Radix Astragali polysaccharide RAP directly protects hematopoietic stem cells from chemotherapy-induced myelosuppression by increasing FOS expression.

Radix Astragali polysaccharide RAP has been reported to play a crucial role in hematopoiesis without a clear mechanism. In this study, RAP's effects to enhance the recovery of cyclophosphamide (Cy)-suppressed bone marrow and blood cells is confirmed in vivo first. Confocal micrographs demonstrated the interesting direct binding of FITC-RAP to hematopoietic stem cells (HSC) in bone marrow. RAP protects both mice and human HSC in terms of cell morphology, proliferation, and apoptosis. RNA-sequencing and shRNA approaches revealed FOS to be a key regulator in RAP's protection. These evidences provide an unreported mechanism that RAP directly protects hematopoietic stem cells from chemotherapy-induced myelosuppression by increasing FOS expression.

In vivo pathology: seeing with molecular specificity and cellular resolution in the living body.

The emerging tools of in vivo molecular imaging are enabling dynamic cellular and molecular analyses of disease mechanisms in living animal models and humans. These advances have the potential to dramatically change a number of fields of study, including pathology, and to contribute to the development of regenerative medicine and stem cell therapies. The new tools of molecular imaging, which have already had a tremendous impact on preclinical studies, hold great promise for bringing important and novel information to the clinician and the patient. These approaches are likely to enable early diagnosis, rapid typing of molecular markers, immediate assessment of therapeutic outcome, and ready measures of the extent of tissue regeneration after damage. However, the full impact of these new techniques will be determined by our ability to translate them to the clinic and to develop a general strategy that integrates them with other advances in molecular diagnostics and molecular medicine.

Supplementation of conventional freezing medium with a combination of catalase and trehalose results in better protection of surface molecules and functionality of hematopoietic cells.

Our previous studies had shown that a combination of the bio-antioxidant catalase and the membrane stabilizer trehalose in the conventional freezing mixture affords better cryoprotection to hematopoietic cells as judged by clonogenic assays. In the present investigation, we extended these studies using several parameters like responsiveness to growth factors, expression of growth factor receptors, adhesion assays, adhesion molecule expression, and long-term culture-forming ability. Cells were frozen with (test cells) or without additives (control cells) in the conventional medium containing 10% dimethylsulfoxide (DMSO). Experiments were done on mononuclear cells (MNC) from cord blood/fetal liver hematopoietic cells (CB/FL) and CD34(+) cells isolated from frozen MNC. Our results showed that the responsiveness of test cells to the two early-acting cytokines, viz. interleukin-3 (IL-3) and stem cell factor (SCF) in CFU assays was better than control cells as seen by higher colony formation at limiting concentrations of these cytokines. We, therefore, analyzed the expression of these two growth factor receptors by flow cytometry. We found that in cryopreserved test MNC, as well as CD34(+) cells isolated from them, the expression of both cytokine receptors was two- to three-fold higher than control MNC and CD34(+) cells isolated from them. Adhesion assays carried out with CB/FL-derived CD34(+) cells and KG1a cells showed significantly higher adherence of test cells to M210B4 than respective control cells. Cryopreserved test MNC as well as CD34(+) cells isolated from them showed increased expression of adhesion molecules like CD43, CD44, CD49d, and CD49e. On isolated CD34(+) cells and KG1a cells, there was a two- to three-fold increase in a double-positive population expressing CD34/L-selectin in test cells as compared to control cells. Long-term cultures (LTC) were set up with frozen MNC as well as with CD34(+) cells. Clonogenic cells from LTC were enumerated at the end of the fifth week. There was a significantly increased formation of CFU from test cells than from control cells, indicating better preservation of early progenitors in test cells. Our results suggest that use of a combination of catalase and trehalose as a supplement in the conventional freezing medium results in better protection of growth factor receptors, adhesion molecules, and functionality of hematopoietic cells, yielding a better graft quality.

Cell surface antigen CD109 is a novel member of the alpha(2) macroglobulin/C3, C4, C5 family of thioester-containing proteins.

