Reprogramming of mice primary hepatocytes into insulin-producing cells by transfection with multicistronic vectors

Abstract

The neogenesis of insulin-producing cells (IPCs) from non-beta-cells has emerged as a potential method for treating diabetes mellitus (DM). Many groups have documented that activation of pancreatic transcription factor(s) in hepatocytes can improve the hyperglycemia in diabetic mice. In the present study, we explored a novel protocol that reprogrammed primary hepatocytes into functional IPCs by using multicistronic vectors carrying pancreatic and duodenal homeobox-1 (Pdx1), neurogenin 3 (Ngn3), and v-musculoaponeurotic fibrosarcoma oncogene homolog A (MafA). These triple-transfected cells activated multiple beta-cell genes, synthesized and stored considerable amounts of insulin, and released the hormone in a glucose-regulated manner in vitro. Furthermore, when transplanted into streptozotocin-induced diabetic mice, the cells markedly ameliorated glucose tolerance. Our results indicated that ectopic expression of Pdx1, Ngn3, and MafA facilitated hepatocytes-to-IPCs reprogramming. This approach may offer opportunities for treatment of DM.

Figure 1

Flow cytometry quantitation of GFP-positive cells demonstrates that treatment with GFP results in a 16-fold higher number, compared with those without GFP; n = 3.

Figure 2

Gene expression profiles in transfected hepatocytes. Total RNA was isolated from hepatocytes treated with pPdx1 (P), pNgn3 (N), pMafA (M), pNgn3+Pdx1 (NP), pMafA+Pdx1 (MP), pMafA+Ngn3 (MN), pMafA+Ngn3+Pdx1 (MNP), and null vector (NV).

Figure 3

Relative levels to mice islet of endogenous pancreatic markers in 5-day cultured hepatocytes, evaluated by real-time PCR; n = 3. *P < 0.05 versus MNP.

Figure 4

Protein levels of Ins1 and Ins2 in hepatocytes on day 5 after transfection, evaluated by Western blotting, with mice islet as positive control.

Figure 5

Evaluation of insulin synthesis and secretary ability in transfected cells. (a) Measurements of intracellular C-peptide were conducted by an ELISA kit; n = 3. (b) Glucose-induced insulin secretion from transfected hepatocytes. Values are mean ± SD of insulin in 3 different experiments, relative to 0 mM glucose. *P < 0.05.

Figure 6

Effects on blood glucose levels by transplanting IPCs into diabetic mice at different times. A glucose tolerance test was performed, and glucose levels were determined from blood drawn from the tail vein. (a) Fasting blood glucose of mice until day 19. ((b) and (c)) Glucose levels during a glucose tolerance test of mice on days 7 and 19. Data are presented as mean ± SD. *P > 0.05 versus NV;  ^# P > 0.05 versus nondiabetes.