含人AGM区、胎肝及骨髓基质细胞培养体系程序化诱导小鼠胚胎干细胞向造血干细胞的分化

背景：前期已分别制备人主动脉-性腺-中肾区基质细胞系及胎肝基质细胞系,发现前者可促进小鼠胚胎干细胞定向分化为造血干细胞。 目的：模拟胚胎发育过程中永久造血发育的时空顺序,探讨人主动脉-性腺-中肾(AGM)区、胎肝(FL)及骨髓(BM)基质细胞对小鼠胚胎干细胞体外诱导分化为造血干细胞的支持作用,以寻求更佳的诱导条件。 方法：将小鼠E14胚胎干细胞诱导为拟胚体(EB),并利用Transwell非接触共培养体系依次在人主动脉-性腺-中肾区、胎肝及骨髓基质细胞饲养层上进一步诱导分化,按不同诱导阶段分为拟胚体对照、EB/AGM、EB/AGM+FL和EB/AGM+FL+BM共4组。共培养6 d后分别收获各组拟胚体来源细胞,以流式细胞仪检测Sca-1+c-Kit+细胞含量,进行各系造血细胞集落形成单位分析并观察细胞形态。 结果与结论：①EB/AGM+FL组和EB/AGM+FL+BM组收获细胞涂片均发现原始造血细胞。②拟胚体来源细胞经AGM区基质细胞诱导后Sca-1+c-Kit+ 细胞明显升高(P < 0.05)。③拟胚体对照组造血细胞集落形成单位低于其他各组(P < 0.05), 而EB/AGM+FL、EB/AGM+FL+BM组造血细胞集落形成单位计数亦较EB/AGM组明显增高。提示AGM+FL和AGM+FL+骨髓基质细胞微环境对原始造血干细胞的扩增效应均明显高于单纯主动脉-性腺-中肾饲养层。     

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。     

人主动脉-性腺-中肾区基质细胞诱导胚胎干细胞分化为造血干细胞及体内造血重建

背景：研究证实多种造血生长因子、基质细胞饲养层及其条件培养液可促进胚胎干细胞向造血干细胞分化。 目的：以人主动脉-性腺-中肾(aorta-gonad-mesonephros,AGM)区基质细胞为饲养层体外诱导小鼠胚胎干细胞分化为造血干细胞,并比较不同移植途径对造血干细胞体内造血重建能力的影响。 方法：将小鼠E14 胚胎干细胞诱导为拟胚体,采用Transwell非接触共培养体系在人AGM区基质细胞饲养层上诱导6 d,接种NOD-SCID小鼠检测体内致瘤性。再将诱导后的拟胚体细胞移植经致死量60Co γ射线辐照的BALB/C雌鼠,受鼠随机分为静脉移植组、骨髓腔移植组、照射对照组及正常对照组。 结果与结论：拟胚体细胞经人AGM区基质细胞诱导后Sca-1⁺c-Kit⁺细胞占(13.12±1.30)%。NOD-SCID小鼠皮下接种经人AGM区基质细胞诱导的拟胚体细胞可出现畸胎瘤,经骨髓腔接种未见肿瘤形成。静脉移植组动物全部死亡,骨髓腔移植组生存率为55.6%,移植后21 d外周血象基本恢复,存活受鼠检测到供体来源Sry基因。提示小鼠胚胎干细胞经人AGM区基质细胞诱导分化的造血干细胞通过骨髓腔移植安全并具有一定的造血重建能力。     

