University of Groningen
Development of MAPC derived induced endodermal progenitors Sambathkumar, Rangarajan
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Sambathkumar, R. (2017). Development of MAPC derived induced endodermal progenitors: Generation of pancreatic beta cells and hepatocytes. University of Groningen.
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Chapter 2
Rationale and Objectives
Chapter 2
Rationale and objectives for studies in chapter 3 and 4
Human MAPCs have extended proliferative potential (>70 population doublings). They differentiate in vitro to osteogenic, chondrogenic, adipogenic, and smooth muscle cells and unlike classical MSC (mesenchymal stem cells), also into endothelial cells [1-‐3]. However, by applying growth factors used for rodent MAPCs or human ESCs to differentiate hMAPCs to pancreatic endocrine β-‐cells and hepatocytes [4-‐7], differentiation is not robust.
The discovery of induced pluripotent stem cells (iPSCs) demonstrated that the differentiation state of mature cells can be manipulated and reprogrammed to a pluripotent state [8] from which mature functional pancreatic endocrine cells [9] and hepatocyte like cells [10] can be created. In addition, numerous studies have demonstrated that in mouse, rat and more recently in human, multiple different mature cells by using TFs alone or combined with growth factors and /or multiple small molecules can be converted to cells with at least some characteristics of β-‐cells [11-‐23] [24-‐31] or hepatocytes [32-‐36] and this by using TFs alone or combined with growth factors and/or multiple small molecules.
To overcome the restricted differentiation of human MAPCs towards pancreatic endodermal cells and hepatocytes, I tested the hypothesis that by using a complement of TFs, chosen based on insights from early endoderm development and differentiation to pancreatic β-‐cell and hepatocyte fate specifications, it might be possible to transdifferentiate hMAPC to an induced endoderm progenitor (iENDO) state that then could be differentiated towards pancreatic β-‐cells or hepatocytes. To address this hypothesis, I formulated the following objectives.
Objective 1: To determine if reprogramming with 16 (or fewer) TFs can create iENDO
cells, defined as cells expressing (mes) endodermal transcription factors, not mature endodermal genes, that can be expanded in vitro, and can then be committed to maturing endodermal progeny (pancreatic and hepatic).
Objective 2: Determine the in vivo differentiation potential and tumorogenicity of
iENDO cells by grafting these cells under the kidney capsule of immunodeficient mice.
Objective 3: Determine if iENDO cells can be differentiated in vitro into pancreatic
endocrine and hepatocyte like cells using established protocols obtained from PSC differentiation studies.
Objective 4: Determine the in vivo differentiation potential and tumorogenicity of
iENDO cells pre-‐differentiated to the hepatocyte and β-‐cell lineage, by grafting under the kidney capsule of immunodeficient mice.
Figure.1 Schematic representation of research hypothesis of studies in Chapter 3 and 4.
2.1 Rationale for the study (related to chapter 5)
A number of studies have demonstrated that small molecule DNA methyltransferase inhibitors 5’Azacytidine (5’AZA) and Histone deacetylase inhibitor (HDACi) Trichostatin-‐A, (TSA) can change cell fate into the pancreatic cell lineage, both in the setting of differentiation from lineage specific progenitors and transdifferentiation [37-‐43]. Therefore, I tested if a combination of the epigenetic modifiers 5AZA and TSA with combination of chromatin remodeling medium (CRM) could reprogram primary human skin and BJ1 foreskin fibroblasts to an endodermal and pancreatic endodermal cell fate.
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