Ex vivo expansion of cord blood mononuclear cells on mesenchymal stem cells

BACKGROUND: Cord blood (CB) cells are being used increasingly as a source of hematopoietic cells to support high dose chemotherapy. However, CB units contain low numbers of cells, including CD34+ cells, and thus their use is associated with significant delays in engraftment of neutrophils and platelets. Exvivo expansion of CB has been proposed to increase the numbers of cells available. We and others have reported the requirement of CD34 selection for optimal expansion of CB products'; however, the selection of frozen CB products in clinical trials results in significant loss of CD34+ cells, with a median recovery of 50, but less than 40% recovery in more than one-third of products. In the present studies we evaluated the potential of mesenchymal stem cells (MSC) to support ex vivo expansion of unselected CB products. METHODS: Mononuclear cells (MNC) from CB products were isolated and cultured on preformed MSC layers in T150 flasks containing 50 mL Stemline II media plus hematopoietic growth factors. Various culture conditions were compared for optimal expansion of the CB MNC. RESULTS: Ex vivo expansion of CB MNC on MSC resulted in 10- to 20-fold expansion of total nucleated cells, seven- to 18-fold expansion of committed progenitor cells, two- to five-fold expansion of primitive progenitor cells and 16- to 37-fold expansion of CD34+ cells. DISCUSSION: These studies demonstrated significant expansion of CB products without CD34 cell selection using culture conditions that are clinically applicable. Our current focus is to initiate clinical trials to evaluate the in vivo potential of CB cells expanded with these conditions.     

Ex vivo expansion of neutrophil precursor cells from mobilized peripheral blood cells: similar results in cancer patients and normal donors

BACKGROUND: Infusion of ex vivo differentiated myeloid progenitors may reduce or abrogate severe neutropenia following mobilized peripheral blood transplantation. We compared the ex vivo expansion of myeloid progenitor cells starting from cancer patients (CP) and from normal donors (ND) and evaluated the influence of the CD34(+) cell mobilization on the capacities of cells to be expanded. METHODS: The ex vivo-expanded cells were evaluated for their phenotype, the presence of primary and secondary granules and their functional capacities (oxidative burst activity and phagocytosis). RESULTS: We did not observe significant differences between ND and CP for the total leukocyte and CD34(+) cell expansions nor for the myeloid progenitor production. In CP as well as in ND, the expanded cells were functionally competent. DISCUSSION: This suggests that the capacities of CD34(+) cells to proliferate and differentiate ex vivo are not impaired by prior chemotherapy and/or disease status. On the other hand, we did not observe any significant correlation between the number of mobilized CD34(+) cells before apheresis and the cell expansion. In conclusion, the ex vivo expansion of CP and ND cells is comparable and achievable even with a low CD34(+) cell number in mobilized peripheral blood.     

Pre-clinical development of cord blood-derived progenitor cell graft expanded ex vivo with cytokines and the polyamine copper chelator tetraethylenepentamine

BACKGROUND: We have previously demonstrated that the copper chelator tetraethylenepentamine (TEPA) enables preferential expansion of early hematopoietic progenitor cells (CD34+CD38-, CD34+CD38-Lin-) in human umbilical cord blood (CB)-derived CD34+ cell cultures. This study extends our previous findings that copper chelation can modulate the balance between self-renewal and differentiation of hematopoietic progenitor cells. METHODS: In the present study we established a clinically applicative protocol for large-scale ex vivo expansion of CB-derived progenitors. Briefly, CD133+ cells, purified from CB using Miltenyi Biotec's (Bergisch Gladbach, Germany) CliniMACS separation device and the anti-CD133 reagent, were cultured for 3 weeks in a clinical-grade closed culture bag system, using the chelator-based technology in combination with early-acting cytokines (SCF, thrombopoietin, IL-6 and FLT-3 ligand). This protocol was evaluated using frozen units derived from accredited cord blood banks. RESULTS: Following 3 weeks of expansion under large-scale culture conditions that were suitable for clinical manufacturing, the median output value of CD34+ cells increase by 89-fold, CD34+CD38- increase by 30-fold and CFU cells (CFUc) by 172-fold over the input value. Transplantation into sublethally irradiated non-obese diabetic (NOD/SCID) mice indicated that the engraftment potential of the ex vivo expanded CD133+ cells was significantly superior to that of unexpanded cells: 60+/-5.5% vs. 21+/-3.5% CD45+ cells, P=0.001, and 11+/-1.8% vs. 4+/-0.68% CD45+CD34+ cells, P=0.012, n=32, respectively. DISCUSSION: Based on these large-scale experiments, the chelator-based ex vivo expansion technology is currently being tested in a phase 1 clinical trial in patients undergoing CB transplantation for hematological malignancies.     

