Hematopoietic repopulating ability of cord blood CD34(+) cells in NOD/Shi-scid mice

Although umbilical cord blood (CB) is increasingly being used as an alternative to bone marrow (BM) as a source of transplantable hematopoietic stem cells (HSC), information on the hematopoietic repopulating ability of CB HSC is still limited. We recently established a xenotransplantation system in NOD/Shi-scid mice to evaluate human stem cell activity. In the present study, we transplanted 5 to 10 x 10(4) CB CD34(+) cells into six NOD/Shi-scid mice treated with anti-asialo GM1 antiserum to investigate the hematopoietic repopulating ability of CB. The BM of all recipients contained human CD45(+) cells 10 to 12 weeks after the transplantation (43.8 +/- 17.7%). Clonal culture of the recipient BM cells revealed the formation of various types of human hematopoietic colonies, including myelocytic, erythroid, megakaryocytic, and multilineage colonies, indicating that CB HSC can differentiate into hematopoietic progenitors of various lineages. However, the extent of the differentiation and maturation differed with each lineage. CD13(+)/CD14(+)/CD33(+) myelocytic cells were mainly repopulated in BM and peripheral blood (PB). While CD41(+) megakaryocytic cells and platelets were present, few glycophorin A(+)CD71(+) or hemoglobin alpha-containing erythroid cells were detected. CD19(+) B cells were the most abundantly repopulated in NOD/Shi-scid mice, but their maturational stage differed among the hematopoietic organs. Most of the BM CD19(+) cells were immature B cells expressing CD10 but not surface immunoglobulin (Ig) M, whereas more mature CD19(+)CD10(-) surface IgM(+) B cells were predominantly present in spleen and PB. CD3(+) T cells were not detected even in the recipient thymus. The transplantation to the NOD/Shi-scid mouse may provide a useful tool for evaluating the repopulating ability of transplantable human HSC.     

Immunophenotype and functional characteristics of human primitive CD34-negative hematopoietic stem cells: the significance of the intra-bone marrow injection

The biology of hematopoietic stem cell (HSC) is a current topic of interest which has important implications for clinical HSC transplantation as well as for the basic research of HSC. The most primitive HSCs in mammals, including mice and humans, have long been believed to be CD34 antigen (Ag)-positive (CD34(+)) cells. In fact, bone marrow (BM), peripheral blood (PB), and cord blood (CB) stem cell transplantation studies indicate that a CD34(+) subpopulation in the BM, PB, or CB can provide durable long-term donor-derived lymphohematopoietic reconstitution. Therefore, CD34 Ag was used to identify/purify immature HSCs. However, Osawa et al. reported that murine long-term lymphohematopoietic reconstituting HSCs are lineage marker-negative (Lin(-)) c-kit(+)Sca-1(+)CD34-low/negative (CD34(low/-)), which are called CD34(low/-) KSL cells. Recently, human CB-derived CD34(-) HSCs, a counterpart of murine CD34(low/-) KSL cells, were successfully identified using an intra-bone marrow injection (IBMI) method. This review will update the concept of the immunophenotype and the functional characteristics of human primitive CD34(-) HSCs. In addition, the significance of the application of the IBMI technique in clinical HSC transplantation is also discussed. Recent rapid advances in understanding the biological nature of HSCs may make it possible to fully characterize the most primitive class of human HSCs in the near future.     

Expression of adhesion molecules on CD34(+) cells in peripheral blood of non-hodgkin's lymphoma patients mobilized with different growth factors

