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1.
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. 相似文献
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Background aimsCord blood is considered to be a superior source of hematopoietic stem and progenitor cells for transplantation, but clinical use is limited primarily because of the low numbers of cells harvested. Ex vivo expansion has the potential to provide a safe, effective means of increasing cell numbers. However, an absence of consensus regarding optimum expansion conditions prevents standard implementation. Many studies lack clinical applicability, or have failed to investigate the combinational effects of different parameters.MethodsThis is the first study to characterize systematically the effect of growth factor combinations across multiple oxygen levels on the ex vivo expansion of cord blood CD34+ hematopoietic cells utilizing clinically approvable reagents and methodologies throughout.ResultsOptimal fold expansion, as assessed both phenotypically and functionally, was greatest with thrombopoietin, stem cell factor, Flt-3 ligand and interleukin-6 at an oxygen level of 10%. With these conditions, serial expansion showed continual target population expansion and consistently higher expression levels of self-renewal associated genes.ConclusionsThis study has identified optimized fold expansion conditions, with the potential for direct clinical translation to increase transplantable cell dose and as a baseline methodology against which future factors can be tested. 相似文献
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Dimitriou H Bakogeorgou E Kampa M Notas G Stiakaki E Kouroumalis E Kalmanti M Castanas E 《Cytotherapy》2006,8(4):367-374
BACKGROUND: Opioid agonists have been shown to exert an inhibitory action on a number of malignant and non-malignant cell types. However, there are no reports dealing with their effect on hemopoietic progenitors. Based upon our previous experience of opioid agonists we examined whether opioids could interfere with the growth of CFU-GM from CD133(+) cord blood cells. METHODS: Cord blood samples were subjected to CD133(+) column selection, with subsequent exposure to opioid agonists and antagonists or both, in semi-solid cultures for CFU-GM growth. Colonies of day 7 of culture were replated in fresh medium in the absence of opioids. The colonies were evaluated at 7 and 14 days of culture. RT-PCR was performed for the detection of opioid and somatostatin receptors. Apoptosis tests and immunophenotypic evaluations were employed in liquid cultures in conditions identical to those of the semi-solid ones. RESULTS AND DISCUSSION: Our results suggest that opioids can induce a significant inhibition of CFU-GM growth, which is reversible and not mediated through opioid or somatostatin receptors, while apoptosis is not implicated. Whether this finding could be used for clinical intervention remains to be examined. 相似文献
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Objectives: Ex vivo expansion is a feasible strategy, which may overcome limitation of the very low frequency of haematopoietic stem/progenitor cells, in umbilical cord blood (UCB). However, both quality of cells and safety of expanded population are critical issues to be addressed for their clinical application. Hence, in this study, we evaluated genetic stability of UCB‐derived CD34+ cells during ex vivo culture, based on karyotype analysis, as well as its effect on cell proliferation characteristics. Materials and methods: CD34+ cells were isolated from human UCB samples by immunomagnetic separation and were expanded ex vivo over a 28‐day period. Expansion of total nucleate cells, CD34+ cells and CD34+ CD38? cells was investigated. Karyotype analysis of the expanded cells from six randomly selected UCB samples was performed to evaluate their genetic stability. Results: Chromosomal abnormality of expanded cells mainly appeared by day 14, but was seldom sustained until day 28. None of the chromosomal abnormal samples displayed neoplastic proliferation, and expanded cells with altered chromosomes did not show obvious transformation phenomena according to soft agar assay. Conclusions: Ex vivo expansion could lead to occurrence of chromosomal abnormality, although here it did not produce excessive proliferative advantage of the expended cells. Importantly, chromosomal alteration seemed not to be inheritable and unlikely to result in malignant transformation. However, further in‐depth evaluation of potential clinical risks of chromosomal abnormality is warranted. 相似文献
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Andrade PZ da Silva CL dos Santos F Almeida-Porada G Cabral JM 《Journal of cellular biochemistry》2011,112(7):1822-1831