体外培养脐血单个核细胞与CD34+富集细胞

对比MNC和CD34 +富集细胞在SCF +IL 3+IL 6 +FL +Tpo细胞因子组合下的体外扩增特性 ,发现 :CD34 +富集细胞具有很高的扩增潜力 ,在本实验条件下其总细胞持续扩增了 8周 ,扩增倍数达 312 70 9± 86 40 5倍 ;而MNC在培养至第 4周扩增就已呈现下降趋势 ,最大仅扩增了 5 3 3± 6 2倍。对比集落和CD34 +细胞的扩增发现 ,MNC的集落密度和CD34 +细胞含量由第 0天至第 7天有一个上升的过程 ,而CD34 +富集细胞在培养过程中 ,集落密度和CD34 +细胞含量却始终呈下降趋势。在体外培养过程中 ,CD34 +富集细胞的CFU GM和CD34 +细胞最大分别扩增了 185 7± 14 1和 191 7± 188 8倍 ,明显高于MNC的 12 4± 3 2和 5 0 6± 33 2倍 ;而CD34 +富集细胞和MNC的BFU E则只实现了少量扩增 ,分别为 7 2± 5 2和 10 1± 3 4倍。结果显示 ,从CD34 +富集细胞出发扩增造血干 祖细胞 ,可以得到更多的CD34 +细胞和CFU GM集落形成细胞     

Expansion of CD34+ cells from human umbilical cord blood by FL and/or TPO gene transfected human marrow stromal cell lines

To elucidate the effect of gene transfected marrow stromal cell on expansion of human cord blood CD34+ cells, a culture system was established in which FL and TPO genes were transfected into human stromal cell line HFCL. To establish gene transfected stromal cells co-culture system, cord blood CD34+ cells were purified by using a magnetic beads sorting system. The number of all cells and the number of CD34+ cells and CFC (CFU-GM and BFU-E) were counted in different culture systems. The results showed that in all 8 culture systems, SCF+IL-3+HFT manifested the most potent combination, with the number of total nucleated cells increasing by (893.3±52.1)-fold, total progenitor cells (CFC) by (74.5±5.2)-fold and CD34+ cells by 15.7-fold.Maximal expansions of CFC and CD34+ cells were observed at the end of the second week of culture. Within 14 days of culture, (78.1 ± 5.5)-fold and (57.0 ± 19.7)-fold increases in CFU-GM and BFU-E were obtained. Moreover, generation of LTC-IC from amplified CD34+ cells within 28 days was found only in two combinations, I.e. SCF+IL-3+FL+TPO and SCF+IL-3+HFT, and there was no significant difference between these two groups statistically. These results suggest that human umbilical cord blood CD34+ cells can be extensively expanded ex vivo by using gene transfected stromal cells along with cytokines.     

Autologous cell therapy as a new approach to treatment of radiation-induced bone marrow aplasia: preliminary study in a baboon model

The sparing of viable hematopoietic stem and progenitor cells located in underexposed bone marrow territories associated with the relative radioresistance of certain stem cell populations is the rationale for autologous cell therapy consisting of ex vivo expansion of residual cells after collection postirradiation. The feasibility of this treatment mainly depends on time constraints and hematopoietic cell threshold. We showed in this study that in the absence of early-acting mobilizing agent administration, subliminar amounts of CD34+ cells can be collected (1 x 10(6) CD34+ cells/100 mL bone marrow or for 1 L apheresis) from 6-Gy gamma globally irradiated baboons. Residual CD34+ cells were successfully expanded in serum-free medium in the presence of antiapoptotic cytokine combination (stem cell factor + FLT-3 ligand + thrombopoietin + interleukin 3, 50 ng/mL each, i.e., 4F): KCD34+ = x2.8 and x13.7 (n = 2). Moreover, we demonstrated the short-term neutrophil engraftment potential of a low-size mixed expanded graft (1.5 x 106 final CD34+cells/kg) issued from the coculture of unirradiated (20%) and 2.5-Gy in vitro irradiated (80%) CD34+ cells on an allogeneic stromal cell layer in the presence of 4F. Further preclinical research needs to be performed to clearly establish this therapeutic approach that could be optimized by the early administration of antiapoptotic cytokines.     

