Substance P and calcitonin gene-related neuropeptides as novel growth factors for ex vivo expansion of cord blood CD34+ hematopoietic stem cells

There is little evidence on roles of growth factors other than cytokines in expansion of cord blood (CB) stem cells. We aimed to explore a novel approach for expansion, using Substance P (SP) and calcitonin gene-related peptide (CGRP) neuropeptides.

CB CD34⁺ cells were cultured in different concentrations of SP and/or CGRP in combination with a cytokine cocktail. Phenotypic and functional analysis was performed by flowcytometry and colonogenic assay.

Our results show a significant improvement of total expansion of neuropeptide treated cells. There was a selective effect of CGRP on CD34⁺ CD133⁺ cells, SP on CD34⁺ CD45^dim cells, and 10^{? 9} M SP and/or CGRP on expansion of CD34⁺ CD38^? cells. There was also a tendency for erythroid and granulocyte–myeloid colony formation in SP and CGRP treated cultures, respectively.

Supplementation of cytokines with other growth factors, such as neuropeptides, might enable us to overcome the difficulties of ex vivo expansion of CB cells.     

Primary cells as feeder cells for coculture expansion of human hematopoietic stem cells from umbilical cord blood--a comparative study

Although umbilical cord blood (UCB) has been widely accepted as an alternative source of hematopoietic stem cells (HSC) for transplantation, its use in adults is restricted because of low absolute HSC numbers. To overcome this obstacle, expansion of HSC in coculture with feeder cells is a promising possibility. In this study, we compared the potential of three human primary cell types, namely, mesenchymal stem cells (MSC), human umbilical cord vein endothelial cells (HUVEC), and Wharton's jelly cells (WJC), for use as feeder cells in a potentially clinically applicable coculture system. In first experiments, we evaluated procedures needed to obtain feeder cells, the possibility to separate them from cells derived from CD34(+) cells after coculture, their ability to activate allogeneic T cells, and their survival in CD34(+)-adapted medium. Finally, we compared their support for UCB-derived CD34(+) expansion. MSC and WJC were superior to HUVEC in terms of ease and reliability of isolation procedures needed. None of the potential feeder cells expressed CD34 or CD45, thus providing markers for cell sorting after coculture. Other markers (CD31, CD90, CD105, CD166) were expressed differently on feeder cell types. While MSC in higher concentrations did not activate allogeneic T cells, those were stimulated by lower concentrations of MSC as shown by CD25, CD69, and CD71 expression. In contrast, HUVEC and WJC were proven to activate T cells at all ratios tested. Feeder cells survived a 7-day culture in CD34(+)-adapted medium. In cocultures of UCB CD34(+)cells with primary feeder cells, mononuclear cell expansion was 30- to 60-fold, colony-forming cell expansion 20- to 40-fold, and cobblestone area-forming cell expansion 10- to 50-fold. We conclude that after a careful further evaluation especially of their immunological properties, all three primary cell types might possibly be suitable for use in a potentially clinically applicable system for expansion from UCB CD34(+)cells, with WJC being best choice and MSC still superior to HUVEC.     

Induction of various immune modulatory molecules in CD34(+) hematopoietic cells

Lipopolysaccharide (LPS) has been shown to induce proliferation of human T-lymphocytes only in the presence of monocytes and CD34(+) hematopoietic cells (HCs) from peripheral blood. This finding provided evidence of an active role of CD34(+) HCs during inflammation and immunological events. To investigate mechanisms by which CD34(+) HCs become activated and exert their immune-modulatory function, we used the human CD34(+) acute myeloid leukemia cell line KG-1a and CD34(+) bone marrow cells (BMCs). We showed that culture supernatants of LPS-stimulated mononuclear cells (SUP(LPS)) as well as tumor necrosis factor alpha (TauNF-alpha), but not LPS alone, can activate nuclear factor-kappaB in KG-1a cells. By cDNA subtraction and multiplex polymerase chain reaction, we revealed differential expression of cellular inhibitor of apoptosis protein-1, inhibitor of kappaB (IkappaB)/IkappaBalpha (MAD-3), and intercellular adhesion molecule-1 (ICAM-1) in SUP(LPS)-stimulated KG-1a cells and up-regulation of interferon (IFN)-inducible T cell-chemoattractant, interleukin (IL)-8, macrophage-inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, RANTES, CD70, granulocyte macrophage-colony stimulating factor, and IL-1beta in stimulated KG-1a cells and CD34(+) BMCs. Although monokine induced by IFN-gamma, IFN-inducible protein 10, and IFN-gamma were exclusively up-regulated in KG-1a cells, differential expression of monocyte chemoattractant protein-1 (MCP-1), macrophage-derived chemokine, myeloid progenitor inhibitory factor-2, and IL-18 receptor was only detectable in CD34(+) BMCs. More importantly, CD34(+) BMCs stimulated by TNF-alpha also showed enhanced secretion of MCP-1, MIP-1alpha, MIP-1beta, and IL-8, and increased ICAM-1 protein expression could be detected in stimulated KG-1a cells and CD34(+) BMCs. Furthermore, we revealed that T cell proliferation can be induced by TNF-alpha-stimulated KG-1a cells, which is preventable by blocking anti-ICAM-1 monoclonal antibodies. Our results demonstrate that CD34(+) HCs have the potential to express a variety of immune-regulatory mediators upon stimulation by inflammatory cytokines including TNF-alpha, which may contribute to innate- and adaptive-immune processes.     

