The role of mesenchymal stem cells in allogeneic hematopoietic stem cell transplantation for patients with refractory severe aplastic anemia

<正>Objective To evaluate the efficacy and safety ofmesenchymal stem cells in allogeneic hematopoietic stemcell transplantation for patients with refractory severeaplastic anemia(R-SAA).MethodsThe clinical data of25 R-SAA patients receiving co-transplantation of mesen-chymal stem cells combined with peripheral blood stem     

Allografting in patients with severe, refractory aplastic anemia using peripheral blood stem cells and a fludarabine-based conditioning regimen: the Mexican experience

We studied the effectiveness of a fludarabine/cyclophosphamide-based conditioning regimen without anti-thymocyte globulin in 23 aplastic anemia patients who had no response to previous conventional pharmacologic treatment. Patients received oral busulphan 4 mg/kg/day/2 days, IV cyclophosphamide 350 mg/m(2)/day/3 days, and fludarabine 30 mg/m(2)/day/3 days. For GVHD prophylaxis, patients received MTX 5 mg/m(2) days +1, +3, +6, and +11 and oral cyclosporin A (CyA) 5 mg/kg/day, starting on day -1. Peripheral blood stem cell products were used with a median dose of 5.5 x 10(6) CD34(+)/kg. The patients were followed for an average of 25 months. By a median of day +11, an ANC > 0.5 x 10(9)/L was reached; and by day +12, the platelet count had reached >20,000 x 10(9)/L. Acute grade I-II GVHD occurred in 4 patients, whereas limited chronic GVHD presented in 6 cases. Twenty-one patients (91.3%) achieved engraftment. Two patients failed to engraft, and 4 developed late rejection; 2 of these individuals died, 2 have survived with high transfusion requirements, whereas 2 received a second peripheral blood stem cell infusion and achieved sustained engraftment. Currently 21 (91%) of the 23 patients are alive, whereas 19 of 21 (90%) remain in complete remission. The average cost was about USD 15,000 for this kind of reduced-intensity allotransplant. Reduced-intensity stem cell transplantation represents an affordable alternative to traditional more cytotoxic conditioning for severe aplastic anemia (SAA) patients. Long-term effects however, remain to be evaluated.     

非清髓无关供体外周血干细胞移植治疗重型再生障碍性贫血

目的 探讨无关供体造血干细胞移植治疗重型再生障碍性贫血(SAA)的方法 和疗效.方法 对1例SAA的患者进行了无关供体HLA高分辨4/6相合的外周血干细胞移植.采用环磷酰胺(100 mg/kg) 氟达拉宾(150 mg/m2) 抗人淋巴细胞球蛋白(100 mg/kg)的非清髓性预处理后,回输粒细胞集落刺激因子(G-CSF)动员的外周血干细胞,共输注单个核细胞(MNC)6.77×108/kg,CD 34细胞1.95×106/kg.预防移植物抗宿主病(GVHD)采用环胞菌素A(CsA)联合短疗程甲氨蝶呤(MTX)的基础上加用霉酚酸酯(MMF)的方案.结果 患者移植后造血恢复顺利,于移植后第6天WBC植入,第8天PLT植入,第30天行患者骨髓STR-PCR检测显示为完全供者的基因型,第150天血型转变为供者型(O→A).未发生急性GVHD(aGVHD)及慢性GVHD(cGVHD),随访至移植后8个月,造血功能恢复良好,仍在继续随访中.结论 以氟达拉宾、环磷酰胺和抗人淋巴细胞球蛋白组成的非清髓性预处理方案用于无关供体外周血干细胞移植治疗SAA,能够获得稳定的植入,且并发症少,是有效移植方法之一.     

T-cell suppression mediated by mesenchymal stem cells is deficient in patients with severe aplastic anemia

