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.
Highlights
The unlimited proliferation potential of human pluripotent stem cells is both an opportunity as well as a challenge: it provides a renewable source of cells for cell replacement for degenerative disorders such as diabetes, but it is a risk for the formation of growths in cell transplants
We found that protein levels of PDX1, NKX6.1, v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MAFA), and Proinsulin were all upregulated in GFP-positive cells isolated from APH-treated clusters compared with GFP-positive cells in control clusters (Figure 4, G and H)
We found that the expression of CDK inhibitor 1A (CDKN1A, a cell cycle progression inhibitor) was upregulated, and the expression of cyclin D1 (CCND1) and CDK4 was downregulated in APH-treated insulin-positive cells (Figure 5B and Supplemental Figure 7B)
Summary
The unlimited proliferation potential of human pluripotent stem cells is both an opportunity as well as a challenge: it provides a renewable source of cells for cell replacement for degenerative disorders such as diabetes, but it is a risk for the formation of growths in cell transplants. Limitations in proliferation potential are established in parallel to limitations in differentiation potential; cells of the adult pancreas have largely stable identities and a very low proliferative and regenerative potential. When forced into the cell cycle, adult β cells and neurons frequently undergo apoptosis [6, 7], suggesting a compromised ability to progress and complete S phase. Whether these limitations in S phase progression play a functional role in establishing cell-intrinsic limitations in cell proliferation and are important to establish the terminally differentiated state is not known
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