CD34+和CD34-细胞在NOD/SCID小鼠体内的造血重建

利用非肥胖糖尿病型重症联合免疫缺陷型(NOD/SCID)小鼠模型, 比较了新鲜及培养后的CD34+和CD34-细胞在体内植入及重建造血能力。从新鲜脐血及培养后的单个核细胞(MNC)中分离出CD34+和CD34-细胞, 经尾静脉输注入经亚致死剂量照射的NOD/SCID小鼠体内, 6周后处死存活的小鼠, 取其骨髓、脾脏和外周血细胞, 分别进行细胞表型分析、造血集落形成单位和人特异性基因的检测。经检测, 输注CD34+细胞和混合细胞的小鼠, 其体内CD45+细胞及人源各系血细胞的含量相近, 两者均远远高于输注CD34-细胞的小鼠。输注培养后CD34-细胞的小鼠饲养6周后全部死亡,输注培养后CD34+细胞的小鼠存活率约为66.7%, 而输注培养后混合细胞的小鼠全部存活, 且在两组存活的小鼠体内均能检测到CD45+细胞及人源各系血细胞。结果表明: 无论是新鲜还是培养后的CD34+细胞均具有在NOD/SCID小鼠体内植入和重建造血能力, 而CD34-细胞不具有该能力, 但CD34-细胞与CD34+细胞同时输注有助于提高小鼠的存活率, 说明其对CD34+细胞在小鼠体内发挥植入和造血重建能力有一定的辅助作用。     

人脐血CD34+细胞在NOD/SCID小鼠免疫重建及其抗HBV作用的初步探讨

目的：探讨NOD／SCID（nonobese diabetic／severe combined immunodeficient）小鼠移植人脐带血（human umbilical cord blood,HUCB）CD34＋细胞后免疫重建的特性。建立hu—NOD／SCID人鼠嵌合模型并观察其人源化免疫细胞在小鼠体内的生长分化特性、存活时间及其对HBV感染的清除作用。方法：1．NOD／SCID小鼠于C0603．5Gy照射后24h内尾静脉输注HUCBCD34＋细胞;2．以流式细胞技术鉴定小鼠外周血中CD45＋,CD3＋,CD19＋,CD56＋等人源化细胞的比例;3．NOD／SCID小鼠于移植后第4wk注射HBV感染者血清并以未移植的NOD／SCID小鼠作为对照,注射同等量的患者血清;4．于感染后1、7、10、15天采血,免疫荧光定量PCR方法分别检测其HBV—DNA含量。结果：1．HUCBCD34＋细胞移植后第2wk,在小鼠外周血中检测出的CD3＋CD8＋T细胞、CD3＋CD4＋T细胞、CD19＋B细胞、CD56＋NK细胞的比例分别为18．6％、16．1％、13．1％和27．8％。各细胞比例随小鼠周龄而变化。所有移植小鼠存活时间均达9wk;2．移植后小鼠感染HBV血清后,病毒仅在感染后第一天检出,随后消失;未移植CD34＋细胞的小鼠外周血HBV—DNA一直维持在103水平：结论：1．NOD／SCID小鼠经射线照射后移植HUCBCD34＋细胞,在不加任何刺激因子的情况下小鼠可以长时间存活并重建免疫;2．hu—NOD／SCID人鼠嵌合模型小鼠免疫成功重建后,对HBV感染有快速的清除作用。     

急性淋巴细胞白血病CD34+CD38-细胞在NOD/SCID小鼠体内的自我更新与增值能力

目的 探索急性淋巴细胞白血病(ALL)患者CD34+ CD38-细胞移植到NOD/SCID小鼠体内建立白血病的可行性、自我更新与增殖潜能.方法 分选并鉴定ALL患者骨髓CD34+ CD38-细胞及对照CD34- CD38+细胞后,经尾静脉分别注射104个细胞于亚致死剂量射线照射的NOD/SCID小鼠体内,连续监测小鼠状态以及外周血血象改变,对濒死或死亡小鼠进行骨髓检查、肝脾病理学检查.结果 接种从ALL患者分选的CD34+ CD38-细胞到NOD/SCID小鼠体内后4周,小鼠外周血白细胞上升,到8周左右达高峰,约15×109～20× 109/L,原始及幼稚淋巴细胞明显增多.骨髓象显示以原始及幼稚淋巴细胞增生为主,约为40％,且肝脾组织也有白细胞浸润,明显高于接种了对照组CD34- CD38+细胞的NOD/SCID小鼠.结论 ALL患者CD34+CD38-细胞可以成功移植NOD/SCID 小鼠,在小鼠体内增殖形成白血病,说明该群细胞具有自我更新和增殖的潜能,可作为探索白血病起始细胞研究的重要载体.     