OBJECTIVE: To compare the suppressive effect of mesenchymal stem cells (MSC), derived from normal individuals or severe aplastic anemia patients (SAA), on T-cell activation. PATIENTS AND METHODS: We studied bone marrow MSC from 19 healthy donors and 23 SAA patients in different phases of the disease: at diagnosis (n = 3), following immunosuppressive therapy (IS) (n = 16), or after a bone marrow transplant (BMT) (n = 4). MSC were tested for T-cell suppression in the following assays: mixed lymphocyte reaction (MLR), phytohemaglutinin (PHA)-primed cultures, activation surface markers, gamma-IFN production, hematopoietic colony formation (CFC), production of cyclic ADP-ribose (cADPR). RESULTS: The abnormalities of SAA MSC included: 1) significantly lower suppression of T-cell proliferation induced by alloantigens (p = 0.009) or PHA (p = 0.006); 2) impaired capacity to suppress CD38 expression on PHA-primed T cells (p = 0.001); 3) impaired ability to suppress gamma-IFN production in PHA cultures, resulting in an 11-fold higher gamma-IFN concentration; 4) no preventive effect on T cell-mediated inhibition of CFC; and 5) significantly reduced (p = 0.009) production of cADPR, a universal calcium mobilizer. MSC-mediated suppression of PHA-induced T-cell proliferation was restored to control levels in 3 of 4 patients post-BMT. CONCLUSION: The ability of MSC to downregulate T-cell priming, proliferation, and cytokine release is deficient in patients with SAA, persists indefinitely after immunosuppressive therapy, but seems to be restored after BMT. Whether these abnormalities are relevant to the pathogenesis of aplastic anemia remains to be determined.     

Non-radiotherapy conditioning with stem cell transplantation from alternative donors in children with refractory severe aplastic anemia

Conditioning including total body/lymphoid irradiation is widely used to prevent graft rejection in patients with refractory severe aplastic anemia (SAA) undergoing hemopoietic cell transplantation (HCT) from alternative donors and or after graft manipulation. To reduce regimen-related toxicity we transplanted three children with refractory SAA after conditioning with radiotherapy-free regimens. Conditioning included fludarabine 175-180 mg/m2 in all patients. In addition, patient 1 (failing two previous grafts) received thiotepa 10 mg/kg and Campath-1H 60 mg/m2; patient 2 cyclophosphamide 120 mg/kg, thiotepa 15 mg/kg and OKT-3 0.1 mg/kg/day for 4 weeks; and patient 3 cyclophosphamide 120 and ATG 90 mg/kg. Stem cell source was unmanipulated marrow from the same unrelated donor as for the two previous transplantations in patient 1 and CD34+-purified peripheral blood stem cells from an HLA-matched unrelated donor and from the haploidentical mother in patients 2 and 3. Only patient 1 received graft-versus-host disease (GVHD) prophylaxis with cyclosporine A and mycophenolate mofetil. Follow-up is now 30, 51, and 15 months. None of the patients developed GVHD. All patients have normal counts with complete donor chimerism. Fludarabine-based conditioning is powerfully immunosuppressive and may be used for children with refractory SAA undergoing HCT from alternative donors even after rejection following previous HCT.     

脐带华通胶间充质干细胞对急性再生障碍性贫血T淋巴细胞亚群的影响

目的体外观察脐带华通胶间充质干细胞（UCWJMSCs）对急性再生障碍性贫血（AAA）患者T淋巴细胞（TLC）亚群的调节作用。方法收集21例AAA患者和10例健康志愿者外周血,制备单个核细胞（MNCs）悬液,UC-WJMSCs和MNCs以一定比例混合共育一定时间后,采用流式细胞仪检测TLC亚群并进行统计分析。结果 AAA患者TLC亚群呈Th1、Tc1极化状态。混合培养后,健康人的TLC亚群未见明显变化（P〉0.05）;AAA患者的TLC亚群除Th2与Tc2无明显变化外,CD3＋、CD8＋、CD2＋8、Th1和Tc1亚群比例明显降低,而CD4＋、CD4＋/CD8＋、CD5＋6、CD2＋5和CD1＋03亚群比例明显升高（P〈0.05）。调节作用24 h就已产生7,2 h时达高峰;调节作用随UCWJMSCs浓度的增高而增强。结论 UCWJMSCs对AAA患者的TLC亚群有明显调节作用,不同个体来源的UCWJMSCs调节作用无区别。     

HLA半相合非清髓性造血干细胞与间充质干细胞共移植治疗重症再生障碍性贫血1例

目的:观察HLA半相合非清髓性造血干细胞与间充质干细胞(MSC)共移植治疗重症再生障碍性贫血(SAA)的疗效及安全性。方法:1例24岁男性SAA患者。应用非清髓性预处理方案,进行HLA半相合异基因外周血造血干细胞和MSC共移植。移植rhG-CSF动员的供者外周血单个核细胞9.22×108/kg,CD34 细胞8.56×106/kg,及体外扩增培养的供者骨髓MSC2.12×105/kg。结果:移植后 12d中性粒细胞数>0.5×109/L, 21d WBC4.5×109/L,Hb99g/L,PLT108×109/L。经HLA配型,红细胞亚型和VNTR检测,为供者型完全嵌合体。随访14个月,无急、慢性移植物抗宿主病(GVHD)发生。结论:HLA半相合非清髓性造血干细胞与MSC共移植治疗SAA是安全有效的方法。     

