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J. Biol. Chem., Vol. 281, Issue 45, 34484-34491, November 10, 2006

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The SHB Adapter Protein Is Required for Normal Maturation of Mesoderm during in Vitro Differentiation of Embryonic Stem Cells^*

Vitezslav Kriz¹, Nina Ågren, Cecilia K. Lindholm, Samuel Lenell, Johan Saldeen, Jaroslav Mares^¶, and Michael Welsh²

From the Department of Medical Cell Biology, Uppsala University, Uppsala 75123, Sweden, Division of Clinical Chemistry, Department of Laboratory Medicine, Karolinska University Hospital, Huddinge 14186, Sweden, and ^¶Institute of Biology and Medical Genetics, Charles University, Prague, 15006, Czech Republic

Definitive mesoderm arises from a bipotent mesendodermal population, and to study processes controlling its development at this stage, embryonic stem (ES) cells can be employed. SHB (Src homology 2 protein in -cells) is an adapter protein previously found to be involved in ES cell differentiation to mesoderm. To further study the role of SHB in this context, we have established ES cell lines deficient for one (SHB^+/-) or both SHB alleles (SHB^-/-). Differentiating embryoid bodies (EBs) derived from these ES cell lines were used for gene expression analysis. Alternatively, EBs were stained for the blood vessel marker CD31. For hematopoietic differentiation, EBs were differentiated in methylcellulose. SHB^-/- EBs exhibited delayed down-regulation of the early mesodermal marker Brachyury. Later mesodermal markers relatively specific for the hematopoietic, vascular, and cardiac lineages were expressed at lower levels on day 6 or 8 of differentiation in EBs lacking SHB. The expression of vascular endothelial growth factor receptor-2 and fibroblast growth factor receptor-1 was also reduced in SHB^-/- EBs. SHB^-/- EBs demonstrated impaired blood vessel formation after vascular endothelial growth factor stimulation. In addition, the SHB^-/- ES cells formed fewer blood cell colonies than SHB^+/+ ES cells. It is concluded that SHB is required for appropriate hematopoietic and vascular differentiation and that delayed down-regulation of Brachyury expression may play a role in this context.

Received for publication, April 28, 2006 , and in revised form, September 11, 2006.

^* This work was supported by the Swedish Research Council (31X-10822), the Swedish Cancer Foundation, The Juvenile Diabetes Research Foundation International, the Swedish Diabetes Foundation, and the Family Ernfors Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1 and Figs. 1 and 2.

¹ Supported by a grant from Henning and Gösta Ankarstrand and by a grant from Erland Wessler.

² To whom correspondence should be addressed: Dept. of Medical Cell Biology, P.O. Box 571, Husargatan 3, SE-751 23, Uppsala, Sweden. Tel.: 46-184714447; Fax: 46-184714059; E-mail: michael.welsh{at}mcb.uu.se.

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