CD34+ selected cells in mismatched stem cell transplantation: a single centre experience of haploidentical peripheral blood stem cell transplantation

Over the past 3 years we have performed 10 haploidentical peripheral blood stem cell transplants in patients with incurable haematological malignancies and no prospect of a matched unrelated donor within an adequate time period. Conditioning consisted of ATG, TBI, thiotepa, cyclophosphamide and additional radioimmunotherapy in five patients. All patients received G-CSF mobilized peripheral blood stem cell grafts. GVHD prophylaxis consisted of T cell depletion by CD34+ selection; no post-transplant immunosuppression was given in nine patients. Stable engraftment was achieved in nine patients; one case of acute graft rejection was observed. Seven patients developed grade I acute GVHD, and six patients have developed chronic GVHD. Infections were the most significant clinical problem post transplant. Two patients have suffered a relapse of their disease and two further patients have died of transplant-related complications. After a median follow-up of 13 months (range 5-37 months) six patients are surviving in remission. We conclude that haploidentical PBSCT is a reasonable alternative to a MUD transplant.     

Large-scale isolation of CD133+ progenitor cells from G-CSF mobilized peripheral blood stem cells

We have evaluated the feasibility of large-scale isolation of CD133+ progenitors from healthy mobilized adult donors for potential clinical use in autologous and allogeneic transplantation. A total of 11 healthy volunteer adult donors were mobilized with G-CSF. CD133+ stem cells were isolated from a single leukapheresis using the Clinimacs method. The median percentage of CD133 before positive selection was 0.75% (range 0.39-2.03%). After selection, the median purity and recovery was 94% (range 85.2-98.0%) and 69% (range 44-100%), respectively. The median log10 T-cell depletion obtained by CD133+ positive selection was 4.2 (range 3.8-4.7). The CD133+ progenitors were highly enriched in colony-forming units (CFU) and transplantation into NOD/SCID mice resulted in a high engraftment rate. Transplantation of sorted CD133+/CD34+ cells into NOD/SCID mice showed a higher engraftment compared to CD133-/CD34+ cells. Mobilized peripheral CD133+ stem cells can be purified in large scale for potential clinical use. The biological function of the cells is not impaired. The majority of the NOD/SCID repopulating cells are within the CD133+/CD34+ subpopulation. Therefore, clinical studies using purified CD133+ stem cells can be envisoned to further clarify the role of CD133+ stem cells in hematopoietic reconstitution after transplantation.     

Generation of CD133+ cells from CD133- peripheral blood mononuclear cells and their properties

OBJECTIVE: CD133 may be the most specific marker of endothelial progenitor cells (EPCs), which are thought to be largely confined to the bone marrow milieu. This study reports on the phenotypic characterization and functional analysis of human CD133+ cells and their generation from cells in the peripheral circulation. METHODS: Adult human CD133+ and CD133- cells were isolated from peripheral blood mononuclear cells, and the generation of CD133+ cells in culture was attempted using different culture combinations. The phenotypic, migratory, adhesive, and angiogenic properties of the native and generated populations were investigated. RESULTS: In adherent and in suspension culture systems, CD133+ cells also expressing CD34 and VEGFR-2 were successfully derived from a previously CD133- population. The migratory potential of CD133+ cells was enhanced by the presence of the CD133- cells. Also, the CD133+ cells derived from the CD133- cells demonstrated improved adhesion to extracellular matrix and endothelial monolayer substrates, and their contribution to in vitro angiogenesis was enhanced compared to freshly isolated CD133+ cells. CONCLUSIONS: These results demonstrate a source of blood CD133+ cells other than direct mobilization from the bone marrow. Cellular interaction was observed between fractions, with CD133+ cells showing better in vitro function in the presence of CD133- cells. These findings provide a novel source for CD133+ cells and a rationale for the investigation of angiogenic cell recruitment or delivery strategies involving more than one cell type at ischemic sites.     

Allogeneic transplantation using peripheral blood stem cells

Over the past 9 years there has been a remarkable increase in the use of peripheral blood stem cells (PBSC) for allogeneic transplantation, primarily for matched sibling transplants but also increasingly for unrelated donor transplantation. In 1999 over 50% of all sibling transplants and over 25% of unrelated donor transplants reported to the European Group for Blood and Marrow Transplantation (EBMT) used PBSC. The major reason for this increasing use of PBSC relates to the rapid haemopoietic recovery seen which mirrors the advantages of using PBSC in autologous transplantation. This improvement in engraftment is a consequence of the larger number of stem cells that can be collected from G-CSF-mobilized peripheral blood compared to bone marrow. Evidence from randomized trials now shows a survival advantage for the use of PBSC in patients with advanced leukaemia. The reason for this improved survival appears primarily to relate to a reduced risk of transplant-related mortality and, possibly, a reduced risk of relapse, However, these randomized studies have also confirmed that there is an increased risk of chronic graft-versus-host disease associated with PBSC transplantation and further follow-up is required to determine the long-term impact on outcome.     

