Generation of beta cells from human pluripotent stem cells: Potential for regenerative medicine

Abstract

The loss of beta cells in Type I diabetes ultimately leads to insulin dependence and major complications that are difficult to manage by insulin injections. Given the complications associated with long-term administration of insulin, cell-replacement therapy is now under consideration as an alternative treatment that may someday provide a cure for this disease. Over the past 10 years, islet transplantation trials have demonstrated that it is possible to replenish beta cell function in Type I diabetes patients and, at least temporarily, eliminate their dependency on insulin. While not yet optimal, the success of these trials has provided proof-of-principle that cell replacement therapy is a viable option for treating diabetes. Limited access to donor islets has launched a search for alternative source of beta cells for cell therapy purposes and focused the efforts of many investigators on the challenge of deriving such cells from human embryonic (hESCs) and induced pluripotent stem cells (hiPSCs). Over the past five years, significant advances have been made in understanding the signaling pathways that control lineage development from human pluripotent stem cells (hPSCs) and as a consequence, it is now possible to routinely generate insulin producing cells from both hESCs and hiPSCs. While these achievements are impressive, significant challenges do still exist, as the majority of insulin producing cells generated under these conditions are polyhormonal and non functional, likely reflecting the emergence of the polyhormonal population that is known to arise in the early embryo during the phase of pancreatic development known as the 'first transition'. Functional beta cells, which arise during the second phase or transition of pancreatic development have been generated from hESCs, however they are detected only following transplantation of progenitor stage cells into immunocompromised mice. With this success, our challenge now is to define the pathways that control the development and maturation of this second transition population from hPSCs, and establish conditions for the generation of functional beta cells in vitro.

Figure 1. Schematic representation of the developmental steps leading to beta cells development

Insulin-producing cells are generated in two distinct phases during embryonic development, known as the first and second transition. The cells produced during the first transition, are predominantly poly-hormonal and do not contribute to the adult islets of Langherans. During the second transition, adult beta cells arise from a multipotent progenitor that expresses pdx1, nkx6-1, sox9 and ptf1a. In the absence of nkx6.1 adult beta cells are not generated. Using hPSCs to model pancreatic development, we have recapitulated the steps leading to the formation of 1^st transition pancreatic progenitors (highlighted in red). Signaling pathways leading to the generation of the progenitors of the second transition and beta cells in vitro still remain to be elucidated (highlighted in orange).

Figure 2. Three-dimensional representation of the signaling requirements for germ layer induction from hPSCs

Endoderm (red blocks) arises in the presence of high concentration of Activin A and low levels of canonical WNT signaling [42, 43]. Short exposure to Activin A and WNT (in the absence of BMP, not in the figure) will lead to the formation of the anterior foregut endoderm (A. Foregut) [48]. A longer period of Activin A signaling will induce posterior foregut endoderm (P. Foregut), whereas intermediate exposure together with increasing canonical WNT signaling will give rise to midgut and hindgut endoderm (Ogawa in preparation)[38, 55]. Lower levels of Activin A signaling will induce anterior (A. Mesoderm) and posterior mesoderm (P. Mesoderm) (orange and yellow blocks) [43]. Ectoderm is induced in the absence of Activin A, WNT (and BMP, not in the figure) (green block) [93].

Figure 3. Schematic representation of the signaling pathways that regulate the development of first transition endocrine cells from hPSCs

Pancreatic lineage specification from hPSC-derived definitive endoderm requires stage specific inhibition of the BMP, SHH, TGF-β and NOTCH pathways. BMP inhibition is required throughout the differentiation while FGF signaling and SHH inhibition are required during endoderm patterning (Definitive Endoderm, DE to Primitive Gut, PG) and pancreatic specification (PG to the Pancreatic Progenitor of the first transition 1^st PP). Canonical WNT is required to pattern definitive endoderm to a pancreatic-competent epithelium (DE to PG) and RA is important for pancreatic specification (PG to 1^st PP). Endocrine lineage commitment is achieved in the presence of BMP/TGF-β/NOTCH inhibition and forskolin treatment (1^st PP to Pancreatic Endocrine, PE).