Co-culture of umbilical cord blood CD34+ cells with human mesenchymal stem cells

Insufficient numbers of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) sometimes limit allogenic transplantation of umbilical cord blood (UCB). Ex vivo expansion may overcome this limitation. Mesenchymal stem cells (MSCs), as non-hematopoietic, well-characterized skeletal and connective-tissue progenitor cells within the bone marrow stroma, have been investigated as support cells for the culture of HSCs/HPCs. MSCs are attractive for the rich environmental signals that they provide and for immunological compatibility in transplantation. Thus far, HSC/MSC co-cultures have mainly been performed in 2-dimensional (2D) configuration. We postulate that a 3-dimensional (3D) culture environment that resembles the natural in vivo hematopoietic compartment might be more conducive for regulating HSC expansion. In this study, we compared the co-culture of HSCs and MSCs in 2D and 3D configurations. The results demonstrated the benefit of MSC inclusion in HSC expansion ex vivo. Direct contact between MSCs and HSCs in 3D cultures led to statistically significantly higher expansion of cord blood CD34+ cells than in 2D cultures (891- versus 545-fold increase in total cells, 96- versus 48-fold increase of CD34+ cells, and 230- versus 150-fold increase in colony-forming cell assay [CFC]). Engraftment assays in non-obese diabetic/severe combined immunodeficiency mice also indicated a high success rate of hematopoiesis reconstruction with these expanded cells.     

Cord blood mesenchymal stem cells: Potential use in neurological disorders

Our previous studies demonstrate enhanced neural protective effects of cord blood (CB) cells in comparison to stem cells from adult marrow. To determine further whether mesenchymal stem cells (MSCs) derived from human umbilical cord blood (hUCB) possess optimal characteristics for neural therapy, we isolated populations of plastic-adherent CB MSCs. These cells generated CD34-, CD45-, CD11b-, CD3-, CD19- cells in culture and failed to produce CFU-M, CFU-GEMM, or CFU-GM hematopoietic colonies in methylcellulose. However, cultured CB MSCs possessed a remarkable ability to support proliferation as well as differentiation of hematopoietic cells in vitro. In addition, supernatants from cultured CB MSCs promoted survival of NT2 N neural cells and peripheral blood mononuclear cells (MNCs) cultured under conditions designed to induce cell stress and limit protein synthesis. After incubation in neural differentiation medium, CB MSCs expressed the neural cell-surface antigen A2B5, the neurofilament polypeptide NF200, the oligodendrocyte precursor marker 04, intermediate filament proteins characteristic of neural differentiation (nestin and vimentin), as well as the astrocyte marker glial fibrillary acidic protein (GFAP) and the neural progenitor marker TUJ-1. We examined the immunomodulatory effects of the CB MSCs after co-culture with murine splenocytes. Whereas spleen cells from normal C57Bl/6 mice exhibited a prominent immunoglobulin M (IgM) response after immunization with the T cell-dependent antigen sheep red blood cells, this response was significantly decreased after incubation with CB MSCs. These data indicate that CB MSCs possess multiple utilities that may contribute to their therapeutic potency in the treatment of neurological disorders.     

Quantity and quality of engrafting cells in cord blood and autologous mobilized peripheral blood.

Cord blood (CB) and autologous mobilized peripheral blood stem/progenitor cells (PBSC) are now used widely for clinical transplantation. We characterized the short-term (<8 weeks) and long-term (>8 weeks) engraftment in NOD/SCID mice resulting from transplanted CD34+ cells from these two sources. We also quantified the frequency of long-term engrafting cells, and the average proliferative capacity of individual engrafting cells by a competitive repopulation assay with binomial variance-covariance modeling. We found that 0.5 million CD34+ CB cells were able to generate sustained, high-level, multilineage human hematopoiesis, whereas a sixfold higher number of CD34+ PBSC (3 million) from cancer patients undergoing chemotherapy generated comparable short-term, but much lower sustained multilineage human hematopoiesis after transplantation. In comparison to CD34+ cells from PBSC from cancer patients, long-term engrafting cells were approximately eightfold enriched in CB CD34+ cells, and each CB long-term engrafting cell had an approximately 15-fold higher multilineage proliferative capacity. Thus, the number and function of transplantable hematopoietic cells were remarkably different between these two sources of stem/progenitor cells.     

