CD34+ testicular stromal cells support long-term expansion of embryonic and adult stem and progenitor cells

Stem cells reside in specialized microenvironments created by supporting stromal cells that orchestrate self-renewal and lineage-specific differentiation. However, the precise identity of the cellular and molecular pathways that support self-renewal of stem cells is not known. For example, long-term culture of prototypical stem cells, such as adult spermatogonial stem and progenitor cells (SPCs), in vitro has been impeded by the lack of an optimal stromal cell line that initiates and sustains proliferation of these cells. Indeed, current methods, including the use of mouse embryonic fibroblasts (MEFs), have not been efficient and have generally led to inconsistent results. Here, we report the establishment of a novel CD34-positive cell line, referred to as JK1, derived from mouse testicular stromal cells that not only facilitated long-term SPC culture but also allowed faithful generation of SPCs and multipotent stem cells. SPCs generated on JK1 maintained key features of germ line stem cells, including expression of PLZF, DAZL, and GCNA. Furthermore, these feeders also promoted the long-term cultivation of other types of primitive cells including multipotent adult spermatogonial-derived stem cells, pluripotent murine embryonic stem cells, and embryonic germ cells derived from primordial germ cells. Stem cells could be passaged serially and still maintained expression of characteristic markers such as OCT4 and NANOG in vitro, as well as the ability to generate all three germ layers in vivo. These results indicate that the JK1 cell line is capable of promoting long-term culture of primitive cells. As such, this cell line allows for identification of stromal-derived factors that support long-term proliferation of various types of stem cells and constitutes a convenient alternative to other types of feeder layers. Disclosure of potential conflicts of interest is found at the end of this article.     

Thymosin-alpha1 stimulates maturation of CD34+ stem cells into CD3+4+ cells in an in vitro thymic epithelia organ coculture model.

The effect of thymosin-alpha1 on thymopoiesis is largely unknown. Thymosin is found in the cortical and medullary thymic epithelia, as well as in nurse cells; thus, it is hypothesized that thymosin may affect both early and late stage of thymocyte maturation. In this study, the effect of thymosin-alpha1 on thymopoiesis was determined by coculturing in vitro CD34+ stem cells (SC) with allogeneic cultured thymic epithelia fragments (CTEF) for 1-4 weeks and analyzing T-cell maturation by flow cytometry. Thymosin-alpha1 significantly enhanced the cell number (e.g., proliferation) of mononuclear cells obtained at 2 and 4 weeks of the SC-CTEF cocultures (P < 0.01 and < 0.05, respectively). In particular, thymosin-alpha1 stimulated expression of CD3+ cells at 3 and 4 weeks (P < 0.05). The predominant subpopulation increased by thymosin stimulation was single positive mature CD4+ cells, which was confirmed to occur within the SC-CTEF thymic organ tissue by laser confocal immunofluorescence microscopy. Thymosin stimulation tended to enhance IL-7 synthesis, critical cytokine in the maturation of thymocytes. In summary, this is the first study to demonstrate that thymosin-alpha1 enhanced thymopoiesis of CD34+ stem cells in humans using an in vitro model of differentiation using stem cells and cultured thymic epithelial fragments cocultures. Furthermore, the thymosin significantly increased expression of CD3+4+ T cells.     

Megakaryothrombopoiesis during ex vivo expansion of human cord blood CD34+ cells using thrombopoietin