Low angiogenic potency induced by the implantation of ex vivo expanded CD117(+) stem cells

Ex vivo expansion of stem cells might be a feasible method of resolving the problem of limited cell supply in cell-based therapy. The implantation of expanded CD34(+) endothelial progenitor cells has the capacity to induce angiogenesis. In this study, we tried to induce angiogenesis by implanting expanded CD117(+) stem cells derived from mouse bone marrow. After 2 wk of culture with the addition of several growth factors, the CD117(+) stem cells expanded approximately 20-fold and had an endothelial phenotype with high expression of CD34 and vascular endothelial-cadherin. However, >70% of these ex vivo expanded cells had a senescent phenotype by beta-galactosidase staining, and their survival and incorporation were poor after implantation into the ischemic limbs of mice. Compared with the PBS injection only, the microvessel density and the percentage of limb blood flow were significantly higher after the implantation of 2 x 10(5) freshly collected CD117(+) cells (P < 0.01) but not after the implantation of 2 x 10(5) expanded CD117(+) cells (P > 0.05). These data indicate that ex vivo expansion of CD117(+) stem cells has low potency for inducing therapeutic angiogenesis, which might be related to the cellular senescence during ex vivo expansion.     

Ex vivo expansion and clonality of CD34+ selected cells from bone marrow and cord blood in a serum-free media

Although umbilical cord blood is increasingly being used in allogeneic marrow transplantation, delayed platelet engraftment is often a concern for cord blood transplant recipients. We evaluated the potential of ex vivo expansion and clonality in CD34+ cells separated from a bone marrow source, and cord blood, in a serum-free Media. The CD34+ cells, selected from bone marrow (BM) and umbilical cord blood (CB), were expanded with hematopoietic growth factors. They were then cultured for burst-forming units of erythrocytes (BFU-E), colony-forming units of granulocytes and monocytes (CFU-GM) and colony-forming units of megakaryocytes (CFU-Mk) at days 0, 4, 7, and 14 under the combination of growth factors, with cell counts. The cytokines included the recombinant human megakaryocyte growth and development (100 ng/ml), interleukin-3 (10 ng/ml), stem cell factor (100 ng/ml), flt-3 ligand (50 ng/ml) and interleukin-11 (200 ng/ml). The CB-selected CD34+ cells showed significantly higher total cell expansion than those from the BM at day 7 (3.0 fold increase than BM), day 14 (2.4 fold), and day 17 (2.6 fold). The colony count of the BFU-E/CFU-E per CD34+ cell at day 0 was 0.14 +/- 0.023 in the CB, which was significantly higher than 0.071 +/- 0.015 in the BM. The CB-selected CD34+ cells produced more BFU-E colonies than the BM on culture days 4, 7, and 14. The BFU-E colonies from the CB cells increased markedly on culture days 4 and 7, with a 4-fold increase at day 14. The colony count of the CFU-Mk per CD34+ cell at day 0 was 0.047 +/- 0.011 in the CB-selected CD34+ cells cultures, which was higher than the 0.026 +/- 0.014 in the BM. The CB-selected CD34+ cells produced more CFU-Mk colonies than the BM on culture days 4, 7 and 14. In conclusion, the ex vivo expansion of the CB cells may be very promising in producing total cellular expansion, CFU-Mk and BFU-E compared with BM, especially at day 7. The ex vivo expansion of the CB may have rationale in making an ex vivo culture for 7 to 14 d.     

