Leprdb diabetic mouse bone marrow cells inhibit skin wound vascularization but promote wound healing

Bone marrow stem cells participate in tissue repair processes and may have roles in skin wound repair. Diabetes is characterized by delayed and poor wound healing, and type 1 diabetes seems to lead to stem cell dysfunction. Hence, stem cell dysfunction could contribute to poor healing, and stem cell-based therapies may be efficacious in diabetic wounds. We investigated the potential of exogenous stem cells to promote skin healing and possible effects of type 2 diabetes on stem cell function. Mouse bone marrow cells from nondiabetic and diabetic mice were enriched for putative stem cells and injected under skin wounds of nondiabetic or type 2 diabetic Leprdb mice. Using histology and morphometry, vascularization and healing in treated and untreated mice were analyzed. We anticipated a correlation between improved wound healing and vascularization, because therapies that increase tissue vascularization tend to enhance wound healing. Our data indicate that exogenous nondiabetic bone marrow-derived cells increase vascularization and improve wound healing in Leprdb mice but have little effect on nondiabetic controls. In contrast, Leprdb-derived marrow cells inhibit vascularization but promote wound healing in Leprdb mice. Thus, adult stem cell function may be impaired by type 2 diabetes; the ability to promote vascularization and wound healing are distinct functions of bone marrow cells; and neovascularization and wound healing may not be tightly coupled. Additionally, we observed little incorporation of injected cells into wound structures, suggesting that improved healing is mediated through mechanisms other than direct differentiation and incorporation of the cells.     

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.     

Effect of human umbilical cord blood CD34+ progenitor cells transplantation in diabetic mice

Shortage of donor organs spurs research into alternative means of generating β cells. Stem cells might represent a potential source of tissues for cell therapy protocols, and diabetes is a candidate disease that may benefit from cell replacement protocols. We examined the effect of transplanted human umbilical cord blood CD34+ cells on some detailed parameters in streptozotocin- (STZ) induced diabetic mice. An experimental study was conducted in the departments of clinical pathology, physiology and pathology of Faculty of Medicine, Suez Canal University. Thirty male albino mice 8–12 weeks were included and subdivided into 3 groups, first group served as normal control group, second group as diabetic control after induction of diabetes with STZ and third group treated diabetic mice by injection of positively selected CD34 progenitor cells from human umbilical cord blood (HUCB) with a dose of one million cells/mouse. Blood glucose and serum insulin were measured at specific time interval and immunohistochemical (IHC) analysis and histopathology on pancreas were conduced. Data were analyzed using chi square between groups. Intravenous injection of CD34+ cells caused significant improvement in blood glucose level (277.9?±?102.5 mg/dl in treated group vs 530.3?±?99 mg/dl in untreated group, p?<?0.01). Blood level of mouse insulin was higher in the treated group as compared with untreated diabetic mice (0.77?±?0.2 ng/ml in treated group versus 0.26?±?0.09 in untreated group, p?<?0.001). IHC analysis for detection of human insulin producing cells in pancreas of treated mice revealed that 33.3% positive cellular staining and 55.6% positive sinusoidal staining were detected. In conclusion, Transplantation of HUCB-CD34+ cells appear to be a modality of stem cell therapy in diabetes mellitus.     

Elastic plasma protein film blended with platelet releasate accelerates healing of diabetic mouse skin wounds

BACKGROUND AND OBJECTIVES: The growth factors derived from platelets and plasma proteins mediate the wound-healing process that is characterized by the sequential migration and differentiation of several cell populations that give rise to angiogenesis, collagen synthesis, wound contraction, and re-epithelialization. To evaluate the efficacy of the blood-derived factors in wound healing, we examined a novel wound dressing consisting of concentrated human plasma proteins and platelet releasate (CPPP). MATERIALS AND METHODS: To generate CPPP, plasma proteins and platelets in the peripheral blood (n = 5) were concentrated with the cold ethanol precipitation method. The thrombin obtained from the same blood unit and calcium chloride (CaCl(2)) were mixed to a concentrate. The CPPP has enough strength to dress cutaneous wounds and contains large amounts of cytokines and fibronectin. We applied the CPPP to excisional skin wounds in genetically healing-impaired model mice (n= 5) and the wounds were evaluated 10 days after the operation. RESULTS: The area of CPPP-treated wounds decreased significantly compared with that of the control wounds (65% vs. 94% of the original size, respectively, P= 0.032). The immunostained section revealed a striking effect of CPPP on vascularization compared with the control wounds (13.2 vs. 2.7 vessels per mm(2) as mean vascular density observed in the sections, respectively, P= 0.013). CONCLUSIONS: Our results suggest that CPPP is a promising biologically active dressing for full-thickness skin wounds. CPPP can be an entirely autologous biological dressing, suggesting that it is free from the risk of transmission of pathogens through blood products.     

