DIFFERENTIATION OF EMBRYONIC STEM CELLS: LESSONS FROM EMBRYONIC DEVELOPMENT
Keywords:
embryonic stem cells, in vitro differentiation, cardiac, neuronalAbstract
Embryonic stem (ES) cells, the undifferentiated cells of early embryos are
established as permanent lines and are characterised by their self-renewal capacity
and the ability to retain their developmental capacity in vivo and in vitro. The
pluripotent properties of ES cells are the basis of gene targeting technologies used
to create mutant mouse strains with inactivated genes by homologous
recombination. There are several methods to induce the formation of EBs. One of
them the formation by aggregating ES cells in hanging drops, using gravity as an
aggregation force. This method presents the advantage of obtaining well-calibrated
EBs almost identical in size. We used at our experiment the mouse ES cell line
KA1/11/C3/C8 with a normal karyotype, at 14th passages. Immunohistochemical
examination was aimed to identify tissue-restricted proteins for the two
differentiated lineages: titin as a cell-specific antigen for cardiac and skeletal
muscle, betaIII-tubulin for the neuronal differentiation, cytokeratin Endo-A
(TROMA) for the presence of mesenchymal progenitor cells, Oct-4 for the presence
of the undifferentiated ES cells. The beating cardiac muscle clumps showed more
synchronous rhythm than those seen in EBs obtained from suspension culture
method, where the beating cardiac muscle clumps appeared later, had a lower
frequency and were uneven. The synaptic networks of neuronal cells were best
developed in EBs from suspension, compared to those observed in EBs from
hanging-drop method.
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