Table 1.
Formulation of serum free differentiation medium containing specific compounds (SFDM).
Fig 1.
A triple combination of transcription factors (TFs) induces transdifferentiation into insulin-producing cells in vitro.
(A) Schematic diagram of the experimental strategy used to optimize the transgene. Adenoviruses encoding bicistronic TFs and nuclear localized GFP (nGFP) linked by an IRES element (I) were used. Each vector containing genes encoding Pdx1, Ngn3, NeuroD1, and MafA is abbreviated as P, N, D, and M, respectively. The combination of P, N, D, and M means the co-infection of each vector. (B) Appearance of nGFP-positive cells on the day after transfection. Scale bar, 200 μm. (C, D, E, F and G) Quantitative real-time polymerase chain reaction (qRT-PCR) analyses stratified according to the combination of TFs that were used were performed using primer sets for Ins1 (C), Ins2 (D), Gcg (E), Sst (F) and Ppy (G). “Pre” represents PBLHCs without transfection. The data are presented as fold-changes in the gene expressions relative to the values in the cells transfected with PNDM (n = 4). *P < 0.05 (one-way ANOVA followed by Dunnett’s test. The values in the cells transfected with PNDM were used as the control.); error bars, S.D. (H) Intracellular c-peptide contents in the transfected cells (n = 4). *P < 0.05 (one-way ANOVA followed by Dunnett’s test. The values in the cells transfected with PNDM were used as control.); error bars, S.D. The results obtained from βTC6, a mouse insulinoma cell line, were excluded from all the statistical analyses, because of the marked deviations from other values. CMV, cytomegaloviral promoter; Ins1, insulin I; Ins2; insulin II; Gcg, glucagon; Sst, somatostatin; Ppy, pancreatic polypeptide.
Fig 2.
The assessment of the synergistic effects between a 3D culture system and serum-free differentiation medium for cellular transdifferentiation.
(A) Adenoviral constructs for the polycistronic expression of three transcription factors (TFs). Dark gray bar, 2A peptide. (B) Schematic diagram of the procedures used to analyze the differences among culture systems for TFs-mediated transdifferentiation. (C) Representative microscopic images of infected cells treated with serum-free differentiation medium (SFDM) in two different culture formats. Arrow, cellular aggregate. Scale bars, 200 μm. (D and E) The analyses of insulin gene expressivity based on the difference in culture system. The expression of insulin I (Ins1: D) and insulin II (Ins2: E) was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) (n = 4). *P < 0.05 (one-way ANOVA followed by Dunnett’s test. The values in the cells treated with AC-SCM were used as control); error bars, S.D. (F) Intracellular c-peptide contents in infected cells (n = 4). *P < 0.05 (one-way ANOVA followed by Dunnett's test. The value of cells treated with AC-SCM was used as a control.); error bars, S.D. The results obtained from the βTC6 cells were excluded from all the statistical analyses, because of the marked deviations from other values. CMV, cytomegalovirus promoter; mCherry, monomeric cherry fluorescent protein; AC-SCM, adherent culture with serum-contained medium; AC-SFDM, adherent culture with serum-free differentiation medium; SC-SCM, suspension culture with serum-contained medium; SC-SFDM, suspension culture with serum-free differentiation medium.
Fig 3.
Comprehensive analyzes about extracellular environment of transdifferentiated cells.
(A) Schematic diagram of the formulation of SFDM. (B and C) The expression of Ins1 (B) and Ins2 (C) of infected cells treated with indicated supplements. The data are presented as fold-changes in the gene expression levels relative to the values in the cells treated with N2 and B27 supplement (n = 4). IH medium indicates the basal medium composed of IMDM and Hamm’s F12. The precise composition is shown in Table 1. *P < 0.05 (one-way ANOVA followed by Dunnett's test. The value of cells treated with N2 and B27 supplements was used as a control.); error bars, S.D. (D) Differences in the intracellular c-peptide contents depending on the supplements added (n = 4). The values in the cells treated with N2 and B27 supplement were used as control. *P < 0.05 (one-way ANOVA followed by Dunnett’s test); error bars, S.D. (E, F, G and H) Influence on expression of insulin gene by individually removing seven molecules contained in SFDM. The Ins1 (E and G) and Ins2 (F and H) gene expressivity was analyzed when each factor was individually removed. M7 contained epidermal growth factor (EGF), exendin-4, forskolin, L685,458, nicotinamide, noggin, and SB431542. M4 contained exendin-4, forskolin, L685,458, and noggin. The data are presented as fold-changes in the gene expression levels relative to the values in the cells treated with M7 (E, F) or M4 (G, H) (n = 4). *P < 0.05 (one-way ANOVA followed by Dunnett's test. The value of cells treated with M7 [E, F] or M4 [G, H] was used as a control.); error bars, S.D. (I) Differences in the intracellular c-peptide contents depending on the supplements added (n = 4). The values in the cells treated with M7 were used as control. M3 contained exendin-4, L685,458 and noggin. *P < 0.05 (one-way ANOVA followed by Dunnett’s test. The values in the cells treated with M7 were used as control); error bars, S.D. The results obtained from the βTC6 cells were excluded from all the statistical analyses, because of the marked deviations from other values. B27, B27 supplement; N2, N2 supplement.
Fig 4.
The effect of simultaneous administration of N2 and M3 for cellular transdifferentiation.
