Generating mESC-derived insulin-producing cell lines through an intermediate lineage-restricted progenitor line |
| |
Authors: | GuoDong Li Ruihua Luo Jiping Zhang Keng Suan Yeo Qizhou Lian Fei Xie Eileen Khia Way Tan Dorothée Caille Oi Lian Kon Manuel Salto-Tellez Paolo Meda Sai Kiang Lim |
| |
Institution: | 1. Cardiovascular Research Institute, National University Medical Institutes, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Singapore;2. Genome Institute of Singapore, ASTAR Singapore;3. Department of Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland;4. National Cancer Center, Singapore;5. Department of Pathology, National University of Singapore, Singapore 117597, Singapore;6. Institute of Medical Biology, ASTAR, Singapore 138648, Singapore;7. Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Singapore;8. School of Biological Sciences, Nanyang Technological University, Singapore |
| |
Abstract: | Generating surrogate insulin-producing cells from embryonic stem cells (ESCs) through in vitro replication of successive steps during pancreatic development has been challenging . Here we describe a novel reproducible protocol to establish homogeneous and scalable insulin-producing cell lines from mouse (m) ESCs via differentiation of the previously described lineage-restricted clonal mESC-derived E-RoSH cells. Unlike their parental mESCs, E-RoSH cells expressed high levels of mesodermal and endodermal genes. Nutrient depletion in the presence of nicotinamide inhibited proliferation of E-RoSH cells and induced differentiation into heterogeneous cultures comprising vascular-like structures that produced detectable levels of insulin and C-peptide in an equimolar ratio. Limiting dilution of these cultures resulted in the isolation of eight independent insulin-producing cell lines in five experiments. All these lines were cloned and shown to be amenable to repeated cycles of freeze and thaw and to replicate for months with a doubling time of 3–4 days. Under such conditions, the cultured cells exhibited genomic, structural, biochemical, and pharmacological properties of pancreatic β cells, including storage of an equimolar ratio of insulin and C-peptide in granules and release of the contents of these organelles through a glucose-sensitive machinery. After transplantation, these cells reversed hyperglycemia in streptozotocin-treated SCID mice and did not form teratomas. |
| |
Keywords: | |
本文献已被 ScienceDirect 等数据库收录! |
|