Distinctive contact between CD34+ hematopoietic progenitors and CXCL12+ CD271+ mesenchymal stromal cells in benign and myelodysplastic bone marrow

Mesenchymal stromal cells (MSCs) support hematopoiesis and are cytogenetically and functionally abnormal in myelodysplastic syndrome (MDS), implying a possible pathophysiologic role in MDS and potential utility as a diagnostic or risk-stratifying tool. We have analyzed putative MSC markers and their relationship to CD34+ hematopoietic stem/progenitor cells (HSPCs) within intact human bone marrow in paraffin-embedded bone marrow core biopsies of benign, MDS and leukemic (AML) marrows using tissue microarrays to facilitate scanning, image analysis and quantitation. We found that CD271+, ALP+ MSCs formed an extensive branching perivascular, periosteal and parenchymal network. Nestin was brightly positive in capillary/arteriolar endothelium and occasional subendothelial cells, whereas CD146 was most brightly expressed in SMA+ vascular smooth muscle/pericytes. CD271+ MSCs were distinct by double immunofluorescence from CD163+ macrophages and were in close contact with but distinct from brightly nestin+ and from brightly CD146+ vascular elements. Double immunofluorescence revealed an intimate spatial relationship between CD34+ HSPCs and CD271+ MSCs; remarkably, 86% of CD34+ HSPCs were in direct contact with CD271+ MSCs across benign, MDS and AML marrows, predominantly in a perivascular distribution. Expression of the intercrine chemokine CXCL12 was strong in the vasculature in both benign and neoplastic marrow, but was also present in extravascular parenchymal cells, particularly in MDS specimens. We identified these parenchymal cells as MSCs by ALP/CXCL12 and CD271/CXCL12 double immunofluorescence. The area covered by CXCL12+ ALP+ MSCs was significantly greater in MDS compared with benign and AML marrow (P=0.021, Kruskal-Wallis test). The preservation of direct CD271+ MSC/CD34+ HSPC contact across benign and neoplastic marrow suggests a physiologically important role for the CD271+ MSC/CD34+ HSPC relationship and possible abnormal exposure of CD34+ HSPCs to increased MSC CXCL12 expression in MDS.     

Growth hormone-induced stimulation of multilineage human hematopoiesis

Growth hormone (GH) has been shown to have significant positive effects on hemato-lymphopoiesis in rodent models and, more recently, to increase thymic mass and circulating na?ve CD4+ T cells in humans infected with the human immunodeficiency virus, type 1. To determine whether the latter effects on human T lymphopoiesis might be due, at least in part, to effects on the bone marrow (BM), we examined the specific effects of GH and its proximal mediator, insulin-like growth factor I (IGF-I), on human multilineage hematopoiesis in fetal BM (FBM). Using in vitro analysis, we found that GH and IGF-I each stimulated the expansion of primitive multilineage CD34+CD38- hematopoietic progenitor cells and increased yields of several hematopoietic subpopulations, including CD34+CD38+CD10+ lymphoid progenitor cells. Additionally, GH and IGF-I had direct effects on FBM stromal elements, inducing the expansion of myeloid-like CD45+CD14+ FBM stromal cells and enhancing production of the hematopoietic cytokine interleukin-3 by fibroblast-like CD45-CD10+ FBM stromal cells. Surface expression of GH and type-I IGF receptors correlated with the observed biologic responses to these hormones. Whereas GH enhanced the proliferation of FBM progenitors and stroma, IGF-I exerted a predominantly antiapoptotic effect. Finally, both GH and IGF-I stimulated the generation of hematopoietic colony forming cells. These findings identify specific targets of GH and IGF-I within human FBM, and demonstrate direct and indirect effects that may contribute to GH-mediated enhancement of human hemato-lymphopoiesis.     

