AGM区来源的基质细胞促进造血干细胞扩增的体外实验研究

目的： 探讨主动脉-性腺-中肾（aorta-gonad-mesonephros，AGM）来源的基质细胞对造血干细胞（HSC）增殖的促进作用，为探寻HSC的体外扩增方法奠定实验基础。 方法： 分别从孕11 d BALB/c小鼠胚胎AGM区及6周龄小鼠骨髓分离、培养基质细胞，流式细胞仪等对基质细胞进行鉴定；利用小鼠胚胎干细胞（ESC）向造血细胞定向分化的模型，结合高增殖潜能集落（HPP-CFC）、原始细胞集落（BL-CFC）形成实验及流式细胞仪分析CD34+、CD34+Sca-1+细胞比例，对比研究AGM及骨髓基质细胞对ESC来源的HSC的扩增作用。 结果： 小鼠AGM和骨髓基质细胞在形态及表型上基本相似，均符合基质细胞的特征。AGM和骨髓基质细胞均可促进ESC来源的HPP-CFC的形成，但AGM基质细胞还可促进ESC来源的 BL-CFC的形成；流式细胞仪检测发现：在骨髓基质细胞支持下，CD34+细胞增加了3-4倍，但CD34+/Sca-1+却无明显增加；而在AGM基质细胞支持下CD34+、CD34+Sca-1+细胞均明显增加了4-5倍。 结论： AGM基质细胞在有效扩增小鼠HSC同时，能很好地维持HSC自我更新及多向分化的潜能。     

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.     

Homing-associated cell adhesion molecules and cell cycle status on the nucleated cells in the bone marrow, mobilized peripheral blood and cord blood

Homing-associated cell adhesion molecules (H-CAM) on the CD34+ cells play an important role for the engraftment process following hematopoietic stem cell transplantation (HSCT). However, it seems that not only CD34+ cells but also other nucleated cells (NCs) with H-CAM could be implicated in the engraftment process and the proliferation of hematopoietic stem cells. We investigated the differences of HCAM and cell cycle status on the NCs in cord blood (CB), bone marrow (BM), and mobilized peripheral blood (PB). The proportions of CXCR4+ cells within the NC populations were greater in CB than in PB or BM (p=0.0493), although the proportions of CXCR4+, CD44+, and CD49d+ cells within the CB CD34+ cell populations were same within BM or PB. A lower proportion of CD34+CD49d+ cells within the CD34+ cell populations was more noted in CB than in PB or BM (p=0.0085). There were no differences in cell cycle status between CB and BM or PB. Our results suggest that the migrating potential of CB would be enhanced with increased CXCR4 expression on the NCs, but the adhesion potential of CB CD34+ cells would be less than that of PB and BM. These findings may help explain why the lower cell dose is required and engraftment is delayed in cord blood stem cell transplantation.     

High efficiency electroporation of human umbilical cord blood CD34+ hematopoietic precursor cells.

Human umbilical cord blood provides an alternative source of hematopoietic cells for purposes of transplantation or ex vivo genetic modification. The objective of this study was to evaluate electroporation as a means to introduce foreign genes into human cord blood CD34+ cells and evaluate gene expression in CD34+/CD38(dim) and committed myeloid progenitors (CD33+, CD11b+). CD34+ cells were cultured in X-VIVO 10 supplemented with thrombopoietin, stem cell factor, and Flt-3 ligand. Electroporation efficiency and cell viability measured by flow cytometry using enhanced green fluorescent protein (EGFP) as a reporter indicated 31% +/- 2% EGFP+ /CD34+ efficiency and 77% +/- 3% viability as determined 48 hours post-electroporation. The addition of allogeneic cord blood plasma increased the efficiency to 44% +/- 5% with no effect on viability. Of the total CD34+ cells 48 hours post-electroporation, 20% were CD38(dim)/EGFP+. CD34+ cells exposed to interleukin-3, GM-CSF and G-CSF for an additional 11 days differentiated into CD33+ and CD11b+ cells, and 9% +/- 3% and 8% +/- 7% were expressing the reporter gene, respectively. We show that electroporation can be used to introduce foreign genes into early hematopoietic stem cells (CD34+/CD38(dim)), and that the introduced gene is functionally expressed following expansion into committed myeloid progenitors (CD33+, CD11b+) in response to corresponding cytokines. Further investigation is needed to determine the transgene expression in functional terminal cells derived from the genetically modified CD34+ cells, such as T cells and dendritic cells.     