Since umbilical cord blood (UCB), contains a limited hematopoietic stem/progenitor cells (HSC) number, successful expansion protocols are needed to overcome the hurdles associated with inadequate numbers of HSC collected for transplantation. UCB cultures were performed using a human stromal‐based serum‐free culture system to evaluate the effect of different initial CD34+ cell enrichments (Low: 24 ± 1.8%, Medium: 46 ± 2.6%, and High: 91 ± 1.5%) on the culture dynamics and outcome of HSC expansion. By combining PKH tracking dye with CD34+ and CD34+CD90+ expression, we have identified early activation of CD34 expression on CD34? cells in Low and Medium conditions, prior to cell division (35 ± 4.7% and 55 ± 4.1% CD34+ cells at day 1, respectively), affecting proliferation/cell cycle status and ultimately determining CD34+/CD34+CD90+ cell yield (High: 14 ± 1.0/3.5 ± 1.4‐fold; Medium:22 ± 2.0/3.4 ± 1,0‐fold; Low:31 ± 3.0/4.4 ± 1.5‐fold) after a 7‐day expansion. Considering the potential benefits of using expanded UCB HSC in transplantation, here we quantified in single UCB units, the impact of using one/two immunomagnetic sorting cycles (corresponding to Medium and High initial progenitor content), and the average CD34+ cell recovery for each strategy, on overall CD34+ cell expansion. The higher cell recovery upon one sorting cycle lead to higher CD34+ cell numbers after 7 days of expansion (30 ± 2.0 vs. 13 ± 1.0 × 106 cells). In particular, a high (>90%) initial progenitor content was not mandatory to successfully expand HSC, since cell populations with moderate levels of enrichment readily increased CD34 expression ex‐vivo, generating higher stem/progenitor cell yields. Overall, our findings stress the importance of establishing a balance between the cell proliferative potential and cell recovery upon purification, towards the efficient and cost‐effective expansion of HSC for cellular therapy. J. Cell. Biochem. 112: 1822–1831, 2011. © 2011 Wiley‐Liss, Inc. 相似文献
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Human umbilical vein endothelial cells increase ex vivo expansion of human CD34(+) PBPC through IL-6 secretion 总被引:2,自引:0,他引:2
Li N Eljaafari A Bensoussan D Wang Y Latger-Cannard V Serrurier B Boura C Kennel A Stoltz J Feugier P 《Cytotherapy》2006,8(4):335-342
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. 相似文献
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Ex vivo expansion and clonality of CD34+ selected cells from bone marrow and cord blood in a serum-free media 总被引:2,自引:0,他引:2
Kim SK Koh SK Song SU Shin SH Choi GS Kim WC Lee MH Seoh JY Park SK Fraser JK 《Molecules and cells》2002,14(3):367-373
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. 相似文献
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Ghaderi M Allameh A Soleimani M Rastegar H Ahmadi-Ashtiani HR 《Mutation research》2011,719(1-2):14-20
This study compared the sensitivity of differentiated hepatocyte-like cells, their progenitor mesenchymal stem cells (MSCs) and CD34(+) stem cells to DNA damage and toxicity induced by aflatoxin B1 (AFB1). The hepatocyte-like cells and their progenitor cells (isolated from umbilical cord blood (UCB)) were each treated with AFB1 on day 15 of differentiation. Cell toxicity and genotoxicity effects were assessed using MTT and alkaline comet assays. AFB1 treatment resulted in a dose- and time-dependent inhibition of cell growth. The IC(50) values of AFB1 for hepatocytes differentiated from CD34(+) and MSCs were within the same range (44.7-46.8μM). The IC(50) calculated for non-differentiated MSCs and CD34(+) cells was slightly lower (42.0-43.4μM) than that calculated for their differentiated counterparts. However, the extent of DNA damage was different in differentiated and non-differentiated cells. The percentages of DNA (% DNA) in comet tails measured in hepatocytes differentiated from MSCs exposed to AFB1 (0, 2.5, 10 and 20μM) for 24h were ~15, 55, 65 and 70%, respectively. In comparison, hepatocytes from CD34(+) cells were more resistant to AFB1-induced DNA damage. Hepatocyte-MSCs were most sensitive to DNA damage, followed by UCB-CD34(+) cells, then UCB-MSCs and finally hepatocyte-CD34(+) cells. These results clearly showed that stem cells from different sources have different sensitivities to DNA damaging agents. These differences can be assigned to the expression levels of cytochrome P450 (CYP) particularly CYP3A4 in non-differentiated and differentiated cells. These data are useful in better understanding the susceptibility/resistance of stem cells in the process of differentiation to environmental toxicants. 相似文献
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Natural Killer (NK)-cells are peripheral blood lymphocytes that represent an important arm of the innate immune system. NK-cells play a critical role in the immune surveillance against tumors and virally infected cells in a major histocompatibiliy complex (MHC)-unrestricted fashion. We have explored such capacities of NK-cells after differentiation from hematopoietic stem and progenitor cells derived from human umbilical cord blood. Several culture conditions have been established supporting proliferation and subsequent differentiation of these cells in terms of receptor expression and specific lysis depending on the growth conditions in the presence and absence of supportive stromal feeders. We show that acquisition of Killer Immunoglobulin Receptor (KIR) as well as NK Cytotoxicity Receptor expressions is independent of culture condition whereas absence of stromal feeders did not support acquisition of CD94/NKG2A expression. Such KIR-positive/NKG2A-negative cells generated under different culture conditions showed strong and specific cytolytic activity which could have impact on further immunotherapeutic strategies. 相似文献
