Closed-channel culture system for efficient and reproducible differentiation of human pluripotent stem cells into islet cells

Abstract

Human pluripotent stem cells (hPSCs) are thought to be a promising cell-source solution for regenerative medicine due to their indefinite proliferative potential and ability to differentiate to functional somatic cells. However, issues remain with regard to achieving reproducible differentiation of cells with the required functionality for realizing human transplantation therapies and with regard to reducing the potential for bacterial or fungal contamination. To meet these needs, we have developed a closed-channel culture device and corresponding control system. Uniformly-sized spheroidal hPSCs aggregates were formed inside wells within a closed-channel and maintained continuously throughout the culture process. Functional islet-like endocrine cell aggregates were reproducibly induced following a 30-day differentiation protocol. Our system shows an easily scalable, novel method for inducing PSC differentiation with both purity and functionality.

Hypoxia Promotes Differentiation of Pure Cartilage from Human iPS Cells

“…Various studies have been conducted regarding improving the degree of purity of tissue differentiation from iPSCs. Hirano et al were able to increase the purity of islet cell differentiation from iPSCs by applying a unique culture method referred to as a closed-channel culture system [12]. In addition, Hwang et al increased the purity of cardiomyocyte differentiation from iPSCs by adding a small molecule compound to the culture environment [13].…”

Generation of Pancreatic β-cells From iPSCs and their Potential for Type 1 Diabetes Mellitus Replacement Therapy and Modelling

“…Direct differentiation of iPSCs into insulin producing cells was conducted by treatment of cultured cells with WNT3A, Activin A for DE formation, and subsequent treatment with FGF10, cyclopamine and retinoic acid for formation of gut tube endoderm and pancreatic progenitors. The endocrine cells were able to respond on glucose stimulation after further differentiation by secreting C-pep- Human skin cells Integrating retroviral reprogramming Generation of insulin-producing islet-like clusters; secretion of C-peptide upon glucose stimulation [25] Adult T1D skin fibroblasts Integrating retroviral reprogramming Differentiation of patient-derived iPSCs into insulin producing cells in vitro [27] Normal mouse skin fibroblasts Integrating retroviral reprogramming Differentiation of T1D -iPSCs patients into insulin secreting β-cells; respond to glucose stimulation under physiological or pathological conditions [26] Adult T1D skin fibroblasts Integration-free transfection of synthetic mRNAs T1D-iPSCs showed upregulations of pancreas-specific microRNAs [63] Human foreskin fibroblast Polycistronic lentiviral vector Differentiation of iPSCs into β-cells promoted by over-expression of miR-375 without growth factors; iPSC-derived β-cells secreted insulin in glucose-dependent manner [33] Adult skin fibroblasts NA Growth factor-free generation of iPSC-derived islet-like cell clusters via transfection of hsa-miR-186 and hsa-miR-375 [32] Diabetes skin fibroblasts Non-integrating Sendai viral vectors Differentiation of T1D -iPSCs patients into β-cells; response to antidiabetic drugs [62] iPSC lines (253G1, 454E2, and RIKEN-2A) obtained from RIKEN cell bank -Generation of uniform population of iPSC-derived pancreatic endocrine cells via 3D approach cultured on agarose microwell plates [34] CD34 + cord blood cells EBNA-based episomal system Efficient generation of iPSC-derived pancreatic islet cells cultured on a closed channel-based culture system [35] T1D somatic cells NA Generation of high number of iPSC-derived glucose-sensitive and insulin producing cells via highly efficient 3D protocol based on demethylation strategy [36] Diabetes fibroblasts lentiviral inducible transduction system Efficient differentiation of iPSCs into glucose-responsive insulin producing cells; presence of mature endocrine secretory granules [31] tide. Jeon et al [28] generated iPSCs both from NOD mouse embryonic fibroblasts and NOD mouse pancreas-derived epithelial cells and developed a method for stepwise differentiation of NOD-iPSCs into functional pancreatic β-cells.…”

“…However, the levels of key transcription factors (NKX6.1 and MAFA) were low. Hirano et al [35] constructed a closed channel culture device in order to prevent culturing iPSCs from bacterial and fungal contamination resulting in higher differentiation efficacy. Following a 30-day differentiation protocol, the endodermal lineage developed during the first 3 days and the pancreatic endoderm during the following 7 days.…”

Beta‐cell replacement strategies for diabetes

“…For instance, human pancreatic progenitor cells seem to mature differently in mice vs rats, and altered levels of thyroid hormone can impair the formation of β‐cells, indicating that the tissue environment can influence pancreatic progenitor maturation. More advanced scalable differentiation protocols have been developed, and cells generated, while not fully mature β‐cells, can more rapidly reverse diabetes in mice compared with pancreatic progenitors. Additional studies are required to verify if the cells function more consistently in variable host conditions in order to safely and effectively regulate blood glucose levels.…”