Thrombopoietin (TPO) is one of the most promising stimulants for ex vivo expansion of haematopoietic stem cells. Previously, we have found that TPO induces a characteristic pattern of apoptosis during ex vivo expansion of human cord blood (CB) CD34+ cells and that the TPO-induced apoptotic cells belong to megakaryocyte (MK) lineage. In this study, we have examined the maturation of MK and platelet production in association with the TPO-induced apoptosis. CD34+ cells, purified from human CB, were expanded in serum-free conditions stimulated with TPO. Apoptosis was confirmed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labelling (TUNEL) assay and electron microscopy (EM). Simultaneous measurement of DNA content and immunophenotyping revealed that the cells with higher DNA content (>8 N) constituted less than 5% of the CD41+ fractions until day 14, implying premature apoptosis of MKs before full polyploidization. Nevertheless, EM observation showed not only platelet territories but also newly produced platelets in which granules and microfilaments could be identified. Furthermore, flow cytometry demonstrated that the platelet fraction expressed P-selectin and an activation motif on GPIIb/IIIa recognized by monoclonal antibody PAC-1 upon stimulation with adenosine diphosphate (ADP). In addition, periodic acid-Schiff (PAS)-positive materials and nonspecific esterase activities could be demonstrated. Therefore, it is suggested that platelet production and the accompanying processes, rather than apoptosis only, be hastened during the ex vivo expansion of CB CD34+ cells when using TPO.     

早期作用造血因子对人脐血CD34+细胞的体个扩增作用

为了观察早期作用造血细胞因子SCF、FL、IL-3、IL-6、TPO单独及联合应用,对脐血CD34+细胞的体外扩增作用.我们用吸附单克隆抗体-磁珠分离系统富集人脐血CD34+细胞,在体外液体培养体系中加入不同的细胞因子扩增4周,每周取样计数有核细胞总数及集落形成细胞(CFC)数.结果表明:用磁性细胞分离仪富集脐血CD34+细胞纯度为80%～87%;一些细胞因子有明显的协同效应,其联合应用的扩增作用显著高于单因子作用;SCF+FL存在下,IL-3是有效扩增有核细胞总数及CFC的关键因子;细胞因子SCF+FL+IL-3和SCF+FL+IL-3+IL-6组合对有核细胞总数及CFC均有良好的扩增效应,培养2周时对CFC的扩增倍数分别为38.3±4.4 和29.6±2.7倍,可满足成人移植及基因治疗等的需要.     

Osteopetrotic mouse stroma with thrombopoietin, c-kit ligand, and flk-2 ligand supports long-term mobilized CD34+ hematopoiesis in vitro

OP-9 cells are stromal cells derived from macrophage colony-stimulating factor (M-CSF)-deficient osteopetrotic mice. To evaluate the OP-9 capability to sustain long-term hematopoiesis, we reported the expansion of granulocyte colony-stimulating factor (G-CSF)-mobilized human peripheral blood (PB) CD34(+) cells in co-culture with murine OP-9 and MS-5 stromal cells, either transfected with various combinations of adenovectors (Ad) expressing c-kit ligand (KL) (either soluble or transmembrane form), thrombopoietin (TPO), flt-3/flk2 ligand (FL), and granulocyte-macrophage (GM)-CSF or with weekly addition of these cytokines. Expression of TPO as well as association of TPO, FL, and KL increased progenitor cell and week-6 cobblestone area forming cell (CAFC) production in all stromal co-cultures. Similar progenitor expansion was obtained by weekly addition of soluble cytokine. Five weeks of co-culture with OP9 and TPO, FL + KL resulted in the greatest expansion of progenitor cells and week-6 CAFC as measured by secondary assay on MS-5. In contrast to MS-5 and TPO or TPO + FL + KL cultures where hematopoiesis declined by week 4, progenitor as well as week-6 CAFC expansion continued for over 3 months in TPO + FL + KL OP9 cocultures. This was associated with decrease of CD14(+) macrophage production. The addition of human macrophage (M)-CSF or CD14(+) cells to the co-culture decrease progenitor and stem cell expansion; however, murine M-CSF to OP-9 co-cultures did not decrease progenitor expansion. High levels of stromal-derived factor-1 (SDF-1) production by MS-5 and low or absent production by OP-9 may account for stem cell adhesion and CAFC formation in the former cultures and the predominance of stem and progenitor cells in the nonadherent fraction in the latter cultures.     

The expansion of megakaryocyte progenitors from CD34+-enriched mobilized peripheral blood stem cells is inhibited by Flt3-L.

