Islet-Like Structures Generated In Vitro from Adult Human Liver Stem Cells Revert Hyperglycemia in Diabetic SCID Mice

Abstract

A potential therapeutic strategy for diabetes is the transplantation of induced-insulin secreting cells. Based on the common embryonic origin of liver and pancreas, we studied the potential of adult human liver stem-like cells (HLSC) to generate in vitro insulin-producing 3D spheroid structures (HLSC-ILS). HLSC-ILS were generated by a one-step protocol based on charge dependent aggregation of HLSC induced by protamine. 3D aggregation promoted the spontaneous differentiation into cells expressing insulin and several key markers of pancreatic β cells. HLSC-ILS showed endocrine granules similar to those seen in human β cells. In static and dynamic in vitro conditions, such structures produced C-peptide after stimulation with high glucose. HLSC-ILS significantly reduced hyperglycemia and restored a normo-glycemic profile when implanted in streptozotocin-diabetic SCID mice. Diabetic mice expressed human C-peptide and very low or undetectable levels of murine C-peptide. Hyperglycemia and a diabetic profile were restored after HLSC-ISL explant. The gene expression profile of in vitro generated HLSC-ILS showed a differentiation from HLSC profile and an endocrine commitment with the enhanced expression of several markers of β cell differentiation. The comparative analysis of gene expression profiles after 2 and 4 weeks of in vivo implantation showed a further β-cell differentiation, with a genetic profile still immature but closer to that of human islets. In conclusion, protamine-induced spheroid aggregation of HLSC triggers a spontaneous differentiation to an endocrine phenotype. Although the in vitro differentiated HLSC-ILS were immature, they responded to high glucose with insulin secretion and in vivo reversed hyperglycemia in diabetic SCID mice.

A Versatile Model of Microfluidic Perifusion System for the Evaluation of C-Peptide Secretion Profiles: Comparison Between Human Pancreatic Islets and HLSC-Derived Islet-Like Structures

“…We hypothesize that such oscillations in the second phase of secretion were likely caused by a lack of enough "reserve pool" of secretory granules [19]. These data confirm the fairly immature secretion profile observed before undergoing further maturation after transplantation in diabetic mice [3]. Future studies will be performed to investigate the possibility to induce in vitro maturation of HLSC-ILS by using different culture media or adding differentiation factors [24].…”

“…Recently, our lab has described a simple charge-based single-step protocol to differentiate human liver stem cells (HLSC) into islet-like structures [3]. These islet-like structures, not only express several genetic markers of β-cell differentiation, but were also glucose-responsive and, most importantly, were able to restore euglycemia in STZ-induced SCID diabetic mice when implanted at day 14 post-differentiation [3]. By using our MPS we were able to characterize the dynamic secretion profile of HLSC-ILS at day seven post-differentiation.…”

“…After validation of the perifusion protocols in terms of reproducibility of the glucose concentration determined in each stimulation step, we evaluated whether the MPS could be suitable to assess the C-peptide secretion profiles of both human PI and stem-cell-derived islets (HLSC-ILS). For this purpose, we used human PIs derived from clinically different donors (healthy, obese, and type 2 diabetic) ( Table 2), and HLSC-ILS recently described in our lab [3]. First, we performed the SPGP protocol (Figure 2A) to evaluate C-peptide secretion in response to an HG concentration (28 mM), and the complete degranulation in response to a high-potassium concentration (50 mM).…”

“…Unravelling the relationship between glucose stimulus and the production of insulin represented a substantial step towards understanding both normal and impaired functions of pancreatic islets (PI). Independent of the source of insulin-producing cells (either enzymatically isolated islets [1], β-cell lines [2], or stem-cell-derived islet-like structures [3]), two methodologies (based on the format by which the stimuli is provided: static or dynamic) have been platform and the flow-rate sensors were connected to a flow-rate regulator platform (eight channels, Flowboard; Fluigent; France) which allows real-time monitoring and control of the perifusion solutions using the Maesflo software (version 3.3.4, Fluigent, Okabé, France). The reservoirs containing the perifusion solutions (LG and HG concentration) were connected to a microreactor chip (two inlet and one outlet, 330 mm channel length, 0.3 µL internal volume, Micronit, Overijssel, Nederland) and this was connected to the cell culture chamber made of transparent PET membrane (300 µL internal volume, OCC three-layer layer, Micronit, Overijssel, Nederland).…”

Intrahepatic Administration of Human Liver Stem Cells in Infants with Inherited Neonatal-Onset Hyperammonemia: A Phase I Study

“…HLSCs express some hepatocyte markers such as albumin, cytokeratin 8 and alpha-fetoprotein, some mesenchymal stem cells markers (CD73, CD29, CD90, CD105) and some embryonic markers (Nanog, Oct 3/4, Musashi1) [11,12]. In vitro, HLSCs showed multiple differentiation potentials, including differentiation into mature hepatocyte [12] and pancreatic islet-like organoid differentiation [13]. In vivo, HLSCs were shown to increase survival in a lethal model of fulminant liver failure and to restore liver function [12].…”