Thymic repopulation by CD34(+) human cord blood cells after expansion in stroma-free culture

Thymic repopulation by transplanted hematopoietic progenitor cells (HPC) is likely to be important for long-term immune reconstitution and for successful gene therapy of diseases affecting the T-cell lineage. However, the T-cell progenitor potential of HPC, cultured in vitro for cell number expansion and gene transfer remains largely unknown. Here, we cultured highly purified human umbilical cord blood (CB) CD34(+)CD38(-) or CD34(+)CD38(+) cells for up to 5 weeks in stroma-free cultures supplemented with various combinations of the cytokines thrombopoietin (TPO), stem cell factor (SCF), flt3/flk-2 ligand (FL), interleukin-3 (IL-3), and IL-6 and investigated thymus-repopulating ability of expanded cells in vitro and in vivo. After up to 5 weeks of culture in IL-3 + SCF + IL-6 or TPO + FL + SCF supplemented medium, the progeny of CD34(+)CD38(-) CB cells generated T cells and natural killer cells in the thymus. Limiting dilution experiments demonstrated increase in the number of T-cell progenitors during culture. After 3 weeks of culture, gene marked CD34(+)CD38(-) CB cells injected in the human thymus fragment transplanted in severe combined immunodeficient (SCID) mice (SCID-hu) generated thymocytes expressing the retroviral encoded marker gene GFP in vivo. Thus, our results show that the progeny of CD34(+)CD38(-) CB cells cultured for extensive periods, harbor thymus-repopulating cells that retain T-cell progenitor potential after expansion and gene transfer.     

Characterization of dendritic cell differentiation pathways from cord blood CD34(+)CD7(+)CD45RA(+) hematopoietic progenitor cells

To better characterize human dendritic cells (DCs) that originate from lymphoid progenitors, the authors examined the DC differentiation pathways from a novel CD7(+)CD45RA(+) progenitor population found among cord blood CD34(+) cells. Unlike CD7(-)CD45RA(+) and CD7(+)CD45RA(-) progenitors, this population displayed high natural killer (NK) cell differentiation capacity when cultured with stem cell factor (SCF), interleukin (IL)-2, IL-7, and IL-15, attesting to its lymphoid potential. In cultures with SCF, Flt3 ligand (FL), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor (TNF)-alpha (standard condition), CD7(+)CD45RA(+) progenitors expanded less (37- vs 155-fold) but yielded 2-fold higher CD1a(+) DC percentages than CD7(-)CD45RA(+) or CD7(+)CD45RA(-) progenitors. As reported for CD34(+)CD1a(-) thymocytes, cloning experiments demonstrated that CD7(+)CD45RA(+) cells comprised bipotent NK/DC progenitors. DCs differentiated from CD7(-)CD45RA(+) and CD7(+)CD45RA(+) progenitors differed as to E-cadherin CD123, CD116, and CD127 expression, but none of these was really discriminant. Only CD7(+)CD45RA(+) or thymic progenitors differentiated into Lag(+)S100(+) Langerhans cells in the absence of exogenous transforming growth factor (TGF)-beta 1. Analysis of the DC differentiation pathways showed that CD7(+)CD45RA(+) progenitors generated CD1a(+)CD14(-) precursors that were macrophage-colony stimulating factor (M-CSF) resistant and CD1a(-)CD14(+) precursors that readily differentiated into DCs under the standard condition. Accordingly, CD7(+)CD45RA(+) progenitor-derived mature DCs produced 2- to 4-fold more IL-6, IL-12, and TNF-alpha on CD40 ligation and elicited 3- to 6-fold higher allogeneic T-lymphocyte reactivity than CD7(-)CD45RA(+) progenitor-derived DCs. Altogether, these findings provide evidence that the DCs that differentiate from cord blood CD34(+)CD7(+)CD45RA(+) progenitors represent an original population for their developmental pathways and function. (Blood. 2000;96:3748-3756)     

Identification of parameters required for efficient lentiviral vector transduction and engraftment of human cord blood CD34(+) NOD/SCID-repopulating cells

