Ex vivo generation of CD34(+) cells from CD34(-) hematopoietic cells

The human Lin(-)CD34(-) cell population contains a newly defined class of hematopoietic stem cells that reconstitute hematopoiesis in xenogeneic transplantation systems. We therefore developed a culture condition in which these cells were maintained and then acquired CD34 expression and the ability to produce colony-forming cells (CFC) and SCID-repopulating cells (SRCs). A murine bone marrow stromal cell line, HESS-5, supports the survival and proliferation of Lin(-)CD34(-) cells in the presence of fetal calf serum and human cytokines thrombopoietin, Flk-2/Flt-3 ligand, stem cell factor, granulocyte colony-stimulating factor, interleukin-3, and interleukin-6. Although Lin(-)CD34(-) cells do not initially form any hematopoietic colonies in methylcellulose, they do acquire the colony-forming ability during 7 days of culture, which coincides with their conversion to a CD34(+) phenotype. From 2.2% to 12.1% of the cells became positive for CD34 after culture. The long-term multilineage repopulating ability of these cultured cells was also confirmed by transplantation into irradiated NOD/SCID mice. These results represent the first in vitro demonstration of the precursor of CD34(+) cells in the human CD34(-) cell population. Furthermore, the in vitro system we reported here is expected to open the way to the precise characterization and ex vivo manipulation of Lin(-)CD34(-) hematopoietic stem cells.     

Proliferation and differentiation of myelodysplastic CD34+ cells: phenotypic subpopulations of marrow CD34+ cells

In a search for a mechanism to explain the impaired growth of progenitor cells in patients with myelodysplastic syndromes (MDS), marrow CD34+ cells were purified up to 94.9% +/- 4.2% for normal individuals and 88.1% +/- 17.6% for MDS patients, using monoclonal antibodies and immunomagnetic microspheres (MDS CD34+ cells). Phenotypic subpopulations of these CD34+ cells were analyzed for CD38, HLA-DR, CD33, CD13, CD14, CD41 and CD3 plus CD19, in association with proliferative and differentiative capacities. The 15 studies performed included 12 MDS patients. Coexpression rate of CD13 significantly increased in the MDS CD34+ cell population with a value of 91.4% +/- 11.6% and ranging from 60.3% to 100%, and exceeded 99% in four studies, whereas that of normal CD34+ cells was 49.9% +/- 15.8%, ranging from 28.2% to 70.1% (P < .001). Coexpression rate of CD38, HLA-DR, CD33, CD14, and CD3 plus CD19 in MDS CD34+ cells did not significantly differ from that of normal CD34+ cells. The total number of colonies and clusters grown from 100 normal marrow CD34+ cells was 40.4 +/- 8.6, the range being from 27.2 to 50.3; this varied in MDS marrow CD34+ cells with a value of 34.0 +/- 28.7, the range being 0 to 95.9. The lineage of colonies and clusters promoted by MDS marrow CD34+ cells was predominantly committed to nonerythroid with impaired differentiation in 13 of 15 studies (87%). CD13 is first expressed during hematopoiesis by colony-forming unit granulocyte-macrophage and is absent in erythroid progenitors. Therefore, this study provides direct evidence for the lineage commitment of MDS CD34+ cells to nonerythroid with impaired differentiation and explains the mechanism of nil or low colony expression of MDS progenitor cells to erythroid lineage.     

