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Pluripotent Stem Cells Induced from Mouse Neural Stem Cells and Small Intestinal Epithelial Cells by Small Molecule Compounds

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Pluripotent Stem Cells Induced from Mouse Neural Stem Cells and Small Intestinal Epithelial Cells by Small Molecule Compounds Cell Research (2016) 26:34-45. npg © 2016 IBCB, SIBS, CAS All rights reserved 1001-0602/16 $ 32.00 ORIGINAL ARTICLE www.nature.com/cr Pluripotent stem cells induced from mouse neural stem cells and small intestinal epithelial cells by small molecule compounds Junqing Ye1, 2, *, Jian Ge1, 2, *, Xu Zhang1, 2, Lin Cheng1, 2, Zhengyuan Zhang1, 2, Shan He 1, 2, Yuping Wang4, Hua Lin4, Weifeng Yang5, Junfang Liu1, 2, Yang Zhao1, 2, 3, Hongkui Deng1, 2 1Shenzhen Stem Cell Engineering Laboratory, Key Laboratory of Chemical Genomics, Peking University Shenzhen Graduate School, Shenzhen, Guangdong 518055, China; 2The MOE Key Laboratory of Cell Proliferation and Differentiation, College of Life Sciences, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China; 3Department of Cell Biology, School of Basic Medical Sciences, Peking University Stem Cell Research Center, State Key Laboratory of Natural and Biomimetic Drugs, Peking University Health Science Center, Beijing 100191, China; 4Department of Gynecology and Obstetrics, China-Japan Friendship Hospital, Beijing 100029, China; 5BeijingVitalstar Biotechnology Co., Ltd., Beijing 100012, China Recently, we reported a chemical approach to generate pluripotent stem cells from mouse fibroblasts. Howev- er, whether chemically induced pluripotent stem cells (CiPSCs) can be derived from other cell types remains to be demonstrated. Here, using lineage tracing, we first verify the generation of CiPSCs from fibroblasts. Next, we demon- strate that neural stem cells (NSCs) from the ectoderm and small intestinal epithelial cells (IECs) from the endoderm can be chemically reprogrammed into pluripotent stem cells. CiPSCs derived from NSCs and IECs resemble mouse embryonic stem cells in proliferation rate, global gene expression profile, epigenetic status, self-renewal and differen- tiation capacity, and germline transmission competency. Interestingly, the pluripotency gene Sall4 is expressed at the initial stage in the chemical reprogramming process from different cell types, and the same core small molecules are required for the reprogramming, suggesting conservation in the molecular mechanism underlying chemical repro- gramming from these diverse cell types. Our analysis also shows that the use of these small molecules should be fine- tuned to meet the requirement of reprogramming from different cell types. Together, these findings demonstrate that full chemical reprogramming approach can be applied in cells of different tissue origins and suggest that chemical reprogramming is a promising strategy with the potential to be extended to more initial types. Keywords: reprogramming; CiPSC; cell type; fine-tuning Cell Research (2016) 26:34-45. doi:10.1038/cr2015.142; published online 25 December 2015 Introduction proposed that iPSCs are predominantly derived from a rare cell population [4-6], numerous advances have been Somatic cells can be reprogrammed into pluripotent made in extending the application of iPS technology to stem cells by ectopic expression of defined transcription other defined cell types, including keratinocytes [7], neu- factors [1-3]. Induced pluripotent stem cells (iPSCs) are ral stem cells [8-10], hepatocytes [11] and hematocytes commonly generated by Oct4, Sox2, Klf4 and c-Myc [12-15]. (OSKM) overexpression. Although several studies have Recently, we have developed a novel approach to re- program mouse somatic cells into pluripotent stem cells using only small molecules [16]. This chemical approach *These two authors contributed equally to this work. is different from the traditional genetic approach, as it Correspondence: Hongkui Denga, Yang Zhaob offers more flexible control of the complex signaling a E-mail: [email protected] networks and epigenetic status in the cells during re- bE-mail: [email protected] Received 19 July 2015; revised 9 November 2015; accepted 13 November programming [17]. Small molecules are cell permeable; 2015; published online 25 December 2015 their effect is functionally reversible, and the treatment- Junqing Ye et al. npg 35 can be easily standardized. Thus, precise cell fate control MEFs were separated into two subpopulations of tdTo- can be rendered by manipulating the concentrations, mato-positive and non-fluorescent cells. After treatment treatment duration, and combinations of chemical com- with the chemical cocktail as we previously reported pounds. Because of these advantages, the chemically [16], both tdTomato-positive and non-fluorescent CiP- induced pluripotent stem cell (CiPSC) approach provides SCs were generated, suggesting that both fibroblasts and a unique