Improved Differentiation of hESC-Derived Pancreatic Progenitors by Using Human Fetal Pancreatic Mesenchymal Cells in a Micro‐scalable Three-Dimensional Co-culture System

Zahra Ghezelayagh,Bagher Larijani,Babak Arjmand,Massoud Vosough,Anne Grapin-Botton,Carla A C Gonçalves,Hamid Reza Aghayan,Hossein Baharvand,Mahsa Zabihi,Newsha Hagh-Parast,Ibrahim Zarkesh,Michael Larsen,Yaser Tahamtani,Mohammad Pakzad

doi:10.1007/s12015-021-10266-z

Abstract

Mesenchymal cells of diverse origins differ in gene and protein expression besides producing varying effects on their organ-matched epithelial cells' maintenance and differentiation capacity. Co-culture with rodent's tissue-specific pancreatic mesenchyme accelerates proliferation, self-renewal, and differentiation of pancreatic epithelial progenitors. Therefore, in our study, the impact of three-dimensional (3D) co-culture of human fetal pancreatic-derived mesenchymal cells (hFP-MCs) with human embryonic stem cell-derived pancreatic progenitors (hESC-PPs) development towards endocrine and beta cells was assessed. Besides, the ability to maintain scalable cultures combining hFP-MCs and hESC-PPs was investigated. hFP-MCs expressed many markers in common with bone marrow-derived mesenchymal stem cells (BM-MSCs). However, they showed higher expression of DESMIN compared to BM-MSCs. After co-culture of hESC-PPs with hFP-MCs, the pancreatic progenitor (PP) spheroids generated in Matrigel had higher expression of NGN3 and INSULIN than BM-MSCs co-culture group, which shows an inductive impact of pancreatic mesenchyme on hESC-PPs beta-cells maturation. Pancreatic aggregates generated by forced aggregation through scalable AggreWell system showed similar features compared to the spheroids. These aggregates, a combination of hFP-MCs and hESC-PPs, can be applied as an appropriate tool for assessing endocrine-niche interactions and developmental processes by mimicking the pancreatic tissue.

Highlights

Knowledge on normal pancreas development [1,2] and findings on in-vitro differentiation [3] and coculture studies [4,5] can help us develop novel cell- and micro-tissue-based therapeutic/modeling approaches for pancreatic degenerative diseases
After co-culture of hESC-PPs with human fetal pancreatic-derived mesenchymal cells (hFP-MCs), the pancreatic progenitor (PP) spheroids generated in Matrigel had higher expression of NGN3 and INSULIN than bone marrow-derived mesenchymal stem cells (BM-MSCs) co-culture group, which shows an inductive impact of pancreatic mesenchyme on hESC-PPs beta-cells maturation
During our stepwise pancreatic progenitor (PP)-directed differentiation protocol, Human pluripotent stem cells (hPSCs) were induced on Matrigel through 14 days in five consecutive steps (Fig. 1a)

Summary

Introduction

Knowledge on normal pancreas development [1,2] and findings on in-vitro differentiation [3] and coculture studies [4,5] can help us develop novel cell- and micro-tissue-based therapeutic/modeling approaches for pancreatic degenerative diseases. Recapitulating signaling mechanisms involved in different stages of pancreas development has led to the development of multistep protocols to direct hPSCs differentiation to β-cells [7,8,3]. The insulin-producing cells differentiated in the early two-dimensional (2D) culture systems were more similar to fetal than adult β-cells [9,10]. These protocols have been continuously improved, including three-dimensional (3D) culture, but the glucose-responsiveness and functionality of adult β-cells are still challenging to reach [10,11,12]. Pancreas development is affected by extrinsic stimuli and paracrine signals of different cell types in the milieu, providing cues for organogenesis; more attention should be paid to interactions between the pancreatic epithelium and its surrounding niche cells, notably the mesenchyme, for the in-vitro reconstruction of pancreatic cells [15,10]

Methods

Results

Conclusion