Informace o publikaci

In vitro developmental potential of human embryonic stem cell lines evaluated by gene expression analysis of a new set of differentiation markers

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Rok publikování 2013
Druh Konferenční abstrakty
Fakulta / Pracoviště MU

Fakulta informatiky

Popis The ability to direct differentiation of human embryonic stem cells (hESCs) into specific cell types presents great promise for regenerative medicine. For example in vitro generated hematopoietic progenitors could provide an alternative to hematopoietic stem cells used for transplantations. However, the current limiting factor is the low efficiency of differentiation protocols and functional defects of derived blood progenitors. One of the issues to address these obstacles is to focus on the selection of original cells, as significant differences in the ability to differentiate into various germ layers and into hematopoietic cells were found among hESC lines. The aim of this study was to design the methodology to determine hESC differentiation potential with focus to hematopoietic differentiation and to evaluate this parameter in several hESC lines. Gene expression of twelve selected markers was monitored by RT-qPCR technology using hydrolysis probes from the Universal ProbeLibrary in multiplex assay with the Universal ProbeLibrary Reference Gene. hESC lines CCTL-12, CCTL-14, and HUES-1 were evaluated during their spontaneous differentiation induced by embryoid body formation using hanging drop method, while undifferentiated hESCs and cells from days three, seven, eleven, fifteen, and twenty-one of differentiation were analysed. A set of differentiation markers was established representing three germ layers: mesoderm (Brachyury T, MIXL1), endoderm (AFP, SOX17) and ectoderm (NEFH, PAX6); hemangioblasts and hematopoietic stem cells (KDR, PECAM1, PROM1, CD34); and pluripotent cells (NANOG, POU5F1) and including two housekeeping genes (G6PD, TBP). Expression pattern of these markers during hESC differentiation revealed their developmental potential. While CCTL-12 cell line demonstrated the potential to differentiate into all three germ layers, HUES-1 cell line showed skewed differentiation to endoderm layer and CCTL-14 cell line was characterised by preferential expression of ectoderm and mesoderm markers making this line the most suitable one for directed hematopoietic differentiation. Derivation of hematopoietic precursors defined by CD34 expression is inefficient during spontaneous differentiation of hESC, which was demonstrated by very low expression of this marker in all three developing cell lines in our study. Optimal RT-qPCR conditions were determined for twelve newly designed assays enabling the sensitive gene expression analysis of selected differentiation markers. Using this set of markers developing pluripotent stem cells can be monitored under various conditions, enabling to evaluate the differentiation potential of these cells.
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