Cell-replacement therapy for diabetes: Generating functional insulin-producing tissue from adult human liver cells

Abstract

Shortage in tissue availability from cadaver donors and the need for life-long immunosuppression severely restrict the large-scale application of cell-replacement therapy for diabetic patients. This study suggests the potential use of adult human liver as alternate tissue for autologous beta-cell-replacement therapy. By using pancreatic and duodenal homeobox gene 1 (PDX-1) and soluble factors, we induced a comprehensive developmental shift of adult human liver cells into functional insulin-producing cells. PDX-1-treated human liver cells express insulin, store it in defined granules, and secrete the hormone in a glucose-regulated manner. When transplanted under the renal capsule of diabetic, immunodeficient mice, the cells ameliorated hyperglycemia for prolonged periods of time. Inducing developmental redirection of adult liver offers the potential of a cell-replacement therapy for diabetics by allowing the patient to be the donor of his own insulin-producing tissue.

Fig. 1.

PDX-1 activates the insulin promoter in human liver cells in vitro. Representative phase contrast morphology (A, C, and E), and green fluorescence (B, D, and F) of the same field of adult (A and B) and fetal (C and D) human liver cells infected by Ad-RIP-GFP-CMV-PDX-1. Arrows indicate the fluorescing cells. Ad-CMV-GFP-infected adult liver cells (E and F) represent the yield of infection at 500 moi used for all viral treatments in this study. Original magnifications are ×200 (A and B) and ×100 (C–F).

Fig. 2.

The promoting effect of SF on pancreatic hormones gene expression, insulin content, and secretion in AHL cells treated by Ad-CMV-PDX-1. (a) Quantitative real-time RT-PCR analyses of insulin, glucagon, and somatostatin gene expression levels. C_T (threshold cycle) values are normalized to β-actin gene expression within the same cDNA sample (n ≥ 30 in five different experiments). (b) Insulin content (n ≥ 10) and insulin secretion (n ≥ 25) by static incubations for 48 h. Ad-CMV-hIns-infected cells (500 moi) serve as constitutive control for human (pro)insulin production and secretion. Results are presented as fold of increase of the mean ± SD compared with untreated control liver cells.

Fig. 3.

TAHL express a wide repertoire of pancreatic gene expression. Quantitative RT-PCR analyses of pancreatic genes in THAL cells compared with untreated AHL and human islets demonstrated in ethidium bromide staining of agarose-separated PCR products (a) and quantitative analyses levels (b). C_T (threshold cycle) values are normalized to β-actin gene expression within the same cDNA sample. Results are presented as fold increase compared with untreated AHL cells, arbitrary set at 1.

Fig. 4.

TAHL cells produce, store, and secrete insulin and C-peptide in a glucose regulated manner. (a) Immunofluorescent staining for insulin (cytoplasmatic, red) and Pdx-1 (nuclear, green) in untreated AHL (A) and TAHL (B) cells. Nuclei are stained in blue (DAPI). Original magnifications are ×600 (A) and ×1,000 (B). (b) Electron microscopy of insulin ImmunoGold histochemistry in untreated AHL (A) and in TAHL (B and C) cells. C is a further magnification of an area in B. Arrows, ImmunoGold particles concentrated in granules, which appear in TAHL cells. (Scale bar, 0.25 μm.) (c) Static incubation of glucose or 2-deoxy-glucose (2-DOG) dose–response (0–30 mM) of C-peptide secretion. Results are presented as the mean ± SD; n = 30 in four different experiments.

Fig. 5.

TAHL cells ameliorate hyperglycemia in NOD-SCID mice. (a) Diabetic NOD-SCID mice were implanted under the kidney capsule with 7 × 10⁶ TAHL cells (n = 15) or with untreated AHL cells (n = 9). Glucose levels at the indicated time points after implantation are given as mean ± SE in mg percentage. Asterisks denote a significant difference (*, P < 0.05; **, P < 0.01) between the glucose levels of mice implanted by TAHL cells and these implanted by AHL cells. Dashed lines indicate glucose levels measured after nephrectomy (Nx) at the indicated time points. (b) Serum human C-peptide levels in mice implanted by TAHL cells (n = 10) or with untreated AHL cells (n = 7). (c) Immunohistochemical analysis of Pdx-1 (A) and insulin (B) in the kidney capsule sections, 10 days after transplantation of TAHL cells. (B) Enlarged magnification of insulin-positive liver cells. (C) Insulin staining of the same NOD-SCID mouse pancreas. Arrows indicate positive cell staining. Original magnifications are ×400 (A and B) and ×200 (C). (d) Glucose tolerance test in normoglycemic NOD-SCID controls (n = 3) and in mice implanted with TAHL cells (n = 3), 25–35 days after implantation (*, P < 0.1; **, P < 0.01).