用微血管内皮细胞体外扩增的骨髓造血干细胞重建大剂量化疗后的？…

目的 探索造血干细胞体外培养扩增的新方法。 方法 采用磁性细胞分离技术纯化小鼠骨髓血管内皮细胞,将其与骨髓造血细胞一起培养。培养的骨髓细胞移植给大剂量化疗后的小鼠,观察其造血重建功能。 结果 纯化的血管内皮细胞具有鹅卵石样细胞形态,表达多种内皮细胞相关抗原如ＣＤ３１,ＣＤ３４,ＩＣＡＭ－１,ＶＣＡＭ－１和凝集素ＢＳ－１结合位点。将内皮细胞与骨髓造血细胞共同培养后,骨髓细胞数明显增加,其中６０％为原     

诱导体外扩增的小鼠骨髓CD34+造血细胞分化为树突状细胞

诱导体外培养扩增后的小鼠骨髓ＣＤ３４＋造血细胞分化为树突状细胞（ＤＣ）。方法Ｃ５７ＢＬ／６ｊ小鼠骨髓ＣＤ３４＋细胞采用血管内皮细胞共培养方法进行体外扩增，扩增后的ＣＤ３４＋细胞经磁性细胞分选技术纯化，并用ＧＭ┐ＣＳＦ等细胞因子诱导其向ＤＣ分化。结果小鼠骨髓ＣＤ３４＋造血细胞不论是否经过体外培养均可被诱导分化为ＤＣ，后者具有典型的树突状形态特征，表达高水平的ＭＨＣⅠ、Ⅱ类抗原和ＣＤ８６共刺激分子，在混合白细胞反应（ＭＬＲ）中能有效地刺激静息期Ｔ淋巴细胞增殖。结论小鼠骨髓ＣＤ３４＋细胞经体外培养扩增后仍然具有向ＤＣ分化的能力，通过骨髓细胞体外培养扩增可以显著提高ＤＣ产量。     

体外扩增的小鼠骨髓CD34＋和c—kit^＋细胞具有长期造血功能

目的 应用小鼠骨髓微血管内皮细胞能够在体外大量扩增骨髓造血细胞。本文旨在进一步研究（１）内皮细胞分泌的造血细胞因子；（２）优化培养条件，提高骨髓细胞的扩增指数；（３）扩增细胞的长期造血重建功能。方法 采用ＲＴ－ＰＣＲ和生物活性测定技术分析内皮细胞表达的造血细胞因子；小鼠骨髓细胞经与内皮细胞共２方法进行扩增，扩增的骨髓在剂量化疗后的异基因小鼠，然后检测外周血中供体细胞的比例。结果 （１）内皮细胞能     

人骨髓,脐血CD34^＋干细胞体外扩增的树突状细胞的表型及 …

目的：从人骨髓造血前体细胞体外培养扩增树突状细胞（ＤＣｓ）,测定其表型及Ｔ细胞刺激活性。方法：采用Ｍｉｎｉ－ＭＡＣＳ分离技术,从正常人骨髓、脐血分离ＣＤ３４＾＋造血干细胞,体外以重组ｈＧＭ－ＣＳＦ,ｈＴＮＦ－α,ｈＩＬ－３诱导培养２周,流式细胞术检测扩增细胞的表面表型及细胞内ＩＬ－１２的表达,体外同种混合淋巴细胞反应检测扩增ＤＣｓ的Ｔ细胞刺激活性。结果：从正常人骨髓、脐血分离得到高纯度（〉９０％）     

体外扩增的小鼠造血祖细胞重建大剂量化疗后的骨髓造血系统

在体内,正常的造血过程是通过骨髓造血微环境来维持的.研究表明,这种微环境是一个复杂的三维结构.由骨髓基质细胞如纤维母细胞、血管内皮细胞、巨噬细胞、脂肪细胞等组成.这些基质细胞能分泌造血细胞因子,并能维持造血细胞在体外长期生存.根据这一原理,我们建立了一种新颖的小鼠HPC培养系统.该系统以骨髓微血管内皮细胞(BMEC)作为生长支持介质,通过加入重组人粒细胞集落刺激因子(G-CSF)来促进HPC增殖.实验结果表明,将氟脲嘧啶(5-Fu,150mg/kg) 处理的C57小鼠骨髓细胞置于BMEC细胞层上,并加入25ng/ml G-CSF培养2周,细胞数量增加21.2倍.流式细胞分析提示,扩增后的细胞中60%表达祖细胞相关抗原CD117,40%表达髓系细胞分化抗原Gr-1.而培养前的骨髓细胞中上述二种细胞的比例分别为5%和     

