Selection for CD49A+ and CD26- cells in pancreatic islet-like clusters differentiated from human pluripotent stem cells improves their therapeutic activity in diabetic mice
Authors: Molakandov Kfir , Berti Denise A. , Beck Avital , Elhanani Ofer , Walker Michael D. , Soen Yoav , Yavriyants Karina , Zimerman Michal , Volman Ella , Toledo Yitzhak , Eruhimovich Anna , Levy Alon , Hasson Arik , Itskovitz-Eldor Joseph , Chebath Judith , Revel Michel 
Published: April 2020

Abstract
BackgroundCell therapy of diabetes aims at restoring the physiological control of blood glucose by transplantation of functional pancreatic islet cells. Human islets from post-mortem donations have shown efficiency but the demand for islets vastly exceeds the availability of donations. A potentially unlimited source of cells for such transplantations would be islet cells derived from in vitro differentiation of human pluripotent stem cells (hPSC), such as embryonic stem cells (hESC). The islet-like clusters (ILC) produced by the known differentiation protocols contain various cell populations. Among these, the beta cells that express both insulin and the transcription factor Nkx6.1 seem to be the most efficient to restore normoglycemia in diabetes animal models. Our aim was to find markers allowing selection of these efficient cells.MethodsFunctional Cell-Capture Screening (FCCS), using an array of antibodies to cell surface proteins, was used to identify markers that preferentially capture the cells expressing insulin, or expressing both insulin and Nkx6.1, from hESC-derived ILC cells. In order to test whether selection for such markers could improve cell therapy in diabetic mouse models, we used ILC produced from a clinical-grade line of hESC by a refined differentiation protocol adapted to up-scalable bioreactors. The ILC, dissociated to single cells, were fractionated by Magnetic Activated Cell Sorting (MACS) for presence of the marker. The sorted cells, re-aggregated into clusters, were encapsulated in microspheres made of alginate modified to reduce foreign body reaction. Implantation was done intraperitoneally in C57BL/6 immuno-competent mice that were made diabetic by prior injections of Streptozotocin (STZ).ResultsCD49A (integrin alpha1) was identified by FCCS as a marker for cells double positive (DP) for insulin (and C-peptide) as well as Nkx6.1 in ILC derived by hESC differentiation. After sorting by MACS with CD49A antibodies, the ILC fraction enriched in CD49A+ cells rapidly reduced glycemia when implanted in the diabetic mice, whereas mice receiving the CD49A depleted population remained highly diabetic. CD49A-enriched ILC cells also produced significantly higher levels of human C-peptide in mouse blood. Another marker, CD26 (DPP4, dipeptidyl peptidase-4), was identified by FCCS as binding insulin-expressing cells which are Nkx6.1-negative. Depletion of CD26+ cells followed by enrichment for CD49A+ cells increased DP cells to over 70%. After this double selection, the CD26 depleted/CD49A enriched ILC were more active than non-sorted ILC to reduce glycemia in the diabetic mice.ConclusionsRefining the composition of ILC differentiated from hPSC by negative selection to remove cells expressing CD26 and positive selection for CD49A expressing cells can enable more effective cell therapy of diabetes.