Browsing by Subject "cell differentiation"

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  • Bogacheva, Mariia S.; Khan, Sofia; Kanninen, Liisa K.; Yliperttula, Marjo; Leung, Alan W.; Lou, Yan-Ru (2018)
    Definitive endoderm (DE) is the first stage of human pluripotent stem cell (hPSC) differentiation into hepatocyte-like cells. Developing human liver cell models for pharmaceutical applications is highly demanding. Due to the vast number of existing protocols to generate DE cells from hPSCs, we aimed to compare the specificity and efficiency of selected published differentiation conditions. We differentiated two hPSC lines (induced PSC and embryonic stem cell) to DE cells on Matrigel matrix using growth factors (Activin A and Wnt-3a) and small molecules (sodium butyrate and IDE 1) in different combinations. By studying dynamic changes during 6 days in cell morphology and the expression of markers for pluripotency, DE, and other germ layer lineages, we found that Activin A is essential for DE differentiation, while Wnt-3a and sodium butyrate are dispensable. Although sodium butyrate exerted rapid DE differentiation kinetics, it caused massive cell death and could not generate sufficient cells for further differentiation and applications. We further discover that IDE 1 could not induce DE as reported previously. Hereby, we compared different conditions for DE induction and found an effective six day-protocol to obtain DE cells for the further differentiation and applications.
  • Tolkachov, Alexander; Fischer, Cornelius; Ambrosi, Thomas H.; Bothe, Melissa; Han, Chung-Ting; Muenzner, Matthias; Mathia, Susanne; Salminen, Marjo; Seifert, Georg; Thiele, Mario; Duda, Georg N.; Meijsing, Sebastiaan H.; Sauer, Sascha; Schulz, Tim J.; Schupp, Michael (2018)
    The transcription factor GATA2 is required for expansion and differentiation of hematopoietic stem cells (HSCs). In mesenchymal stem cells (MSCs), GATA2 blocks adipogenesis, but its biological relevance and underlying genomic events are unknown. We report a dual function of GATA2 in bone homeostasis. GATA2 in MSCs binds near genes involved in skeletal system development and colocalizes with motifs for FOX and HOX transcription factors, known regulators of skeletal development. Ectopic GATA2 blocks osteoblastogenesis by interfering with SMAD1/5/8 activation. MSC-specific deletion of GATA2 in mice increases the numbers and differentiation capacity of bone-derived precursors, resulting in elevated bone formation. Surprisingly, MSC-specific GATA2 deficiency impairs the trabecularization and mechanical strength of bone, involving reduced MSC expression of the osteoclast inhibitor osteoprotegerin and increased osteoclast numbers. Thus, GATA2 affects bone turnover via MSC-autonomous and indirect effects. By regulating bone trabecularization, GATA2 expression in the osteogenic lineage may contribute to the anatomical and cellular microenvironment of the HSC niche required for hematopoiesis.
  • Veistinen, Lotta K.; Mustonen, Tuija; Hasan, Md. Rakibul; Takatalo, Maarit; Kobayashi, Yukiho; Kesper, Dorthe A.; Vortkamp, Andrea; Rice, David P. (2017)
    Loss-of-function mutations in GLI3 and IHH cause craniosynostois and reduced osteogeneiss, respectively. In this study, we show that ihh ligand, the receptor Ptch1 and Gli transcription factors are differentialyy expressed in embryonic mouse calvaria osteogenic condenstions. We show that in both ihh(-/-) and Gli3(Xt-J/Xt-J) embroyonic mice, the normal gene expression architecture is lost and this results in disorganized calvarial bone developement. RUNX2 is a master regulatory transciption factor controlling osteogenesis. In the absence of Gli3, RUNX2 isoform II and IHH are upregulated, and RUNX2 isoform I downregulated. This is consistent with the expandeed and aberant osteogenesis observed in Gli3Xt-J/Xt-J mice, and consistent RunX2-t expression by relatively immature osteoprogenitors. ihh-/- mice exhibited small calvarial bones HH target genes, Ptch1 and Gli1, were absent. This indicates that IHH is the functional HH ligand, and that it is not compensated by another HH ligand. To decipher the roles and potential interaction of Gli3 and ihh. we generated ihh-/-; gli3Xt-J/Xt-J compound mutant mice. Even in the absence of ihh, Gli3 deletion was sufficient to induce aberrant precocious ossification across the developing suture, indicating that the carniosyostosis pehnotype of Gli3Xt-J/Xt-J mice is not dependent on IHH ligand. Also we found that ihh was not required for Runx2 expression as the expression of RUNX2 target genes was unaffected by deletion of Ihh. To test whether RUNX2 has a role upstream of IHH we performed RUNX2 siRNA knock down experiements in WT calvarial osteoblasts and explants and found that Ihh expression is suppressed. Our results show that IHH is the functional HH ligand in the embroynic mouse calvaria osteogenic condensations, where it regulates the progression of osteoblastic differentation. As GLI3 represses the expression of Runx2-II abd Ihh, and also elevats the Runx2-I expression, and as IHH may be regulated by RUNX2 these results raise the possibility of a regualtory feedback circuit to control calvarial osteogenesis and suture patency. Taken together RUNX2-controlled osteoblastic cell fate is regulated by IHH through concomitant inhibition of GLI3-repressor formation and activation of downstreams targets.