OX1 Receptors

was supported by a Kraft Family Fellowship and a JDRF postdoctoral fellowship (1-PNF-2016-320-S-B), S

was supported by a Kraft Family Fellowship and a JDRF postdoctoral fellowship (1-PNF-2016-320-S-B), S.A. for human pluripotent stem cell (hPSC)-derived islet cells using these three antibodies, leading to the formation of islet-like clusters with improved glucose-stimulated insulin secretion and reduced growth upon transplantation. This strategy should facilitate large-scale production of functional islet clusters from stem cells for disease modeling and cell replacement therapy. differentiation of human pluripotent stem cells (hPSCs) (Nair et?al., 2020). Several multistage strategies leading to the production of functional human cells from hPSCs have been reported (Nair et?al., 2019b; Pagliuca et?al., 2014; Rezania et?al., 2014; Russ et?al., 2015; Velazco-Cruz et?al., 2019). Although these protocols generate functional insulin-producing cells, the differentiated cultures can also contain additional populations of cells, such as multihormonal cells, endocrine progenitors, or other undesirable proliferating cell types that may CCR4 antagonist 2 interfere with islet function or graft safety. Notably, the maturation and safety of hPSC-derived cell differentiations can be further increased by recapitulating endocrine CCR4 antagonist 2 cell clustering to form enriched clusters (eBCs) (Nair et?al., 2019b). This method enables the generation of mature cells by sorting based on the expression of insulin at the immature -like stage. The purification method reported in this study, however, requires cell sorting of a transgenic hPSC line in which a Green Fluorescent Protein (GFP) reporter gene has been inserted into the endogenous human insulin locus, which limits its use for cell therapy. We thus aimed to replicate the same strategy but using cell surface markers expressed on immature cells instead of relying on a reporter cell line. Although surface markers may serve as an alternative to purify relevant cell populations during cell differentiation, identifying Rabbit Polyclonal to MCL1 antibodies capable of specifically isolating large numbers CCR4 antagonist 2 of hPSC-derived cells remains challenging. Antibody-mediated cell sorting has been used to isolate anterior definitive endoderm (Mahaddalkar et?al., 2020) or pancreatic progenitors that can differentiate into cells (Ameri et?al., 2017; Cogger et?al., 2017; Kelly et?al., 2011). The clusters generated with these approaches had a higher proportion of cells than unsorted clusters, but the resulting percentage of insulin-expressing cells at the -like stage still remained below 50%. A method to enrich for cells using a cell surface antibody against CD49a was also reported (Veres et?al., 2019). Although the typical yield of cells recovered after CCR4 antagonist 2 purification with this antibody was low ( 10% of cells), the sorting enhanced functional maturation of hPSC-derived cells, suggesting that antibody-based magnetic CCR4 antagonist 2 sorting of -like cells could provide the same benefits as GFP-based fluorescence-activated cell sorting (FACS) but with greater scalability. Here, we generated more than a thousand monoclonal antibodies (mAbs) against cell surface markers that selectively label stem cell-derived endocrine cell subsets. A high-throughput screening assay was employed to identify promising antibodies, including three clones that mark a high fraction of cell progenitors derived from hPSCs. A scalable magnetic sorting method was developed to enrich for hPSC-derived -like cells using these antibodies, leading to the formation of islet-like clusters with increased maturation and safety. These antibodies thus selectively isolate islet cell populations from hPSCs differentiated using a scalable magnetic sorting approach, facilitating the large-scale production of functional islet-like clusters from stem cell cultures. Results Generation of mAbs recognizing cell surface antigens of hPSC-derived pancreatic cells Previous studies have shown that the insulin secretory profile of hPSC-derived cells can be improved by enriching for immature -like cells followed by reaggregation of the cells (Nair et?al., 2019b; Veres et?al., 2019). However, these methods either require the use of cell sorters, which limits the total numbers of cells that can be processed at once, or had low recovery of insulin-expressing cells. In addition, although the generation of antibodies directed against pancreatic antigens has enabled isolation of mature, adult cells from primary islets (Dorrell et?al., 2008, 2016), these antibodies do not efficiently enrich for C-peptide+ cells from immature stem cell-derived -like cells (Figures S1A and S1B). To develop alternative endocrine clustering strategies that are more efficient and scalable, we thus set out to generate new antibodies against cell surface markers present in hPSC-derived immature islet cell populations generated using our published protocol (Nair et?al., 2019b). We employed a subtractive immunization strategy to generate mouse anti-human mAbs against surface proteins present on day (d) 19C20 insulin-expressing cells derived from insulin (INS)-GFP human embryonic stem cells (hESCs) (Figures 1A and S1C). These insulin-expressing cells include immature , , and cells (Nair et?al., 2019b; Russell et?al., 2020). This approach led to the production of 1 1,248 hybridoma clones that were screened by high-throughput flow cytometry for their specificity to cell surface epitopes present on d19C20 insulin-expressing cells. The gating strategy for the screen is.