A widely adaptable approach to generate integration-free iPSCs from non-invasively acquired human somatic cells
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Human pluripotent stem cells including human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are cells displaying abilities of unlimited self-renewal and differentiation into any somatic cell type. These unique properties make them increasingly attractive for novel applications in disease modeling, drug discovery, and cell therapy (Buganim et al., 2014; Liu et al., 2011; Liu et al., 2012; Sanchez Alvarado and Yamanaka, 2014). Moreover, iPSCs hold great potential for personalized cell therapy as they avoid some of the ethical concerns as well as the immunological rejection issues ascribed to ESCs. To date, iPSCs can be generated from various cell sources that range from commonly used skin fibroblasts to rarely employed blood cells, mostly through virus-based reprogramming methods with different efficiencies (Patel and Yang, 2010). However, the isolation of these original human cells often involves an invasive sample acquisition procedure. Therefore, human somatic cells that can be conveniently obtained in a non-invasive manner, including human endometrium cells (EMCs) and human urine-derived cells (UCs), may represent more convenient and promising sources for the generation of iPSCs. This is especially crucial for patients hypersensitive to any invasive operation (i.e. patients with Diabetes mellitus (DM) and patients with hemophilia). On the other hand, non-integrative reprogramming methods including those mediated by episomal vectors (Okita et al., 2011), Sendai virus vectors (Seki et al., 2012) and small chemicals (Hou et al., 2013; Lin et al., 2009), are increasingly regarded as superior alternatives to viral approaches. Recently, an elegant study has demonstrated that iPSCs could be established by an episomal system comprised of miR-302–367 from human UCs (Xue et al., 2013). Here we report on the generation of integration-free human endometrium-derived iPSCs (emiPSCs) and urine-derived iPSCs (uiPSCs) by a modified episomal reprogramming system. Subsequently, we show that these iPSCs can robustly differentiate into several valuable lineage-specific cell types, including pancreatic progenitor cells (PPs) and neural stem cells (NSCs).
We further examined the in vitro and in vivo differentiation potentials of the generated iPSCs. The in vitro spontaneous differentiation potential of iPSCs was investigated by embryoid body (EB)-based differentiation assays. As shown in Fig. 1G, iPSCs effectively differentiated into TUJ1 (ectoderm), SMA (mesoderm), as well as FOXA2 and ALB (endoderm)-positive cells upon spontaneous differentiation. Furthermore, after injection into immune-deficient NOD/SCID mice, iPSCs were able to develop into teratomas composed of cells from all three germ-layers marked by SMA, AFP, PDX1, and Nestin expressions (Fig. 1H).
Next, we investigated the potency of the iPSCs in directed cell differentiations. PPs and NSCs were chosen because of their value in potential applications. In order to generate PPs, iPSCs were firstly treated with a combination of activin A and Wnt3a to initiate the definitive endoderm cell (DE) differentiation, a step resulting in 95% endodermal progenitors co-expressing FOXA2 and SOX17 (Fig. 1I and 1J). After treating the endodermal progenitors with Retinoic acid, FGF10, Noggin, and Cyclopamine-KAAD, we obtained a highly pure population of PPs positive for the pancreatic progenitor marker PDX1 (Fig. 1I and 1J). As anticipated, these PPs also expressed NKX6.1 and NGN3. An upregulation of insulin gene (INS) expression at transcriptional level was also observed (Fig. 1I and 1J). Additionally, we differentiated iPSCs toward NSCs by treatment with a chemically defined medium comprising of hLIF, SB431542, CHIR99021, Compound E, and dorsomorphin (Liu et al., 2012). These resulting NSCs expressed typical neural progenitor markers SOX2, PAX6, Musashi, and Nestin (Fig. 1K). As a characteristic feature of neural progenitor cells, these cells were able to efficiently differentiate into MAP2 and TUJ1-positive neurons when exposed to a differentiation medium containing ascorbic acid, dbcAMP, BDNF and GDNF for 14 days (Fig. 1K).
In summary, we demonstrated here that integration-free iPSCs can be efficiently and consistently obtained from non-invasively acquired human EMCs and UCs, even in a defective disease context (i.e. type II DM) (Fig. 1L). Given the fact that a similar strategy has been proven to be effective in reprograming diseased somatic cells inherently resistant to the regular cell reprogramming procedure (Liu et al., 2014), we speculate that this methodology could be universally adaptable for various applications. The autologous PP and NSC derivatives generated from emiPSCs and uiPSCs could be further subjected to animal transplantation studies (Sui et al., 2013; Zhang et al., 2009), and even potentially be applied to human regenerative medicine settings. The robustness and non-invasive nature of our system to generate iPSCs from human samples may enormously fuel the application of human iPSCs towards novel cell therapy as well as applications in disease modeling and drug discovery in the near future.
This work was supported by National Basic Research Program (973 program) (Nos. 2015CB964800, 2014CB910500, and 2014CB964600), the Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDA01020312), National Natural Science Foundation of China (Grant Nos. 81300677, 81330008, 81271266, 31222039, 31201111, 81371342, 81471414, 81401159, 81300261, 81422017, and 21331001), Key Research Program of the Chinese Academy of Sciences (No. KJZD-EW-TZ-L05), National Science and Technology Major Project (No. 2011ZX09307-001-08), Beijing Natural Science Foundation (Nos. 7141005 and 5142016), the Thousand Young Talents Program of China, National Laboratory of Biomacromolecules (Nos. 2013kf05, 2013kf11, and 2014kf02), and State Key Laboratory of Drug Research (No. SIMM1302KF-17). JCIB was supported by G. Harold and Leila Y. Mathers Charitable Foundation, and The Leona M. and Harry B. Helmsley Charitable Trust grant (2012-PG-MED002).
Zhichao Ding, Lina Sui, Ruotong Ren, Yanjun Liu, Xiuling Xu, Lina Fu, Ruijun Bai, Tingting Yuan, Ying Hao, Weiqi Zhang, Huize Pan, Wensu Liu, Han Yu, Concepcion Rodriguez Esteban, Xiaobing Yu, Ze Yang, Jian Li, Xiaomin Wang, Juan Carlos Izpisua Belmonte, Guang-Hui Liu, Fei Yi, and Jing Qu declare that they have no conflict of interest.
All institutional and national guidelines for the care and use of laboratory animals were followed. The individuals in this manuscript have signed written informed consent for donating EMCs and UCs for somatic cell reprogramming and iPSC study.
- Mali P, Chou BK, Yen J, Ye Z, Zou J, Dowey S, Brodsky RA, Ohm JE, Yu W, Baylin SB et al (2010) Butyrate greatly enhances derivation of human induced pluripotent stem cells by promoting epigenetic remodeling and the expression of pluripotency-associated genes. Stem cells 28:713–720CrossRefPubMedCentralPubMedGoogle Scholar
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