Generating mESC-derived insulin-producing cell lines through an intermediate lineage-restricted progenitor line.
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 beta 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.
Li
GuoDong
G
Cardiovascular Research Institute, National University Medical Institutes, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. nmiligd@nus.edu.sg
Luo
Ruihua
R
Zhang
Jiping
J
Yeo
Keng Suan
KS
Lian
Qizhou
Q
Xie
Fei
F
Tan
Eileen Khia Way
EK
Caille
Dorothée
D
Kon
Oi Lian
OL
Salto-Tellez
Manuel
M
Meda
Paolo
P
Lim
Sai Kiang
SK
D000818:Animals; D002096:C-Peptide / Q000032:analysis; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D019070:Cell Lineage; D017690:Cell Transplantation; D053595:Embryonic Stem Cells / Q000166:cytology*; D004707:Endoderm; D006943:Hyperglycemia / Q000628:therapy; D007328:Insulin / Q000032:analysis; D050417:Insulin-Secreting Cells / Q000166:cytology*; D008648:Mesoderm; D051379:Mice; D016513:Mice, SCID; D016896:Treatment Outcome
D002096:C-Peptide; D007328:Insulin