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 6

Reducing replication fork speed and S phase entry establishes cell-intrinsic limitations in cell proliferation.

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Reducing replication fork speed and S phase entry establishes cell-intri...
(A) In vivo imaging of mice transplanted with control and APH cells. Tx, transplantation. (B) Growth of grafted cells in mice after transplantation quantified by the bioluminescence intensity. Differentiation efficiency and graft growth in controls are variable but consistent in APH treatment. Black and blue lines show mice transplanted with control clusters (black, approximately 20% differentiation efficiency, n = 4; blue, >60%, n = 3). (C) Fold change of graft growth in control and APH mice. *Mann-Whitney U test with P < 0.05. (D) Grafts in the legs of mice transplanted with equal number of cells in control and APH group at 20 weeks (black circles, graft location). (E and F) Cell composition determined by immunostaining in mice transplanted with control and APH cells. *cysts. (Pictures with white line: close-ups.) Scale bar: 100 μm. (G) Quantification of MAFA- and INS-positive cells (control: 2 grafts from 2 mice from 1 batch of differentiation, INS+ cells counted from 3 sections; APH: 2 grafts from 2 mice from 2 batches of differentiation, INS+ cells counted from 3 sections) and KI67-positive cells (control: 2 grafts from 2 mice from 1 batch of differentiation, cells counted from 5 sections; APH: 3 grafts from 3 mice from 2 batches of differentiation, cells counted from 6 sections) (Supplemental Table 6). Two-tailed paired t test *P < 0.05. (H) Representative day 34 cell cycle distribution with day 15–day 27 APH pretreatment (n = 2). (I) Cell growth in mice after transplantation of cells pretreated with APH measured by bioluminescence intensity. Two-way ANOVA *P < 0.05 (0.1APH vs. 1APH); ****P < 0.0001 (0.1, 0.25, 1APH vs. control). (J) Human C-peptide secretion in mice transplanted with APH-treated cells. Two-way ANOVA **P < 0.05 (0.1APH vs. 0.25APH in black; 0.25APH vs. control in color ); ****P < 0.0001 (0.1APH vs. 1APH in black; 1APH vs. control in color).