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 2

Inhibiting replication fork progression promotes endocrine differentiation independent of apoptosis.

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Inhibiting replication fork progression promotes endocrine differentiati...
(A) Quantification of NKX6.1 or C-peptide–positive cells on day 15 (n = 3) and on day 27 (B) with different concentrations of APH. (C) Quantification of C-peptide–positive cells in control and 1 μM APH on day 27 (n = 12). Two-tailed paired t test with ****P < 0.0001. (D) Quantification of C-peptide/glucagon-positive cells (n = 6) or C-peptide/somatostatin-positive cells (n = 5). (E) Immunostaining of APH-treated clusters for C-peptide, NKX6.1, glucagon, and somatostatin on day 27. Scale bar: 100 μm. Insets: 6.25× higher magnification. (F) A schematic diagram indicates APH duration. (G) Flow cytometry quantification of C-peptide–positive and C-peptide/NKX6.1-positive cells in indicated conditions on day 27 (n = 3). One-way ANOVA test, *P < 0.05; **P < 0.01; ***P < 0.001. (H) Flow cytometry profile of cell cycle progression on days 15, 18, 20, and 27 without and with APH, indicated by the percentage of cells positive for C-peptide and EdU. (I) Quantification of total C-peptide–positive cells on days 20 and 27. One-way ANOVA test, *P < 0.05; **P < 0.01. (J) Total cell numbers on days 20 and 27. (K) Immunostaining on day 27 clusters for TUNEL and C-peptide (6.25× higher magnification in inset). Scale bar: 100 μm. Quantification of TUNEL-positive and C-peptide–negative cells on day 27. (L) Quantification of C-peptide– and TUNEL-positive cells on days 17, 20, and 27 (n = 3). (M) Timing of insulin-GFP expression. (N) NGN3 expression determined by quantitative real-time PCR (RT-PCR). (O) Schematic diagram of experimental conditions. (P) Flow cytometry analysis of day 20 cells for NKX6.1 and C-peptide when day 13 pancreatic progenitors were cultured in progenitor medium with and without APH (day 20 control no APH: 41.5% ± 13.44%; day 20 progenitor medium no APH: 13% ± 1.4%; day 20 progenitor medium plus APH: 52% ± 9.9%, n = 2). (Q) Quantitative RT-PCR for NGN3 expression of day 15 cells cultured in basal endocrine medium with and without APH (n = 3). Two-tailed unpaired t test, *P < 0.05. DBZ, γ-secretase inhibitor; T3, thyroid hormone; LDN, LDN193189; KGF, keratinocyte growth factor.