Comparison of hematopoietic activities of human bone marrow and umbilical cord blood CD34 positive and negative cells.

Although the hematopoietic activities of human CD34+ bone marrow (BM) and cord blood (CB) cells have been well characterized, the phenotype of nonobese-diabetic severe combined immunodeficient (NOD/SCID) mice repopulating cells (SRCs) in CB and BM has not yet been fully examined. To address this issue, various hematopoietic activities were compared in terms of total and CD34+ CB and BM cells. Clonal culture of fluorescence-activated cell sorter (FACS) CD34+ CB and BM cells revealed a higher incidence of colony-forming cells with greater proliferation capacity in CB over BM CD34+ cells. CB CD34+ cells also demonstrated higher secondary plating efficiency over BM cells. In addition, we demonstrated that mice transplanted with CB mononuclear cells (MNCs) showed significantly higher levels of chimerism than those transplanted with BM MNCs. However, recipients of FACS-sorted CD34+ CB cells showed significantly lower levels of chimerism than those that received total CB MNCs, suggesting a role of facilitating cells in the CD34- cell population. To further analyze the role of CD34- cells, the NOD/SCID repopulating ability of FACS-sorted CB CD34-c-kit+Lin- and CD34-c-kit-Lin- cells were examined. However, SRCs were not detected in those cells. Taken together, these data suggest that CB is a better source of hematopoietic stem cells and that there are cells in the CD34- fraction that facilitate repopulation of hematopoiesis in the NOD/SCID environment.     

A combination of stem cell factor and granulocyte colony-stimulating factor enhances the growth of human progenitor B cells supported by murine stromal cell line MS-5

We have developed a long-term culture system using the murine bone marrow stromal cells MS-5 to support the growth of progenitor B cells with CD34^–, CD10⁺, CD19⁺, and cytoplasmic μ chain (Cμ)-negative surface phenotype from human CD34⁺ cells purified from umbilical cord blood (CB). When 10³ CB CD34⁺ cells/well were seeded on MS-5 stromal cells at the beginning of culture in the absence of exogenously added cytokines, progenitor B cells first appeared after 14 days, and the maximal cell production was achieved during the 6th week of culture. Intriguingly, the addition of recombinant human stem cell factor (rhSCF) and granulocyte colony-stimulating factor (rhG-CSF), but not rhIL-7, strikingly enhanced the growth of progenitor B cells from CB CD34⁺ population cultured on MS-5 stromal cells. The culture of progenitor B cells could be maintained until the 6th week of culture when some cells were revealed to have a Cμ⁺ phenotype, and a small number of cells had immunoglobulin μ chain on their cell surface in the presence of both rhSCF and rhG-CSF. When CD34⁺ cells were cultured physically separated from the stromal layer by membrane, supportive effects of MS-5 stromal cells for the growth of progenitor B cells were not observed. These results suggest that the present culture system could generate progenitor B cells to proliferate from CB CD34⁺ cells, that some of these progenitor B cells could differentiate into immature B cells in conjunction with rhSCF and rhG-CSF, and that a species-cross-reactive membrane-bound factor(s), which stimulates early human B lymphopoiesis, may exist in MS-5 stromal cells. Further studies are required to investigate the mechanism how rhG-CSF acts on progenitor B cells to allow their proliferation and differentiation.     