一种新型生物反应器在造血干/祖细胞体外扩增中的应用

针对造血干/祖细胞体外扩增对培养环境的需求, 结合静/动态培养的特点, 开发了一种新型的生物反应器用于造血干/祖细胞的体外扩增。在该生物反应器内, 采用SCF+TPO+Flt-3细胞因子组合, 比较了静态和循环培养两种方式体外扩增脐血CD34+细胞的效果。培养7 d后, 总细胞分别扩增了(13.86 ± 4.26)和(7.23 ± 2.67)倍, 显示静态培养有利于总细胞的扩增; CD34+细胞扩增倍数、培养物中CD34+细胞含量均相近, 无显著性差异; 而CD34+CD38-细胞扩增倍数以及培养物中CD34+CD38?细胞的百分含量分别为(1.82 ± 0.58)和(3.90 ± 0.85)倍以及(9.45 ± 4.85)和(37.47 ± 14.06)%, 循环培养明显高于静态培养。可见, 在该生物反应器内, 采用静态和循环两种培养方式, 均能实现造血干/祖细胞的体外扩增, 但静态培养促使造血干细胞向定向祖细胞分化, 而循环培养则更有利于早期造血干细胞的扩增。     

一种新型生物反应器在造血干/祖细胞体外扩增中的应用

针对造血干/祖细胞体外扩增对培养环境的需求, 结合静/动态培养的特点, 开发了一种新型的生物反应器用于造血干/祖细胞的体外扩增.在该生物反应器内, 采用SCF TPO Flt-3细胞因子组合, 比较了静态和循环培养两种方式体外扩增脐血CD34 细胞的效果.培养7 d后, 总细胞分别扩增了(13.86 ± 4.26)和(7.23 ± 2.67)倍, 显示静态培养有利于总细胞的扩增; CD34 细胞扩增倍数、培养物中CD34 细胞含量均相近, 无显著性差异; 而CD34 CD38-细胞扩增倍数以及培养物中CD34 CD38-细胞的百分含量分别为(1.82 ± 0.58)和(3.90 ± 0.85)倍以及(9.45 ± 4.85)和(37.47 ± 14.06)%, 循环培养明显高于静态培养.可见, 在该生物反应器内, 采用静态和循环两种培养方式, 均能实现造血干/祖细胞的体外扩增, 但静态培养促使造血干细胞向定向祖细胞分化, 而循环培养则更有利于早期造血干细胞的扩增.     

In vitro characterization of two lineage-negative CD34+ cell-enriched hematopoietic cell populations from human UC blood

BACKGROUND: During the last few years there has been increasing interest, from both biologic and clinical points of view, in the ex vivo expansion of umbilical cord blood (UCB)-derived hematopoietic cells. This has brought about the need to characterize different cell populations present in UCB, and to explore different ex vivo approaches for the culture, expansion and biologic manipulation of these cells. METHODS: By using a negative-selection method, two UCB cell populations were obtained that were enriched for primitive lineage-negative (Lin-) cells, including those expressing the CD34 Ag (35-93% of the total cells in each fraction). Population I was enriched for CD34+ Lin- cells, whereas population II was enriched for CD34+ CD38- Lin- cells. Both populations were cultured in serum-free liquid cultures supplemented with different combinations of early and late-acting recombinant cytokines (all of them added at 10 ng/mL). Every 5-7 days proliferation, expansion and differentiation capacities of each population were determined, for a total period of 25-42 days. RESULTS: Both cell populations showed extensive proliferation and expansion capacities; however, population II [2300- and 232-fold increase in nucleated and colony-forming cell (CFC) numbers, respectively] was clearly superior in both parameters compared with population I (1120- and 20-fold increase in nucleated and CFC numbers, respectively). Depending on the cytokine combination used, granulocytes, macrophages and erythroblasts were preferentially produced. We also observed that both populations were highly sensitive to the inhibitory effects of tumor necrosis factor-alpha, even in the presence of stimulatory cytokines. DISCUSSION: This study demonstrates that the two progenitor cell-enriched populations obtained by negative selection possess extensive proliferation and expansion potentials in vitro, generating significant numbers of both primitive and mature cells. These cells may be a good alternative to purified CD34+ cells, obtained by positive selection, for pre-clinical and clinical protocols aimed at the ex vivo expansion of UCB cells.     