Comparison of the distribution of progenitor cells in G-CSF-mobilized peripheral blood and steady-state bone marrow after counterflow centrifugal elutriation.

Blood-derived progenitor cells obtained following mobilization with granulocyte colony-stimulating factor (MoPBSC) are increasingly being used as an alternative to bone marrow (BM) in allogeneic stem cell transplantation. The higher numbers of mature T lymphocytes in MoPBSC grafts may increase the risk of (chronic) graft-vs.-host disease. Counterflow centrifugal elutriation (CCE) is an effective method for T-cell depletion of BM grafts. The elutriation characteristics of steady-state BM and MoPBSC were compared using a CCE procedure in which fractions were obtained after small incremental increases in flow rate with constant centrifugal force. Counterflow centrifugal elutriation experiments with MoPBSC from six healthy volunteers showed that 54% of all cells collected were recovered in the < or = 15 mL/minute fractions, whereas experiments with mononuclear BM cells from five healthy volunteers resulted in recovery of 52% of collected cells from the > or = 19 mL/minute fractions. The peak concentrations of CD34+ cells were found in the same fraction (18 mL/minute), but more CD34+ cells from MoPBSC were recovered from the small (< or = 16 mL/minute) fractions (54% for MoPBSC, 26% for BM; p = 0.08). The small CD34+ cells from BM were more frequently lacking CD38 and human leucocyte antigen-DR expression than the small CD34+ cells from MoPBSC. Mature T-cells (CD3+) in BM and MoPBSC samples had similar CCE features, as did early (long-term culture initiating cells, high-proliferative potential colony-forming cells) and more mature (colony-forming units granulocyte/macrophage, BFU-e) hematopoietic progenitor cells. The results of this study suggest that T-cell depletion by CCE of MoPBSC as compared to BM products, may lead to a greater loss of CD34+ cells, but not of immature hematopoietic progenitor cells.     

Small peptide analogue of SDF-1alpha supports survival of cord blood CD34+ cells in synergy with other cytokines and enhances their ex vivo expansion and engraftment into nonobese diabetic/severe combined immunodeficient mice

The SDF-1/CXCR4 axis has been implicated in the chemotaxis, homing, mobilization, and expansion of hematopoietic stem and progenitor cells. We studied the effects of a SDF-1 peptide analogue CTCE-0214 on the survival of cord blood CD34+ cells in culture, expansion, and engraftment of expanded cells in the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model. Our results demonstrated that CTCE-0214 synergized with thrombopoietin (TPO), stem cell factor (SCF), or flt-3 ligand (FL) on the survival of stem and progenitor cells in culture. Adding CTCE-0214 at a low concentration (0.01 ng/ml) for 4 days together with TPO, SCF, and FL significantly enhanced ex vivo expansion of CD34+ cells to subsets of primitive (CD34+CD38- cells, colony-forming unit-mixed [CFU-GEMMs]), erythroid (CFU-Es), myeloid (CFU-GMs), and megakaryocytic (CD61+CD41+ cells, CFU-MKs) progenitors, as well as their multilineage engraftment in NOD/SCID mice. Interestingly, the short exposure of expanded cells to CTCE-0214 (100 and 500 ng/ml) for 4 hours did not increase the quantity of progenitor cells but enhanced their engraftment capacity. The proportion of CD34+ cells expressing surface CXCR4 was decreased, but the overall number of this population increased upon expansion. The small peptide analogue of SDF-1 could be developed for ex vivo expansion and improving engraftment of cord blood transplantation.     

Hematopoietic differentiation of embryoid bodies derived from the human embryonic stem cell line SNUhES3 in co-culture with human bone marrow stromal cells

Human embryonic stem (ES) cells can be induced to differentiate into hematopoietic precursor cells via two methods: the formation of embryoid bodies (EBs) and co-culture with mouse bone marrow (BM) stromal cells. In this study, the above two methods have been combined by co-culture of human ES-cell-derived EBs with human BM stromal cells. The efficacy of this method was compared with that using EB formation alone. The undifferentiated human ES cell line SNUhES3 was allowed to form EBs for two days, then EBs were induced to differentiate in the presence of a different serum concentration (EB and EB/high FBS group), or co- cultured with human BM stromal cells (EB/BM co-culture group). Flow cytometry and hematopoietic colony-forming assays were used to assess hematopoietic differentiation in the three groups. While no significant increase of CD34+/CD45- or CD34+/CD38- cells was noted in the three groups on days 3 and 5, the percentage of CD34+/CD45- cells and CD34+/ CD38- cells was significantly higher in the EB/BM co-culture group than in the EB and EB/high FBS groups on day 10. The number of colony-forming cells (CFCs) was increased in the EB/BM co-culture group on days 7 and 10, implying a possible role for human BM stromal cells in supporting hematopoietic differentiation from human ES cell-derived EBs. These results demonstrate that co-culture of human ES-cell-derived EBs with human BM stromal cells might lead to more efficient hematopoietic differentiation from human ES cells cultured alone. Further study is warranted to evaluate the underlying mechanism.     

