Inverse relationship between patient peripheral blood CD34+ cell counts and collection efficiency for CD34+ cells in two automated leukapheresis systems

BACKGROUND: The purpose of this study was to analyze the CD34 cell collection efficiency (CE) of automated leukapheresis protocols of two blood cell separators (Spectra, COBE [AutoPBSC protocol] and AS104, Fresenius [PBSC-Lym, protocol]) for peripheral blood progenitor cell (PBPC) harvest in patients with malignant diseases. STUDY DESIGN AND METHODS: PBPCs were collected by the Spectra AutoPBSC protocol in 95 patients (123 collections) and the AS104 PBSC-Lym protocol in 87 patients (115 harvests). Patients underwent a median of one (range, 1-4) conventional-volume apheresis procedure of 10.8 L (9.0-13.9) to obtain a target cell dose of > or =2.5 x 10(6) CD34+ cells per kg. RESULTS: The median overall CD34 CE was significantly better on the AS104 than on the Spectra: 55.8 percent versus 42.4 percent (p = 0.000). This was also true below (59.2% vs. 50.1%; p = 0.022) and above (51.2% vs. 41.3%; p = 0.001) the preleukapheresis threshold of 40 CD34+ cells per microL needed to collect a single-apheresis autograft. However, at > or =40 circulating CD34+ cells per microL, both cell separators achieved the target of > or =2.5 x 10(6) CD34+ cells per kg. The CD34 CE dropped significantly, from 59.2 percent at <40 cells per microL to 51.2 percent at > or =40 cells per microL on the AS104 (p = 0.017) and from 50.1 percent to 41.3 percent on the Spectra (p = 0.033). CONCLUSION: Whereas the CD34 CE was significantly different with the AS104 and the Spectra, the CD34 CE of both machines correlated inversely with peripheral blood CD34+ cell counts, showing a significant decline with increasing numbers of circulating CD34+ cells. Nevertheless, at > or 40 preapheresis CD34+ cells per microL, sufficient hematopoietic autografts of > or =2.5 x 10(6) CD34+ cells per kg were harvested by a single conventional-volume (11 L) leukapheresis on both cell separators.     

CD34+ cells and CD34+CD38- subset from mobilized blood show different patterns of adhesion molecules compared to those from steady-state blood, bone marrow, and cord blood

As suggested previously, a down-regulation of some cellular adhesion molecules (CAMs) on CD34(+) hematopoietic progenitor cells (HPC) may contribute to their egress from bone marrow (BM) to peripheral blood (PB) by decreasing their adhesion to BM stromal cells. Besides counting the percentage of CAM-positive cells, we decided to define clearly the antigen density (AgD) of the CAM on mobilized- and steady-state CD34(+) HPC using QIFIKIT calibration beads. Five sources of cells were compared: PB and BM from normal donors (nPB, nBM) cord blood (CB), mobilized PB obtained from leukapheresis products (LKP), and mobilized BM (mBM) samples. In our study the CAM-AgD was the lowest on CD34(+) cells in LKP which, on the contrary, contained the highest percentage of CD117(+), CD54(+), CD58(+) cell subsets. As for CB, a greater proportion of CD44(+) and CD62L(+) cells was observed in LKP than in other products. The LKP-CD34(+) cell population contained a greater percentage of CD11a(+) cells when compared to mBM, but the lowest percentage of CD49d(+) and CD49e(+) cells when compared to all products. The proportion of the CD34(+)CD38(-) immature subset expressing CD11a, CD44, CD54, or CD62L was greater in LKP than in mBM; the CD62L-AgD was higher in LKP than in mBM. This quantitative analysis clearly showed a downregulation of all CAM on LKP-CD34(+). The CD44, CD62L, CD11a, and CD54 AgD decrease appears to be specifically involved in the egress of the CD34(+) subsets into PB. The control of antigen density of these adhesion molecules is likely to be clinically important for effective mobilization of HPC as well as for rapid engraftment following HPC transplant.     