Different growth factor requirements for the ex vivo amplification of transplantable human cord blood cells in a NOD/SCID mouse model

The growth factor combination containing early acting cytokines FLT-3 ligand (FL), Stem Cell Factor (SCF) and thrombopoietin (TPO) is able to maintain, for an extended culture period, early stem cells, defined as long-term repopulating NOD/SCID mice (Scid Repopulating Cell-SRC) contained in cord blood (CB). In this culture system, the role of IL-6 and IL-3 has not been clearly established. Using a combination of FL+TPO+SCF with or without IL-6, we were able to form CB CD34+ cells for 30 weeks. The CB CD34+ cells cultured in this system engrafted NOD/SCID mice after 6 weeks of culture; the cells from primary recipients were also able to engraft secondary NOD/SCID mice. When CB CD34+ cells were cultured in the presence of IL-3 in the place of IL-6 we observed an even better expansion of cells and a similar clonogenic progenitor output in the first 8 weeks of culture. However, more primitive LTC-IC output increased up to week 6 with the growth factor combination containing IL-3 and then decreased and disappeared, while with the growth factor combination with or without IL-6 increased up to week 23. Cells cultured for 4 weeks with the 4-factor combination containing IL-3 engrafted NOD/SCID mice less efficiently. Repopulation of NOD/SCID mice was no longer observed when ex vivo expansion was performed for 6 weeks. This study provides some evidence that no differences could be detected in long-term maintenance and even expansion of human primitive cord blood cells cultured with FL+TPO+SCF in the presence or absence of IL-6. Under the culture conditions employed in this study, the presence of IL-3 reduced the repopulating potential of expanded CB CD34+ cells.     

Ex vivo expanded human CD4+CD25+Foxp3+ regulatory T cells prevent lethal xenogenic graft versus host disease (GVHD)

Mouse studies demonstrated that infusion of CD4+CD25+ regulatory T cells (Tregs) prevented graft versus host disease (GVHD) lethality after bone marrow transplantation (BMT). But the potential impact of human Tregs on GVHD has not been well demonstrated. In this study, we demonstrated that human Tregs enriched from peripheral blood of healthy donors could be expanded ex vivo to clinically relevant cell numbers in 2-3 weeks while maintaining Foxp3, CD25, CTLA-4, and CD62L expression as well as in vitro suppressive function. Furthermore, injection of human PBL into NOD/SCID mice induced lethal xenogenic GVHD, but co-transfer of expanded human Tregs with human PBL significantly enhanced survival, reduced GVHD symptoms, and inhibited human IgG/IgM production in the NOD/SCID mice. These results demonstrated that ex vivo expanded human Tregs retained their in vivo suppressive activity and prevented lethal xenogeneic GVHD, revealing the therapeutic potential of expanded human Tregs for GVHD.     

Ex vivo expansion and lentiviral transduction of Macaca nemestrina CD4+ T cells

Background  Macaca nemestrina is a nonhuman primate used as a model in preclinical studies of hematopoietic stem cell transplantation and adoptive transfer of T cells. Adoptive T cell transfer studies typically require ex vivo expansion of substantial numbers of T cells prior to their reinfusion into the subject.
Methods  Pigtailed macaque peripheral blood CD4⁺ cells were expanded using CD3 and CD28 antibody-coated beads. These cells were transformed using Herpesvirus saimiri and were also transduced with HIV-1 based lentiviral vectors.
Results  We report an efficient method for the ex vivo expansion of CD4⁺ T cells from Macaca nemestrina peripheral blood. With this protocol, primary CD4⁺ T cells can be expanded between 300- to 6000-fold during 24-day period and can be efficiently transduced with lentiviral vectors. Furthermore, these T cells can be transformed by Herpesvirus saimiri and maintained in culture for several months. The transformed T cell lines can be productively infected with the simian immunodeficiency virus (SIV) strain SIV_mac239.
Conclusions  We have established methods for the expansion and transformation of primary M. nemestrina CD4⁺ T cells and demonstrated the utility of these methods for several applications.     