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     

Circulating CD34+ cells in cord blood and mobilized blood have a different profile of adhesion molecules than bone marrow CD34+ cells

Abstract: The expression of adhesion molecules was studied on CD34+ hematopoietic precursors in cord blood, bone marrow and mobilized blood. The samples were labeled in a double immunofluorescence procedure with a CD34 monoclonal antibody and with antibodies against maturation and differentiation antigens and adhesion molecules. Myeloid precursors formed the majority of the CD34+ cells in all samples. In bone marrow a separate cluster of B-cell precursors with low forward scatter was present. Nearly all CD34+ cells in normal bone marrow expressed VLA-4 and VLA-5, PECAM-1, LFA-3 and HCAM. The majority of the CD34+ cells also had LFA –1 and L-selectin on the surface membrane. A small subset was VLA-2, VLA-3, ICAM-1 or Mac-1 positive. CD34+ cells expressing the vitronectin receptor or the CD11c antigen were rare. Cord blood and mobilized blood CD34+ cells had a lower expression of VLA-2, VLA-3 and VLA-5 and a higher expression of LFA-1, ICAM-1 and L-selectin than bone marrow CD34+ cells. Except for LFA-1, this was not due to the presence of more myeloid precursors in these samples. Low β₁ integrin expression may lead to less adhesion to the extracellular matrix. High expression of L-selectin may facilitate interaction with endothelial cells. Therefore, this phenotype may favour mobilization.     

Ex vivo expanded mobilized peripheral blood CD34+ cells accelerate haematological recovery in a baboon model of autologous transplantation

To address the value of ex vivo expanded haematopoietic cells for shortening cytopenia in autologous haematopoietic transplantation, we designed an ex vivo expansion protocol based on a cocktail of early acting cytokines and short-term culture and tested it in a baboon model. Expansion involved enriched CD34+ peripheral blood haematopoietic cells cultured for 6 d with a combination of FLT3-L, stem cell factor (SCF), thrombopoietin (TPO) and interleukin (IL)-3 (50 ng/ml each); CD34+ cells, granulocyte-macrophage colony-forming units (GM-CFU) and megakaryocytic colony-forming units (MK-CFU) were amplified, respectively, 10.5-, 20.5- and 17.9-fold. Baboons were submitted to a myeloablative regimen consisting of cyclophosphamide plus total body irradiation (TBI; 6 Gy) and were then grafted with either 2 x 106/kg unmanipulated CD34+ cells (control group, n = 4) or cells cultured from 2 x 106/kg CD34+ cells (expansion group, n = 4). No cytokines were administered after transplantation. All the animals engrafted. The mean times to white blood cell (WBC), granulocyte and platelet recovery were significantly shorter in the expansion group than in the control group: WBC (> 1 x 109/l) and neutrophil (> 0.5 x 109/l) recovery occurred on days 8 (range 6-9) and 9 (range 6-11), respectively, compared with days 12 (range 10-15) and 14 (range 11-16); platelets recovered (> 20 x 109/l) on day 9 (range 7-12) compared with day 13 (range 11-15) in the control group (P < 0.05). No toxicity was observed after reinfusion. No secondary hypoplasia was observed during more than 12 months of follow-up. Functions of both neutrophils and platelets produced from expanded cells were normal in terms of oxidative metabolism, chemotaxis and the bleeding time. This study shows that in comparison with unmanipulated cells peripheral blood haematopoietic cells expanded from similar doses of CD34+ cells, under the conditions defined here, accelerated both neutrophil and platelet recovery without impairing long-term haematopoiesis.     