GFP在人脐血CD34~+细胞中的表达及意义

目的探讨慢病毒载体在CD34+脐血细胞(CBCs)中的基因转导效率,为基因治疗的临床应用提供关键材料。方法应用1型人类免疫缺陷病毒(HIV-1)改造而成的第三代自身失活(self-inactivating,SIN)慢病毒载体(lentiviralvector)系统,通过流式细胞术检测基因导入细胞百分比,评价该载体系统在人CBCs中的基因转导效率。结果 HIV载体的转导效率在95%以上。结论基于慢病毒载体基因转导的高效性,该载体系统可作为CD34+CBCs基因转导的极好工具。     

阵发性睡眠性血红蛋白尿症患者骨髓CD34+CD59+细胞的体外扩增

目的研究阵发性睡眠性血红蛋白尿症(PNH)患者CD34+CD59+细胞的分离、纯化及其体外扩增的条件和性能,为探索PNH新的治疗途径提供实验依据.方法利用免疫磁珠-流式细胞仪二步分选法,从PNH患者骨髓中分选出CD34+CD59+细胞,然后对CD34+CD59+细胞在不同造血生长因子组合条件下,进行体外扩增培养2周.结果体外扩增的最适宜的生长因子组合为SCF+IL-3+IL-6+FL+Tpo+Epo,最适宜的扩增时机为第7天,在此条件下,CD34+CD59+细胞的扩增倍数为22.42±3.73倍.CD34+CD59+细胞在扩增以后,仍保持较好的形成集落形成单位的能力,但是其向多系分化的潜能有所下降.结论 PNH患者CD34+CD59+细胞能够进行体外扩增.按照最佳扩增条件,在对PNH患者进行自体骨髓移植或自体外周血干细胞移植时有一定的应用价值.     

Clinical applications of ex vivo cultured CD34+ cells and myeloid progenitors

After extensive preclinical work, hematopoietic cellular therapy has recently entered a new era of clinical trials involving ex vivo cultured cells. The evolution of hematopoietic cell culture from clonogenic assays to large-scale static culture systems and bioreactors, and the identification and production of hematopoietic growth factors, have in part made this possible. In addition, murine models have demonstrated encouraging results with regard to the feasibility of infusing cultured cells, as well as to the potential efficacy. Several trials have recently been published utilizing ex vivo generated hematopoietic progenitors and myeloid progenitors, and are reviewed here. The field of clinical hematopoietic cellular therapy, while still in its infancy, is progressing rapidly, and promises to offer improved therapeutic options.     

Suppressive effects of TNF-alpha, TGF-beta1, and chemokines on megakaryocytic colony formation in CD34+ cells derived from umbilical cord blood compared with mobilized peripheral blood and bone marrow

CD34+ cells from human umbilical cord blood (CB) were isolated and investigated for megakaryocytic (MK) colony formation in response to recombinant human (rh) stimulatory and suppressive cytokines and compared with their counterparts in normal BM and G-CSF-mobilized peripheral blood (mPBL). First, we observed that IL-11 by itself at any dosage had no stimulator activity on MK colony formation derived from CD34+ cells in CB, mPBL, and BM. IL-3, steel factor (SLF), or thrombopoietin (Tpo) alone stimulated numbers of colony-forming unit-megakaryocyte (CFU-MK) in a dose-dependent fashion. Maximum growth of MK progenitor cells was noted in the presence of a combination of cytokines: IL-11, IL-3, SLF, and Tpo. The frequency of CFU-MK in CB and mPBL was significantly greater than that in BM, and the size of colonies in CB and mPBL was significantly greater than that in BM, and the size of colonies was larger as well. In addition, an increased number of big mixed colonies containing MK were observed in CB and mPBL. In the presence of IL-11, IL-3, SLF, and Tpo, CFU-MK derived from CB, mPBL, and BM was suppressed by tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta1 (TGF-beta1). CFU-MK derived from normal BM was inhibited by some chemokines evaluated, whereas CFU-MK derived from CB was suppressed only by platelet factor-4 (PF-4), IFN-inducible protein-10 (IP-10), Exodus-1, Exodus-2, and Exodus-3, but to a lesser degree. In CB, unlike granulocyte-macrophage (CFU-GM), erythroid (BFU-E), high-proliferative potential (HPP-CFC), or multipotential (CFU-GEMM) progenitors, at least a subpopulation of MK progenitors are in S-phase. Therefore, CB MK progenitors respond to the suppressive effects of some members of the chemokine family. Similar results were noted for burst-forming unit-MK (BFU-MK). Our results indicate that CB and mPBL are rich sources of MK progenitors and that MK progenitors in CB are responsive to the suppressive effects of TNF-alpha and TGF-beta1 and some members of the chemokine family.     