Serum-free ex vivo expansion of CD34(+) hematopoietic progenitor cells

In an effort to obtain defined culture conditions for ex vivo expansion of hematopoietic stem and progenitor cells which avoid the supplementation of serum, we cultured human CD34(+) hematopoietic progenitor cells in a chemically defined, serum-free medium in the presence of hematopoietic growth factors (HGFs), stem cell factor (SCF), interleukin (IL)-1beta, IL-3, IL-6, and erythropoietin (EPO). A medium, SFM-1, was prepared according to a protocol previously optimized for semisolid progenitor cell assays containing Iscove's Modified Dulbecco's Medium (IMDM) plus cholesterol, bovine serum albumin, transferrin, nucleotides and nucleosides, insulin, and beta-mercaptoethanol. In static cultures seeded with CD34(+)-enriched progenitor cells isolated from human peripheral blood, a mean 76.6-fold expansion of total nucleated cells and a mean 4.6-fold expansion of colony-forming cells (CFC) was recorded after 14 days. Morphological analysis of the expanded cells revealed formation of myeloid, erythroid, and megakaryocytic cells. Flow cytometric analysis indicated that CD34(+) antigen expressing cells were maintained to a limited degree only, and cell populations expressing surface markers for myeloid (CD33, CD14, and CD15) and megakaryocytic (CD41a) lineages predominated. Within SFM-1, bovine serum albumin (BSA), cholesterin, and transferrin represented the most critical components needed for efficient total cell and CFC expansion. Addition of autologous patient plasma (APP) or fetal calf serum (FCS) to SFM-1 resulted in inferior cell amplification and CFC formation compared to controls in SFM-1, indicating that the components used in SFM-1 could replace exogenous serum. Four commercially available serum-free media resulted in either comparable or lower total cell and CFC yields as SFM-1. The transplantation potential of CD34(+) cells after culture in SFM-1 was assayed using limiting dilution analysis on preformed irradiated bone marrow stroma and revealed maintenance of long-term bone marrow culture initiating cell (LTCIC) levels during the culture period. These data indicate that HGF-supported multilineage ex vivo expansion of human CD34(+) hematopoietic progenitor cells is feasible using an IMDM-based culture medium which contains a restricted number of additives, resulting in analogous or improved yields of both primitive and differentiated cells compared to previously established protocols. We suggest that this culture protocol is of advantage when working with pharmaceutical-grade preparations under serum-free conditions.     

Differences amid bone marrow and cord blood hematopoietic stem/progenitor cell division kinetics

Human hematopoietic stem/progenitor cells (HSC) isolated based upon specific patterns of CD34 and CD38 expression, despite phenotypically identical, were found to be functionally heterogeneous, raising the possibility that reversible expression of these antigens may occur during cellular activation and/or proliferation. In these studies, we combined PKH67 tracking with CD34/CD38 immunostaining to compare cell division kinetics between human bone marrow (BM) and cord blood (CB)‐derived HSC expanded in a serum‐free/stromal‐based system for 14 days (d), and correlated CD34 and CD38 expression with the cell divisional history. CB cells began dividing 24 h earlier than BM cells, and significantly higher numbers underwent mitosis during the time in culture. By d10, over 55% of the CB‐cells reached the ninth generation, whereas BM‐cells were mostly distributed between the fifth and seventh generation. By d14, all CB cells had undergone multiple cell divisions, while 0.7–3.8% of BM CD34⁺ cells remained quiescent. Furthermore, the percentage of BM cells expressing CD34 decreased from 60.8 ± 6.3% to 30.6 ± 6.7% prior to initiating division, suggesting that downmodulation of this antigen occurred before commencement of proliferation. Moreover, with BM, all primitive CD34⁺CD38^? cells present at the end of culture arose from proliferating CD34⁺CD38⁺ cells that downregulated CD38 expression, while in CB, a CD34⁺CD38^? population was maintained throughout culture. These studies show that BM and CB cells differ significantly in cell division kinetics and expression of CD34 and CD38, and that the inherent modulation of these antigens during ex vivo expansion may lead to erroneous quantification of the stem cell content of the expanded graft. J. Cell. Physiol. 220: 102–111, 2009. © 2009 Wiley‐Liss, Inc.     

Human umbilical vein endothelial cells increase ex vivo expansion of human CD34(+) PBPC through IL-6 secretion