急性淋巴细胞白血病CD34~+ CD38~-细胞在NOD/SCID小鼠体内的自我更新与增殖能力

目的探索急性淋巴细胞白血病(ALL)患者CD34+CD38-细胞移植到NOD/SCID小鼠体内建立白血病的可行性、自我更新与增殖潜能。方法分选并鉴定ALL患者骨髓CD34+CD38-细胞及对照CD34-CD38+细胞后,经尾静脉分别注射104个细胞于亚致死剂量射线照射的NOD/SCID小鼠体内,连续监测小鼠状态以及外周血血象改变,对濒死或死亡小鼠进行骨髓检查、肝脾病理学检查。结果接种从ALL患者分选的CD34+CD38-细胞到NOD/SCID小鼠体内后4周,小鼠外周血白细胞上升,到8周左右达高峰,约15×109～20×109/L,原始及幼稚淋巴细胞明显增多。骨髓象显示以原始及幼稚淋巴细胞增生为主,约为40%,且肝脾组织也有白细胞浸润,明显高于接种了对照组CD34-CD38+细胞的NOD/SCID小鼠。结论 ALL患者CD34+CD38-细胞可以成功移植NOD/SCID小鼠,在小鼠体内增殖形成白血病,说明该群细胞具有自我更新和增殖的潜能,可作为探索白血病起始细胞研究的重要载体。     

人胎盘间充质干细胞对脐血CD34~+细胞迁移作用的影响

人胎盘作为间充质干细胞的新来源在调控造血干/祖细胞(CD34+细胞)发育与分化方面具有重要作用,但其在移植中是否对CD34+细胞迁移起作用尚不清楚.为此,本研究通过胰酶消化法和密度梯度离心法,单克隆筛选培养出人胎盘来源间充质干细胞,RT-PCR检测其与造血和趋化相关的细胞因子和黏附分子的表达;进一步利用transwell体系检测其对脐血CD34+细胞的迁移作用.结果表明,PDMSCs呈现典型的成纤维样细胞,表达间充质干细胞相关表面抗原CD73,CD90和CD105,不表达造血及内皮细胞表面抗原CD34,CD45,CD31等,PDMSCs具有诱导成脂肪、成骨和成软骨的能力.RT-PCR检测结果显示,PDMSCs表达IL-6,LIF,G-CSF,GM-CSF,M-CSF,FL,SCF,SDF-1,VEGF等多种造血与趋化因子,Integrinβ1,Integrinα5,ICAM-1,ICAM-2,ICAM-3,VCAM-1等多种黏附分子;将PDMSCs按照不同密度接种在transwell体系,4h后脐血CD34+细胞迁移率随着PDMSCs的密度增加而增加;在PDMSCs与SDF-1对CD34+细胞的趋化迁移比较实...     

CD34^—造血干细胞研究进展

新近研究发现,在小鼠和人体内存在一类CD34抗原阴性的造血干细胞,它们以其更广泛的增殖和分化潜能,向临床造血干细胞移植提出了新的挑战和选择,也引起了人们的关注,本文对CD34^-造血干细胞目前的研究进展加以阐述。     