Adhesion molecules on CD34(+) cells were implicated in the process of peripheral blood stem cell (PBSC) mobilization and homing. We studied the mobilization of CD34(+)Thy1(+) cells, CD34(+) very late-acting antigen (VLA)4(+) cells, and CD34(+)L-selectin(+) cells in non-Hodgkin's lymphoma patients mobilized with cyclophosphamide plus G-CSF, GM-CSF, or GM-CSF followed by G-CSF. The mean percentage of CD34(+) cells in the bone marrow (BM) expressing Thy1 was 23.6% +/- 11% and 17.8% +/- 8% in the PB before mobilization, and was markedly decreased to 4.5% +/- 3.3% in the apheresis collections. Similarly, the mean percentage of CD34(+) cells expressing L-selectin was 35.8% +/- 4.3% in the BM, 21.6% +/- 4.1% in the PB before mobilization and was markedly decreased to 9.1% +/- 2.5% in the apheresis collections. Patients in the three arms of the study had a similar pattern of CD34(+)Thy1(+) and CD34(+)L-selectin(+) cell mobilization. Also, a similar pattern of coexpression of CD34(+)Thy1(+) and CD34(+)L-selectin(+) cells was observed when the patients were regrouped as "good mobilizers" (> or =2 x 10(6) CD34(+)CD45(dim) cells/kg, in four collections) and "poor mobilizers" (<0.4 x 10(6) CD34(+)CD45(dim) cells/kg, in two collections). The mean percentage of CD34(+) cells expressing VLA-4 in the BM and PB was relatively high (73.4% +/- 12% and 65.4% +/- 6.6%, respectively) and dropped considerably in the PBSC collections to 43.5% +/- 7.1% with a similar pattern observed for patients in arms A, B, and C. However, when the patients were regrouped as "good mobilizers" and "poor mobilizers," a higher percentage of CD34(+) cells expressing VLA-4 was observed in the PBSC of the pooled "good mobilizers" (50.5% +/- 9% versus 36.3% +/- 6.4%; p = 0.01). We conclude that release of CD34(+) cells to the PB involves a general downregulation of Thy1, L-selectin and VLA-4 on CD34(+) cells, irrespective of the growth factor used for mobilization. However, good mobilizers had a relatively higher percentage of CD34(+) cells expressing the VLA-4 antigen.     

In vitro proliferation potential of AC133 positive cells in peripheral blood

AC133 antigen is a novel marker for human hematopoietic stem/progenitor cells. In this study, we examined the expression and proliferation potential of AC133(+) cells obtained from steady-state peripheral blood (PB). The proportion of AC133(+) cells in the CD34(+) subpopulation of steady-state PB was significantly lower than that of cord blood (CB), although that of cytokine-mobilized PB was higher than that of CB. The proliferation potential of AC133(+)CD34(+) and AC133(-)CD34(+) cells was examined by colony-forming analysis and analysis of long-term culture-initiating cells (LTC-IC). Although the total number of colony-forming cells was essentially the same in the AC133(+)CD34(+) fraction as in the AC133(-)CD34(+) fraction, the proportion of LTC-IC was much higher in the AC133(+)CD34(+) fraction. Virtually no LTC-IC were detected in the AC133(-)CD34(+) fraction. In addition, the features of the colonies grown from these two fractions were quite different. Approximately 70% of the colonies derived from the AC133(+)CD34(+) fraction were granulocyte-macrophage colonies, whereas more than 90% of the colonies derived from the AC133(-)CD34(+) fraction were erythroid colonies. Furthermore, an ex vivo expansion study observed expansion of colony-forming cells only in the AC133(+)CD34(+) population, and not in the AC133(-)CD34(+) population. These findings suggest that to isolate primitive hematopoietic cells from steady-state PB, selection by AC133 expression is better than selection by CD34 expression.     