Kinetics of engraftment of CD34(-) and CD34(+) cells from mobilized blood differs from that of CD34(-) and CD34(+) cells from bone marrow

OBJECTIVE: Mobilized peripheral blood (PB) progenitors are increasingly used in autologous and allogeneic transplantation. However, the short- and long-term engraftment potential of mobilized PB or bone marrow (BM) has not been directly compared. Although several studies showed that BM-derived Lin(-)CD34(-) cells contain hemopoietic progenitors, no studies have addressed whether Lin(-)CD34(-) cells from mobilized PB contain hemopoietic progenitors. Here, we compared the short- and long-term engraftment potential of CD34(+) cells and Lin(-)CD34(-) cells in BM and PB of normal donors who received 5 days of granulocyte colony-stimulating factor (G-CSF). MATERIALS AND METHODS: 35 x 10(3) CD34(+) or Lin(-)CD34(-) cells from G-CSF mobilized BM and PB of normal donors were transplanted in 60-day-old fetal sheep. Animals were evaluated 2 and 6 months after transplantation for human hemopoietic cells. In addition, cells recovered after 2 months from fetal sheep were serially passaged to secondary and tertiary recipients to assess long-term engrafting cells. RESULTS: Mobilized PB CD34(+) cells supported earlier development of human hemopoiesis than BM CD34(+) cells. When serially transferred to secondary and tertiary recipients, earlier exhaustion of human hematopoiesis was seen for PB than BM CD34(+) cells. A similar degree of chimerism was seen for Lin(-)CD34(-) cells from PB or BM in primary recipients. We again observed earlier exhaustion of human hemopoiesis with serial transplantation of PB than BM Lin(-)CD34(-) cells. CONCLUSIONS: Differences exist in the short- and long-term repopulating ability of cells in PB and BM from G-CSF mobilized normal donors, and this is independent of the phenotype. Studies are ongoing to examine if this reflects intrinsic differences in the repopulating potential between progenitors from PB and BM, or a lower frequency of long-term repopulating cells in PB than BM CD34(+) and Lin(-)CD34(-) cells, that may not be apparent if larger numbers of cells are transplanted.     

Transplantation of allogeneic CD34+ blood cells

Pluripotent stem cells of hematopoiesis and lymphopoiesis are among the CD34+ cells in blood or bone marrow. After granulocyte-colony stimulating factor (G-CSF) treatment, 1% to 2% of the mononuclear cells in blood are CD34+ cells, which can be procured by leukapheresis. We investigated the potential of CD34+ blood cells for reconstituting hematopoiesis and lymphopoiesis after allogeneic transplantation. HLA- identical sibling donors of 10 patients with hematologic malignancies were treated with G-CSF (filgrastim), 5 microgram/kg subcutaneously twice daily for 5 to 7 days. CD34+ cells were selected from the apheresis concentrates by immunoadsorption, concomitantly the number of T cells was reduced 100- to 1,000-fold. After transplantation, five patients received cyclosporine A for graft-versus-host disease (GvHD) prophylaxis (group I); five patients additionally received methotrexate (group II). G-CSF and erythropoietin were given to all patients. Mean numbers of 7.45 x 10(6) CD34+ and 1.2 x 10(6) CD3+ cells per kilogram were transplanted. In group I, the median times of neutrophil recovery to 100, 500, and 1,000 per mm3 were 10, 10, and 11 days, respectively. Group II patients reached these neutrophil levels after 10, 14, and 15 days, respectively. Platelet transfusions were administered for a median of 18 days in group I and 30 days in group II, and red blood cells for 9 and 12 days, respectively. Between day 30 and 60, lymphocytes reached levels of 353 +/- 269 cells per mm3. The median grades of acute GvHD were III in group I and I in group II. Two patients in group I died from acute GvHD. Two leukemic relapses occurred in group II. Complete and stable donor hematopoiesis was shown in all patients with a median follow up of 370 (45 to 481) days. Allogeneic blood CD34+ cells can successfully reconstitute hematopoiesis and lymphopoiesis. Reduction of T cells by CD34+ blood cell enrichment and cyclosporine A alone might not be sufficient for prophylaxis of severe acute GvHD.     

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.     

