Expression of Pax4 in embryonic stem cells promotes differentiation of nestin-positive progenitor and insulin-producing cells.
Mouse embryonic stem (ES) cells differentiate into cells of all three primary germ layers including endodermal cells that produce insulin in vitro. We show that constitutive expression of Pax4 (Pax4(+)), and to a lesser extent Pdx1 (Pdx1(+)), affects the differentiation of ES cells and significantly promote the development of insulin-producing cells. In Pax4 overexpressing R1 ES cells, isl-1, ngn3, insulin, islet amyloid polypeptide, and glucose transporter 2 (Glut-2) mRNA levels increase significantly. The number of nestin-expressing (nestin+) cells also increases. Constitutive Pax4 expression combined with selection of nestin+ cells and histotypic culture conditions give rise to spheroids containing insulin-positive granules typical of embryonal and adult beta cells. In response to glucose, Pax4(+) and wild-type ES-derived cells release insulin. Transplantation of these cells into streptozotocin-treated diabetic mice results in a normalization of blood glucose levels. We conclude that constitutive expression of Pax4 in combination with histotypic cultivation facilitates ES cell differentiation into the pancreatic lineage, which leads to the formation of islet-like spheroid structures that produce increased levels of insulin.
Blyszczuk
Przemyslaw
P
In Vitro Differentiation Group, Institute of Plant Genetics and Crop Plant Research, D-06466 Gatersleben, Germany.
Czyz
Jaroslaw
J
Kania
Gabriela
G
Wagner
Martin
M
Roll
Ursula
U
St-Onge
Luc
L
Wobus
Anna M
AM
D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D003921:Diabetes Mellitus, Experimental; D004622:Embryo, Mammalian / Q000166:cytology*; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors; D005934:Glucagon / Q000096:biosynthesis; D005947:Glucose / Q000378:metabolism; D051273:Glucose Transporter Type 2; D018398:Homeodomain Proteins / Q000096:biosynthesis*; D007118:Immunoassay; D007150:Immunohistochemistry; D007328:Insulin / Q000096:biosynthesis*; D007381:Intermediate Filament Proteins / Q000096:biosynthesis*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins; D064231:Nestin; D051761:Paired Box Transcription Factors; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism*; D013997:Time Factors; D014157:Transcription Factors / Q000096:biosynthesis*
D018076:DNA, Complementary; D051273:Glucose Transporter Type 2; D018398:Homeodomain Proteins; D007328:Insulin; D007381:Intermediate Filament Proteins; D009004:Monosaccharide Transport Proteins; D009419:Nerve Tissue Proteins; C577485:Nes protein, mouse; D064231:Nestin; D051761:Paired Box Transcription Factors; C110218:Pax4 protein, mouse; D012333:RNA, Messenger; D014157:Transcription Factors; D005934:Glucagon; D005947:Glucose