Conditions for glucose control by implants of human pluripotent stem cell-derived beta cell preparations in mice
Kaat De Groot MD1, Ines De Mesmaeker PhD1, Krista Suenens1, Geert Stangé1, Jolien Nijns1, Zhidong Ling MD PhD1, Bart Keymeulen MD PhD1, Daniel Jacobs-Tulleneers-Thevissen MD PhD1, Daniel Pipeleers MD PhD1.
1Diabetes Research Center, Vrije Universiteit Brussel, Brussels, Belgium
INTRODUCTION
Studies in immune-compromised mice help identify the potential of human pluripotent stem cell (hPSC)-derived implants as beta cell replacement therapy. Formation of beta cells is shown by appearance of plasma human (hu)-C-peptide. Glucose-inducible hu-C-peptide levels illustrate responsiveness of the cells to their major regulator. Additionally, the capacity of the formed beta cell mass to exert glucose control is demonstrated when basal and glucose-induced glycemia is controlled by the human beta cell glucosensor, without participation by that of rodent beta cells.
We use normoglycemic mice to assess the implant’s capacity for glucose control. Compared to diabetic recipients, this model avoids prolonged exposure of implanted cells to excessively high glucose levels. Mouse basal glycemia is moderately higher than in non-diabetic humans and in the range of that in insulin-treated type 1 diabetes patients. It thus allows assessment of control by the human beta cell mass.
Subcutaneous implants of device-encapsulated hPSC-derived pancreatic endoderm (PE)* were previously found to establish glucose control from post-transplant (PT) week 20 onwards when achieving and maintaining plasma hu-C-peptide levels ≻6 ng/ml following intraperitoneal glucose-loading test (IPG) (1). Recipients exhibited low or undetectable mouse C-peptide release.
AIM
The present study examined whether differentiation to a stage with insulin-positive (INS+) cells generates implants that establish metabolic control at an earlier time and/or whether this time is related to their insulin content.
METHODS
Stage 6 cells, differentiated from the CyT49 cell line** according to published methods (2,3), exhibited ≻80% chromogranin-positive cells, including ≻40% INS+ cells that were functionally immature (low insulin content, 6 μg insulin/106 INS+ cells, no glucose-induced insulin release). Grafts containing 106 INS+ cells were implanted, non-encapsulated, in the fat pad of normoglycemic Scid/Beige mice. Glycemia, human and mouse C-peptide levels at basal and after IPG were followed over 20 weeks, before retrieving implants for analysis of insulin content and cellular composition.
RESULTS
Hu-C-peptide was detectable at PT-week 5 in 35/39 recipients; its basal and glucose-stimulated levels increased with time reaching ≻6 ng/ml (IPG) in 24/39 recipients at PT-week 20. These mice then exhibited the characteristics of glucose control by the human beta cell mass, which was found to contain, on average, 3.8-fold more insulin than the grafts at start. Mice not achieving this hu-C-peptide level were not under glucose control by their implant, which did not increase its insulin content over that of the grafts.
CONCLUSION
These observations demonstrate that extraportal implants of hPSC-derived beta cells can establish glucose control in mice. For a beta cell dose of 40x106 cells/kg BW at start, this capacity developed over 20 weeks involving both growth and functional differentiation of the beta cell mass.
This study shows the utility of normoglycemic mice to seek conditions that increase dose/efficacy of this cell therapy product to achieve and maintain glucose control.
This study has been supported by a grant from the European Commission (H2020 681070). The hPSC-PE* cells and CyT49 cell line** were provided by dr. Kroon, ViaCyte, Inc within this project. Kaat De Groot is holder of a PhD grant fundamental research of the Research Foundation - Flanders (11B5623N)..
[1] Robert T, et al. Stem Cell Reports 2018;10(3):739-750.
[2] Pagliuca FW, et al. Cell 2014;159(2):428-39.
[3] Schulz TC, et al. PLoS One 2012;7(5):e37004.