多药耐药基因在人骨髓CD34+细胞中的表达及对化疗药物的耐受性

目的　探讨人多药耐药 (MDR1)基因过表达能否提高骨髓造血干 /祖细胞对化疗药物的耐受性。方法　应用免疫磁性分选系统 (miniMACS)体外分离、纯化骨髓CD34 细胞并进行扩增 ;采用脂质体介导的基因转移方法 ,将人MDR1基因转染骨髓CD34 细胞 ,并运用流式细胞术检测基因转导前后造血干 /祖细胞中MDR1基因的编码产物 Pl70糖蛋白表达和功能变化。MTT法检测基因转导前后造血干 /祖细胞对化疗药物耐受性的改变。结果　MDR1基因转导后 4 8h骨髓造血干 /祖细胞Pl70抗原表达为 (2 3 6± 2 34) % ,明显高于转导前 (11 2± 2 2 ) % (P <0 0 1)。P170的功能活性被Rh 12 3的摄取和排除试验证实。转基因后细胞表现为典型的多药耐药表型 ,对P170谱的多种化疗药的耐受性提高了约 2～ 8倍 ,对非P170谱的顺铂、氨甲喋呤耐受性没有改变。结论　人多药耐药基因能提高骨髓造血干 /祖细胞对多种化疗药物的耐受性 ,表现为典型的多药耐药表型。     

人骨髓基质细胞促进脐血CD34+细胞的体外扩增和对NOD/SCID小鼠的植入

目的 研究人骨髓基质细胞(MSC)促进脐血CD34+细胞体外扩增及植入能力的作用.方法 分离、培养正常人的MSC作为滋养层细胞.在TPO、SCF、FL和G-CSF刺激下,比较有、无MSC滋养层细胞对扩增脐血CD34+细胞后,CD34+细胞和CFU数的增加倍数,以及植入非肥胖性糖尿病/重症联合免疫缺陷(NOD/SCID)小鼠的能力.结果 以骨髓MSC为滋养层细胞的培养体系可以更加有效地扩增脐血CD34+细胞.体外扩增1周总细胞数(TNC)、CD34+细胞和CFU的均数分别增加111.6、19.3和58.0倍;体外扩增2周后TNC、CD34+细胞和CFU的均数分别增加532.8、41.3和563.5倍.脐血细胞输入NOD/SCID小鼠6周后,移植未扩增脐血细胞的对照组小鼠骨髓细胞中人CD45+细胞比例仅为1.2%～3.7%;移植单纯细胞因子刺激扩增组小鼠骨髓中,人CD45+细胞比例为7.6%～12.1%;以MSC为滋养层扩增的脐血细胞移植组,人CD45+细胞比例达到45.3%～59.1%.结论 以人骨髓MSC为作为饲养层细胞,不但可以更加有效扩增脐血CD34+细胞,而且可以促进脐血细胞植入NOD/SCID小鼠的能力,有潜在的临床应用价值.     

Hematopoietic recovery in cancer patients after transplantation of autologous peripheral blood CD34+ cells or unmanipulated peripheral blood stem and progenitor cells

BACKGROUND: A study of CD34+ cell selection and transplantation was carried out with particular emphasis on characteristics of short- and long-term hematopoietic recovery. STUDY DESIGN AND METHODS: Peripheral blood stem and progenitor cells (PBPCs) were collected from 32 patients, and 17 CD34+ cell-selection procedures were carried out in 15 of the 32. One patient in whom two procedures failed to provide 1 × 10(6) CD34+ cells per kg was excluded from further analysis. After conditioning, patients received CD34+ cells (n = 10, CD34 group) or unmanipulated (n = 17, PBPC group) PBPCs containing equivalent amounts of CD34+ cells or progenitors. RESULTS: The yield of CD34+ cells was 53 percent (18–100) with a purity of 63 percent (49–82). The CD34+ fraction contained 66 percent of colony-forming units-granulocyte- macrophage (CFU-GM) and 58 percent of CFU of mixed lineages, but only 33 percent of burst-forming units-erythroid (BFU-E) (p < 0.05). Early recovery of neutrophils and reticulocytes was identical in the two groups, although a slight delay in platelet recovery may be seen with CD34+ cell selection. Late hematopoietic reconstitution, up to 1.5 years after transplant, was also similar. The two groups were thus combined for analyses of dose effects. A dose of 40 × 10(4) CFU-GM per kg ensured recovery of neutrophils to a level of 1 × 10(9) per L within 11 days, 15 × 10(4) CFU of mixed lineages per kg was associated with platelet independence within 11 days, and 100 × 10(4) BFU-E per kg predicted red cell independence within 13 days. However, a continuous effect of cell dose well beyond these thresholds was apparent, at least for neutrophil recovery. CONCLUSION: CD34+ cell selection, despite lower efficiency in collecting BFU-E, provides a suitable graft with hematopoietic capacity comparable to that of unmanipulated PBPCs. In both groups, all patients will eventually show hematopoietic recovery of all three lineages with 1 × 10(6) CD34+ cells per kg or 5 × 10(4) CFU-GM per kg, but a dose of 5 × 10(6) CD34+ cells or 40 × 10(4) CFU-GM per kg is critical to ensure rapid recovery.     

