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PubMed2001

Inverse relationship between patient peripheral blood CD34+ cell counts and collection efficiency for CD34+ cells in two automated leukapheresis systems.

BACKGROUND: The purpose of this study was to analyze the CD34 cell collection efficiency (CE) of automated leukapheresis protocols of two blood cell separators (Spectra, COBE [AutoPBSC protocol] and AS104, Fresenius [PBSC-Lym, protocol]) for peripheral blood progenitor cell (PBPC) harvest in patients with malignant diseases. STUDY DESIGN AND METHODS: PBPCs were collected by the Spectra AutoPBSC protocol in 95 patients (123 collections) and the AS104 PBSC-Lym protocol in 87 patients (115 harvests). Patients underwent a median of one (range, 1-4) conventional-volume apheresis procedure of 10.8 L (9.0-13.9) to obtain a target cell dose of > or =2.5 x 10(6) CD34+ cells per kg. RESULTS: The median overall CD34 CE was significantly better on the AS104 than on the Spectra: 55.8 percent versus 42.4 percent (p = 0.000). This was also true below (59.2% vs. 50.1%; p = 0.022) and above (51.2% vs. 41.3%; p = 0.001) the preleukapheresis threshold of 40 CD34+ cells per microL needed to collect a single-apheresis autograft. However, at > or =40 circulating CD34+ cells per microL, both cell separators achieved the target of > or =2.5 x 10(6) CD34+ cells per kg. The CD34 CE dropped significantly, from 59.2 percent at <40 cells per microL to 51.2 percent at > or =40 cells per microL on the AS104 (p = 0.017) and from 50.1 percent to 41.3 percent on the Spectra (p = 0.033). CONCLUSION: Whereas the CD34 CE was significantly different with the AS104 and the Spectra, the CD34 CE of both machines correlated inversely with peripheral blood CD34+ cell counts, showing a significant decline with increasing numbers of circulating CD34+ cells. Nevertheless, at > or 40 preapheresis CD34+ cells per microL, sufficient hematopoietic autografts of > or =2.5 x 10(6) CD34+ cells per kg were harvested by a single conventional-volume (11 L) leukapheresis on both cell separators.

Adolescent↗
PubMed2005

CD34+ cells home, proliferate, and participate in capillary formation, and in combination with CD34- cells enhance tube formation in a 3-dimensional matrix.

OBJECTIVE: Emerging evidence suggests that human blood contains bone marrow (BM)-derived endothelial progenitor cells that contribute to postnatal neovascularization. Clinical trials demonstrated that administration of BM-cells can enhance neovascularization. Most studies, however, used crude cell populations. Identifying the role of different cell populations is important for developing improved cellular therapies. METHODS AND RESULTS: Effects of the hematopoietic stem cell-containing CD34+ cell population on migration, proliferation, differentiation, stimulation of, and participation in capillary-like tubule formation were assessed in an in vitro 3-dimensional matrix model using human microvascular endothelial cells. During movement over the endothelial monolayer, CD34+ cells remained stuck at sites of capillary tube formation and time- and dose-dependently formed cell clusters. Immunohistochemistry confirmed homing and proliferation of CD34+ cells in and around capillary sprouts. CD34+ cells were transduced with the LNGFR marker gene to allow tracing. LNGFR gene-transduced CD34+ cells integrated in the tubular structures and stained positive for CD31 and UEA-1. CD34+ cells alone stimulated neovascularization by 17%. Coculture with CD34- cells led to 68% enhancement of neovascularization, whereas CD34- cells displayed a variable response by themselves. Cell-cell contact between CD34+ and CD34- cells facilitated endothelial differentiation of CD34+ cells. CONCLUSIONS: Our data suggest that administration of CD34+-enriched cell populations may significantly improve neovascularization and point at an important supportive role for (endogenous or exogenous) CD34- cells.

Antigens, CD34↗
PubMed1994

Stem cell factor (c-kit ligand) enhances the interleukin-9-dependent proliferation of human CD34+ and CD34+CD33-DR- cells.