Identification of a massive reserve of hematopoietic progenitors in mice

Previous studies have demonstrated that mice null (-/-) for either CD34 or c-mpl are viable and have greatly decreased numbers of multipotential (CFU-Mix), erythroid (BFU-E), and granulocytemacrophage (CFU-GM) progenitor cells in the bone marrow (BM), spleen (Spl) and peripheral blood (PB), without noticeable decreases in the nucleated cellularity of these organs. To evaluate the significance of these two proteins further, mice null for both CD34 and c-mpl were assessed for hematopoietic progenitor cells (HPC) and nucleated cellularity and compared with these cells in CD34-/- and c-mpl-/- mice. The following progenitors were assessed: CFU-GM, BFU-E, CFU-Mix with an erythroid component, CFU-Mix with erythroid and megakaryocyte components, nonerythroid CFU with a megakaryocyte (Meg) component and pure CFU-Meg. Results demonstrated significant decreases in progenitors in the BM of dual CD34/c-mpl-/- mice compared to decreases from CD34-/- or c-mpl-/- mice; progenitor numbers in CD34/c-mpl-/- mice were decreased by 83-99.3% compared to that in wild-type littermate control mice. Decreases in progenitors in spleens of c-mpl-/- mice (89-96%) were more drastic than those of CD34-/- mice (50-78%) whereas those of dual CD34/c-mpl-/- mice were equal to or lower than that of c-mpl-/- mice (93-98%). Decreases in PB progenitors were seen in the c-mpl-/- and dual CD34/c-mpl-/- mice (75-90%). Whereas progenitor cells in BM, Spl and PB were drastically reduced in dual CD34/c-mpl-/- mice compared to controls, absolute numbers of nucleated cells in these organs were essentially not reduced. These studies demonstrate that CD34 and c-mpl have non-redundant effects on maintenance of steady-state hematopoiesis and highlight how few progenitor cells are required in steady-state conditions to populate and maintain the BM, Spl, and PB with nucleated cells.     

A study on the incidence of ABL gene deletion on derivative chromosome 9 in chronic myelogenous leukemia by interphase fluorescence in situ hybridization and its association with disease progression

Fluorescence in situ hybridization for the BCR/ABL rearrangement in 138 bone marrow specimens from 59 Philadelphia(+) (Ph(+)) chronic myelogenous leukemia (CML) patients, 35 Ph(+) acute lymphoblastic leukemia (ALL) patients, and 57 Ph(-) ALL patients was used. Sixteen (27.1%) of the 59 CML patients had deletions of the residual ABL gene on the derivative chromosome 9. During the study period, 32 of the 59 CML patients progressed to blast crisis or accelerated phase. Of these, nine patients had residual ABL gene deletions on the derivative chromosomes 9 and 23 patients had no deletions. The mean duration from first diagnosis to blast crisis or accelerated phase for the nine patients with ABL deletions was 32.8 months, and for the 23 patients without ABL deletions, it was 62.4 months (P = 0.017). The overall survival time for the 16 patients with deletions was 32.8 months, and for the 43 patients without deletions, it was 60.1 months (P = 0.164). ABL deletions were not detected among the 35 ALL patients (17 with major BCR/ABL, 18 with minor BCR/ABL), and it appears that this deletion occurs rarely or not at all in Ph(+) ALL patients, which is in contrast to the CML patients (27.1%). However, we detected two ALL cases with ABL deletion but without BCR/ABL rearrangement among 49 Ph(-) ALL and 66 Ph(-) AML patients. In conclusion, patients with ABL deletions progress to blast crisis or accelerated phase in a significantly shorter time than do those without such deletions. It is therefore suggested that the ABL deletion is an indicator of a poor prognosis in CML.     

Comparison of CD34+ subsets and clonogenicity in human bone marrow, granulocyte colony-stimulating factor-mobilized peripheral blood, and cord blood