Combined transplantation of allogeneic bone marrow and CD34+ blood cells

Allogeneic peripheral blood progenitor cells (PBPCs) were transplanted after immunoselection of CD34+ cells. Two patient groups were studied: group I patients received immunoselected blood CD34+ cells and unmanipulated marrow cells from the same donor. Group II patients were given immunoselected blood and bone marrow (BM) CD34+ cells. One to 6 weeks before bone marrow transplantation (BMT), PBPCs from HLA- identical and MLC- sibling donors were mobilized with granulocyte colony-stimulating factor (G-CSF) (5 micrograms/kg twice daily subcutaneously) for 5 days. Aphereses were performed at days 4 and 5 of G-CSF application. CD34+ cells were separated from the pooled PBPC concentrates by immunoadsorption onto avidin with the biotinylated anti- CD34 monoclonal antibody 12.8 and then stored in liquid nitrogen. BM was procured on the day of transplantation. Patients were conditioned with either busulfan (16 mg/kg) or total body irradiation (12 Gy) followed by cyclophosphamide (120 mg/kg). Cyclosporin A and short methotrexate were used for graft-versus-host disease (GVHD) prophylaxis. After transplantation, all patients received 5 micrograms G-CSF/kg/d from day 1 until greater than 500 neutrophils/microL were reached and 150 U erythropoietin/kg/d from day 7 until erythrocyte transfusion independence for 7 days. Group I consisted of patients with acute myeloid leukemia (AML) (n = 2), chronic myeloid leukemia (CML) (n = 2), and T-gamma-lymphoproliferative syndrome and BM aplasia (n = 1). The patients received a mean of 3.3 x 10(6) CD34+ and 3.7 x 10(5) CD3+ cells/kg body weight of PBPC origin and 4.5 x 10(6) CD34+ and 172 x 10(5) cells/kg body weight of BM origin. Group II consisted of five patients (two AML, two CML, one non-Hodgkin's lymphoma). They received a mean of 3.3 x 10(6) CD34+ and 3.2 x 10(5) CD3+ cells/kg from PBPC and 1.4 x 10(6) CD34+ and 0.6 x 10(5) CD3+ cells from BM. A matched historical control group (n = 12) transplanted with a mean of 5.2 x 10(6) CD34+ and 156 x 10(5) CD3+ cells/kg from BM alone was assembled for comparison. In group I, the median time to neutrophil recovery to > 100, > 500, and > 1,000/microL was 12, 15, and 17 days, respectively. Patients from group II reached these neutrophil levels at days 13, 15 and 17 post BMT. Neutrophil recovery in the control patient group occurred at days 17, 18, and 20 respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Clinical applications of CD34+ cell-selected peripheral blood stem cells

Peripheral blood stem cells (PBSC) are increasingly used for stem cell transplantation after high dose chemotherapy. CD34+ cell selection has also been done for use in autologous transplantation studies Bone marrow (BM) may contain tumor cells at the time of harvesting, and on re-infusion, these cells could contribute to a subsequent relapse. Similarly, tumor cell contamination of PBSC collections has been found in a number of studies. Therefore, purging contaminating tumor cells may prevent cases of relapse. As most tumor cell types do not express CD34 antigen, one of the most widespread applications of CD34+ cell selection is likely to be in tumor cell purging. Similarly, CD34+ cell selection has aided allogeneic transplantation studies. Acute graft-versus-host disease (aGVHD) is a major cause of morbidity and mortality in cases of allogeneic transplantation. As aGVHD is mediated by donor T cells, removal of T cells from the graft by CD34+ cell selection may ensure prophylaxis against aGVHD. Further, high-dose immunosuppression followed by CD34+ cell-selected stem cell rescue is theoretically reasonable as a therapeutic tool for patients with autoimmune disease resistant to conventional therapy. However, patients given T cell-depleted transplantation have an increased risk of opportunistic infection as well as malignancies related to immunosuppression; therefore, close monitoring is warranted. We describe here clinical applications of CD34+ cell-selected PBSC for a variety of diseases, with special emphasis on the efficacy as well as drawbacks of this novel technique.     

In vitro expansion of human peripheral blood CD34+ cells

To elucidate the role of recombinant human colony-stimulating factors (CSFs) for expanding peripheral blood (PB) CD34+ cells, these cells were purified up to 94.5% +/- 1.3% and the effects of individual and combined CSFs on the proliferation and differentiation of these cells were studied in a 7-day suspension culture. The majority of CD34+ cells coexpressed CD38 (81.8% +/- 5.1%), but was negative for CD33 (88.5% +/- 3.4%). Among the individual CSFs examined, recombinant interleukin-3 (rIL-3) was identified as the most potent factor for expanding PB progenitor cells and increased nonerythroid progenitor cells 13- +/- 4- fold (P < .01). Recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF), recombinant granulocyte-CSF (rG-CSF), recombinant macrophage-CSF (rM-CSF), rIL-6, rIL-11, and recombinant stem cell factor (rSCF) did not alone expand nonerythroid progenitor cells. A combination of 5 CSFs, ie, rIL-3, rIL-6, rGM-CSF, rG-CSF, and rSCF, was identified as the most potent combination of those tested and increased nonerythroid progenitor cells 57- +/- 11-fold. After a 7-day suspension culture of CD34+ cells with these 5 CSFs, CD34+ cells expanded 14.5- fold, and CD34+/CD33- cells and CD34+/CD33+ cells were also expanded 2.9-fold and 307-fold, respectively. Most secondary colonies derived from expanded cells were small; however, the absolute number of large- sized colonies expanded 5.9- +/- 3.3-fold. Thus, the combination of CSFs can achieve a degree of amplification of PB CD34+ cells. The capability of in vitro expansion of PB CD34+ cells as an adjunct to PB stem cell transplantation is worthy of consideration.     