Age and stress related phenotypical changes in bone marrow CD34+ cells

Objective. Phenotypical changes in the human bone marrow (BM) due to age and stress have not so far been properly addressed in the literature. In the present study, we compared CD34⁺ BM cells between older and young volunteers. The influence of stress on CD34⁺ cell phenotype in older patients was investigated in an age‐matched group with acute myocardial infarction (AMI). Cytokines thought to influence BM CD34⁺ cell homeostasis were also analysed. Material and methods. BM mononuclear cells of 10 older volunteers and of 7 young volunteers (18–25 years), as well as 22 AMI patients, were analysed by flow cytometry for the following markers: CD34, CD38, CD117 (c‐kit) and CD133. Blood samples were analysed for CRP, IL‐6, MCP‐1, IL‐8, MMP‐9, TIMP‐1 and TNFα by ELISA methods. Results. Significantly higher numbers of CD34⁺ CD38^? cells (both absolute and relative) were observed in older volunteers than in young volunteers and AMI patients. Higher numbers of immature progenitors, namely CD34⁺CD38^? cells and CD34⁺CD38^?CD117⁺CD133⁺ cells, were observed among older volunteers compared to the other groups. However, the relative number of CD34⁺ cells lacking CD38 expression or expressing CD133 was higher in the old volunteers and AMI patients. None of the circulating factors investigated correlated with any of the cell population yields. Conclusion. In this study, we found that the absolute and relative numbers of BM CD34⁺CD38^? progenitor cells increase with age. The increment is attenuated in patients with AMI.     

人骨髓基质细胞协同外源性细胞因子对脐血CD34+细胞的体外扩增作用

目的　探讨人骨髓基质细胞 (hBMSC)协同以干细胞因子和FL为主的细胞因子对脐血CD3 4 + 细胞的体外扩增作用。方法　采用免疫磁珠法分选脐血CD3 4 + 细胞 ,以SCF +IL 3+IL 6 +FL +EPO组合高效扩增CD3 4 +细胞[1] ,并结合该细胞因子组合接种到预先照射 (2 0Gy)的hBMSC上 ,d10结束培养 ,收获细胞分别作细胞计数、集落培养和流式细胞术检测CD3 4 + 细胞数。结果　本法获得的脐血CD3 4 + 细胞纯度较高 (92± 0 .0 4 ) % ,在hBMSC组培养的d2 ,造血细胞几乎都粘附到hBMSC上 ,随着培养时间的延长 ,CD3 4 + 细胞比例不断下降。hBMSC组与无hBMSC组相比 ,除细胞总数扩增倍数外 ,CFU GM、BFU E、CD3 4 + 细胞扩增倍数差异有显著性意义 (P <0 .0 5 )。结论　①脐血来源的CD3 4 + 细胞粘附于滋养层上形成造血灶 ,且 10d后造血细胞仍具有体外集落形成能力 ,表明骨髓基质细胞可支持并维系体外造血 ;②hBMSC协同外源性细胞因子可能是扩增造血干 /祖细胞的较理想方案     

Selective survival of peripheral blood lymphocytes in children with HIV-1 following delivery of an anti-HIV gene to bone marrow CD34(+) cells.