体外培养脐血单个核细胞与CD34+富集细胞

对比MNC和CD34 +富集细胞在SCF +IL 3+IL 6 +FL +Tpo细胞因子组合下的体外扩增特性 ,发现 :CD34 +富集细胞具有很高的扩增潜力 ,在本实验条件下其总细胞持续扩增了 8周 ,扩增倍数达 312 70 9± 86 40 5倍 ;而MNC在培养至第 4周扩增就已呈现下降趋势 ,最大仅扩增了 5 3 3± 6 2倍。对比集落和CD34 +细胞的扩增发现 ,MNC的集落密度和CD34 +细胞含量由第 0天至第 7天有一个上升的过程 ,而CD34 +富集细胞在培养过程中 ,集落密度和CD34 +细胞含量却始终呈下降趋势。在体外培养过程中 ,CD34 +富集细胞的CFU GM和CD34 +细胞最大分别扩增了 185 7± 14 1和 191 7± 188 8倍 ,明显高于MNC的 12 4± 3 2和 5 0 6± 33 2倍 ;而CD34 +富集细胞和MNC的BFU E则只实现了少量扩增 ,分别为 7 2± 5 2和 10 1± 3 4倍。结果显示 ,从CD34 +富集细胞出发扩增造血干 祖细胞 ,可以得到更多的CD34 +细胞和CFU GM集落形成细胞     

低氧对CD34~ 造血干/祖细胞增殖分化及其对细胞因子依赖性的影响

采用免疫磁珠法分离脐血CD34 造血干 /祖细胞 ,进行低氧和常氧条件下单个核细胞 (MNC)及CD34 细胞的半固体及液体培养 ,计细胞总数和集落产率 ,并通过流式细胞仪检测细胞表型和细胞周期 ,以探讨造血干/祖细胞在低氧环境下增殖分化性能的改变及其对细胞因子反应性的变化。结果显示 :CD34 细胞在低氧条件下生成的BFU E集落数 ( 32 4 8± 41 4/10 4 细胞 )明显增多 (对照为 191 2± 34 5 /10 4 细胞 ,P <0 0 1) ;在无细胞因子存在的液体培养体系中 ,低氧组的BFU E产率 ( 15 2 4± 2 2 6 /10 4 细胞 )明显高于常氧组 ( 74 2± 9 3/10 4 细胞 ,P <0 0 1) ;低氧培养细胞中CD34 细胞的比例高于对照 2 5± 1 2倍 (P <0 0 5 )。但MNC生成的BFU E在常氧和低氧条件下无显著差异。这些结果表明 :体外低氧环境能显著增加CD34 造血干 /祖细胞形成红系祖细胞的产率 ,且使其对细胞因子的依赖性降低 ,并对早期红系祖细胞的维持有增强作用 ,但对粒系祖细胞的增殖则有抑制作用     

人脐血CD34+细胞来源的树突状细胞疫苗在SCID小鼠体内的抗肿瘤免疫作用

目的观察脐血CD34 干细胞来源的树突状细胞(dendritic cells, DC)疫苗在严重联合免疫缺陷(severecombined immunity deficiency,SCID)小鼠体内对人肝癌细胞的免疫治疗和免疫保护作用.方法采用微磁珠分选系统(Mini MACS)从脐血单个核细胞中分离CD34 干细胞.重组人粒细胞-巨噬细胞集落刺激因子(recombined human granulocyte-macrophage colony-stimulating factor, rhGM-CSF)、重组人肿瘤坏死因子(recombined human tumor necrosis factor,TNF)-α诱导脐血CD34 细胞向DC分化,相差显微镜下观察DC分化过程中细胞的形态及数量变化.用人肝癌细胞BEL-7402裂解物冲击DC制备DC疫苗.在SCID小鼠体内观察DC疫苗致敏的淋巴细胞对人肝癌细胞的免疫治疗和免疫保护作用.结果脐血CD34 细胞在细胞因子诱导下,细胞形态由小变大、由圆形逐渐变为不规则形;细胞分裂扩增过程中,数量逐渐增多,形成细胞集落.经过细胞因子联合诱导2周的DC胞质突起丰富,具有典型的树枝状形态.在体内的抗肿瘤实验中,经DC疫苗致敏的人外周血淋巴细胞治疗荷瘤小鼠,肿瘤生长速度、瘤体积和重量明显小于未致敏淋巴细胞治疗组(P<0.05).与未致敏淋巴细胞免疫组和DC疫苗致敏的淋巴细胞治疗组比较,经DC疫苗致敏的淋巴细胞免疫SCID小鼠,肝癌细胞攻击后,肿瘤的发病率降低(P<0.05),发病潜伏期延长(P<0.01),肿瘤体积和重量减小(P<0.05).结论人脐血CD34 细胞来源的DC疫苗能在一定程度上抑制人肝癌细胞在小鼠体内的生长,抵抗肿瘤细胞的攻击,对机体具有相应的抗肿瘤免疫治疗和免疫保护作用.     

