Reversal of diabetes following transplantation of an insulin-secreting human liver cell line: Melligen cells

Abstract

As an alternative to the transplantation of islets, a human liver cell line has been genetically engineered to reverse type 1 diabetes (TID). The initial liver cell line (Huh7ins) commenced secretion of insulin in response to a glucose concentration of 2.5 mmol/l. After transfection of the Huh7ins cells with human islet glucokinase, the resultant Melligen cells secreted insulin in response to glucose within the physiological range; commencing at 4.25 mmol/l. Melligen cells exhibited increased glucokinase enzymatic activity in response to physiological glucose concentrations, as compared with Huh7ins cells. When transplanted into diabetic immunoincompetent mice, Melligen cells restored normoglycemia. Quantitative real-time polymerase chain reaction (qRT-PCR) revealed that both cell lines expressed a range of β-cell transcription factors and pancreatic hormones. Exposure of Melligen and Huh7ins cells to proinflammatory cytokines (TNF-α, IL-1β, and IFN-γ) affected neither their viability nor their ability to secrete insulin to glucose. Gene expression (microarray and qRT-PCR) analyses indicated the survival of Melligen cells in the presence of known β-cell cytotoxins was associated with the expression of NF-κB and antiapoptotic genes (such as BIRC3). This study describes the successful generation of an artificial β-cell line, which, if encapsulated to avoid allograft rejection, may offer a clinically applicable cure for T1D.

Figure 1

Insulin secretion from liver cell lines: Insulin secretion from (a) Huh7ins and (b) Melligen cells in response to increasing concentrations of glucose: 1.5–20 mmol/l. (c) Insulin secretion from Melligen cells in response to increasing concentrations of glucose: 3.75–5.0 mmol/l. Values are expressed as means ± SE (n = 6).

Figure 2

Reversal of diabetes in NOD/scid mice following transplantation of Huh7ins and Melligen cells: (a) Blood glucose levels of NOD/scid mice transplanted with Huh7ins and Melligen cells and diabetic controls. Grafts of Huh7ins and Melligen cells were removed at 27 days. (b) Intraperitoneal glucose tolerance test in mice transplanted with Huh7ins and Melligen cells, normal and diabetic controls. Values are expressed as means ± SE (n = 8).

Figure 3

Expression of pancreatic hormones following reversal of diabetes in NOD/scid mice following transplantation of Melligen cells. (a–c) Photomicrographs of triple anti-insulin (INS), anti-somatostatin (SST), and anti-glucagon (GLUC) staining of (a) Melligen graft, (b) normal mouse pancreas, and (c) diabetic mouse pancreas; original magnification, 400×. (d) Transmission electron micrograph showing secretory vesicles with dense granules (g) (bar = 250 nm) in Melligen grafts, 30 days after transplant. (e) Immunoelectron micrograph showing localization of insulin in Melligen grafts, 30 days after transplant (bar = 300 nm).

Figure 4

Protein expression of glucokinase in liver cell lines. Western blot analysis for (a) human glucokinase in Huh7ins cells (lane 1), Huh7 ins cells with pIRlSpuro3 vector only (lane 2), Melligen cells (lane 3), and human islets (lane 4) and (b) GADPH. Human glucokinase generated a band at 52 kDa and GADPH a band of 36 kDa. GADPH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 5

Enzyme activity of glucokinase in liver cell lines. Glucokinase activity of Huh7, Huh7ins, Huh7ins (with pIRlSpuro3 vector only), and Melligen cells in the presence of (a) 20 mmol/l glucose and (b) 20 mmol/l glucose plus 10 mmol/l glucose-6-phosphate. Mean ± SEM, n = 6.

Figure 6

The effect of cytokine treatment on glucose responsiveness in MIN6, Huh7ins, and Melligen cells. Glucose responsiveness was determined using 20 mmol/l glucose stimulus with and without cytokine treatment for (a) MIN6 cells, (b) Huh7ins cells, and (c) Melligen cells after 10 days incubation with triple cytokine culture medium (TCCM). The 10-day cytokine treatment did not affect (d) Huh7ins and Melligen cell responsiveness to glucose in the millimolar range with and without the TCCM treatment. Results are expressed as mean ± SE, n = 3 independent experiments.

Figure 7

β-cell transcription factors and pancreatic hormones expressed in liver cell lines. (a) RT-PCR detection of mouse β-cell transcription factors (Pdx1, Neurod1, Neurog3, Nkx2-2, Nkx6-1, Pax 4, Pax 6, MAFA, MAFB), the pancreatic endocrine hormones: insulin (Ins), somatostatin (Sst), glucagon (Gcg) and pancreatic polypeptide (Ppy); GLUT 2 (Slc2a2) and liver glucokinase (L-Gck); islet glucokinase (I-Gck), insulin proconvertases 1 and 2 (Pc1 and Pc2); exocrine marker p48 and Actb in Huh7 cells (lane 1), Huh7ins cells (lane 2), Melligen cells (lane 3) and human islets (lane 4). (b) RT-PCR analysis of human insulin and human islet glucokinase in transplanted Huh7ins cells (lane 1), transplanted Melligen cells (lane 2) and human islets (lane 3); (c) RT-PCR analysis of cytokine receptor expression, IFNR1, IFNR2, IL1R1, IL1R2, TNFR1 and TNFR2, in human islets (lane 1), Huh7 cells (lane 2), Huh7ins cells (lane 3), and Melligen cells (lane 4). RT-PCR, real-time polymerase chain reaction.