way to generate iPSCs and is a promising strat- non-fibroblast cells can yield CiPSCs (Figure 1A-1D). egy for widespread use in the future. Chimeric mice were obtained from fibroblast-derived In the previous report, we generated CiPSCs from fi- CiPSCs (Figure 1E). broblasts, including mouse embryonic fibroblasts (MEFs), neonatal skin fibroblasts and adult lung fibroblasts [16]. Generation of CiPSCs from NSCs As fibroblasts are a heterogeneous population of cells To test whether chemical reprogramming is appli - [18], and the reprogramming efficiency of our approach cable to cell types from other germ layers, we applied was relatively low [16], the possibility remains that there our chemical cocktail to NSCs from the ectodermal exists within fibroblast culture a specific cell subpopu- lineage, which exhibit distinct gene expression profiles lation with propensity for reprogramming. Moreover, from MEFs. NSCs were isolated from embryonic mouse MEFs, neonatal skin fibroblasts and adult lung fibro- brains of Oct4-GFP transgenic mice using the standard blasts are cells from the mesoderm. It is unknown wheth- protocol and then cultured in standard NSC medium er other cell types, including cells derived from ectoderm with bFGF and EGF in low attachment plates [20-22]. and endoderm, can also be reprogrammed by entirely Established NSC lines could self-renew and form neuro- chemical conditions. It has been reported that different spheres for up to 8 passages in vitro (4-5 days/ passage) cell types require modulations of different signaling (Figure 2A). Cultured NSCs expressed neural stem cell pathways to achieve efficient reprogramming, possibly markers SOX2 and NESTIN at high levels (Figure 2B). due to their intrinsic properties [19]. It is thus interesting Gene ontology analysis showed that the most highly ex- to explore if fundamentally different small molecules are pressed genes in cultured NSCs were enriched in neural required to reprogram different cell types, which is a key lineages (Supplementary information, Figure S1A, Table question for the extension of the chemical reprogram- S1) [23]. ming approach. To induce pluripotency from NSCs, we initially In this study, we tested the chemical reprogramming treated NSCs by using the reported chemical protocol strategy on different initial cell types. We show that neu- for generation of MEF-derived CiPSCs. As the original ral stem cells (NSCs) from the ectoderm and small intes- chemical reprogramming cocktail “VC6TF” (V, VPA; C, tinal epithelial cells (IECs) from the endoderm can also CHIR99021 or CHIR; 6, 616452; T, tranylcypromine; F, be reprogrammed by small molecules. Moreover, the forskolin) [16] was unable to induce pluripotency from same core small molecules can be applied for chemical NSCs, we attempted to boost the reprograming by add- reprogramming of different cell types, and fine-tuning of ing EPZ 004777 (EPZ, E), an inhibitor of H3K79 histone these small molecules is critical to achieve pluripotency. methyltransferase DOT1L [24], and Ch 55 (5), a retinoid acid receptor (RAR) agonist (Supplementary informa- Results tion, Figure S1B). Although neither epithelioid colonies nor CiPSC colonies were generated with the new cock- Lineage-tracing verifies generation of CiPSCs from fi- tail “VC6TFE5” with the same drug concentrations as broblasts we reported for MEF, we found, surprisingly, that after To determine whether CiPSCs are derived from a reducing the concentration of 616452 from 10 µM to 2 specific cell type within fibroblast culture, we performed µM, epithelial clusters emerged on day 8 (Figure 2C), fibroblast specific protein 1 (Fsp1) tracing during the and proliferated with time. After the addition of DZNep chemical reprogramming process. We crossed mice car- (Z), an S-adenosylhomocysteine (SAH) hydrolase inhib- rying an Fsp1-Cre (Fsp1 promoter-driven CRE expres- itor, from day 20, compact and epithelioid colonies were sion) transgene with mice carrying a conditional Rosa- observed on day 32 (Figure 2C). The Oct4-GFP reporter 26LoxP reporter locus containing a “floxed stop cassette” was gradually turned on after the colonies were switched located in front of the Tomato gene (Rosa26RtdTomato). The to 2i-medium with dual inhibition (2i) of mitogen-ac- offspring were crossed with Oct4-GFP (Oct4 promot- tivated protein kinase (MAPK) signaling and glycogen er-driven GFP expression, OG) transgenic mice to create synthase kinase-3 (GSK3) [25] from day 40, and mouse Fsp1-Cre: Rosa26RtdTomato: Oct4-GFP progeny mice. As embryonic stem cell (mESC)-like OG-positive colonies Fsp1 is specifically expressed in fibroblasts, the initial with clear-cut edges were observed (Figure 2D and 2I). www.cell-research.com | Cell Research npg CiPSCs generated from NSCs and IECs 36 Figure 1 Generation of CiPSCs from both fibroblasts and non-fibroblast cells. (A, B) A primary CiPSC colony generated from Fsp1-Cre-tdTomato
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