Human T-cell lymphotropic virus type 1 infection of CD34+ hematopoietic progenitor cells induces cell cycle arrest by modulation of p21(cip1/waf1) and survivin

Human T-cell lymphotropic virus type 1 (HTLV-1) is an oncogenic retrovirus and the etiologic agent of adult T-cell leukemia (ATL), an aggressive CD4(+) malignancy. HTLV-2 is highly homologous to HTLV-1; however, infection with HTLV-2 has not been associated with lymphoproliferative diseases. Although HTLV-1 infection of CD4(+) lymphocytes induces cellular replication and transformation, infection of CD34(+) human hematopoietic progenitor cells (HPCs) strikingly results in G(0)/G(1) cell cycle arrest and suppression of in vitro clonogenic colony formation by induction of expression of the cdk inhibitor p21(cip1/waf1) (p21) and concurrent repression of survivin. Immature CD34(+)/CD38(-) hematopoietic stem cells (HSCs) were more susceptible to alterations of p21 and survivin expression as a result of HTLV-1 infection, in contrast to more mature CD34(+)/CD38(+) HPCs. Knockdown of p21 expression in HTLV-1-infected CD34(+) HPCs partially abrogated cell cycle arrest. Notably, HTLV-2, an HTLV strain that is not associated with leukemogenesis, does not significantly modulate p21 and survivin expression and does not suppress hematopoiesis from CD34(+) HPCs in vitro. We speculate that the remarkable differences in the activities displayed by CD34(+) HPCs following infection with HTLV-1 or HTLV-2 suggest that HTLV-1 uniquely exploits cell cycle arrest mechanisms to establish a latent infection in hematopoietic progenitor/hematopoietic stem cells and initiates preleukemic events in these cells, which eventually results in the manifestation of ATL.     

Human umbilical cord blood-derived stromal cells are superior to human umbilical cord blood-derived mesenchymal stem cells in inducing myeloid lineage differentiation in vitro

Stromal cells and mesenchymal stem cells (MSCs), 2 important cell populations within the hematopoietic microenvironment, may play an important role in the development of hematopoietic stem/progenitor cells. We have successfully cultured human umbilical cord blood-derived stromal cells (hUCBDSCs). It has been demonstrated that MSCs also exist in hUCB. However, we have not found any reports on the distinct characteristics of hUCBDSCs and human umbilical cord blood-derived mesenchymal stem cells (hUCBDMSCs). In this study, hUCBDSCs and hUCBDMSCs were isolated from the cord blood of full-term infants using the same density gradient centrifugation and cultured in the appropriate medium. Some biological characteristics and hematopoietic supportive functions were compared in vitro. hUCBDSCs were distinct from hUCBDMSCs in morphology, proliferation, cell cycle, passage, immunophenotype, and the capacity for classical tri-lineage differentiation. Finally, quantitative real-time polymerase chain reaction analysis revealed that granulocyte colony-stimulating factor (G-CSF) gene expression was higher in hUCBDSCs than that in hUCBDMSCs. Enzyme-linked immunosorbent assay revealed that the secretion of G-CSF, thrombopoietin (TPO), and granulocyte macrophage colony-stimulating factor (GM-CSF) by hUCBDSCs was higher than that by hUCBDMSCs. After coculture, the granulocyte/macrophage colony-forming units (CFU-GM) of hematopoietic cells from the hUCBDSC feeder layer was more than that from the hUCBDMSC feeder layer. Flow cytometry was used to detect CD34(+) hematopoietic stem/progenitor cell committed differentiation during 14 days of coculture; the results demonstrated that CD14 and CD33 expression in hUCBDSCs was significantly higher than their expression in hUCBDMSCs. This observation was also true for the granulocyte lineage marker, CD15. This marker was expressed beginning at day 7 in hUCBDSCs. It was expressed earlier and at a higher level in hUCBDSCs compared with hUCBDMSCs. In conclusion, hUCBDSCs are different from hUCBDMSCs. hUCBDSCs are superior to hUCBDMSCs in supporting hematopoiesis stem/progenitor cells differentiation into myeloid lineage cells at an early stage in vitro.     

