Investigation of a pancreatic derived progenitor cell population for use in regenerative medicine

Stevenson, Karen Sara (2010) Investigation of a pancreatic derived progenitor cell population for use in regenerative medicine. PhD thesis, University of Glasgow.

Abstract

Diabetes mellitus remains a significant cause of morbidity and mortality despite best current treatment with insulin replacement therapy. Pancreatic stem cells or progenitor
cells have been proposed as an attractive therapy either as a source of cells for in vitro expansion and differentiation to islets for transplantation, or as a direct in vivo cell therapy. Cellular senescence is a state of irreversible growth arrest that can be induced by
multiple factors, including telomere dysfunction associated with replication and oxidant stress. Senescence of stem cell, or progenitor, populations has been proposed as a factor in impaired tissue homeostasis and a mechanism of ageing. In addition, cellular senescence may be a significant limiting factor to any potential therapeutic application of pancreatic stem cells that would require a period of in vitro culture for expansion.
A progenitor cell population from adult rat pancreas, termed pancreas-derived progenitor cells (PDPCs) has previously been isolated in the Shiels laboratory. The current study has sought to determine the character of these PDPCs and determine their potential for therapeutic use. The heterogeneous population of PDPCs were characterised by cell surface markers and RT-PCR. In addition, serial in vitro cultures was performed and cell
separation, via a magnetic activated cell sorting (MACS) protocol, on the basis of Thy1.1 expression, was performed. The expression of cell surface markers between the Thy1.1
positive and Thy1.1 negative cell populations was similar, but not identical. Both cell lines expressed CD147, CD44 and CD49f. Both showed low levels of CD71 expression and did not express the haematopoietic markers CD31, CD34, CD45 and c-kit. However,in contrast to the Thy1.1 positive PDPC population, the Thy1.1 negative PDPC population was positive for CD24.
The common developmental origin of the liver and pancreas suggests that these organs may share common stem/progenitor cell populations. The in vitro potential of both subpopulations of PDPCs (Thy1.1 positive and Thy1.1 negative) to undergo pancreatic and hepatic differentiation was investigated. Using a serum free Fibroblast Growth Factor 4 (FGF4) containing differentiation medium, a change in morphology and gene expression over a 28 day period was observed, indicative of hepatic lineage differentiation for the Thy1.1 positive sub-population. This included induction of gene expression of albumin,cytokeratin 19 (CK 19) and Hepatic Nuclear Factor-1alpha (HNF-1α).Immunocytochemical analysis confirmed functional differentiation with expression of albumin protein and glycogen storage capacity. Both Thy1.1 sub-populations expressed Pdx-1. However, only Thy1.1 positive PDPCs express GATA4, Hepatocyte Nuclear Factor 3 beta (HNF3b) and αFP in an undifferentiated state.The Thy1.1 positive PDPCs exhibited bi-potentiality for differentiation into both pancreatic and hepatic cell types, both morphologically and by RT-PCR. Thy1.1 positive PDPCs could be induced by serum free nicotinamide containing media to form islet-like clusters and demonstrate insulin and glucagon gene expression. No morphological evidence of islet-like clusters was observed using the Thy1.1 negative population.
However, insulin gene transcription was induced by growing Thy1.1 negative cells in pancreatic differentiation medium. Glucagon was not expressed by the undifferentiated Thy1.1 negative cells, nor was it induced in vitro after differentiation.
A human PDPC population from cadaveric pancreatic tissue was isolated using the same protocol. Initial cell surface characterisation and magnetic activated cell sorting on the
basis of Thy1.1 was performed. The Thy1.1 positive population expressed Thy1.1 and CD44 but did not express c-kit, CD49f, CD34, or CD24. The Thy1.1 negative human
PDPC population also expressed CD44 and CD49f, but did not express c-kit, CD34, or CD24. Human PDPCs differed from those derived from rat. Rat-derived Thy 1.1 positive
PDPCs expressed CD49f, whereas human Thy1.1 positive PDPCs did not, while ratderived Thy 1.1 negative PDPCs also expressed CD24 which human Thy1.1 negative PDPCs did not.
Previous work in this laboratory had demonstrated that intravenous delivery of unsorted rat PDPCs mitigated the effects of Streptozotocin (STZ) induced diabetes in mice. The efficacy of intravenous injection of unsorted, Thy1.1 positive and Thy1.1 negative sorted populations of rat PDPCs were investigated and the efficacy of the human PDPCs
assessed in the same model. Normoglycaemia was restored and maintained for up to 100 days following the induction of diabetes in all PDPC treated animals. Normal pancreatic
histology also appeared to be restored and treated diabetic animals gained body weight.Significantly the regeneration observed by immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH) was overwhelmingly mouse in origin.
Rat PDPCs were further assessed in long-term serial culture and in an in vitro acute oxidant stress induced senescence model. After an initial elevation in senescence
biomarkers in vitro there was no evidence of replicative senescence, or shortening of telomeres in long-term culture of PDPCs. However, they were susceptible to acute
oxidant stress and underwent p21WAF/CIP1 dependent, telomere independent senescence. These results demonstrate the feasibility of deriving a functional cell population for use in regenerative medicine strategies. They demonstrate that this is achievable using intravenous administration of adult cells to regenerate pancreatic tissue. Critically, they enhance our understanding of the mechanisms relating to such repair and suggest a means for novel therapeutic intervention in diabetes. Furthermore, they demonstrate that in vitro expansion and endodermal differentiation of progenitors is feasible and thus
PDPCs may be suitable for use in both in vivo transplant and toxicology studies.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Due to copyright restrictions the full text of this thesis cannot be made available online. Access to the printed version is available once any embargo periods have expired
• Keywords: Pancreatic progenitors Thy1.1 and pancreatic progenitors Hwpatic differntiation of pancreatic progenitors Senescence Pancreatic regeneration
• Subjects: R Medicine > R Medicine (General); R Medicine > RZ Other systems of medicine; Q Science > Q Science (General)
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cancer Sciences
• Supervisor's Name: Shiels, Dr. Paul G.
• Date of Award: 2010
• Embargo Date: 17 March 2013
• Depositing User: Dr Karen S Stevenson
• Unique ID: glathesis:2010-1659
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 13 Apr 2010
• Last Modified: 10 Dec 2012 13:44
• URI: https://theses.gla.ac.uk/id/eprint/1659