Ex vivo expansion of CD34+/CD41+ late progenitors from enriched peripheral blood CD34+ cells

 In our experience, patients with neuroblastoma who undergo transplantation with CD34+ cells following high-dose chemotherapy have prolonged delays in platelet recovery. In vitro expansion of megakaryocyte (MK) cells may provide a complementary transplant product able to enhance platelet production in the recipient. We investigated the ability of a combination of various hematopoietic growth factors to generate ex vivo MK progenitors. Immunoselected CD34+ cells from peripheral blood stems cells (PBSCs) were cultured in media with or without serum, supplemented by IL-3, IL-6, IL-11, SCF, TPO, Flt-3 ligand, and MIP-1α. In terms of MK phenotypes, we observed a maximal expansion of CD61+, CD41+, and CD42a of 69-, 60-, and 69-fold, respectively, i.e., 8–10 times greater than the expansion of total cell numbers. Whereas the absolute increment of CD34+ cells was slightly elevated (fourfold) we showed increases of 163-, 212-, and 128-fold for CD34+/CD61+, CD34+/CD41+, and CD34+/CD42a+ cells, respectively. We obtained only a modest expansion of CFU-MKs after only 4 days of culture (fourfold) and similar levels of CFU-MKs were observed after 7 days (fivefold). Morphology and immunohistochemistry CD41+ analyses confirmed expansion of a majority of CD41+ immature cells on days 4 and 7, while on day 10 mature cells began to appear. These results show that primarily MK progenitors are expanded after 4 days of culture, whereas MK precursor expansion occurs after 7 days. When we compared the two culture media (with and without serum) we observed that increases of all specific phenotypes of the MK lineage were more elevated in serum-free culture than in medium with serum. This difference was especially marked for CD34+/CD61+ and CD34+/CD41+ (163 vs 42 and 212 vs 36, respectively). We contaminated CD34+ cells with a neuroblastoma cell line and we observed no expansion of malignant cells in our culture conditions (RT-PCR for tyrosine hydroxylase positive at day 4 and negative at day 7). With our combination of hematopoietic growth factors we are able to sufficiently expand ex vivo MK late progenitor cells to be used as complementary transplant products in neuroblastoma patients who undergo transplantation with CD34+ cells. It is possible that these committed MK late progenitors could accelerate short-term platelet recovery in the recipient until more primitive progenitor cells have had time to engraft. Received: February 1, 1999 / Accepted: June 1, 1999     

Increase of CD34 positive cells in polycythaemia vera

Abstract: The purpose of the present work was to evaluate the proliferative character of polycythaemia vera (PV). Therefore, in 15 patients with different stages of PV we assessed the level of CD34 positive (CD34+) cells in peripheral blood and bone marrow, erythroid colony growth of bone marrow cells and plasma erythropoietin (EPO). The mean concentration of CD34+ cells in blood was significantly increased in PV patients (9.0±11.2×10³/mL) compared to healthy controls (2.0±1.7×10³/mL). In aspirated bone marrow no such difference between PV and control subjects was present. Six patients with splenomegaly and/or requirement for chemotherapy had significantly higher mean blood levels of CD34+ cells compared to the remaining PV patients. All PV patients presented EPO independent erythroid colonies. Three PV patients with anaemia and long disease duration had high EPO levels.     

Production of soluble CD34 by human myeloid cells

CD34, a glycophosphoprotein present in lymphohaematopoietic stem and progenitor cells, as well as in other cell types, exists in both transmembrane and intracytoplasmic forms. Transmembrane CD34 expression, which is high in the earliest haematopoietic precursors, decreases as cells mature. However, to our knowledge, there is no information on whether a decrease in transmembrane CD34 can also predict a release of the molecule from the cell membrane into the extracellular fluid. To investigate the above possibility, we studied conditions (incubation time, cell density and proliferative status) in human myeloid cells (lines KG-1a, KG-1 and cord blood-derived cells) that may cause a decrease in surface CD34 and the generation of a soluble form of the molecule. The latter, as demonstrated by Western blot analysis, adds more complexity to the proposed structural features and functional properties of CD34 in myeloid cells.     