Cell surface antigen CD109 is a glycosylphosphatidylinositol (GPI)-linked glycoprotein of approximately 170 kd found on a subset of hematopoietic stem and progenitor cells and on activated platelets and T cells. Although it has been suggested that T-cell CD109 may play a role in antibody-inducing T-helper function and it is known that platelet CD109 carries the Gov alloantigen system, the role of CD109 in hematopoietic cells remains largely unknown. As a first step toward elucidating the function of CD109, we have isolated and characterized a human CD109 cDNA from KG1a and endothelial cells. The isolated cDNA comprises a 4335 bp open-reading frame encoding a 1445 amino acid (aa) protein of approximately 162 kd that contains a 21 aa N-terminal leader peptide, 17 potential N-linked glycosylation sites, and a C-terminal GPI anchor cleavage-addition site. We report that CD109 is a novel member of the alpha 2 macroglobulin (alpha 2M)/C3, C4, C5 family of thioester-containing proteins, and we demonstrate that native CD109 does indeed contain an intact thioester. Analysis of the CD109 aa sequence suggests that CD109 is likely activated by proteolytic cleavage and thereby becomes capable of thioester-mediated covalent binding to adjacent molecules or cells. In addition, the predicted chemical reactivity of the activated CD109 thioester is complement-like rather than resembling that of alpha 2M proteins. Thus, not only is CD109 potentially capable of covalent binding to carbohydrate and protein targets, but the t(1/2) of its activated thioester is likely extremely short, indicating that CD109 action is highly restricted spatially to the site of its activation.

The "vanishing counting bead" phenomenon: effect on absolute CD34+ cell counting in phosphate-buffered saline-diluted leukapheresis samples.

BACKGROUND: Using a single-platform protocol to count absolute CD34+ hematopoietic precursor cell (HPC) levels with different reference microbeads, we recorded occasionally artifactually high CD34+ HPC counts in some leukapheresis bags, whereas dual-platform calculations were always consistent. Abnormal countings were observed only when phosphate-buffered saline (PBS)-diluted leukapheresis samples were vortexed before analysis. A large series of blood samples analyzed similarly for CD34+ and CD4+ absolute counts did not show any sample or vortexing effect. With the volumetric absolute counting cytometer Partec-PAS, lower counts were also observed when different reference beads were vortexed before the instrument checking procedures. The counting abnormality was caused by a drop in microbead concentration (the "vanishing bead phenomenon"). This phenomenon reduced the total and relative bead event number in experimental and routine samples and in calibration procedures. This altered the bead denominator used to calculate absolute CD34+ HPC levels and it also reduced the concentration of standard calibration beads. METHODS: Using the Partec-PAS to measure volumetrically the actual bead concentration, we studied the vanishing bead phenomenon. Different types of counting and reference microbeads were resuspended in media with or without proteins or cells. Replicates were submitted either to gentle manual mixing or to vortexing before counting. RESULTS: Vortex agitation almost invariably induced the vanishing bead phenomenon when beads were resuspended in saline media or when an insufficient protein concentration was present, such as in diluted leukapheresis samples. Different bead types showed various degrees of sensitivity to vortexing. The bead disappearance was not caused by bubble formation or disruption. The addition of small amounts of protein completely prevented the vanishing bead phenomenon. The causative effect of the electrostatic charging of tube induced by vortexing is hypothesized. CONCLUSIONS: Sample suspensions containing counting beads for single-platform analysis must be resuspended in media with protein supplements to prevent the vanishing bead phenomenon and to ensure accurate counting.

Role of the alpha1 and alpha2 integrin cytoplasmic domains in cell morphology, motility and responsiveness to stimulation by the protein kinase C pathway.

The alpha1beta1 and alpha2beta1 integrins, extracellular matrix receptors for collagens and/or laminins, have similarities in structure and ligand binding. Recent studies suggest that the two receptors mediate distinct post-ligand binding events and are not simply redundant receptors. To discern the mechanisms by which the two receptors differ, we focused on the roles of the cytoplasmic domains of the alpha subunits. We expressed either full-length alpha1 integrin subunit cDNA (X1C1), full-length alpha2 integrin subunit cDNA (X2C2), chimeric cDNA composed of the extracellular and transmembrane domains of alpha2 subunit and the cytoplasmic domain of alpha1 (X2C1), chimeric cDNA composed of the extracellular and transmembrane domains of alpha1 subunit and the cytoplasmic domain of alpha2 (X1C2), alpha1 cDNA truncated after the GFFKR sequence (X1C0) or alpha2 cDNA truncated after the GFFKR sequence (X2C0) in K562 cells. Although the cytoplasmic domains of the alpha1 and alpha2 subunits were not required for adhesion, the extent of adhesion at low substrate density was enhanced by the presence of either the alpha1 or alpha2 cytoplasmic tail. Spreading was also influenced by the presence of an alpha subunit cytoplasmic tail. Activation of the protein kinase C pathway with phorbol dibutyrate-stimulated motility that was dependent upon the presence of the alpha2 cytoplasmic tail. Both the phosphatidylinosotide-3-OH kinase and the mitogen-activated protein kinase pathways were required for phorbol-activated, alpha2-cytoplasmic tail-dependent migration.