Two HIV-1-infected children on antiretroviral therapy were enrolled into a clinical study of retroviral-mediated transfer of a gene that inhibits replication of HIV-1, targeting bone marrow CD34+ hematopoietic stem/progenitor cells. Two retroviral vectors were used, one encoding a "humanized" dominant-negative REV protein (huM10) that is a potent inhibitor of HIV-1 replication and one encoding a nontranslated marker gene (FX) to serve as an internal control for the level of gene marking. Peripheral blood mononuclear cells (PBMC) containing the huM10 gene or FX gene were detected by quantitative PCR at frequencies of approximately 1/10,000 in both subjects for the first 1-3 months following re-infusion of the gene-transduced bone marrow, but then were at or below the limits of detection (<1/1,000,000) at most times over 2 years. In one patient, a reappearance of PBMC containing the huM10 gene, but not the FX gene, occurred concomitant with a rise in the HIV-1 viral load during a period of nonadherence to the antiretroviral regimen. Unique clones of gene-marked PBMC were detected by LAM-PCR during the time of elevated HIV-1 levels. These findings indicate that there was a selective survival advantage for PBMC containing the huM10 gene during the time of increased HIV-1 load.     

阵发性睡眠性血红蛋白尿症患者骨髓CD34+CD59+细胞与CD34+CD59-细胞生物学性能的研究

目的 探讨阵发性睡眠性血红蛋白尿症(PNH)患CD34^ CD59^ 细胞的特性及PNH克隆呈优势造血的可能原因，以探索PNH发病的内在机制。方法 用免疫磁珠吸附法富集纯化CD34^ 细胞，再用流式细胞仪分选出PNH患的CD34^ CD59^ 细胞、CD34^ CD59^ 细胞及正常对照CD34^ 细胞。分别进行体外扩增液体培养2周，并对扩增前、后的细胞进行半固体培养。结果 ①PNH患CD34^ CD59^ 细胞与正常对照CD34^ 细胞形成集落形成单位(CFU)均在第7天达到扩增高峰，并且扩增后的细胞仍能保持CD59抗原，无GPI锚连蛋白的丢失。②正常对照的CD34^ 细胞在生存、增殖、形成CFU及扩增能力上均明显强于FHN患的CD34^ CD59^ 细胞及CD34^ CD59^-细胞.③PNH患CD34^ CD59^ 细胞及CD34^ CD59^ 细胞体外半固体培养，其形成CFU的能力无明显差异。④PNH患CD34^ CD59^ 细胞及CD34^ CD59^ 细胞在SCF IL3 IL6 FL Tpo及SCF IL3 IL6 FL Tpo Epo组合下液体培养，其生存、增殖、扩增等能力上均无明显差异。但在SCF IL3 IL6 FL Tpo Epo GM-CSF组合下液体培养，CD34^ CD59^ 细胞的生存、增殖、扩增能力均明显强于CD34^ CD59^ 细胞。结论 ①正常对照的CD34^ 细胞在生存、增殖、形成CFU及扩增能力上均明显强于PNH患的CD34^ CD59^ 细胞。②PNH患CD34^ CD59^ 细胞及CD34^ CD59^ 细胞体外半固体培养，以及在SCF IL3 IL6 LF Tpo及SCF IL3 IL6 FL Tpo Ep组合下液体培养，其生存、增殖、扩增等能力上均无明显差异，说明CD34^ CD59^ 细胞在造血能力上并无内在的优势。GM—CSF或许是使PNH克隆呈造血优势的原因之一。     

IL-3 or IL-7 increases ex vivo gene transfer efficiency in ADA-SCID BM CD34+ cells while maintaining in vivo lymphoid potential.

To improve maintenance and gene transfer of human lymphoid progenitors for clinical use in gene therapy of adenosine deaminase (ADA)-deficient SCID we investigated several gene transfer protocols using various stem cell-enriched sources. The lymphoid differentiation potential was measured by an in vitro clonal assay for B/NK cells and in the in vivo SCID-hu mouse model. Ex vivo culture with the cytokines TPO, FLT3-ligand, and SCF (T/F/S) plus IL-3 or IL-7 substantially increased the yield of transduced bone marrow (BM) CD34(+) cells purified from ADA-SCID patients or healthy donors, compared to T/F/S alone. Moreover, the use of IL-3 or IL-7 significantly improved the maintenance of in vitro B cell progenitors from ADA-SCID BM cells and allowed the efficient transduction of B and NK cell progenitors. Under these optimized conditions transduced CD34(+) cells were efficiently engrafted into SCID-hu mice and gave rise to B and T cell progeny, demonstrating the maintenance of in vivo lymphoid reconstitution capacity. The protocol based on the T/F/S + IL-3 combination was included in a gene therapy clinical trial for ADA-SCID, resulting in long-term engraftment of stem/progenitor cells. Remarkably, gene-corrected BM CD34(+) cells obtained from one patient 4 and 11 months after gene therapy were capable of repopulating the lymphoid compartment of SCID-hu hosts.     

