Fibronectin and pellet suspension culture promote differentiation of human mesenchymal stem cells into insulin producing cells.
Multipotential mesenchymal stem cells (MSCs) isolated from bone marrow can differentiate into multiple mesenchymal tissues and exhibit a neuronal phenotype under appropriate induction conditions. Methods promoting neural differentiation have been adapted to derive insulin producing cells (IPCs) from embryonic stem cells, but it remains unclear whether neuronal cell-based differentiation method will be able to derive IPCs from MSCs. Using a four-stage differentiation protocol which contains neuronal differentiation factor and IPC-conversion reagent-nicotinamide, the potential of human MSCs to differentiate into IPCs was evaluated by means of reverse transcription-polymerase chain reaction, immunostaining, and functional analysis. MSCs in monolayer spontaneously expressed genes for islet transcription factors, Nkx6.1 and Ngn3, but did not express insulin after treatment in this protocol. Pellet suspension culture and the addition of fibronectin enhanced pancreatic differentiation with increase in insulin and Glut2 gene expression. Switching of cells to high-glucose culture further increased immunostaining for proinsulin and insulin. IPCs secreted insulin in response to elevated glucose concentration, which was regulated by reagents that increase cyclic AMP production or modify calcium influx. Our data suggest that MSCs in the monolayer do not undergo IPC differentiation and pellet suspension culture with fibronectin promotes IPCs derived from MSCs.
Chang
Ching-Fang
CF
Stem Cell Laboratory, Department of Medical Research and Education, Veterans General Hospital-Taipei, Taiwan.
Hsu
Ke-Hsun
KH
Chiou
Shih-Hwa
SH
Ho
Larry Low-Tone
LL
Fu
Yu-Show
YS
Hung
Shih-Chieh
SC
D002449:Cell Aggregation / Q000187:drug effects; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects*; D004475:Ectoderm / Q000166:cytology / Q000187:drug effects; D005353:Fibronectins / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000166:cytology*; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects*; D008648:Mesoderm / Q000166:cytology / Q000187:drug effects; D009474:Neurons / Q000166:cytology / Q000187:drug effects; D011384:Proinsulin / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D005353:Fibronectins; D007328:Insulin; D014157:Transcription Factors; D011384:Proinsulin; D005947:Glucose