In vivo functioning and transplantable mature pancreatic islet-like cell clusters differentiated from embryonic stem cell.
BACKGROUND
Although the differentiation of embryonic stem (ES) cells to islet like clusters using differentiation method without employing gene transfer technique has been recently reported, neither endocrine granules in the cytoplasm nor in vivo function of differentiated islet like clusters has been demonstrated.


OBJECTIVE
To investigate whether ES cells could be differentiated to mature islet like clusters which show in vivo function after transplantation as well as retain endocrine granules in the cytoplasm by electron microscopic observation.


METHODS
In this experiment, using mouse embryonic stem (mES) cells as a model system for lineage specific differentiation, we tried to differentiate mES cells to pancreatic islet-like cell clusters (PICCs) through a series of treatments (4-step procedure). Differentiated PICCs were analyzed and characterized by various techniques, such as RT-PCR, immunohistochemistry, electron microscopic observation, in vitro static incubation test, and in vivo transplantation to diabetic animals.


RESULTS
Differentiated islet-like cell clusters from ES cells using our newly developed method (four-step procedure) showed strong expression of essential specific genes to the endocrine pancreas and also specific genes to the exocrine pancreas demonstrating that these islet-like clusters were mature from the developmental biologic point of view. These differentiated cells clearly revealed many mature insulin secretory granules of pleomorphic shape in the cytoplasm as well as well-developed rough endoplasmic reticulum. In vitro study indicated that differentiated cells retain a potent insulin secretory responsiveness to glucose stimulation. Furthermore, the islet-like cell clusters significantly decreased high blood glucose levels almost to normal levels when grafted to streptozotocin-induced diabetic mice without induction of any teratoma formation after transplantation.


CONCLUSIONS
Our results provide evidence that ES cells could differentiate to functioning and transplantable mature pancreatic islet-like cell clusters using our newly developed differentiation method without employing gene transfer technique. This study may lead to a basis for production of indefinite sources of islets that could be applicable for future clinical trial.
Kim
Dohoon
D
Department of Organ Reconstruction, Institute for Frontier Medical Sciences, Kyoto University, 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.
Gu
Yuanjun
Y
Ishii
Michiyo
M
Fujimiya
Mineko
M
Qi
Meirigeng
M
Nakamura
Naoto
N
Yoshikawa
Toshikazu
T
Sumi
Shoichiro
S
Inoue
Kazutomo
K
D000818:Animals; D001786:Blood Glucose / Q000378:metabolism; D001835:Body Weight; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D003921:Diabetes Mellitus, Experimental / Q000097:blood / Q000628:therapy; D004622:Embryo, Mammalian / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D007150:Immunohistochemistry; D007328:Insulin / Q000032:analysis / Q000378:metabolism; D000078790:Insulin Secretion; D016381:Islets of Langerhans Transplantation / Q000379:methods*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008815:Mice, Inbred Strains; D008819:Mice, Nude; D008854:Microscopy, Electron; D010179:Pancreas / Q000378:metabolism / Q000503:physiopathology / Q000648:ultrastructure; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure
D001786:Blood Glucose; D007328:Insulin