(L-456) Codevelopment of Endothelium and Pancreatic Tissues from iPSCs

Introduction:: Diabetes is a major health issue worldwide due to its high prevalence, economic burden, and serious complications. Restoring islet function through islet transplantation is one of the most promising treatments for type-1 and type-2 diabetes patients. Currently, islet transplantation relies upon mainly cadaveric donors, limiting the availability of this treatment to most patients. Hence, in vitro generated functional islets that can be used for transplantation is highly desired. The pancreas is a highly vascularized glandular organ in the digestive system. Thus, the existence of endothelial cells is critical for in vitro islet maturation and function. Previous stem cell research mainly focuses on single lineage differentiation. The models which could achieve the co-development of endothelium and islet lineages are lacking. Herein, we developed a new co-differentiation system to coordinate the cell lineage specification of both endothelium and pancreatic tissues from induced pluripotent stem cells (iPSCs).


Materials and Methods:: The differentiation strategy for the generation of pancreatic progenitors and endothelial cells is illustrated in Figure 1a. A human iPSC line, IMR90 was used for this study. A Wnt signaling inhibitor C59 and an activator CHIR99021 were added to the differentiation medium to regulate Wnt signal during definitive endoderm (DE) and mesoderm co-differentiation of stage I (Fig. 1a). Gene and protein expressions were characterized by quantitative real-time PCR, immunofluorescence microscopy, and flow cytometry. RNA sequencing will be done to further investigate the effect of the interaction between human islets and endothelium on organogenesis and to develop the techniques for in vitro co-differentiation of multiple tissues.


Results, Conclusions, and Discussions:: We discovered the role of Wnt signaling in mesodermal and endodermal co-specification. The degree of Wnt signaling activation regulates the co-development of pancreatic progenitor and endothelium tissues (Fig. 1e and f). We found that CHIR promotes both endothelial and pancreatic cell lineage commitment. The duration and dosage of CHIR played a critical role in balancing cell lineage specification between endothelium and pancreatic lineages. The generation of both SOX17⁺ FOXA2⁺ subsets of cells suggested the success in endoderm induction, whereas the emerging of NCAM1⁺ subsets of cells in Stage 1 indicated the commitment of mesoderm (Fig. 1b). PDX1⁺ and NKX6.1⁺ pancreatic progenitors and VE-Cadherin⁺ and PECAM1⁺ endothelial cells were generated at stage III stepwise differentiation (Fig. 1a and c). Furthermore, we observed that the PECAM1⁺ cells form a vascular-like structure (Fig. 1d).

In this study, we found that Wnt signal activation is critical during induction of multiple lineage differentiation. By changing the dose and timing of the Wnt signal activator CHIR99021, we are able to co-differentiate iPSCs into endothelial cell and pancreatic progenitors simultaneously. In conclusion, we created a multilineage differentiation system capable of coordinating co-development of mesoderm-derived PECAM1⁺ endothelial cells and endoderm-derived PDX1⁺ pancreatic progenitors within a single differentiation of iPSCs via temporal specific tuning of WNT signaling.

In the future, we will continue to generate intra-islet vascularized organoids based on this work. The intra-islets vascularization will reduce the center necrotic loss and support the maintenance of the islet organoids in an extended culture (< 40 days), which is critical to drug screening and diabetes pathophysiological study. In addition, this study will decode rules that govern islet development and vascularization. The data obtained from high-throughput RNA-seq will provide new insights into endoderm and mesoderm specification, and organ vascularization. The knowledge can be used to design more robust and more in vivo embryonic development-like organ development systems.


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