Outcome of unrelated donor stem cell transplantation for children with severe aplastic anemia

For children with severe aplastic anemia (SAA) who fail immunosuppressive therapy and lack a human leucocyte antigen (HLA)-matched sibling donor, unrelated donors provide a source of hematopoietic stem cells. Data from 195 children with acquired SAA who underwent unrelated donor transplantation between 1989 and 2003 were analyzed. Neutrophil recovery (86% at day-28) was higher with total body irradiation-containing conditioning regimen and in younger recipients (aged ≤16 years) receiving grafts from older donors (aged >40 years). Recovery was lower after mismatched transplants and transplantations prior to 1997. Mortality rates were higher after mismatched transplants, in recipients with a poor performance score, and when the interval between diagnosis and transplantation was longer than 4 years. When restricted to donor-recipient pairs with allele-level HLA typing (8-loci; n  = 118), mortality rates were also higher after mismatched transplants and older recipients receiving grafts from older donors; 5-year probabilities of overall survival after HLA-A, -B, -C, -DRB1 matched and mismatched transplants adjusted for donor and recipient age were 57% and 39%, respectively ( P  = 0·008). The data suggest that unrelated donor transplantation is an acceptable alternative for children; early referral for transplantation and identification of an HLA-matched (allele-level) donor offers the best outcome.     

非血缘异基因脐血移植治疗SAA可行性研究

目的评价非血缘脐血移植(UCBT)治疗急性重型再生障碍性贫血(SAA)的疗效。方法SAA患者6例,采用环磷酰胺和抗胸腺细胞球蛋白预处理方案;移植方式为HLA不全相合UCBT;应用环孢素A和骁悉预防移植物抗宿主病(GVHD)。随访时间为60~1358d。结果移植后l2~38d造血重建。短串联重复序列-聚合酶链反应分析显示移植后l7~150d基因型表现为完全性患者型,未发生GVHD。血常规、骨髓象检查正常。结论非血缘HLA配型不合的脐血用于治疗SAA是可行的。     

Successful sustained engraftment after reduced-intensity umbilical cord blood transplantation for adult patients with severe aplastic anemia

We retrospectively analyzed 12 consecutive adult severe aplastic anemia patients who received unrelated umbilical cord blood transplantation after a reduced-intensity conditioning regimen (RI-UCBT). The conditioning regimen consisted of 125 mg/m2 fludarabine, 80 mg/m2 melphalan, and 4 Gy of total body irradiation. The median infused total nucleated cell number and CD34(+) cell number were 2.50 × 10?/kg and 0.76 × 10?/kg, respectively. Eleven of the 12 patients achieved primary neutrophil and platelet engraftment. All patients who achieved engraftment had complete hematologic recovery with complete donor chimerism, except for one patient who developed late graft failure 3 years after RI-UCBT. Two of the 12 patients died of idiopathic pneumonia syndrome, and the remaining 10 patients are alive, having survived for a median of 36 months. Our encouraging results indicate that RI-UCBT may become a viable therapeutic option for adult severe aplastic anemia patients who lack suitable human leukocyte antigen-matched donors and fail immunosuppressive therapy.     

Supplemental peripheral blood stem cells to decrease marrow rejection in adult patients with severe aplastic anemia

Twenty-two multi-transfused patients with a long duration of severe aplastic anemia (SAA) received a transplant from an HLA-matched donor after cyclophosphamide (CY) plus antithymocyte globulin plus procarbazine using CD34(+) enriched blood stem cells + fresh marrow. Peripheral blood stem cells (PBSC) were collected on days 5 and 6 of G-CSF (10 microg/kg/day), and T cells were depleted using an immunoadsorption column (n = 15) or magnetic cell sorting (n = 7). Marrow harvesting was performed 48 hr after the last leukapheresis. Two patients (9.1%) that developed graft failure had a successful engraftment again using unpurged PBSC. Median time to neutrophils > or = 0.5 x 10(9)/l and platelets > or = 20 x 10(9)/l without platelet transfusions were 12 days and 17 days, respectively. Acute graft-versus-host disease (GVHD) grade II occurred in four of 22 patients. No patient developed grade III or IV acute GVHD. Four of the evaluable 21 patients had chronic GVHD. One patient developed extensive disease. Three patients (13.6%) died from CY-induced heart failure, extensive-type chronic GVHD, and sepsis of unknown cause. The Kaplan-Meier estimate of survival was 83.9% (95% CI, 70.1-95.2%) with a median follow-up duration of 33.5 (6-44) months. CD34(+)-enriched PBSC in combination with unmanipulated marrow seem to play a role in overcoming the sensitization to histocompatibility antigens without an apparent increase in GVHD. The stem cell component therapy may be feasible for the high-risk SAA adult patients.     

