Reduced replication fork speed promotes pancreatic endocrine differentiation and controls graft size
Lina Sui, Yurong Xin, Qian Du, Daniela Georgieva, Giacomo Diedenhofen, Leena Haataja, Qi Su, Michael V. Zuccaro, Jinrang Kim, Jiayu Fu, Yuan Xing, Yi He, Danielle Baum, Robin S. Goland, Yong Wang, Jose Oberholzer, Fabrizio Barbetti, Peter Arvan, Sandra Kleiner, Dieter Egli
Lina Sui, Yurong Xin, Qian Du, Daniela Georgieva, Giacomo Diedenhofen, Leena Haataja, Qi Su, Michael V. Zuccaro, Jinrang Kim, Jiayu Fu, Yuan Xing, Yi He, Danielle Baum, Robin S. Goland, Yong Wang, Jose Oberholzer, Fabrizio Barbetti, Peter Arvan, Sandra Kleiner, Dieter Egli
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Research Article Cell biology Stem cells

Reduced replication fork speed promotes pancreatic endocrine differentiation and controls graft size

Abstract

Limitations in cell proliferation are important for normal function of differentiated tissues and essential for the safety of cell replacement products made from pluripotent stem cells, which have unlimited proliferative potential. To evaluate whether these limitations can be established pharmacologically, we exposed pancreatic progenitors differentiating from human pluripotent stem cells to small molecules that interfere with cell cycle progression either by inducing G1 arrest or by impairing S phase entry or S phase completion and determined growth potential, differentiation, and function of insulin-producing endocrine cells. We found that the combination of G1 arrest with a compromised ability to complete DNA replication promoted the differentiation of pancreatic progenitor cells toward insulin-producing cells and could substitute for endocrine differentiation factors. Reduced replication fork speed during differentiation improved the stability of insulin expression, and the resulting cells protected mice from diabetes without the formation of cystic growths. The proliferative potential of grafts was proportional to the reduction of replication fork speed during pancreatic differentiation. Therefore, a compromised ability to enter and complete S phase is a functionally important property of pancreatic endocrine differentiation, can be achieved by reducing replication fork speed, and is an important determinant of cell-intrinsic limitations of growth.

Authors

Lina Sui, Yurong Xin, Qian Du, Daniela Georgieva, Giacomo Diedenhofen, Leena Haataja, Qi Su, Michael V. Zuccaro, Jinrang Kim, Jiayu Fu, Yuan Xing, Yi He, Danielle Baum, Robin S. Goland, Yong Wang, Jose Oberholzer, Fabrizio Barbetti, Peter Arvan, Sandra Kleiner, Dieter Egli

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Figure 4

Single-cell transcriptome analysis shows an increase of endocrine cells and decrease of nonendocrine cells in the APH condition.

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Single-cell transcriptome analysis shows an increase of endocrine cells ...
(A) Identified cell populations in stem cell–derived islet cells with and without APH during differentiation (samples were collected from 4 independent wells of 1 experiment in each condition). (B) Identified cell populations in stem cell–derived islet cells compared with primary human islet cells. (C) Quantification of indicated cell populations between control and APH groups. *Wilcoxon’s test with P < 0.05. (D) The upregulated and downregulated genes after cells were treated with APH compared with control in SC-β 1 cells and (E) SC-β 2 cells. (F) Insulin-GFP–positive and –negative cells were sorted from control and APH groups for downstream analysis. (G) Protein expressions of sorted cells by a representative Western blot analysis for PDX1, NKX6.1, MAFA, Proins, and Vinculin (Supplemental Figure 9, A and B). (H) Quantification of Western blot band intensity and normalized to α-tubulin (n = 3). Two-tailed paired t test *P < 0.05; **P < 0.01. (I) Proinsulin biosynthesis of an iPSC line–derived β cell clusters with and without APH treatment (Supplemental Figure 9C). (J) Static glucose stimulated insulin secretion (GSIS) (a stimulation index is determined by the fold changes of insulin secretions of nonsorted cells incubated in 2 mM and 20 mM glucose). Mann-Whitney U test *P < 0.05. (K) Basal insulin secretion levels of nonsorted cells normalized to insulin content. (L) Dynamic analysis of insulin secretion of nonsorted cells stimulated sequentially by 2 mM glucose, 20 mM glucose, 150 μM tolbutamide.