To compare the clonogenicity and distribution of CD34+ subsets in bone marrow (BM), granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood (PB) and cord blood (CB), we analyzed in vitro colony formation and CD34+ cells co-expressing differentiation molecules (CD38, HLA-DR), myeloid associated molecules (CD13, CD33), a T-cell associated molecule (CD3), and a B-cell associated molecule (CD19) from mononuclear cells (MNCs) in the three compartments. The proportions of CD34+CD38- cells (BM: 4.4+/-2.8%, PB: 5.3+/-2.1%, CB: 5.9+/-3.9%) and CD34+HLA-DR cells (BM: 4.7+/-3.4%, PB: 5.5+/-2.3%, CB: 6.1+/-3.7%) did not differ significantly among the compartments. In contrast, a significantly higher proportion of CD34 cells of PB and CB co-expressed CD13 (75.0+/-11.4%, 77.7+/-17.3%) and CD33 (67.1 +/-5.7%, 56.8+/-10.3%) compared with those of BM (43.0+/-6.3%, 27.6+/-5.1%) and a significantly higher number of granulocyte-macrophage colony-forming units (CFU-GM) and erythroid burst-forming units (BFU-E) were detected in MNCs derived from PB and CB compared with those from BM (p<0.01). The proportion of CD34+CD19+ cells was higher in BM (34.9+/-11.9%) than those in PB (5.6+/-3.0%) and CB (4.7=2.1%) (p<0.05). The proportion of CD34+CD3+ was comparable in all three compartments. In conclusion, our findings show that MNCs of mobilized PB and CB display similar phenotypic profiles of CD34+ subsets and clonogenicity, different from those of BM.     

Molecular status of individual CFU-GM colonies derived from chemotherapy-mobilised peripheral blood stem cells in chronic myeloid leukaemia

Following chemotherapy in chronic myeloid leukaemia (CML), some peripheral blood (PB) cells may be Philadelphia (Ph) chromosome negative. The BCR-ABL mRNA status of residual Ph⁺ progenitors is not known. We examined the BCR-ABL mRNA status of individual colony-forming-unit granulocyte-macrophage (CFU-GM) colonies derived from PB harvested following chemotherapy. Seven patients were treated with 200 mg/m²/day cytarabine and 20 mg/m²/day Idarubicin and followed by Lenograstim. PB collections commenced daily when the white blood cell count reached 0.6 × 10⁹/l and continued until at least 5 × 10⁸/kg nucleated cells were obtained. CD34⁺ cells, Ph status, and CFU-GM were estimated at each harvest. For each patient, up to 24 individual CFU-GM colonies were analysed for BCR-ABL status. Two cases were BCR-ABL negative on all colonies and completely Ph⁻, and another case was BCR-ABL positive in all colonies and completely Ph⁺. In contrast, in two patients all colonies were BCR-ABL negative, despite virtually complete Ph⁺ metaphases. The final assessible case had five of nine BCR-ABL negative colonies, despite 94% Ph⁺ metaphases. After chemotherapy priming, the PB may contain Ph⁺ CFU-GM that do not express BCR-ABL. Genes Chromosom. Cancer 18:292–298, 1997. © 1997 Wiley-Liss, Inc.     

Plerixafor Plus Granulocyte Colony-Stimulating Factor versus Placebo Plus Granulocyte Colony-Stimulating Factor for Mobilization of CD34(+) Hematopoietic Stem Cells in Patients with Multiple Myeloma and Low Peripheral Blood CD34(+) Cell Count: Results of a Subset Analysis of a Randomized Trial

Preapheresis peripheral blood (PB) CD34(+) cell count is a strong predictor of hematopoietic stem cell (HSC) mobilization and is routinely used to optimize the timing, cost, and success of HSC collection in patients with multiple myeloma. However, a uniform PB CD34(+) cell count that predicts mobilization failure has not been defined, resulting in the development of institute-specific algorithms for mobilization, particularly regarding the decision of when to use the novel stem cell mobilization agent plerixafor. In this post hoc analysis, we evaluated the mobilization efficacy of plerixafor plus granulocyte colony-stimulating factor (G-CSF) versus placebo plus G-CSF in patients with multiple myeloma, stratified by preapheresis PB CD34(+) cell count: <10, <15, <20, and ≥20 cells/μL. Regardless of the PB CD34(+) cell count, the total yield of CD34(+) cells from apheresis was significantly higher in the plerixafor group than in the placebo group, and significantly more patients in the plerixafor group collected the minimum (≥2?×?10(6) cells/kg) and optimum (≥6?×?10(6) cells/kg) stem cell yields on each day of apheresis. As a corollary, the greater stem cell collection in plerixafor-treated patients resulted in the need for significantly fewer days of apheresis to reach minimum and optimum cell doses across all cell count groups. For all CD34(+) cell count groups, the proportion of patients proceeding to transplantation and the median time to platelet and neutrophil engraftment were similar in the plerixafor and placebo groups. Our findings demonstrate that in patients with multiple myeloma who might be predicted to fail mobilization based on low PB CD34(+) cell count, the addition of plerixafor to G-CSF allows for collection of the minimal and optimal cell doses in a greater proportion of patients compared with G-CSF alone. In addition, plerixafor plus G-CSF significantly improves the likelihood of optimal HSC collection in patients with higher preapheresis PB CD34(+) cell counts (≥20 cells/μL) compared with placebo plus G-CSF. Collectively, this analysis of predicted poor mobilizers validates the superiority of plerixafor plus G-CSF compared with G-CSF alone, which had been demonstrated previously in the overall patient population.     

