FOXF1 Mediates Endothelial Progenitor Functions and Regulates Vascular Sprouting.

Endothelial colony forming cells (ECFC) or late blood outgrowth endothelial cells (BOEC) have been proposed to contribute to neovascularization in humans. Exploring genes characteristic for the progenitor status of ECFC we have identified the forkhead box transcription factor FOXF1 to be selectively expressed in ECFC compared to mature endothelial cells isolated from the vessel wall. Analyzing the role of FOXF1 by gain- and loss-of-function studies we detected a strong impact of FOXF1 expression on the particularly high sprouting capabilities of endothelial progenitors. This apparently relates to the regulation of expression of several surface receptors. First, FOXF1 overexpression specifically induces the expression of Notch2 receptors and induces sprouting. Vice versa, knock-down of FOXF1 and Notch2 reduces sprouting. In addition, FOXF1 augments the expression of VEGF receptor-2 and of the arterial marker ephrin B2, whereas it downmodulates the venous marker EphB4. In line with these findings on human endothelial progenitors, we further show that knockdown of FOXF1 in the zebrafish model alters, during embryonic development, the regular formation of vasculature by sprouting. Hence, these findings support a crucial role of FOXF1 for endothelial progenitors and connected vascular sprouting as it may be relevant for tissue neovascularization. It further implicates Notch2, VEGF receptor-2, and ephrin B2 as downstream mediators of FOXF1 functions.

Ex-vivo expanded umbilical cord blood stem cells retain capacity for myocardial regeneration.

BACKGROUND: Umbilical cord blood (UCB) is a source of human hematopoietic precursor cells (HPCs), a stem cell (SC) type that has been used in several trials for myocardial repair. A certain minimal number of cells is required for measurable regeneration and a major challenge of SC-based regenerative therapy constitutes ex-vivo expansion of the primitive cell compartment. The aim of this study was to investigate the ex-vivo expansion potential of UCB-derived HPCs and the ability of these expanded cells to migrate to the site of damage and improve ventricular function in a rodent model of myocardial infarction (MI). METHODS AND RESULTS: UCB-derived HPCs, defined by coexpression of CD133 and CD34, were expanded using various cytokine combinations. MI was induced by left anterior descending artery ligation in nude rats. Cells were injected intravenously 2 days after infarction. The combination of SC factor, thrombopoietin, flt3-ligand and interleukin-6 was found to be the most effective for inducing proliferation of HPCs. The migratory capacity of expanded HPCs was similar to that of non-expanded HPCs and improvement of ejection fraction was significant in both groups, with a relative increase of >60%. CONCLUSIONS: UCB-derived HPCs can be reproducibly expanded ex-vivo and retain their potential to improve cardiac function post-MI. (Circ J 2010; 74: 188 - 194).

Gene expression profiling of human mesenchymal stem cells derived from bone marrow during expansion and osteoblast differentiation.

BACKGROUND: Human mesenchymal stem cells (MSC) with the capacity to differentiate into osteoblasts provide potential for the development of novel treatment strategies, such as improved healing of large bone defects. However, their low frequency in bone marrow necessitate ex vivo expansion for further clinical application. In this study we asked if MSC are developing in an aberrant or unwanted way during ex vivo long-term cultivation and if artificial cultivation conditions exert any influence on their stem cell maintenance. To address this question we first developed human oligonucleotide microarrays with 30.000 elements and then performed large-scale expression profiling of long-term expanded MSC and MSC during differentiation into osteoblasts. RESULTS: The results showed that MSC did not alter their osteogenic differentiation capacity, surface marker profile, and the expression profiles of MSC during expansion. Microarray analysis of MSC during osteogenic differentiation identified three candidate genes for further examination and functional analysis: ID4, CRYAB, and SORT1. Additionally, we were able to reconstruct the three developmental phases during osteoblast differentiation: proliferation, matrix maturation, and mineralization, and illustrate the activation of the SMAD signaling pathways by TGF-beta2 and BMPs. CONCLUSION: With a variety of assays we could show that MSC represent a cell population which can be expanded for therapeutic applications.

The lipid droplet enzyme Tgl1p hydrolyzes both steryl esters and triglycerides in the yeast, Saccharomyces cerevisiae.

Based on sequence homology to mammalian acid lipases, yeast reading frame YKL140w was predicted to encode a triacylglycerol (TAG) lipase in yeast and was hence named as TGL1, triglyceride lipase 1. A deletion of TGL1, however, resulted in an increase of the cellular steryl ester content. Fluorescently labeled lipid analogs that become covalently linked to the enzyme active site upon catalysis were used to discriminate between the lipase and esterase activities of Tgl1p. Tgl1p preferred single-chain esterase inhibitors over lipase inhibitors in vitro. Under assay conditions optimal for acid lipases, Tgl1p exhibited steryl esterase activity only and lacked any triglyceride lipase activity. In contrast, at pH 7.4, Tgl1p also exhibited TAG lipase activity; however, steryl ester hydrolase activity was still predominant. Tgl1p localized exclusively to lipid droplets which are the intracellular storage compartment of steryl esters and triacylglycerols in the yeast S. cerevisiae. In a tgl1 deletion mutant, the mobilization of steryl esters in vivo was delayed, but not abolished, suggesting the existence of additional enzymes involved in steryl ester mobilization.

Proteomic profiling of human stem cells derived from umbilical cord blood.

CD34+ preparations from five different umbilical cord samples were compared with respect to their proteome profile using 2-D gel electrophoresis. Fifty-two protein spots were found to match in all preparations referring to the high heterogeneity of such samples indicating a not fully developed (or instable) proteome of stem cells. All matching spots were subjected to in-gel digestion and nano-LC-MS/MS sequence analysis, from which 22 proteins were unambiguously identified.

Preliminary 2-D chromatographic investigation of the human stem cell proteome.

Stem cells represent a promising tool for the treatment of various hematopoietic diseases. In order to identify stem cell-specific proteins, the proteome of human stem cells from umbilical cord blood was explored for the first time. For this purpose, the crude lysate of 4 x 10(5) CD34+ cells was subjected to in solution trypsin digestion. The resulting peptides were then separated via cation exchange followed by reversed phase chromatography and analyzed by nanospray MS/MS. Database search revealed a total of 215 proteins which could be reliably identified. To obtain a more complete picture of the human stell cell proteome and to also access low abundant proteins, pooling of more than one CD34+ preparations seems necessary in order to increase the cell number and thus the protein content.