骨髓增生异常综合征异常克隆起源、分布与疾病进展

 骨髓增生异常综合征（MDS）是一组异质性克隆性疾病,主要特征为骨髓衰竭和发展为急性白血病（AL）。MDS异常克隆起源于多向祖细胞或其以上的干细胞。新近研究发现某些类型的MDS干细胞来源于正常的干细胞。对于不同染色体异常的患者,异常克隆在各细胞系列的分布不同。随着异常克隆比例的增加疾病恶化。MDS异常克隆的研究,对于了解MDS发病机制,进行临床诊断、治疗和判断预后有很大的帮助。     

Aberrant marker expression patterns on the CD34+CD38- stem cell compartment in acute myeloid leukemia allows to distinguish the malignant from the normal stem cell compartment both at diagnosis and in remission.

Acute myeloid leukemia (AML) is generally regarded as a stem cell disease. In CD34-positive AML, the leukemic stem cell has been recognized as CD38 negative. This CD34+CD38- population survives chemotherapy and is most probable the cause of minimal residual disease (MRD). The outgrowth of MRD causes relapse and MRD can therefore serve as a prognostic marker. The key role of leukemogenic CD34+CD38- cells led us to investigate whether they can be detected under MRD conditions. Various markers were identified to be aberrantly expressed on the CD34+CD38- population in AML and high-risk MDS samples at diagnosis, including C-type lectin-like molecule-1 and several lineage markers/marker-combinations. Fluorescent in situ hybridization analysis revealed that marker-positive cells were indeed of malignant origin. The markers were neither expressed on normal CD34+CD38- cells in steady-state bone marrow (BM) nor in BM after chemotherapy. We found that these markers were indeed expressed in part of the patients on malignant CD34+CD38- cells in complete remission, indicating the presence of malignant CD34+CD38- cells. Thus, by identifying residual malignant CD34+CD38- cells after chemotherapy, MRD detection at the stem cell level turned out to be possible. This might facilitate characterization of these chemotherapy-resistant leukemogenic cells, thereby being of help to identify new targets for therapy.     

NOD/SCID mice transplanted with marrow from patients with myelodysplastic syndrome (MDS) show long-term propagation of normal but not clonal human precursors

Sublethally irradiated NOD/SCID mice were transplanted with hematopoietic progenitor cells obtained from the marrow of patients with myelodysplastic syndromes (MDS). Engraftment of MDS cells, as determined by flow cytometry, was delayed compared to marrow from normal donors. Human CD38(+)CD34(-) cells were prominent in marrows and spleens of MDS chimeras. CD34(+)CD38(-), CD34(+)CD38(+) and T cells were also easily detected. Human myeloid cells (CD33(+); CD15(+)) were present in low proportions. No clonal precursors were identified by fluorescent in situ hybridization (FISH) or by molecular analysis of polymorphic X-linked markers in mice with documented engraftment of human cells more than 2 months after transplantation. These data indicate that human cells present in murine MDS chimeras, at the levels of sensitivity of our assays, were derived from residual normal cells in human MDS marrow, and suggest that the NOD/SCID environment was not conducive to the expansion of clonal MDS precursors. This model may allow identification of factors relevant for sustaining or expanding clonal precursors.     

骨髓增生异常综合征骨髓微环境的改变

骨髓微环境是一个由造血干／祖细胞（HSPC）、非造血细胞以及细胞外基质和多种细胞因子等组成的复杂网状结构,其各成分相互作用以支持正常的造血。近来研究发现,骨髓微环境中的间充质干细胞（MSC）、髓源抑制性细胞、细胞因子功能紊乱以及造血干细胞的表观遗传学改变都可以影响正常造血,导致血液系统恶性疾病如骨髓增生异常综合征（MDS）的发生。已有动物模型证实了骨髓微环境在造血系统恶性疾病的发生和维持中起着重要作用。     