Isolation and expansion of synovial CD34(-)CD44(+)CD90(+) mesenchymal stem cells: comparison of an enzymatic method and a direct explant technique

Synovium-derived mesenchymal stem cells (MSCs) offer a promising therapeutic option for cartilage regeneration. The conventional method of MSC isolation involves single-cell suspensions using collagenases. Recently, a nonenzymatic explant technique was developed to isolate MSCs. We compared these techniques in the isolation of functional MSCs. MSCs were isolated from human fibrous and adipose synovium of osteoarthritic patients using explants or enzymatic methods. Total cell number, percentage of MSCs, and surface marker expression of MSCs were measured following expansion. Multipotentiality was determined using a MSC functional identification kit. MSCs isolated from fibrous or adipose synovium using these two techniques expressed similar levels of the surface markers CD44, CD90, and CD105, and displayed similar multipotentiality in generating adipocytes, osteoblasts, and chondrocytes. Total cell number and number of CD34(-)CD44(+)CD90(+) MSCs after 10-day expansion were similar in each culture, regardless of the source and method used, although the percentage of MSCs was slightly higher in explant cultures. There were no correlations between MSC yield and patient age, Hospital for Special Surgery score, and degree of deformity under all culture conditions. Both the enzymatic and explant techniques yielded similar yields of MSCs with similar characteristics. Because the explant technique is simpler and less invasive, it may be preferred over enzymatic techniques for isolating MSCs from the synovium of osteoarthritic patients for cartilage regeneration.     

Bone marrow stromal cells act as feeder cells for tendon fibroblasts through soluble factors

Feeder effects of bone marrow stromal cells (BMSCs) on tendon fibroblasts were investigated using a co-culture method for the application of ligament or tendon tissue engineering and cell therapy. BMSCs had significant effects on enhancing cell proliferation, the ability of cells to migrate, and cell adhesivity but little effect on the extracellular matrix (ECM) synthesis of tendon fibroblasts without cell-cell contact. Furthermore, the conditioned medium from BMSCs, despite the existence of fibroblasts, significantly increased the number of fibroblasts. Based on these results, the mechanism of the feeder effects is considered to be a certain signal of soluble factors from BMSCs to the fibroblasts. Comparative proteome analysis of the conditioned medium from co-culture of fibroblasts and BMSCs revealed less expression of plasminogen, which showed inhibitory effects on fibroblast proliferation. With regard to the relationships between plasminogen and BMSCs in the co-culture system, we speculate that BMSCs allow resolution of plasminogen or its cleavage activity in the medium via some mechanism.     

Therapeutic effect of RANTES-KDEL on inhibition of HIV-1 in CD34(+) human hematopoietic stem cells (hHSC)

