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

Viacyte, Inc., Athens, Georgia, United States of America.
PLoS ONE (Impact Factor: 3.23). 05/2012; 7(5):e37004. DOI: 10.1371/journal.pone.0037004
Source: PubMed


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.

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    • "These additional manipulations of signaling pathways resulted in a protocol that was effective for many different stem cell lines. Based on these studies, a number of groups have succeeded in generating insulin-producing cells with an efficiency of about 25% (Kelly et al, 2011; Nostro et al, 2011; Kunisada et al, 2012; Rezania et al, 2012, 2013; Schulz et al, 2012; Hua et al, 2013; Shang et al, 2014). However, none of them reported the derivation of glucoseresponsive and monohormonal insulin-positive cells. "
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    ABSTRACT: Bjarki Johannesson1, Lina Sui2, Donald O Freytes1, Remi J Creusot3 and Dieter Egli*,1,21The New York Stem Cell Foundation Research Institute, New York, NY, USA2Naomi Berrie Diabetes Center & Department of Pediatrics, College of Physicians and Surgeons, Columbia University, New York, NY, USA3Columbia Center for Translational Immunology, Department of Medicine and Naomi Berrie Diabetes Center, Columbia University, New York, NY, USA↵*Corresponding author. Tel: +1 212 851 4890; E‐mail: de2220{at}cumc.columbia.edu Abstract The discovery of insulin more than 90 years ago introduced a life‐saving treatment for patients with type 1 diabetes, and since then, significant progress has been made in clinical care for all forms of diabetes. However, no method of insulin delivery matches the ability of the human pancreas to reliably and automatically maintain glucose levels within a tight range. Transplantation of human islets or of an intact pancreas can in principle cure diabetes, but this approach is generally reserved for cases with simultaneous transplantation of a kidney, where immunosuppression is already a requirement. Recent advances in cell reprogramming and beta cell differentiation now allow the generation of personalized stem cells, providing an unlimited source of beta cells for research and for developing autologous cell therapies. In this review, we will discuss the utility of stem cell‐derived beta cells to investigate the mechanisms of beta cell failure in diabetes, and the challenges to develop beta cell replacement therapies. These challenges include appropriate quality controls of the cells being used, the ability to generate beta cell grafts of stable cellular composition, and in the case of type 1 diabetes, protecting implanted cells from autoimmune destruction without compromising other aspects of the immune system or the functionality of the graft. Such novel treatments will need to match or exceed the relative safety and efficacy of available care for diabetes. beta cellscell replacement therapystem cellstype 1 diabetesReceived November 27, 2014.Revision received January 13, 2015.Accepted January 22, 2015.© 2015 The Authors
    The EMBO Journal 01/2015; · 10.43 Impact Factor
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    • "The most important aspects of stem cells are their ability to self-renew and to differentiate into many different kinds of cells. These properties have contributed to the use of stem cells in various ways such as cell replacement therapies (Schulz et al.2012; Nelander et al.2013; Sillence et al.2012), tissue engineering (He and Callanan 2013), and pharmacology or toxicology screens (Desbordes and Studer 2013; Jonsson et al.2012). Each of these applications requires a large number of cells of high quality, which requires quick cell expansion. "
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    ABSTRACT: Stem cells have emerged as important players in the generation and maintenance of many tissues. However, the accurate in vitro simulation of the native stem cell niche remains difficult due at least in part to the lack of a comprehensive definition of the critical factors of the stem cell niche based on in vivo models. Three-dimensional (3D) cell culture systems have allowed the development of useful models for investigating stem cell physiology particularly with respect to their ability to sense and generate mechanical force in response to their surrounding environment. We review the use of 3D culture systems for stem cell culture and discuss the relationship between stem cells and 3D growth matrices including the roles of the extracellular matrix, scaffolds, soluble factors, cell-cell interactions and shear stress effects within this environment. We also discuss the potential for novel methods that mimic the native stem cell niche in vitro as well as the current associated challenges.
    SpringerPlus 02/2014; 3(1):80. DOI:10.1186/2193-1801-3-80
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    • "The differentiation of human embryonic stem cells (hESCs) has been extensively studied to generate specific cell types for cell-based therapies and drug screening tests, and to study the early development of human embryos [Thomson et al., 1998; Reubinoff et al., 2000]. Several cell types, such as cardiomyocytes [Mummery et al., 2012], neurons [Zeng et al., 2004; Hong et al., 2008; Zhu et al., 2012], retinal pigment epithelium [Idelson et al., 2009; Hu et al., 2012] and pancreatic β cells [D'Amour et al., 2006; Kroon et al., 2008; Schulz et al., 2012], were successfully generated from hESCs through specific differentiation protocols. Recently, Schwartz et al. [2012] showed that hESCs could be differentiated into functional retinal pigment epithelial cells that were subsequently transplanted into a patient to treat macular degeneration; in this preliminary report, researchers successfully used cells derived from hESCs in a clinical setting. "
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    ABSTRACT: Human neural progenitor cells (hNPCs) are the starting material required for neuronal subtype differentiation. Proliferation of hNPCs allows researchers to study the mechanistic complexities and microenvironments present during neural differentiation and to explore potential applications for hNPCs in cell therapies. The use of enzymatic dissociation during hNPC proliferation causes dissociation-induced apoptosis; therefore, in the present study, we examined the effect of the p-160-Rho-associated coiled-coil kinase (ROCK) inhibitor Y-26732 on dissociation-induced apoptosis of hNPCs. We generated hNPCs via embryoid body formation using serum-free culture medium supplemented with noggin. The established hNPCs were characterized and the effect of the ROCK inhibitor on hNPC dissociation was studied. We demonstrated that supplementation of the culture media with 10 μM Y-26732 efficiently reduced apoptosis of dissociated hNPCs; this supplementation was effective when the inhibitor was applied either at (i) 24 h before dissociation of the cells and at 24 h after plating the cells or (ii) at 24 h after plating of the cells only. In addition to reducing apoptosis, both supplementation conditions with Y-26732 enhanced the proliferation of dissociated hNPCs. Our findings provide the optimal time window for ROCK treatment of hNPC dissociation in respect to apoptosis and cell proliferation. © 2013 S. Karger AG, Basel.
    Cells Tissues Organs 10/2013; 198(2):127-138. DOI:10.1159/000354031 · 2.14 Impact Factor
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