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Ahrens I Domeij H Topcic D Haviv I Merivirta RM Agrotis A Leitner E Jowett JB Bode C Lappas M Peter K 《PloS one》2011,6(8):e23210
Endothelial progenitor cells (EPCs) can be purified from peripheral blood, bone marrow or cord blood and are typically defined by a limited number of cell surface markers and a few functional tests. A detailed in vitro characterization is often restricted by the low cell numbers of circulating EPCs. Therefore in vitro culturing and expansion methods are applied, which allow at least distinguishing two different types of EPCs, early and late EPCs. Herein, we describe an in vitro culture technique with the aim to generate high numbers of phenotypically, functionally and genetically defined early EPCs from human cord blood. Characterization of EPCs was done by flow cytometry, immunofluorescence microscopy, colony forming unit (CFU) assay and endothelial tube formation assay. There was an average 48-fold increase in EPC numbers. EPCs expressed VEGFR-2, CD144, CD18, and CD61, and were positive for acetylated LDL uptake and ulex lectin binding. The cells stimulated endothelial tube formation only in co-cultures with mature endothelial cells and formed CFUs. Microarray analysis revealed highly up-regulated genes, including LL-37 (CAMP), PDK4, and alpha-2-macroglobulin. In addition, genes known to be associated with cardioprotective (GDF15) or pro-angiogenic (galectin-3) properties were also significantly up-regulated after a 72 h differentiation period on fibronectin. We present a novel method that allows to generate high numbers of phenotypically, functionally and genetically characterized early EPCs. Furthermore, we identified several genes newly linked to EPC differentiation, among them LL-37 (CAMP) was the most up-regulated gene. 相似文献
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《Cell communication & adhesion》2013,20(3):45-55
AbstractNatural Killer (NK)-cells are peripheral blood lymphocytes that represent an important arm of the innate immune system. NK-cells play a critical role in the immune surveillance against tumors and virally infected cells in a major histocompatibiliy complex (MHC)-unrestricted fashion. We have explored such capacities of NK-cells after differentiation from hematopoietic stem and progenitor cells derived from human umbilical cord blood. Several culture conditions have been established supporting proliferation and subsequent differentiation of these cells in terms of receptor expression and specific lysis depending on the growth conditions in the presence and absence of supportive stromal feeders. We show that acquisition of Killer Immunoglobulin Receptor (KIR) as well as NK Cytotoxicity Receptor expressions is independent of culture condition whereas absence of stromal feeders did not support acquisition of CD94/NKG2A expression. Such KIR-positive/NKG2A-negative cells generated under different culture conditions showed strong and specific cytolytic activity which could have impact on further immunotherapeutic strategies. 相似文献
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Functional small-diameter neovessels created using endothelial progenitor cells expanded ex vivo 总被引:62,自引:0,他引:62
Kaushal S Amiel GE Guleserian KJ Shapira OM Perry T Sutherland FW Rabkin E Moran AM Schoen FJ Atala A Soker S Bischoff J Mayer JE 《Nature medicine》2001,7(9):1035-1040
Arterial conduits are increasingly preferred for surgical bypass because of inherent functional properties conferred by arterial endothelial cells, especially nitric oxide production in response to physiologic stimuli. Here we tested whether endothelial progenitor cells (EPCs) can replace arterial endothelial cells and promote patency in tissue-engineered small-diameter blood vessels (4 mm). We isolated EPCs from peripheral blood of sheep, expanded them ex vivo and then seeded them on decellularized porcine iliac vessels. EPC-seeded grafts remained patent for 130 days as a carotid interposition graft in sheep, whereas non-seeded grafts occluded within 15 days. The EPC-explanted grafts exhibited contractile activity and nitric-oxide-mediated vascular relaxation that were similar to native carotid arteries. These results indicate that EPCs can function similarly to arterial endothelial cells and thereby confer longer vascular-graft survival. Due to their unique properties, EPCs might have other general applications for tissue-engineered structures and in treating vascular diseases. 相似文献
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Kamonnaree Chotinantakul Chavaboon Dechsukhum Duangnapa Dejjuy Wilairat Leeanansaksiri 《Cellular & molecular biology letters》2013,18(2):263-283
