Insulin-Producing Endocrine Cells Differentiated In Vitro From Human Embryonic Stem Cells Function in Macroencapsulation Devices In Vivo

Abstract

The PEC-01 cell population, differentiated from human embryonic stem cells (hESCs), contains pancreatic progenitors (PPs) that, when loaded into macroencapsulation devices (to produce the VC-01 candidate product) and transplanted into mice, can mature into glucose-responsive insulin-secreting cells and other pancreatic endocrine cells involved in glucose metabolism. We modified the protocol for making PEC-01 cells such that 73%-80% of the cell population consisted of PDX1-positive (PDX1+) and NKX6.1+ PPs. The PPs were further differentiated to islet-like cells (ICs) that reproducibly contained 73%-89% endocrine cells, of which approximately 40%-50% expressed insulin. A large fraction of these insulin-positive cells were single hormone-positive and expressed the transcription factors PDX1 and NKX6.1. To preclude a significant contribution of progenitors to the in vivo function of ICs, we used a simple enrichment process to remove remaining PPs, yielding aggregates that contained 93%-98% endocrine cells and 1%-3% progenitors. Enriched ICs, when encapsulated and implanted into mice, functioned similarly to the VC-01 candidate product, demonstrating conclusively that in vitro-produced hESC-derived insulin-producing cells can mature and function in vivo in devices. A scaled version of our suspension culture was used, and the endocrine aggregates could be cryopreserved and retain functionality. Although ICs expressed multiple important β cell genes, the cells contained relatively low levels of several maturity-associated markers. Correlating with this, the time to function of ICs was similar to PEC-01 cells, indicating that ICs required cell-autonomous maturation after delivery in vivo, which would occur concurrently with graft integration into the host.

Significance: Type 1 diabetes (T1D) affects approximately 1.25 million people in the U.S. alone and is deadly if not managed with insulin injections. This paper describes the production of insulin-producing cells in vitro and a new protocol for producing the cells, representing another potential cell source for a diabetes cell therapy. These cells can be loaded into a protective device that is implanted under the skin. The device is designed to protect the cells from immune rejection by the implant recipient. The implant can engraft and respond to glucose by secreting insulin, thus potentially replacing the β cells lost in patients with T1D.

Keywords: Cell therapy; Diabetes; Drug delivery systems; Embryonic stem cells; Insulin-secreting cells; Pancreas development.

Figure 1.

Digital RNA analysis of cell aggregates for pancreatic gene expression during the PEC-01 and IC protocols. Representative results from three or more experiments are shown. The two bars depicted for each sample represent biological replicates. RNA levels are shown for PP markers (PDX1, PTF1A, NKX6.1, and SOX9) (A) and endocrine markers (CHGA, INS, GCG, and SST) (B) on day 13 (experiment 8). (C): RNA levels of NGN3 over days 5–13 (stages 3 and 4; experiment 8). (D): NGN3 expression during stage 5 (days 13–15) and into stage 6 (day 18) of the IC protocol (experiment 6). Treatments were (+) GSI, GSI added during stage 5; (−) GSI, no GSI added. Abbreviations: GSI, γ-secretase inhibitor; IC, islet-like cells; NGN3, neurogenin 3; PEC, pancreatic endoderm cells derived from the PEC-01 protocol; PP, pancreatic progenitor.

Figure 2.

RNA analysis of IC aggregates during stages 5–7 of differentiation (experiment 7) showing early endocrine (NGN3, PAX4, and ARX), hormone (INS, GCG, and SST), and mature β cell (GCK, IAPP, and G6PC2) markers. Representative results from three or more experiments are shown. The two bars depicted for each sample represent biological replicates. Abbreviations: GCG, glucagon; INS, insulin; NGN3, neurogenin 3; SST, somatostatin.

Figure 3.

RNA analysis of IC aggregates at stage 7 of differentiation (experiments 2 and 3) comparing pancreatic gene expression in native ICs (IC), native ICs that had been cryopreserved and thawed (Cryo), and reaggregated ICs (RA) to purified human islets. Representative results from three or more experiments are shown. The two bars depicted for each sample represent biological replicates. Abbreviations: Expt., experiment; GCG, glucagon; IC, islet-like cells; INS, insulin; SST, somatostatin.

Figure 4.

Immunostaining of stage 7 islet-like cell (IC) aggregates (experiment 3, day 27). (A, B): Immunofluorescence for colocalization of INS, NKX6.1, and PDX1 in native IC aggregates (A) and reaggregated ICs (B). (C, D): Immunofluorescence for INS, GCG, and SST in native IC aggregates (C) and reaggregated ICs (D). Scale bars = 50 μm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; GCG, glucagon; INS, insulin; SST, somatostatin.

Figure 5.

Proinsulin processing by stage 7 IC aggregates and human islets. Samples tested were three lots of purified human islets, designated a, b, and c, as IC-a (experiment 9, day 34), IC-b (experiment 10, day 28), and IC-c (experiment 10, day 33). (A): Enzyme-linked immunosorbent assay (ELISA) for C-peptide content (in micrograms) normalized to DNA content (in micrograms). (B): Percentage of proinsulin processed, calculated using the ratio of C-peptide to C-peptide plus proinsulin (both determined by ELISA). Error bars represent the SEM of C-peptide. Abbreviations: C-PEP, C-peptide; IC, islet-like cells.

Figure 6.

Enzyme-linked immunosorbent assay of human C-peptide levels in sera of implanted mice. Mice implanted with Encaptra-loaded aggregates were analyzed at the indicated postengraftment times (8, 12, or 16 weeks) for human C-peptide at fasting and 60 minutes after intraperitoneal glucose administration. Human C-peptide released by IC implants was compared with our historical data from mice with VC-01 implants. The x-axis labels for IC implants are as follows: IC (native ICs, experiment 2), Cryo (native ICs that had been cryopreserved and thawed, experiment 3), RA-1 (reaggregated ICs, experiment 2), RA-2 (reaggregated ICs, experiment 3). C-peptide values are shown both in picomoles per liter and nanograms per milliliter. Horizontal bars show median serum C-peptide content. Abbreviation: IC, islet-like cells.

Figure 7.

Immunostaining of IC grafts in Encaptra devices at 22 weeks after implantation (experiment 2). (A, B): Immunofluorescence for colocalization of INS, NKX6.1, and PDX1 in grafts of native ICs (A) and reaggregated ICs (B). (C, D): Immunofluorescence for INS, GCG, and SST in grafts of native ICs (C) and reaggregated ICs (D). Scale bars = 50 μm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; GCG, glucagon; IC, islet-like cells; INS, insulin; SST, somatostatin.