含人AGM区基质细胞培养体系定向诱导胚胎干细胞为造血干细胞的实验研究

目的： 体外模拟胚胎早期AGM区造血微环境，诱导胚胎干细胞（ESCs）分化为造血干细胞（HSCs）。方法：将小鼠E14 ESCs在含BMP-4及VEGF的半固体培养基中诱导为拟胚体（EB），分别于3、6、9、12、15 d时收获EB，流式细胞术检测Flk-1+细胞含量。取Flk-1+ 细胞处于高峰期的EB细胞，在人AGM区基质细胞饲养层上进一步诱导分化，并设无饲养层对照，分别于3、6、9、12 d时收获细胞计数、流式细胞术检测Sca-1+c-kit+ 细胞含量，并分析造血细胞集落形成能力。结果：诱导E14细胞形成EB过程中添加BMP4+VEGF的因子组Flk-1+细胞在第9 d达峰值（27.53%± 2.84％），与未添加因子组（8.77%± 1.12％）比较差异显著(P<0.05)。将培养9 d的EB细胞在hAGMS3、hAGMS4饲养层上进一步诱导分化，第6 d时Sca-1+c-kit+细胞达峰值，分别为7.31%±1.21％、7.62%±1.52％，其绝对数分别扩增(2.57±0.48)倍、(2.35±0.36)倍，与无饲养层组比较显著差异（P<0.05）。该分化阶段的Sca-1+c-kit+细胞具有形成各系造血细胞集落的能力。结论：人胚早期AGM区基质细胞能促进小鼠ESCs定向分化为HSCs，为研究ESCs分化为HSCs的分子机制提供了实验模型。     

成人骨髓间充质干细胞对造血干细胞体外增殖的影响

目的研究骨髓间充质干细胞（MSC）对脐带血（CB）CD34^＋细胞体外增殖和造血重建能力的影响。方法取人骨髓单个核细胞贴壁培养．梭形细胞完全融合后传代，用流式细胞仪检测免疫表型；将CBCD34^＋细胞接种到MSC或其他培养液中．比较不同培养条件对造血干细胞扩增能力、集落形成能力及黏附分子表达的影响。结果在加入IL-3的培养体系中．在MSC和细胞因子作用下，CD34^＋细胞扩增7d和14d后，有核细胞（NC）、CD34^＋细胞和CDl33^＋细胞数，实验组均显著多于对照组。CD34＋细胞在未加入IL-3的培养体系中培养8d后，实验组NC、CD34^＋细胞、CD34^＋CD38-细胞和造血祖细胞集落扩增倍数均显著高于对照组。扩增后CD34^＋细胞的ALCAM、VLA-α4、VLA-α5、VLA-β1、HCAM、PECAM和LFA-1表达较扩增前无显著变化。结论MSC可为造血干细胞（HSC）体外扩增提供适宜的微环境，有助于CD34^＋细胞体外增殖并抑制HSC分化，保持其造血重建潜能和归巢能力。     

人AGM区及胎肝基质细胞促进小鼠胚胎干细胞向造血干细胞分化和体内造血重建

目的: 探讨小鼠胚胎干细胞(ESCs)经人主动脉-性腺-中肾(AGM)区及胎肝(FL)基质细胞程序诱导后,向造血干细胞(HSCs)分化的效率及其造血功能。方法: 将E14 ESCs诱导为拟胚体(EB),并在人AGM区及FL基质细胞饲养层上进一步诱导分化,培养6 d后收集细胞检测Sca-1⁺c-Kit⁺细胞含量、分析造血细胞集落形成能力及致瘤性。再将不同诱导阶段的EB来源细胞移植经致死量 γ射线辐照的BALB/c雌鼠,观察生存率、植入状况和造血重建。结果: (1)EB来源细胞经人AGM区及FL基质细胞程序诱导后Sca-1⁺c-Kit⁺细胞含量为(21.96±2.54)%,造血集落总数为(520±52)/10⁵cells,明显优于诱导前及人AGM区基质细胞初步诱导者(P＜0.05)。(2)NOD-SCID小鼠接种经人AGM区及FL基质细胞诱导的ESCs未见畸胎瘤。(3)BALB/c雌鼠移植经人AGM区及FL基质细胞诱导的EB来源细胞后生存率77.8%,14 d外周血细胞计数明显改善,存活受鼠均检测到供体来源sry基因,而移植人AGM区基质细胞诱导的EB细胞者15 d内全部死亡。结论: 人AGM区及FL基质细胞能促进小鼠ESCs定向分化为HSCs,有效重建体内造血功能。     