Successful peripheral blood stem cell mobilization using pegfilgrastim in allogeneic stem cell transplantation

Pegfilgrastim is produced by binding a 20,000-dalton polyethylene glycol molecule to granulocyte colony-stimulating factor (G-CSF), increasing the mass of the compound, and resulting in a longer-lasting form of G-CSF. This makes it more convenient to use pegfilgrastim as a single-day injection. This study was a prospective phase II single-center trial. Fifteen normal related donors received pegfilgrastim 12 mg subcutaneously to mobilize peripheral blood stem cells (PBSC) for allogeneic stem cell transplantation. Leukapheresis was planned to start 3 days after injection. All harvests were successful. Median number of leukapheresis was 2 days (range 1–3 days). There were 7/15 donors who only required single leukapheresis. The maximum concentration of white blood cells (WBC) and circulating CD34 cells occurred 3 days after pegfilgrastim injection (WBC: median 62,200/μl; CD34: median 69.76/μl). The median yield of CD34 cells was 6.78 × 10⁶/kg recipient weight. The median CD3 cells was 1.89 × 10⁸/kg recipient weight. The main adverse events were bone pain and headache. Median neutrophil and platelet engraftments in the recipients occurred on day 12 and day 13, respectively, after transplantation. PBSC mobilization with single-day injection of pegfilgrastim in normal donor is feasible. Further comparisons of this protocol to standard G-CSF for allogeneic stem cell mobilization should be conducted in future.     

CD34~+ CD38~-细胞对异基因造血干细胞移植的影响研究

目的观察CD34+CD38-细胞对异基因造血干细胞移植术后造血重建和移植物抗宿主病(GVHD)的影响。方法分析2004年1月至2009年12月河南省人民医院血液科全相合异基因外周血干细胞造血干细胞移植78例,CD34+、CD34+CD38-细胞输入量与血缘全相合异基因外周血造血干细胞移植术后造血重建及GVHD发生率间的相关性。结果粒细胞、血小板恢复时间与CD34+CD38-细胞输入量呈负相关(r分别为-0.521、-0.448,P<0.01),与CD34+细胞输入量也呈负相关(r分别为-0.405、-0.371,P<0.05)。急性GVHD、慢性GVHD的发生与CD34+、CD34+CD38-、CD3+、CD4+、CD8+细胞输入量无相关性。结论输入高数量的CD34+CD38-细胞有利于移植术后的粒细胞、血小板快速恢复;对于预测术后造血恢复,CD34+CD38-细胞亚群输入量可能优于CD34+细胞总数。     

Autologous CD34+ and CD133+ stem cells transplantation in patients with end stage liver disease

AIM:To assess the utility of an autologous CD34 + and CD133 + stem cells infusion as a possible therapeutic modality in patients with end-stage liver diseases.METHODS:One hundred and forty patients with endstage liver diseases were randomized into two groups.Group 1,comprising 90 patients,received granulocyte colony stimulating factor for five days followed by autologous CD34 + and CD133 + stem cell infusion in the portal vein.Group 2,comprising 50 patients,received regular liver treatment only and served a...     

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.     

Successful peripheral blood stem cell transplantation for myelodysplastic syndrome

Wilms' tumor (WT1) gene expression is increased in patients with leukemia as well as myelodysplastic syndrome (MDS) and is useful for detection of minimal residual disease (MRD). A 47-year-old man given a diagnosis of refractory anemia with excess of blasts in transformation (RAEB-T) received myeloablative therapy followed by autologous peripheral blood stem cell transplantation (PBSCT). MRD by WT1 expression was not detected in the graft. The patient has been in CR for 25 months after PBSCT. These observations suggest that PBSCT is feasible for patients with RAEB-T and analysis of WT1 expression can be applied for patients with high risk MDS.     