This study aimed to determine the optimal growth factor combination for expansion of megakaryocyte (Mk) progenitors with clonogenic potential from CD34+-enriched mobilized peripheral blood stem cells (PBSC). Mobilized PBSC were monocyte depleted and CD34+ enriched, then cultured with various combinations of interleukin-3 (IL-3), IL-6, IL-11, Flt3 ligand (Flt3-L), stem cell factor (SCF), granulocyte-macrophage colonystimulating factor (GM-CSF), and erythropoietin (EPO), using a 2(7-3) IV fractional factorial design. Expansion of Mk committed progenitors (CD41+) and primitive precursors (CD61+ CD34+) was determined using FACS and colony-forming assays. Amplification of Mk progenitor production was attributed to IL-3 (p < 0.002), SCF (p < 0.001), and GM-CSF (p < 0.05). Flt3-L inhibited the production of total CD61+ cells (p < 0.05), CD61+CD34+ cells (p < 0.03), and total CD41a+ cells (p < 0.01). Addition of Flt3-L to the optimum growth factor combination of megakaryocyte growth and development factor (MGDF), SCF, IL-3, and GM-CSF caused the greatest increase in total nucleated cells but reduced Mk progenitor expansion. There was also a 20% reduction in Mk+ colonies from cells expanded in the presence of Flt3-L. Factorial analysis identified the optimal combination of growth factors required to expand Mk precursors with clonogenic potential. The addition of Flt3-L to the optimal combination of MGDF, SCF, IL-3, and GM-CSF reduced both the fold expansion of Mk progenitors and Mk colony numbers.     

In vitro expansion of adipose-derived adult stromal cells in hypoxia enhances early chondrogenesis

Cartilage is an avascular tissue, and chondrocytes in vivo experience a severely hypoxic environment. Using a defined in vitro model of early chondrogenesis, we attempted to enrich for cells with an enhanced ability for chondrogenic differentiation by pre-exposure of mouse adipose-derived adult stromal cells (ADASs) to a hypoxic (2% oxygen) environment. ADASs were subsequently expanded in 2% or 21% oxygen environments, resulting in 2 groups of cells, and then early chondrogenic differentiation was induced at 21% oxygen tension using a 3-dimensional micromass culture system. ADAS chondrogenesis was assessed using Alcian Blue staining for proteoglycans and quantification of sulfated glycosaminoglycans. Osteogenesis of the 2 cell groups was also studied. Two percent oxygen tension profoundly increased the proliferation of ADASs. ADASs expanded in 2% oxygen tension exhibited enhanced early chondrogenic differentiation and diminished osteogenesis, suggesting that the reduced oxygen environment may favor chondroprogenitors. Gene expression analysis suggested that matrix metalloproteinase synthesis was inhibited in cells expanded in 2% oxygen. Furthermore, re-oxygenation of the 2% oxygen-expanded ADASs before differentiation did not significantly affect early chondrogenesis. Thus, priming ADASs with 2% oxygen may have selected for chondrogenic progenitors with an enhanced ability to survive and differentiate. This study is relevant for the future application of cell-based therapies involving cartilage tissue regeneration.     

