Certain anti-CD34 monoclonal antibodies induce homotypic adhesion of leukemic cell lines in a CD18-dependent and a CD18-independent way

CD34 is a highly glycosylated type I membrane protein expressed by early hematopoietic progenitor cells as well as by endothelial cells and a subset of bone marrow stromal cells. CD34 is thought to play an important role during early hematopoiesis, although its function is unknown. We demonstrate that triggering of CD34 results in a rapid and vigorous homotypic adhesion in CD34⁺ cell lines, thereby providing evidence for a cell-cell adhesion function of CD34. The cellular adhesion event, induced by only two anti-CD34 mAb, (Immu-133 and QBend-10) was dependent on metabolic energy, an intact cytoskeleton and the presence of divalent cations. Analysis of antibody inhibition experiments indicated that the aggregation process partially involved the CD18 molecule.     

Characterization of TEK receptor tyrosine kinase and its ligands, Angiopoietins, in human hematopoietic progenitor cells

TEK, or TIE-2, is a receptor tyrosine kinase (RTK) that is known as a functioning molecule of vascular endothelial cells. TEK comprises a subfamily of RTK with TIE, and these two receptors play critical roles in vascular maturation, maintenance of integrity and remodeling. We generated mAb against the extracellular domain of human TEK protein to elucidate its expression pattern in human hematopoietic cells. Flow cytometric analysis of bone marrow cells revealed that TEK was expressed in 27% of CD34+ cells, 20% of c-KIT+ cells and 26% of CD34+CD38- cells, indicating that TEK is expressed in a subset of primitive hematopoietic stem cells (HSC). TEK was also expressed in 20% of CD19+ B lymphocytes but not in other lineage-committed cells. Progenitor assays in methylcellulose culture showed that CD34+TEK+ cells formed significantly less BFU-E and CFU-Mix than CD34+TEK- cells, but there was no difference in the number of CFU-GM between these two populations. Two recently identified TEK ligands, termed Angiopoietin-1 and -2, bound to TEK with similar affinities, and Angiopoietin-1 effectively induced TEK phosphorylation in hematopoietic cells. Angiopoietin-2 also induced a low level of TEK phosphorylation and weakened the phosphorylation induced by Angiopoietin-1, suggestive of an elaborate regulator of the TEK-TEK ligand signaling pathway. Although neither ligands affected the proliferation of TEK-transfected hematopoietic cells or the colony formation of CD34+TEK+ bone marrow cells, both promoted the adhesion of TEK-transfected hematopoietic cells to a collagen matrix or a layer of bone marrow stromal cells. These findings indicate that the TEK-TEK ligand signaling pathway is regulated in a refined manner and is involved in hematopoietic cell- microenvironment interaction.      

胎肝条件培养液体外诱导人骨髓间充质干细胞定向分化为造血细胞的研究

目的：探讨人骨髓间充质干细胞（hMSCs）体外造血分化潜能。方法： 选用孕12.5-14.5 d（12.5-14.5 dpc）的昆明小鼠，分别制备小鼠胎肝基质细胞条件培养液（FLSC-CM）及胚胎成纤维细胞饲养层（FD），将体外扩增的CD34-CD45-hMSCs分别接种于含FLSC-CM、FD和IL-6及SCF组合的培养体系中，培养7 d后，通过形态学、表型、粒-单/巨噬细胞系集落培养（CFU-GM）对分化细胞进行鉴定。结果： hMSCs与FLSC-CM共培养组产生的非贴壁细胞明显增多，形态类似于单核或小淋巴细胞，部分细胞可表达人造血细胞特异性表面分子（CD34和CD45），在含人粒-单集落刺激因子（GM-CSF）的甲基纤维素培养体系中能够形成CFU-GM，而FD和IL-6+SCF诱导组无上述作用。结论： FLSC-CM可诱导CD34-CD45-hMSCs分化为造血细胞，提示hMSCs具有体外造血分化潜能。     

Simian immunodeficiency virus infection of hematopoietic stem cells and bone marrow stromal cells

