Optimum storage conditions for cord blood-derived hematopoietic progenitor cells prior to isolation

Optimum storage conditions of cord blood-derived hematopoietic progenitor cells before isolation remain unknown. We therefore evaluated CD34+ cells isolated from cord blood units (n=57) within 1 h after collection and following storage for 24, 48 and 72 h at either room temperature (RT) or 4 degrees C. Isolated CD34+ cells were analyzed for their cell count, immunophenotype, apoptosis rate, clonogenicity and transmigration capacity in response to stroma-derived factor 1alpha using direct-paired comparisons (n=27). CD34+, CD133+ and CD45+ positivity after isolation remained the same under all conditions. After 24 h, CD34+ cell counts and numbers of CFU-GM colonies dropped regardless of the storage temperature. After 48 h, the number of CD34+ cells increased compared to 24 h, if the cord blood had been stored at RT resulting in almost three times more CD34+ cells than at 4 degrees C. These cells had a lower early apoptosis rate and formed four times more BFU-E than those stored at 4 degrees C with equivalent plating efficiencies. CD34+ cells kept at RT for 48 h had the highest transmigration capacities, which paralleled an increased CXCR-4 expression. Cord blood should be stored at RT before CD34+ isolation and a storage time for 48 h should be preferred to 24 h.     

Human placenta-derived mesenchymal progenitor cells support culture expansion of long-term culture-initiating cells from cord blood CD34+ cells

OBJECTIVE: Allogeneic transplantation with umbilical cord blood (UCB) in adult recipients is limited mainly by a low CD34+ cell dose. To overcome this shortcoming, human placenta as a novel source of human mesenchymal progenitor cell (MPC) was incorporated in an attempt to expand CD34+ cells from UCB in vitro. MATERIALS AND METHODS: Human placenta MPC was isolated and characterized by morphologic, immunophenotypical, and functional analysis. UCB CD34+ cells were expanded by coculture with placental MPC. Suitable aliquots of cells were used to monitor cell production, clonogenic activity, and long-term culture-initiating culture (LTC-IC) output. Finally, the immunoregulatory effect of placental MPC was evaluated by T-cell proliferation assay. RESULTS: In its undifferentiated state, placental MPC displayed fibroblastoid morphology; was CD73, CD105, CD29, CD44, HLA-ABC, and CD166 positive; produced fibronectin, laminin, and vimentin; but was negative for CD14, CD31, CD34, CD45, HLA-DR, and alpha-smooth muscle actin. Functionally, it could be induced into adipocytes, osteocytes, and chondrocytes. In vitro expansion of UCB hematopoietic cells, when cocultured with placental MPC in the presence of cytokines, was significantly enhanced: CD34+ cells by 14.89 +/- 2.32 fold; colony-forming cell (CFC) by 36.73 +/- 5.79 fold; and LTC-IC by 7.43 +/- 2.66 fold. Moreover, placental MPC could suppress T-cell proliferation induced by cellular stimuli. CONCLUSION: These results strongly suggest that human placental MPC may be a suitable feeder layer for expansion of hematopoietic progenitors from UCB in vitro.     

Cocultivation of umbilical cord blood CD34^＋ cells with retro-transduced hMSCs leads to effective amplification of long-term culture-initiating cells

AIM:To establish a novel coculture system for ex vivoexpansion of umbilical cord blood(UCB)hematopoieticprogenitors using thrombopoietin(TPO)/Flt-3 ligand(FL)-transduced human marrow-derived mesenchymalstem cells(tfhMSCs)as feeder.METHODS:UCB CD34~ cells were isolated and culturedusing four culture systems in serum-containing or serum-free medium.Suitable aliquots of cultured cells wereused to monitor cell production,clonogenic activity,and long-term culture-initiating culture(LTC-IC)output.Finally,the severe-combined immunodeficient(SCID)mouse-repopulating cell(SRC)assay was performed toconfirm ability of the cultured cells to reconstitute long-term hematopoiesis.RESULTS:There were no significant differences in thenumber of total nucleated cells among different culturesystems in serum-containing medium during 21-dculture.However,on d 14,the outputs of CD34~ cells,CFU-C and CFU-GEMM in ffhMSCs coculture system weresignificantly enhanced.LTC-IC assay demonstrated thatthe tfhMSCs coculture system had the most powerfulactivity.The severe-combined immunodeficient(SCID)mouse repopulating cell(SRC)assay confirmed extensiveability of the expanded cells to reconstitute long-termhematopoiesis.Furthermore,PCR analysis demonstratedthe presence of human hematopoietic cells in the bonemarrow and peripheral blood cells of NOD/SCID mice. CONCLUSION:The TPO/FL-transduced hMSCs,incombination with additive cytokines,can effectivelyexpand hematopoietic progenitors from UCB in vitro andthe tfhMSCs coculture system may be a suitable systemfor ex vivo manipulation of primitive progenitor cellsunder contact culture conditions.     