OBJECTIVE: Human cord blood (CB) is a potential source of hematopoietic stem cells (HSC) for gene therapy to treat patients with hematopoietic disorders. However, limited numbers of CB CD34(+) cells, low transduction efficiency with lentiviral vectors (LVs), and low engraftment efficiency of nonobese diabetic/severe combined immunodeficient (NOD/SCID) repopulating cells (SRC), a measure of HSC, are blocks to this procedure. To optimize culture and transduction conditions, we compared various lengths of time for prestimulation before transduction, transduction duration, and posttransduction cell culture. MATERIALS AND METHODS: We used a LV to transduce human CB CD34(+) cells followed by engraftment into NOD/SCID mice. We evaluated the effects of prestimulation and transduction time and optimized ex vivo cell culture duration before transplantation. RESULTS: We were able to achieve up to 40% transduction efficiency and up to 50% engraftment efficiency of SRC in CB CD34(+) cells when CB CD34(+) cells were either not prestimulated or prestimulated in 1% fetal bovine serum medium for 1 hour, followed by 5 hours transduction and 3 days culture in a cocktail of growth factors after transduction. No apparent functional changes of CB CD34(+) cells were noted under these conditions. CONCLUSION: This gene-transduction/cell-expansion protocol is the first systematic study to optimize prestimulation time, transduction time, and, very importantly, ex vivo culture time after transduction, and may be of use for LV gene transduction in a gene therapy setting.     

Ex vivo expansion of human umbilical cord blood and peripheral blood CD34(+) hematopoietic stem cells

The proliferation and expansion of human hematopoietic stem cells (HSC) in ex vivo culture was examined with the goal of generating a suitable clinical protocol for expanding HSC for patient transplantation.HSC were derived from umbilical cord blood (UCB) and adult patient peripheral blood stem cell collections. HSC were stimulated to proliferate ex vivo by a combination of two growth factors, flt-3 ligand (FL) and thrombopoietin/c-mpl ligand (TPO/ML), and assessed for expansion by flow cytometry.Ex vivo expansion cultures of UCB were maintained for prolonged periods (up to 16 weeks), and sufficient HSC were generated for adult transplantation. In contrast to UCB, FL + TPO/ML did not significantly increase CD34(+) peripheral blood stem cell (PBSC) numbers.UCB-HSC can be expanded in culture to numbers theoretically adequate for safe, rapid engraftment of adult patients. Additional studies are needed to establish the functional activity of expanded UCB-HSC.     

Differentiation and maintenance of mast cells from CD34(+) human cord blood cells

OBJECTIVE: Establishment of a stable umbilical cord blood CD34(+) (UCB CD34(+)) cell culture system and identification of the cells in the cobblestone area differentiated from UCB CD34(+) long-term culture cells. MATERIALS AND METHODS: Human UCB CD34(+) cells were cultured on MS-5 mouse stroma cells in the presence of stem cell factor (SCF), flt-3 ligand (FL), and thrombopoietin (TPO) for 4 to 16 weeks. Cells in the culture medium and in the cobblestone area were collected and characterized by flow cytometry and microscopy. RESULTS: CD34(+) cells were stably expanded by culturing on MS-5 stroma cells in the presence of SCF, FL, and TPO for more than 4 months. Cells highly expressing CD117 (c-kit) appeared in the cobblestone area after 2 weeks and stably expanded. Isolation of cells highly expressing CD117 by fluorescence-activated cell sorter (FACS) revealed the cells were tryptase-positive and Fc epsilon receptor 1-negative mast cells. They showed typical mast cell morphology and released histamine upon stimulation by substance P or compound 48/80 in vitro. CONCLUSION: Human UCB CD34(+) cells were stably expanded on MS-5 stroma cells in the presence of SCF, FL, and TPO. Under this condition, multipotent CD34(+) cells and mast cells differentiated from UCB CD34(+) cells were expanded in the cobblestone area. The expanded mast cells showed histamine release after substance P or compound 48/80 stimulation. These human mast cells will be useful as a source of human cells for evaluating the allergic effects of drugs.     