Sustained and stable hematopoietic donor-recipient mixed chimerism after unrelated cord blood transplantation for adult patients with severe aplastic anemia

We evaluated the engraftment of donor cells from unrelated cord blood into adult patients with severe aplastic anemia (SAA) and the outcome of allo-CBSCT (cord blood stem cell transplantation). Nine patients were conditioned with decreased dosage of immunosuppressive agents of CTX (60 mg/kg) and ALG (120 mg/kg). The prophylaxis of GVHD consisted of standard CsA and MTX. Patients have a media age of 25.3 yr (range: 15-37), and a median weight of 57.2 kg (range: 52.5-60) at the time of transplantation. Cord blood searches were all conducted at Guangzhou Cord Blood Bank. The engraftment state of the donor cells into recipients was confirmed by microsatellite DNA fingerprinting and fluorescent quantitative PCR analysis. Engrafted evidence has been found in seven patients involved by biomolecular analyses showing donor-recipient mixed chimerism post-transplant which was stable and persistent. After a median follow up of 32.2 months (range: 4-69), seven patients were alive and disease free. This study shows that durable donor-recipient stable mixed chimerism can be achieved by unrelated CBSCT in patients with SAA. Umbilical cord blood could be employed as a source of hematopoietic stem cell for adult transplantation.     

Cotransplantation of haploidentical hematopoietic and umbilical cord mesenchymal stem cells with a myeloablative regimen for refractory/relapsed hematologic malignancy

Human leukocyte antigen haploidentical hematopoietic stem-cell transplantation (haplo-HSCT) is associated with an increased risk of graft failure and severe graft-versus-host disease (GVHD). Mesenchymal stromal cells (MSCs) have been shown to support in vivo normal hematopoiesis and to display potent immunesuppressive effects. We cotransplanted the culture-expanded third-party donor-derived umbilical cord MSCs (UC-MSCs) in 50 people with refractory/relapsed hematologic malignancy undergoing haplo-HSCT with myeloablative conditioning. We observed that all patients given MSCs showed sustained hematopoietic engraftment without any adverse UC-MSC infusion-related reaction. The median times to neutrophil >0.50?×?10⁹/L and platelet >20?×?10⁹/L engraftment were 12.0 and 15.0 days, respectively. We did not observe an increase in severe acute GVHD (aGVHD) and extensive chronic GVHD (cGVHD), too. Grade II–IV aGVHD was observed in 12 of 50 (24.0 %) patients. cGVHD was observed in 17 of 45 (37.7 %) patients and was extensive in 3 patients. Additionally, only five patients (10.0 %) experienced relapse at a median time to progression of 192 days. The probability that patients would attain progression-free survival at 2 years was 66.0 %. The results indicate that this new strategy is effective in improving donor engraftment and reducing severe GVHD, which will provide a feasible option for the therapy of high-risk hematologic malignancy.     

Syngeneic peripheral blood stem cell transplantation with brief immunosuppression for severe aplastic anemia

We report the cases of two severe aplastic anemia (SAA) patients who were successfully treated with syngeneic peripheral blood stem cell transplantation (PBSCT) using immunosuppression without high-dose chemotherapy or irradiation for conditioning. A 21-year-old woman with SAA of 6 years duration had been transfused heavily before transplantation and had developed refractory thrombocytopenia, chronic hepatitis and secondary hematochromatosis. Syngeneic PBSCT with immunosuppression using ATG, methylprednisolone, and cyclosporin-A was eventually performed without high-dose chemotherapy in September 1997. The second syngeneic PBSCT with the same immunosuppression was successfully performed in a 35-year-old male patient who had had SAA for 3 months in November 1998. Haemopoietic engraftment was rapid and sustained. There was no infection or mucositis during the syngeneic PBSCT. The patients are currently 9 to 22 months post-PBSCT without rejection. Our experience suggests that syngeneic PBSCT with brief immunosuppression is an effective alternative to pretransplant high-dose chemotherapy conditioning for SAA patients having syngeneic transplantation. Bone Marrow Transplantation (2000) 25, 337-339.     

