Abnormal development of mouse embryoid bodies lacking p27^Kip1 cell cycle regulator

VÍTZSLAV BRYJA ¹, LUKÁ AJÁNEK ², JIÍ PACHERNÍK ³, ANITA C. HALL ⁴, VIKTOR HORVÁTH ⁵, PETR DVOÁK ⁶, ALE HAMPL ^6*

¹ Center for Cell Therapy and Tissue Repair, Charles University, Prague, Czech Republic; Department of Molecular Embryology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic; Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden
² Center for Cell Therapy and Tissue Repair, Charles University, Prague, Czech Republic
³ Center for Cell Therapy and Tissue Repair, Charles University, Prague, Czech Republic; Laboratory of Molecular Embryology, Mendel University Brno, Brno, Czech Republic
⁴ Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden
⁵ Laboratory of Cytokinetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic
⁶ Center for Cell Therapy and Tissue Repair, Charles University, Prague, Czech Republic; Department of Molecular Embryology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic; Laboratory of Molecular Embryology, Mendel University Brno, Brno, Czech Republic

^* To whom correspondence should be addressed. E-mail: hampl{at}mendelu.cz.

	Abstract

Cultures of three-dimensional aggregates of embryonic stem (ES) cells called embryoid bodies (EBs) provide a valuable system for analyzing molecular mechanisms that regulate differentiation of this unique cell type. Cyclin dependent kinase inhibitor p27^Kip1 (p27) becomes elevated during the differentiation of mouse ES (mES) cells. In this study, various aspects of differentiation of EBs produced from normal and p27-deficient mES cells were analyzed to address biological significance of this elevation. It was found that EBs lacking p27 grew significantly bigger but this was not accompanied by detectable abnormalities in the activities of cyclin dependent kinases (CDKs). In the majority of EB cells, down-regulation of activating cyclins rather than up-regulation of inhibiting p27 is probably responsible for lowering the activity of their CDKs. Abnormalities in the development of specific cell lineages were also observed in p27-deficient EBs. These included: a) elimination of cells positive for cytokeratin endo-A (TROMA-I) and b) increased proliferation and formation of cavities originating from cells positive for Lewis-X. Our data also suggest that although two different pools of Lewis-X-expressing cells, cluster forming (ES cell like) and cavity forming (neural progenitors), normally exist in EBs, the absence of p27 leads to the enhancement of only the neural pool. No failure was found when the neurogenic capacity of p27-deficient mES cells was tested using various protein markers. Together, our data point to a dual role of p27 in mES cells, with one role being in the regulation of proliferation and the other role in establishing some other aspects of a differentiated phenotype.

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