Using a well-characterized fetal rhesus monkey model, simian immunodeficiency virus (SIV) infection of hematopoietic stem cells (HSCs) and stromal cells was investigated to characterize bone marrow abnormalities in SIV-infected animals in vivo. Fetuses (n = 4) were inoculated in utero with pathogenic SIVmac251 using established techniques at 65 days; gestation (early second trimester), then harvested for tissues 65 days after inoculation (late third trimester), and compared with findings from controls of a comparable age (n = 11). Sorted bone marrow CD34CD38, CD34CD38, and marrow stromal cells were analyzed for the SIV genome. Results indicate that CD34CD38 HSCs were not infected, whereas the SIV genome was detected in the CD34CD38 hematopoietic cell population by PCR analysis. The SIV proviral sequence was not detected in stromal cells from SIV-infected fetuses, although SIVmac251 was found to readily infect these cells when assessed in vitro. Flow cytometric analysis revealed that stromal cells express low levels (1 in 600 total cells) of CD4 and CCR5 viral receptors, whereas CXCR4 expression was not observed. These data suggest the following: (1) SIV infection of HSCs requires some degree of differentiation for viral entry similar to findings for humans infected with HIV type 1, and (2) SIV can infect bone marrow stromal cells that express CD4 and CCR5 in vitro, but infected stromal cells are not readily found in vivo.     

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。     

体外培养慢性粒细胞白血病患者骨髓间充质干细胞的生物学特性

背景：课题组从胎儿骨髓间充质干细胞的培养体系中鉴定出一类贴壁细胞,已证实此类细胞在单细胞水平可以向造血及内皮细胞分化。 目的：检测骨髓间充质干细胞的生物学特性,为慢性粒细胞白血病的治疗提供相关的依据。 方法：以慢性粒细胞白血病患者骨髓为研究对象,体外培养并扩增原始间充质干细胞,检测其BCR/ABL融合基因的表达、免疫学特性和生长曲线,使用RT-PCR和FISH的方法检测其BCR/ABL融合基因的表达情况。 结果与结论：慢性粒细胞白血病患者骨髓来源的间充质干细胞呈成纤维样生长,大部分细胞处于G0/G1期,并且高表达Flk1,CD13,CD29,CD44,用RT-PCR和FISH的方法能够检测出BCR/ABL融合基因的表达。提示慢性粒细胞白血病的白血病基因转化可能发生在比造血干细胞更高的骨髓间充质干细胞水平上,对慢性粒细胞白血病的疾病起源及干细胞移植治疗有重要的意义。     

体外定向诱导小鼠胚胎干细胞发育为造血干/祖细胞方法的初步研究

目的:探讨体外定向诱导胚胎干细胞(ESC)发育为造血干细胞(HSC)的方法。方法:将小鼠E14胚胎干细胞在含干细胞生长因子(SCF)和血管内皮生长因子(VEGF)的甲基纤维素培养基中首先诱导发育为胚胎体(EB),再将EB置于均含SCF、VEGF、IL-3、IL-6及促红细胞生成素(EPO)的3种不同培养体系中定向分化为HSC,并观察HSC表面标志性抗原、造血集落形成及瑞氏-姬姆萨染色的结果。结果:经两阶段诱导ESC分化为HSC,发现在甲基纤维素半固体培养体系中HSC发育缓慢,分化14d后CD34+/Sca-1+细胞数最高为(31.5±4.7)%;而在骨髓基质细胞饲养层上HSC发育较快,细胞数量较多,分化第10dCD34+/Sca-1+细胞数即达到峰值,为(47.8±6.3)%;骨髓基质细胞饲养层+胎肝基质细胞上清培养体系中HSC发育同样迅速,所产生的CD34+/Sca-1+细胞数量在3个体系中最高,为(53.6±7.2)%。经瑞氏-姬姆萨染色证实上述细胞为早期造血细胞,均有形成各系造血细胞集落的能力。结论:使用骨髓基质细胞饲养层+胎肝基质细胞上清培养体系及SCF、VEGF、IL-3、IL-6及EPO等细胞因子,通过两阶段诱导分化,可从小鼠ESC获得较高比例的HSC。     

