Supplementary MaterialsSupplemental data jci-130-130767-s254

Supplementary MaterialsSupplemental data jci-130-130767-s254. exhibit high levels of characteristic mDA markers, produce and secrete dopamine, and exhibit electrophysiological features common of mDA cells. Transplantation of these cells into rodent models of PD robustly restores motor function and reinnervates host brain, while showing no evidence of tumor formation or redistribution of the implanted cells. We propose that this platform is suitable for the successful implementation of human personalized autologous cell therapy for PD. = 5. * 0.05; ** 0.01, 1-way ANOVA with Tukeys post test. (E and F) Time course of OCR (E) and ECAR (F) in hDFs infected with Y4F, miR-302s, and/or miR-200c. Mean SD. = 3. * 0.05; ** 0.01; *** 0.005, 2-way ANOVA with Tukeys post test. (G) Percentage of TRA-1-60+ colonies among AP+ colonies following lentiviral contamination encoding Y4F, Y4F+3, or Y4F+3+2. Mean SD. = 6. *** 0.005, 2-way ANOVA with Tukeys post RGFP966 test. (H) Percentage of TRA-1-60+ colonies among AP+ colonies following transfection with episomal vectors encoding Y4F, Y4F+3, or Y4F+3+2. Mean SD. = 4. ** 0.01, 2-way ANOVA with Tukeys post test. We next tested to determine whether this combination (Y4F+3+2) could generate high-quality hiPSCs using nonviral vectors. We developed 2 episomal vectors harboring Y4F on 1 vector (pY4F; Supplemental Physique 2C) and miR-302s and miR-200c clusters around the other (p3+2; Supplemental Physique 2D). Because of the known transformation activity RGFP966 of c-Myc (26), we RGFP966 replaced it with L-MYC on pY4F. We thus established an episomal reprogramming protocol using single transfection with these 2 vectors (Supplemental Physique 2E) that efficiently reprogrammed hDFs to hiPSC colonies that were more than 90% AP+TRA-1-60+ (Physique 1H). We selected hiPSC lines with hESC-like morphology generated by Y4F, Y4F+3, and Y4F+3+2, passaged them more than 20 occasions, and characterized their properties. As shown in Physique 2, A and B, their morphologies and expression levels of pluripotency markers closely resembled those of H9 hESC. Interestingly, H9 and hiPSCs generated by Y4F+3+2 differentiated RGFP966 equally well to all 3 germ layer lineages, while differentiation of those generated by Y4F or Y4F+3 was skewed toward mesodermal lineage, as evidenced by (a) staining with antibodies against the 3 germ layer markers and (b) gene expression of lineage-specific markers (Physique 2, C and D). These results suggest that the Y4F+3+2 combination enables the generation of higher quality hiPSCs from both newborn and adult human fibroblasts with less biased differentiation potential, regardless of the delivery vector, compared with conventional methods (Y4F or Y4F+3) (Supplemental Desk 1). Open up in another window Body 2 Top quality hiPSC Rabbit Polyclonal to MASTL lines generated from our improved reprogramming technique.(A) Heatmaps depicting gene expression degrees of pluripotency markers among established hiPSC lines weighed against the initial hDFs and an hESC line (H9). = 3. (B) Immunostaining of hiPSC lines generated by different combos with particular antibodies against pluripotency markers (e.g., OCT4, NANOG, TRA-1-60, and SOX2) along with Hoechst 33342 nuclear staining (insets). Size pubs: 100 m. (C) Immunostaining for lineage-specific markers for ectoderm (OTX2), mesoderm (BRACHYURY), and endoderm (SOX17) following spontaneous differentiation for 7 days. Level bars: 100 m. (D) Heatmaps depicting gene expression levels of early differentiation markers of ectoderm (PAX6 and MAP2), endoderm (FOXA2, SOX17, and CK8), and mesoderm markers (MSX1, MYL2A, and COL6A2) in hiPSC lines generated by pY4F, pY4F+3, or pY4F+3+2. = 2. Genomic integrity and somatic mutations in hiPSCs. To determine whether our reprogramming method can reliably generate clinical grade hiPSCs, we attempted to generate hiPSC lines using adult hDFs from multiple sources, including 9 fibroblast lines from your Coriell Institute (3 familial PD, 3 sporadic PD, and 3 healthy subjects) and 4 samples from new skin biopsies (3 healthy subjects and 1 sporadic PD patient). As shown in Supplemental Table 2 and Supplemental Physique 3, A and B, our method generated multiple hiPSC lines from all of these fibroblasts using a 1-time transfection with pY4F and p3+2 (Supplemental Physique 2E), all displaying hESC-like morphology and prominent expression of pluripotent markers, including OCT4, TRA-1-60, NANOG, and SSEA-4. Focusing on personalized cell therapy, we further characterized hiPSC clones made from skin biopsy of a sporadic PD patient (MCL540 in Supplemental Table 2). A fundamental criterion for clinical grade hiPSCs is usually maintenance of genomic integrity and absence of harmful (e.g., reported malignancy causing) mutation(s) (7, 17). As an example, we tested 5 impartial hiPSC clones that were passaged approximately 20 occasions since the initial isolation from MCL540 (N17, C4, N3, C11, and C5) as well as control cells (parental fibroblasts and H9) for potential integration of vector DNAs into.