Human adipose-derived stromal cells efficiently support hematopoiesis in vitro and in vivo: a key step for therapeutic studies

Adipose-derived stromal cells (ADSCs) are close relatives of bone marrow mesenchymal stromal cells (BM-MSCs). The ease of access to subcutaneous fat pad and the abundance of stromal precursors make fat tissue an attractive source of stromal cells for clinicians. However, their ability to support hematopoietic stem cells in vitro and in vivo has not been established definitively. Thus, their usefulness in supporting hematopoietic stem cell engraftment is not as clear as with BM-MSCs. In this article, we show that human ADSCs, cultured with a good manufacturing practice medium, maintain in vitro human early and committed hematopoietic progenitors and support their complete differentiation toward myeloid and lymphoid lineages. Compared with BM-MSCs, ADSCs elicit a more precocious early progenitor formation and faster proliferation and differentiation of hematopoietic progenitors. Further, in vivo, when co-injected in NOD.Cg-Prkdc(scid) Il2(rgtm1Wjl)/SzJ (NSG) mice with a low number of human CD34(+) cells, ADSCs enabled the higher production of immature human hematopoietic progenitors and CD45(+) cells when compared with BM-MSCs. As a whole, our results indicate that human ADSCs, isolated and expanded under clinical-grade conditions, support hematopoiesis in vitro and in vivo and thus provide the rationale for their use in supporting hematopoietic reconstitution in clinical settings.     

人骨骼肌源性血管内皮细胞对造血干/祖细胞的体外支持

背景:人骨骼肌源性血管内皮细胞位于血管壁,共表达肌肉干细胞和血管内皮细胞的标记(CD56+CD34+CD144+CD45-).研究显示,人肌血管内皮细胞与间充质干细胞存在相似性,表达间充质干细胞表面标记物,具有多向分化潜能.目的:建立人肌血管内皮细胞作为滋养层与人脐血CD34+细胞体外培养体系,以培养前后CD34+细胞...     

CDCP1 identifies a broad spectrum of normal and malignant stem/progenitor cell subsets of hematopoietic and nonhematopoietic origin

CUB-domain-containing protein 1 (CDCP1) is a novel transmembrane molecule that is expressed in metastatic colon and breast tumors as well as on the surface of hematopoietic stem cells. In this study, we used multiparameter flow cytometry and antibodies against CDCP1 to analyze the expression of CDCP1 on defined hematopoietic cell subsets of different sources. In addition, CDCP1 expression on leukemic blasts and on cells with nonhematopoietic stem/progenitor cell phenotypes was determined. Here we demonstrate that a subset of bone marrow (BM), cord blood (CB), and mobilized peripheral blood (PB) CD34+ cells expressed this marker and that CDCP1 was detected on CD34(+)CD38- BM stem/progenitor cells but not on mature PB cells. Analysis of leukemic blasts from patients with acute lymphoblastic leukemia, acute myeloid leukemia, and chronic myeloid leukemia in blast crisis revealed that CDCP1 is predominantly expressed on CD34(+)CD133+ myeloid leukemic blasts. However, CDCP1 was not strictly correlated with CD34 and/or CD133 expression, suggesting that CDCP1 is a novel marker for leukemia diagnosis. Stimulation of CD34+ BM cells with CDCP1-reactive monoclonal antibody CUB1 resulted in an increased (approximately twofold) formation of erythroid colony-forming units, indicating that CDCP1 plays an important role in early hematopoiesis. Finally, we show that CDCP1 is also expressed on cells phenotypically identical to mesenchymal stem/progenitor cells (MSCs) and neural progenitor cells (NPCs). In conclusion, CDCP1 is not only a novel marker for immature hematopoietic progenitor cell subsets but also unique in its property to recognize cells with phenotypes reminiscent of MSC and NPC.     