Purging of mammary carcinoma cells during ex vivo culture of CD34+ hematopoietic progenitor cells with recombinant immunotoxins.

Tumor cells have been found in autologous hematopoietic cell transplants used after high-dose chemotherapy. To specifically eliminate contaminating mammary tumor cells during ex vivo expansion of CD34+ hematopoietic progenitor cells, we used recombinant immunotoxins (ITs) directed against cell-surface antigens expressed on mammary carcinoma cells. ITs were expressed from fusion cDNAs combining a single-chain antibody fragment (scFv) directed against the Erb-B2 or epidermal growth factor (EGF) receptors with a truncated Pseudomonas exotoxin A fragment devoid of its cell-binding domain. CD34+ hematopoietic progenitor cells did not express Erb-B2 and EGF receptors as detected by Western blotting. Ex vivo expansion of total hematopoietic cells or of colony-forming cells from CD34+ progenitors in the presence of stem-cell factor (SCF), interleukin-1 (IL-1), IL-3, IL-6, and erythropoietin (Epo) was not affected when ITs were added to the cultures. In contrast, MDA-MB 453 and MCF-7 mammary carcinoma cells were depleted in a dose- and time-dependent manner by more than 3 log in coculture with CD34+ cells over a period of 7 days in the presence of 100 to 1,000 ng/mL of anti-Erb-B2 IT. This included elimination of the subpopulations with regrowth potential. Similarly, addition of either anti-Erb-B2 or anti-EGF receptor ITs to primary breast cancer cells isolated from patients with metastatic disease resulted in elimination of cytokeratin-positive cells in seven of seven samples. ITs are highly efficient and convenient to use for the depletion of mammary tumor cells during ex vivo expansion of hematopoietic progenitor-cell autografts.

Analysis of platelet recovery after autologous transplantation with G-CSF mobilized CD34+ cells purified from leukapheresis products.

We studied platelet recovery in relation to graft content in CFUs and CD34+ cells in 31 patients with multiple myeloma (21) or non-Hodgkin lymphoma (10) receiving marrow-ablative therapy followed by autologous transplantation with G-CSF mobilized CD34+ cells purified from leukapheresis products. Twelve patients had prolonged post-transplantation thrombopenia (> or = 14 days): their graft contents in CD34+ cells, CFU-GM and BFU-E were significantly inferior to those of patients with rapid platelet recovery. Although numbers of infused CD34+ cells and CFU-GM or BFU-E were well correlated, the graft content in CD34+ cells was the only parameter predictive of platelet recovery (r = -0.38, p = 0.04), with a threshold of 2.5 x 10(6) CD34+ cells/kg. However, because rapid platelet reconstitution was obtained for 4 of 16 patients re-infused with < 2.5 x 10(6) CD34+ cells/kg, we investigated whether the graft CFU-MK content might be a better predictor of platelet reconstitution than the CD34+ cell content. Eighteen CD34 grafts were studied for CFU-MK content: CD34 and CFU-MK contents were weakly correlated (r = 0.52, p = 0.03), but there was no correlation between numbers of infused CFU-MK and time to platelet recovery. We conclude that, for autologous CD34 grafts, CFU-MK assays, like CFU-GM or BFU-E assays, cannot be used to predict platelet recovery. A CD34+ cell content > or = 2.5 x 10(6)/kg remains the only reliable indicator of the platelet reconstitution capacity of a CD34 graft.

Cloning and characterization of fetal liver phosphatase 1, a nuclear protein tyrosine phosphatase isolated from hematopoietic stem cells.