Cobblestone area-forming cells,long-term culture-initiating cells and NOD/SCID repopulating cells in human neonatal blood: a comparison with umbilical cord blood

Our prior study demonstrated that neonatal blood (NB) contained hematopoietic stem and progenitor cells that declined rapidly after birth. To validate that NB is a source of functional stem cells, we characterized this population in terms of cobblestone area-forming cells (CAFC), long-term culture-initiating cells (LTC-IC) and NOD/SCID mouse repopulating cells (SRC) in NB and umbilical cord blood (CB). Our data demonstrated that the frequencies of CAFC (30.2 vs 37.1, P = 0.14) and LTC-IC (28.6 vs 31.0, P = 0.49) in 1 x 10(5) mononuclear cells (MNC) of NB and CB were similar, suggesting that these cells were preserved in the circulation of the neonates shortly after birth. Sublethally irradiated NOD/SCID mice were transplanted with CD34(+) cells enriched from thawed NB and CB. At 6 weeks post transplant, human (hu)CD45(+) cells were detected in the bone marrow (BM), spleen and peripheral blood (PB) of the mice as demonstrated by flow cytometric and DNA analysis. Levels of huCD45(+)cells and colony forming units (CFU) appeared to be dependent on the infusion cell dose and were higher in animals receiving CB cells when compared with those of the NB group. The transplanted cells were capable of differentiation into multi-lineage progenitor cells (CD34(+) cells and differential CFU), as well as mature myeloid (CD14(+), CD33(+)), B lymphoid (CD19(+)) and megakaryocytic (CD61(+)) cells in the recipients. NB cells, subjected to ex vivo culture in an optimized preclinical condition, were significantly expanded to early and committed progenitor cells. Expanded NB contained SRC at a reduced quantity but with high proportions of CD14(+) cells and CD33(+) cells. Our study confirms that NB contains pluripotent hematopoietic stem and progenitor cells capable of homing and engrafting the NOD/SCID mice.     

Proliferation, differentiation, and cytokine secretion of human umbilical cord blood-derived mononuclear cells in vitro

OBJECTIVE: Human umbilical cord blood (hUCB)-derived mononuclear cells were previously shown to exert therapeutic effects in a number of animal models of nervous system impairment. However, the mechanisms underlying the structural and functional improvements are still unclear. As cell replacement seems to be a rare or absent event in vivo, we suggest secondary mechanisms, by which the therapeutic effect of transplanted mononuclear cells might be mediated. We investigated the potential of hUCB-derived mononuclear cells in vitro to proliferate, differentiate, and to secrete factors possibly beneficial for the host brain tissue in vivo. METHODS: Using a succession of distinct culture media, mononuclear cells were stimulated by growth factor combinations, e.g., epidermal growth factor (EGF)/fibroblast growth factor-2 (FGF-2) or nerve growth factor (NGF)/retinoic acid (RA). Expression of hematological and neural marker proteins was investigated by immunoblotting, immunocytochemistry, and fluorescence-activated cell analysis. Secretion of proteins was assayed using a human cytokine antibody array, and quantified via enzyme-linked immunosorbent assay. RESULTS: Mononuclear cells were shown to undergo proliferation in the presence of EGF/FGF-2. When cells were cultured in NGF/RA-containing medium, neuronal and glial marker proteins were expressed, indicating differentiation. In the presence of either growth factor combination, cells in vitro secrete interleukins, growth factors, and chemotactic proteins. CONCLUSION: Although capable of incipient differentiation, cytokine secretion of hUCB-derived mononuclear cells envisages the potential of an indirect effect in vivo. Most factors detected in conditioned medium are renowned for their anti-inflammatory, neuroprotective, angiogenic, or chemotactic actions, thus, providing the means for a therapeutic outcome mediated by secondary effects.     