造血干细胞的体外扩增及其临床应用

ＣＤ３４＾＋造血干细胞已显示有巨大的临床应用价值，本文概述了造血干细胞移植时的剂量要求，ＣＤ３４＾＋细胞的纯化手段和扩增技术，并简介ＣＤ３４＾＋细胞纯化扩增技术的临床应用情况。     

体外长期培养骨髓基质细胞对造血调控的实验研究

体外长期培养骨髓基质细胞对造血调控的实验研究王兰琴，曲垣瑞，崔小平骨髓体外长期培养（ＬＴＢＭＣ）是研究骨髓造血的理想模型。我们对７例正常骨髓细胞的ＬＴＢＭＣ生长特点及内源性ＣＳＦ活性进行观察，将结果报导如下：材料与方法１．骨髓细胞来源３例取自胸外科手...     

短程大剂量格拉诺赛特动员APBSC移植(附1例报告)

目的：报导短程大剂量格拉诺赛特（ｒｈＧ－ＣＳＦ）动员外周血造血干细胞,进行自体外周血造血干细胞移植（ＡＰＢＳＣＴ）治疗急性白血病。方法：ｒｈＧ－ＣＳＦ,５００μｇ／ｄ皮下注射４天,第５、６天单采外周血单个核细胞（ＭＮＣ）,行ＣＦＵ－ＧＭ培养,ＣＤ３４＾＋细胞由流式细胞仪检测。于ＭＡＣ方案处理,回输自体外周围血造血干细胞（ＡＰＢＳＣ）。结果：动员后ＣＦＵ－ＧＭ明显增高,ＣＤ３４＾＋细胞增多,骨髓造血     

体外液体培养联合IL—2净化的自体骨髓移植治疗恶性血液病

目的：提高自体骨髓移植疗效,减少移植后疾病的复发。方法：应用液体体外培养联合白细胞介素２（ＩＬ－２）激活的骨髓净化骨髓细胞,进行了１０例恶性血液病的自体骨髓移植（ＡＢＭＴ）。结果：净化后骨髓有核细胞和ＣＦＵ－ＧＭ的回收率分别为４９．８％和３６．４％。移植后所有患者均顺利地重建了骨髓造血功能,７例患者的中位持续完全缓解（ＣＣＲ）时间达２７．６个月,２例患者移植后１０个月死于非病意外,１例ＣＣＲ７个月     

自体外周血造血干细胞动员及采集时机的探讨（附12例临床分析）

自体外周血造血干细胞移植（ＡＰＢＳＣＴ）是治疗肿瘤的重要手段。能克服大剂量化疗所致的骨髓衰竭，重建病人的造血功能和免疫功能，并逐渐有取代骨髓移植的趋势。然而自体外周血干细胞（ＡＰＢＳＣ）在体内动员扩增及采集时机的正确选择，目前仍有争议。我们院于１９９...     

Mesenchymal cells generated from patients with myelodysplastic syndromes are devoid of chromosomal clonal markers and support short- and long-term hematopoiesis in vitro

Myelodysplastic syndromes (MDS) are clonal malignant stem cell disorders characterized by inefficient hematopoiesis. The role of the marrow microenvironment in the pathogenesis of the disease has been controversial and no study has been performed so far to characterize mesenchymal cells (MC) from MDS patients and to analyse their ability to support hematopoiesis. To this end, we have isolated and characterized MC at diagnostic marrow samples (n=12) and have purified their CD34+CD38- and CD34+CD38+ counterparts (n=7) before using MC as a short- and long-term hematopoietic support. We show that MC can be readily isolated from MDS marrow and exhibit a major expansion potential as well as an intact osteoblastic differentiation ability. They do not harbor the abnormal marker identified by FISH in the hematopoietic cells and they stimulate the growth of autologous clonogenic cells. Conversely, highly purified stem cells and their cytokine-expanded progeny harbor the clonal marker with variable frequencies, and both normal and abnormal long-term culture-initiating cell-derived progeny can be effectively supported by autologous MC. Thus, we demonstrate that MDS marrow is an abundant source of MC appearing both cytogenetically and functionally noninvolved by the malignant process and able to support hematopoiesis, suggesting their possible usefulness in future cell therapy approaches.     