人胎盘CD133~+细胞具有高增殖潜能集落形成细胞特性

目的 通过对人胎盘CD133+细胞群中高增殖潜能集落形成细胞(HPP-CFC)检测与生物学特性的分析,证明人胎盘存在早期造血干/祖细胞(HSPC). 方法 采用机械法制备人胎盘组织(PT)单细胞悬液,用Histopaque-1007分离出单个核细胞(MNC),经磁式分选(MACS)富集CD133+细胞,培养28 d后观察HPP-CFC集落形成能力,用流式细胞仪(FCM)对分选的细胞组份和HPP-CFC进行表型分析,实验全程用脐带血(UCB)作平行比较分析. 结果 培养28 d后,PT-CD133+与UCB-CD133+细胞组份分别扩增了266和362倍,前者低于后者(P<0.01);PT-CD133+与UCB-CD133+细胞中HPP-CFC分别为(32.4±11.2)/5×103、(17.7±5.7)/5×103,前者形成的HPP-CFC数量明显高于后者(P<0.01);PT.CD133+、UCB-CD133+细胞培养至28 d时,除UCB-CD133+组的CD133+CD34-亚群比例无明显改变外,CD133+CD34+、CD133-CD34+和CD133+CD34-(PT-CD133+组)亚型均比培养前减少. 结论 人胎盘组织CD133+细胞中存在HPP-CFC,说明胎盘CD133+细胞群中存在早期HSPC.     

CTLA4Ig基因修饰的BMSCs支持CD34+细胞扩增的实验研究

目的：通过腺病毒介导CTLA4Ig在骨髓基质细胞（BMSCs)中的表达，探讨CTLA4Ig基因修饰的BMSCs支持CD34^ 细胞扩增的功能变化，为该基因修饰的BMSCs联合造血干细胞移植（HSCT），达到预防移植物抗宿主病（GVHD）和纠正预处理损伤的造血微环境（HIM）积累实验依据。方法：以CTLA4Ig-重组腺病毒按感染复数（Multiplicity of infection,MOI)50转染BMSCs，以RT－PCR检测目的基因转录；免疫磁珠（MACS）阳性选择分选骨髓CD34^ 细胞，流式细胞仪检测其纯度；通过骨髓基质细胞支持CD34^ 扩增的细胞总数及集落形成细胞数（CFC）的变化，比较转染和未转染BMSCs在支持CD34^ 细胞扩增方面的差异。结果：RT－PCR在转染组及转染后传两代BMSCs中检测到CTLA4Ig基因的转录；通过观察扩增后细胞总数及CFC数的变化，发现CTLA4Ig基因转染组与未转染组BMSCs支持CD34^ 细胞扩增的能力也无显著差异（P＞0．05）。结论：CTLA4Ig－重组腺病毒有效介导目的基因对BMSCs的转染，在MOI为50时对其支持造血的功能无显著影响。     

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。     

乙型肝炎患者骨髓造血干细胞来源的树突状细胞表型分析

目的 检测乙型肝炎患者骨髓造血干细胞（HSC）诱导的树突状细胞（DC）的表面分子的表达,评价其相关因素及临床意义.方法 收集慢性乙型肝炎患者的骨髓液9例,健康者7例后用磁珠分离仪分离纯化骨髓液CD34＋细胞,在含有干细胞生长因子（SCF）、酪氨酸激酶受体家族Ⅲ的配体（FLT3）、促血小板生成素（TPO）、IL-3和10%FBS的IMDM培养基中孵育并进行扩增,在干细胞扩增基础上,在GM-CSF和IL-4作用下诱生DC,通过流式细胞仪分析其表面分子的表达并对DC进行形态学观察.结果 乙型肝炎患者HSC经诱导为DC后,其免疫表型CD80、CD86、CD1a的表达均低于正常对照组,差异有统计学意义：CD1a（t=3.94,P＜0.05）,CD80（t=7.08,P＜0.01）,CD86（t=3.65,P＜0.05）,而HLA-DR表达与正常组比较差异无统计学意义（t=0.34,P＞0.05）.结论 慢性乙型肝炎患者HSC来源的部分DC的表面分子表达低下可能与HBV感染HSC后出现病态的免疫细胞分化有关.     