Cell surface receptors, such as the CCR5 chemokine receptors, represent key determinants of the human immunodeficiency virus type 1 (HIV-1) entry into target cells. The CC-chemokine, RANTES (regulated upon activation, normal T-cell expressed and secreted), a ligand for CCR5, have been targeted to the lumen of endocytoplasmic reticulum (ER) using a KDEL (ER-retention signal) fusion termed RANTES-KDEL and this construct was found to prevent effectively transport of newly synthesized CCR5 to the cell surface. Lentiviral vectors have emerged as potent and versatile tools of gene transfer for basic and applied research are able to transduce nondividing cells and maintain sustained long-term expression of transgenes. For this reason, an HIV-based lentiviral vector expressing RANTES-KDEL, pLenti6/V5-R-K, was constructed and then cotransfected with the ViraPower Packaging Mix (pLP1, pLP2, and pLP/VSVG) into 293FT cells to produce a replication-incompetent lentivirus stock. The lentiviral stock was titrated using HeLa cells, and the expression of the gene of interest, RANTES, was detected by indirect immunofluorescence. Based on the above results, the lentiviral stock was transduced into CD34(+) human hematopoietic stem cells (hHSC) separated magnetically from the cord blood (the purity was 96.8% evaluated by flow cytometry). Finally, the levels of p24 in the cultures of pLenti6/V5-R-K-transduced CD34(+) hHSC were detected after infection by HIV-1 DP1 (a R5-tropic HIV-1 strain, which was isolated by the Centers for Disease Control and Prevention of China in Henan province in 2000 from a Chinese man who had asymptomatic HIV-1 infection with a history of blood transfusions). It was shown that pLenti6/V5-R-K transduction inhibited expression of the DP1 p24 antigen by 51%, 58% and 60% on the 4th, 7th and 10th day respectively (P<0.05).     

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.     

Intramyocardial transplantation of circulating CD34+ cells: source of stem cells for myocardial regeneration

This study was designed to investigate the increase in the number of circulating CD34+ cells after acute myocardial infarction (MI) and the differentiation of these cells to cardiomyocytes after transplantation into infarcted myocardium. The study involved five donor groups: MI (n=27), sham (n=26), granulocyte-colony stimulating factor (GCSF) (n=26), MI+GCSF (n=25), and control (n=25). Acute MI was induced by ligating the left anterior descending coronary arteries (LAD) of donor rats, and LAD of recipient rats were ligated on the same day. Seven days after ligation, CD34+ cells in donor rats were counted and then were directly injected into the infarcted myocardium of recipient rats. Eight weeks after the transplantation, significant differences (p<0.001) were observed in the CD34+cell counts among the 5 donor groups with the greatest increase in the MI+GCSF donor group. In rats receiving CD34+ cells, the size of the scar area smaller (p<0.001) and the thickness of the scar was greater (p=0.001) than in CD34- and saline-transplanted rats. The transplanted CD34+ cells differentiated into cardiomyocytes in the scar. This study suggests that CD34+ cells may be a potential source of stem cells and that they may be useful in strategies aimed at the regeneration of infarcted myocardium.     

Lentivirus-mediated transduction of PKR into CD34(+) hematopoietic stem cells inhibits HIV-1 replication in differentiated T cell progeny.

Previous studies from this laboratory evaluated the role of p68 kinase (PKR) in the control of HIV-1 replication via retrovirus-mediated gene transfer. PKR was studied because it is a key component of the interferon (IFN)-associated innate antiviral defense pathway in mammalian cells. In this study, CD34(+) hematopoietic stem cells (HSC) were transduced with an HIV-1-based lentiviral vector encoding the PKR transgene (pHIV-PIB) and cultured under conditions that support in vitro differentiation. With high-titer pseudotyped vector stocks, the histogram suggests 100% transduction of the HSC because the cells were blasticidin resistant. Analysis of transduced cells by hybridization revealed an average proviral vector copy number of 1.8 and 2.1 copies of vector sequence per cell. Increased PKR expression and activity (phosphorylation of eukaryotic initiation factor 2alpha [eIF2alpha]) were demonstrated in PKR-transduced, differentiated HSC. There was minimal reduction in cell viability and no induction of apoptosis after transduction of PKR. HSC transduced with the pHIV-PIB lentiviral vector demonstrated normal differentiation into CD34-derived T cell progeny. Two days after HIV-1 infection, lentivirus-mediated transduction of PKR inhibited HIV-1 replication by 72% in T cell progeny compared with cells transduced with the empty vector control (pHIV-IB). By days 5 and 7 post-HIV-1 infection, the surviving PKR-transduced cells were protected from HIV-1 infection, as evidenced by a decrease in p24 antigen expression of at least two orders of magnitude. Our results demonstrate that PKR can be effectively delivered to HSC by a lentiviral vector and can protect CD34-derived T cell progeny from HIV-1 infection. These results provide support for application of the innate antiviral defense pathway in a gene therapy setting to the treatment of HIV-1 infection.     