Gene-expression profiling of CD34+ cells from various hematopoietic stem-cell sources reveals functional differences in stem-cell activity

The replacement of bone marrow (BM) as a conventional source of stem cell (SC) by umbilical cord blood (UCB) and granulocyte-colony stimulating factor-mobilized peripheral blood SC (PBSC) has brought about clinical advantages. However, several studies have demonstrated that UCB CD34(+) cells and PBSC significantly differ from BM CD34(+) cells qualitatively and quantitatively. Here, we quantified the number of SC in purified BM, UCB CD34(+) cells, and CD34(+) PBSC using in vitro and in vivo assays for human hematopoietic SC (HSC) activity. A cobblestone area-forming cell (CAFC) assay showed that UCB CD34(+) cells contained the highest frequency of CAFC(wk6) (3.6- to tenfold higher than BM CD34(+) cells and PBSC, respectively), and the engraftment capacity in vivo by nonobese diabetic/severe combined immunodeficiency repopulation assay was also significantly greater than BM CD34(+), with a higher proportion of CD45(+) cells detected in the recipients at a lower cell dose. To understand the molecular characteristics underlying these functional differences, we performed several DNA microarray experiments using Affymetrix gene chips, containing 12,600 genes. Comparative analysis of gene-expression profiles showed differential expression of 51 genes between BM and UCB CD34(+) SC and 64 genes between BM CD34(+) cells and PBSC. These genes are involved in proliferation, differentiation, apoptosis, and engraftment capacity of SC. Thus, the molecular expression profiles reported here confirmed functional differences observed among the SC sources. Moreover, this report provides new insights to describe the molecular phenotype of CD34(+) HSC and leads to a better understanding of the discrepancy among the SC sources.     

The impaired number of circulating granulocyte/macrophage progenitors (CFU-GM) in human immunodeficiency virus-type 1 infected subjects correlates with an active HIV-1 replication

Summary In this paper we investigated the role played by human immuno-deficiency virus type 1 (HIV-1) in the pathogenesis of peripheral blood (PB) cytopenias of AIDS patients. The in vitro growth of PB granulocyte/macrophage progenitors (CFU-GM) was investigated in 45 HIV-1 seropositive (+) individuals at different stages of the disease. The number of circulating CFU-GM was significantly (p<0.01) lower in AIDS patients (stages WR V-VI) than in HIV-1(+) asymptomatic individuals (stages WR I-II). Moreover, the presence of gag p 24 in the plasma and/or viral isolation from PB mononuclear cells of HIV-1(+) individuals was inversely correlated (p<0.01) with the number of circulating CFU-GM, irrespectively with the stage of the disease. Viral isolates obtained from one asymptomatic and four symptomatic HIV-1(+) individuals were tested on the in vitro growth of normal hematopoietic progenitor (CD34⁺) cells, purified from PB of healthy donors. All the different viral isolates showed a dose-dependent inhibition of CD34⁺ cells, in the absence of either productive or latent infection. This suppressive effect was completely reversed by prein-cubating the different viral isolates with a polyclonal anti-gp 120 antibody before adding to normal CD34⁺ cells. These findings suggest a direct involvement of active viral replication products in the progressive impairment of hematopoiesis, characteristic of HIV-1(+) individuals in spite of the lack of a productive or latent infection of CD34⁺ hematopoietic progenitors.     

