Growth inhibitors promote differentiation of insulin-producing tissue from embryonic stem cells

Abstract

The use of embryonic stem cells for cell-replacement therapy in diseases like diabetes mellitus requires methods to control the development of multipotent cells. We report that treatment of mouse embryonic stem cells with inhibitors of phosphoinositide 3-kinase, an essential intracellular signaling regulator, produced cells that resembled pancreatic beta cells in several ways. These cells aggregated in structures similar, but not identical, to pancreatic islets of Langerhans, produced insulin at levels far greater than previously reported, and displayed glucose-dependent insulin release in vitro. Transplantation of these cell aggregates increased circulating insulin levels, reduced weight loss, improved glycemic control, and completely rescued survival in mice with diabetes mellitus. Graft removal resulted in rapid relapse and death. Graft analysis revealed that transplanted insulin-producing cells remained differentiated, enlarged, and did not form detectable tumors. These results provide evidence that embryonic stem cells can serve as the source of insulin-producing replacement tissue in an experimental model of diabetes mellitus. Strategies for producing cells that can replace islet functions described here can be adapted for similar uses with human cells.

Fig 1.

Similarities of gene expression and mitotic status in pancreatic β cells and insulin-producing cells comprising stage 5NL IPCCs. (A–C) Dithizone staining of isolated pancreatic islets (A), stage 5NL IPCCs (B), and stage 5NB IPCCs (C). Magnification in A–C is equal. Images in D–R were obtained by confocal microscopy and are representative of at least 12 samples for each immunohistochemical probe. Samples were processed identically and in parallel for each immunohistochemical probe. Shown is simultaneous immunofluorescent detection of insulin and glucagon (D–F), α-fetoprotein (α-FP) (G–I), MAP2 (J–L), or β tubIII (M–O). In mature islets, there is localized expression of insulin and glucagon (D) but no detectable expression of α-FP (G) or the neuronal markers MAP2 (J) and β tubIII (M). Stage 5NL IPCCs cellular homogeneity (B, E, H, K, and N) contrasts with cellular heterogeneity of stage 5NB IPCCs (F, I, L, and O). (P–R) Immunofluorescent detection of Ki67, a nuclear marker of proliferating cells. Stage 5NL IPCCs (Q) are predominantly nonproliferating, similar to mature pancreatic islets (P), whereas stage 5NB culture conditions result in significant numbers of proliferating cells (R). Magnification in D–R is equal.

Fig 2.

Stage 5NL IPCCs express several characteristic pancreatic β cell markers. Images in A–J were obtained by confocal microscopy and are representative of at least 12 samples for each immunohistochemical probe. Samples were processed identically and in parallel for each immunohistochemical probe. Immunofluorescent staining of adult pancreatic islets and stage 5NL IPCCs for insulin and C-peptide (A and B), insulin and proinsulin (C and D), Pdx1 (E and F), GLUT2 (G and H), and glucokinase (I and J). (K) RT-PCR analysis of IPCC gene expression during stages 1–4, and in handpicked stage 5NB and stage 5NL IPCCs, and wild-type pancreatic islets. Islet-1 and neurogenin-3 are transcription factors required for islet formation in mice (29, 30) and nestin is an intermediary filament protein (2). In stage 5 IPCCs, we detected carboxypeptidase A but not pancreatic amylase, both products of pancreatic exocrine (acinar) cells. Amylase transcripts are routinely detected by RT-PCR in pancreatic islet preparations because of adherent acinar cells. Other gene products are described in the text. (L) insulin release by IPCCs on exposure to 25 mM glucose. Results show insulin release by 10 IPCCs per time point.

Fig 3.

IPCC graft analysis. (A) Teratoma (white arrows) formation 3 weeks after transplantation of 300 stage 5NB IPCCs under the left kidney capsule (pink tissue to left of tumor). (B) Lack of tumor growth after renal transplantation of 300 stage 5NL IPCCs (graft indicated by dashed lines and white arrow). (C) Immunohistochemical detection of insulin (green) and glucagon (red) expression in stage 5NL IPCCs 3 weeks after engraftment. Cell nuclei appear as black central spots surrounded by insulin, indicating cytoplasmic immunostaining. (D) Immunohistochemical detection of insulin (green) and C-peptide (red) expression in stage 5NL IPCCs 3 weeks after engraftment. (E–G) Hematoxylin and eosin staining of sectioned stage 5NL IPCC before transplantation (E) and sectioned stage 5NL IPCC grafts recovered from subcapsular renal transplantation site (F), compared with a sectioned adult pancreatic islet (G). Magnification in E–G is equal.

Fig 4.

Transplanted stage 5NL IPCCs function in vivo to rescue and ameliorate diabetic phenotypes. (A) Kaplan–Meier survival distribution after stage 5NL IPCC transplantation (n = 7, black lines), pancreatic islet transplantation (n = 3, blue lines), or sham transplant (n = 17, red lines). IPCC graft removal 3 weeks after engraftment resulted in increased mortality, in contrast to unilateral nephrectomy in surviving control sham-transplanted mice. (B) Random-fed blood glucose levels in STZ-treated mice with transplantation of stage 5NL IPCCs (n = 7, black lines), of 40 pancreatic islets (n = 3, blue lines), or in surviving sham-transplanted mice (n = 8, red lines). Comparing sham-transplanted mice to IPCC-transplanted mice: **, P < 0.01; ***, P < 0.005. In sham-transplanted mice that did not survive, average blood glucose on the day preceding death was 532 ± 33 mg/dl (n = 4). IPCC graft removal 3 weeks after transplantation resulted in comparable hyperglycemia (674 ± 117 mg/dl, n = 4). (C) i.p. glucose challenge in fasted mice 21 days after transplant with IPCCs (n = 7; black squares) or sham-transplant (n = 14; red circles). Blood glucose level was 135 ± 12 mg/dl in untreated control mice (n = 6) during random-feeding, and 85 ± 10 mg/dl (n = 6) after 15 h overnight fasting. **, P < 0.01; *, P < 0.05.