High frequency of long-term culture-initiating cells retain in vivo repopulation and self-renewal capacity

OBJECTIVE: We wished to test if the long-term culture initiating cell (LTC-IC) assay measures primitive hematopoietic stem cells. An LTC-IC is defined by its ability to repopulate a stromal layer by forming colonies of myeloid cells. A negative well should never have received a stem cell, whereas a positive well should have been initiated by a stem cell. If these colonies were derived from stem cells, then a subset of the positive wells should retain stem cell activity. MATERIALS AND METHODS: Limiting dilution cultures were initiated on the stromal cell line S17. Individual clonal cultures from LTC-IC assays were assessed for repopulation capacity in W(14)W(41) mice. RESULTS: In long-term repopulation experiments, little activity was found in the negative wells, whereas 50% of the positive wells contained repopulating stem cells. The diverse in vivo repopulation patterns of the clonally derived stem cells suggest that this assay detects the full spectrum of stem cell types. Secondary transfers show that the clonally derived stem cells have self-renewal capacity. Experiments with mixtures of genetically distinguished cells showed that most (>90%) of the cultures were clonal. CONCLUSIONS: Our data present the first formal link between LTC-IC and repopulating stem cells. Moreover, the culture system presents a new way of generating a high frequency of clonally repopulating stem cells.     

Detection and quantitation of long-term culture-initiating cells in normal human peripheral blood.

The objective of this study was to utilize the Dexter long-term culture system to characterize and quantitate early hematopoietic cells in normal human blood. Peripheral blood mononuclear cells were depleted of T cells. B cells, and monocytes, yielding a population (null cells) highly enriched for hematopoietic precursors. One to 2 x 10(7) null cells containing an average of 5.4 x 10(4) granulocyte-macrophage progenitors (granulocyte-macrophage colony-forming units, CFU-GM) were cultured on irradiated allogeneic marrow stromal layers in T-25 flasks. Nonadherent progenitors were detected for an average of 6.8 +/- 0.7 (standard error of the mean) weeks and exceeded 1 x 10(4) per flask for 4 weeks. Null cells were treated with 5-fluorouracil (5-FU) to determine whether long-term growth was due to survival of CFU-GM or to maturation of more primitive precursors. When 5-FU treatment killed greater than 99% of CFU-GM, an increase in progenitors was observed after 12 days of cultivation with stroma. CFU-GM cloning efficiency of 5-FU-treated cells peaked after 24 days of incubation, whereas controls peaked after 7 days. The total number of nonadherent CFU-CM in 5-FU-treated and control cultures converged during the 5-8 weeks of incubation. The frequency of long-term culture-initiating cells (LTCIC) in peripheral blood was determined by limiting dilution analysis in micro-stromal cultures. In two donors, the frequencies of LTCIC were 6.3 and 19.6 per 1 x 10(5) null cells. The CFU-GM:LTCIC ratios were 12.6:1 and 12.8:1. We conclude that normal peripheral blood contains primitive hematopoietic precursors demonstrable as 5-FU-resistant precursors of CFU-GM or as LTCIC. The frequency of the latter is 13-fold lower than day-14 CFU-GM.     

Stromal derived factor-1-induced chemokinesis of cord blood CD34(+) cells (long-term culture-initiating cells) through endothelial cells is mediated by E-selectin

Homing of hematopoietic stem cells to the bone marrow (BM) involves sequential interaction with adhesion molecules expressed on BM endothelium (BMEC) and chemokine stromal derived factor-1 (SDF-1). However, the mechanism whereby adhesion molecules regulate the SDF-1-induced transendothelial migration process is not known. E-selectin is an endothelial-specific selectin that is constitutively expressed by the BMEC in vivo. Hence, we hypothesized that E-selectin may mediate SDF-1-induced transendothelial migration of CD34(+) cells. We show that CD34(+) cells express both E-selectin ligand and fucosyltransferase-VII (FucT-VII). Soluble E-selectin-IgG chimera binds avidly to 75% +/- 10% of CD34(+) cells composed mostly of progenitors and cells with long-term culture-initiating cell (LTC-IC) potential. To assess the functional capacity of E-selectin to mediate CD34(+) cell migration in a transendothelial migration system, CD34(+) cells were placed on transwell plates coated with interleukin-1beta-activated BMEC. In the absence of SDF-1, there was spontaneous migration of 7.0% +/- 1.4% of CD34(+) cells and 14.1% +/- 2.2% of LTC-IC. SDF-1 induced migration of an additional 23.0% +/- 4.4% of CD34(+) cells and 17.6% +/- 3.6% of LTC-IC. Blocking MoAb to E-selectin inhibited SDF-1-induced migration of CD34(+) cells by 42.0% +/- 2.5% and LTC-IC by 90.9% +/- 16.6%. To define the mechanism of constitutive expression of E-selectin by the BMEC in vivo, we have found that vascular endothelial growth factor (VEGF(165)) induces E-selectin expression by cultured endothelial cells. VEGF-stimulated endothelial cells support transendothelial migration of CD34(+) cells that could be blocked by MoAb to E-selectin. These results suggest that trafficking of subsets of CD34(+) cells with LTC-IC potential is determined in part by sequential interactions with E-selectin and SDF-1.     

