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.