再生障碍性贫血患者骨髓间充质干细胞成脂和成骨分化能力的变化

目的 通过观察再生障碍性贫血(再障)患者间充质干细胞(MSCs)成脂肪和成骨能力的变化,研究骨髓MSCs成脂分化的异常在再障患者红髓脂肪化中的作用.方法 分离培养再障患者及正常人的骨髓,观察其一般生物学特性,并在体外诱导其向脂肪、成骨细胞分化,同时用RT-PCR方法检测成脂肪、成骨特异基因的表达时间,比较再障患者和正常对照MSCs向脂肪细胞、成骨分化能力的不同.结果 原代培养7 d,再障组贴壁细胞克隆形成率为(19.30±4.77)/(5×105 MNCs),较正常对照组明显降低(47.72±3.46)/(5×105 MSCs)(P＜0.05).在培养初期,再障组MSCs与正常对照MSCs增殖能力相似,但在连续传8代后,其增殖能力降低.再障组MSCs体外诱导成脂滴早,诱导分化的脂肪细胞leptin(瘦素)基因表达早.再障组MSCs体外诱导成骨形成钙化结节少,碱性磷酸酶活性低,诱导的成骨细胞osteocalcin(骨钙素)基因表达晚.结论 再障患者MSCs成脂分化能力增强而成骨分化能力降低,可能在再障的病程中起一定作用.     

Unrelated peripheral blood stem cell transplantation with 'megadoses' of purified CD34+ cells in three children with refractory severe aplastic anemia

Three children with refractory severe aplastic anemia were transfused with high numbers of unrelated matched (n = 2) or C-locus haploidentical mismatched (n = 1) CD34-selected peripheral blood stem cells in the absence of an HLA-identical family donor. Two leukaphereses of the donors yielded a median number of 10.1 x 10(10) nucleated cells (range 9.7-15.4) with a median number of 9.89 x 10(8) CD34+ cells (range 7.46-26.1) and a median percentage of CD34+cells of 0.98% (range 0.77-1.7). After positive selection by magnetic cell sorting the patients received a median of 14.3 x 10(6) CD34+ cells/kg (range 11.7-24.3) and of 1.3 x 10(4) CD3+ cells/kg (range 0.57-5.8). Median time to ANC >/=0.5 x 10(9)/l was 7 days (range 7-12) and to platelets >/=20 x 10(9)/l 13 days (range 13-27). Chimerism analysis of peripheral blood after transplantation revealed permanent 100% donor hematopoiesis in all patients. The patient with the C-locus haploidentical mismatch presented with acute GVHD (grade III-IV) of the skin, liver and lower gastrointestinal tract (onset day +40) and died despite intensive immunosuppressive treatment on day +238. The two survivors developed lymphopoietic recovery of B and T lymphocytes within 3 months after transplantation. To our knowledge this experience represents the first report of transplantation with unrelated CD34+ enriched peripheral blood stem cell in children with refractory severe aplastic anemia. Bone Marrow Transplantation (2000) 25, 513-517.     

Umbilical cord blood transplant for adult patients with severe aplastic anemia using anti-lymphocyte globulin and cyclophosphamide as conditioning therapy

Allo-CBSCT (cord blood stem cell transplant) has been applied in six adult patients with severe aplastic anemia (SAA). Anti-lymphocyte globulin (ALG) 40 mg kg(-1) d(-1) x 3 days combined with cyclophosphamide (CTX) 20 mg kg(-1) d(-1) x 3 days constituted a lower intensive conditioning regimen. The prophylaxis of GVHD consisted of standard CsA and MTX. Patients are all male having a mean age of 26.5 years (range 22-38), and a median weight of 55.6 kg (range 52-60 kg). Cord blood searches were all conducted at Guangzhou Cord Blood Bank. Three of six patients in our study received one unit of cord blood in a procedure, whereas for another three patients, two units of cord blood (double units) were infused at the same time in a transplant protocol. The nine units of umbilical cord blood (UCB) infused contained 1.6-10.7 x 10(7) nucleated cells/kg body weight of the recipient after thawing. HLA antigens were identical in one unit, 1 antigen mismatched in seven, 2 antigens mismatched in 1. As of February 2003, after a median follow up of 20 months (range 7-50), four patients are alive and disease free. Five patients engrafted with molecular biology analyses showing donor-recipient mixed chimerism post transplant which is stable and persistent. One patient died of severe infection in the third month from transplant and another patient died in the early stage post transplant of serious aspergillus infection without evidence of engraftment.