Directed differentiation of human pluripotent stem cells (hPSCs) has the potential to produce human cell types that can be used for disease modeling and cell transplantation. Two key challenges in the differentiation from hPSCs to \(\beta\) cells are the specification from pancreatic progenitors to insulin-expressing \((INS^+
)\) cells and the maturation of \(INS^+\) cells into glucose responsive β cells. To address the first, two high-content chemical screens identified PKC inhibitors as inducers of \(INS^+\) cells from pancreatic progenitors. PKC inhibition generated up to tenfold more \(INS^+\) cells while PKC agonists blocked differentiation into \(INS^+\) cells. Transplantation of \(PKC\beta\) inhibitor-treated pancreatic progenitors, containing higher
proportions of endocrine progenitors and endocrine cells, resulted in mature \(\beta\) cells showing higher levels of glucose-stimulated human c-peptide production in vivo. This indicates that in vitro derived \(INS^+\)
cells might be competent to mature into functional \(\beta\) cells. To address the second challenge, we first studied mouse and human \(\beta\) cell maturation in vivo. Postnatal mouse \(\beta\) cell maturation was marked by an increase in the glucose threshold for insulin secretion and by expression of the gene urocortin 3. To study human \(\beta\) cell maturation, a Method for Analyzing RNA following Intracellular Sorting (MARIS) was developed and used for transcriptional profiling of sorted human fetal and adult \(\beta\) cells. Surprisingly, transcriptional differences between human fetal and adult \(\beta\) cells did not resemble differences between mouse fetal and adult \(\beta\) cells, calling into question inter-species homology at the late stages of development. A direct comparison between hPSC-derived \(INS^+\) cells, and \(\beta\) cells produced during human development is essential to validate directed differentiation and provide a roadmap for maturation of hPSC-derived \(INS^+\) cells. Genome-wide transcriptional analysis of sorted \(INS^+\) cells derived from three hPSC-lines suggest that different lines produce highly similar \(INS^+\) cells, confirming robustness of directed differentiation protocols. Furthermore, nonfunctional hPSC-derived \(INS^+\) cells resemble human fetal \(\beta\) cells, which are distinct from adult \(\beta\) cells. We therefore suggest that in vitro directed differentiation mimics normal human development and reveal differences in gene expression that may account for the functional differences between hPSC-derived \(INS^+\) cells and true \(\beta\) cells.
| Identifer | oai:union.ndltd.org:harvard.edu/oai:dash.harvard.edu:1/10433470 |
| Date | 18 March 2013 |
| Creators | Hrvatin, Sinisa |
| Contributors | Melton, Douglas A. |
| Publisher | Harvard University |
| Source Sets | Harvard University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis or Dissertation |
| Rights | open |
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