Co-culture of umbilical cord blood CD34+ cells with human mesenchymal stem cells

Insufficient numbers of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) sometimes limit allogenic transplantation of umbilical cord blood (UCB). Ex vivo expansion may overcome this limitation. Mesenchymal stem cells (MSCs), as non-hematopoietic, well-characterized skeletal and connective-tissue progenitor cells within the bone marrow stroma, have been investigated as support cells for the culture of HSCs/HPCs. MSCs are attractive for the rich environmental signals that they provide and for immunological compatibility in transplantation. Thus far, HSC/MSC co-cultures have mainly been performed in 2-dimensional (2D) configuration. We postulate that a 3-dimensional (3D) culture environment that resembles the natural in vivo hematopoietic compartment might be more conducive for regulating HSC expansion. In this study, we compared the co-culture of HSCs and MSCs in 2D and 3D configurations. The results demonstrated the benefit of MSC inclusion in HSC expansion ex vivo. Direct contact between MSCs and HSCs in 3D cultures led to statistically significantly higher expansion of cord blood CD34+ cells than in 2D cultures (891- versus 545-fold increase in total cells, 96- versus 48-fold increase of CD34+ cells, and 230- versus 150-fold increase in colony-forming cell assay [CFC]). Engraftment assays in non-obese diabetic/severe combined immunodeficiency mice also indicated a high success rate of hematopoiesis reconstruction with these expanded cells.     

Extended in vitro expansion of adult, mobilized CD34+ cells without significant cell senescence using a stromal cell coculture system with single cytokine support

The macrophage colony-stimulating factor-deficient bone marrow stromal cell line OP9, derived from osteopetrotic mice, is known to support hematopoietic stem cell (HSC) expansion as well as hematopoietic differentiation of embryonic stem cells. Coculture of HSC in the OP9 system requires cytokine support to achieve significant cell expansion. Recently, we reported extensive expansion without cell senescence of cord blood (CB)-derived HSC cocultured with OP9 stromal cells for more than 18 weeks with a single cytokine support using human thrombopoietin (TPO). In this study, we evaluated the efficiency of the OP9/TPO coculture system to sustain long-term hematopoiesis of adult, granulocyte colony-stimulating factor mobilized human peripheral blood (PB) CD34(+) cells. Maximum cell expansion was attained during the first 4 weeks of coculture. At the same time, the maximum progenitor cell expansion was demonstrated by the production of colony-forming cells and cobblestone area-forming cells. In contrast to the expansion of CB CD34(+) cells, PB CD34(+) cells showed termination of cultures after 8 weeks, independent of the cell expansion rates attained. The evaluation of cell senescence by assessing the telomere length in most cultures showed no relevant telomere shortening, despite rapid decrease in telomerase activity. Interestingly, increases in telomere length were demonstrated. In conclusion, OP9/TPO system provides extensive stem cell expansion without concomitant telomere erosion for both CB and adult CD34(+) cells. Termination of adult CD34(+) cell cocultures seems to be independent of telomere length.     

Immunophenotype and functional characteristics of human primitive CD34-negative hematopoietic stem cells: the significance of the intra-bone marrow injection

The biology of hematopoietic stem cell (HSC) is a current topic of interest which has important implications for clinical HSC transplantation as well as for the basic research of HSC. The most primitive HSCs in mammals, including mice and humans, have long been believed to be CD34 antigen (Ag)-positive (CD34(+)) cells. In fact, bone marrow (BM), peripheral blood (PB), and cord blood (CB) stem cell transplantation studies indicate that a CD34(+) subpopulation in the BM, PB, or CB can provide durable long-term donor-derived lymphohematopoietic reconstitution. Therefore, CD34 Ag was used to identify/purify immature HSCs. However, Osawa et al. reported that murine long-term lymphohematopoietic reconstituting HSCs are lineage marker-negative (Lin(-)) c-kit(+)Sca-1(+)CD34-low/negative (CD34(low/-)), which are called CD34(low/-) KSL cells. Recently, human CB-derived CD34(-) HSCs, a counterpart of murine CD34(low/-) KSL cells, were successfully identified using an intra-bone marrow injection (IBMI) method. This review will update the concept of the immunophenotype and the functional characteristics of human primitive CD34(-) HSCs. In addition, the significance of the application of the IBMI technique in clinical HSC transplantation is also discussed. Recent rapid advances in understanding the biological nature of HSCs may make it possible to fully characterize the most primitive class of human HSCs in the near future.     

