Pluripotent stem cells induced from mouse neural stem cells and small intestinal epithelial cells by small molecule compounds

Abstract

Recently, we reported a chemical approach to generate pluripotent stem cells from mouse fibroblasts. However, 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 demonstrate 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 differentiation 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 reprogramming 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.

Figure 1

Generation of CiPSCs from both fibroblasts and non-fibroblast cells. (A, B) A primary CiPSC colony generated from Fsp1-Cre-tdTomato fluorescent MEFs (A), and passaged CiPSCs (B). (C, D) A primary CiPSC colony generated from Fsp1-Cre-tdTomato non-fluorescent MEFs (C), and passaged CiPSCs (D). (E) A chimeric mouse embryo (E13.5) derived from Fsp1-Cre-tdTomato fluorescent CiPSCs. For A-D, Scale bar, 100 μm.

Figure 2

Generation of CiPSCs from mouse NSCs. (A) Neurosphere formation of isolated NSCs. (B) Immunofluorescence staining of SOX2 and NESTIN in embryonic NSCs (passage 2). (C-E) Epithelial clusters at day 8 (D8) after chemical treatment (C), a compact, epithelioid colony at day 32 (D32) after treatment (C), a primary CiPS colony on day 50 (D50) (D), and passaged CiPSC colonies (P1) (E). (F) Optimization of 616452 concentration in the first 20 days during chemical reprogramming (error bars, mean ± SD, n = 3). (G) Numbers of SOX2- and NESTIN-positive cells in 500 FACS-sorted SSEA1-positive NSCs. (H) A primary CiPSC colony generated from SSEA1-positive NSCs. (I) Schematic diagram for the chemical reprogramming of NSCs. V, VPA; C, CHIR 99021; 6, 616452; T, tranylcypromine; F, forskolin; E, EPZ004777; 5, Ch 55; Z, DZNep. For A and C-E, scale bar, 100 μm. For B, scale bar, 20 μm. See also Supplementary information, Figure S1.

Figure 3

Generation of CiPSCs from mouse IECs. (A) Immunofluorescence staining of intestinal epithelial cell marker KERATIN20 (KRT20) expression in primary embryonic IECs. (B, C) Morphology of colonies during chemical reprogramming process, and a primary CiPS colony on day 42 (D42) (B), and passaged CiPSC colonies (P1) (C). (D, E) Lineage tracing of the isolated IECs (red) from pVillin-Cre: Rosa26R^tdTomato mice (D), and a passaged CiPSC colony derived from pVillin-Cre-tdTomato-positive IECs (E). (F) Optimization of 616452 concentration in the first 12 days during chemical reprogramming (error bars, mean ± SD, n = 3). (G) Schematic diagram for the chemical reprogramming of IECs. V, VPA; C, CHIR 99021; 6, 616452; T, tranylcypromine; F, forskolin; A, AM 580; Z, DZNep. For A-E, scale bar, 100 μm. See also Supplementary information, Figure S2.

Figure 4

Characterization of CiPSCs derived from NSCs and IECs. (A, B) Pluripotency gene expression profile of NSC-CiPSCs (A) and IEC-CiPSCs (B) as revealed by quantitative real-time PCR (error bars, mean ± SD, n = 3). (C, D) Immunofluorescence staining for the indicated pluripotency markers in CiPSCs derived from NSCs (C) and IECs (D). (E) Hematoxylin and eosin staining of teratoma derived from NSC-CiPSCs (left) and IEC-CiPSCs (right). (F) Number of chromosomes in NSC-CiPSCs and IEC-CiPSCs by karyotype analysis (clone IEC-CiPSC-4 is derived from pVillin-Cre-tdTomato-positive IECs). (G) Chimeric mice and F2 offspring derived by NSC-CiPSCs (left) and IEC-CiPSCs (right). For C and D, scale bar, 50 μm. For E, scale bar, 100 μm. See also Supplementary information, Figure S3.

Figure 5

Initial gene activation was conserved in chemical reprogramming from different cell types. (A) Expression of Sall4, Gata6 and Sox17 at the early stage of chemical reprogramming from NSCs (day 0 (D0), day 4 (D4) and day 16 (D16), respectively) measured by quantitative real-time PCR (error bars, mean ± SD, n = 3). (B) Expression of Sall4, Gata6 and Sox17 genes at day 16 of the reprogramming by the chemical cocktail with different concentration of 616452 (error bars, mean ± SD, n = 3). (C) Expression of Sall4, Gata4 and Sox17 genes at the early stage of chemical reprogramming from IECs (day 0 (D0), day 4 (D4) and day 16 (D16), respectively) measured by quantitative real-time PCR (error bars, mean ± SD, n = 3). (D) Expression of Sall4, Gata4 and Sox17 genes at day 20 of the reprogramming by the chemical cocktail with different concentration of 616452 (error bars, mean ± SD, n = 3). (E) Expression of pluripotency genes Sall4, Lin28, Esrrb, Dppa2 and Oct4 during the chemical reprogramming from NSCs (day 0 (D0), day 4 (D4), day 12 (D12), day 16 (D16) and day 20 (D20)) measured by quantitative real-time PCR (error bars, mean ± SD, n = 3). (F) Schematic diagram illustrating the step-wise chemical reprogramming of different cell types.