Engineered vasculature induces functional maturation of pluripotent stem cell-derived islet organoids

Abstract

Blood vessels play a critical role in pancreatic islet health and function, yet current culture methods to generate islet organoids from human pluripotent stem cells (SC-islets) lack a vascular component. Here, we engineered 3D vascularized SC-islet organoids by assembling SC-islet cells, human primary endothelial cells (ECs) and fibroblasts both in a non-perfused model and a microfluidic device with perfused vessels. Vasculature improved stimulus-dependent Ca2+ influx into SC-β-cells, a hallmark of β-cell function that is blunted in non-vascularized SC-islets. We show that an islet-like basement membrane is formed by vasculature and contributes to the functional improvement of SC-β-cells. Furthermore, cell-cell communication networks based on scRNA-seq data predicted BMP2/4-BMPR2 signaling from ECs to SC-β-cells. Correspondingly, BMP4 augmented the SC-β-cell Ca2+ response and insulin secretion. These vascularized SC-islet models will enable further studies of crosstalk between β-cells and ECs and can serve as in vivo-mimicking platforms for disease modeling and therapeutic testing.

Validating expression of beta cell maturation-associated genes in human pancreas development

“…Protocol A was derived from our previously published protocol (Xu et al, 2011), with modifications in the late stages of differentiation based on (Rezania et al, 2014) and (Pagliuca et al, 2014). Protocol B was further modified using Stages 1-4 from Nostro et al (2015) to derive an improved pancreas progenitor population, and with factors such as BayK 8644 and MK-801 added to the end stages influenced by (Zhu et al, 2016)and (Huang et al, 2017). Although SC-islets from both protocols, Protocol A and Protocol B, have relatively low glucoseresponsive function, we find that major gene expression differences between these two protocols warrant the use of these SC-islets for further study.…”

“…Once SC-islets are transplanted into a suitable environment, they experience myriad interactions and signals that are absent in vitro , including vascularization, frequent stimulation by feeding cycles, circadian entrainment, contact with the in vivo microenvironment including ECM-signaling and cell-cell interactions with endothelial, mural, neuronal, and immune cells. The effects that some of these individual elements have on islet and SC-islet health and function have been explored in vitro through many different studies, and each have been shown to have influence on the identity and function of the cells ( Alvarez-Dominguez et al, 2020 ; Singh et al, 2020 ; Nguyen-Ngoc et al, 2022 ; Tremmel et al, 2022 ). Together, it is possible that these elements of the tissue microenvironment provide combined cues to stimulate the final push towards maturation that cells require.…”

“…Understanding these gene expression profiles for normal beta cell development has been key to recapitulating those developmental stages in vitro. For example, the discovery that PDX1 and NKX6.1 expression must precede NGN3 induction in order to successfully produce endocrine progenitors has allowed for the generation of more highly enriched endocrine cell and beta cell populations at the end of the differentiation protocol (Nostro et al, 2015). The identification of similar markers at the transition from immature to mature beta cells would provide an efficient way to isolate and purify functionally mature SC-islets from heterogeneous populations, and advance our understanding of the biology underlying maturation.…”

“…Although rare in HFP at gestational week 17 and older (Riedel et al, 2012), we observed the presence of INS + GCG + cells that also co-expressed UCN3 (Figures 3A-H). Polyhormonal cells are also generated by some SC-islet differentiation protocols, and are considered to be an undesirable immature cell type (Nostro et al, 2015;Russ et al, 2015). INS + GCG + UCN3 + cells were also found in Protocol B (Figures 3B-H).…”

“…Cell culture and differentiation H1 (WA01, WiCell) cells were differentiated toward a pancreatic endocrine fate using protocols based on Xu et al (2011), Rezania et al (2014, Pagliuca et al (2014), Nostro et al (2015 and Zhu et al (2016). Media components for each stage of differentiation for both Protocol A and Protocol B are included in detail in Supplemental Tables S1-3.…”