Characterization of serum-free ex vivo-expanded hematopoietic stem cells derived from human umbilical cord blood CD133(+) cells

The development of ex vivo expansion of hematopoietic stem cells (HSCs) is a promising approach to restore the required bone marrow function of patients with hematological disorders. Previously, we have reported the development of an optimized serum-free and cytokines-limited defined medium using statistic methodology for umbilical cord blood-derived HSC expansion. The aim of this study was to analyze further the characteristics and functions of cells in vitro and in vivo when cultured in this defined medium. After a 7-day batch culture, the average absolute fold expansions for CD133(+) cells, CD34(+)CD133(+) cells, CD34(+)CD38() cells, CD133(+)CD38(-) cells, CD34(+)CXCR4(+) cells, CD133(+)CXCR4(+) cells, and long-term culture-initiating cells were 21-, 20-, 723-, 618-, 160-, 384-, and 8-fold, respectively. The high enrichment of CD38(-) cells and CXCR4(+) cells of the CD34(+) subpopulation provided a very early uncommitted HSC proliferation and homing ability. Furthermore, the expanded cells showed a high level of telomerase activity to maintain their telomere length and repopulated the lethally irradiated NOD/SCID mice in vivo. These results indicated that the cytokines limited expanded cells from CD133(+) cells could substantially support simultaneous expansion of various stem/progenitor cells and engraft with the expanded cells from a low number of HSCs initially.     

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.     

Mesenchymal stem cells secreting angiopoietin-like-5 support efficient expansion of human hematopoietic stem cells without compromising their repopulating potential

Clinical and preclinical applications of human hematopoietic stem cells (HSCs) are often limited by scarcity of cells. Expanding human HSCs to increase their numbers while maintaining their stem cell properties has therefore become an important area of research. Here, we report a robust HSC coculture system wherein cord blood CD34(+) CD133(+) cells were cocultured with mesenchymal stem cells engineered to express angiopoietin-like-5 in a defined medium. After 11 days of culture, SCID repopulating cells were expanded ~60-fold by limiting dilution assay in NOD-scid Il2rg(-/-) (NSG) mice. The cultured CD34(+) CD133(+) cells had similar engraftment potential to uncultured CD34(+) CD133(+) cells in competitive repopulation assays and were capable of efficient secondary reconstitution. Further, the expanded cells supported a robust multilineage reconstitution of human blood cells in NSG recipient mice, including a more efficient T-cell reconstitution. These results demonstrate that the expanded CD34(+) CD133(+) cells maintain both short-term and long-term HSC activities. To our knowledge, this ~60-fold expansion of SCID repopulating cells is the best expansion of human HSCs reported to date. Further development of this coculture method for expanding human HSCs for clinical and preclinical applications is therefore warranted.     

Microencapsulated feeder cells as a source of soluble factors for expansion of CD34(+) hematopoietic stem cells

Expansion of hematopoietic stem cells (HSCs) from cord blood is highly desired for treatment and transplantation of adult patients for hematologic diseases. For efficient proliferation of HSCs, CD34(+) cells from cord blood were co-cultured with microencapsulated murine stromal cells (HESS-5) or immortalized human mesenchymal stem cells (MSCs) in their conditioned media (CM). Bioactive substances for HSC proliferation in CM at the onset of culture are likely consumed by HSCs with time, and co-culturing with microencapsulated feeder cells ensures a continuous supply. The cell number of CD34(+) cell progeny efficiently increased under these culture conditions, and progeny were analyzed by flow cytometry, the colony assay and the cobblestone area-forming cell (CAFC) assay. Total nucleated cells and CD34(+) cell number increased 194- and 7.4-fold, respectively, in the presence of microencapsulated HESS-5 in CM. Colony forming cells and CAFCs were well maintained. The effective expansion of total cells and maintenance of primitive progenitor cells suggest that transfusion of the progeny obtained from CD34(+) cell culture with microencapsulated HESS-5 in CM could shorten the time to engraftment by bridging the pancytopenic period and support functional hematopoietic repopulation.