We have studied the effects of recombinant human interleukin-9 (IL-9), alone and combined with stem cell factor (SCF, c-kit ligand), IL-3, and granulocyte-macrophage colony-stimulating factor (GM-CSF) on the clonogenic proliferation of highly enriched human hematopoietic CD34+ and CD34+CD33-DR- progenitor cells. Colony assays were performed under serum-containing and serum-free conditions. IL-9, as a single agent, did not support colony formation. The addition of erythropoietin (Epo) to IL-9 induced the growth of erythroid progenitors (BFU-E) derived from both CD34+ and CD34+CD33-DR- cells. The IL-9-dependent growth of BFU-E derived from CD34+ cells was increased in an additive manner by SCF and, to a lesser extent, by IL-3, whereas CD34+CD33-DR- erythroid precursors were also responsive to GM-CSF in combination with IL-9. The addition of SCF to IL-9 did stimulate the development of CD34+ and CD34+CD33-DR- macroscopic, multicentered BFU-E and multilineage colonies (CFU-GEMM). When IL-9 was used in serum-free conditions, the growth of CD34+ and CD34+CD33-DR- BFU-E was observed in the presence of Epo. Moreover, a marked synergy on BFU-E colony formation was evident when IL-9 was combined with SCF, and their activity was enhanced by the addition of IL-3. IL-9 showed a negligible proliferative activity on colony-forming units-granulocyte/macrophage (CFU-GM). However, it increased the number of CD34+CD33-DR- CFU-GM responsive to IL-3 (37% of the colonies generated by phytohemagglutinin-stimulated lymphocyte conditioned medium [PHA-LCM]). The effects of IL-9 on CD34+CD33-DR- cells were also studied in a short-term suspension culture system, which evaluates the proliferation of progenitors earlier than day 14 CFU-C (Delta assay). In this system, IL-9 had a minimal activity on its own. In combination with SCF, however, it induced a nine-fold expansion of CD34+CD33-DR- cells, which generated a greater number of CFU-GM than BFU-E in secondary methylcellulose cultures. These experiments indicate that IL-9 induces the proliferation of very primitive human erythroid cells, and this effect is potentiated by SCF and other cytokines. Furthermore, IL-9 synergizes in vitro with the c-kit ligand in expanding the pool of early pluripotent hematopoietic progenitor cells.

Antigens, CD↗
PubMed1996

Peripheral blood CD34+ cells differ from bone marrow CD34+ cells in Thy-1 expression and cell cycle status in nonhuman primates mobilized or not mobilized with granulocyte colony-stimulating factor and/or stem cell factor.

Granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) have been shown to stimulate the circulation of hematopoietic progenitor cells in both mice and nonhuman primates. We evaluated the immunophenotype and cell cycle status of CD34+ cells isolated from the bone marrow (BM) and leukapheresis product of cytokine-mobilized nonhuman primates. CD34+ cells were isolated from rhesus macaques that had received no cytokine therapy, 100 micrograms/kg/d G-CSF, 200 micrograms/kg/d SCF, or a combination of both 100 micrograms/kg/d G-CSF and 200 micrograms/kg/d SCF as a subcutaneous injection for 5 days. BM was aspirated before (day 0) and on the last day (day 5) of cytokine administration. On days 4 and 5, peripheral blood (PB) mononuclear cells were collected using a novel method of leukapheresis. Threefold more PB mononuclear cells were collected from animals receiving G-CSF alone or G-CSF and SCF than from animals that had received either SCF alone or no cytokine therapy. CD34+ cells were positively selected using an immunoadsorptive system from the BM, PB, and/or leukapheresis product. Threefold and 10-fold more CD34+ cells were isolated from the leukapheresis product of animals receiving G-CSF or G-CSF and SCF, respectively, than from animals receiving no cytokine therapy or SCF alone. The isolated CD34+ cells were immunophenotyped using CD34-allophycocyanin, CD38-fluorescein isothiocyanate, and Thy-1-phycoerythrin. These cells were later stained with 4', 6-diamidino-2-phenylindole for simultaneous DNA analysis and immunophenotyping. BM-derived CD34+ cells did not differ significantly in cell cycle status and Thy-1 or CD38 phenotype before or after G-CSF and/or SCF administration. Similarly, CD34+ cells isolated from the leukapheresis product did not differ significantly in immunophenotype or cell cycle status before or after G-CSF and/or SCF administration. However, there were consistent differences in both immunophenotype and cell cycle status between BM- and PB-derived CD34+ cells. CD34+ cells isolated from the PB consistently had a smaller percentage of cells in the S+G2/M phase of the cell cycle and had a higher percentage of cells expressing Thy-1 than did CD34+ cells isolated from the BM. A greater proportion of PB-derived CD34+ cells were in the S+G2/M phase of the cell cycle after culture in media supplemented with interleukin-6 and SCF, However, culturing decreased the proportion of CD34+ cells expressing Thy-1.