(A, B, C and D) The expression of Ins1 (A), Ins2 (B), Pcsk1 (C), and Pcsk2 (D) of infected cells treated with indicated supplements. The data are presented as fold-changes in the gene expression levels relative to the values in the untreated cells (n = 4). M3 contained exendin-4, L685,458, and noggin. IH medium indicates the basal medium composed of IMDM and Hamm's F12. The detailed composition is listed in Table 1. *P < 0.05 (one-way ANOVA followed by Dunnett’s test. The values in the untreated cells were used as control.); error bars, S.D. (E) Intracellular c-peptide contents in infected cells treated with indicated supplements (n = 4). *P < 0.05 (one-way ANOVA followed by Dunnett’s test. The value of untreated cells was used as a control.); error bars, S.D. (F and G) Immunostaining with c-peptide and Ngn3 antibody for transdifferentiated cells treated with N2 and M3 (F) or without (G). Scale bars, 200 μm. (H and I) Immunostaining with c-peptide antibody in dispersed single-cells from cellular aggregates treated (H) or not treated (I) with N2 and M3. Scale bars, 200 μm. (J) Measurement of the percentage of c-peptide-positive cells in the presence or absence of N2+M3 treatment (n = 5 independent experiments). *P < 0.05 (t-test); error bars, S.D. (K) Insulin secretion by transdifferentiated cells in response to the indicated concentrations of glucose (n = 4). *P < 0.05 (t-test); error bars, S.D. The results obtained from the βTC6 cells were excluded from all the statistical analyses, because of the marked deviations from other values. N2, N2 supplement; Pcsk1, proprotein convertase subtilisin/kexin type 1; Pcsk2, proprotein convertase subtilisin/kexin type 2; C-P, c-peptide; Ngn3, neurogenin 3.
Fig 5.
Expressions of pancreas-related genes in the transdifferentiated cells.
The data are presented as the fold-change in gene expression relative to the value in the transfected cells not treated with N2+M3 (n = 4). M3 contained exendin-4, L685,458, and noggin. IH medium indicates the basal medium composed of IMDM and Hamm's F12. The detailed composition is listed in Table 1. *P < 0.05 (t-test or Mann-Whitney U test, as appropriate); error bars, S.D. The results obtained from the βTC6 cells were excluded from all the statistical analyses, because of the marked deviations from other values. N2, N2 supplement; VI, viral infection; Ins1, insulin I; Ins2, insulin II; Pcsk1, proprotein convertase subtilisin/kexin type 1; Pcsk2, proprotein convertase subtilisin/kexin type 2; Abcc8, the ATP-binding cassette sub-family C member 8; Slc2a2, solute carrier family 2 member 2; NeuroD1, neurogenic differentiation 1; Pax4, paired box gene 4; Pax6, paired box gene 6; Isl1, islet 1; Sst, somatostatin; Ppy, pancreatic polypeptide; Pdx1, pancreas duodenal homeobox 1; Ngn3, neurogenin 3; MafA, v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog A; Arx, aristaless related homeobox.
Fig 6.
The gene expressions of liver-related genes in the transdifferentiated cells.
The data are presented as fold-changes in the gene expression levels relative to the values of the transfected cells not treated with N2+M3 (n = 4). M3 contained exendin-4, L685,458, and noggin. IH medium indicates the basal medium composed of IMDM and Hamm's F12. The detailed composition is listed in Table 1. *P < 0.05 (t-test or Mann-Whitney U test, as appropriate); error bars, S.D. The results obtained from the βTC6 cells were excluded from all the statistical analyses, because of the marked deviations from other values. N2, N2 supplement; VI, viral infection; Alb, albumin; Cyp1a2, cytochrome P450 1A2; Cebpb, CCAAT/enhancer-binding protein β; Trf, transferrin; Krt19, keratin 19; Afp, alpha-fetoprotein.
Fig 7.
Transdifferentiated cells ameliorate hyperglycemia in diabetic mouse.
(A and B) The non-fasting blood glucose levels (A) and body weight (B) in diabetic mice. The red line with squares indicates the values obtained from the mice transplanted with N2 and M3-treated transdifferentiated cells (n = 5), and the blue line with circles indicates those obtained from the untreated control mice (n = 5). M3 contained exendin-4, L685,458, and noggin. *P < 0.05 (t-test); error bars, S.D. (C and D) Immunostaining with insulin antibody for transplanted transdifferentiated cells treated with N2 and M3. Arrow, insulin-positive cells. Scale bar, 50 μm. STZ, streptozotocin; Tx, transplantation; N2, N2 supplement; INS, insulin.
Fig 8.
Analysis of the time-course of changes in the insulin-producing ability of the N2+M3-treated transdifferentiated cells.
(A and B) The expression levels of Ins1 (A) and Ins2 (B) in the N2+M3-treated transdifferentiated cells at different time-points. The data are presented as fold-changes in the gene expression levels relative to the values in the cells measured on day 7 days after re-seeding (n = 4). M3 contained exendin-4, L685,458, and noggin. IH medium indicates the basal medium composed of IMDM and Hamm's F12. The precise composition is listed in Table 1. *P < 0.05 (one-way ANOVA followed by Dunnett’s test. The values in the cells on day 7 days after re-seeding were used as the control.); error bars, S.D. (C) Intracellular c-peptide contents in the N2+M3-treated transdifferentiated cells at different time-points (n = 4). *P < 0.05 (one-way ANOVA followed by Dunnett’s test. The values in the cells on day 7 after re-seeding were used as the control.); error bars, S.D. (D to K) Representative microscopic images (D, E, F, and G) and immunostaining of the transdifferentiated cells treated continually with N2 and M3 for c-peptide and Ngn3 (H, I, J, and K) at four different time-points. The results obtained from βTC6 were excluded from all the statistical analyses, because of the marked deviations from other values. N2, N2 supplement; C-P, c-peptide; Ngn3, neurogenin 3.