Establishing an optimized platform for human pluripotent stem cell differentiation into islet organoid

Abstract

Diabetes mellitus (DM) is characterized by prolonged elevation of blood glucose. It was estimated that by 2040 there are 642 million adults with DM. The prevalent treatment for DM is insulin administration which, however, requires life-long insulin injection and only offers suboptimal control of blood glucose. Alternatively, the transplantation of cadaveric islets restores normoglycemia without exogenous hormone. Nonetheless, the scarce of cadaveric islets limits its pervasion. Human pluripotent stem cells (hPSCs) are self-renewable and can differentiate into any cell type. In the past decade, efforts have been made in developing in vitro differentiation regime for pancreatic progenitors (PPs) or mature β-cells. However, many of these cells resembled fetal, rather than adult, β-cells. Moreover, the insulin-producing cells were different from the native adult β-cells revealed by transcriptomic analyses. Although progress has been made recently in generating islet organoids from hPSCs, the protocols are time-consuming, and the formation of organoids needs manual assistance. Thus, a platform is needed to efficiently generate human stem cell-derived islets (SC-islets) with superior comparability to native human islets. Also, devising a protocol to maintain PPs is essential for accelerating the SC-islets production. Here, by establishing and employing a PDX1-EGFP human embryonic stem cell (hESC) line to monitor the expression of crucial PP marker PDX1 during the differentiation in real-time, a protocol to efficiently generate PPs from hPSCs was established. These cells strongly expressed PDX1, NKX6.1 and SOX9. Combinations of extracellular matrix (ECM) proteins and medium supplements were tested on their ability to maintain PPs. However, none of these maintained PPs because significant loss of critical PP markers such as PDX1 and increase in maturation marker INS were observed when culturing PPs in these conditions. When hPSC-derived mesenchymal stem cells (MSCs) were used to replace ECM coating, PPs were maintained on these MSCs for 5 passages, or 35 days. As hPSC-derived MSCs not only increase the complexity of the differentiation system but also compromise its stability and reproducibility, chemically defined conditions would be much preferred. Such method could help to accelerate the SC-islet production by initiating the differentiation from PPs rather than hPSCs. Next, I established an optimized platform to produce SC-islets, which allowed the spontaneous formation of SC-islets with high efficiency. The organoids were transferred to 3D suspension culture for maturation, after which they released significant amount of insulin in response to elevated glucose level. They also possess mature insulin and glucagon secretory granules, as well as β-cell identity, functionality, and maturation markers such as CHGA, ISL1, and NEUROD1. Pseudotime analysis revealed the dynamic expression of key β-cell maturation, functionality, and developmental genes on single-cell RNA sequencing data from different time points, indicating the recapitulation of natural β-cell maturation process. These organoids were better clustered to native islets than the published ones. Improvements in cell-cell and cell-matrix interactions, cytoskeletal organization, and cell polarity during the maturation stage were reflected in GSEA results, from which both SC-islet integrity and functionality were benefited. Again, a chemically defined protocol should be generated for clinical applications.