Nonmyeloablative stem cell transplantation with CD8-depleted or CD34-selected peripheral blood stem cells

To decrease the incidence of graft-versus-host disease (GVHD) observed after nonmyeloablative stem cell transplantation (NMSCT), we studied the feasibility of CD8-depleted or CD34-selected NMSCT followed by CD8-depleted preemptive donor lymphocyte infusion (DLI) given in incremental doses on days 40 and 80. Fourteen patients with high-risk malignancies and an HLA-identical sibling (n = 8) or alternative donor (n = 6) but ineligible for a conventional transplant were included. Nonmyeloablative conditioning regimen consisted in 2 Gy total body irradiation (TBI) alone, 2 Gy TBI and fludarabine (previously untreated patients) or cyclophosphamide and fludarabine (patients who had previously received > or =12 Gy TBI). Patients 1-4 (controls) received unmanipulated peripheral blood stem cells (PBSC) and DLI and patients 5-14 CD8-depleted or CD34-selected PBSC followed by CD8-depleted DLI. Post-transplant immunosuppression was carried out with cyclosporine A (CsA) and mycophenolate mofetil (MMF). Initial engraftment was seen in all patients, but 1 patient (7%) later rejected her graft. The actuarial 180-day incidence of grades II-IV acute GVHD was 75% for patients 1-4 versus 0% for patients 5-14 (p = 0.0019). Five of 14 patients were in complete remission (CR) 180 days after the transplant and 6/14 had partial responses. The 1-year survival rate was 69%, and nonrelapse and relapse mortality rates were 16 and 18%, respectively. We conclude that CD8-depleted or CD34-selected NMSCT followed by CD8-depleted DLI is feasible and considerably decreases the incidence of acute GVHD while preserving engraftment and apparently also the graft-versus-leukemia (GVL) effect. Further studies are needed to confirm this encouraging preliminary report.     

Preincubation with endothelial cell monolayers increases gene transfer efficiency into human bone marrow CD34(+)CD38(-) progenitor cells

Retroviral gene transfer studies targeting bone marrow CD34(+)CD38(-) stem cells have been disappointing because of the rarity of these cells, their G(0) cell cycle status, and their low or absent expression of surface retroviral receptors. In this study, we examined whether preincubation of bone marrow CD34(+)CD38(-) stem cells with a hematopoietically supportive porcine microvascular endothelial cell line (PMVECs) could impact the cell cycle status and expression of retroviral receptors in pluripotent CD34+CD38- cells and the efficiency of gene transfer into these primitive target cells. PMVEC coculture supplemented with GM-CSF + IL-3 + IL-6 + SCF + Flt-3 ligand induced >93% of the CD34(+)CD38(-) population to enter the G(1) or G(2)/S/M phase while increasing this population from 1.4% on day 0 to 6.5% of the total population by day 5. Liquid cultures supplemented with the identical cytokines induced 73% of the CD34(+)CD38(-) population into cell cycle but did not maintain cells with the CD34(+)CD38(-) phenotype over time. We found no significant increase in the levels of AmphoR or GaLVR mRNA in PMVEC-expanded CD34(+)CD38(-) cells after coculture. Despite this, the efficiency of gene transfer using either amphotropic vector (PA317) or GaLV vector (PG13) was significantly greater in PMVEC-expanded CD34(+)CD38(-) cells (11.4 +/- 5.6 and 10.9 +/- 5.2%, respectively) than in either steady state bone marrow CD34(+)CD38(-) cells (0.6 +/- 1.7 and 0.2 +/- 0.6%, respectively; p < 0.01 and p < 0.01) or liquid culture-expanded CD34(+)CD38(-) cells (1.4 +/- 3.5 and 0.0%, respectively; p < 0.01 and p < 0.01). Since PMVEC coculture induces a high level of cell cycling in human bone marrow CD34(+)CD38(-) cells and expands hematopoietic cells capable of in vivo repopulation, this system offers potential advantages for application in clinical gene therapy protocols.