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.     

Decreased circulating CD34+ cells are associated with progression of diabetic nephropathy

Aims Circulating progenitor cells such as CD34+ cells play a key role in maintenance of vascular endothelial function and neovascularization, and a decrease in the number of CD34+ cells is associated with cardiovascular disease. However, the contribution of circulating progenitor cells to microvascular disease, such as diabetic nephropathy, is unclear. This study was therefore designed to clarify the association between diabetic nephropathy and circulating CD34+ cells. Methods We measured circulating CD34+ cell numbers in 85 Type 2 diabetic patients aged 40–70 years with normo‐ and microalbuminuria and determined the association with urinary albumin excretion rate (UAER). Results The number of circulating CD34+ cells significantly correlated with log UAER (r = –0.289, P = 0.008). Furthermore, in patients with low numbers of CD34+ cells (0.68 > cells/µl, lowest quartile of CD34+ cell number) UAER increased significantly after 12 months compared with baseline [from 34.3 ± 7.0 to 53.6 ± 10.8 mg/g creatinine (gCr), P < 0.05], whereas in patients with a high number of CD34+ cells (1.0 < cells/µl, highest quartile of CD34+ cell number) UAER did not change (from 16.7 ± 4.8 to 20.1 ± 3.0 mg/gCr). Conclusions These results suggest that a decreased number of circulating CD34+ cells is involved in the progression of diabetic nephropathy and may be a predictor of the disease.     

Caspase-3在脐血CD34+造血干/祖细胞中的表达及意义

目的：探讨脐血CD34^＋干／祖细胞在不同细胞因子支持下的体外扩增过程中Caspase-3表达及意义，方法：采用RT－PCR、Wester blot和流式细胞仪分析技术测定脐血CD34^ 细胞在体外扩增过程中的生物学特性及Caspase-3的表达。结果：Caspase-3 mRNA在新鲜分离的脐血CD34^ 细胞中低水平表达，在细胞因子支持下体外增养3d，扩增的CD34^ 细胞中Caspase-3 mRNA和蛋白质表达上调，但在该两种细胞中仅能检测到分子量为32000的无活性酶原形式的Caspase-3，随着体外培养时间的延长，在IL－3、IL－6和GM－CSF组合条件下，Caspase-3被激活，可检测到分子量为20000的裂解片段。结论：虽然造血干细胞的凋亡是个复杂的过程，但在脐血CD34^ 干／祖细胞体外扩增过程中，Caspase-3参与了凋亡事件并发挥着重要的作用。     

Phenotypic characterization of immunomagnetically purified umbilical cord blood CD34+ cells

This study describes the multilineage differentiation pattern of purified CD34+ stem cells obtained from human umbilical cord blood. CD34+ cells were collected from 49 umbilical cord blood samples. Following immunomagnetic purification, cells were double stained with anti CD34 and CD71, CD61, CD7, CD19, CD33, CD36 and triple stained with anti CD34, CD38 and HLA-DR. Analysis were performed using a FACScan flow cytometer. After purification, the mean CD34+ cells' purity was 85.49 +/- 7.08%. Several subpopulations of umbilical cord blood CD34+ cells were identified indicating different lineage commitment. The majority of CD34+ cells expressed both CD38 and HLA-DR (91.74 +/- 3.76%), while those lacking CD38 were 3.43 +/- 2.12% (CD38-DR+) and 1.81 +/- 1.54% (CD38-DR-). These data were compared to the expression of lineage commitment markers on purified CD34+ cells from 5 mobilized peripheral blood samples. The percentage of peripheral blood CD34+CD38-DR+) and CD34+CD38-DR- cells was significantly lower than umbilical cord blood, 0.24 +/- 0.18% and 0.04 +/- 0.03% respectively. The knowledge and standardized of umbilical cord blood CD34+ cells phenotype is critical since umbilical cord blood volume is limited. The homogeneity of CD34+ subpopulation phenotype suggests that monitoring of lineage differentiation antigens may not be relevant for clinical use of umbilical cord blood samples. However, the observed higher percentage of pluripotent CD34+38- stem cells in umbilical cord blood compared to peripheral blood, that might explain the successful clinical use of umbilical cord blood even when low number of cells are used, candidates these antigens as the predictive parameter for clinical use of umbilical cord blood samples.     