Rhodamine 123 efflux in human subpopulations of hematopoietic stem cells: comparison between bone marrow, umbilical cord blood and mobilized peripheral blood CD34+ cells

Hematopoietic stem cells (HSC) can be identified by the expression of the CD34 molecule. CD34+ cells are found in bone marrow (BM), umbilical cord blood (UCB) and in mobilized peripheral blood (PB). CD34+ cells express P-glycoprotein (Pgp), a product of the multidrug resistance (MDR) gene. Pgp activity can be measured by the efflux of the dye Rhodamine 123 (Rho 123) and can be blocked by verapamil. Transport activity in HSC suggests that Pgp could have a functional role in stem cell differentiation. This study compared the number of CD34+ cells with Pgp activity measured by efflux of Rho 123 in the hematopoietic population obtained from different sources. Samples were analysed for their content of CD34+ cells, and BM had a significantly higher amount of CD34+ cells compared to UCB, mobilized PB and normal PB. When the frequency of Rholow cells was studied among the CD34+ population, an enrichment of cells with Pgp activity was observed. The frequency in BM was significantly lower than that in UCB and mobilized PB. The low retention of Rho 123 could be modified by verapamil, indicating that the measurements reflected dye efflux due to Pgp activity. Although UCB and mobilized PB had a lower number of CD34+ cells compared to BM, the total number of CD34+ cells with Pgp activity was similar in the three tissues. The different profiles may indicate the existence of subpopulations of stem cells or different stages of cellular differentiation detected by the extrusion of the dye Rho 123.     

Studies of complex Ph translocations in cases with chronic myelogenous leukemia and one with acute lymphoblastic leukemia

The BCR/ABL gene fusion, the hallmark of chronic myelogenous leukemia (CML) is generated in 2-10% of patients by a variant Ph translocation involving 9q34, 22q11.2, and one or more additional genomic regions. The objective of this study was the characterization by conventional and molecular cytogenetics of complex variant Ph translocations present at diagnosis. FISH studies were performed in 7 cases using the LSI BCR/ABL ES probe allowing the detection of the fusion BCR/ABL gene on the Ph chromosome in all of them and 9q34 deletions in 2 cases. Three cryptic complex rearrangements were detected by FISH studies. The third and the fourth chromosome regions involved in the 8 complex variant translocations were: 1q21, 1p36, 5q31, 11q13, 12q13, 12p13, and 20q12. In conclusion, FISH studies have been useful in the detection of the BCR/ABL rearrangements and 9q34 deletions, and to identify complex rearrangements that differ from the ones previously established by conventional cytogenetics.     

DEK regulates hematopoietic stem engraftment and progenitor cell proliferation

DEK is a biochemically distinct protein that is generally found in the nucleus, where it is vital to global heterochromatin integrity. However, DEK is also secreted by cells (eg, macrophages) and influences other adjacent cells (eg, acts as a chemoattractant for certain mature blood cells). We hypothesized that DEK may modulate functions of hematopoietic stem (HSCs) and progenitor (HPCs) cells. C57Bl/6 mice were used to demonstrate that absolute numbers and cycling status of HPCs (colony forming unit-granulocyte macrophage [CFU-GM], burst forming unit-erythroid [BFU-E], and colony forming unit-granulocyte erythroid macrophage megakaryocyte [CFU-GEMM]) in bone marrow (BM) and spleen were significantly enhanced in DEK -/- as compared with wild-type (WT) control mice. Moreover, purified recombinant DEK protein inhibited colony formation in vitro by CFU-GM, BFU-E, and CFU-GEMM from WT BM cells and human cord blood (CB) cells in a dose-dependent fashion, demonstrating that DEK plays a negative role in HPC proliferation in vitro and in vivo. Suppression was direct acting as determined by inhibition of proliferation of single isolated CD34(+) CB cells in vitro. In contrast, DEK -/- BM cells significantly demonstrated reduced long term competitive and secondary mouse repopulating HSC capacity compared with WT BM cells, demonstrating that DEK positively regulates engrafting capability of self-renewing HSCs. This demonstrates that DEK has potent effects on HSCs, HPCs, and hematopoiesis, information of biological and potential clinical interest.     