ADP-ribosyl Cyclase↗
PubMed1996

Identification of CD34+ subsets after glycoprotease selection: engraftment of CD34+Thy-1+Lin- stem cells in fetal sheep.

Epitopes on the CD34 molecule detected by some CD34 antibodies can be cleaved by a unique glycoprotease from Pasteurella haemolytica, which cleaves only glycoproteins rich in O-linked glycans. A method to isolate CD34+ cells from adult bone marrow was developed subsequently, in which CD34+ cells were isolated in high purity and yield following immunomagnetic bead selection and detachment with the glycoprotease. Using a variety of other cell-surface markers shown here to be insensitive to glycoprotease, committed progenitors of T lymphoid, B lymphoid, monomyeloid, megakaryoblastic, or erythroid lineages could be identified. Significantly, candidate hematopoietic stem cells (HSC) that are contained within a CD34+Lin- (CD2-, CD14-, CD15-, CD16-, CD19-) (or CD34+CD38-) subset expressing the Thy-1 antigen (CDw90), c-kit receptor (CD117), and CDw109 but lacking expression of CD71 and HLA-DR antigens also were detected. Functionally distinct subsets of glycoprotease-selected CD34+ cells were identified and subfractionated using flow cytometry and fluorescence-activated cell sorting (FACS). These subsets included candidate HSCs expressing the CD34+Thy-1+Lin- phenotype, which were sorted from a CD34+ fraction of a mobilized peripheral blood (MPB) sample. In a fetal sheep model, when CD34+Thy-1+Lin- cells were injected intraperitoneally, they were capable of homing to the marrow, where they generated long-term multilineage hematopoiesis and maintained human CD34+ cells, indicating that candidate HSC subsets of CD34+ cells selected with this highly specific enzyme were capable of engraftment in vivo. The ability to identify and purify virtually any phenotypically defined subset of glycoprotease-selected CD34+ stem/progenitor cells should facilitate the study of hematopoiesis in vitro and in animal models in vivo as well as the development of novel genetic techniques for the correction of specific blood cell disorders in humans.

Adult↗
PubMed2001

Comparison of processing four and five times the patients' blood volume during peripheral blood stem cell collection and analysis of CD34+38--and CD34+49d+ subsets during apheresis.

PURPOSE: We investigated whether increasing the patients' processed blood volume (BV) during peripheral blood stem cell collection (PBSCC) from four to five times leads to a greater yield of CD34+ cells. We also studied the kinetics of CD34+38- and CD34+49d+ subsets and compared the amount of transfused cells with engraftment. METHODS: All patients ( n=20) received chemotherapy followed by G-CSF for PBSC mobilization. Samples from the patients' peripheral blood and the PBSC harvests were taken after processing 1-, 4-, and 5 times the patients' calculated BV. RESULTS: The mean total yields of CD34+, CD34+38-, and CD34+49d+ cells were 15.69-, 1.13- and 4.17 x 10(6)/kg body weight, respectively. The mean increase for these subsets between 4- and 5 BV was 10%, 8%, and 21%, respectively. Based on the mean number of 2.25 (range 2-3) planned courses of high-dose chemotherapy (HDC) per patient, the mean yield of CD34+ cells per kg body weight and intended course of HDC after 4- and 5 BV was 6.31- and 6.97 x 10(6) ( P=0.014). Twenty HDC were evaluable for engraftment. There was some correlation between the number of transfused CD34+ and CD34+38- cells and WBC engraftment ( r = -0.66 and --0.69; P<0.01) and CD34+ cells and platelet engraftment ( r = -0.56; P= 0.013). No toxicity occurred during PBSCC, although the mean platelet count dropped by 50% which must be kept in mind regarding the additional application of anti-coagulants and the fact that most patients had large indwelling catheters. CONCLUSION: Processing 4 BV is sufficient to collect >5 x 10(6) CD34+ cells/kg body weight and intended course of HDC in most patients, although extension to 5 BV further increases the total yield of CD34+ cells.

ADP-ribosyl Cyclase↗
PubMed1998

The role of the different CD34 epitopes in detection and positive selection of CD34+ bone marrow and peripheral blood stem cells.