流式细胞术分析冻存前后脐血CD34+细胞的分布

目的探讨低温冻存对脐血CD34+细胞的影响.方法采用流式细胞仪分析冻存前后脐血CD34+细胞百分率、CD45+细胞和CD34+细胞的荧光强度变化及死细胞群的分布情况.结果冻存后CD34+细胞占CD45+细胞的百分率[(0.84±0.39)%]明显高于冷冻前[(0.51±0.24)%](P<0.01),冻存前后CD34+细胞绝对数无明显变化[(9.372±6.072) ×106/L和(9.246±6.132)×106/L](P>0.05),冻存前后CD34+细胞百分率呈正直线相关(r=0.564, P<0.01).冻存后CD45+细胞荧光强度减弱(P<0.01),CD34+细胞荧光强度无明显变化(P>0.05);中性粒细胞比例下降,淋巴细胞和单核细胞比例增高.死细胞组分中以中性粒细胞为主,占81.52%;活细胞组分中以淋巴细胞为主,占59.44%.结论冻存后CD34+细胞占CD45+细胞的百分率增高,但低温冻存对CD34+细胞绝对数量影响不大.死细胞主要为较成熟的粒细胞,冻存后CD34+细胞的分析需排除死细胞的干扰.     

Identification of human T-lymphoid progenitor cells in CD34+ CD38low and CD34+ CD38+ subsets of human cord blood and bone marrow cells using NOD-SCID fetal thymus organ cultures

In contrast to myeloid and B-lymphoid differentiation, which take place in the marrow environment, development of T cells requires the presence of thymic stromal cells. We demonstrate in this study that human CD34+, CD34+ CD38+ and CD34+ CD38(low) cells from both cord blood and adult bone marrow reproducibly develop into CD4+ CD8+ T cells when introduced into NOD-SCID embryonic thymuses and further cultured in organotypic cultures. Such human/mouse FTOC fetal thymic organ culture) thus represents a reproducible and sensitive system to assess the T-cell potential of human primitive progenitor cells. The frequency of T-cell progenitors among cord-blood-derived CD34+ cells was estimated to be 1/500. Furthermore, the differentiation steps classically observed in human thymus were reproduced in NOD-SCID FTOC initiated with cord blood and human marrow CD34+ cells: immature human CD41(low) CD8- sCD3- TCR alphabeta- CD5+ CD1a+ T cells were mixed with CD4+ CD8+ cells and more mature CD4+ CD8- TCR alphabeta+ cells. However, in FTOC initiated with bone marrow T progenitors, <10% double-positive cells were observed, whereas this proportion increased to 50% when cord blood CD34+ cells were used, and most CD4+ cells were immature T cells. These differences may be explained by a lower frequency of T-cell progenitors in adult samples, but may also suggest differences in the thymic signals required by bone marrow versus cord blood T progenitors. Finally, since cytokine-stimulated CD34+ CD38(low) cells retained their ability to generate T cells, these FTOC assays will be of value to monitor, when combined with other biological assays, the influence of different expansion protocols on the potential of human stem cells.     

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.     

A comparative study of CD34+ cells, CD34+ subsets, colony forming cells and cobblestone area forming cells in cord blood and bone marrow allografts

Cord blood (CB) has become an alternative source of hematopoietic progenitor cells (HPCs) for allogeneic transplantation. We have developed a new efficient protocol for CB collection. Using this method an average of 17.7 x 10(8) [range (6.8-29.6) x 10(8), n = 13] total nucleated cells (TNCs) were harvested. Based on recent Eurocord data, which have shown safe engraftment using a threshold dose of 0.37 x 10(8) CB TNCs/kg body weight (BW), we calculated that six out of thirteen CB grafts collected by this method were sufficient to engraft adults. The CB derived CD34+ population contained two-fold higher numbers of committed HPCs (CFU-GM, BFU-E) and six-fold higher numbers of pluripotent HPCs [CD34+/CD38- cells, wk 5 and wk 8 cobblestone area forming cells (CAFCs)] than the CD34+ population of BM. Extrapolation revealed that BM grafts providing the threshold dose for allogeneic transplantation of 2 x 10(8) TNCs/kg BW contained nearly 3 times more pluripotent HPCs than CB grafts providing the Eurocord threshold dose. The assessment of CD34+/CD38(-) cell numbers in CB grafts was highly reproducible and correlated well with the in vitro performance of pluripotent HPCs, i.e. numbers of CAFCs. We conclude that CB grafts providing high numbers of TNCs have the potential to engraft adults and that the enumeration of pluripotent HPCs by flow cytometry may be a useful tool to define the ultimate threshold dose for CB transplantation.