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Resource table. Resource details Macaca fascicularis iPS rad51 inhibitor ATCi-MF1 were generated from adult skin-derived fibroblasts by transduction with non-integrative Sendai viruses coding for the reprogramming factors OCT3/4, KLF4, SOX2 and c-MYC (Ban et al. 2011; Zapata-Linares et al. 2016). Transduced fibroblasts were maintained in fibroblast medium (DMEM, 15% Fetal Bovine Serum (FBS) for 1week, then cells were detached and re-plated on irradiated mouse embryonic fibroblasts (iMEF) in iPS cell medium (KO-DMEM, 15% Knock-out Serum Replacement (KSR) with 20ng/ml bFGF) and maintained for 3–4weeks, until iPS-like colonies appeared. Individual colonies were picked up, partially digested with Accutase and re-plated on iMEFs in iPS cell medium. Cells were splitted 1:5–1:8 with partial digestion with collagenase type IV and mechanical fragmentation every 5–7days. From passage 1–2, colonies acquired a flat shaped morphology, typical of primate iPS cells (Fig. 1A). Cells were routinely checked for mycoplasma contamination. Cells at passage 21 were negative for mycoplasma, as shown by a ratio readB/readA<1 as measured with MicoAlert R Sample Kit (Cambrex). Karyotype analysis was run on 22 metaphases of cells at passage 14, all showing a normal 42, XX chromosome number (Fig. 1B). Cells at passage 12 were Sendai virus and transgenes-free as tested by RT-PCR with specific primers (Fig. 1C); additionally, PCR of Macaca fascicularis microsatellites (Higashino et al. 2009) was performed to unequivocally prove their origin from the parental cell line (Fig. 1D). Endogenous expression of pluripotency-associated markers such as OCT3/4, NANOG, SOX2 and SSEA-4 was confirmed by immunofluorescence staining. Expression of SSEA1, rodent-specific pluripotency marker, was negative as expected (Fig. 1E). Furthermore, cells showed alkaline phosphatase activity (AP), that in combination with the expression of the above-mentioned genes is hallmark of pluripotency (Fig. 1F). Finally, pluripotency was tested in vivo by teratoma formation assay by injecting undifferentiated fragmented colonies into the hind limb of Rag2−/− ƔC−/− mice. After 8weeks, formed teratomas contained differentiated tissues from the three germ layers, as shown by histological analysis on tissue sections (Fig. 1G).
Materials and methods
Author disclosure statement
Acknowledgments We thank Gloria Gonzalez-Aseguinolaza and David Salas from Gene Therapy and Regulation of Gene Expression Program, CIMA, Pamplona, Spain, for providing the skin biopsies from Macaca fascicularis. This work was supported by the Ramón y Cajal State Program (MINECO, RYC-2015-17233) personal grant to XLA; by “Retos de la Sociedad” State Program (MINECO SAF2015-64224-R) project grant to XLA, by Juan de la Cierva Formación (FJCI-2014-22909) personal grant to GC and the Institute of Health Carlos III RETIC (RD12/0019/0031) project grant to FP.
Resource table Resource details An integration-free, virus-free rhesus macaque (Macacca mulata) induced pluripotent stem cell (riPSC) line was generated from rhesus embryonic fibroblasts (REFs). REFs were expanded from samples of embryonic skin, which was removed from the torso of an embryonic day 47 rhesus embryo at Carnegie stage 23 (embryo #34990). The REFs in this study were called REF90, and were reprogrammed using the non-infectious, non-viral self-replicating RNA originally derived from the Venezuelan equine encephalitis (VEE) virus (Yoshioka et al., 2013) and now commercially available as the product called Simplicon™ RNA reprogramming technology. The RNA replicon produces a synthetic RNA containing the reprograming factors OCT4, KLF4, SOX2, and GLIS1 (OKS-iG) as a polycistronic transcript. Using this technology, we reprogrammed REF90 cells with VEE-OKS-iG RNA and B18R RNA in the presence of human recombinant B18R protein for eighteen days (Fig. 1A). Pioneering riPSC colonies first appeared between day 14–18, and were picked on day 21 (Fig. 1A). Picked colonies were expanded in iPSC media on mytomycin C inactivated mouse embryonic fibroblasts (MEFs) and split every 5–7days. The established riPSC line which originated from a single colony was called riPSC90 to reflect its origin from REF90 (Fig. 1B). The picked colony was not grown from a single cell. riPSC90 (poor resolution) and REF90 (good resolution) were both normal male 42, XY karyotype (Fig. 1C). STR profiles for the parent fibroblast line, REF90 and the generated riPSC90 line were identical (Fig. S1A), but differed from the REF89/riPSC89 pair, which instead were identical to each other (Fig. S1B). Non-integration was confirmed by genomic PCR, through the absence of the virus-specific nonstructural protein 2 (NSP2) (Fig. 1D). riPSC90 expressed markers of self-renewal, such as OCT4, NANOG, SSEA4 and TRA-1-81 (Fig. 1E). To assess pluripotency, we performed a teratoma analysis by injecting undifferentiated colonies of riPSC90 into severe combined immunodeficient-beige (SCID-bg) mice. Using this assay, it was discovered that riPSC90 was competent to form teratomas, with evidence of ectoderm, mesoderm and endoderm by histology (Fig. 1F).