Positive selection of CD34+ cells is an attractive approach to reduce tumour cell contamination in bone marrow (BM) and peripheral blood progenitor cell (PBPC) autografts in malignancies not expressing CD34. All current selection methods use monoclonal antibodies (MoAbs) specific for the class I or class II CD34 epitopes, while for detection most investigators use class III MoAbs. Since the distribution of the different CD34 epitopes on haematopoietic progenitors differs, we studied their significance in CD34+ selection procedures. Testing MoAbs against class I, II and III CD34 epitopes on normal BM we observed that +/- 23% of class III positive cells was class I negative. A higher expression of the class III epitope compared with classes I or II was observed on the KG1 cell line, whereas no differences in binding capability were found. The class III epitope anti-CD34, 561, was compared with the class I epitope anti-CD34, BI-3C5, both coupled to M450 Dynabeads. The yield of CD34+ cells obtained with the 561 beads was 1.7% of the mononuclear cells versus 0.95% using the class I epitope, a 1.95-fold increase (1.3-2.7), whereas the purity was similar (96% in both cases). The absolute number of CD34+ cells was therefore twofold higher after 561 selection, including cells with a more mature phenotype. In single cell assay comparable numbers of highly proliferative progenitors but higher numbers of differentiated colonies per phenotypic subfraction were measured. In conclusion, M450 beads coated with the 561 anti-class III CD34 epitope are more efficient in isolating CD34+ cells from bone marrow, probably due to a broader distribution of the class III epitope.

Antibodies, Monoclonal↗
PubMed2006

CD34(+) CD38(-) and CD34(+) HLA-DR(-) cells in BM stem cell grafts correlate with short-term engraftment but have no influence on long-term hematopoietic reconstitution after autologous transplantation.

BACKGROUND: Prior studies have demonstrated that relatively immature hematopoietic stem cells, including CD34(+) CD38(-) and CD34(+) HLA-DR(-) subsets, correlate with short-term hematopoietic reconstruction (SHR) after transplantation. The aim of this study was to investigate whether these immature CD34(+) subsets also correlate with long-term hematopoietic reconstitution (LHR) in recipients of ABMT. METHODS: We examined stem cell grafts from 58 patients with B-cell lymphoma or CLL who underwent ABMT after myeloablative conditioning. We determined whether total mononuclear cell dose (MNC), colony-forming unit-granulocyte-monocyte (CFU-GM), CD34(+) cell dose and CD34(+) cell subsets (CD34(+) CD38(-) and CD34(+) HLA-DR(-) were associated with SHR and/or LHR. Time to neutrophil engraftment (TNE) and time to platelet engraftment (TPE) were used to measure SHR, while platelet counts at day 100 and 1 year post-ABMT were used as indicators for LHR. RESULTS AND DISCUSSION: CD34(+) cell dose and CD34(+) cell subsets were significantly associated with SHR. However, at day 100 and 1 year post-transplant only total CD34(+) cell dose was associated with LHR. The association of total CD34(+) cell dose with LHR persisted after adjusting for age, sex and disease. None of the CD34(+) cell subsets analyzed showed evidence of significant association with LHR.

ADP-ribosyl Cyclase 1↗
PubMed1999

Targeting retroviral vectors to CD34-expressing cells: binding to CD34 does not catalyze virus-cell fusion.

We have attempted to engineer murine leukemia virus (MuLV)-based retroviral vectors to specifically transduce cells expressing human CD34, an antigen present on the surface of undifferentiated hematopoietic stem cells. A number of chimeric ecotropic MuLV envelope (Env) proteins were constructed that contained anti-CD34 single-chain antibody variable fragments (scFvs). The scFv-Env proteins were generated either by replacing the receptor-binding domain of Env with the scFv or by inserting the scFv into the N terminus of the Env protein. Only chimeric Env proteins with scFv insertions between amino acids 6 and 7 were incorporated into viral particles, and coexpression of native MuLV Env did not rescue incorporation-defective proteins. In addition, the efficiency of incorporation varied with the specific anti-CD34 scFv that was used. Retroviral vectors containing the scFv-Env proteins bound to CD34+ cells and transduced NIH 3T3 cells expressing human CD34 (3T3-CD34 cells) at approximately twice the efficiency of the parental NIH 3T3 cells. However, the introduction of the mutation D84K, which prevents binding to the ecotropic MuLV receptor mcat-1, prevented transduction of both NIH 3T3 and 3T3-CD34 cells. Complementation cell-cell fusion assays [Zhao et al. (1997). J. Virol. 71, 6967-6972] in 3T3-CD34 cells revealed that although the scFv-Env proteins could contribute postbinding entry functions when bound to mcat-1, they were unable to do so when bound to CD34. Taken together, these data suggest that although the interaction with CD34 effectively increased the concentration of virus on 3T3-CD34 cells, entry could occur only through an interaction with mcat-1; CD34 alone was not capable of triggering the appropriate postbinding changes that lead to viral entry.