We report the isolation of cDNAs encoding protein tyrosine phosphatases (PTPs) from highly purified hematopoietic stem cell populations. One such cDNA encodes a novel PTP, designated fetal liver phosphatase 1 (FLP1), which consists of one PTP domain followed by a carboxy terminal domain of 160 amino acids. Northern blot and in situ hybridization analysis showed that expression of FLP1 mRNA is restricted to thymus in 15.5-day-old and 17.5-day-old mouse embryos and to kidney and hematopoietic tissues in adult mice. Furthermore, polymerase chain reaction-based analysis shows that FLP1 is expressed in hematopoietic stem cells as well as in more mature hematopoietic cells. Peptide antisera against FLP1 immunoprecipitated a 48-kD protein that is localized in the nuclei of Ba/F3 lymphoid cells. We have analyzed the effects of overexpressing either wild-type FLP1 or a functionally inactive mutant of FLP1 in hematopoietic cells. In the progenitor K562 cell line, cells ectopically expressing functional FLP1 differentiated normally to megakaryocytes after induction with tetradecanoyl phorbol acetate (TPA). In contrast, when K562 transfectants expressing an inactive mutant FLP1 protein were treated with TPA, the characteristic cell spreading and substrate adhesion that accompany megakaryocytic differentiation did not occur. We show that, in these cells, the induction of the differentiation marker alphaIIb beta3 is not affected. However, both constitutive and TPA-induced expression of alpha2 integrin, a late megakaryocytic marker, are inhibited. These results suggest that the expression of an inactive form of FLP1 affects late signaling events of K562 megakaryocytic differentiation.

Positive selection of CD34+ cells from cryopreserved peripheral blood stem cells after thawing: technical aspects and clinical use.

Autologous stem cell transplantation has become an important therapy in lymphoma, multiple myeloma and solid tumors. The rationale for the selection of CD34+ cells from peripheral blood or bone marrow progenitor cell collections is based on the observation that contaminating tumor cells can be depleted approximately 3 to 6 logs. This procedure may be limited because of lack of sufficient numbers of progenitor cells in the leukapheresis concentrates. The use of frozen/thawed peripheral blood mononuclear cell (PBMC) samples makes it possible to pool two or even more stem cell harvests collected at different time points to increase the total number of CD34+ progenitor cells. We report in this work the feasibility of frozen/thawed peripheral blood CD34+ positive cell selection, using the large-scale (Ceprate SC) and the lab-scale avidin-biotin immunoadsorption system (Ceprate LC). This procedure consists of a washing step and a positive selection step. Our results show that frozen/thawed CD34+ cells were obtained with a purity of 86.68 +/- 3.62%, a viability of 97.94 +/- 0.97% and a recovery of 91.85 +/- 10.84% (range 80 to 112%). The CFU-GM assays were performed in a methylcellulose based medium; 89.13 +/- 19.63 colonies were obtained for 10(3) cells plated. Two patients were grafted with peripheral blood CD34+ cells selected after freezing. Our clinical data show that these cells are capable of rapidly reconstituting hematopoiesis after high-dose chemotherapy.

Purified CD34+ Lin- Thy+ stem cells do not contain clonal myeloma cells.

High-dose therapy with autologous marrow or peripheral blood stem cell (PBSC) rescue has been extensively applied in the treatment of multiple myeloma (MM) patients during the past 10 years resulting in improved event-free and overall survival when compared with standard chemotherapy. However, relapses are common and cure is unlikely in the majority of patients. Because both bone marrow and PBSCs are contaminated with myeloma cells it is conceivable that relapse after autotransplantation originates at least in part from autografted tumor cells. In this study, mobilized PBSCs were examined for the presence of myeloma cells based on immunophenotyping and sensitive polymerase chain reaction (PCR)-based techniques. In addition, CD34+ Lin- Thy+ stem cells were purified from mobilized PBSC harvests of 10 MM patients by sequentially using counterflow elutriation centrifugation, treatment with phenylalanine methylester, and flow sorting, using 5-parameter gating (propidium iodide, forward scatter, side scatter, CD34+ v Lin- and CD34+ v Thy+). Virtually all mobilized unsorted PBSC preparations contained myeloma cells in sufficient quantities (range, < 0.01 to > 10%) potentially causing a disease relapse. Stem cell purification led to an overall enrichment by about 50-fold in all 10 patients; approximately 90% of the final cell population expressed CD34+ Lin- Thy+ with no evidence of myeloma cell contamination based on flow cytometric analysis of CD38bright cells (< 0.1%). Quantitative PCR amplification of patient-specific complementarity determining region III (CDRIII) DNA sequences showed depletion of clonal B cells by 2.7 to 7.3 logs, with the highest log reduction noted in the samples initially containing the most tumor cells. Our results show that purification of CD34+ Lin- Thy+ cells depletes myeloma cells to undetectable levels from up to 10% present in unsorted PBSCs, thus offering a tool to investigate whether MM relapse after autotransplantation can be reduced markedly.

Molecular cloning of a novel receptor tyrosine kinase gene, STK, derived from enriched hematopoietic stem cells.