Studies of the site and distribution of CD34+ cells in idiopathic myelofibrosis

The frequency and distribution of CD34+ cells in the bone marrow (BM) of patients with idiopathic myelofibrosis (IM) were determined using an immunohistochemical technique. The percentage and absolute number of circulating CD34+ cells were enumerated. Patients with IM exhibited a continuum of number of BM CD34+ cells ranging from 1 to 85 per 5 mm(2). The frequency of BM CD34+ cells was inversely related to the number of circulating CD34+ cells. The BM biopsy specimens obtained from 4 patients with IM who underwent allogeneic stem cell transplantation were examined sequentially. Quantitative measurement revealed that the reticulin fiber volume was progressively reduced after allogeneic stem cell transplantation. All 4 patients had normocellular marrow with normal numbers of BM CD34+ cells after transplantation. These findings suggest that the BM fibrosis and abnormal hematopoietic stem cell distribution in patients with IM is a consequence of the progeny of a malignant hematopoietic stem cell clone.     

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.     

Clonogenic haematopoietic progenitor assays from clinically normal horses

Clonal assays for haematopoietic progenitors were performed on mononuclear cells isolated from bone marrow samples collected from the sternebrae of normal horses. Colony-forming units-erythroid (CFUE), burst-forming units-erythroid (BFU-E), colony-forming units-granulocyte/monocyte (CFU-GM) and colony-forming units-fibroblastic (CFU-F) were assayed, and reference values for clinically normal horses were established. The mean numbers of CFU-E, BFU-E and CFU-GM per 5 × 10⁴ cells were 329 ± 48, 30 ± 5 and 131 ± 13, respective. The mean number of CFU-F was 49 ± 6 per 2 ± 10 cells. The numbers of CFU-E and BFU-E were linearly related to the concentrations of fetal bovine serum (FBS), bovine serum albumin (BSA) and the cell number plated. The number of CFU-E were increased in the presence of erythropoietin (EPO) but its presence was not required for colony growth. BFU-E had an absolute requirement for EPO but the number of BFU-E was not linearly related to dose of EPO. Methods for clonal assays of equine hematopoietic progenitors are described.     

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.     

体外分化胚胎干细胞为造血干细胞重建造血功能的研究

目的：探讨体外定向分化胚胎干细胞（ESCs）为造血干细胞（HSCs）对体内造血功能的重建作用。方法：将小鼠E14.1胚胎干细胞采用“三步诱导法”在体外分化发育为HSCs，造血克隆形成(CFU)实验观察其体外造血集落形成情况，免疫磁珠分选纯化HSCs移植给经亚致死剂量γ射线照射的雌性SCID小鼠，观察其植入及小鼠造血功能恢复情况。结果: 经过分阶段诱导，多种造血刺激因子联合应用能有效促进ESCs定向分化发育为HSCs,流式细胞仪检测HSCs特异性表面标志物CD34+/Sca-1+表达最高为（58.64±4.20）%，CFU培养能形成较多的红系、粒系/巨噬细胞系及混合细胞集落, Wright-Giemsa 染色显示为原始的造血细胞。此阶段的HSCs经分选纯化后移植给经γ射线照射后的小鼠，移植组小鼠+10 d造血功能开始恢复，观察40 d后除血小板恢复较慢外，白细胞、红细胞、血红蛋白等指标已接近正常，植入率为71.4%，存活率为43.0%，染色体检测证实已由受体鼠的XX转为供体鼠的XY。结论: 采用分阶段诱导的方法，可在体外定向诱导小鼠ESCs分化发育为HSCs，此来源的HSCs可以有效重建体内造血功能。     