人T细胞急性淋巴细胞白血病小鼠动物模型的建立

目的:通过建立一理想的动物模型来模拟T细胞急性淋巴细胞白血病的发病状态,为探索急性淋巴细胞白血病全新的治疗方法提供平台。方法:采用抗鼠-CD122抗体注射NOD/SCID小鼠进行预处理,通过尾静脉注射9例不同病例的白血病细胞,以及1株T-ALL细胞系。通过检测小鼠体内白血病细胞及脏器白血病细胞浸润情况,观察白血病细胞植入。将白血病细胞进行二次移植,观察移植稳定性。对白血病动物模型进行药物处理,观察小鼠生存期,模拟人体治疗反应。结果:有4例病例的细胞及T-ALL细胞株成功植入。流式细胞检测显示受体小鼠体内较多比例人CD45+细胞表达,免疫组化显示CD45+细胞浸润小鼠不同脏器,系列移植也获得成功。体内药物处理显示地塞米松能延长小鼠的生存期,与临床观察相一致。结论:成功建立经抗鼠CD122单抗预处理的人T细胞急性淋巴细胞白血病NOD/SCID小鼠模型,具有原发疾病的所有主要特征。     

Generation of Dendritic Cells from Fresh and Frozen Cord Blood CD34Cells

Dendritic cells (DCs) are professional antigen-presenting cells that are required for the initiation of the immune response. DCs have been shown to be generated from CD34⁺pluripotent hematopoietic progenitor cells in the bone marrow and cord blood (CB), but relatively little is known about the effect of cryopreservation on functional maturation of DCs from hematopoietic stem cells. In this work we report the generation of DCs from cryopreserved CB CD34⁺cells. CB CD34⁺cells were cryopreserved at −80°C for 2 days. Cryopreserved CB CD34⁺cells as well as freshly isolated CB CD34⁺cells cultured with granulocyte—macrophage colony-stimulating factor (GM-CSF)/stem cell factor (SCF)/tumor necrosis factor-α (TNF-α) for 14 days gave rise to CD1a⁺/CD4⁺/CD11c⁺/CD14⁻/CD40⁺/CD80⁺/CD83⁺/CD86⁺/HLA-DR⁺cells with dendritic morphology. DCs derived from cryopreserved CB CD34⁺cells showed a similar endocytic capacity for fluorescein isothiocyanate-labeled dextran and lucifer yellow when compared with DCs derived from freshly isolated CB CD34⁺cells. Flow cytometric analysis revealed that two CC chemokine receptors (CCRs), CCR-1 and CCR-3, were expressed on the cell surface of DCs derived from both cryopreserved and freshly isolated CB CD34⁺cells, and these DCs exhibited similar chemotactic migratory capacities in response to regulated on activation normal T-cell expressed and secreted. DCs derived from cryopreserved as well as freshly isolated CB CD34⁺cells were more efficient than peripheral blood mononuclear cells in the primary allogeneic T-cell response. These results indicate that frozen CB CD34⁺cells cultured with GM-CSF/TNF-α/SCF gave rise to dendritic cells which were morphologically, phenotypically and functionally similar to DCs derived from fresh CB CD34⁺cells.     

Loss of CD34 Expression in Aging Human Choriocapillaris Endothelial Cells

Structural and gene expression changes in the microvasculature of the human choroid occur during normal aging and age-related macular degeneration (AMD). In this study, we sought to determine the impact of aging and AMD on expression of the endothelial cell glycoprotein CD34. Sections from 58 human donor eyes were categorized as either young (under age 40), age-matched controls (> age 60 without AMD), or AMD affected (>age 60 with early AMD, geographic atrophy, or choroidal neovascularization). Dual labeling of sections with Ulex europaeus agglutinin-I lectin (UEA-I) and CD34 antibodies was performed, and the percentage of capillaries labeled with UEA-I but negative for anti-CD34 was determined. In addition, published databases of mouse and human retinal pigment epithelium-choroid were evaluated and CD34 expression compared between young and old eyes. Immunohistochemical studies revealed that while CD34 and UEA-I were colocalized in young eyes, there was variable loss of CD34 immunoreactivity in older donor eyes. While differences between normal aging and AMD were not significant, the percentage of CD34 negative capillaries in old eyes, compared to young eyes, was highly significant (p = 3.8×10⁻⁶). Endothelial cells in neovascular membranes were invariably CD34 positive. Published databases show either a significant decrease in Cd34 (mouse) or a trend toward decreased CD34 (human) in aging. These findings suggest that UEA-I and endogenous alkaline phosphatase activity are more consistent markers of aging endothelial cells in the choroid, and suggest a possible mechanism for the increased inflammatory milieu in the aging choroid.     