3T3 Cells↗
PubMed2004

Heart infarct in NOD-SCID mice: therapeutic vasculogenesis by transplantation of human CD34+ cells and low dose CD34+KDR+ cells.

Hematopoietic (Hem) and endothelial (End) lineages derive from a common progenitor cell, the hemangioblast: specifically, the human cord blood (CB) CD34+KDR+ cell fraction comprises primitive Hem and End cells, as well as hemangioblasts. In humans, the potential therapeutic role of Hem and End progenitors in ischemic heart disease is subject to intense investigation. Particularly, the contribution of these cells to angiogenesis and cardiomyogenesis in myocardial ischemia is not well established. In our studies, we induced myocardial infarct (MI) in the immunocompromised NOD-SCID mouse model, and monitored the effects of myocardial transplantation of human CB CD34+ cells on cardiac function. Specifically, we compared the therapeutic effect of unseparated CD34+ cells vs. PBS and mononuclear cells (MNCs); moreover, we compared the action of the CD34+KDR+ cell subfraction vs. the CD34+KDR- subset. CD34+ cells significantly improve cardiac function after MI, as compared with PBS/MNCs. Similar beneficial actions were obtained using a 2-log lower number of CD34+KDR+ cells, while the same number of CD34+KDR- cells did not have any effects. The beneficial effect of CD34+KDR+ cells may mostly be ascribed to their notable resistance to apoptosis and to their angiogenic action, since cardiomyogenesis was limited. Altogether, our results indicate that, within the CD34+ cell population, the CD34+KDR+ fraction is responsible for the improvement in cardiac hemodynamics and hence represents the candidate active CD34+ cell subset.

Animals↗
PubMed2006

Comparative quantification of umbilical cord blood CD34+ and CD34+ bright cells using the ProCount-BD and ISHAGE protocols.

The total number of CD34+ cells is the most relevant clinical parameter when selecting human umbilical cord blood (HUCB) for transplantation. The objective of the present study was to compare the two most commonly used CD34+ cell quantification methods (ISHAGE protocol and ProCount - BD) and analyze the CD34+ bright cells whose 7-amino actinomycin D (7AAD) analysis suggests are apoptotic or dead cells. Twenty-six HUCB samples obtained at the Placental Blood Program of New York Blood Center were evaluated. The absolute numbers of CD34+ cells evaluated by the ISHAGE (with exclusion of 7AAD+ cells) and ProCount (with exclusion of CD34+ bright cells) were determined. Using the ISHAGE protocol we found 35.6 +/- 19.4 CD34+ cells/microL and with the ProCount method we found 36.6 +/- 23.2 CD34+ cells/microL. With the ProCount method, CD34+ bright cell counts were 9.3 +/- 8.2 cells/microL. CD34+ bright and regular cells were individually analyzed by the ISHAGE protocol. Only about 1.8% of the bright CD34+ cells are alive, whereas a small part (19.0%) is undergoing apoptosis and most of them (79.2%) are dead cells. Our study showed that the two methods produced similar results and that 7AAD is important to exclude CD34 bright cells. These results will be of value to assist in the correct counting of CD34+ cells and to choose the best HUCB unit for transplantation, i.e., the unit with the greatest number of potentially viable stem cells for the reconstitution of bone marrow. This increases the likelihood of success of the transplant and, therefore, the survival of the patient.

Antigens, CD34↗
PubMed2000

Isolation and characterization of human CD34(-)Lin(-) and CD34(+)Lin(-) hematopoietic stem cells using cell surface markers AC133 and CD7.

Recent evidence indicates that human hematopoietic stem cell properties can be found among cells lacking CD34 and lineage commitment markers (CD34(-)Lin(-)). A major barrier in the further characterization of human CD34(-) stem cells is the inability to detect this population using in vitro assays because these cells only demonstrate hematopoietic activity in vivo. Using cell surface markers AC133 and CD7, subfractions were isolated within CD34(-)CD38(-)Lin(-) and CD34(+)CD38(-)Lin(-) cells derived from human cord blood. Although the majority of CD34(-)CD38(-)Lin(-) cells lack AC133 and express CD7, an extremely rare population of AC133(+)CD7(-) cells was identified at a frequency of 0.2%. Surprisingly, these AC133(+)CD7(-) cells were highly enriched for progenitor activity at a frequency equivalent to purified fractions of CD34(+) stem cells, and they were the only subset among the CD34(-)CD38(-)Lin(-) population capable of giving rise to CD34(+) cells in defined liquid cultures. Human cells were detected in the bone marrow of non-obese/severe combined immunodeficiency (NOD/SCID) mice 8 weeks after transplantation of ex vivo-cultured AC133(+)CD7(-) cells isolated from the CD34(-)CD38(-)Lin(-) population, whereas 400-fold greater numbers of the AC133(-)CD7(-) subset had no engraftment ability. These studies provide novel insights into the hierarchical relationship of the human stem cell compartment by identifying a rare population of primitive human CD34(-) cells that are detectable after transplantation in vivo, enriched for in vitro clonogenic capacity, and capable of differentiation into CD34(+) cells. (Blood. 2000;95:2813-2820)