用微血管内皮细胞体外扩增的骨髓造血干细胞重建大剂量化疗后的小鼠造血功能

目的探索造血干细胞体外培养扩增的新方法。方法采用磁性细胞分离技术纯化小鼠骨髓血管内皮细胞,将其与骨髓造血细胞一起培养。培养的骨髓细胞移植给大剂量化疗后的小鼠,观察其造血重建功能。结果纯化的血管内皮细胞具有鹅卵石样细胞形态,表达多种内皮细胞相关抗原如ＣＤ３１,ＣＤ３４,ＩＣＡＭ１,ＶＣＡＭ１和凝集素ＢＳ１结合位点。将内皮细胞与骨髓造血细胞共同培养后,骨髓细胞数明显增加,其中６０％为原始的造血细胞,后者移植后能有效重建血功能。结论骨髓微血管内皮细胞能促进造血干细胞体外扩增,并有效阻止其分化。扩增的造血干细胞具有造血重建功能。     

Biological purging of breast cancer cells using an attenuated replication-competent herpes simplex virus in human hematopoietic stem cell transplantation

Autologous hematopoietic stem cell transplantation after myelosuppressive chemotherapy is used for the treatment of high-risk breast cancer and other solid tumors. However, contamination of the autologous graft with tumor cells may adversely affect outcomes. Human hematopoietic bone marrow cells are resistant to herpes simplex virus type 1 (HSV-1) replication, whereas human breast cancer cells are sensitive to HSV-1 cytotoxicity. Therefore, we examined the utility of G207, a safe replication-competent multimutated HSV-1 vector, as a biological purging agent for breast cancer in the setting of stem cell transplantation. G207 infection of human bone marrow cells had no effect on the proportion or clonogenic capacity of CD34+ cells but did enhance the proliferation of bone marrow cells in culture and the proportion of CD14+ and CD38+ cells. On the other hand, G207 at a multiplicity of infection of 0.1 was able to purge bone marrow of contaminating human breast cancer cells. Because G207 also stimulates the proliferation of human hematopoietic cells, it overcomes a limitation of other purging methods that result in delayed reconstitution of hematopoiesis. The efficient infection of human bone marrow cells in the absence of detected toxicity suggests that HSV vectors may also prove useful for gene therapy to hematopoietic progenitor cells.     

Inappropriate notch activity and limited mesenchymal stem cell plasticity in the bone marrow of patients with myelodysplastic syndromes

Myelodysplastic syndromes (MDSs) are a heterogeneous group of hematological disorders characterized by ineffective hematopoiesis, enhanced bone marrow apoptosis and frequent progression to acute myeloid leukemia. Several recent studies suggested that, besides the abnormal development of stem cells, microenvironmental alterations are also present in the MDS bone marrow. In this study, we have examined the relative frequencies of stem and progenitor cell subsets of MDS and normal hematopoietic cells growing on stromal cell layers established from MDS patients and from normal donors. When hematopoietic cells from MDS patients were co-cultured with normal stromal cells, the frequency of either early or late cobblestone area-forming cells (CAFC) was significantly lower compared to the corresponding normal control values in 4 out of 8 patients. In the opposite situation, when normal hematopoietic cells were incubated on MDS stromal cells, the CAFC frequencies were decreased in 5 out of 6 patients, compared to normal stromal layer-containing control cultures. Moreover, a soluble Notch ligand (Jagged-1 protein) was an inhibitor of day-35-42 CAFC when normal hematopoietic cells were cultured with normal or MDS stromal cells, but was unable to inhibit MDS stem and early progenitor cell growth (day-35-42 CAFC) on pre-established stromal layers. These findings suggest that in early hematopoietic cells isolated from MDS patients the Notch signal transduction pathway is disrupted. Furthermore, there was a marked reduction in the plasticity of mesenchymal stem cells of MDS patients compared with those of normal marrow donors, in neurogenic and adipogenic differentiation ability and hematopoiesis supporting capacity in vitro. These results are consistent with the hypothesis that when alterations are present in the myelodysplastic stroma environment along with intrinsic changes in a hematopoietic stem/progenitor cell clone, both factors might equally contribute to the abnormal hematopoiesis in MDS.     