To identify the novel receptor tyrosine kinases (RTKs) critical to the proliferation of hematopoietic stem cells, we performed polymerase chain reaction-based cloning from highly purified murine hematopoietic stem cells. Lineage marker-negative, c-KIT-positive, and Ly6A/E- or Sca-1-positive (Lin-c-KIT+Sca-1+) cells were sorted by a fluorescence-activated cell sorter. Two sets of degenerate oligonucleotide primers were directed to the conserved sequences of the catalytic domain, and were used to amplify cDNAs that encode protein tyrosine kinases (PTKs). One hundred cDNA clones were sequenced and 8 RTKs were identified, as well as 12 non-RTKs and 2 serine/threonine kinases. Sixteen cDNAs were identical to the known kinase genes (PKC beta, JAK-1, JAK-2, TYK-2, HCK, FGR, FYN, BLK, c-FES, FER, c-ABL, c-KIT, FLK-1, FLK-2, IGF1R, and ECK). Six novel cDNA sequences (stk series) were identified. However, three of them turned out to be BPK, RYK, and TEK. The remaining three showed high homology to S6 kinase II, JAK-2, and v-SEA/c-MET, respectively. Characterization of full-length cDNA sequence of the v-SEA/cMET-related gene showed that this was a novel RTK gene and we named this gene STK (stem cell-derived tyrosine kinase). We identified two distinct forms of STK cDNA; the short one encoded a putative truncated protein that lacked most of the extracellular domain. STK was expressed at various stages of hematopoietic cells, including stem cells, but we could not detect any apparent expression in other adult tissues. The expression of the truncated form of mRNA was more predominant than that of the complete form. STK was assigned by fluorescent in situ hybridization to the R-positive F1 band of chromosome 9, the same region to which hepatic growth factor-like protein has been assigned. Characterization of these PTKs, including STK, will be helpful to elucidate the molecular mechanism of the growth regulation of hematopoietic stem cells.

Interleukin-3 (IL-3) receptors on rhesus monkey bone marrow cells: species specificity of human IL-3, binding characteristics, and lack of competition with GM-CSF.

The relative affinity of recombinant human interleukin-3 (IL-3) binding to normal rhesus monkey bone marrow cells was found to be 25- to 50-fold less than that of homologous IL-3, which explained the species specificity of human IL-3 observed when tested in Macaca species. In contrast, only a small difference was found between human and rhesus monkey IL-3 in relative binding affinity for receptors on human acute myelogenous leukemia (AML) cells, which confirmed that the species specificity of IL-3 is largely unidirectional. The biological significance of the findings was demonstrated by direct in vivo comparison of the effects of high-dose recombinant rhesus monkey and human IL-3. The binding characteristics of IL-3 receptors on rhesus monkey bone marrow and peripheral blood cells were further characterized by specific binding of radiolabeled rhesus monkey IL-3. Scatchard analysis of two bone marrow samples demonstrated high-affinity IL-3 receptors (25 and 80 sites/cell, respectively; equilibrium dissociation constants [Kd] of 8 and 3 pM/L) as well as low-affinity receptors (1070 and 1290 sites/cell; equilibrium dissociation constants of 2600 and 1200 pM/L). In addition, IL-3 receptor expression was also detected on purified CD34-positive bone marrow cells. Competition by human granulocyte-macrophage colony-stimulating factor (GM-CSF) of IL-3 binding to high- or low-affinity receptors on rhesus monkey peripheral blood and bone marrow cells could not be demonstrated, which may indicate that the growth factor-specific alpha-subunits of the GM-CSF and IL-3 receptors are expressed predominantly on different cell types.

Large volume leukapheresis to maximize peripheral blood stem cell collection.

Large volume leukapheresis, defined as the processing of greater than three volumes of blood at one sitting, can be safely and easily performed using a variety of cell separators in a range of patients. This paper describes the history, technique, outcome, complications, and proposed uses for this procedure.

Gene transfer into human hematopoietic progenitor cells: a review of current clinical protocols.

Recent reviews by Clay Smith (1992) in this journal and by W. French Anderson (1990) and A. Dusty Miller (1992) have described the technologies used for gene transfer, the potential applications of the approach, and the safety issues that require consideration. This review will have a narrower focus. It will deal specifically with current and forthcoming clinical protocols for gene transfer into hemopoietic progenitor cells, because a major goal of gene therapy for malignant and nonmalignant disease is to transfer and express genes on a long-term basis in these cells or their progeny.