Neural cell adhesion molecule contributes to hemopoiesis-supporting capacity of stromal cell lines

To clarify mechanisms underlying cell-to-cell interactions between hemopoietic stem cells (HSCs) and stromal cells, we established a stromal cell line (FMS/PA6-P) from day-16 fetal bone marrow (BM) adherent cells using an anti-PA6 monoclonal antibody (mAb) specific for BM stromal cells. Importantly, this FMS/PA6-P cell line, showing homogenous fibroblastic morphology, is absent from hematolymphoid and endothelial lineage markers and maintains a high level of expression of PA6 molecule, recognized by the anti-PA6 mAb, for approximately 20 passages. Further, the cell line expressing a high level of PA6 molecule has a better hemopoiesis-supporting capacity in vitro than other stromal cell lines such as PA6 and MS-5. In fact, the PA6 molecule is closely related to the hemopoiesis-supporting capacity of the stromal cells because the proliferation of HSCs was suppressed to a great extent by the anti-PA6 mAb. Affinity chromatography and mass peptide fingerprinting revealed that the protein reacting with the anti-PA6 mAb is neural cell adhesion molecule (NCAM). The frequencies of long-term cobblestone area-forming cells and long-term culture-initiating cells were significantly suppressed by repression of NCAM in the FMS/PA6-P cells using NCAM small interfering RNA. Our findings clearly indicate that NCAM functions on the maintenance of HSCs.     

骨髓基质细胞的造血支持作用等生物学特性的研究

目的研究人骨髓基质细胞体外长期培养的生物学特性和造血支持功能。方法①采用静置贴壁细胞培养法,体外长期培养胎儿、儿童和成人的骨髓基质细胞。②采用免疫细胞化学染色法和流式细胞仪检测法,分析细胞的表型。③将不同发育阶段的骨髓基质细胞体外培养,并扩增脐血造血干细胞。结果①建立了成纤维肌样细胞系,可传至10代,维持6个月,同时还培养出内皮细胞和巨噬细胞。②儿童骨髓基质肌样细胞的染色特征为波形纤维蛋白(viementin)呈阳性,第VIII因子呈阴性;儿童骨髓基质细胞的表型为CD33     

胸腺基质细胞支持下骨髓CD34~+细胞向T细胞体外定向分化

目的：研究人骨髓CD34^＋细胞体外向T细胞定向分化的方法，为研究造血细胞淋系造血活性及T细胞发育和分化提供技术平台。方法：免疫磁珠法分离骨髓CD34^＋细胞，在骨髓基质细胞条件培养液构建的微环境下，在胸腺基质细胞的支持下，使其体外向T细胞定向分化，收集培养的非贴壁细胞，免疫荧光染色后经流式细胞术检测培养不同时间CD1^-CD3^＋细胞、CD3^＋CD4^＋CD8^-细胞及CD3^＋CD4^-CD8^＋细胞比例。结果：培养1周时，培养细胞中以不成熟的CD1^＋CD3^-细胞、CD1^＋CD3^＋细胞为主，可检测到少量CD1^-CD3^＋细胞，随培养时间延长，不成熟细胞比例逐渐减少，而成熟的CD1^-CD3^＋细胞比例逐渐增加；在CD3^＋细胞中，培养初期以不成熟的双阳性细胞CD4^＋CD8^＋为主，而成熟的单阳性CD^＋CD8^-细胞及CD4^-CD8^＋细胞占极小比例，随培养时间延长，双阳性细胞比例逐渐减少，而成熟的单阳性细胞比例逐渐增高；而无胸腺基质细胞支持的CD34＋细胞仅在培养初期检测到成熟细胞存在，而培养后4周基本检测不到成熟T细胞的存在。结论：在骨髓基质细胞及胸腺基质细胞的支持下，骨髓CD34^＋细胞可体外发育为成熟的CD1^-CD3^＋细胞及单阳性T细胞，其中胸腺基质细胞的支持对于造血细胞向T细胞的体外定向分化极其重要。     