Lentiviral Transduction of CD34+ Cells Induces Genome-Wide Epigenetic Modifications

Epigenetic modifications may occur during in vitro manipulations of stem cells but these effects have remained unexplored in the context of cell and gene therapy protocols. In an experimental model of ex vivo gene modification for hematopoietic gene therapy, human CD34⁺ cells were cultured shortly in the presence of cytokines then with a gene transfer lentiviral vector (LV) expected to transduce cells but to have otherwise limited biological effects on the cells. At the end of the culture, the population of cells remained largely similar at the phenotypic level but some epigenetic changes were evident. Exposure of CD34⁺ cells to cytokines increased nuclear expression of epigenetic regulators SIRT1 or DNMT1 and caused genome-wide DNA methylation changes. Surprisingly, the LV caused additional and distinct effects. Large-scale genomic DNA methylation analysis showed that balanced methylation changes occurred in about 200 genes following culture of CD34⁺ cells in the presence of cytokines but 900 genes were modified following addition of the LV, predominantly increasing CpG methylation. Epigenetic effects resulting from ex vivo culture and from the use of LV may constitute previously unsuspected sources of biological effects in stem cells and may provide new biomarkers to rationally optimize gene and cell therapy protocols.     

促红细胞生成素基因修饰的胎肝基质细胞促进脐血CD34 +细胞向红系分化

通过重组慢病毒系统感染人胎肝基质细胞(fetal liver stromal cells,FLSCs),建立了能够稳定高效表达促红细胞生成素(erythropoietin,EPO)的细胞株EPO/FLSCs.从胎儿肝脏克隆EPO基因,构建重组慢病毒EPO的表达载体,感染FLSCs,根据荧光表达强弱进行流式分选,获得能够继续稳定传代的高表达EPO基因的FLSCs,RT-PCR和ELISA结果证实,细胞株中的EPO基因稳定表达.RT-PCR结果显示,FLSCs的EPO在mRNA水平的表达分别是未转染FLSCs和转染空载体FLSCs的5.63倍和5.71倍.ELISA法检测了转染重组慢病毒EPO表达载体的FLSCs EPO蛋白表达水平,结果显示EPO蛋白的表达水平也明显升高.收集EPO/FLSCs的条件培养基,体外诱导脐血CD34+细胞向造血细胞分化,结果显示向红系定向分化的细胞比例明显居多,有可能为临床细胞治疗提供稳定、高质量的细胞来源.     

Transplantation of Immortalized CD34+ and CD34- Adipose-Derived Stem Cells Improve Cardiac Function and Mitigate Systemic Pro-Inflammatory Responses