AC133 Antigen↗
PubMed1991

Effects of interleukin-4 and interleukin-6 on the proliferation of CD34+ and CD34- blasts from acute myelogenous leukemia.

We studied the effects of interleukin-4 (IL-4) and IL-6 on the growth of leukemic blasts from 40 patients with acute myelogenous leukemia (AML). Patients were selected on the basis of negativity for a series of B-cell antigens including CD10 and CD19. Twenty-one cases were CD34-positive (CD34+) (greater than 15% of blasts) and the remaining 19 were CD34-negative (CD34-) (less than 3% of blasts). IL-4 alone (100 U/ml) could stimulate either DNA synthesis (with greater than 2.0 stimulation index) or leukemic blast colony formation in 24 of 40 AML patients. In the presence of other growth factors, IL-4 showed divergent effects on IL-3-, granulocyte-macrophage colony-stimulating factor-, granulocyte colony-stimulating factor-, or erythropoietin-dependent colony formation. These effects of IL-4 were observed in both CD34+ and CD34- AML cases. IL-6 (100 U/mL) alone could not stimulate DNA synthesis and blast colony formation except for one CD34+ case. On the other hand, IL-6 showed synergistic effects on IL-3- and IL-4-dependent blast colony formation in 10 of 12 and 7 of 9 CD34+ AML cases, respectively. Among CD34- AML cases, such synergism was seen only in 1 of 12 cases for IL-3-dependent colony formation and in 3 of 7 cases for IL-4-dependent colony formation. The divergent effect of IL-4 and the synergistic effect of IL-6 were also observed in purified CD34+ leukemic blast populations, indicating that these phenomena are not mediated by accessory cells. The present study suggests that IL-4, alone or in combination with other growth factors, has divergent effects on the growth of AML progenitors irrespective of the CD34 expression, and that IL-6 acts synergistically with IL-3 or IL-4 on the growth of leukemic progenitors preferentially in CD34+ AML.

Adolescent↗
PubMed1994

A comparative study of the phenotype and proliferative capacity of peripheral blood (PB) CD34+ cells mobilized by four different protocols and those of steady-phase PB and bone marrow CD34+ cells.

Peripheral blood (PB) CD34+ cells from four commonly used mobilization protocols were studied to compare their phenotype and proliferative capacity with steady-state PB or bone marrow (BM) CD34+ cells. Mobilized PB CD34+ cells were collected during hematopoietic recovery after myelosuppressive chemotherapy with or without granulocyte-macrophage colony-stimulating factor (GM-CSF) or granulocyte colony-stimulating factor (G-CSF) or during G-CSF administration alone. The expression of activation and lineage-associated markers and c-kit gene product were studied by flow cytometry. Proliferative capacity was measured by generation of nascent myeloid progenitor cells (granulocyte-macrophage colony-stimulating factor; CFU-GM) and nucleated cells in a stroma-free liquid culture stimulated by a combination of six hematopoietic growth factors (interleukin-1 (IL-1), IL-3, IL-6, GM-CSF, G-CSF, and stem cell factor). G-CSF-mobilized CD34+ cells have the highest percentage of CD38- cells (P < .0081), but otherwise, CD34+ cells from different mobilization protocols were similar to one another in their phenotype and proliferative capacity. The spectrum of primitive and mature myeloid progenitors in mobilized PB CD34+ cells was similar to their steady-state counterparts, but the percentages of CD34+ cells expressing CD10 or CD19 were lower (P < .0028). Although steady-state PB and chemotherapy-mobilized CD34+ cells generated fewer CFU-GM at day 21 than G-CSF-mobilized and steady-state BM CD34+ cells (P < .0449), the generation of nucleated cells and CFU-GM were otherwise comparable. The presence of increased or comparable numbers of hematopoietic progenitors within PB collections with equivalent proliferative capacity to BM CD34+ cells is not unexpected given the rapid and complete hematopoietic reconstitution observed with mobilized PB. However, all four types of mobilized PB CD34+ cells are different from steady-state BM CD34+ cells in that they express less c-kit (P < .0002) and CD71 (P < .04) and retain less rhodamine 123 (P < .0001). These observations are novel and suggest that different mobilization protocols may act via similar pathways involving the down-regulation of c-kit and may be independent of cell-cycle status.

