Engineered human pluripotent stem cell lines for following differentiation into pancreatic islets and addressing their maturation
Year: 2023
Type: dissertation
Abstract: Human pluripotent stem cells (hPSC) have the potential to generate all the differentiated cell types of the body and this is why they represent a promising option for cell therapies for diseases such as diabetes.Patients with diabetes Type 1 have an absolute or relative lack of insulin-secreting pancreatic β-cells and this results in inability to normalize blood glucose levels.The treatment is based on lifetime exogenous insulin administration, but this does not fully replicate the function of the endogenous β-cells.Transplantation of the full pancreas or islets could be an alternative, but this is limited by the need for immunosuppression as well as the scarcity and quality of donor islets.Hence to provide these patients with insulin independence, hPSC represent an abundant alternative supply of cells for mass production of uniform hPSC-derived islet-like clusters (SC-islets).Tremendous progress has been achieved in the differentiation of hPSC into SC-islets.Currently, hPSC can be differentiated to definitive endoderm and subsequently to pancreatic progenitors with high efficiencies.However, current procedures still need optimization because they still give rise to polyhormonal β-cells and cells partially lack function.We have developed a procedure, adapted from the Kieffer lab, which consists of seven stages and includes a transition from a 2D culture of pancreas progenitor cells to a 3D cluster culture in micropatterned wells.This approach enables us to produce a large number of uniform SC-islets that contain functional β-cells.To efficiently screen for conditions enhancing endocrine specification and maturation, I developed several hPSC reporter lines using CRISPR/Cas9-mediated homologous recombination.In particular, with the INS eGFP and the INS eGFP /GCG mCherry lines we will be able to follow the efficiency of the differentiation into monohormonal cells.We will use the INS eGFP /MAFA mCherry line to monitor the maturation of differentiated β-cells, whereas with the INS GCaMP6 and PHOGRIN CLIPeGFP we will evaluate the functionality of the hPSC derived β-cells.I showed that, after the genetic modification, all lines retained their pluripotency by strongly and uniformly expressing the key pluripotent markers OCT4, SOX2 and SSEA4.The reporter lines efficiently differentiated into definitive endoderm cells and subsequently into pancreatic progenitors with similar efficiency to the H1 parent line.Up to 60% of the pancreas progenitors co-expressed the specific markers PDX1, SOX9 and NKX6.1.After clustering, PP cells, derived from the reporter lines, differentiated into pancreas endocrine progenitors expressing high levels of the endocrine progenitor markers NEUROG3 and its downstream effector NEUROD1, like the H1 wild type derived cells.Similar to the SC-islets derived from the parent line, reporter line derived SC-islets showed high expression of the hormones INS, GCG and SST as well as of the β-cell markers, PDX1 and NKX6.1.SC-islets derived from all reporter lines contained functional β-cells that responded to glucose in a similar manner to that VII
Author: Zanfrini, Elisa
Language: English
Subfield: Endocrinology, Diabetes and Metabolism
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W7009417040