Stromal fibroblast heparan sulfate is required for cytokine-mediated ex vivo maintenance of human long-term culture-initiating cells

We have recently demonstrated that 50% of primitive human long-term culture-initiating cells (LTC-IC) are maintained for up to 8 weeks in stroma-dependent cultures in which progenitor-stroma contact is prevented (stroma noncontact), or when progenitors are cultured in medium conditioned by stromal feeders. This indicates that factors responsible for LTC-IC maintenance are present in soluble form in stromal supernatant (SN). Although the picogram concentrations of cytokines present in stromal SN can induce the differentiation of CD34+/HLA-DR- (DR-) cells to clonogenic cells (colony forming cells; CFC), they maintain only 10% of LTC-IC for 5 weeks, suggesting that factors other than these cytokines are required for LTC-IC maintenance. To characterize the factor(s) in stromal SN responsible for LTC-IC maintenance, we purified glycoproteins and proteoglycans (PG) from the SN of the LTC-IC supportive murine marrow stromal fibroblast cell line M2-10B4 by ion exchange high performance liquid chromatography (HPLC). Culture of DR- cells in a combination of M2-10B4-derived PG, but not glycoproteins and picogram concentrations of recombinant human interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), leukemia inhibitory factor (LIF), granulocyte- macrophage colony-stimulating factor (GM-CSF), and macrophage inflammatory protein-1alpha (MIP-1alpha) resulted in the recovery of 96% +/- 8% of LTC-IC maintained in cultures supplemented with unfractionated stromal SN. LTC-IC maintenance was largely retained after digestion of the PG-rich fraction with proteinase K and after dissociative gel filtration chromatography, but was completely abolished following treatment with nitrous acid, which digests heparan sulfate glycosaminoglycans (HS GAG). As for M2-10B4-derived HS GAG, high concentrations of bovine kidney HS GAG, but not bovine tracheal chondroitin sulfate, significantly improved cytokine-mediated LTC-IC maintenance. Maintenance of LTC-IC by these nonmarrow-derived HS GAG was, however, significantly lower than that seen with M2-10B4-derived HS. These studies demonstrate a role for marrow stroma-derived HS GAG in the long-term in vitro maintenance of human LTC-IC. Further structure-function analysis of these HS GAG may have important implications for ex vivo stem cell expansion and gene transfer into hematopoietic progenitors.     

体外不同诱导条件对人脐血间充质干细胞分化为神经细胞的研究

目的观察体外不同诱导条件对人脐血间充质干细胞(MSCs)向神经细胞分化的作用,探讨最佳诱导方法。方法取第3代脐血MSCs进行诱导,分为化学诱导剂组、生长因子诱导组、丹参素联合生长因子诱导组。采用免疫细胞化学方法和免疫荧光方法检测诱导前后神经元特异性标志小鼠抗神经元核抗原(NeuN)、兔抗微管蛋白(β-TubulinⅢ)和星形胶质细胞特异性标志神经胶质纤维酸性蛋白(GFAP)表达的变化。结果 MSCs经3种方法诱导后出现类似神经元样细胞的形态改变,伸出长突起。免疫组织化学和免疫荧光方法鉴定显示,诱导后的细胞能特异性表达NeuN和β-TubulinⅢ,而GFAP阳性细胞较少。丹参素联合生长因子诱导组β-TubulinⅢ及NeuN的阳性细胞率均明显高于生长因子诱导组和化学诱导剂组,而GFAP阳性细胞率明显低于生长因子诱导组,差异均有统计学意义。结论丹参素联合表皮生长因子和碱性成纤维细胞生长因子在体外可定向诱导人脐血MSCs分化为神经元,诱导效果最佳。     