ADP-ribosyl Cyclase↗
PubMed1995

Expression of adhesion molecules on CD34+ cells: CD34+ L-selectin+ cells predict a rapid platelet recovery after peripheral blood stem cell transplantation.

Adhesion molecules play a role in the migration of hematopoietic progenitor cells and regulation of hematopoiesis. To study whether the mobilization process is associated with changes in expression of adhesion molecules, the expression of CD31, CD44, L-selectin, sialyl Lewisx, beta 1 integrins very late antigen 4 (VLA-4) and VLA-5, and beta 2 integrins lymphocyte function-associated 1 and Mac-1 was measured on either bone marrow (BM) CD34+ cells or on peripheral blood CD34+ cells mobilized with a combination of granulocyte colony-stimulating factor (G-CSF) and chemotherapy. beta 1 integrin VLA-4 was expressed at a significantly lower concentration on peripheral blood progenitor cells than on BM CD34+ cells, procured either during steady-state hematopoiesis or at the time of leukocytapheresis. No differences in the level of expression were found for the other adhesion molecules. To obtain insight in which adhesion molecules may participate in the homing of peripheral blood stem cells (PBSCs), the number of CD34+ cells expressing these adhesion molecules present in leukocytapheresis material was quantified and correlated with hematopoietic recovery after intensive chemotherapy in 27 patients. The number of CD34+ cells in the subset defined by L-selectin expression correlated significantly better with time to platelet recovery after PBSC transplantation (r = -.86) than did the total number of CD34+ cells (r = -.55). Statistical analysis of the relationship between the number of CD34+L-selectin+ cells and platelet recovery resulted in a threshold value for rapid platelet recovery of 2.1 x 10(6) CD34+ L-selectin+ cells/kg. A rapid platelet recovery (< or = 14 days) was observed in 13 of 15 patients who received > or = 2.1 x 10(6) CD34+ L-selectin+ cells/kg (median, 11 days; range, 7 to 16 days), whereas 10 of 12 patients who received less double positive cells had a relative slow platelet recovery (median, 20 days; range, 13 to 37 days). The L-selectin+ subpopulation of CD34+ cells also correlated better with time to neutrophil recovery (r = -.70) than did the total number of reinfused CD34+ cells (r = -.51). However, this latter difference failed to reach statistical significance. This study suggests that L-selectin is involved in the homing of CD34+ cells after PBSC transplantation.

Adult↗
PubMed1999

Cytokine-induced expansion of human CD34+ stem/progenitor and CD34+CD41+ early megakaryocytic marrow cells cultured on normal osteoblasts.

Thrombocytopenia remains a significant cause of morbidity in cancer patients undergoing allogeneic bone marrow transplantation (BMT), which consumes millions each year for frequent platelet transfusions. Using a novel culture system containing appropriate cytokine(s) on a layer of normal human osteoblasts, we investigated the expansion of early megakaryocytic progenitor cells while maintaining the number of CD34+ stem/progenitor marrow cells in an attempt to provide an effective solution for the problem of post-transplant thrombocytopenia. After seven days of culture, normal human osteoblasts alone without cytokines significantly increased the number of CD34+ and CD34+CD41+ marrow cells. Among the various cytokine combinations tested, both stem cell factor (SCF), interleukin 3 (IL-3)+IL-11 and SCF+IL-3+IL-11+thrombopoietin (TPO) emerged as the most effective in expanding early CD34+CD41+ megakaryocytic cells. Early CD34+CD41+ megakaryocytic cells have increased by 3.1- and 4.7-fold compared with day 7 control cultures, and by 62- and 94-fold, respectively, compared with day 0 input, respectively. Also, late CD41+ megakaryocytic cells have increased by 15.4- and 27.5-fold compared with day 7 control cultures in the presence of the same two combinations. In addition, the same cytokine combinations achieved 17.6- and 13.3-fold increases in the number of CD34+ marrow cells after the same seven days of culture on a layer of human osteoblasts. The combination (SCF+IL-3+IL-11+TPO) achieved the highest expansion of CD34+CD41+ early megakaryocytic cells from human marrow CD34+ cells reported so far in the literature. Recently, transplantation of SCF+IL-1+IL-3+TPO ex vivo expanded megakaryocytic progenitor cells as a supplement has been shown to accelerate platelet recovery by three to five days in mice. Therefore, the clinical use of the combination (SCF+IL-3+IL-11+TPO) for ex vivo expansion of CD34+ and megakaryocytic progenitor cells from a portion of the donor's marrow harvest is warranted in allogeneic BMT. Such a protocol would accelerate platelet recovery and shorten the period of hospitalization after allogeneic BMT. The present study has confirmed the role of human osteoblasts in supporting the proliferation and maintenance of human CD34+ stem/progenitor marrow cells. Given the facilitating role of osteoblasts shown previously in several allogeneic BMT studies in mice, it is possible to envisage a future role for donor osteoblasts in clinical BMT. Transplantation of the cultured donor osteoblasts together with the ex vivo expanded CD34+ marrow cells as a supplement might not only accelerate platelet recovery but also prevent acute graft-versus-host disease in allogeneic BMT. The present novel culture system should have useful clinical application in allogeneic BMT.

