[Preservation, thawing and infusion of hematopoietic stem cells].

Peripheral blood stem cells can be stored by the following 3 methods: liquid storage, non-rate controlled freezing and rate controlled freezing. Methods of processing of these cells including thawing, ex vivo purging and infusion are described in detail.

Stage-specific expression of mouse BST-1/BP-3 on the early B and T cell progenitors prior to gene rearrangement of antigen receptor.

Human bone marrow stromal cell antigen 1 (BST-1) was identified as a glycosylphosphatidyl-inositol-anchored ectoenzyme expressed on bone marrow stromal or synovial cell lines and having the ability to facilitate pre-B cell line growth. The analysis of the expression of mouse BST-1/BP-3 on the surface of lymphoid cells in the bone marrow and thymus revealed that it was very transiently expressed on both B and T cell progenitors undergoing gene rearrangement of the antigen receptor. Among CD45R+ CD43+ B cell progenitors in the bone marrow, BST-1 expression appeared on the CD24 (heat stable antigen)+, CD19+ or CD117 (c-kit)+ population. In the thymus, BST-1 was expressed on CD4-CD8-CD3- [triple negative (TN)] CD90 (Thy-1)+ cells. In TN thymocytes, the majority of CD25+ cells and CD44(10)/- cells expressed BST-1. In fetuses, BST-1+ cells appeared in the thymus and liver at day 14 and 16 of gestation respectively. The expression level of BST-1 by fetal thymus was maximal and > 60% of thymocytes were positive for BST-1 at day 15 or 16 and the proportion then gradually decreased during development. Among day 15 fetal thymocytes, BST-1 was negative on the CD44+ CD25- fraction, very slightly positive on the CD44+ CD25+ fraction, and strongly positive on the CD44(10)/- CD25+ and CD44-CD25- fractions. These results showed that murine BST-1 is a useful marker for lymphoid progenitor cells initiating gene rearrangement of their antigen receptors.

Elevated levels of the soluble form of bone marrow stromal cell antigen 1 in the sera of patients with severe rheumatoid arthritis.

OBJECTIVE: Bone marrow stromal cell antigen 1 (BST-1) is a novel glycosyl phosphatidylinositol-anchored ectoenzyme, which is overexpressed on bone marrow stromal and synovial cell lines derived from patients with rheumatoid arthritis (RA). To investigate the pathophysiologic roles of BST-1 in RA, we established an enzyme-linked immunosorbent assay (ELISA) system to detect the soluble form of BST-1 (sBST-1) and examined levels of sBST-1 in the sera of RA patients. METHODS: Concentrations of sBST-1 in sera from healthy donors and from patients with RA, osteoarthritis, Sjögren's syndrome, and systemic lupus erythematosus were measured with the ELISA. RESULTS: In 7% of the RA patient samples (10 of 143), concentrations of serum sBST-1 were higher (approximately 30-50-fold) than in non-RA samples. Serum sBST-1 concentrations showed no correlation with age, C-reactive protein level, or rheumatoid factor level. All RA patients with high concentrations of serum sBST-1 had severe disease with involvement of several large joints. CONCLUSION: We believe the measurement of serum sBST-1 may have prognostic value, but further analysis is necessary to clarify the clinical significance of elevated sBST-1 in RA.

Molecular cloning and chromosomal mapping of a bone marrow stromal cell surface gene, BST2, that may be involved in pre-B-cell growth.

Bone marrow stromal cells regulate B-cell growth and development through their surface molecules and cytokines. In this study, we generated a mAb, RS38, that recognized a novel human membrane protein, BST-2, expressed on bone marrow stromal cell lines and synovial cell lines. We cloned a cDNA encoding BST-2 from a rheumatoid arthritis-derived synovial cell line. BST-2 is a 30- to 36-kDa type II transmembrane protein, consisting of 180 amino acids. The BST-2 gene (HGMW-approved symbol BST2) is located on chromosome 19p13.2. BST-2 is expressed not only on certain bone marrow stromal cell lines but also on various normal tissues, although its expression pattern is different from that of another bone marrow stromal cell surface molecule, BST-1. BST-2 surface expression on fibroblast cell lines facilitated the stromal cell-dependent growth of a murine bone marrow-derived pre-B-cell line, DW34. The results suggest that BST-2 may be involved in pre-B-cell growth.

Expression of GATA-binding transcription factors in rat hepatocytes.

Recently, we demonstrated that a DNA-binding protein(s) is involved in transcriptional repression of the rat serine dehydratase gene in fetal liver. Here, we report that a GAT(A/T) motif is the target sequence for the DNA-binding protein. By screening a fetal liver cDNA library, we isolated a rat homolog of GATA-1. Rat GATA-1 expressed as a GST-fusion protein in E. coli bound to the GAT(A/T) motif in the serine dehydratase gene. Northern analysis show that GATA-1 and GATA-4 mRNAs are expressed in fetal hepatocytes.

Molecular cloning of murine BST-1 having homology with CD38 and Aplysia ADP-ribosyl cyclase.

Human BST-1, a bone marrow stromal cell surface antigen, is a glycosyl-phosphatidylinositol-anchored protein that facilitates the growth of pre-B cells. We report here the molecular cloning of murine BST-1 cDNA. The deduced amino acid sequence of murine BST-1 had 71% similarity with human BST-1 and 30 and 25% similarity with CD38 and Aplysia adenosine diphosphate-ribosyl cyclase, respectively. Murine BST-1 mRNA was expressed in the bone marrow, spleen and thymus in lymphoid organs, and the lung, kidney and heart in non-lymphoid organs. Restriction fragment length polymorphism (RFLP) was shown in BALB/c, DBA/2 and NZB vs C57BL/6, A/J, CBA/N, NZW, BXSB and MRL/lpr. RFLP was mapped to the 5' portion of the murine BST-1 gene.

Developmental regulation of rat serine dehydratase gene expression: evidence for the presence of a repressor in fetal hepatocytes.

To determine why the rat serine dehydratase gene becomes transcriptionally activated just after birth, we examined the interactions of DNA binding proteins of fetal and adult rat livers with the serine dehydratase gene promoter by DNase I protection analysis and gel mobility shift assay. Several binding regions of nuclear proteins were found to be common to fetal and adult livers and interaction of factors with the characteristics of Sp1 or NF-Y was suggested. Two additional regions, named regions B and I, were specific to fetal liver. These regions contain GATA-like sequences and competition experiments by gel mobility shift assay suggested that the fetal liver-enriched factor binds to the GATA-like sequences. The function of the regions B and I in transcription regulation was investigated in fetal and adult hepatocytes by transient DNA transfer experiments with serine dehydratase-chloramphenicol acetyltransferase fusions. These experiments showed that these regions functioned as negative cis-acting elements in fetal hepatocytes, but not in adult hepatocytes.