胸腺基质细胞支持下骨髓CD34~+细胞向T细胞体外定向分化

目的：研究人骨髓CD34^＋细胞体外向T细胞定向分化的方法，为研究造血细胞淋系造血活性及T细胞发育和分化提供技术平台。方法：免疫磁珠法分离骨髓CD34^＋细胞，在骨髓基质细胞条件培养液构建的微环境下，在胸腺基质细胞的支持下，使其体外向T细胞定向分化，收集培养的非贴壁细胞，免疫荧光染色后经流式细胞术检测培养不同时间CD1^-CD3^＋细胞、CD3^＋CD4^＋CD8^-细胞及CD3^＋CD4^-CD8^＋细胞比例。结果：培养1周时，培养细胞中以不成熟的CD1^＋CD3^-细胞、CD1^＋CD3^＋细胞为主，可检测到少量CD1^-CD3^＋细胞，随培养时间延长，不成熟细胞比例逐渐减少，而成熟的CD1^-CD3^＋细胞比例逐渐增加；在CD3^＋细胞中，培养初期以不成熟的双阳性细胞CD4^＋CD8^＋为主，而成熟的单阳性CD^＋CD8^-细胞及CD4^-CD8^＋细胞占极小比例，随培养时间延长，双阳性细胞比例逐渐减少，而成熟的单阳性细胞比例逐渐增高；而无胸腺基质细胞支持的CD34＋细胞仅在培养初期检测到成熟细胞存在，而培养后4周基本检测不到成熟T细胞的存在。结论：在骨髓基质细胞及胸腺基质细胞的支持下，骨髓CD34^＋细胞可体外发育为成熟的CD1^-CD3^＋细胞及单阳性T细胞，其中胸腺基质细胞的支持对于造血细胞向T细胞的体外定向分化极其重要。     

Ad-hLIF转基因饲养层细胞对CD34+造血干/祖细胞增殖和分化的影响

目的 用腺病毒载体介导的人白血病抑制因子基因(Ad-hLIF)感染WI-38人胚肺成纤维细胞制备饲养层细胞,体外观察转基因细胞对CD34+造血干/柑细胞增殖和分化的影响,体内研究对辐射损伤SCID小鼠造血功能恢复的效果.方法 用RT-PCR和ELISA法鉴定Ad-hLIF转基因饲养层细胞目的 基因的表达后,将经免疫磁珠法分离和流式细胞术检测后的CD34+细胞在饲养层和/或细胞因子培养体系中扩增28 d,检测不同时间点的单个核细胞(MNC)数量及CD34+细胞阳性率;扩增后的MNC经CFDA SE荧光标记后移植入辐射损伤SCID小鼠体内,RT-PCR和细胞荧光标记法检测小鼠内含Alu基因人源细胞的门巢情况.结果 感染50MOI(multiplicity of infection)Ad-hLIF的饲养层细胞均有绿色荧光,RT-PCR和ELISA法结果 显示hLIF目的 基因能在WI-38饲养层细胞中表达,免疫磁珠法分离的CD34+造血干/祖细胞经流式细胞术检测其纯度可达95.60%±2.58%,MNC在Ad-hLIF转基因饲养层培养体系持续扩增,最高可达356.95±0.87倍,其中CD34+细胞仪在0～14 d能维持较高水平,最高可扩增52.11±1.13倍,以后逐渐降低.将其移植辐射损伤SCID小鼠后,可明显提高小鼠存活率,4周内小鼠骨髓中不仅可观察到CFDA SE荧光标记的细胞,而且经RT-PCR法搭定后.还可检测到表达Alu人源基因的人脐血造血归巢细胞.结论 成功建立的Ad-hLIF转基因饲养层细胞不仅体外可以有效地扩增CD34+造血干/祖细胞,并延缓其分化.扩增的CD34+细胞对辐射损伤SCID小鼠具有造血功能恢复的功能.     

转FL、GM-CSF基因的人骨髓基质细胞促进脐血CD34+细胞体外扩增

研究转FL、GM-CSF基因的基质细胞对脐血CD34+细胞的扩增效应.将转FL、GM-CSF基因的入骨髓基质细胞系与脐血CD34+细胞共培养,观察细胞总数、CD34+细胞数、CFU-GM的变化情况.培养到第4周时,第(4)组(SCF+IL-3+IL-6+GM-CSF+FL)和第(8)组(HFCL/hGM-CSF+HFCL/hFL+SCF+IL-3+IL-6)的细胞总数增加到最大,分别扩增了717±24.47和709±63.63,第1周,第(5)组(HFCL+SCF+IL-3+IL-6)扩增了10.5±2.08倍,较第(8)组减少(P＜0.05).第1周时,CD34+细胞总数第(4)组和第(8)组分别扩增了8.44倍和11.5倍(P＜0.05),CD34+细胞百分率第(7)组(FCL/hFL+SCF+IL-3+II,-6)为50.2%,第(6)组(HFCL/hGM-CSF+SCF+IL-3+IL-6)为28.95%(P＜0.01).第2周,各组CFU-GM增加显著,以第(4)组和第(8)组增加最为明显,以后随扩增时间延长,造血细胞集落数、集落体积逐渐减少.表明转FL、GM-CSF基因的基质细胞,能有效的协同其他细胞因子对脐血CD34+细胞产生明显的扩增作用,能显著改变基质细胞造血功能.     