Stable transduction of quiescent CD34(+)CD38(-) human hematopoietic cells by HIV-1-based lentiviral vectors

We compared the efficiency of transduction by an HIV-1-based lentiviral vector to that by a Moloney murine leukemia virus (MLV) retroviral vector, using stringent in vitro assays of primitive, quiescent human hematopoietic progenitor cells. Each construct contained the enhanced green fluorescent protein (GFP) as a reporter gene. The lentiviral vector, but not the MLV vector, expressed GFP in nondivided CD34(+) cells (45.5% GFP+) and in CD34(+)CD38(-) cells in G0 (12.4% GFP+), 48 hr after transduction. However, GFP could also be detected short-term in CD34(+) cells transduced with a lentiviral vector that contained a mutated integrase gene. The level of stable transduction from integrated vector was determined after extended long-term bone marrow culture. Both MLV vectors and lentiviral vectors efficiently transduced cytokine-stimulated CD34(+) cells. The MLV vector did not transduce more primitive, quiescent CD34(+)CD38(-) cells (n = 8). In contrast, stable transduction of CD34(+)CD38(-) cells by the lentiviral vector was seen for over 15 weeks of extended long-term culture (9.2 +/- 5.2%, n = 7). GFP expression in clones from single CD34(+)CD38(-) cells confirmed efficient, stable lentiviral transduction in 29% of early and late-proliferating cells. In the absence of growth factors during transduction, only the lentiviral vector was able to transduce CD34(+) and CD34(+)CD38(-) cells (13.5 +/- 2.5%, n = 11 and 12.2 +/- 9.7%, n = 4, respectively). The lentiviral vector is clearly superior to the MLV vector for transduction of quiescent, primitive human hematopoietic progenitor cells and may provide therapeutically useful levels of gene transfer into human hematopoietic stem cells.     

Apoptosis and megakaryocytic differentiation during ex vivo expansion of human cord blood CD34+ cells using thrombopoietin

Thrombopoietin (TPO), the primary regulator of megakaryocytopoiesis, plays important roles in early haematopoiesis. Previously, we have demonstrated that TPO induces a characteristic pattern of apoptosis during ex vivo expansion of cord blood (CB) CD34+ cells. In this study, we have demonstrated that the TPO-induced apoptotic cells belong to the megakaryocytic (MK) lineage and that initially expanding MK progenitors declined along with the appearance of TPO-induced apoptosis. Human CB CD34+ cells were expanded in serum-free conditions with TPO. Multidimensional flow cytometry using simultaneous measurement of apoptosis and immunophenotyping showed that the TPO-induced apoptotic cells appeared in CD61+ fractions. Immunocytochemical analysis of the fluorescent activated cell-sorted fractions showed that the apoptosis-associated CD44low fraction expressed CD61. Clonogenic assay revealed 7.4 +/- 0.50-fold increase of total megakaryocyte colony-forming units (CFU-MKs) during the initial 9 d. Thereafter, the number of CFU-MKs decreased in parallel with the increase of apoptosis. When the MK colonies were subdivided according to size, the proportion of large colonies progressively decreased, while that of medium and small colonies increased. In particular, from d 6 small colonies became predominant. These results suggested that the MK progenitors matured as they expanded during ex vivo expansion with TPO and then proceeded to apoptosis.     

Codevelopment of dendritic cells along with erythroid differentiation from human CD34(+) cells by tumor necrosis factor-alpha

OBJECTIVE: Tumor necrosis factor-alpha (TNF-alpha) inhibits erythropoiesis and enhances nonerythroid colony formation. The present study examines the nature of these nonerythroid cells and investigates their physiologic role in relation to erythroid progenitor cells. MATERIALS AND METHODS: Highly purified human CD34(+) cells underwent erythroid differentiation in the presence of multiple cytokines, including stem cell factor (SCF), interleukin-3 (IL-3), and erythropoietin (EPO), with and without TNF-alpha. We enumerate colony-forming unit-erythroid (CFU-E) and glycophorin A (GPA; a specific marker for erythroid lineage) positive cells in semisolid phase as well as in liquid suspension culture. The character and roles of codeveloping nonerythroid cells in the presence of TNF-alpha were analyzed using fluorescent activating cell sorter, enzyme immunohistochemistry, and confocal microscopy. RESULTS: TNF-alpha inhibited the generation of GPA(+) cells and conversely enhanced the generation of GPA(-) cells. The GPA(-) cells were comprised of cells with excentric cell shape and were positive for HLA class I, HLA class II, CD1a, CD4, CD11c, CD14, CD40, CD80, CD83, and CD86, but not for CD3, CD8, CD19, CD20, and CD56, indicating the codevelopment of dendritic cells (DC) along with erythroid differentiation. Developing DC/DC precursors were detected within 3 days of culture. Only in the presence of TNF-alpha did CD34(+) cells proliferate by forming aggregates where both GPA(+) and CD11c(+) DC/DC precursors were present. During culture period, immature CD11c(+) DC were capable of endocytosing damaged GPA(+) cells. CONCLUSIONS: GPA(-) cells cogenerated from human CD34(+) cells during erythroid differentiation in the presence of IL-3/SCF/EPO and TNF-alpha express DC phenotypes. The CD11c(+) DC subset physically and selectively associates with developing immature erythroid cells and damaged self-GPA(+) cells and then obtains and captures self-substances.     