Interphase fluorescence in situ hybridization studies for the detection of 9q34 deletions in chronic myelogenous leukemia: a practical approach to clinical diagnosis

Chronic myelogenous leukemia (CML) is characterized by the Philadelphia chromosome (Ph) in more than 90% of cases. Recent studies using fluorescence in situ hybridization (FISH) have shown that in a subset of patients with CML, deletions of 9q34 involving the argininosuccinate synthetase region occur at the time of the Philadelphia translocation and are associated with a poor prognosis. We performed interphase FISH studies in 152 cases of CML using a dual-color, dual-fusion probe system with a third probe directed at 9q34. Cytogenetic studies showed a simple (typical) Ph in 124/152 (82%), a cryptic Ph in 11/152 (7%), and a variant Ph chromosome with a complex translocation in 17/152 (11%) of cases. Interphase FISH studies showed single BCR/ABL fusion patterns in 48/152 (32%) of cases. Deletions of 9q34 were observed in 14% of all the cases and were present in 46% of cases with single BCR/ABL fusion pattern. All the 9q34 deletions occurred in cases with single BCR/ABL fusion signal. However, a single-fusion pattern is not specific for 9q34 deletions, and cases should be routinely screened for the presence of this prognostically significant abnormality by using a third probe directed specifically at 9q34.     

体外培养慢性粒细胞白血病患者骨髓间充质干细胞的生物学特性

背景：课题组从胎儿骨髓间充质干细胞的培养体系中鉴定出一类贴壁细胞,已证实此类细胞在单细胞水平可以向造血及内皮细胞分化。 目的：检测骨髓间充质干细胞的生物学特性,为慢性粒细胞白血病的治疗提供相关的依据。 方法：以慢性粒细胞白血病患者骨髓为研究对象,体外培养并扩增原始间充质干细胞,检测其BCR/ABL融合基因的表达、免疫学特性和生长曲线,使用RT-PCR和FISH的方法检测其BCR/ABL融合基因的表达情况。 结果与结论：慢性粒细胞白血病患者骨髓来源的间充质干细胞呈成纤维样生长,大部分细胞处于G0/G1期,并且高表达Flk1,CD13,CD29,CD44,用RT-PCR和FISH的方法能够检测出BCR/ABL融合基因的表达。提示慢性粒细胞白血病的白血病基因转化可能发生在比造血干细胞更高的骨髓间充质干细胞水平上,对慢性粒细胞白血病的疾病起源及干细胞移植治疗有重要的意义。     

The generation of human innate lymphoid cells is influenced by the source of hematopoietic stem cells and by the use of G‐CSF

NK cells play a central role in the haploidentical HSC transplantation (HSCT) to cure high‐risk leukemias. Other innate lymphoid cells (ILCs) have been proposed to exert a protective role in graft‐versus‐host disease and could also contribute to anti‐microbial defence and to lymphoid tissue remodeling. Thus, we investigated the ILC differentiation potential of HSCs isolated from BM, mobilized peripheral blood (PB), and umbilical cord blood (UCB). BM CD34⁺ cells are enriched in lymphoid‐committed precursors, while PB CD34⁺ cells preferentially contain myeloid precursors. In vitro differentiation experiments revealed that the highest and the lowest CD56⁺CD161⁺ ILC recovery was detected in UCB and PB HSC cultures, respectively. Among CD56⁺CD161⁺ ILCs, the ratio between NK cells and ILC3s was similar for all HSC analyzed. ILC recovery in PB CD34⁺ cultures was lower for G‐CSF‐mobilized HSCs (good mobilizers) than for G‐CSF+plerixafor‐mobilized HSC (poor mobilizers). Moreover, G‐CSF inhibited in vitro ILC recovery and the degree of inhibition was proportional to the time of exposure to the cytokine. Thus, although all common sources of HSC for transplant differentiate towards ILCs, substantial differences exist among different sources and G‐CSF may influence ILC recovery. These data offer new clues for a better understanding of the immune reconstitution after HSCT.     