Mitochondrial DNA sequence heterogeneity of single CD34+ cells after nonmyeloablative allogeneic stem cell transplantation

We applied a single-cell method to detect mitochondrial DNA (mtDNA) mutations to evaluate the reconstitution of hematopoietic stem cells (HSCs) and committed progenitor cells after nonmyeloablative allogeneic stem cell transplantation in humans. In a total of 1,958 single CD34(+) cells from six human leukocyte antigen-matched sibling donor and recipient pairs, individual CD34(+) clones were recognized based on the observed donor- or recipient-specific mtDNA sequence somatic alteration. There was no overall reduction of mtDNA heterogeneity among CD34(+) cells from the recipient after transplantation. Samples collected from two donors over time showed the persistence of certain CD34(+) clones marked by specific mutations. Our results demonstrate the feasibility of distinguishing donor and recipient individual CD34(+) clones based on mtDNA mutations during engraftment. HSCs were not limited in number, and similar mtDNA heterogeneity levels suggested representation of the total stem cell compartment during rapid hematopoietic reconstitution in the recipient. Disclosure of potential conflicts of interest is found at the end of this article.     

CD4+ T cell-independent maintenance and expansion of memory CD8+ T cells derived from in vitro dendritic cell activation

CD4+ T cells are essential for the maintenance of CD8+ memory T (Tm) cells following acute infection, but the importance of CD4+ T cells for the maintenance and expansion of CD8+ Tm cells to non-infectious antigens remains mostly unknown. Here, we showed that ovalbumin (OVA)-specific CD8+ Tm cell precursors derived from in vitro stimulation of TCR transgenic OT I CD8+ T cells with OVA protein-pulsed bone marrow-derived dendritic cells (DCOVA) can give rise to functional CD8+ Tm cells after adoptively transferred into mice. These CD8+ Tm cells can be maintained and remain fully functional in CD4+ T cell-absent environments in vivo. Furthermore, CD4+ T cells are not essential for the expansion of these CD8+ Tm cells. Finally, these in vitro DCOVA-activated CD8+ Tm cells maintained in CD4-deficient mice are also able to confer fully protective immunity against a later challenge of OVA-expressing tumor cells. Collectively, these findings demonstrate that in contrast to acute infections, maintenance and expansion of CD8+ Tm cells after priming with OVA protein-pulsed dendritic cells are independent of CD4+ T cells.     

Inhibition of thymopoiesis of CD34+ cell maturation by HIV-1 in an in vitro CD34+ cell and thymic epithelial organ culture model

The mechanisms by which HIV-1 affects thymopoiesis were determined by preincubating CD34+ cells or cultured thymic epithelial (CTE) cells with lymphotropic (T-) and monotropic (M-) strains of HIV-1 in an in vitro CTE organ and CD34+ cell coculture model that allows for analysis of development of thymocytes and mature T cells. When purified CD34+ cells were precultured with either T- or M-tropic strains of HIV-1, thymopoiesis was impaired in a two-week coculture manifested by decreased cell number of thymocytes generated. However, the percentages of thymocyte subpopulations were comparable to control uninfected cocultures. Furthermore, HIV infection of thymocytes was predominantly observed in the CD44+CD3- population. However, in a four-week coculture experiment, HIV infection and depletion of more mature thymocytes were also observed. When CTE cells were preincubated with T- and M-tropic strains of HIV before addition of CD34+ cells, the number of thymocytes and subpopulations of thymocytes at early and later stages of maturation were markedly decreased. Furthermore, CD34+ and CD44+CD3- cells become HIV-infected. In summary, HIV-1 infection inhibited thymocyte maturation at early stages of thymocyte maturation CD44+CD25-CD3-. In addition, HIV also depleted later stages of CD4+ thymocyte subpopulations.     