Immunophenotypic and functional characterization of CD33(+)CD34(+) cells in human cord blood of preterm neonates

OBJECTIVE: To characterize CD33(+)CD34(+) cells, a major population in human cord blood (CB) CD34(+) cells of preterm neonates. MATERIALS: The proportion of CD33(+) cells was analyzed on CB CD34(+) cells from preterm and full-term neonates. CD33(+)CD34(+) cells were purified by cell sorting and analyzed on their clonogenic activity, proliferative activity in short-time liquid suspension culture, and GATA-2 mRNA expression by RT-PCR and Southern blot. RESULTS: The absolute numbers and proportion of CD34(+) cells in mononuclear cells inversely correlated with gestational age. CD33 was expressed on a majority of CB CD34(+) cells of preterm neonates but on only a minor population of them in full-term neonates. In addition, CD33 was dominantly expressed on CD38(-)CD34(+) cells or CD117(low)CD34(+) cells in CB of preterm neonates. CD33(+)CD34(+) cells of preterm cord blood had high proliferative and reproducible potentials compared with CD33(-)CD34(+) cells. CD33(+)CD34(+) cells as well as CD33(-)CD34(+) cells from preterm CB highly expressed GATA-2, in contrast to those from BM. CONCLUSIONS: These results suggest that CD33(+)CD34(+) cells, which are a major population in CB CD34(+) cells of preterm neonates, do not simply represent relatively mature myeloid lineage hematopoietic progenitor cells as those in adult BM CD34(+) cells, and may contain hematopoietic stem cells or primitive progenitor cells as in fetal liver.     

CD13/N-aminopeptidase is involved in the development of dendritic cells and macrophages from cord blood CD34(+) cells

Expression of CD13/N-aminopeptidase may reflect cell activation and growth. We examined its role regarding cell growth in cultures of cord blood CD34(+) cells with stem cell factor/Flt-3 ligand/granulocyte-macrophage colony-stimulating factor/tumor necrosis factor-alpha. Indeed, 82% +/- 6% of cells from culture day 5 were CD13(hi), 25% +/- 8% of which were still Lin-. About 50% of CD13(hi)Lin- cells, which comprise progenitors of dendritic cells (DC), monocytes/macrophages and granulocytes, and 30% of CD13(lo)Lin- cells were CD34(+). Sorted CD34(+)CD13(hi)Lin- cells, cultured further for 7 days with the same cytokines, expanded 31-fold and CD34(-)CD13(hi)Lin- cells 7-fold, but CD34(+)CD13(lo)Lin- and CD34(-)CD13(lo)Lin- cells did not grow. Thus, cell growth correlated with CD13 expression, all the more so that cells were CD34(+). Actinonin, the most potent N-aminopeptidase inhibitor, was used to engage CD13 on sorted CD13(hi)Lin- cells and on culture day-7 bulk cells. In both cases, this resulted in reversible cell growth arrest, with 30% to 60% fewer cells in the G2/S-M phase than in controls. Interestingly, similar effects were noted with CD13 monoclonal antibody TUK1, which does not inhibit N-aminopeptidase activity, but not with N-aminopeptidase-blocking antibodies WM15 and F23. All cycling cells appeared susceptible to actinonin, which induced cell apoptosis at the same time as Bcl-2 was downregulated and caspase-3 activity increased, but finally percentages and yields of DC and macrophage precursors were affected more than those of granulocytic cells. Thus, through engagement of N-aminopeptidase enzymatic site but possibly also of an independent determinant, CD13 plays a role in the growth of DC/macrophage progenitors and precursors. (Blood. 2000;95:453-460)     

