PubMed · 11524773
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K Remes. 1998. [Cell therapy].. https://pubmed.ncbi.nlm.nih.gov/11524773/
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We previously showed that heparan sulfate (HS) is required for in vitro cytokine + chemokine-mediated maintenance of primitive human hematopoietic progenitors. However, HS preparations are mixtures of polysaccharide chains of varying size, structure, and protein-binding abilities. Therefore, we examined whether the long-term culture-initiating cells (LTC-IC) supportive capability of HS is attributable to an oligosaccharide of defined length and protein-binding ability. Oligosaccharides of a wide range of sizes were prepared, and their capability to support human marrow LTC-IC maintenance in the presence of low-dose cytokines and a single chemokine, macrophage inflammatory protein-1alpha (MIP-1alpha), was examined. LTC-IC supportive capability of HS oligosaccharides correlated directly with size and MIP-1alpha binding ability. A specific MIP-1alpha-binding HS oligosaccharide preparation of M(r) 10 kDa that optimally supported LTC-IC maintenance was identified. This oligosaccharide had the structure required for MIP-1alpha binding, which we have recently described. The present study defines the minimum size and structural features of LTC-IC supportive HS.
BACKGROUND: We previously developed a method for counting CD34(+) cells in unlysed whole blood. This method was applied to normal human bone marrow, peripheral blood after mobilization of progenitor cells, leukapheresis products, and cord blood and was validated with two different lyse-no wash methods. However, the main advantage that we described, erythrocyte discrimination using nucleic acid staining, was also the main restriction because additional markers for the immunologic characterization of CD34(+) cells cannot be included. METHODS: We used SYTO-13 and fluorescein isothiocyanate (FITC)-CD45 staining (FL1) instead of SYTO-13 and phycoerythrin (PE)Cy5-CD45 staining (FL3) to leave the third and fourth fluorescence parameters available for further characterization of CD34(+) cells. The new method was validated by applying it to cord blood samples (n = 20). RESULTS: FITC-CD45 antibody gave a 1.7-fold increase in mean fluorescence intensity over SYTO-13 alone. From absolute counts (CD34(+) cells per microliter), we plotted the differences between the methods against their mean, showing that differences fell into acceptable ranges. CONCLUSIONS: No-lyse procedures may represent an advance for cell immunophenotyping and it could be applied to the measurement of additional markers. Cytometry (Clin. Cytometry) 50:249-253, 2002.
The large capillary mass of the newborn lung demands the presence of endothelial cell precursors in lung tissue before development of the pulmonary capillary bed. The objective of this investigation was to isolate and characterize putative endothelial cell precursors from developing human lung. CD34, a cell surface marker for hematopoietic progenitor cells, endothelial precursor cells, and small vessel endothelial cells, was employed as an immunological "handle" for the selection of the desired cells. When CD34+ cells were isolated from midtrimester human fetal lung tissue, then maintained in culture, the isolated cells expressed immunoreactivity for the endothelial cell marker von Willebrand factor and the vascular endothelial growth factor receptors KDR and Flt-1. However, only 5% or fewer of the cells expressed PECAM, an important factor in cell-cell interactions and a marker for endothelial cells associated with vessels. The CD34+ cells endocytosed acetylated low-density lipoprotein and formed capillary-like structures when incubated in a cushion of Matrigel. RT-PCR analysis of mRNA for endothelial cell-related proteins Flt-1, Tie-2, and endothelial nitric oxide synthase demonstrated expression of these mRNAs by the isolated cells for at least 16 cell passages. These observations demonstrate that capillary endothelial cell precursors can be isolated from developing human lung and maintained in cell culture. These cells represent a potentially important tool for investigating the regulation of mechanisms governing development of the air-blood barrier in the human lung.