Impaired clonogenic growth of myelodysplastic bone marrow progenitors in vitro is irrelevant to their apoptotic state

Excessive intramedullary apoptosis has been considered to account for the paradox of hypercellular marrow and refractory cytopenias in myelodysplastic syndrome (MDS). However, a causative relationship of apoptosis to the progenitor's defective clonogenic growth has not been sufficiently demonstrated. We investigated the degree of apoptosis and its contribution to ineffective hematopoiesis in MDS, by assessing the differential clonogenic capacity of purified "apoptotic" and "non-apoptotic" bone marrow progenitors in a short-term semisolid culture system. Although increased apoptosis was indeed detected in MDS bone marrow progenitors, there was no correlation between the existence of apoptosis and culture performance. Non-apoptotic as well as apoptotic CD34+ cells gave similar patterns of growth, both defective compared to normal. The ability of "apoptotic" CD34+ cells to proceed in colony formation as well as the abnormal growth of "non-apoptotic" progenitors are probably pointing towards the need to reconsider the role of apoptosis in the defective clonogenicity of MDS.     

Gene expression profiling of myelodysplastic CD34+ hematopoietic stem cells treated in vitro with decitabine

Abnormal gene promoter methylation contributes to deregulate gene expression of hematopoietic progenitors in myelodysplastic syndromes (MDS). We analyzed the gene expression profile of myelodysplastic and normal CD34+ hematopoietic stem cells (HSCs) treated in vitro with decitabine. We identified a list of candidate tumor suppressor genes, expressed at low levels in MDS HSCs and induced by hypomethylating treatment only in MDS, but not in normal HSCs. Real-time RT-PCR confirmed reduced CD9 expression in MDS CD34+ and bone marrow mononuclear cells, compared to normal controls. CD9 was specifically up-regulated by decitabine treatment in myelodysplastic CD34+ cells.     

Comparative multi-color flow cytometric analysis of cell surface antigens in bone marrow hematopoietic progenitors between refractory anemia and aplastic anemia

Refractory anemia (RA) in myelodysplastic syndrome (MDS) without prominent dysplasia closely resemble the mild type of aplastic anemia (AA) in their hematological features. This sometimes makes it difficult to distinguish clearly between the two diseases. Using the multi-color flow cytometric technique, we compared cell surface antigen expression patterns on bone marrow hematopoietic progenitor cells which were isolated as a CD34 positive- CD45 dull positive with low side scatter intensity (CD34(+)CD45(dull+)SSC(low)) population in flow cytogram between RA (n=12) and AA (n=11). The antigens analyzed in CD34(+)CD45(dull+)SSC(low) mononuclear cells were: CD38 and CD71 for cell growth-related antigens, CD 33 and CD13 for myeloid and monocytoid lineage-associated antigens, CD7 and CD19 for lymphoid lineage, and CD14 for a monocytic lineage specific antigen. The percentages of CD34(+)CD45(dull+)SSC(low) cells in bone marrow non-erythroid mononuclear cells, and the expression frequencies of CD38, CD71, CD33 and CD13 antigens in CD34(+)CD45(dull+)SSC(low) progenitors were all significantly decreased in AA compared to normal bone marrows (n=7) (P<0.005). In contrast, in RA bone marrows the percentages of CD34(+)CD45(dull+)SSC(low) cells showed wide distribution and the cell surface antigen expression patterns varied among each case: some cases showed low frequencies of CD38 and CD71 expression as well as AA, whereas the others showed high expression frequency of specific antigen(s) which may reflect the clonal expansion of an abnormal clone in bone marrow. An MDS patient who had progressed from RA to RAEB showed further projecting pattern of expression of CD38 and CD33 in CD34(+)CD45(dull+)SSC(low) population in accordance with the disease progression. These data suggest that analysis of cell surface antigen expression patterns of CD34(+)CD45(dull+)SSC(low) progenitor cells by multi-color flow cytometry appears to be a useful method for qualitative and quantitative assessment of marrow progenitor states in AA and RA, therefore this method could be helpful for early detection of clonal evolution in MDS.     