Diabetes can impair wound closure, which can give rise to major clinical problems. Most treatments for wound repair in diabetes remain ineffective. This study aimed to investigate the influence on wound closure of treatments using expanded human cord blood CD34+ cells (CB-CD34+ cells), freshly isolated CB-CD34+ cells and a cytokine cocktail. The test subjects were mice with streptozotocin-induced diabetes. Wounds treated with fresh CB-CD34+ cells showed more rapid repair than mice given the PBS control. Injection of expanded CB-CD34+ cells improved wound closure significantly, whereas the injection of the cytokine cocktail alone did not improve wound repair. The results also demonstrated a significant decrease in epithelial gaps and advanced re-epithelialization over the wound bed area after treatment with either expanded CB-CD34+ cells or freshly isolated cells compared with the control. In addition, treatments with both CB-CD34+ cells and the cytokine cocktail were shown to promote recruitment of CD31+-endothelial cells in the wounds. Both the CB-CD34+ cell population and the cytokine treatments also enhanced the recruitment of CD68-positive cells in the early stages (day 3) of treatment compared with PBS control, although the degree of this enhancement was found to decline in the later stages (day 9). These results demonstrated that expanded CB-CD34+ cells or freshly isolated CB-CD34+ cells could accelerate wound repair by increasing the recruitment of macrophages and capillaries and the reepithelialization over the wound bed area. Our data suggest an effective role in wound closure for both ex vivo expanded CB-CD34+ cells and freshly isolated cells, and these may serve as therapeutic options for wound treatment for diabetic patients. Wound closure acceleration by expanded CB-CD34+ cells also breaks the insufficient quantity obstacle of stem cells per unit of cord blood and other stem cell sources, which indicates a broader potential for autologous transplantation. 相似文献
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Kanayasu-Toyoda T Ishii-Watabe A Suzuki T Oshizawa T Yamaguchi T 《The Journal of biological chemistry》2007,282(46):33507-33514
We previously reported that CD31(bright) cells, which were sorted from cultured AC133(+) cells of adult peripheral blood cells, differentiated more efficiently into endothelial cells than CD31(+) cells or CD31(-) cells, suggesting that CD31(bright) cells may be endothelial precursor cells. In this study, we found that CD31(bright) cells have a strong ability to release cytokines. The mixture of vascular endothelial growth factor (VEGF), thrombopoietin (TPO), and stem cell factor stimulated ex vivo expansion of the total cell number from cultured AC133(+) cells of adult peripheral blood cells and cord blood cells, resulting in incrementation of the adhesion cells, in which endothelial nitric oxide synthase and kinase insert domain-containing receptor were positive. Moreover, the mixture of VEGF and TPO increased the CD31(bright) cell population when compared with VEGF alone or the mixture of VEGF and stem cell factor. These data suggest that TPO is an important growth factor that can promote endothelial precursor cells expansion ex vivo. 相似文献
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Kleinman ME Tepper OM Capla JM Bhatt KA Ceradini DJ Galiano RD Blei F Levine JP Gurtner GC 《Lymphatic research and biology》2003,1(4):301-307
Hemangioma is the most common soft-tissue tumor of infancy. Despite the frequency of these vascular tumors, the origin of hemangioma-endothelial cells is unknown. Circulating endothelial progenitor cells (EPCs) have recently been identified as vascular stem cells with the capacity to contribute to postnatal vascular development. We have attempted to determine whether circulating EPCs are increased in hemangioma patients and thereby provide insight into the role of EPCs in hemangioma growth. METHODS AND RESULTS: Peripheral blood mononuclear cells (PBMCs) were isolated from hemangioma patients undergoing surgical resection (N = 5) and from age-matched controls (N = 5) undergoing strabismus correction surgery. PBMCs were stained with fluorescent-labeled antibodies for AC133, CD34, and VEGFR2/KDR. Fluorescent-labeled isotype antibodies served as negative controls. Histologic sections of surgical specimens were stained with the specific hemangioma markers Glut1, CD32, and merosin, to confirm the diagnosis of common hemangioma of infancy. EPCs harvested from healthy adult volunteers were stained with Glut1, CD32, and merosin, to assess whether cultured EPCs express known hemangioma markers. Hemangioma patients had a 15-fold increase in the number of circulating CD34 AC133 dual-staining cells relative to controls (0.78+/-0.14% vs.0.052+/-0.017%, respectively). Similarly, the number of PBMCs that stained positively for both CD34 and KDR was also increased in hemangioma patients (0.49+/-0.074% vs. 0.19+/-0.041% in controls). Cultured EPCs stained positively for the known hemangioma markers Glut1, CD32, merosin. CONCLUSIONS: This is the first study to suggest a role for EPCs in the pathogenesis of hemangioma. Our results imply that increased levels of circulating EPCs may contribute to the formation of this vascular tumor. 相似文献