Skin fibroblasts from patients with type 1 diabetes (T1D) can be chemically transdifferentiated into insulin-expressing clusters: a transgene-free approach

Abstract

The conversion of differentiated cells into insulin-producing cells is a promising approach for the autologous replacement of pancreatic cells in patients with type 1 diabetes (T1D). At present, cellular reprogramming strategies encompass ethical problems, epigenetic failure or teratoma formation, which has prompted the development of new approaches. Here, we report a novel technique for the conversion of skin fibroblasts from T1D patients into insulin-expressing clusters using only drug-based induction. Our results demonstrate that skin fibroblasts from diabetic patients have pancreatic differentiation capacities and avoid the necessity of using transgenic strategies, stem cell sources or global demethylation steps. These findings open new possibilities for studying diabetes mechanisms, drug screenings and ultimately autologous transgenic-free regenerative medicine therapies in patients with T1D.

Figure 1. Original fibroblasts population and chemical transdifferentiation.

(A) Characterization of the original fibroblast population in the initial cell cultures by immunocytochemistry. (B) Time schedule of chemical transdifferentiation protocol. (C) Morphology of chemically treated fibroblast obtained from a type 1 diabetes patient at day 12. (D) Typical image of treated cells at day 24. The cells migrate to the surface of the petri dish and begin to form islet-like clusters. (E) Typical image of the transdifferentiated cells with clusters morphology obtained by the end of the treatment (day 30). (F) Control treated fibroblast without chemical induction. HF1 and HF2: fibroblasts from patients with type 1 diabetes. Transdiferentiated: fibroblasts chemically transdifferentiated for 30 days; Untreated: fibroblasts day 2–5; Control treated: fibroblasts cultured for 30 days without chemical induction (Bar 200 µm). TM: Transdifferentiation Medium.

Figure 2. Gene expression analyses of fibroblasts before and after chemical transdifferentiation.

(A) Gene-expression profiles of transdifferentiated in vitro fibroblasts from patient whit diabetes type 1 (patient HF1, tree replicates) and parental controls fibroblasts by RT-PCR analysis. (B) Gene-expression profiles of transdifferentiated fibroblasts (n = 6 replicates) and untreated control fibroblasts (n = 3 replicates) by quantitative PCR analysis in pancreatic genes (INS, GCG) and fibroblast markers (MEOX, ASPN) (t test, P<0.05). The bars represent the standard errors of independent experiments. (C) Hierarchical clustering of differentially expressed genes. Using normalized value, plotted a heat map of hierarchical clustering on distance similarity for samples and probes. (D) Plotted a scatter plot of expression level between groups. The red dots indicate significant probes (2-Fold). (E) Venn diagram for overlapped upregulated (up) and downregulated (down) gene expression in transdifferentiated cells groups. HF1 and HF2: fibroblasts from patients with type 1 diabetes; Transdifferentiated: fibroblasts chemically transdifferentiated for 30 days; Untreated: fibroblasts day 2–5; Control treated: fibroblasts cultured for 30 days without chemical induction. Rep: Replicates.

Figure 3. Methylation analysis of PDX1, OCT4 and NANOG proximal promoters in fibroblasts before and after chemical transdifferentiation.

(A-C) Hypomethylation of PDX1, OCT4 and NANOG proximal promoters in untreated skin fibroblasts from diabetic patients (HF1 and HF2). Lack of expression of these genes was found for untreated fibroblasts. The bars represents the average and standard deviations of three (PDX1) and two (OCT4 and NANOG) independent sequencing procedures. (D) Direct sequencing analysis for two PDX+ reprogrammed cell lines from the same diabetic patient (HF1). No tendency were found on OCT4 (E) and NANOG (F) proximal promoters. 5mC site: 5 methylation Citocine; Rep: Repetition.

Figure 4. Analyses of transdifferentated cluster before and after transplantation in NUDE mouse.

(A) Typical images of in vitro immunostaining for glucagon in human pancreas control, untreated fibroblasts and in chemically transdifferentiated fibroblasts from patient with type 1 diabetes. (B) G-banding karyotype in HF2 patient before and after chemical transdifferentiation treatment showing normal male karyotypes. (C) Survival and presence of the transplanted cells in the mouse pancreas by qPCR using specific primers for human SRY and β-actin genes. Analyses of streptozotocin (STZ)-treated mice that were transplanted with insulin-expressing cells transdifferentiated from fibroblasts of patients with type 1 diabetes. Mice were inspected daily for weight loss (D) and basal blood glucose levels (E). Human insulin serum was measured at 30 min after intraperitoneal glucose stimulation pre and post STZ treatment (15 and 30 day post implant) (F) and in nonfasting and 30 min after glucose stimulation at day 30 (G, glucose challenge). Error bars indicate s.d. HF1 and HF2: fibroblasts from patients with type 1 diabetes; Transdifferentiated: fibroblasts chemically transdifferentiated for 30 days: Untreated: fibroblasts day 2–5.