Efficient generation of human iPSCs by a synthetic self-replicative RNA

Abstract

The generation of human induced pluripotent stem cells (iPSCs) holds great promise for the development of regenerative medicine therapies to treat a wide range of human diseases. However, the generation of iPSCs in the absence of integrative DNA vectors remains problematic. Here, we report a simple, highly reproducible RNA-based iPSC generation approach that utilizes a single, synthetic self-replicating VEE-RF RNA replicon that expresses four reprogramming factors (OCT4, KLF4, and SOX2, with c-MYC or GLIS1) at consistent high levels prior to regulated RNA degradation. A single VEE-RF RNA transfection into newborn or adult human fibroblasts resulted in efficient generation of iPSCs with all the hallmarks of stem cells, including cell surface markers, global gene expression profiles, and in vivo pluripotency, to differentiate into all three germ layers. The VEE-RF RNA-based approach has broad applicability for the generation of iPSCs for ultimate use in human stem cell therapies in regenerative medicine.

Figure 1. Construction and Persistence of Synthetic VEE-RF RNA Replicons in Primary Human Fibroblasts

(A) Schematic of VEE-RF RNA replicons. 5′ end nsP1-4: non-structural proteins1–4; 3′ end C, E2, E1: Structural proteins. Location of 26S internal promoter, ribosome shifting 2A peptide, IRES sequence, Puromycin (Puro) resistance gene and PCR detection of replicon as indicated. (B) B18R-CM Conditioned Media and puromycin selection are required for persistence of VEE-GFP RNA over 7 days. HFF cells were transfected on day 0 with VEE-GFP RNA and treated as indicated. GFP fluorescence of GFP positive cell population was measured by flow cytometry. (C) B18R-CM and puromycin are required for retention of VEE-GFP RNA. Photographs of GFP expression on day 7 as indicated. Bar, 200 μm. (D) Immunoblot analysis of VEE RNA expressed reprogramming factors expressed in HFFs cells on day 1 versus retrovirus (RV-4Fs: Oct, Sox2, Klf4, cMyc) expression. See also Figure S1.

Figure 2. Generation of iPS cells by VEE-RF RNA

(A) Schematic of epigenetic VEE-RF RNA iPS cell generation protocol. Human fibroblasts were plated on day 0 (d0) and co-transfected (Tfx) with VEE-RF RNA replicon plus B18R mRNA on day 1 (confluent, ~4×10⁵ cells) and treated with puromycin until day 7 (or 10) as indicated. Cells were cultured in B18R-CM until iPS cell colonies were isolated on day 25 (to 30). (B) iPS cell colonies stained with Alkaline Phosphatase were generated with VEE-OKS-iM RNA, but not VEE-OMKS RNA. Transfection was performed on day 1 and 3 (2x Tfx), or 1, 3, 5, 7 and 9 (5x Tfx). (C) Alkaline Phosphatase staining of iPS cell colonies generated from BJ or HFFs from d1, 4, 7, 10 transfection protocol as indicated. (D) Typical images of iPS cell colonies on day 26 by VEE-OKS-iM RNA and day 22 for VEE-OKS-iG RNA from BJ or HFFs fibroblasts as indicated. Bar, 100 μm. (E) Immunohistochemistry staining of pluripotent ES marker genes in isolated iPS cell clones generated as indicated. Similar results obtained for 26 additional iPS cell clones (30 clones total). Bar, 100 μm; insert, 10× amplification. See also Figures S2, S5 and S6.

Figure 3. RT-PCR Analysis for Persistent VEE-RF RNA Replicon in iPS Cell Clones

(A, B) RT-PCR of HFF-OKS-iM iPS cell clones from total RNA prepared from passage 4 (A) and passage 8 (B), as indicated. +, positive control, total RNA was prepared from one day after transfection of OKS-iM-RNA replicon. −, negative control, total RNA was prepared from mock transfected HFFs. See also Table S2.

Figure 4. Characterization of VEE-RF RNA iPS Cell Clones

(A) Expression of ES maker genes by qRT-RCR analysis from indicated BJ and HFF VEE-RF RNA iPS cell clones. (B) DNA methylation analysis of NANOG and OCT4 promoter regions. Solid circle, methylated; Open circle, demethylated. Numbers indicate CpG position relative to transcription start site. (C) Genome-wide mRNA sequence profile scatter plot analysis of BJ-OKS-iM #2 and BJ-OKS-iG #5 compared to parental human BJ fibroblasts and human HUES9 embryonic stem cells with pluripotency NANOG, OCT4, SOX2 indicated. (D) Unsupervised hierarchical dendrogram of genome-wide RNA sequences analysis showing clustering of four independent iPS cell clones with HUES9 compared to BJ fibroblasts. See also Figures S4, S5, S6 and Table S1.

Figure 5. Differentiation assays of VEE-RF RNA iPS Cell Clones

(A) VEE-RF RNA iPS clones were differentiated into cardiomyocytes as described in Experimental Procedures. Contractile embryoid bodies (EBs) were recorded (see supplemental movie S1), and then dissociated and replated onto slides for immunofluorescence staining with mouse anti-Cardiac Troponin or mouse anti-alpha-Actinin, Anti-Mouse IgG Alexa Fluor 488, and DAPI. Bar, 50 μm. (B) Teratoma formation of VEE-RF RNA iPS clones in nude mice. H&E staining; Bar, 100 μm. (C) Immunohistochemistry staining of VEE-RF RNA iPS cell clone teratomas in nude mice. AE1/AE3 (cytokeratin), NF-1 (neuronal cells) and GFAP (neuronal cells) used for markers of ectoderm; Desmin (muscle cells) used for marker of mesoderm; and AFP (primitive and definitive endoderm) used for marker of endoderm. Bar, 100 μm. See also Movie S1 and Table S1.