Lack of self-renewal capacity in Fancc-/- stem cells after ex vivo expansion

Treatments of the hematological manifestation in Fanconi anemia (FA) are first supported by attempts to stimulate hematopoiesis with androgens or hematopoietic growth factors. However, the long-term curative treatment of the hematological manifestation in FA patients is bone marrow (BM) or cord blood stem cell transplantation. The success rate for BM transplantation is fairly high with HLA-matched sibling donors but is, unfortunately, low with HLA-matched unrelated donors. An alternative curative treatment for those patients with no sibling donors might be gene transfer into hematopoietic stem cells. Because FA patients have reduced numbers of stem/progenitor cells, ex vivo expansion of hematopoietic stem cells would be a crucial step in gene transfer protocols. Using the FA mouse model, Fancc-/-, we tested the ability of CD34- hematopoietic stem cells to support ex vivo expansion. We determined that Fancc-/- CD34- stem cells have reduced reconstitution ability and markedly reduced self-renewal ability after culture, as shown by secondary transplants. These results indicate that FA stem cells may not be well suited for ex vivo expansion before gene transfer or transplantation protocols.     

Clinical-scale expansion of a mixed population of bone-marrow-derived stem and progenitor cells for potential use in bone-tissue regeneration

Preclinical and clinical studies have demonstrated the ability of bone marrow derived stem and progenitor cells to regenerate many tissues, including bone. Methods to expand or enrich progenitors from bone marrow are common; however, these methods include many steps not amenable to clinical use. A closed automated cell production culture system was developed for clinical-scale ex vivo production of bone marrow-derived stem and progenitor cells for hematopoietic reconstitution. The current study tested the ability of this bioreactor system to produce progenitor cells, termed tissue repair cells (TRC), possessing osteogenic potential. Three TRC formulations were evaluated: (a) cells cultured without exogenous cytokines (TRC); (b) cells cultured with exogenous cytokines (TRC-C); and (c) an adherent subset of TRC-C (TRC-C(Ad)). Starting human bone marrow mononuclear cells (BM MNC) and TRC products were characterized for the expression of cell surface markers, in vitro colony forming ability, and in vivo osteogenic potential. Results showed significant expansion of mesenchymal progenitors (CD90+, CD105+, and CD166+) in each TRC formulation. In vivo bone formation, measured by histology, was highest in the TRC group, followed by TRC-C(Ad) and TRC-C. The TRC product outperformed starting BM MNC and had equivalent bone forming potential to purified MSCs at the same cell dose. Post hoc analysis revealed that the presence of CD90+, CD105+, and CD166+ correlated strongly with in vivo bone formation scores (r(2) > .95). These results demonstrate that this bioreactor system can be used to generate, in a single step, a population of progenitor cells with potent osteogenic potential. Disclosure of potential conflicts of interest is found at the end of this article.     

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.     

Interleukin 3 improves the ex vivo expansion of primitive human cord blood progenitor cells and maintains the engraftment potential of scid repopulating cells.

In umbilical cord blood (UCB) transplantation, the number of nucleated cells per kilogram is a major predictive and critical factor of hematopoietic recovery. Thus, ex vivo expansion of hematopoietic UCB progenitors could potentially accelerate engraftment. Whereas Flt-3 ligand (FL), stem cell factor (SCF), and thrombopoietin (TPO) are considered indispensable, the role of interleukin 3 (IL-3) is still controversial: it has been reported either to support or abrogate the reconstituting ability of stem cells. By adding IL-3 we aimed to enhance the amplification of early and committed progenitor cells without impairing the long-term engraftment of stem cells. Demonstrating a positive impact of IL-3 on the proliferation of all progenitor subsets, the amplification of CD34+ UCB cells was increased 20.9-fold +/- 5.4 (mean +/- standard error) in serum-free culture with FL, SCF, TPO, and IL-3 as opposed to 9.3-fold +/- 3.2 without IL-3 after 7 days. If IL-3 was included, primitive long-term culture-initiating cells and committed colony-forming cells were expanded 16.3-fold +/- 5.5 and 18.1-fold +/- 2.4, respectively, compared to 12.6-fold +/- 5.6 and 9.1-fold +/- 2.0 without IL-3. Analysis of cultured CD34+ UCB cells in sublethally irradiated nonobese diabetic/severe combined immunodeficient mice confirmed that cultured cells had preserved their repopulating potential. After 6 weeks, all mice showed multilineage engraftment with their bone marrow containing an average of 45% human CD45+ cells of the unmanipulated sample, 43% of cells after culture in the presence of IL-3, and 27% of cells after culture without IL-3. In combination with early acting cytokines, IL-3 therefore improves the ex vivo expansion of UCB stem and progenitor cells without impairing their engraftment potential.