Functional, metabolic and transcriptional maturation of human pancreatic islets derived from stem cells

Diego Balboa, Tom Barsby, Väinö Lithovius, Jonna Saarimäki-Vire, Muhmmad Omar-Hmeadi, Oleg Dyachok, Hossam Montaser, Per-Eric Lund, Mingyu Yang, Hazem Ibrahim, Anna Näätänen, Vikash Chandra, Helena Vihinen, Eija Jokitalo, Jouni Kvist, Jarkko Ustinov, Anni I. Nieminen, Emilia Kuuluvainen, Ville Hietakangas, Pekka Katajisto, Joey Lau, Per-Ola Carlsson, Sebastian Barg, Anders Tengholm, Timo Otonkoski

Contact: timo.otonkoski@helsinki.fi

Abstract

Transplantation of pancreatic islet cells derived from human pluripotent stem cells is a promising treatment for diabetes. Despite progress in the generation of stem cell–derived islets (SC-islets), detailed characterization of their functional properties has not been conducted. Here, we generated functionally mature SC-islets using an optimized protocol and comprehensively benchmarked them against primary adult islets. Biphasic glucose-stimulated insulin secretion developed during in vitro maturation, associated with cytoarchitectural reorganization and increasing presence of alpha cells. Electrophysiology, signaling and exocytosis of SC-islets were similar to those of adult islets. Glucose-responsive insulin secretion was achieved despite differences in glycolytic and mitochondrial glucose metabolism. Single-cell transcriptomics of SC-islets in vitro and throughout 6 months of engraftment in mice revealed a continuous maturation trajectory culminating in a transcriptional landscape closely resembling that of primary islets. Our thorough evaluation of SC-islet maturation highlights their advanced degree of functionality and supports their use in further efforts to understand and combat diabetes.

Figure 5: Single cell transcriptomic profiling of stem cell derived islet cells

(a) Experimental outline for scRNAseq transcriptomic profiling of SC-islets at the end of in vitro culture stages 5 (S5) and 6 (S7w0) and at week 3 (S7w3) and week 6 of S7 culture (S7w6), together with grafts retrieved after 1 (M1), 3 (M3) and 6 months (M6) post-implantation.

(b) Uniform Manifold Approximation and Projection (UMAP) -base embedding projection of an integrated dataset of 46 261 SC-derived endocrine cells and adult human islet cells (from Krentz et al. 2018 and Xin et al. 2018), colored by time and sample of origin. 

(c) Clustering of the dataset in (b) cells into different cell types. 

(d) Relative expression of marker genes for pancreatic progenitor cells (PDX1, NKX6-1, NEUROG3) and alpha- (GCG), delta- (SST) and beta- (INS) cells. Dashed line indicates the beta cell cluster selected for further study.

(e) UMAP projection of the beta cell cluster indicating the relative expression of insulin (INS) and mature beta cell markers G6PC2, MAFA and SIX3.

(f) SC-beta cells and adult primary beta cells average gene expression of beta cell maturation markers. The average expression of the beta cell populations (Fig.1e) coming from each independent sample with different time of origin (S5 to Adult islets) is represented.

(g) Principal component analysis of the beta cell populations from each independent sample. (S7w0: n=3; S7w3: n=3; S7w6: n=2; M1: n=3; M3: n=3; M6: n=2; Adult: n=12).

(h) Heterogeneous distribution of the beta cells from different time of origin within the beta cell cluster (Fig.1e).

(i) Clustering of beta cells according to their transcriptional similarity into early, late and adult beta cluster.

(j) Fractional contribution to each early, late and adult beta clusters of beta cells from different times of origin.

(k) UMAP projection of the beta cell cluster with RNA velocity vectors overlayed. Cells are annotated by latent-time dynamics. Earlier latent-time points, the origin of the trajectory, are indicated in blue, and the later in yellow on the latent-time colour scale.

(l) Pseudotemporal ordering of cells in the beta cell cluster. Earlier pseudotemporal points, the origin of the trajectory, are indicated in blue, and the later in yellow on the pseudotime colour scale.

(m-n) Relative expression levels of example genes that are upregulated (m) or downregulated (n) along the pseudotime trajectory inferred in Fig.5l.