Successful hematologic reconstitution using CD34+ cells positively selected from cryopreserved autologous peripheral blood stem cells in a patient with malignant lymphoma

Clinical use of CD34+ cells positively selected from cryopreserved peripheral blood stem cells (PBSC) has been limited, and there have been only a few reports of this procedure, mainly because clump formation decreases the proportion of CD34+ cells that can be recovered. A 49-year-old Japanese woman with non-Hodgkin's lymphoma (NHL) (follicular mixed, B cell, stage IVA) was treated with seven cycles of conventional chemotherapy and achieved partial remission. During hematopoietic recovery after the seventh course of chemotherapy, PBSC were harvested by continuous leukapheresis and cryopreserved. However, clonal rearrangement of the immunoglobulin heavy chain gene was detected in the PBSC by Southern blot analysis. After high-dose chemotherapy, CD34+ cells were positively immunoselected from the cryopreserved PBSC and infused into the patient at 1.97 x 10(6)/kg. The overall purity and recovery rate of the CD34+ cells were 72.2% and 65.0%, respectively. There were no severe adverse effects after PBSC transplantation, and the time required for recovery of neutrophils to over 0. 5 x 10(9)/l and platelets to over 50 x 10(9)/l was 11 and 21 days, respectively. Transplantation of CD34+ cells positively selected from cryopreserved PBSC provides engraftment ability similar to that of unmanipulated PBSC.     

Allo-PBSCT患者CD4＋CD25＋调节性T细胞的体外研究

目的探讨异基因外周血干细胞移植（Allo-PBSCT）患者外周血CD4＋CD25＋调节性T细胞（Tregs）在协同刺激信号作用下的增殖反应、与CD4＋CD25-T细胞混合淋巴细胞反应及上述两种培养细胞的细胞因子分泌情况。方法对36例Allo-PBSCT患者离体CD4＋CD25＋Tregs在抗CD3-mAbs和抗CD28-mAbs的刺激下行CD4＋CD25＋Tregs培养和CD4＋CD25＋Tregs、CD4＋CD25-T细胞混合淋巴细胞培养72 h,然后加入CCK-8溶液孵育1 h,用酶标仪检测OD450值,同时测量上述两种细胞培养上清液中IL-10、TGF-β和IFN-γ的浓度。另以7例健康者为对照组。结果 CD4＋CD25＋Tregs细胞OD450值低于CD4＋CD25-T细胞（P〈0.01）。CD4＋CD25＋Tregs与CD4＋CD25-T细胞混合OD450值也低于CD4＋CD25-T细胞（P〈0.01）。有移植物抗宿主病（GVHD）和无GVHD患者离体CD4＋CD25＋Tregs在抗CD3-mAbs和抗CD28-mAbs的共刺激下分泌的IL-10、TGF-β和IFN-γ低于CD4＋CD25-T细胞（P〈0.01）,有GVHD和无GVHD患者的两细胞混合组分泌的这3种细胞因子均低于CD4＋CD25-T细胞（P〈0.05）,Allo-PBSCT患者分泌细胞因子情况与对照组类似。结论有效地利用Allo-PBSCT受者外周血CD4＋CD25＋Tregs的抑制功能,则有可能降低Allo-PBSCT后GVHD的发病率。     

CD34+/CD90+ cells infused best predict late haematopoietic reconstitution following autologous peripheral blood stem cell transplantation

This study aimed to identify which graft product subset of cells might be the most predictive of late haematopoietic recovery (three to 12 months) following autologous peripheral blood stem cell transplantation (PBSCT). The relationships between the numbers of reinfused CD34+ cells and their immature subsets such as CD34+/CD90+, CD34+/AC133+, CD34+/CD38- and CD34+/HLA-DR- cells, and haemoglobin, white blood cell (WBC) and platelet counts at 3, 6, 9 and 12 months after PBSCT, were studied in 25 patients with haematological and solid malignancies. The total CD34+ cell number, as well as CD34+/CD90+ and CD34+/AC133+ cell numbers, correlated with platelet counts at 3, 6, 9 and 12 months after PBSCT, but the CD34+/CD90+ cells infused best predicted platelet recovery during the first 12 months after PBSCT (P < 0.0238 at any time-point). The CD34+/AC133+ cell dose also correlated with WBC counts at 3 months post PBSCT. In addition, all patients receiving more than 80 x 10(4) CD34+/CD90+ cells/kg showed platelet counts greater than 100 x 10(9)/l at all points after PBSCT, suggesting that this value of the CD34+/CD90+ cells infused was a threshold dose for durable haematopoietic engraftment after PBSCT.     

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.     

