Matrigel. For clinical applications, a quality-controlled, xenobiotic-free culture system is required to minimize risks from contaminating animal-derived pathogens and immunogens. We previously reported that the pericellular matrix of decidua-derived mesenchymal cells (PCM-DM) is an ideal human-derived substrate on which to maintain hiPSCs/hESCs. In this study, we examined whether PCM-DM could be used for the generation and long-term stable maintenance of hiPSCs. Decidua-derived mesenchymal cells (DMCs) were reprogrammed by the retroviral transduction of four factors (OCT4, SOX2, KLF4, c-MYC) and cultured on PCM-DM. The established hiPSC clones expressed alkaline phosphatase, hESC-specific genes and cell-surface markers, and differentiated into three germ layers in vitro and in vivo. At over 20 passages, the hiPSCs cultured on PCM-DM held the same cellular properties with genome integrity as those at early passages. Global gene expression analysis showed that the GDF3, FGF4, UTF1, and XIST expression levels varied during culture, and GATA6 was highly expressed under our culture conditions; however, these gene expressions did not affect the cells’ pluripotency. PCM-DM can be conveniently prepared from DMCs, which have a high proliferative potential. Our findings indicate that PCM-DM is a versatile and practical human-derived substrate that can be used for the feeder-cell-free generation and long-term stable maintenance of hiPSCs.
Highlights
Induced pluripotent stem cells are generated from various somatic cells by introducing defined transcription factors [1,2], and they have properties similar to those of embryonic stem cells (ESCs). iPSCs are expected to contribute greatly to the realization of regenerative medicine and to understanding the molecular pathogenesis of many currently intractable diseases
No colonies with clear human ESCs (hESCs)-like characteristics were obtained from derived mesenchymal cells (DMCs) cultured on gelatin with NC-hESC medium (Fig. 8, Supplemental Table S5). These findings suggest that pericellular matrix of decidua-derived mesenchymal cells (PCM-DM) could be used to generate human iPSCs (hiPSCs) with a reprogramming efficiency that was almost as good or the same as that of other substrates used with mouse embryonic fibroblasts (MEFs)-CM or NC-hESC medium
Non-conditioned Medium to examine the applicability of hiPSCs generated on PCM-DM without MEF-conditioned medium (MEF-CM), we examined the detailed cellular properties of hiPSCs generated on PCM-DM in NC-hESC medium
Summary
Induced pluripotent stem cells (iPSCs) are generated from various somatic cells by introducing defined transcription factors [1,2], and they have properties similar to those of embryonic stem cells (ESCs). iPSCs are expected to contribute greatly to the realization of regenerative medicine and to understanding the molecular pathogenesis of many currently intractable diseases. The promise of cell-based therapies using human iPSCs (hiPSCs) is generally recognized, and has driven an intense search for good cell sources, reprogramming methods, and cell culture systems. Their clinical application has yet to be realized. HiPSCs/human ESCs (hESCs) are generated and maintained on living feeder cells, such as mouse embryonic fibroblasts (MEFs) [2,3,4] or SNL cells [1,5], or on a feeder-free culture substrate such as Matrigel [6,7,8,9], fibronectin [10,11,12,13], or human recombinant laminin-511 [14,15]. The maintenance activity of PCM-DM is similar to that of Matrigel, and its preparation is easy and reproducible, because decidua-derived mesenchymal cells (DMCs) can be obtained and expanded in large quantity [23]
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