Endothelial cells in the early murine yolk sac give rise to CD41-expressing hematopoietic cells

Hematopoietic and endothelial cells may be derived from a common precursor cell (hemangioblast) during embryogenesis; however, some evidence suggests that hematopoietic cells may emerge from endothelial cells. The onset of definitive hematopoiesis at E8.25 in the murine embryo is marked by high-level CD41 expression. We questioned whether these hematopoietic cells were derived directly from mesoderm cells or emerged from endothelium. At 8.25 days post coitus (dpc), CD41 was coexpressed with CD31, CD34, and Flk1 in some intraluminal round cells that appeared to arise from flattened endothelial cells lining yolk sac capillary vessels. Cell-sorting studies revealed that all subpopulations of cells expressing CD41 possessed hematopoietic activity. Surprisingly, Tie2(+)Flk1(+) cells, a phenotype enriched in adult endothelial progenitors, also displayed some hematopoietic progenitor activity in vitro, but this activity was restricted to the CD41(+) fraction; only endothelial cells were derived from freshly isolated Tie2 (+)Flk1(bright) CD41() cells. Tie2(+)Flk1(dim)CD41() 8.25-dpc yolk sac cells devoid of hematopoietic progenitor activity gave rise to endothelial-like capillary networks in vitro and differentiated upon co-culture with OP9 stromal cells into definitive hematopoietic progenitors. These results demonstrate that CD41-expressing definitive hematopoietic cells appear to arise from endothelial cells lining nascent capillaries in vivo.     

成人脂肪源间充质干细胞在小鼠体内具有血液血管干细胞特性

目的:观察成人脂肪源Flk1+ CD31- CD34-细胞在小鼠体内是否具有血液血管干细胞的特性。方法:将男性成人脂肪源Flk1+ CD31- CD34-细胞经尾静脉输入6-8周龄、接受亚致死剂量射线照射的非肥胖型糖尿病及严重联合免疫缺陷(NOD/SCID)小鼠体内(每只输入剂量为105,悬于0.4m LRPMI-1640培养基中),对照组接受同等剂量的RPMI-1640培养基中。2个月后处死小鼠。用聚合酶链式反应、流式细胞术、三色荧光法及荧光原位杂交法检测脂肪源Flk1+ CD31- CD34-细胞在实验小鼠骨髓及消化系统内的分化情况。结果:脂肪源Flk1+ CD31- CD34-细胞在单细胞水平上在NOD/SCID小鼠体内可分化为内皮细胞和造血细胞。结论:脂肪源Flk1+ CD31- CD34-细胞在体内具有血液血管干细胞的特性,这类细胞有可能作为种子细胞用以治疗血液和血管性疾病。     

从胚胎干细胞诱导发育为浆细胞的体外实验研究

目的：建立胚胎干细胞体外定向诱导分化为浆细胞的体系,为研究造血发生的基因调控及Ｂ淋巴细胞发育过程奠定基础。方法：分阶段培养,第一、二阶段使用半固体培养,分组加入细胞因子、２－巯基乙醇和胚胎细胞条件培养液,用流式细胞仪、免疫组化及Ｗｒｉｇｈｔ’ｓ染色鉴定细胞,第三阶段移入骨髓基质细胞滋养层。结果：第一阶段：加入ＳＣＦ、ＴＰＯ、ＩＬ－６、ＩＬ－３、Ｅｐｏ和Ｇ－ＣＳＦ,４ｄ后细胞大片坏死,仅加入２－巯基     

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.     