Toll-like receptor 3 regulates cord blood-derived endothelial cell function in vitro and in vivo

Circulating endothelial progenitor cells (cEPC) are capable of homing to neovascularisation sites, in which they proliferate and differentiate into endothelial cells. Transplantation of cEPC-derived cells, in particular those isolated from umbilical cord blood (UCB), has emerged as a promising approach in the treatment of cardio-vascular diseases. After in vivo transplantation, these cells may be exposed to local or systemic inflammation or pathogens, of which they are a common target. Because Toll-like receptors (TLR) are critical in detecting pathogens and in initiating inflammatory responses, we hypothesized that TLR may govern UCB cEPC-derived cells function. While these cells expressed almost all TLR, we found that only TLR3 dramatically impaired cell properties. TLR3 activation inhibited cell proliferation, modified cell cycle entry, impaired the in vitro angiogenic properties and induced pro-inflammatory cytokines production. The anti-angiogenic effect of TLR3 activation was confirmed in vivo in a hind-limb ischemic mice model. Moreover, TLR3 activation consistently leads to an upregulation of miR-29b, -146a and -155 and to a deregulation of cytoskeleton and cell cycle regulator. Hence, TLR3 activation is likely to be a key regulator of cEPC-derived cells properties.     

Quantitative and qualitative alterations of long-term culture-initiating cells in patients with acute leukaemia in complete remission

Bone marrow (BM) samples from 24 patients with acute leukaemia (AML 17, ALL seven) in first complete remission were compared to samples from 10 normal donors with regard to their content in long-term culture-initiating cells (LTC-IC) as assessed by a limiting dilution assay and the clonogeneic capacity of these cells, in order to determine whether remission marrow cells displayed any specific defect at the primitive stem cell level. The frequency of LTC-IC in the whole patient group was 1 in 3487 ± 3125 mononuclear cells (MNC) as compared to 1 in 794 ± 492 MNC in normal controls ( P  = 0.0009), with no difference between AML and ALL. Moreover, the clonogeneic capacities were 2.66 ± 0.7 (range 1.8–1.6) and 4.0 ± 1.6 (range 2.2–7.9) CFC per LTC-IC in patients and controls respectively ( P  = 0.0015). These quantitative and qualitative defects were aggravated by treatment with mafosfamide at a dose of 50 μg/10⁷ MNC/ml, where the mean recovery of LTC-IC after in vitro purging was 42%. In nine patients autografted with purged marrow following high-dose radiochemotherapy, no correlation could be detected between the dose of LTC-IC (mean 6742 ± 7877/kg) and the kinetics of recovery of haemopoiesis. We concluded that, in acute leukaemia patients in complete remission, the presumably normal residual stem cell pool was not only quantitatively diminished but also qualitatively altered in its capacity to give rise to clonogeneic progenitor cells.     

Stromal support enhances cell-free retroviral vector transduction of human bone marrow long-term culture-initiating cells.

Gene transfer into hematopoietic stem cells by cell-free virions is a goal for gene therapy of hematolymphoid disorders. Because the hematopoietic microenvironment provided by the stroma is required for stem cell maintenance both in vivo and in vitro, we reasoned that cell-free transduction of bone marrow cells (BMC) may be aided by stromal support. We used two high-titer replication-defective retroviral vectors to differentially mark progenitor cells. The transducing vector was shown to be a specific DNA fragment by polymerase chain reaction of colony-forming cells derived from progenitors maintained in long-term culture (LTC). BMC were infected separately by cell-free virions with or without pre-established, irradiated, allogeneic stromal layers, and in the presence or absence of exogenous growth factors (GF). The GF assessed were interleukin-3 (IL-3) and IL-6 in combination, leukemia inhibitory factor (LIF), mast cell growth factor (MGF), and LIF and MGF in combination. In addition, we developed a competitive LTC system to directly assess the effect of infection conditions on the transduction of clonogenic progenitors as reflected by the presence of a predominate provirus after maintenance in the same microenvironment. The results show gene transfer into human LTC-initiating cells by cell-free retroviral vector and a beneficial effect of stromal support allowing a transduction efficiency of 64.6% in contrast to 15.8% without a supporting stromal layer. A high transduction rate was achieved independent of stimulation with exogenous GF. We propose that autologous marrow stromal support during the transduction period may have application in clinical gene therapy protocols.     