BACKGROUND: Ex vivo expansion of hematopoietic stem cells (HSC) can help reduce cytopenia following transplantation, especially in NHL patients whose BM is deficient because of extensive chemotherapy. We have previously reported that human umbilical vein endothelial cells (HUVEC) can contribute to improved PBPC expansion when used in co-culture with CD34(+) cells. METHODS: We evaluated the roles of direct HUVEC CD34(+) contact and HUVEC-produced soluble factors. We cultured CD34(+) PBPC harvested from NHL patients in four different conditions: (1) liquid culture without HUVEC; (2) co-culture in contact with HUVEC; (3) co-culture with HUVEC but without direct contact; (4) liquid culture with HUVEC-conditioned medium (CM). Thrombopoietin (Tpo), Flk2Flt3 ligand (FL) and c-kit ligand (KL) with or without rhIL-6 were added to these four culture conditions. RESULTS AND DISCUSSION: Our results showed that HUVEC co-culture or addition of HUVEC-CM to Tpo, FL and KL (TFK) improved CD34(+) PBPC expansion compared with liquid culture, as determined by total viable nucleated cells (TNC), colony-forming cell assay (CFC) and week-6 cobblestone area-forming cells (Wk-6 CAFC) expansions. Non-contact culture led to similar PBPC expansion as contact co-culture; moreover, HUVEC-CM improved PBPC expansion. However, when rhIL-6 was added to HUVEC-CM with TFK, no significant difference was observed. Finally, high quantities of IL-6 were detected in HUVEC-CM and addition of anti-IL-6 Ab inhibited the positive effect of HUVEC on PBPC expansion. Our results thus suggest that HUVEC may improve PBPC expansion, at least through IL-6 secretion.     

Mesenchymal stem cells support expansion of in vitro irradiated CD34(+) cells in the presence of SCF, FLT3 ligand, TPO and IL3: potential application to autologous cell therapy in accidentally irradiated victims

Ex vivo expansion of residual autologous hematopoietic stem and progenitor cells collected from victims soon after accidental irradiation (autologous cell therapy) may represent an additional or alternative approach to cytokine therapy or allogeneic transplantation. Peripheral blood CD34+ cells could be a useful source of cells for this process provided that collection and ex vivo expansion of hematopoietic stem and progenitor cells could be optimized. Here we investigated whether mesenchymal stem cells could sustain culture of irradiated peripheral blood CD34+ cells. In vitro irradiated (4 Gy 60Co gamma rays) or nonirradiated mobilized peripheral blood CD34+ cells from baboons were cultured for 7 days in a serum-free medium supplemented with stem cell factor+thrombopoietin+interleukin 3+FLT3 ligand (50 ng/ml each) in the presence or absence of mesenchymal stem cells. In contrast to cultures without mesenchymal stem cells, irradiated CD34+ cells cultured with mesenchymal stem cells displayed cell amplification, i.e. CD34+ (4.9-fold), CD34++ (3.8-fold), CD34++/Thy-1+ (8.1-fold), CD41+ (12.4-fold) and MPO+ (50.6-fold), although at lower levels than in nonirradiated CD34+ cells. Fourteen times more clonogenic cells, especially BFU-E, were preserved when irradiated cells were cultured on mesenchymal stem cells. Moreover, we showed that the effect of mesenchymal stem cells is related mainly to the reduction of apoptosis and involves cell-cell contact rather than production of soluble factor(s). This experimental model suggests that mesenchymal stem cells could provide a crucial tool for autologous cell therapy applied to accidentally irradiated victims.     

Human CD34+ HLA-DR- bone marrow cells contain progenitor cells capable of self-renewal, multilineage differentiation, and long-term in vitro hematopoiesis.

Human bone marrow cells expressing CD34 but not HLA-DR were isolated by immunofluorescence flow cytometric cell sorting. These cells contained a hematopoietic cell (CFU-B1) capable of producing, in an in vitro semisolid culture system, blast-cell-containing colonies, which possessed the capacity for self-renewal and commitment to multipotential differentiation. In addition, CD34+ HLA-DR- marrow cells contained primitive megakaryocyte progenitor cells, the burst-forming unit-megakaryocyte (BFU-MK). A subset of CD34+ HLA-DR- marrow cells lacking the expression of CD15 and CD71 was obtained by flow cytometric cell sorting and was capable of sustaining in vitro hematopoiesis in suspension culture for up to 8 weeks in the absence of a preestablished adherent marrow cell layer. The combination of IL-3 + IL-1 alpha and IL-3 + IL-6 sustained proliferation of these cells for 8 weeks, induced maximal cellular expansion, and increased the numbers of assayable progenitor cells. These studies demonstrate that human CD34+ HLA-DR- marrow cells and their subsets contain primitive multipotential hematopoietic cells capable of self-renewal and of differentiation into multiple hematopoietic lineages.