The retroviral transduction of HOXC4 into human CD34(+) cells induces an in vitro expansion of clonogenic and early progenitors

OBJECTIVE: +HOX genes are expressed in the hematopoietic system and increasing data point to their involvement in the control of proliferation and/or differentiation. Genes belonging to the C cluster are preferentially expressed in developing and differentiated lymphoid lineages. However, recent studies demonstrated, by RT-PCR, that the HOXC4 gene is also actively transcribed in the most undifferentiated hematopoietic cells (CD34(+)38(low)) and in more mature myeloid and erythroid progenitors. We evaluated the expression of HOXC4 protein on human CD34(+) cells and the in vitro effect of its overexpression on proliferation and differentiation. MATERIALS AND METHODS: We assessed the expression of HOXC4 on human CD34(+) cells using a polyclonal antibody raised against the C-terminal portion of the protein expressed using the baculovirus system. Overexpression of HOXC4 in human CD34(+) cells was obtained by retroviral gene transfer; its effect on clonogenic (CFU-GM, BFU-E, and CFU-GEMM) and early progenitors (LTC-IC) was evaluated. RESULTS: The HOXC4 protein is indeed expressed in human CD34(+) cells, and its overexpression in human CD34(+) cells increases the proliferation potential of clonogenic and early progenitors. CFU-GM showed a median threefold expansion (range: 1.1-19.4; p < 0.002) compared with control transduced with the vector alone. The increment of BFU-E was higher (median ninefold, range 2.5-35; p < 0. 0009) and erythroid colonies presented a larger size with normal morphology. An even more marked effect was observed on LTC-IC (median 13, onefold; range 4.1-102.1, p < 0.0001). CONCLUSION: We demonstrate that HOXC4 is expressed in CD34(+) cells and that its overexpression induces an in vitro expansion of committed as well as very early hematopoietic progenitors. The most striking effect was obtained on LTC-IC with an expansion of 13.1-fold. The enforced expression of HOXC4 induced a significant increase (p < 0.009) in the number of erythroid colonies compared with CFU-GM, although without perturbing, at least in vitro, the maturation program of the cells. On the other hand, the effect of the gene overexpression did not induce any skewing in the colony types derived from the myeloid lineage.     

Large-scale feasibility of gene transduction into human CD34+ cell-derived dendritic cells by adenoviral/polycation complex

With a view to using multiple injections of anti-cancer dendritic cell (DC)-based vaccines, we evaluated the feasibility of the adenoviral transduction of large amounts of human CD34+ cell-derived DCs, and analysed the persistence of the transgene expression and the integrity of DC functional activity after the transduction/cryopreservation procedures. Mature DCs generated from highly enriched human CD34+ cells were transduced by a recombinant adenovirus (rAd-MFG) that carried a modified, membrane-exposed, alkaline phosphatase (AP) sequence as the reporter gene. Cationic lipids such as LipofectAmine or poly-L-lysine were mixed with the viral particles before the transduction of the target cells. The highest transduction efficiency was obtained at a multiplicity of infection (MOI) rate of 500 (AP + DCs: 50 +/- 2%, viability =95%) under both small- and large-scale conditions. The addition of poly-L-lysine or LipofectAmine increased the percentage of transduced cells at an MOI of 500 (CD1a+/AP+ cells = 85 +/- 3% and 80 +/- 2% respectively). Polycations made it possible to reduce the amounts of viral particles, with high efficiency of transduction being achieved at a MOI of 100 with 10 microg/ml poly-L-lysine (CD1a+/AP+: 68 +/- 9%) or 30 microg/ml LipofectAmine (CD1a+/AP+: 60 +/- 7%). Evaluation of the immunophenotype of the transduced DCs showed that the lack of a DC subpopulation was more susceptible to adenoviral transduction. Cryopreservation of transduced DCs did not modify the viability or percentage of AP+ cells that maintain antigen-presenting cell (APC) functions. These findings indicate the efficacy of this method for the transduction of large amounts of CD34+ cell-derived DCs using small quantities of adenoviral vector mixed with polycations. Cryopreservation of transduced DCs did not damage their viability or APC functions, thus making it possible to plan multiple injections of engineered DC-based vaccines.     