Extended in vitro expansion of adult, mobilized CD34+ cells without significant cell senescence using a stromal cell coculture system with single cytokine support

The macrophage colony-stimulating factor-deficient bone marrow stromal cell line OP9, derived from osteopetrotic mice, is known to support hematopoietic stem cell (HSC) expansion as well as hematopoietic differentiation of embryonic stem cells. Coculture of HSC in the OP9 system requires cytokine support to achieve significant cell expansion. Recently, we reported extensive expansion without cell senescence of cord blood (CB)-derived HSC cocultured with OP9 stromal cells for more than 18 weeks with a single cytokine support using human thrombopoietin (TPO). In this study, we evaluated the efficiency of the OP9/TPO coculture system to sustain long-term hematopoiesis of adult, granulocyte colony-stimulating factor mobilized human peripheral blood (PB) CD34(+) cells. Maximum cell expansion was attained during the first 4 weeks of coculture. At the same time, the maximum progenitor cell expansion was demonstrated by the production of colony-forming cells and cobblestone area-forming cells. In contrast to the expansion of CB CD34(+) cells, PB CD34(+) cells showed termination of cultures after 8 weeks, independent of the cell expansion rates attained. The evaluation of cell senescence by assessing the telomere length in most cultures showed no relevant telomere shortening, despite rapid decrease in telomerase activity. Interestingly, increases in telomere length were demonstrated. In conclusion, OP9/TPO system provides extensive stem cell expansion without concomitant telomere erosion for both CB and adult CD34(+) cells. Termination of adult CD34(+) cell cocultures seems to be independent of telomere length.     

OP9 stroma augments survival of hematopoietic precursors and progenitors during hematopoietic differentiation from human embryonic stem cells

The cellular mechanism and target cell affected by stromal microenvironments in augmenting hematopoietic specification from pluripotent human embryonic stem cells (hESCs) has yet to be evaluated. Here, in contrast to aorta-gonad-mesonephros-derived S62 stromal cells, OP9 cells inhibit apoptosis and also augment the proliferation of hemogenic precursors prospectively isolated from human embryoid bodies. In addition, OP9 stroma supported cells within the primitive hematopoietic compartment by inhibiting apoptosis of CD45(+)CD34(+) cells committed to the hematopoietic lineage, but have no effect on more mature blood (CD45(+)CD34(-)) cells. Inability of hESC-derived hematopoietic cells cocultured with OP9 stromal cells to engraft in both the adult and newborn NOD/SCID mice after intrafemoral and intrahepatic injection illustrated that although OP9 stromal cells augment hESC-derived hematopoiesis and progenitor output, this optimized environment does not confer or augment repopulating function of specified hematopoietic cells derived from hESCs. OP9 coculture also increases hematopoietic progenitors output from hemogenic precursors overexpressing HOXB4. Our study demonstrates that OP9 cells support both hemogenic precursors and their primitive hematopoietic progeny, thereby providing the first evidence toward understanding the cellular targets and mechanisms underlying the capacity of OP9 stromal cells to support hematopoiesis from ESCs and define the future steps required to achieve the global goal of generating bona fide human hematopoietic stem cells from ESC lines. Disclosure of potential conflicts of interest is found at the end of this article.     

Differential kinetics of primitive hematopoietic cells assayed in vitro and in vivo during serum-free suspension culture of CD34+ blood progenitor cells.