CD34+ cells cultured in stem cell factor and interleukin-2 generate CD56+ cells with antiproliferative effects on tumor cell lines

In vitro stimulation of CD34+ cells with IL-2 induces NK cell differentiation. In order to define the stages of NK cell development, which influence their generation from CD34 cells, we cultured G-CSF mobilized peripheral blood CD34+ cells in the presence of stem cell factor and IL-2. After three weeks culture we found a diversity of CD56+ subsets which possessed granzyme A, but lacked the cytotoxic apparatus required for classical NK-like cytotoxicity. However, these CD56+ cells had the unusual property of inhibiting proliferation of K562 and P815 cell lines in a cell-contact dependent fashion.     

Presence of circulating abnormal CD34+ progenitors in adult Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is related to the proliferation of cells, which are similar to Langerhans cells (LC) but possess many abnormal characteristics. Lesions are widespread and this fact suggests that LCH cells or their precursors are present in the blood of patients. In five adult patients, we have isolated and cultured CD34+ blood progenitors of dendritic cells. We studied their phenotype by flow cytometry and their functional properties in mixed culture with heterologous lymphocytes and with autologous lymphocytes in the presence of tri-nitro-phenyl antigen (TNP). The amount of CD34+ precursors was dramatically higher than controls but a high mortality occurred during the in vitro differentiation. The phenotype of surviving cells was similar to LC phenotype (CD1a+, CD83+, Lag+) but some of them expressed CD2. These cells were able to induce T cell proliferation in mixed culture. They could not initiate primary response to TNP, except in a patient treated with thalidomide. In our hands, these CD34+ cells may be precursors of LCH cells.     

Granulocyte-colony-stimulating factor stimulation of bone marrow mesenchymal stromal cells promotes CD34+ cell migration via a matrix metalloproteinase-2-dependent mechanism

Human hematopoietic stem/progenitor cells (HSPCs) can be mobilized into the circulation using granulocyte-colony stimulating factor (G-CSF), for graft collection in view of hematopoietic transplantation. This process has been related to bone marrow (BM) release of serine proteases and of the matrix metalloproteinase-9 (MMP-9). Yet, the role of these mediators in HSC egress from their niches remains questionable, because they are produced by nonstromal cells (mainly neutrophils and monocytes/macrophages) that are not a part of the niche. We show here that the G-CSF receptor (G-CSFR) is expressed by human BM mesenchymal stromal/stem cells (MSCs), and that G-CSF prestimulation of MSCs enhances the in vitro trans-stromal migration of CD34+ cells. Zymography analysis indicates that pro-MMP-2 (but not pro-MMP-9) is expressed in MSCs, and that G-CSF treatment increases its expression and induces its activation at the cell membrane. We further demonstrate that G-CSF-stimulated migration depends on G-CSFR expression and is mediated by a mechanism that involves MMPs. These results suggest a molecular model whereby G-CSF infusion may drive, by the direct action on MSCs, HSPC egress from BM niches via synthesis and activation of MMPs. In this model, MMP-2 instead of MMP-9 is implicated, which constitutes a major difference with mouse mobilization models.     

