(623be) Production of Islet Cell Progeny From Human Pluripotent Stem Cells In Microcarrier Cultures

Human pluripotent stem cells (hPSCs) can serve as inexhaustible sources of islet cells for pancreatic tissue engineering and diabetes therapies. Essential for the success of stem cell-based diabetes applications is the development of efficient differentiation protocols and bioprocesses for converting large quantities of hPSCs into genetically normal and functional insulin-producing cells. Here, we employed multiple human embryonic stem cell (hESC) lines to develop a protocol for guiding stem cells along pancreatic cell lineages. Furthermore, our differentiation approach was adapted to the scalable culture of hPSCs in stirred-suspension microcarrier bioreactors.    

A stage-wise differentiation strategy was established to sequentially differentiate hESCs into definitive endoderm, primitive gut tube, posterior foregut and pancreatic islet cells. Differentiation was induced in serum-free media with physiological factors involved in embryonic pancreas development while formation of embryoid bodies was avoided. The differentiating hPSC populations were analyzed by quantitative PCR, immunostaining and flow cytometry and ELISA (for hormone secretion). Microcarrier bioreactor cultures of hPSCs were carried out and analyzed (e.g. cell concentration, viability, lactate dehydrogenase activity etc.) as we described^1,2.    

Human PSC monolayers were coaxed to definitive endoderm cells co-expressing FOXA2 and SOX17 as we reported¹. Subsequent incubation with retinoic acid and FGF7 led to the emergence of HNF1B⁺/HNF4A⁺ cells after 4-5 days while the first PDX1⁺/HNF6⁺ cells appeared on day 8. Additional factors were also explored for curtailing cell apoptosis. The fraction of cells transitioning between successive stages was above 70% as assessed by flow cytometry for the co-expression of relevant markers. These cells gave rise to islet cell-like progeny including cells expressing PDX1, NGN3 and NKX6.1. Cells were also immunopositive for C-peptide and insulin while release of these antigens was detected in response to glucose and secretagogues.

The differentiation scheme was also translated to stirred-suspension microcarrier cultures of hPSCs. Cells were expanded on microcarrier bioreactors under non-differentiating conditions as described¹. Then, the differentiation protocol developed in static cultures was applied. Separate experiments were performed to select bioreactor culture conditions (e.g., the initial cell concentration, cell/microcarrier ratio, agitation rate) which allow the propagation of cells without hindering their pancreatogenic commitment. Cells on microcarriers transitioned through the same stages exhibiting appropriate markers leading to a population with 40% PFG cells. Cells further gave rise to islet-like cells expressing PDX1, NKX2.2, NKX6.1 and GLUT2. Over 8% of hPSC-derived cells actively transcribed insulin. These cells were also immunoreactive to insulin and C-peptide. Current efforts focus on increasing the yield of insulin-producing cells in the differentiation of hPSCs and assessing their function  in vivo.    

A protocol was established for the efficient generation of pancreatic islet progeny from hPSCs using physiological factors in the absence of serum and without the formation of embryoid bodies. Moreover, this differentiation strategy was successfully combined with the microcarrier bioreactor culture of hPSCs. This is an important step toward the establishment of bioprocesses for the generation of functional islet cells for pancreatic tissue engineering and diabetes therapies.

 

Acknowledgements: Support for this project was provided by a grant (C024355) to E.S.T. from the New York State Stem Cell Trust (NYSTEM).

References:

1. Lock, L.T. et al. Tissue Eng. Part A 2009, 15:2051-63.

2. Kehoe, D. et al. Tissue Eng. Part A. 2010, 16:405-21.