A severe and consistent deficit in marrow and circulating primitive hematopoietic cells (long-term culture-initiating cells) in acquired aplastic anemia

We examined the stem cell compartment of patients with acquired aplastic anemia (AA) using the long-term culture-initiating cell assay (LTC-IC), in parallel with measurements of CD34+ cells and mature hematopoietic progenitors. Secondary colonies from cells surviving 5 weeks of long-term bone marrow culture (LTBMC) were determined for the peripheral blood (PB) of 68 AA patients and 13 normal controls and for BM of 49 AA patients and 14 controls; because of low cell numbers, formal limiting dilution analysis could only be performed in 10 patients. The relationship of cell input in LTBMC and the output of secondary colonies was linear, allowing quantification of LTC-IC number from bulk cultures. Secondary colony formation was markedly abnormal in severe AA. In contrast to 7.8 colony-forming cells (CFC)/10(5) mononuclear cells in normal BM and 0.14 CFC/10(5) normal PB mononuclear cells, patients with severe disease showed 0.024 CFC/10(5) in BM and 0.0068 CFC/10(5) in PB. Under limiting dilution conditions, patients' cells also showed markedly lower colony-forming ability. In contrast to 4.3 +/- 1 colonies/normal LTC-IC, we obtained only 1.27 +/- 0.09 and 2.0 +/- 0.35 colonies from BM of acute and recovered cases, respectively. These values were used to extrapolate LTC-IC numbers from secondary colony formation in suspension cultures. In PB, calculated LTC-IC were decreased 7.4-fold in new and relapsed severe AA and 2.8- fold in recovered AA. In BM, LTC-IC were decreased 10-fold in new and relapsed AA and sixfold in recovered cases. Compared with measurements obtained on presentation, LTC-IC were lower in post-treatment samples from patients who had failed to recover after intensive immunosuppression and relatively higher in cases at relapse. In recovered patients, LTC-IC number increased but remained below the normal range in 20 of 25. In patients studied serially for 3 to 12 months after treatment, LTC-IC numbers remained stable but low. LTC-IC number correlated with concurrently determined CD34+ cell number and primary hematopoietic colony formation. These results indicate that stem cell numbers, as quantitated by the LTC-IC assay, are markedly diminished in number in all severe AA. Additionally, the function of the stem cell or the stem cell compartment in AA is also abnormal, as inferred from the low clonogenic potential in secondary colony assays. Early hematologic improvement in some patients occurs without increasing numbers of LTC-IC, and a minority of recovered cases show apparent repopulation of the LTC-IC compartment years after treatment.     

Myoendothelial differentiation of human umbilical cord blood-derived stem cells in ischemic limb tissues

Human umbilical cord blood (UCB) contains high numbers of endothelial progenitors cells (EPCs) characterized by coexpression of CD34 and CD133 markers. Prior studies have shown that CD34+/CD133+ EPCs from the cord or peripheral blood (PB) can give rise to endothelial cells and induce angiogenesis in ischemic tissues. In the present study, it is shown that freshly isolated human cord blood CD34+ cells injected into ischemic adductor muscles gave rise to endothelial and, unexpectedly, to skeletal muscle cells in mice. In fact, the treated limbs exhibited enhanced arteriole length density and regenerating muscle fiber density. Under similar experimental conditions, CD34- cells did not enhance the formation of new arterioles and regenerating muscle fibers. In nonischemic limbs CD34+ cells increased arteriole length density but did not promote formation of new muscle fibers. Endothelial and myogenic differentiation ability was maintained in CD34+ cells after ex vivo expansion. Myogenic conversion of human cord blood CD34+ cells was also observed in vitro by coculture onto mouse myoblasts. These results show that human cord blood CD34+ cells differentiate into endothelial and skeletal muscle cells, thus providing an indication of human EPCs plasticity. The full text of this article is available online at http://www.circresaha.org.     