人类CD8+记忆T细胞体外扩增方法的研究

 目的： 研究体外扩增人类CD8+记忆T细胞的新方法,为抗病毒与抗肿瘤的过继性免疫治疗提供新的手段。方法：将anti-CD3抗体、anti-CD28抗体、CD70、白细胞介素（IL）-2、IL-7和IL-15进行排列组合,设计出63种刺激方式,对体外分离得到的正常人外周血CD8+ T细胞进行体外扩增;培养14 d后进行细胞计数,并检测CD8+ T细胞的纯度以及CD8+中枢记忆T细胞（TCM）和CD8+效应记忆T细胞（TEM）所占的比例,进而计算出CD8+ T细胞、CD8+ TCM和CD8+ TEM的体外扩增倍数,从而确定理想的刺激方法。结果：体外扩增CD8+ T细胞、CD8+ TCM和CD8+ TEM的理想刺激方式均为anti-CD3抗体、IL-2和IL-7三者的组合;该刺激方式使3种细胞在培养14 d后分别扩增了13.19、13.28和15.27倍。结论：Anti-CD3抗体、IL-2和IL-7三者的组合,是刺激人类CD8+记忆T细胞体外扩增的相对理想方法。     

Non-CD34+ cells, especially CD8+ cytotoxic T cells and CD56+ natural killer cells, rather than CD34 cells, predict early engraftment and better transplantation outcomes in patients with hematologic malignancies after allogeneic peripheral stem cell transplantation.

The effect of the transplant dose of each cell subset on engraftment kinetics and transplantation outcomes was evaluated in HLA-identical allogeneic peripheral blood stem cell transplantation (PBSCT). Sixty-nine patients were included in this retrospective study. Engraftment kinetics, transplantation outcomes, and immune reconstitution up to 1 year after transplantation were analyzed according to the transplant dose of CD34+ and non-CD34+ cells, including natural killer (NK) cells and CD8+ cytotoxic T (Tc) cells. An accelerated neutrophil engraftment was strongly associated with a higher transplant dose of NK cells (12 versus 16 days, P < .001) and Tc cells (13 versus 16 days, P < .001) but not CD34+ cells (P = .442). Survival analyses revealed a favorable prognosis for patients who received a higher dose of non-CD34+ cell subsets, rather than CD34+ cells, in terms of overall survival (OS; P = .024 for NK cells and .050 for Tc cells) and nonrelapse mortality (NRM; P = .005 for NK cells, .060 for Tc cells). In addition, a higher transplant dose of NK and Tc cells was correlated with a faster lymphoid reconstitution. In multivariate analyses, rapid neutrophil engraftment was correlated with a higher transplant dose of NK cells (P = .001) and Tc cells (P = .004). Moreover, an increased OS was associated with the NK cell dose (P = .007) and chronic graft-versus-host disease (P = .009), whereas a decreased NRM was associated with the NK dose (P = .024). In conclusion, in a PBSCT setting, a higher transplant dose of NK and Tc cells accelerated neutrophil engraftment, improved the immune reconstitution, and decreased NRM, thereby increasing OS after allogeneic PBSCT.     

Platelet-derived growth factor enhances expansion of umbilical cord blood CD34+ cells in contact with hematopoietic stroma

Stem cell expansion remains an elusive but highly desirable goal. Here we show that platelet-derived growth factor (PDGF), along with cultured endothelial or stromal cells, significantly enhances expansion of human CD34+ cells in vitro. In media supplemented with thrombopoietin, stem cell factor, flt-3 ligand, and granulocyte-colony stimulating factor, CD34+ cells, as well as CFU-GM, BFU-CFU-E, CFU-GEMM, and CFU-MK, increased by 34.3-, 138-, 59.7-, 38.4-, and 86.0-fold, respectively. Co-culturing of CD34+ cells with cultured stromal cells or human umbilical cord vein endothelial cells (HUVECs) greatly enhanced expansion efficiency. The presence of PDGF (50 ng/ml) further augmented expansion, such that increases of 77.0-, 262-, 90.0-, 93.0-, and 200-fold, respectively, were achieved. Six weeks after infusion of expanded cells into NOD/SCID mice, human CD45+ cells were detected in recipients' bone marrow, spleen, and peripheral blood. Our results provide a rationale for development of a stem cell expansion protocol for clinical applications.