Sequential expression of CD34 and CD33 antigens on myeloid colony-forming cells

The expression of CD33 and CD34 antigens on human bone marrow cells was examined by fluorescence-activated cell sorting and colony assays. A marked difference of antigen expression was observed on sorted progenitors of the granulocyte/macrophage lineage (CFU-GM) with respect to enrichment and maturation status. Single-color cell sorting revealed no difference of enrichment by anti-CD33 antibody between day-7 and d-14 progenitors, while anti-CD34 antibody preferentially enriched d-14 colony-forming units (CFU). By two-color cell sorting it could be shown that enrichment of d-14 CFU-GM occurred mostly in the CD34+CD33- and to a lesser extent in the double-positive fraction. In contrast, there was no difference in degree of enrichment between d-7 and d-14 CD34-CD33+ CFU-GM. From these data we conclude that, during early myelopoiesis, CD34 antigens are gradually lost while CD33 antigens are acquired.     

骨髓CD34^＋干细胞向内皮细胞转化的诱导方法

目的探讨骨髓CD34^＋细胞向血管内皮细胞转分化的诱导方法。方法采集犬骨髓,经免疫磁珠分离出内皮祖细胞,内皮细胞生长因子（VEGF）诱导分化为内皮细胞并扩增,倒置相差显微镜、免疫细胞化学和摄取DilAc—LDL试验鉴定。将所得细胞种植于人工血管,扫描电镜观察细胞形态,并与MNCs作对比。结果经流式细胞仪测定,分离后的细胞中CD34^＋细胞占78．46％±6．37％;CD34^＋细胞培养2周后细胞基本铺满培养瓶底面,细胞呈“鹅卵石”状排列,CD34^＋和Ⅷ因子免疫细胞化学染色均为阳性。扫描电镜下观察可见内皮细胞平铺于人工血管表面,有伪足伸出并长入血管内表面微孔内。结论通过免疫磁珠方法可分离得到高纯度的骨髓CD34^＋细胞,经体外培养VEGF诱导后可定向分化为内皮细胞。     

In-vitro clonogenicity of mobilized peripheral blood CD34-expressing cells: inverse correlation to both relative and absolute numbers of CD34-expressing cells

The determination of CD34-expressing cells by multiparameter flow cytometry is now widely used to estimate the reconstitution potential of cells harvested by cytapheresis for peripheral blood stem cell and progenitor cell transplantation. There is a correlation between the number of CD34-expressing cells collected and committed progenitor cells (CFU-GM and BFU-E) capable of forming colonies in vitro , but there is considerable variation in the proportion of CD34-expressing cells capable of clonogenic growth. The data in this study of 782 cytapheresis samples indicates that there is a negative correlation between the clonogenicity of the CD34-expressing cells and the absolute number or the proportion of CD34-expressing cells within the harvest. In 116 samples the proportion of CD34-expressing cells co-expressing the CD45-RA-antigen (a  subset of CD34-expressing cells which includes virtually all clonogenic cells in terms of CFU-GM) was determined, but this did not help to identify the clonogenicity of a given sample. These findings may have clinical relevance, particularly when mobilization is judged to be relatively poor or when a good harvest is to be divided for multiple high-dose procedures.     

Reciprocal expression of CD38 and CD34 by adult murine hematopoietic stem cells

The effects of activation of adult murine stem cells on their expression of CD38 were studied using a murine transplantation model. First, the published finding that the majority of long-term engrafting cells from normal adult steady-state marrow are CD38(+) was confirmed. Next, it was determined that the majority of stem cells activated in vivo by injection of 5-fluorouracil (5-FU) or mobilized by granulocyte colony-stimulating factor are CD38(-). Stem cells that were activated in culture with interleukin-11 and steel factor were also CD38(-). Previous studies have shown that expression of CD34 by adult stem cells is also modulated by in vivo or in vitro activation. To determine whether there is reciprocal expression of CD38 and CD34, 4 populations of post-5-FU marrow cells were analyzed. The majority of the stem cells were in the CD38(-)CD34(+) fraction. However, secondary transplantation experiments indicated that when the bone marrow reaches steady state, the majority of the stem cells become CD38(+)CD34(-). In addition, the minority populations of CD34(+) stem cells that occur in steady-state bone marrow are CD38(-). This reversible and reciprocal expression of CD38 and CD34 by murine stem cells may have implications for the phenotypes of human stem cells.     

