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Generation of insulin-producing β-like cells from human iPS cells in a defined and completely xeno-free culture system.

Journal of molecular cell biology (2014-06-28)
Hussain Md Shahjalal, Nobuaki Shiraki, Daisuke Sakano, Kazuhide Kikawa, Soichiro Ogaki, Hideo Baba, Kazuhiko Kume, Shoen Kume
ABSTRACT

Human induced pluripotent stem (hiPS) cells are considered a potential source for the generation of insulin-producing pancreatic β-cells because of their differentiation capacity. In this study, we have developed a five-step xeno-free culture system to efficiently differentiate hiPS cells into insulin-producing cells in vitro. We found that a high NOGGIN concentration is crucial for specifically inducing the differentiation first into pancreatic and duodenal homeobox-1 (PDX1)-positive pancreatic progenitors and then into neurogenin 3 (NGN3)-expressing pancreatic endocrine progenitors, while suppressing the differentiation into hepatic or intestinal cells. We also found that a combination of 3-isobutyl-1-methylxanthine (IBMX), exendin-4, and nicotinamide was important for the differentiation into insulin single-positive cells that expressed various pancreatic β-cell markers. Most notably, the differentiated cells contained endogenous C-peptide pools that were released in response to various insulin secretagogues and high levels of glucose. Therefore, our results demonstrate the feasibility of generating hiPS-derived pancreatic β-cells under xeno-free conditions and highlight their potential to treat patients with type 1 diabetes.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
DAPI, for nucleic acid staining
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3-Isobutyl-1-methylxanthine, ≥99% (HPLC), powder
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Nicotinamide, BioReagent, suitable for cell culture, suitable for insect cell culture
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L-Glutamine, meets USP testing specifications, suitable for cell culture, 99.0-101.0%, from non-animal source
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Nicotinamide, ≥99.5% (HPLC)
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3-Isobutyl-1-methylxanthine, BioUltra, ≥99%
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L-Glutamine, ReagentPlus®, ≥99% (HPLC)
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Niacinamide, Pharmaceutical Secondary Standard; Certified Reference Material
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Nicotinamide, ≥98% (HPLC), powder
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Carbamoylcholine chloride, ≥98% (titration), crystalline
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Niacinamide, meets USP testing specifications
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FGF-2 human, recombinant, expressed in E. coli, ≥95% (SDS-PAGE), ≥95% (HPLC)
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L-Glutamine, BioUltra, ≥99.5% (NT)
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L-Glutamine, γ-irradiated, BioXtra, suitable for cell culture
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