Fibroblast growth factor and ex vivo expansion of hematopoietic progenitor cells

Fibroblast growth factor (FGF) belongs to a family of heparin-binding polypeptides and shows multiple functions including cell proliferation, differentiation, survival and motility. The expression of FGF receptors is widely distributed on different hematopoietic progenitor cells and stromal cells, and FGFs play an important role in hematopoietic stem cell homeostasis. FGFs have been shown to sustain the proliferation of hematopoietic progenitor cells, maintaining their primitive phenotype. Basic FGF (bFGF, FGF-2) stimulates the formation of an adherent stromal cell layer in human long-term bone marrow cultures, and promotes hematopoietic cell development. FGF-2 has also been shown to synergize with other hematopoietic growth factors to enhance in vitro colony formation by several classes of hematopoietic progenitor cells. Results of ex vivo expansion and clinical trials to date suggest that hematopoietic cells cultured under stroma-free cytokine combination conditions may be insufficient to restore hematopoiesis after a myeloablative conditioning regimen, although some recent trials demonstrated an improvement in engraftment and a reduction of the period of pancytopenia, especially neutrophils and platelets, after transplantation. A recent study by our group demonstrated that FGF-2 is effective in supporting the generation of megakaryocytic progenitor cells during ex vivo expansion. These observations could be useful in reducing the long period of severe thrombocytopenia that occurs frequently after umbilical/placental cord blood transplantation. The development of more effective amplifying systems for hematopoietic stem/progenitor cells can be expected since FGFs have multiple functions in hematopoiesis.     

Hematopoiesis capacity, immunomodulatory effect and ex vivo expansion potential of mesenchymal stem cells are not impaired by cryopreservation

The purpose of this study is to investigate whether hematopoiesis capacity, immunomodulatory effect and ex vivo expansion potential of mesenchymal stem cells (MSCs) are affected by cryopreservation. Chronic myeloid leukemia (CML) patients' bone marrow MSCs cryopreserved for 3 months, 6 months, and 1 year were thawed and analyzed. Cryopreserved CML-MSCs have more than 90% viability. Cell-doubling time of cryopreserved CML-MSCs is 42 to 54 hours. Cells have been expanded in culture for more than 30 passages. Under suitable conditions, cryopreserved CML-MSCs have the ability of multiple lineages differentiation, including bone, endothelial, fat and nerve. Furthermore, cryopreserved CML-MSCs express hematopoietic cytokines, and possess hematopoietic supportive ability. The growth of normal long-term culture-initiating cell (LTC-IC) on CML-MSCs (including noncryopreserved and cryopreserved CML-MSCs) was similar to that of normal derived MSCs. Cryopreserved CML-MSCs did not express costimulatory molecules CD40, CD80, and CD86. They can inhibit T lymphocyte proliferation induced by mitogens. The immunosuppressive effect of cryopreserved CML-MSCs on T-cell proliferation was dose dependent. These findings indicate that cryopreserved CML derived MSCs may be a useful tool for clinical application.     

Immortalization of bone marrow-derived human mesenchymal stem cells by removable simian virus 40T antigen gene: analysis of the ability to support expansion of cord blood hematopoietic progenitor cells

Human marrow-derived mesenchymal stem cells (MSC), which have the potential to differentiate into mesenchymal tissues, such as bone, cartilage, adipose and bone marrow stroma, were transduced with a retroviral vector carrying the simian virus 40 large T antigen, hygromycin-resistant gene and herpes simplex virus thymidine kinase gene, that can be excised by Cre/loxP site-specific recombination. This resulted in establishment of an MSC cell line, HMSC-1, which retained original surface characteristics and differentiation potential, and exhibited a higher proliferative capacity than parental cells. HMSC-1 expressed mRNAs of BMP-4, Jagged-1, and SCF that are known to promote hematopoiesis. Human CB CD34+ hematopoietic progenitor cells (HPC) cultured on a layer of HMSC-1 cells showed high expansion of CD34+CD38- immature HPC, capable of reconstituting human hematopoiesis in non-obese diabetic/severe combined immunodeficient disease (NOD/SCID) mice. This cell line may be of value for developing strategies for ex vivo expansion of human HPC.     