Autologous transplantation in chronic myeloid leukaemia using peripheral blood stem cells

Forty-three patients with chronic myeloid leukaemia in first chronic phase were recruited to study intensive chemotherapy (idarubicin plus cytarabine; IdAC) followed by collection of peripheral blood stem cells (PBSC) in the recovery phase. PBSC autografting was performed on 32 patients. One patient died during mobilization and three died following autograft. All procedural deaths occurred in patients who received IdAc more than a year from diagnosis. Nine further patients died, eight following progression of CML. 72% of transplanted patients showed a major cytogenetic response but most cases have returned to Philadelphia-positive haemopoiesis. 62% of autografted patients remain alive (median survival from diagnosis 52 months). Four of the 11 patients who did not receive a transplant remain in chronic phase.     

Viable CD34+/CD133+ blood progenitor cell dose as a predictor of haematopoietic engraftment in multiple myeloma patients undergoing autologous peripheral blood stem cell transplantation

Both CD34 (cluster of differentiation 34) and the more recently described CD133 are markers of primitive stem cells with haematopoietic repopulating ability. Most transplanting centres use a minimum number of CD34+ cells as the requirement for a transplant and consider this a predictor of haematopoietic engraftment. However, transplanted CD34+ cell dose does not always give a close correlation with time to engraftment nor explain delayed engraftment in some patients. We have retrospectively evaluated the potential of measuring viable CD133+ cell numbers in the autograft as an alternative predictor of haematological engraftment after autologous stem-cell transplantation in a cohort of patients with multiple myeloma (MM). We found an average 32% loss of viability of CD34+ cells in the post-thaw sample compared with the fresh sample. Of the original estimated CD34+ cell numbers transplanted per kg, 43% of the thawed samples were double positive for CD34+/CD133+. In this patient group, the CD34+/CD133+ subset gave the closest statistical correlation with time to neutrophil engraftment (p < 0.05), particularly for patients given above median (1.8 × 10⁶/kg) dose of the double-positive cells. The CD34+/CD133+ population was the only parameter to give a significant correlation with white cell engraftment in this patient cohort (p < 0.05). There was no significant correlation between CD34+, viable CD34+ or viable CD34+/CD133+ cells/kilogram with platelet engraftment. Determination of viable CD34+/CD133+ progenitor cell dose in the autograft may be a useful tool to predict neutrophil recovery after autologous transplantation than conventional assessment of CD34+ numbers. These results warrant further investigation of the role of CD133 in haematopoietic engraftment.     

Selection and expansion of peripheral blood CD34+ cells in autologous stem cell transplantation for breast cancer

Cytopenia after high-dose chemotherapy and autologous stem cell reinfusion is a major cause of morbidity. Ex vivo cultured expansion and differentiation of CD34+ peripheral blood progenitor cells (PBPC) to neutrophil precursors may shorten the neutropenic period further. We explored the use of these ex vivo cultured PBPCs in nine patients with metastatic breast cancer. All underwent PBPC mobilization with cyclophosphamide, VP-16, and G-CSF. Subsequently, they underwent four to five apheresis procedures. One apheresis product from each patient was prepared using the Isolex 300 Magnetic Cell Separation System (Baxter Immunotherapy, Irvine, CA) to obtain CD34+ cells. These cells were then cultured in gas permeable bags containing serum-free X-VIVO 10 (BioWhittaker, Walkersville, MD) medium supplemented with 1% human serum albumin and 100 ng/mL PIXY321. At day 12 of culture the mean fold expansion was 26x with a range of 6 to 64x. One patient's cells did not expand because of a technical difficulty. The final cell product contained an average of 29.3% CD15+ neutrophil precursors with a range of 18.5% to 48.1%. The patients underwent high-dose chemotherapy with cyclophosphamide, carboplatin, and thiotepa. On day 0, the cryopreserved PBPCs were reinfused and on day +1 the 12-day cultured cells were washed, resuspended, and reinfused into eight of nine patients. One patient was not infused with cultured cells. The mean number of cultured cells reinfused was 44.6 x 10(6) cells/kg with a range of 0.8 to 156.6 x 10(6) cells/kg. No toxicity was observed after reinfusion. The eight patients have recovered absolute neutrophil counts > 500/microL on a median of 8 days (range 8 to 10 days); the median platelet transfusion independence occurred on day 10 (range 8 to 12 days) and platelet counts > 50,000/microL were achieved by day 12 (range 9 to 14) for the seven patients whose platelet counts could be determined. Expanded CD34+ selected PBPC can be obtained and safely reinfused into patients.     

Granulocyte colony-stimulating factor after autologous CD34+ immunoselected peripheral blood stem cell transplantation

Granulocyte colony-stimulating factor (G-CSF) can be administered after a peripheral blood stem cell transplantation with the aim of accelerating neutrophil recovery. In a randomized, single-blind study we studied a new administration schedule of G-CSF in this context.