Extended in vitro expansion of adult, mobilized CD34+ cells without significant cell senescence using a stromal cell coculture system with single cytokine support

The macrophage colony-stimulating factor-deficient bone marrow stromal cell line OP9, derived from osteopetrotic mice, is known to support hematopoietic stem cell (HSC) expansion as well as hematopoietic differentiation of embryonic stem cells. Coculture of HSC in the OP9 system requires cytokine support to achieve significant cell expansion. Recently, we reported extensive expansion without cell senescence of cord blood (CB)-derived HSC cocultured with OP9 stromal cells for more than 18 weeks with a single cytokine support using human thrombopoietin (TPO). In this study, we evaluated the efficiency of the OP9/TPO coculture system to sustain long-term hematopoiesis of adult, granulocyte colony-stimulating factor mobilized human peripheral blood (PB) CD34(+) cells. Maximum cell expansion was attained during the first 4 weeks of coculture. At the same time, the maximum progenitor cell expansion was demonstrated by the production of colony-forming cells and cobblestone area-forming cells. In contrast to the expansion of CB CD34(+) cells, PB CD34(+) cells showed termination of cultures after 8 weeks, independent of the cell expansion rates attained. The evaluation of cell senescence by assessing the telomere length in most cultures showed no relevant telomere shortening, despite rapid decrease in telomerase activity. Interestingly, increases in telomere length were demonstrated. In conclusion, OP9/TPO system provides extensive stem cell expansion without concomitant telomere erosion for both CB and adult CD34(+) cells. Termination of adult CD34(+) cell cocultures seems to be independent of telomere length.     

CDCP1 identifies a broad spectrum of normal and malignant stem/progenitor cell subsets of hematopoietic and nonhematopoietic origin

CUB-domain-containing protein 1 (CDCP1) is a novel transmembrane molecule that is expressed in metastatic colon and breast tumors as well as on the surface of hematopoietic stem cells. In this study, we used multiparameter flow cytometry and antibodies against CDCP1 to analyze the expression of CDCP1 on defined hematopoietic cell subsets of different sources. In addition, CDCP1 expression on leukemic blasts and on cells with nonhematopoietic stem/progenitor cell phenotypes was determined. Here we demonstrate that a subset of bone marrow (BM), cord blood (CB), and mobilized peripheral blood (PB) CD34+ cells expressed this marker and that CDCP1 was detected on CD34(+)CD38- BM stem/progenitor cells but not on mature PB cells. Analysis of leukemic blasts from patients with acute lymphoblastic leukemia, acute myeloid leukemia, and chronic myeloid leukemia in blast crisis revealed that CDCP1 is predominantly expressed on CD34(+)CD133+ myeloid leukemic blasts. However, CDCP1 was not strictly correlated with CD34 and/or CD133 expression, suggesting that CDCP1 is a novel marker for leukemia diagnosis. Stimulation of CD34+ BM cells with CDCP1-reactive monoclonal antibody CUB1 resulted in an increased (approximately twofold) formation of erythroid colony-forming units, indicating that CDCP1 plays an important role in early hematopoiesis. Finally, we show that CDCP1 is also expressed on cells phenotypically identical to mesenchymal stem/progenitor cells (MSCs) and neural progenitor cells (NPCs). In conclusion, CDCP1 is not only a novel marker for immature hematopoietic progenitor cell subsets but also unique in its property to recognize cells with phenotypes reminiscent of MSC and NPC.