In vivo characterization of transplanted human embryonic stem cell-derived pancreatic endocrine islet cells

Abstract

Objectives: Islet-like clusters (ILCs), differentiated from human embryonic stem cells (hESCs), were characterized both before and after transplantation under the kidney capsule of streptozotocin-induced diabetic immuno-incompetent mice.

Materials and methods: Multiple independent ILC preparations (n = 8) were characterized by immunohistochemistry, flow cytometry and cell insulin content, with six preparations transplanted into diabetic mice (n = 42), compared to controls, which were transplanted with either a human fibroblast cell line or undifferentiated hESCs (n = 28).

Results: Prior to transplantation, ILCs were immunoreactive for the islet hormones insulin, C-peptide and glucagon, and for the ductal epithelial marker cytokeratin-19. ILCs also had cellular insulin contents similar to or higher than human foetal islets. Expression of islet and pancreas-specific cell markers was maintained for 70 days post-transplantation. The mean survival of recipients was increased by transplanted ILCs as compared to transplanted human fibroblast cells (P < 0.0001), or undifferentiated hESCs (P < 0.042). Graft function was confirmed by secretion of human C-peptide in response to an oral bolus of glucose.

Conclusions: hESC-derived ILC grafts continued to contain cells that were positive for islet endocrine hormones and were shown to be functional by their ability to secrete human C-peptide. Further enrichment and maturation of ILCs could lead to generation of a sufficient source of insulin-producing cells for transplantation into patients with type 1 diabetes.

Figure 1

Photomicrographs of hESC‐derived ILCs after 36 days of in vitro differentiation and immediately prior to transplantation. Serial sections stained by immunohistochemistry for: (a) insulin, (b) glucagon, (c) human C‐peptide, (d) pro‐hormone convertase 1/3, (e) pro‐hormone convertase 2, (f) cytokeratin‐19 and (g) representative isotype control. Magnification ×200; scale bar indicates 100 microns.

Figure 2

Photomicrographs of a hESC‐derived ILC graft‐bearing kidney removed 1‐day post‐transplantation. Serial sections double stained with anti‐insulin (green) and antiglucagon (red) or double stained with antihuman‐C‐peptide (red) and anti‐insulin (green), specific antibodies as indicated. Magnification ×400; scale bar indicates 50 microns.

Figure 3

Electron photomicrographs of hESC‐derived ILCs after 36 days of in vitro differentiation and prior to transplantation. β‐like cells within the ILC appear well granulated, structurally intact containing both mature (solid arrow) and immature (open arrow) secretory granules, typical for adult islet β‐cells. Scale bar indicates 1 micron.

Figure 4

Mean survival (a) and the Kaplan Meier curve of survival (b) of mice transplanted with either hESC derived ILCs (n = 42), a human fibroblast cell line (hFCs, n = 21) or undifferentiated hESC (uhESC, n = 6). For the Kaplan Meier curve (4B), the log‐rank (Mantel‐Cox) test of the equality of survival distributions for the different levels of group denotes P < 0.0001. Of the 42 animals transplanted with ILCs, 7 were found dead at various time points, 19 healthy mice were euthanized at defined time points after 50 days post‐transplant then 3 and 2 mice were euthanized due to illness before or after 50 days post‐transplant, respectively. Lastly, 11 healthy mice in this group were euthanized at predetermined time points before 50 days post transplant and were not included in this graph. Human serum C‐peptide was also determined in mice transplanted with ILCs (n = 12) or control mice implanted with hFCs (n = 9, c). *P < 0.0001, †P < 0.042.

Figure 5

Morphological assessment of differentiated ILC‐derived grafts in mice following at least 40 days post‐transplantation. Data are expressed as percentage of grafts with positive staining for specific pancreatic markers from a total of 23 mice.

Figure 6

Photomicrographs of differentiated ILC‐derived grafts at 56 days post‐transplantation. (a) Insulin, (b) glucagon, (c) human C‐peptide, (d) pro‐hormone convertase 1/3, (e) pro‐hormone convertase 2, (f) somatostatin, (g) synaptophysin, (h) cytokeratin‐19, (I) PDX‐1 and (j) representative isotype control. Arrows indicate areas of cytokeratin‐19 staining which are comparable to the ductal human pancreas. Magnification ×200; scale bar indicates 100 microns.