So far, blood progenitor cells (BPC) expanded ex vivo in the absence of stromal cells have not been demonstrated to reconstitute hematopoiesis in myeloablated patients. To characterize the fate of early hematopoietic progenitor cells during ex vivo expansion in suspension culture, human CD34(+)-enriched BPC were cultured in serum-free medium in the presence of FLT3 ligand (FL), stem cell factor (SCF) and interleukin 3 (IL-3). Both CD34 surface expression levels and the percentage of CD34+ cells were continuously downregulated during the culture period. We observed an expansion of colony-forming units granulocyte-macrophage (CFU-GM) and BFU-E beginning on day 3 of culture, reaching an approximate 2-log increase by days 5 to 7. Limiting dilution analysis of primitive in vitro clonogenic progenitors was performed through a week 6 cobblestone-area-forming cell (CAFC) assay, which has previously been shown to detect long-term bone marrow culture-initiating cells (LTC-IC). A maintenance or a slight (threefold) increase of week 6 CAFC/LTC-IC was found after one week of culture. To analyze the presence of BPC mediating in vivo engraftment, expanded CD34+ cells were transplanted into preirradiated NOD/SCID mice at various time points. Only CD34+ cells cultured for up to four days successfully engrafted murine bone marrow with human cells expressing myeloid or lymphoid progenitor phenotypes. In contrast, five- and seven-day expanded human BPC did not detectably engraft NOD/SCID mice. When FL, SCF and IL-3-supplemented cultures were performed for seven days on fibronectin-coated plastic, or when IL-3 was replaced by thrombopoietin, colony forming cells and LTC-IC reached levels similar to those of control cultures, yet no human cell engraftment was recorded in the mice. Also, culture in U-bottom microplates resulting in locally increased CD34+ cell density had no positive effect on engraftment. These results indicate that during ex vivo expansion of human CD34+ cells, CFC and LTC-IC numbers do not correlate with the potential to repopulate NOD/SCID mice. Our results suggest that ex vivo expanded BPC should be cultured for limited time periods only, in order to preserve bone-marrow-repopulating hematopoietic stem cells.     

Generation of functional and mature dendritic cells from cord blood and bone marrow CD34+ cells by two-step culture combined with calcium ionophore treatment

The object of this study is to explore a culture method to generate a large number of functional and mature dendritic cells (DC) from human CD34+ hematopoietic progenitor cells. In the present study, we used a two-step method combined with calcium ionophore to induce DC from cord blood (CB) or normal human bone marrow (BM) CD34+ progenitor cells. The two-step method consists of 10 days of first step culture for the expansion and proliferation of CD34+ hematopoietic progenitor cells in the presence of SCF, IL-3, IL-6, G-CSF, and 7--11 days of second step culture for the induction of DC in the presence of GM-CSF, IL-4 and TNF-alpha. By the two-step culture, total nucleated cells were increased 208+/-66 (+/-SD, n=13), or 94+/-29 (n=5)-fold in the culture of CB or BM cells, respectively, compared with the number of CD34+ cells at the time of starting culture. Out of the total nucleated cells, 23 +/-10.4% of cells in CB cell culture and 25 +/-5% of cells in the BM cell culture acquired DC characteristic phenotypes, which were marked expressions of CD1a, HLA-DR, co-stimulatory molecules such as CD80, CD40, and adhesion molecule such as CD58. In allogeneic mixed leukocyte reaction (MLR), two-step cultured cells showed potent allo-stimulatory capacity. With this two-step culture, the absolute number of CD1a+ cells that co-expressed HLA-DR, CD80, CD40 and CD58 was enhanced approximately 3 times in CB cell culture and 1.9 times in BM cell culture, compared with the commonly used one-step culture method for the generation of DC from CD34+ cells using SCF, GM-CSF and TNF-alpha. However, on these DC generated in the two-step culture, the expressions of co-stimulatory molecule CD86 and mature DC marker CD83 were not sufficient. By the treatment of two-step cultured cells with calcium ionophore agent (A23187), the expression of co-stimulatory molecules such as CD86 and CD80 (especially CD86) was up-regulated. Besides, the expression of mature DC marker CD83 was remarkably induced by treatment with A23187 for a short duration (24 h). Consistent with the up-regulation of surface molecules CD86, CD80 and CD83, the two-step cultured cells treated with A23187 also showed a stronger allo-stimulatory capacity compared with the cells without A23187 treatment. In conclusion, the present study demonstrated that the two-step culture method effectively improved the yield of CD1a+ DC generated from CD34+ cells, and the phenotypes and functions of these CD1a+ DC could be enhanced efficiently by treatment with a calcium ionophore agent.     

