A Scalable System for Production of Functional Pancreatic Progenitors from Human Embryonic Stem Cells

Abstract

Development of a human embryonic stem cell (hESC)-based therapy for type 1 diabetes will require the translation of proof-of-principle concepts into a scalable, controlled, and regulated cell manufacturing process. We have previously demonstrated that hESC can be directed to differentiate into pancreatic progenitors that mature into functional glucose-responsive, insulin-secreting cells in vivo. In this study we describe hESC expansion and banking methods and a suspension-based differentiation system, which together underpin an integrated scalable manufacturing process for producing pancreatic progenitors. This system has been optimized for the CyT49 cell line. Accordingly, qualified large-scale single-cell master and working cGMP cell banks of CyT49 have been generated to provide a virtually unlimited starting resource for manufacturing. Upon thaw from these banks, we expanded CyT49 for two weeks in an adherent culture format that achieves 50–100 fold expansion per week. Undifferentiated CyT49 were then aggregated into clusters in dynamic rotational suspension culture, followed by differentiation en masse for two weeks with a four-stage protocol. Numerous scaled differentiation runs generated reproducible and defined population compositions highly enriched for pancreatic cell lineages, as shown by examining mRNA expression at each stage of differentiation and flow cytometry of the final population. Islet-like tissue containing glucose-responsive, insulin-secreting cells was generated upon implantation into mice. By four- to five-months post-engraftment, mature neo-pancreatic tissue was sufficient to protect against streptozotocin (STZ)-induced hyperglycemia. In summary, we have developed a tractable manufacturing process for the generation of functional pancreatic progenitors from hESC on a scale amenable to clinical entry.

Blastema cells derived from New Zealand white rabbit’s pinna carry stemness properties as shown by differentiation into insulin producing, neural, and osteogenic lineages representing three embryonic germ layers

“…Although induced SLCs were able to produce and secrete insulin, they did not respond to glucose stimulation. This was in line with previous reports indicating that induced ESCs might present immature b-like cells and more induction is required to reach a higher degree of differentiation (Gao et al 2008;Kroon et al 2008;Rezania et al 2012;Schulz et al 2012). To further study differentiated SLCs, expression of Ins, Pax6 and Glut4 was assayed by RT-PCR.…”

Immunogenicity of human embryonic stem cell-derived beta cells

“…For each series of experiments, cells were thawed and cultured for 72 h in db-N50-K50-E50 medium [7]. A fraction of the cells was sent to Leiden in db medium [1] and the remaining cells were transplanted in Brussels.…”

“…hESC-derived beta cells are well on the way to clinical translation with recent publications on improved safety and scaling of a protocol being made by Kroon and colleagues [1,7,8].…”

A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation

“…They therefore represent highly promising cell sources for numerous biomedical applications, such as cell replacement therapies (4,5), tissue and organ engineering (6), and pharmacology and toxicology screens (7,8). However, these applications require large numbers of cells of high quality (4,(6)(7)(8). For instance, ∼10 5 surviving dopaminergic (DA) neurons, ∼10 9 cardiomyocytes, or ∼10 9 beta cells are likely required to treat a patient with Parkinson disease (PD), myocardial infarction (MI), or type I diabetes, respectively (9).…”