脐血干细胞转化为肝细胞的研究进展

自进行第1例脐血干细胞移植以来,各国科研机构相继开展对于脐血干细胞的研究,脐血库也纷纷建立．随着再生医学的发展,众多国内外学者在尝试进行非肝源性细胞向肝细胞分化方面的研究,并已经证实脐血干细胞在特定的微环境下可以在体内和体外转化为肝样细胞,为脐血干细胞在肝脏疾病中的应用奠定了基础．本文就脐血干细胞向肝细胞转化的最新研究进展作一综述．     

Human umbilical cord blood-derived stromal cell, a new resource of feeder layer to expand human umbilical cord blood CD34+ cells in vitro

Allogeneic transplantation with human umbilical cord blood (hUCB) in adult recipients is mainly limited by a low CD34+ cell dose. To break the limit, hUCB as a novel source of hUCB-derived stromal cells was incorporated in an attempt to expand CD34+ cells from hUCB in vitro. Cord blood CD34 cells were separated by MACS system. HUCB-derived stromal cells were cultured by the Dexter system and characterized by morphologic, immunophenotypical, and functional analysis. We studied the effects of hUCB-derived stromal cells, cytokines, and hUCB-derived stromal cells combined with cytokines on expansion of hUCB CD34 cells. The CD34+ cells were assessed for the degree of expansion and the number of colony-forming units in semisolid culture. Our research found that hUCB-derived stromal cells were mainly composed of three kinds of cell components, with CD106, CD29, CD44, CD45, CD50, CD68, CD31, Fn, Lm, and collagen IV positive, but CD34 negative immunophenotype. Functionally, it was discovered by cell cycle and growth curve analyses that the capability of colony and parietal layer formation of hUCB-derived stromal cells was poorer than that of BM stromal cells, and the doubling time of hUCB-derived stromal cells was longer than that of BM stromal cells. It was indicated by ELISA and RT-PCR that hUCB-derived stromal cells express higher level of TPO and less GM-CSF and SCF than BM stromal cell. Adherent layer of hUCB-derived stromal cells alone or combining with cytokines, increased CD34+ cell expansion. In vitro formation of CFUs by expanded CCD34 cells was significantly higher than that of unexpanded CD34+ cells (P < 0.05). When cocultured with hUCB-derived stromal cells in the presence of cytokines, cell growth was significantly enhanced: CD34 cells by 8.02 +/- 0.96-fold, CFU-GM by 217.60 +/- 6.72-fold, CFU-E by 1940.80 +/- 52.78-fold, and CFU-Mg by 142.60 +/- 4.39-fold. HUCB-derived stromal cells have significant superiority on the expansion of CFU-Mg (P < 0.05). The results indicate that human umbilical cord blood-derived stromal cells may be a suitable feeder layer for expansion of hematopoietic progenitors from hUCB in vitro.     

人脐带血浆细胞样树突状细胞亚群的体外诱导及鉴定

目的建立浆细胞样树突状细胞(pDC)的体外培养方法。方法采集2014年6月-2015年2月于湖南中医药大学第一附属医院就诊的健康产妇脐带血40 ml,分离脐带血单个核细胞(CBMC),用含重组人FMS样酪氨酸激酶3配体(Flt3-L)100ng/ml、白细胞介素(IL)3 10 ng/ml的RPMI1640完全培养基连续培养7 d,每隔1天半换液。第8天加入2μg/ml的CpG ODN,24 h后收集细胞行流式检测,同时检测pDC培养上清干扰素(IFN)α水平。在培养细胞的第1、3、5、7、8天,观察培养孔中的pDC形态特征变化。结果 CBMC培养2 h后可见圆形扁平细胞布满视野,24 h后细胞开始贴壁,细胞质伸展开,体积有所增大,圆形,透亮,并可见散在的小集落形成;培养第3~4天,细胞体积较前继续增大,多数为圆形,部分细胞表面可见小的突起,并可见少量梭形、蝌蚪状、星形或其他不规则形的细胞,培养液中集落较前明显增多、增大;培养第5~8天,集落数量及集落内细胞数逐渐减少,而培养液中圆形或具有小突起的悬浮细胞逐渐增多。流式细胞仪检测发现CD123、BDCA-2、BDCA-4均表达阳性的细胞,该细胞为pDC。在培养过程中,pDC比例不断增加,其在培养开始时仅占CBMC总数的1.08%,第4天升至5.32%,第8天时达到高峰,为19.8%。培养第8天时,pDC培养上清IFNα水平达(11 302.61±1745.31)pg/ml。结论以人CBMC为培养初始细胞,利用Flt3-L与IL-3联合,可成功体外诱导出pDC。