PAX4 enhances beta-cell differentiation of human embryonic stem cells.
BACKGROUND
Human embryonic stem cells (HESC) readily differentiate into an apparently haphazard array of cell types, corresponding to all three germ layers, when their culture conditions are altered, for example by growth in suspension as aggregates known as embryoid bodies (EBs). However, this diversity of differentiation means that the efficiency of producing any one particular cell type is inevitably low. Although pancreatic differentiation has been reported from HESC, practicable applications for the use of beta-cells derived from HESC to treat diabetes will only be possible once techniques are developed to promote efficient differentiation along the pancreatic lineages.


RESULTS
Here, we have tested whether the transcription factor, Pax4 can be used to drive the differentiation of HESC to a beta-cell fate in vitro. We constitutively over-expressed Pax4 in HESCs by stable transfection, and used Q-PCR analysis, immunocytochemistry, ELISA, Ca(2+) microfluorimetry and cell imaging to assess the role of Pax4 in the differentiation and intracellular Ca(2+) homeostasis of beta-cells developing in embryoid bodies produced from such HESC. Cells expressing key beta-cell markers were isolated by fluorescence-activated cell sorting after staining for high zinc content using the vital dye, Newport Green.


CONCLUSIONS
Constitutive expression of Pax4 in HESC substantially enhances their propensity to form putative beta-cells. Our findings provide a novel foundation to study the mechanism of pancreatic beta-cells differentiation during early human development and to help evaluate strategies for the generation of purified beta-cells for future clinical applications.
Liew
Chee Gee
CG
Centre for Stem Cell Biology, Department of Biomedical Science, The University of Sheffield, Sheffield, United Kingdom.
Shah
Nadia N
NN
Briston
Sarah J
SJ
Shepherd
Ruth M
RM
Khoo
Cheen Peen
CP
Dunne
Mark J
MJ
Moore
Harry D
HD
Cosgrove
Karen E
KE
Andrews
Peter W
PW
D015220:Calcium Channels / Q000378:metabolism; D020013:Calcium Signaling; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D018398:Homeodomain Proteins / Q000235:genetics / Q000502:physiology*; D006801:Humans; D007150:Immunohistochemistry; D015640:Ion Channel Gating; D007515:Islets of Langerhans / Q000166:cytology* / Q000378:metabolism; D051761:Paired Box Transcription Factors / Q000235:genetics / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D015854:Up-Regulation
D015220:Calcium Channels; D005456:Fluorescent Dyes; D018398:Homeodomain Proteins; C491709:PAX4 protein, human; D051761:Paired Box Transcription Factors; D012333:RNA, Messenger