Characterization of CD34+, CD13+, CD33- cells, a rare subset of immature human hematopoietic cells

BACKGROUND AND OBJECTIVES: Hematopoietic progenitor cells that express CD34 are heterogeneous in their lineage affiliation and degree of maturation. Expression of CD13 and CD33 antigens indicates myeloid lineage association, but the precise sequence of expression of these two markers during differentiation is unclear. We noted the presence of CD34+ cells expressing CD13 but lacking CD33, a subset of cells not yet well characterized. In this report we describe the prevalence and the immunophenotype of this cell subset. DESIGN AND METHODS: We studied the immunophenotype of immature myeloid cells in human bone marrow samples from 11 healthy transplantation donors and in 4 cord blood samples. We used four-color flow cytometry and a large panel of monoclonal antibodies directed against lineage and differentiation-associated antigens. Three additional bone marrow samples were analyzed after immunomagnetic sorting of CD34+ cells. We focused our analysis on the subset of cells defined by the expression of CD34 and CD13 and the lack of CD33. RESULTS: We found CD34+, CD13+, CD33- cells in all 11 bone marrow and 4 cord blood samples studied. These cells represented 0.5 0.5% (mean SD) and 0.8 1.2% of mononucleated cells, respectively. CD34+, CD13+, CD33- cells appeared to be more immature than those expressing CD33 because of their light scatter characteristics (smaller size and lower granularity), the expression of markers associated with early hematopoietic cells (CD90, CD133 and CD117), and the absence of lineage-associated markers. INTERPRETATION AND CONCLUSIONS: These findings suggest that the expression of CD13 precedes that of CD33 during myeloid differentiation, and that CD34+, CD13+, CD33- cells are at an early stage of human myeloid cell differentiation.     

Relationships between total CD34+ cells reinfused, CD34+ subsets and engraftment kinetics in breast cancer patients

BACKGROUND AND OBJECTIVE: The aim of the present study was to evaluate the correlation between the number of CD34+ cells transfused and the duration of hypoplasia, and the relationship between various CD34+ subsets (CD34+/33-; CD34+/38-; CD34+/ HLA-DR-; CD34+/Thy-1+) and engraftment kinetics in a series of patients with breast cancer treated with high doses of thiotepa and melphalan. DESIGN AND METHODS: We treated 42 consecutive patients: 19 in an adjuvant context (>= 4 positive axillary nodes) and 23 for metastatic disease. A combination of thiotepa 600 mg/m(2) and melphalan 140-160 mg/m(2) was administered as the conditioning regimen. All patients received peripheral blood progenitor cells (PBPC) and growth factors for hematopoietic rescue. RESULTS: In univariate analysis, we found a significant relationship between the number of CD34+ cells reinfused and the time to hematologic recovery and the duration of hospital stay. We observed an inverse correlation between the number of CD34+ cells reinfused and the units of platelets transfused. Cox multivariate analysis confirmed that the number of CD34+ cells reinfused is the most effective predictor of time to hematologic recovery. CFU-GM resulted to be a better predictor of the duration of hospitalization. INTERPRETATION AND CONCLUSIONS: We found a significant relationship between the number of PBPC reinfused and the time to hematologic recovery after high doses of thiotepa and melphalan. In our experience, the numbers of subsets of CD34+ cells infused did not give compared additional information to that provided by the total number of CD34+ cells infused.