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 G 1 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 G 1 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.

Characterization of stem-cell-derived islets during differentiation and after implantation

“…In terms of chemical methods of off-target elimination, the DNA polymerase inhibitor aphidicolin inhibits G1-to-S phase transition, and it has been shown that addition during endocrine progenitor stage differentiation has a substantial effect on b cell maturation and prevention of teratoma formation (Sui et al, 2021;Ben-David and Benvenisty, 2014). Inhibition of the prooncogene survivin with YM155 has also demonstrated targeted hiPSC cell death (Lee et al, 2013).…”

“…SC-islets should lack non-endocrine contaminant cells that may lead to tumorigenicity or complications. Regardless of the cell population transplanted (stage 4 [PPs] versus stage 6 [SC-islets]), upon transplantation and in vivo maturation, SC-islets generated with the current protocols have insulin biogenesis capacity and glucose-responsive insulin release (Sui et al, 2021;Velazco-Cruz et al, 2019, 2020Millman et al, 2016;Millman and Pagliuca, 2017;Aghazadeh et al, 2022;Cogger et al, 2017). Evidently, generation of SC-islets in vitro followed by maturation in vivo upon transplantation enables improved b-like cell functional efficiency and insulin secretion capacity compared with transplantation of PPs (Hogrebe et al, 2020;Rezania et al, 2014;Pagliuca et al, 2014).…”

Harnessing Proliferation for the Expansion of Stem Cell-Derived Pancreatic Cells: Advantages and Limitations

“…The authors of this study tested the CDK inhibitor roscovitine during endocrine progenitor differentiation and found that endocrine progenitor commitment was not affected by CDK inhibition (48). In contrast, disrupting the cell cycle using the compound aphidicolin throughout endocrine progenitor and b-like cell commitment, which arrested endocrine progenitor cells at G1 and inhibited the completion of S phase, improved the differentiation of endocrine progenitor cells to b-like cells, but this effect was not seen in the same degree with CDK inhibition alone (49). This may suggest that compounds that disrupt cell cycle progression in endocrine progenitors enhance differentiation to end-stage b-like cells.…”

Stem Cell-Derived β Cells: A Versatile Research Platform to Interrogate the Genetic Basis of β Cell Dysfunction

“…It is possible that future improvements in differentiation protocols will enhance SC-β maturity bringing the proliferation rate close to that of adult β cells. Indeed, this tradeoff between β cell proliferation and maturity is reported in SC-β and murine β cells [ 333 , 334 , 335 ]. SC-β platforms for screening approaches might represent a cost-effective alternative when compared with human islets and will also bypass the problems associated with wide heterogeneity in human samples.…”