CD164--a novel sialomucin on CD34+ cells

Hematopoiesis in adult bone marrow is a tightly regulated process involving interactions between cytokine and adhesion receptors on hematopoietic progenitor cells and their cognate ligands in the immediate microenvironment. These interactions control hematopoietic stem cell self-renewal, quiescence, commitment and migration. Recently, sialomucins have assumed some importance in hematopoiesis, with six of these receptors, CD34, PSGL-1, CD43, PCLP, CD45RA and CD164, having been identified on primitive hematopoietic precursor cells and/or their associated stromal/endothelial elements. This article reviews the cloning, expression and function of the recently identified sialomucin, CD164, which is highly expressed by primitive hematopoietic progenitor cells. The CD164 receptor is implicated in mediating or regulating hematopoietic precursor cell adhesion to stroma, and may serve as a potent negative regulator of hematopoietic progenitor cell proliferation.     

Functional cloning of IGFBP-3 from human microvascular endothelial cells reveals its novel role in promoting proliferation of primitive CD34+CD38- hematopoietic cells in vitro

Ex vivo expansion of hematopoietic stem cells (HSCs) has been investigated as a means of enhancing engraftment of transplantation therapies, but current ex vivo expansion methods typically result in a loss of functional stem cell activity. Factors that can selectively expand human HSCs remain elusive. Recently we have isolated three functionally distinct human brain microvascular endothelial cells (HBMVECs) that differ greatly in their ability to support in vitro proliferation of human umbilical cord blood (UBC) CD34+CD38-cells. Using these distinct HBMVEC populations, we have devised a cell-based functional cloning assay to identify a molecule(s) capable of facilitating expansion of HSCs in vitro. A gene encoded for IGFBP-3 (insulin-like growth factor binding protein-3) has been identified. IGFBP-3 mRNA and protein are differentially expressed in distinct HBMVEC populations. In vitro cell proliferation assay and CD34+CD38- immunophenotype analysis showed that the addition of an exogenous IGFBP-3 to cultures of purified CD34+/-CD38-Lin- cells (CD2/CD3/CD14/CD16/CD19/CD24/CD56/CD66b/GlyA depleted) enhanced proliferation of primitive hematopoietic cells with CD34+CD38- phenotype, suggesting that IGFBP-3 is capable of expanding primitive human blood cells. These expanded primitive blood cells were illustrated to maintain ability to generate functional progenitors. IGFBP-3 belongs to a family of high-affinity IGFBPs, which binds to IGFs and modulates their actions. IGFBP-3 appears to have intrinsic bioactivity that is independent of IGF binding. We are currently exploring the underlying mechanism by which IGFBP-3 modulates proliferation of primitive hematopoietic cells, and the potential of IGFBP-3 to expand pluripotent human repopulating cells capable of hematopoietic reconstitution of irradiated NOD/SCID recipients.     

The role of Smad signaling in hematopoiesis

The TGF-beta family of ligands, including TGF-beta, bone morphogenetic protein (BMP) and activin, signal through Smad pathways to regulate the fate of hematopoietic progenitor and stem cells during development and postnatally. BMP regulates hematopoietic stem cell (HSC) specification during development, while TGF-beta1, 2 and 3 are not essential for the generation of HSCs. BMP4 can increase proliferation of human hematopoietic progenitors, while TGF-beta acts as a negative regulator of hematopoietic progenitor and stem cells in vitro. In contrast, TGF-beta signaling deficiency in vivo does not affect proliferation of HSCs and does not affect lineage choice either. Therefore, the outcome of Smad signaling is very context dependent in hematopoiesis and regulation of hematopoietic stem and progenitor cells is more complicated in the bone marrow microenvironment in vivo than is seen in liquid cultures ex vivo. Smad signaling regulates hematopoiesis by crosstalk with other regulatory signals and future research will define in more detail how the various pathways interact and how the knowledge obtained can be used to develop advanced cell therapies.     