Impairment in immuno-modulatory function of Flk1(+)CD31(-)CD34(-) MSCs from MDS-RA patients

OBJECTIVE: Myelodysplastic syndromes are a group of hematopoietic disorders characterized by hematopoietic stem cell dysregulation and abnormalities in the immune system. Mesenchymal stem cells (MSCs) and their derived stromal cells constitute a bone marrow microenvironment, which is the niche for hematopoiesis and a key compartment for immune development and regulation. Existing evidence has shown that MSCs from MDS patients have impaired capacity in supporting hematopoiesis. Here, we conducted an investigation to determine whether the immuno-modulatory function of MSCs is also impaired in MDS-RA (refractory anemia) patients. METHOD: Flk1(+)CD31(-)CD34(-) MSCs were isolated from 15 MDS-RA patients and cultured for testing biological and immunological characteristics. RESULTS: MDS-RA patient-derived Flk1(+)CD31(-)CD34(-) MSCs showed normal morphology, phenotype and karyotype but appeared impaired in immuno-modulatory function. The capacity of patient Flk1(+)CD31(-)CD34(-) MSCs to inhibit T lymphocyte activation and proliferation was impaired in vitro. In conclusion, MDS-RA patient-derived MSCs have impaired immuno-modulatory functions, suggesting that the dysregulation of hematopoiesis and immune response may originate from MSCs rather than HSCs. MSCs might be a potential target for developing efficacious cures for MDS.     

髓源性抑制细胞在骨髓增生异常综合征中的研究进展

骨髓增生异常综合征(MDS)以造血细胞发育异常和无效造血为特征,骨髓造血微环境内髓源性抑制细胞(MDSC)的异常扩增和激活可能是重要原因。MDSC细胞扩增与激活导致自然杀伤细胞、CD8+T细胞功能低下与耗竭,并募集炎症细胞及因子,导致MDS患者遗传异常的进一步积累,致使MDS疾病进展。肿瘤环境炎症因子的积累诱导程序性死亡受体1(PD-1)在造血干、祖细胞上的表达和MDSC细胞程序性死亡受体配体1(PD-L1)过表达,PD-1/PD-L1的相互作用导致MDS造血祖细胞的凋亡和无效造血。靶向MDSC的试验及临床研究证实,纠正或逆转MDS免疫失调的骨髓微环境是恢复有效造血功能的治疗策略。     

Expression of DLK1 in hematopoietic cells results in inhibition of differentiation and proliferation

The Delta-like (DLK1) gene is overexpressed in CD34+ cells from myelodysplastic syndrome (MDS) patients. DLK1 encodes an EGF-like homeotic transmembrane protein homologous to the notch/delta/serrate family. Although exogenous DLK1 promotes maintenance of murine hematopoietic stem cells, the functional effects of DLK1 overexpression in hematopoietic cells are unknown. We show that ectopically expressed DLK1 significantly inhibits differentiation and proliferation of human promyelocytic HL-60 cells. Unlike preadipocytes, where proteolytic processing of membrane-bound protein and release of a soluble form mediates differentiation inhibition, proteolytic release of the extracellular domain was not required for inhibition of hematopoietic cell differentiation. However, intracellular domain interactions were critical to this DLK1 function. We conclude that DLK1 overexpression in hematopoietic cells has important functional consequences. Our studies identify novel molecular mechanisms and indicate that DLK1 has activity both as a soluble and a transmembrane expressed protein. Our results support further investigation of the role of DLK1 in abnormal hematopoiesis in MDS.     

