Changes in gene expression and morphology of mouse embryonic stem cells on differentiation into insulin-producing cells in vitro and in vivo.
BACKGROUND
Embryonic stem (ES) cells have the potential to produce unlimited numbers of surrogate insulin-producing cells for cell replacement therapy of type 1 diabetes mellitus. The impact of the in vivo environment on mouse ES cell differentiation towards insulin-producing cells was analysed morphologically after implantation.


METHODS
ES cells differentiated in vitro into insulin-producing cells according to the Lumelsky protocol or a new four-stage differentiation protocol were analysed morphologically before and after implantation for gene expression by in situ reverse transcription polymerase chain reaction and protein expression by immunohistochemistry and ultrastructural analysis.


RESULTS
In comparison with nestin positive ES cells developed according to the reference protocol, the number of ES cells differentiated with the four-stage protocol increased under in vivo conditions upon morphological analysis. The cells exhibited, in comparison to the in vitro situation, increased gene and protein expression of Pdx1, insulin, islet amyloid polypeptide (IAPP), the GLUT2 glucose transporter and glucokinase, which are functional markers for glucose-induced insulin secretion of pancreatic beta cells. Renal sub-capsular implantation of ES cells with a higher degree of differentiation achieved by in vitro differentiation with a four-stage protocol enabled further significant maturation for the beta-cell-specific markers, insulin and the co-stored IAPP as well as the glucose recognition structures. In contrast, further in vivo differentiation was not achieved with cells differentiated in vitro by the reference protocol.


CONCLUSIONS
A sufficient degree of in vitro differentiation is an essential prerequisite for further substantial maturation in a beta-cell-specific way in vivo, supported by cell-cell contacts and vascularisation.
Institute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany.
Ortwin
O
Naujok
Flavio
F
Francini
Sally
S
Picton
Clifford J
CJ
Bailey
Sigurd
S
Lenzen
Anne
A
Jörns
D000682:Amyloid / Q000378:metabolism; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000502:physiology*; D003921:Diabetes Mellitus, Experimental / Q000628:therapy; D003922:Diabetes Mellitus, Type 1 / Q000628:therapy*; D053595:Embryonic Stem Cells / Q000166:cytology / Q000637:transplantation*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000502:physiology; D005941:Glucokinase / Q000378:metabolism; D051273:Glucose Transporter Type 2 / Q000378:metabolism; D018398:Homeodomain Proteins / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000166:cytology* / Q000378:metabolism; D007381:Intermediate Filament Proteins / Q000378:metabolism; D058228:Islet Amyloid Polypeptide; D008297:Male; D051379:Mice; D009419:Nerve Tissue Proteins / Q000378:metabolism; D064231:Nestin; D033581:Stem Cell Transplantation / Q000379:methods*; D015534:Trans-Activators / Q000378:metabolism
D000682:Amyloid; D015415:Biomarkers; D051273:Glucose Transporter Type 2; D018398:Homeodomain Proteins; D007328:Insulin; D007381:Intermediate Filament Proteins; D058228:Islet Amyloid Polypeptide; D009419:Nerve Tissue Proteins; C577485:Nes protein, mouse; D064231:Nestin; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein; D005941:Glucokinase