Effect of HIV-1-related protein expression on cardiac and skeletal muscles from transgenic rats

Background

Human embryonic stem (hES) cells hold considerable promise for cell replacement and gene therapies. Their remarkable properties of pluripotency, self-renewal, and tractability for genetic modification potentially allows for the production of sizeable quantities of therapeutic cells of the hematopoietic lineage. Dendritic cells (DC) arise from CD34+ hematopoietic progenitor cells (HPCs) and are important in many innate and adaptive immune functions. With respect to HIV-1 infection, DCs play an important role in the efficient capture and transfer of the virus to susceptible cells. With an aim of generating DCs from a renewable source for HIV-1 studies, here we evaluated the capacity of hES cell derived CD34+ cells to give rise to DCs which can support HIV-1 infection.

Results

Undifferentiated hES cells were cultured on S17 mouse bone marrow stromal cell layers to derive CD34+ HPCs which were subsequently grown in specific cytokine differentiation media to promote the development of DCs. The hES derived DCs (hES-DC) were subjected to phenotypic and functional analyses and compared with DCs derived from fetal liver CD34+ HPC (FL-DC). The mature hES-DCs displayed typical DC morphology consisting of veiled stellate cells. The hES-DCs also displayed characteristic phenotypic surface markers CD1a, HLA-DR, B7.1, B7.2, and DC-SIGN. The hES-DCs were found to be capable of antigen uptake and stimulating naïve allogeneic CD4+ T cells in a mixed leukocyte reaction assay. Furthermore, the hES-DCs supported productive HIV-1 viral infection akin to standard DCs.

Conclusion

Phenotypically normal and functionally competent DCs that support HIV-1 infection can be derived from hES cells. hES-DCs can now be exploited in applied immunology and HIV-1 infection studies. Using gene therapy approaches, it is now possible to generate HIV-1 resistant DCs from anti-HIV gene transduced hES-CD34+ hematopoietic progenitor cells.     

Characterization of chemokine receptors expressed in primitive blood cells during human hematopoietic ontogeny

Chemokines are capable of regulating a variety of fundamental processes of hematopoietic cells that include proliferation, differentiation, and migration. To evaluate potential chemokine signaling pathways important to the regulation of primitive human hematopoietic cells, we examined chemokine receptor expression of highly purified subpopulations of uncommitted human blood cells. CXCR1-, CXCR2-, CXCR4-, and CCR5-expressing cells were detected by flow cytometry among human blood subsets depleted of lineage-restricted cells (Lin(-)) derived from adult bone marrow, mobilized peripheral blood, cord blood (CB), and circulating fetal blood. Although these chemokine receptors could be detected on Lin(-) cells throughout human development, only CXCR4 could be detected in CD34(-)CD38(-)Lin(-) and CD34(+)CD38(-)Lin(-) subfractions enriched for stem cell function, suggesting that independent of ontogeny, CXCR4-mediated signals are critical to primitive hematopoiesis. Distinct to other stages of human hematopoietic development, primitive CB cells expressed higher levels of CXCR1, CXCR2, CCR5, and CXCR4 on both CD34(-)CD38(-)Lin(-) and CD34(+)CD38(-)Lin(-) subsets. Isolation of these fractions revealed expression of additional chemokine receptors CCR7, CCR8, and Bonzo (STRL133), whereas BOB (GPR15) could not be detected. Our study illustrates that rare uncommitted hematopoietic cells express chemokine receptors not previously associated with primitive human blood cells. Based on these results, we suggest that signaling pathways mediated by chemokine receptors identified here may play a fundamental role in hematopoietic stem cell regulation and provide alternative receptor targets for retroviral pseudotyping for genetic modification of repopulating cells.