MicroRNA target sites as genetic tools to enhance promoter-reporter specificity for the purification of pancreatic progenitor cells from differentiated embryonic stem cells.
Stem Cell Rev Rep
; 9(5): 555-68, 2013 Oct.
Article
in En
| MEDLINE
| ID: mdl-23111459
ABSTRACT
Pluripotent cells hold great promise for cell replacement therapies in regenerative medicine. All known protocols for directed in vitro differentiation of pluripotent cells did not yield pure populations complicating the characterization of the derived cells. In addition, the risk of tumor formation due to residual undifferentiated cells is a serious unresolved problem. In the present study the tissue-specific mouse Pdx1 promoter was used to control the expression of the reporter gene GFP2 in mouse ES cells in order to purify them via FACS during in vitro differentiation. The background fluorescence of transduced ES cells hampered the purification of Pdx1-positive cells due to a contaminating population of partially undifferentiated cells. MicroRNAs (mir) are important regulators of gene expression and were used to enhance promoter specificity during differentiation towards pancreatic progenitor cells. The mouse mmu-mir-294 was found to be mainly expressed during pluripotency, whereas the expression of the mir-302 cluster was increased during early differentiation. Integration of a microRNA target site for the mmu-mir-294 into the lentiviral vector reduced the background fluorescence specifically during pluripotency and permitted re-occurrence of GFP2 expression upon differentiation. A combination of the microRNA target site with the Pdx1 promoter fragment allowed the purification of pancreatic progenitors from differentiated ES cells. This population reflected an early pancreatic progenitor population without other contaminating cell lineages. In conclusion, microRNA target sites are efficient regulatory elements to control transgene expression and to enhance tissue specificity as presented in this study facilitating the sorting and purification of Pdx1-positive pancreatic progenitor cells.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Stem Cells
/
Promoter Regions, Genetic
/
MicroRNAs
/
Embryonic Stem Cells
Type of study:
Guideline
Limits:
Animals
/
Humans
Language:
En
Journal:
Stem Cell Rev Rep
Year:
2013
Type:
Article
Affiliation country:
Germany