Efficient priming of protein antigen-specific human CD4(+) T cells by monocyte-derived dendritic cells

Dendritic cells (DCs) have the unique ability to initiate an immune response in vivo by capturing antigens (Ags) in peripheral tissues and migrating to secondary lymphoid organs, where they sensitize naive CD4(+) T cells. To mimic this process in vitro, previous studies have shown that DCs directly isolated from peripheral blood can be used to elicit primary responses to neoantigens (neoAgs). In other studies, when monocyte-derived DCs have been utilized to sensitize total CD4(+) T cells in vitro, only secondary proliferation to neoAgs could be elicited. In the present study, the relative abilities of CD40 ligation, protein kinase C activation, and culture in tumor necrosis factor alpha (TNF-alpha) to induce functional and phenotypic maturation of human DCs from monocyte precursors were compared. Optimal TNF-alpha-induced maturation of DCs required a prolonged 4-day culture. It was then found that loading immature DCs with the neoAgs keyhole limpet hemocyanin or human immunodeficiency virus-1 p24 gag prior to TNF-alpha-induced maturation, rather than after maturation, was crucial to sensitize CD4(+) T cells to new Ags. This primary proliferation to neoAgs was initiated from the CD4(+) CD45RA(+) naive T-cell population. Finally, it was found that monocyte-derived DCs acquired the ability to secrete interleukin-12 p70, after contact with Ag-specific T cells. The ability to prime and expand Ag-specific CD4(+) T cells ex vivo to neoAgs in serum-free conditions has potential application for cellular vaccination and adoptive immunotherapy.     

IL-6 precludes the differentiation induced by IL-3 on expansion of CD34+ cells from cord blood

BACKGROUND AND OBJECTIVES: Ex vivo expansion of hematopoietic progenitor cells (HPC) from umbilical cord blood (UCB) is an interesting strategy to obtain a sufficient number of transplantable cells for adults. To define the optimal culture conditions allowing the generation of HPC that retain their proliferative capacity without loss of long-term culture-initiating cells (LTC-IC), the effect of different cytokine combinations on the expansion of CD34+ cells from UCB was assessed. DESIGN AND METHODS: CD34+ cells were cultured in serum-free culture medium with four cytokine combinations: stem cell factor plus thrombopoietin plus flk2/flt3 ligand (STF), STF plus interleukin-3 (IL-3), STF plus interleukin-6 (IL-6) and STF plus IL-6 plus IL-3. After a 1-week culture, the number of CD34+ and CD133+ cells, colony forming units (CFU), LTC-IC and telomerase activity were determined. RESULTS: The addition of IL-6 or IL-3 to the combination of STF significantly enhanced the expansion of CD34+, CD133+ cells and CFU. All cytokine combinations tested induced a slight increase in LTC-IC number except that composed by STF plus IL-3. The greatest induction of telomerase activity was observed with the combination of STF plus IL-3 or plus IL-3 plus IL-6. Decay of the activity along time was observed when the combination of STF plus IL-3 was used, and this effect was reverted by the addition of IL-6. INTERPRETATION AND CONCLUSIONS: Our results demonstrate that the inclusion of IL-6 in a serum-free short-term culture has a beneficial effect on HPC expansion from UCB, and precludes the negative effects induced by IL-3 on LTC-IC expansion and telomerase activity.     

In vitro differentiation of natural killer T cells from human cord blood CD34+ cells

Natural killer T (NKT) cells are involved in innate immune defence and also in the regulation of adaptive immune responses. However, the development of NKT cells in vitro has not been fully characterized and culture conditions have not been fully optimized. In the present study, we found that an NKT cell fraction developed during the in vitro culture of cord blood (CB) CD34+ cells, and this was subsequently characterized both phenotypically and morphologically. CD34+ cells purified from 10 human CB were cultured in the presence of several cytokines and analysed by flow cytometry, light microscopy and electron microscopy. The NKT cell fraction, defined phenotypically (CD3+CD16+CD56+CD94+) as expressing the invariant T-cell receptor Valpha24 and Vbeta11, appeared in the CD56hi fractions. Intracytoplasmic staining demonstrated that interferon-gamma and interleukin 4 (IL-4) were detected in the CD56hi fractions. IL-15 was essential and, in combination with either flt3-ligand (FL) or stem cell factor (SCF), was sufficient to induce the development of NKT cells. The phenotype of the NKT cell fraction was CD45RO+CD45RA- and CD4+CD8alpha+. Morphologically, they were very large, with either round or oval nuclei, moderately condensed chromatins, voluminous weakly basophilic cytoplasm and various cytoplasmic granules such as dense core granules, multivesicular bodies, and intermediate form granules. When CD34+ cells purified from bone marrow (BM) were compared with those from CB, the latter were consistently more efficient at generating CD56hi NKT cell fractions. In conclusion, IL-15 in combination with FL and/or SCF can induce the differentiation of NKT cells from human CB CD34+ cells.     