CD47在初诊正常核型急性髓系白血病患者中的表达及其临床意义

目的 探讨CD47在初诊正常核型急性髓系白血病(AML)患者中的表达水平及临床意义.方法 选取137例初诊正常核型AML患者及3名健康志愿者.采用流式细胞术及实时荧光定量聚合酶链反应(qPCR)对健康志愿者骨髓造血干细胞(HSC)和造血祖细胞(MPP)及AML患者骨髓单个核细胞(MNC)和白血病干细胞(LSC,Lin-CD34+CD38-CD90-)的CD47表达水平进行检测;采用基因组分析平台检测患者FMS样酪氨酸激酶3内部串联重复(FLT3-ITD).采用超高速流式分选系统分选CD34+CD38-CD47lo和CD34+CD38-CD47hi细胞.将两组细胞均用MethoCult H4445培养液培养接种于含有琼脂糖的甲基纤维素板,12 d后计数MPP集落形成单位(CFU),同时将1×105个CD34+CD38-CD47lo和CD34+CD38-CD47hi细胞分别移植到经280 cGy照射的NSG(NOD-SCID IL-2Rγnull)小鼠,8周后处死小鼠,流式细胞术检测人CD45+细胞比例.结果 CD47的表达在初诊正常核型AML患者中高于健康志愿者,CD47在AML各法、美、英协作组(FAB)亚型中均有表达,相对表达量比较差异无统计学意义(F=0.545,P>0.05).137例患者中CD34+CD38-CD47hi 37例,其中17例(46%)FLT3-ITD阴性,20例(54%)为FLT3-ITD阳性;CD34+CD38-CD47lo的患者100例,其中63例(63%)FLT3-ITD阴性,37例(37%)FLT3-ITD阳性,CD34+CD38-CD47hi与CD34+CD38-CD47lo患者相比较,FLT3-ITD阳性率差异无统计学意义(χ2=3.79,P>0.05).将FACS分选的CD34+CD38-CD47lo和CD34+CD38-CD47hi接种于含有琼脂糖的甲基纤维素板12 d后,仅CD34+CD38-CD47lo细胞可以形成CFU.NSG小鼠移植实验显示CD34+CD38-CD47lo细胞可重建造血,CD34+CD38-CD47hi植入失败.结论CD34+CD38-CD47hi富集LSC,可作为AML患者化疗后微小残留病的监测指标.     

Persisting multilineage transgene expression in the clonal progeny of a hematopoietic stem cell.

Many applications of hematopoietic gene therapy require selection for clones with active transgene expression. However, it was unclear whether the clonal progeny of a retrovirally transduced hematopoietic stem cell would be capable of maintaining transgene expression through serial repopulation and multilineage differentiation. Such investigations require simultaneous analyses of clonality, multilineage activity and transgene copy numbers. Using a mouse model, the present study demonstrates that a single hematopoietic stem cell expressing a marker gene from one or two insertions of a simple retroviral vector actively maintains multilineage transgene expression in the vast majority (80-99%) of bone marrow and peripheral blood cells. Gene expression persisted through serial transplantations for at least 97 weeks post gene transfer and was observed in the lymphoid (B, T and NK cells), myeloid (CD11b(+), Gr-1(+)), erythroid (Ter119(+), mature red blood cells) and megakaryocytic (as indicated by platelets) progeny. Therefore, a single immunoselection for hematopoietic stem cells expressing the transgene in vivo was sufficient to establish a completely chimeric hematopoiesis. These observations imply that the retroviral vectors used in this study contain cis-elements that mediate expression through massive clonal expansion and multilineage differentiation, provided the insertion occurred in genetic loci permissive for expression in hematopoietic stem cells.     