Antigens, CD34↗
PubMed1994

CD34 selection for purging in multiple myeloma and analysis of CD34+ B cell precursors.

Selection of CD34+ hematopoietic progenitor cells from autografts may be performed in multiple myeloma (MM) to minimize contamination with tumor cells. This approach is based on the assumption that the malignant cells do not express the CD34 antigen. Therefore, we first compared the CD34+/CD10+ and CD34+/CD19+ subpopulations from bone marrow (BM) and peripheral blood (PB) of fourteen MM patients and five normal controls. No difference between the respective early B cell subsets of both groups could be observed. Using tricolor flow cytometry, the CD19 expression on CD34+/CD10+ cells in BM was found to increase continuously from CD19- to CD19dim. In contrast, circulating CD34+/CD10+ cells did not coexpress the CD19 antigen. This population may contain myeloid progenitor cells or bipotential progenitor cells of the myeloid and lymphoid lineage as suggested by data obtained with fetal liver cells. Further functional studies are required. Enrichment of CD34+ cells with immunomagnetic beads was performed from BM of three MM patients and four normal donors. The CD34+ cells were selected with the HPCA-1 antibody and detached from the beads with chymopapain. Compared with the starting cell preparation, a 3.97 +/- 0.48 log (mean +/- SE) reduction of plasma cells could be achieved after CD34 selection. On morphological examination, 84% +/- 4% of the cells in the CD34+ fraction (MM) were immature blasts. The plating efficiency for hematopoietic colony forming cells was 9.7% +/- 2.8% in the CD34 selected fraction of the MM group, reflecting a 51-fold increase as compared with the starting population.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, CD↗
PubMed2001

Hematopoietic capability of CD34+ cord blood cells: a comparison with CD34+ adult bone marrow cells.

The characteristics of hematopoietic progenitor and stem cell (HPC/HSC) populations in mammals vary according to their ontogenic stage. In humans, HPC/HSCs from umbilical cord blood (CB) are increasingly used as an alternative to HPC/HSCs from adult bone marrow (BM) for the treatment of various hematologic disorders. How the hematopoietic activity of progenitor and stem cells in CB differs from that in adult BM remains unclear, however. We compared CD34+ cells, a hematopoietic cell population, in CB with those in adult BM using phenotypic subpopulations analyzed by flow cytometry, the colony-forming activity in methylcellulose clonal cultures, and the repopulating ability of these cells in NOD/Shi-scid (NOD/SCID) mice. Although the proportion of CD34+ cells was higher in adult BM than in CB mononuclear cells, the more immature subpopulations, CD34+ CD33- and CD34+ CD38- cells, were present in higher proportions in CD34+ CB cells. Clonal culture assay showed that more multipotential progenitors were present in CD34+ CB cells. When transplanted into NOD/SCID mice. CD34+ adult BM cells could not reconstitute human hematopoiesis in recipient BM, but CD34+ CB cells achieved a high level of engraftment, indicating that CD34+ CB cells possess a greater repopulating ability. These results demonstrated that human hematopoiesis changes with development from fetus to adult. Furthermore, CD34+ CB cells contained a greater number of primitive hematopoietic cells, including HSCs, than did adult BM, suggesting the usefulness of CD34+ CB cells not only as a graft for therapeutic HSC transplantation but also as a target cell population for ex vivo expansion of transplantable HSCs and for gene transfer in gene therapy.

Adult↗