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 5

Transient APH treatment reduces growth potential and increases stability of insulin expression by upregulating cell cycle inhibitors.

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Transient APH treatment reduces growth potential and increases stability...
(A) Cell cycle profile of untreated and treated cells on day 27 indicated by flow cytometry combined with Hoechst staining for DNA content and KI67 labeling. (B) The differential expression of cell cycle genes between control and APH-treated cells using single-cell RNA sequencing. Violin plots show probability density of gene expression of given single cells. (C–E) Schematic diagrams of cell cycle progression experiments with indicated conditions. (E) APH-treated cells were released and labeled with EdU for 2 hours on either day 17 (n = 3 in each condition) or 26 (n = 4 in each condition), and analyzed 1 day later (day 18 or 27) for cell cycle distribution. Cells without APH treatment (C) and unreleased from APH (D) were analyzed in parallel. (F) A schematic diagram represents the timeline of APH addition, release from cell culture, and cell cycle progression analysis after 2-hour EdU incubation on day 34. Created with BioRender.com (G) Cell cycle distribution (n = 4) and (H) percentage of EdU- and C-peptide–positive cells on day 34 with (n = 4) and without APH (n = 4) from days 15 to 27. Two-tailed unpaired t test *P < 0.05. (I) Microscopic pictures of cells on day 34 after releasing cells from APH from day 27 to 34. Scale bar: 100 μm. (J) Quantification of C-peptide– and NKX6.1-positive cells and C-peptide–positive cells on days 27 and 34 before and after APH releasing. One-way ANOVA test **P < 0.01; ****P < 0.0001.