Myelodysplastic syndrome hematopoietic stem cell

Myelodysplastic syndromes (MDSs) are clonal hematopoietic stem cell (HSC) malignancies that are characterized by ineffective hematopoiesis and frequent progression to acute myeloid leukemia (AML). Thus far, few treatments can actually alter the natural history of this disease. Allogeneic stem‐cell transplantation for high‐risk MDS is becoming the only curative therapy probably because of the improvement of bone marrow transplant procedures. The lack of other options underscores the urgent need to develop new therapy. The prevailing model suggests that genetic and/or epigenetic alterations that occur in HSCs or HSC niche compromise HSC function, resulting in MDS; therefore, MDS HSCs are likely the ideal targets for MDS treatment. Recent encouraging advances—capturing a molecular portrait of the whole genome of MDS CD34⁺ cells, including identifying altered signaling pathways and altered microRNAs—have improved our understanding of MDS pathogenesis and provided novel potential clinical targets for MDS. Here, I will briefly review the characteristics of MDS HSCs and discuss the therapeutic promise of targeting MDS HSCs.     

Down regulation of stem cell colony formation by purified CD8 lymphocytes and CD8 conditioned medium: potential importance for bone marrow transplantation in leukemia.

A methodology for selection of the CD8 cell subset from the peripheral blood and bone marrow mononuclear cells was developed using anti-T8 (CD8) antibody and magnetic microspheres coated with anti-mouse IgG. Following optimization of antibody:cell binding ratio and microsphere:cell ratios, CD8(+)-cells in the peripheral blood and bone marrow were effectively removed, with an overall final recovery of 34.9% +/- 8.6%, and 56% +/- 8.5% respectively with complete recovery of stem cells and very little contamination with effector cells. CD8(+)-depleted cell preparations demonstrated a 3-4 fold increase in CFU-C and CFU-E colony formation over non-depleted preparations when stimulated with G-CSF, GM-CSF or IL-3 and erythropoietin. The largest increase in colony formation was evident when IL-3 was used to stimulate colony formation. Purified autologous CD8+ T-cells or culture supernatant from in vitro cultures of purified autologous CD8+ T-cells added back to CD8 depleted preparations, induced 20%-90% suppression of CFU-C and CFU-E colony formation in a dose dependent manner. Colony formation by CD34+ cells, purified by anti CD34 antibodies and magnetic microspheres, were also inhibited by either pure CD8(+)-cell populations or CD8-culture supernatant. Preliminary fractionation studies indicate that the inhibitory factor is a protein of > 50 kd. In contrast, when purified autologous CD4+ cells were added to purified CD34+ stem cells, an increase (< 50%) in colony formation was observed. These results, taken together, suggest that CD8+ T-cells are negative effectors of normal hematopoiesis whereas CD4+ T-cells function as positive effectors.     

Clonality of the stem cell compartment during evolution of myelodysplastic syndromes and other bone marrow failure syndromes.

Clonal hematopoiesis, observed in certain forms of marrow failure including aplastic anemia (AA), may be due to stem cell depletion. Alternatively, oligoclonality may be a result of recruitment of a preexisting defective clone, such as in paroxysmal nocturnal hemoglobinuria (PNH) or myelodysplastic syndromes (MDS). In PNH, exogenous permissive factors may be required for dominance of the abnormal clone, while in MDS, stem cells undergo transformation steps leading to a growth advantage. Stem or multipotent progenitor cell involvement in PNH is evidenced by long-term persistence of a clonal defect and its presence in all blood cells. In MDS, some clonal aberrations may have a 'founder-effect' and additional defects are secondary. Metaphase cytogenetics measures the proportion of clonal cells within dividing progenitor but not mature cells. Owing to low resolution, lesions can be found in only approximately 50% of MDS patients. This shortcoming may be overcome by application of newer technologies such as comparative genomic hybridization and SNP array-based karyotyping (SNP-A). SNP-A facilitates identification of cryptic lesions in bone marrow failure patients with normal or abnormal cytogenetics and allows for detection of loss of heterozygosity as a result of uniparental disomy, a lesion frequently found in MDS.