Biological purging of breast cancer cells using an attenuated replication-competent herpes simplex virus in human hematopoietic stem cell transplantation

Autologous hematopoietic stem cell transplantation after myelosuppressive chemotherapy is used for the treatment of high-risk breast cancer and other solid tumors. However, contamination of the autologous graft with tumor cells may adversely affect outcomes. Human hematopoietic bone marrow cells are resistant to herpes simplex virus type 1 (HSV-1) replication, whereas human breast cancer cells are sensitive to HSV-1 cytotoxicity. Therefore, we examined the utility of G207, a safe replication-competent multimutated HSV-1 vector, as a biological purging agent for breast cancer in the setting of stem cell transplantation. G207 infection of human bone marrow cells had no effect on the proportion or clonogenic capacity of CD34+ cells but did enhance the proliferation of bone marrow cells in culture and the proportion of CD14+ and CD38+ cells. On the other hand, G207 at a multiplicity of infection of 0.1 was able to purge bone marrow of contaminating human breast cancer cells. Because G207 also stimulates the proliferation of human hematopoietic cells, it overcomes a limitation of other purging methods that result in delayed reconstitution of hematopoiesis. The efficient infection of human bone marrow cells in the absence of detected toxicity suggests that HSV vectors may also prove useful for gene therapy to hematopoietic progenitor cells.     

Multipotent neural precursors express neural and hematopoietic factors, and enhance ex vivo expansion of cord blood CD34+ cells, colony forming units and NOD/SCID-repopulating cells in contact and noncontact cultures.

In view of the possible crosstalks between hematopoiesis and neuropoiesis, we evaluated two microenvironments, murine neonatal neural cell line C17.2 and primary embryonic aorta-gonad-mesonephros (AGM) stromal cells, on the ex vivo expansion of CD34+ cells from human cord blood. In a contact culture system, C17.2 or AGM cells significantly enhanced the expansion of CD34+ cells to a panel of early and committed hematopoietic progenitor cells. In a noncontact transwell system, pre-established C17.2 cells significantly increased the expansion of total nucleated cells, CD34+ cells and multilineage colony forming cells (P<0.01). Expanded cells were infused into nonobese diabetic/severe-combined immunodeficient mice. The engraftment of human (hu)CD45+ cells in the bone marrow of these mice was consistently higher in all the 10 experiments conducted with the support of C17.2 cells when compared with those in respective control groups (11.9 vs 2.43%, P=0.03). Using RT-PCR and Southern blot analysis, we showed that AGM and C17.2 cells expressed a panel of hematopoietic, bone morphogenetic and neurotrophic factors. Our data provided the first evidence on the promoting effects of a neural progenitor cell line on hematopoiesis at a noncontact condition. The mechanism could be mediated by the expression of multilineage regulatory factors.     

Inappropriate notch activity and limited mesenchymal stem cell plasticity in the bone marrow of patients with myelodysplastic syndromes

Myelodysplastic syndromes (MDSs) are a heterogeneous group of hematological disorders characterized by ineffective hematopoiesis, enhanced bone marrow apoptosis and frequent progression to acute myeloid leukemia. Several recent studies suggested that, besides the abnormal development of stem cells, microenvironmental alterations are also present in the MDS bone marrow. In this study, we have examined the relative frequencies of stem and progenitor cell subsets of MDS and normal hematopoietic cells growing on stromal cell layers established from MDS patients and from normal donors. When hematopoietic cells from MDS patients were co-cultured with normal stromal cells, the frequency of either early or late cobblestone area-forming cells (CAFC) was significantly lower compared to the corresponding normal control values in 4 out of 8 patients. In the opposite situation, when normal hematopoietic cells were incubated on MDS stromal cells, the CAFC frequencies were decreased in 5 out of 6 patients, compared to normal stromal layer-containing control cultures. Moreover, a soluble Notch ligand (Jagged-1 protein) was an inhibitor of day-35-42 CAFC when normal hematopoietic cells were cultured with normal or MDS stromal cells, but was unable to inhibit MDS stem and early progenitor cell growth (day-35-42 CAFC) on pre-established stromal layers. These findings suggest that in early hematopoietic cells isolated from MDS patients the Notch signal transduction pathway is disrupted. Furthermore, there was a marked reduction in the plasticity of mesenchymal stem cells of MDS patients compared with those of normal marrow donors, in neurogenic and adipogenic differentiation ability and hematopoiesis supporting capacity in vitro. These results are consistent with the hypothesis that when alterations are present in the myelodysplastic stroma environment along with intrinsic changes in a hematopoietic stem/progenitor cell clone, both factors might equally contribute to the abnormal hematopoiesis in MDS.