Interaction between marrow-derived human mesenchymal stem cells and peripheral blood mononuclear cells in endothelial cell differentiation.

BACKGROUND AND AIMS: In adult connective tissues, mesenchymal stem cells (MSCs) play a key role in normal tissue turnover and repair. MSCs can participate in these processes not only through proliferation and differentiation but also through paracrine/autocrine functions. These characteristics make MSCs the optimal target in the development of cell-based therapies. This study describes a novel interaction between human MSC and blood mononuclear cells (MNCs), resulting in formation of blood vessel-like structures. MATERIALS AND METHODS: Human marrow-derived MSCs and peripheral blood MNCs were co-cultured in monolayer cultures as well as in bovine collagen sponge up to 20 days. No exogenously supplied growth factors were applied. Morphological changes and formations of three dimensional structures were detected by light microscopy. The process was further stu-died for the expression of different endothelial cell markers. The expression of PECAM-1 and endoglin was studied by immunohistochemistry and the expression of vascular endothelial growth factor receptors 1 and 2 using quantitative real time PCR. RESULTS: In co-cultures of human MSCs and MNCs, the previously nonadherent cells attached and started to elongate and formed tube-like structures within one week. At day 10, elongated PECAM-1 and endoglin expressing cells were detected in co-cultures. At day 20, PECAM-1 and endoglin-positive vessel-like structures were observed. VEGFR1 was up-regulated in co-cultures after 10 days, and expression levels increased with time. No PECAM-1, endoglin or VEGFR1 expressing cells were discovered in MSC-cultures without MNCs at any time point. CONCLUSIONS: This study demonstrates induction of endothelial differentiation in co-cultures of human MSCs and MNCs, indicating a mechanism by which local application of MSCs could induce angiogenesis in vivo.

Differential nuclear localization and kinase activity of alternative ErbB4 intracellular domains.

Cleavable isoforms of the ErbB4 receptor tyrosine kinase release a soluble intracellular domain (ICD) that may translocate to the nucleus and regulate signaling. However, ErbB4 gene is alternatively spliced generating CYT-1 and CYT-2 isoforms with different cytoplasmic tails. Here, we addressed whether the two alternative ErbB4 ICDs of either CYT-1 (ICD1) or CYT-2 (ICD2) type differ in signaling to the nucleus. Confocal microscopy and extraction of nuclear cell fractions indicated that significantly more ICD2 translocated to the nuclei when compared to ICD1. Unlike the membrane-anchored 80 kDa fragments derived from full-length ErbB4 isoforms, the two ICDs did not differ from each other in metabolic stability or ubiquitylation. However, ICD2 was phosphorylated at tyrosine residues to a higher extent and demonstrated greater in vitro kinase activity than ICD1. Mutating the ATP-binding site within ICD2 kinase domain (ICD2 K751R) blocked its tyrosine phosphorylation and significantly reduced its nuclear translocation. When expressed in the context of full-length ErbB4, ICD2 was also more efficient than ICD1 in promoting transcriptional activation of the STAT5 target gene beta-casein. These findings indicate that the two alternative ICDs of ErbB4 differ in their nuclear accumulation, and that the mechanism involves differential kinase activity but not ubiquitin-regulated ICD stability.