Expansion of human embryonic stem cell (hESC)-derived pancreatic progenitors (PP) and their differentiation to ß-cells
PhD student: Luka Jarc
Supervisor at TUD: Anthony Gavalas
Supervisor at KCL: Aileen King
Start date: 01.09.2017 Date of defense: 08.07.2021 PhD
Diabetes mellitus is a group of metabolic disorders that are characterized by chronic hyperglycaemia. There are currently over 460 million people living with diabetes and the incidence rate for the most common types is sharply on the rise. The hyperglycaemia and subsequently the majority of diabetic side-effects can be cured by ß-cell transplantation. There is a severe lack of donor tissue for clinical transplantations, hence ß-cells derived from human pluripotent stem cells (hPSCs) offer an attractive option for obtaining the necessary cells for a ß-cell therapy. The current ß-cell differentiation protocols are lengthy, expensive and relatively inefficient. Therefore, it remains unrealistic to scale up production to obtain the necessary 1 billion cells per patient to achieve normoglycaemia. The exponential expansion of an intermediate pancreatic progenitor (PP) population would allow for significant reduction of the cost and time necessary for the production of ß-cells in vitro and, more importantly, it would allow to relatively easily obtain the number of cells necessary for clinical applications. Thus far, the reproducible expansion of suitable hPSC-derived PPs in a chemically-defined, feeder-free culture condition has not been reported.
Our lab has found that a previously reported medium suitable for the temporary expansion of reprogrammed fibroblast-derived PPs could occasionally, in 20% of the cases, mediate expansion of PSC derived PPs. To elucidate the requirements for reproducible expansion, I analysed transcriptomic data from PSC-derived PPs before and following expansion. Several regulated signalling pathways were identified and from these data, I formulated nine candidate expansion conditions (C0-C8) to test. Five of these conditions were able to reproducibly expand hPSC-derived PPs. Of those five conditions, C6 was found to mediate the fastest expansion with a doubling time of 2.2 days, maintained a stable expression of the crucial bipotent trunk progenitor transcription factor (TF) markers PDX1, NKX6.1, SOX9 and FOXA2 and minimized the expression of the hepatic and intestinal markers AFP and CDX2. The C6-expanded PPs were able to further differentiate into pancreatic endocrine progenitors (PEPs) in three-dimensional (3-D) islet-sized clusters formed in micropatterned wells. Micropatterned wells offer the additional advantage of size-controlled, uniform clusters and low culture volumes compared to suspension culture bioreactors proposed for the large-scale production of such clusters, but the use of micropatterned wells has not been reported for such an application thus far. These PEPs showed strong upregulation of the crucial endocrine specific TF NGN3 and around 90% were positive for NEUROD1, a downstream effector of NGN3, as determined by flow cytometry analysis. The PEPs were subsequently differentiated into insulin (INS)-producing ß-cells at around 20% efficiency. The ß-cells were also strongly expressing functionality genes such as zinc transporter 8 (ZnT8), glucokinase (GCK), prohormone convertase 1/3 (PC1/3) and sulfonylurea receptor 1 (SUR1, a subunit of KATP channel) and around 10% of them were expressing the crucial maturation marker MAFA, as determined by flow cytometry analysis of ß-cells derived from the H1INS1-GFP/MAFA-mCHERRY double reporter line generated in the lab.
In summary, I have established a feeder-free, chemically defined and ‘good manufacturing practice’ (GMP)-compatible culture condition for the exponential expansion of hPSC-derived PPs that allows for the production of ß-cells at a fraction of the cost of conventional differentiation protocols. Ongoing work is being done to further optimise the expansion conditions to completely eliminate hepatic and intestinal markers and to optimise the subsequent differentiation steps in micropatterned wells to achieve a high-efficiency differentiation towards functional ß-cells.
Publications:
Metabolic and Non-Metabolic Peripheral Neuropathy: Is there a Place for Therapeutic Apheresis? R. Straube, G. Muller, K. Voit-Bak, S. Tselmin, U. Julius, U. Schatz, H. Rietzsch, H. Reichmann, G.P. Chrousos, A. Schurmann, L. Jarc, T. Ziemssen, T. Siepmann, S.R. Bornstein. Horm Metab Res. 2019;51:779-784.
Fragmentation of Pooled PCR Products for Highly Multiplexed TILLING. A. Tramontano A, L. Jarc, J. Jankowicz-Cieslak, B.J. Hofinger, K. Gajek, M. Szurman-Zubrzycka, I. Szarejko, I. Ingelbrecht, B.J. Till. G3. 2019;9:2657-2666.