CD34免疫亲合柱在脐血造血干/祖细胞分离与扩增中的应用

目的 :观察CD34免疫亲合柱对脐血造血干 祖细胞分离纯化的效果及纯化后CD34 细胞的增殖分化特性。方法 :采用CD34免疫亲合柱分离脐血单个核细胞 (MNC)中的CD34 细胞 ,流式细胞技术 (FACS)进行细胞表面标志测定。将分离前后的细胞加入造血生长因子进行液态扩增培养和多向祖细胞集落 (CFU GEMM)培养。结果 :经CD34免疫亲合柱分离后脐血中CD34 细胞为 49 6 2 %± 17 6 9% ,明显高于脐血MNC(1 17%± 0 6 8% ) ,细胞回收率为 5 4 38%± 11 91%。分离后CD34 细胞和脐血MNC经造血生长因子刺激培养 2 0d分别扩增 5 6 1 0 0倍和 44 44倍。培养至 12d时 ,免疫亲合柱分离组CD34 细胞阳性率为 5 3 38% ,对照组为 7 91%。分离组CFU GEMM产率明显高于对照组 (P <0 0 0 1)。结论 :CD34免疫亲合柱应用于脐血造血干 祖细胞的分离可充分富集CD34 细胞 ,且分离后的CD34 细胞具有明显的增殖效应和CFU GEMM形成能力。     

Chromosome analysis after ex vivo expansion of CD34(+) cells from human cord blood

Ex vivo expansion of cord blood hematopoietic progenitors is an attractive way to prepare a sufficient number of transplantable cells for cord blood transplantation in adult patients. The expanded cells need to have genetic stability. Karyotypic analysis of the expanded cells from cord blood CD34(+) cells by 7-day culture with stem cell growth factor, interleukin (IL)-3, IL-6, granulocyte colony-stimulating factor, and erythropoietin was performed. Several-fold increases in total cell number and CFU-GM were 186.7 +/- 62.1 and 27.1 +/- 9.4 (mean +/- standard deviation of data from 6 samples), respectively. Karyotypes were analyzed in 600 expanded cells in total, and all of them showed normal karyotypes. This observation suggests that multifactor supported expansion of cord blood cells may not induce karyotypic abnormality, although a limited number of ex vivo expanded cells were tested.     

Human hematopoietic (CD34+) stem cells possess high-affinity receptors for adenovirus type 11p

Gene transfer into human hematopoietic stem cells using Ad5 is inefficient due to lack of the primary receptor CAR and the secondary receptors alphavbeta3 integrin and alphavbeta5 integrin, and due to the high seroprevalence of Ad5 antibodies in most adults, resulting in diminished gene transduction. In the present study, we screened six species (species A-F) of adenovirus, displaying different tropisms for interaction with CD34+ cells, at the level of virus attachment and expression. Virus particles were biotinylated and their binding capacity was determined by FACS analysis using streptavidin-FITC. Ad11p, Ad35, and Ad3 (species B) showed high binding affinity, while Ad7, Ad11a (species B), and Ad37 (species D) displayed intermediate affinity. Virions of Ad4 (species E), Ad5 (species C), Ad31 (species A), and Ad41 (species F) hardly bound to hematopoietic progenitor cells. Using a double-labeling system, we demonstrated that adenoviruses bind to quiescent CD34+ cells. Ad11p virions showed the highest affinity among the adenoviruses detected. We further confirmed that virus fiber-specific receptors were present on the hematopoietic progenitor cell surface, because both recombinant fiber of Ad11p and specific antiserum against rfiber could block virus attachment. The ability of the adenoviruses to infect hematopoietic cells was studied by immunofluorescence staining. The adenoviruses from species B and Ad37 showed higher infectivity than Ad31, Ad5, Ad4, and Ad41. Among the studied species B adenoviruses, Ad11p manifested a superior infectivity. Thus, we have confirmed that these cells have high-affinity receptors for species B:2 human adenovirus, Ad11p, and this virus may be used as candidate vector to target therapeutic genes to hematopoietic stem cells.     

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.     

Cytokine-induced expansion of human CD34+ stem/progenitor and CD34+CD41+ early megakaryocytic marrow cells cultured on normal osteoblasts