Immunophenotypic features of uterine stromal cells

CD34 is a myeloid progenitor cell antigen present in endothelial cells and some other mesenchymal cells, including perivascular and periadnexal dermal fibroblasts. It was evaluated immunohistochemically in uterine stromal tissue and in 4 aggressive angiomyxomas and 6 endometrial stromal sarcomas with potentially related and similar stromal tissues. The stromal cells in normal endocervix and endocervical polyps were strongly CD34 positive irrespective of the cycle phase, and negative for muscle actins. Ectocervical stroma was variably but generally less CD34 reactive. In the endometrium, the CD34 reactivity was limited to the stromal cells of the basal endometrium and was found only in 4 of 20 from proliferative endometria and 1 of 8 from secretory endometria. The uterine cervical and myometrial smooth muscle tissues showed CD34 positive cells only between the muscle bundles and around the vessels. In pelvic aggressive angiomyxomas and endometrial stromal sarcomas the tumour cells were CD34 negative and only the vascular endothelial cells were positive. Endothelial cell-specific antigen, CD31, was identified only in endothelial cells and was not present in the endocervical stroma. These results illustrate the particular immunohistochemical profile of endocervical stromal tissue, namely the strong CD34 expression. The CD34 reactivity of the endocervical tissues should be noted and not confused with neoplasms known to be strongly CD34 positive, such as angiosarcomas, Kaposi's sarcomas and some other spindle cell sarcomas.     

颅骨来源骨髓基质干细胞的分离培养方法

背景：从小鼠颅骨提取的一类骨髓基质干细胞称为PA6细胞,研究者发现当PA6细胞与其他干细胞共培养时可向神经细胞分化,此特性可被应用于神经损伤部位的修复。所以,PA6细胞越来越受到研究者的关注,但目前国内对类似骨髓基质干细胞PA6的提取方法鲜见报道。 目的：分离培养SD大鼠颅骨来源的骨髓基质干细胞,体外观察细胞形态并免疫荧光鉴定。 方法：无菌条件下,取新生的SD大鼠颅骨将其剪碎,用含体积分数为10%胎牛血清的DMEM培养液冲洗颅骨碎片,反复吹打制成单细胞悬液,将其置于培养瓶中培养,多次换液培养纯化细胞。取第2代生长均一的细胞,倒置显微镜下观察细胞形态,并用免疫荧光染色法进行细胞表面标记鉴定。 结果与结论：原代细胞培养24 h后,骨髓基质干细胞开始贴壁;3 d后细胞数量增多,形态呈不规则形、多角形、三角形和扁平形;细胞传代后,形态基本均一,细胞排列呈放射状和束状,贴壁能力较强,部分细胞呈聚集生长,增殖速度比原代有明显的加快。免疫荧光染色检测CD105、CD73、CD44、CD90表达呈阳性,CD45、CD34、CD14和HLA-DR表达呈阴性,证实了提取的细胞为骨髓基质干细胞。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

灯盏花素注射液对骨髓间充质干细胞的诱导分化

目的探索灯盏花素注射液体外诱导大鼠骨髓间充质细胞(BMSCs)分化为神经元和胶质细胞的可行性。方法贴壁法分离纯化SD大鼠骨髓间充质细胞。第4代细胞行表型鉴定后,用灯盏花素注射液诱导,每6h倒置相差显微镜观察形态变化,免疫细胞化学染色鉴定诱导后细胞的神经元特异性稀醇化酶(NSE)、神经胶质纤维酸性蛋白(GFAP)的表达情况,四甲基偶氮唑盐(MTT)检测不同浓度灯盏花素注射液诱导后细胞的活力,流式细胞术及RT-PCR检测诱导前后细胞中NSE、GFAP mRNA的表达变化。结果BMSCs表型鉴定为CD44+、CD54+、CD34-,诱导18h后BMSCs胞体开始收缩,有突起伸出,24h后突起增多形成网络结构。免疫细胞化学染色,NSE阳性表达率为(48.7±3.4)%,GFAP阳性表达为(56.8±4.2)%,流式细胞仪检测诱导24h后的细胞NSE及GFAP蛋白表达量均较未诱导组升高,RT-PCR检测诱导后细胞表达NSE、GFAP mRNA,未诱导的细胞则不表达。结论灯盏花素注射液可诱导大鼠骨髓间充质细胞在体外分化为神经元和神经胶质细胞。