Derivation of functional insulin-producing cell lines from primary mouse embryo culture.
We have previously described the derivation of insulin-producing cell lines from mouse embryonic stem cells (mESCs) by differentiation of an intermediate lineage-restricted E-RoSH cell line through nutrient depletion in the presence of nicotinamide followed by limiting dilution. Here we investigated whether insulin-producing cell lines could be similarly derived directly from mouse embryo cells or tissues. Using a similar approach, we generated the RoSH2.K and MEPI-1 to -14 insulin-producing cell lines from the 5.5-dpc embryo-derived E-RoSH-analogous RoSH2 cell line and a 6.0-dpc mouse embryo culture, respectively. Insulin content was approximately 8 microg/10(6) MEPI-1 cells and 0.5 microg/10(6) RoSH2.K cells. Like insulin-producing mESC-derived ERoSHK cell lines, both MEPI and RoSH2.K lines were amenable to repeated cycles of freeze and thaw, replicated for months with a doubling time of 3-4 days, and exhibited genomic, structural, biochemical, and pharmacological properties of pancreatic beta-cells, including storage and release of insulin and C-peptide in an equimolar ratio. Transplantation of these cells also reversed hyperglycemia in streptozotocin-treated SCID mice and did not induce teratoma. Like ERoSHK cells, both RoSH2.K and MEPI-1 cells also induced hypoglycemia in the mice. Therefore, our protocol is robust and could reproducibly generate insulin-producing cell lines from different embryonic cell sources.
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
Xie
Fei
F
Way Tan
Eileen Khia
EK
Caille
Dorothée
D
Que
Jianwen
J
Kon
Oi Lian
OL
Salto-Tellez
Manuel
M
Meda
Paolo
P
Lim
Sai Kiang
SK
D000818:Animals; D002096:C-Peptide / Q000096:biosynthesis; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002460:Cell Line; D017690:Cell Transplantation; D004622:Embryo, Mammalian; D006943:Hyperglycemia / Q000139:chemically induced / Q000628:therapy*; D007328:Insulin / Q000032:analysis / Q000096:biosynthesis; D050417:Insulin-Secreting Cells / Q000166:cytology* / Q000637:transplantation; D051379:Mice; D016513:Mice, SCID; D013311:Streptozocin; D016896:Treatment Outcome
D002096:C-Peptide; D007328:Insulin; D013311:Streptozocin