Thrombocytopenia remains a significant cause of morbidity in cancer patients undergoing allogeneic bone marrow transplantation (BMT), which consumes millions each year for frequent platelet transfusions. Using a novel culture system containing appropriate cytokine(s) on a layer of normal human osteoblasts, we investigated the expansion of early megakaryocytic progenitor cells while maintaining the number of CD34+ stem/progenitor marrow cells in an attempt to provide an effective solution for the problem of post-transplant thrombocytopenia. After seven days of culture, normal human osteoblasts alone without cytokines significantly increased the number of CD34+ and CD34+CD41+ marrow cells. Among the various cytokine combinations tested, both stem cell factor (SCF), interleukin 3 (IL-3)+IL-11 and SCF+IL-3+IL-11+thrombopoietin (TPO) emerged as the most effective in expanding early CD34+CD41+ megakaryocytic cells. Early CD34+CD41+ megakaryocytic cells have increased by 3.1- and 4.7-fold compared with day 7 control cultures, and by 62- and 94-fold, respectively, compared with day 0 input, respectively. Also, late CD41+ megakaryocytic cells have increased by 15.4- and 27.5-fold compared with day 7 control cultures in the presence of the same two combinations. In addition, the same cytokine combinations achieved 17.6- and 13.3-fold increases in the number of CD34+ marrow cells after the same seven days of culture on a layer of human osteoblasts. The combination (SCF+IL-3+IL-11+TPO) achieved the highest expansion of CD34+CD41+ early megakaryocytic cells from human marrow CD34+ cells reported so far in the literature. Recently, transplantation of SCF+IL-1+IL-3+TPO ex vivo expanded megakaryocytic progenitor cells as a supplement has been shown to accelerate platelet recovery by three to five days in mice. Therefore, the clinical use of the combination (SCF+IL-3+IL-11+TPO) for ex vivo expansion of CD34+ and megakaryocytic progenitor cells from a portion of the donor's marrow harvest is warranted in allogeneic BMT. Such a protocol would accelerate platelet recovery and shorten the period of hospitalization after allogeneic BMT. The present study has confirmed the role of human osteoblasts in supporting the proliferation and maintenance of human CD34+ stem/progenitor marrow cells. Given the facilitating role of osteoblasts shown previously in several allogeneic BMT studies in mice, it is possible to envisage a future role for donor osteoblasts in clinical BMT. Transplantation of the cultured donor osteoblasts together with the ex vivo expanded CD34+ marrow cells as a supplement might not only accelerate platelet recovery but also prevent acute graft-versus-host disease in allogeneic BMT. The present novel culture system should have useful clinical application in allogeneic BMT.     

Hybrid HIV/MSCV LTR enhances transgene expression of lentiviral vectors in human CD34(+) hematopoietic cells

HIV-based lentiviral vectors can transduce nondividing cells, an important advantage over murine leukemia virus (MLV)-based vectors when transducing slowly dividing hematopoietic stem cells. However, we find that in human CD34(+) hematopoietic cells, the HIV-based vectors with an internal cytomegalovirus (CMV) promoter express transgenes 100- to 1,000-fold less than the MLV-based retroviral vector murine stem cell virus (MSCV). To increase the expression of the integrated lentivirus, we replaced CMV promoter with that of the Rous sarcoma virus or MSCV and obtained a modest augmentation in expression. A more dramatic effect was seen when the CMV enhancer/promoter was removed and the HIV long-terminal repeat (LTR) was replaced by a novel HIV/MSCV hybrid LTR. This vector retains the ability to transduce nondividing cells but now expresses its transgene (enhanced green fluorescent protein) 10- to 100-fold greater than the original HIV-based vector. When compared under identical conditions, the HIV vector with the hybrid LTR transduced a higher percentage of CD34(+) cells than the MSCV-based retroviral vector (19.4% versus 2.4%). The number of transduced cells and level of transgene expression remain constant over 5-8 weeks as determined by long-term culture-initiating cells, fluoresence-activated cell sorting, and nonobese diabetic/severe combined immunodeficiency repopulation assay.     

Generation of antigen-presenting cells for soluble protein antigens ex vivo from peripheral blood CD34+ hematopoietic progenitor cells in cancer patients

Peripheral blood CD34⁺ hematopoietic progenitor cells (PBPC) mediate hematopoietic reconstitution in cancer patients after autologous transplantation and can be expanded ex vivo in the presence of colony-stimulating factors. This study shows that functionally active antigen-presenting cells (APC) for soluble proteins are generated and expanded in these PBPC cultures. CD34⁺ cells were cultured ex vivo in medium containing stem cell factor, interleukin-1β (IL-1β), IL-3, IL-6, and erythropoietin (EPO). The cells from these cultures developed into very potent APC of tetanus toxid and purified derivative of tuberculin for autologous T cells in vitro. The antigen-presenting capacity of these cells was maintained for at least 38 days of culture. These APC resembled immature cells of the myelomonocytic cell lineage by surface marker, immunocytochemistry and ultrastructural analysis. Such APC might be able to present antigens from certain tumors to the immune system.     

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.