Functional maturation of myeloid cells during in vitro differentiation from human cord blood CD34+ cells

CD34+ cells purified from human cord blood were expanded in the presence of several cytokines. The cultured cells were assayed for myeloid effector functions, including phagocytic activity, respiratory burst and microbicidal activity. The results showed that phagocytic activity was observed as early as day 6, irrespective of the type of cytokines used. By contrast, respiratory burst and microbicidal activity peaked on day 15 and were dependent on the type of cytokines used. In particular, granulocyte-colony stimulating factor and interleukin-6 markedly enhanced respiratory burst and microbicidal activity.     

In vitro expansion of human peripheral blood CD34+ cells

To elucidate the role of recombinant human colony-stimulating factors (CSFs) for expanding peripheral blood (PB) CD34+ cells, these cells were purified up to 94.5% +/- 1.3% and the effects of individual and combined CSFs on the proliferation and differentiation of these cells were studied in a 7-day suspension culture. The majority of CD34+ cells coexpressed CD38 (81.8% +/- 5.1%), but was negative for CD33 (88.5% +/- 3.4%). Among the individual CSFs examined, recombinant interleukin-3 (rIL-3) was identified as the most potent factor for expanding PB progenitor cells and increased nonerythroid progenitor cells 13- +/- 4- fold (P < .01). Recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF), recombinant granulocyte-CSF (rG-CSF), recombinant macrophage-CSF (rM-CSF), rIL-6, rIL-11, and recombinant stem cell factor (rSCF) did not alone expand nonerythroid progenitor cells. A combination of 5 CSFs, ie, rIL-3, rIL-6, rGM-CSF, rG-CSF, and rSCF, was identified as the most potent combination of those tested and increased nonerythroid progenitor cells 57- +/- 11-fold. After a 7-day suspension culture of CD34+ cells with these 5 CSFs, CD34+ cells expanded 14.5- fold, and CD34+/CD33- cells and CD34+/CD33+ cells were also expanded 2.9-fold and 307-fold, respectively. Most secondary colonies derived from expanded cells were small; however, the absolute number of large- sized colonies expanded 5.9- +/- 3.3-fold. Thus, the combination of CSFs can achieve a degree of amplification of PB CD34+ cells. The capability of in vitro expansion of PB CD34+ cells as an adjunct to PB stem cell transplantation is worthy of consideration.     

脐血造血干细胞向巨核细胞诱导分化的研究

目的:建立脐血CD34+造血干细胞向巨核细胞诱导分化的体系,探讨最佳的扩增方法。方法:免疫磁珠法分离获得CD34+细胞培养在无血清无基质培养液中,采用TPO加SCF加IL-3加IL-6、TPO加SCF加IL-3、TPO加SCF3种不同因子组合对其诱导分化及扩增。收集3、7、10、14d的扩增产物,运用荧光显微镜检测巨核细胞的表面标志;流式细胞术(FCM)检测巨核细胞的凋亡;并对巨核细胞形成单位(CFU-MK)及DNA含量进行检测。结果:分离获得的CD34+细胞在体外可以有效扩增,随培养时间的延长CD34+/CD41+细胞数第7天达最高值,之后逐渐下降;而CD41+、CD42b+、CD61+细胞随培养时间的延长表达量逐渐增高。加入IL-3和IL-6后,Annexin Ⅴ阳性细胞由(8.26±2.49)%降至(3.51±1.24)%。CFU-MK的数量在第10天时最高,且8倍体及8倍体以上的巨核细胞所占的的百分比增加,即成熟产板型巨核细胞增加。结论:脐血CD34+造血干细胞在体外可向巨核细胞诱导分化及有效扩增。3种因子组合中TPO加SCF加IL-3加IL-6组扩增效率最高。