Adipose-derived stem cells (ADSCs) have the potential to differentiate into various cell lineages and they are easily obtainable from patients, which makes them a promising candidate for cell therapy. However, a drawback is their limited life span during in vitro culture. Therefore, hTERT-immortalized CD34+ and CD34- mouse ADSC lines (mADSCs^hTERT) tagged with GFP were established. We evaluated the proliferation capacity, multi-differentiation potential, and secretory profiles of CD34+ and CD34- mADSCs^hTERT in vitro, as well as their effects on cardiac function and systemic inflammation following transplantation into a rat model of acute myocardial infarction (AMI) to assess whether these cells could be used as a novel cell source for regeneration therapy in the cardiovascular field. CD34+ and CD34- mADSCs^hTERT demonstrated phenotypic characteristics and multi-differentiation potentials similar to those of primary mADSCs. CD34+ mADSCs^hTERT exhibited a higher proliferation ability compared to CD34- mADSCs^hTERT, whereas CD34- mADSCs^hTERT showed a higher osteogenic differentiation potential compared to CD34+ mADSCs^hTERT. Primary mADSCs, CD34+, and CD34- mADSCs^hTERT primarily secreted EGF, TGF-β1, IGF-1, IGF-2, MCP-1, and HGFR. CD34+ mADSCs^hTERT had higher secretion of VEGF and SDF-1 compared to CD34- mADSCs^hTERT. IL-6 secretion was severely reduced in both CD34+ and CD34- mADSCs^hTERT compared to primary mADSCs. Transplantation of CD34+ and CD34- mADSCs^hTERT significantly improved the left ventricular ejection fraction and reduced infarct size compared to AMI-induced rats after 28 days. At 28 days after transplantation, engraftment of CD34+ and CD34- mADSCs^hTERT was confirmed by positive Y chromosome staining, and differentiation of CD34+ and CD34- mADSCs^hTERT into endothelial cells was found in the infarcted myocardium. Significant decreases were observed in circulating IL-6 levels in CD34+ and CD34- mADSCs^hTERT groups compared to the AMI-induced control group. Transplantation of CD34- mADSCs^hTERT significantly reduced circulating MCP-1 levels compared to the AMI control and CD34+ mADSCs^hTERT groups. GFP-tagged CD34+ and CD34- mADSCs^hTERT are valuable resources for cell differentiation studies in vitro as well as for regeneration therapy in vivo.     

Mobilization of CD34+-Progenitor Cells in Patients with Severe Trauma

Circulating CD34+ progenitor cells () gained importance in the field of regenerative medicine due to their potential to home in on injury sites and differentiate into cells of both endothelial and osteogenic lineages. In this study, we analyzed the mobilization kinetics and the numbers of CD34+, CD31+, CD45+, and CD133+ cells in twenty polytrauma patients (n = 13 male, n = 7 female, mean age 46.5±17.2 years, mean injury severity score (ISS) 35.8±12.5 points). In addition, the endothelial differentiation capacity of enriched CD34+cells was assessed by analyzing DiI-ac-LDL/lectin uptake, the expression of endothelial markers, and the morphological characteristics of these cells in Matrigel and spheroid cultures. We found that on days 1, 3, and 7 after a major trauma, the number of CD34+cells increased from 6- up to 12-fold (p<0.0001) over the number of CD34+cells from a control population of healthy, age-matched volunteers. The numbers of CD31+ cells were consistently higher on days 1 (1.4-fold, p<0.01) and 7 (1.3-fold, p<0.01), whereas the numbers of CD133+ cell did not change during the time course of investigation. Expression of endothelial marker molecules in CD34+cells was significantly induced in the polytrauma patients. In addition, we show that the CD34+ cell levels in severely injured patients were not correlated with clinical parameters, such as the ISS score, the acute physiology and chronic health evaluation II score (APACHE II), as well as the sequential organ failure assessment score (SOFA-2). Our results clearly indicate that pro-angiogenic cells are systemically mobilized after polytrauma and that their numbers are sufficient for the development of novel therapeutic models in regenerative medicine.     

Cross Talk with Hematopoietic Cells Regulates the Endothelial Progenitor Cell Differentiation of CD34 Positive Cells

Introduction

Despite the crucial role of endothelial progenitor cells (EPCs) in vascular regeneration, the specific interactions between EPCs and hematopoietic cells remain unclear.

Methods

In EPC colony forming assays, we first demonstrated that the formation of EPC colonies was drastically increased in the coculture of CD34⁺ and CD34⁻ cells, and determined the optimal concentrations of CD34⁺ cells and CD34⁻ cells for spindle-shaped EPC differentiation.

Results

Functionally, the coculture of CD34⁺ and CD34⁻ cells resulted in a significant enhancement of adhesion, tube formation, and migration capacity compared with culture of CD34⁺ cells alone. Furthermore, blood flow recovery and capillary formation were remarkably increased by the coculture of CD34⁺ and CD34⁻ cells in a murine hind-limb ischemia model. To elucidate further the role of hematopoietic cells in EPC differentiation, we isolated different populations of hematopoietic cells. T lymphocytes (CD3⁺) markedly accelerated the early EPC status of CD34⁺ cells, while macrophages (CD11b⁺) or megakaryocytes (CD41⁺) specifically promoted large EPC colonies.

Conclusion

Our results suggest that specific populations of hematopoietic cells play a role in the EPC differentiation of CD34⁺ cells, a finding that may aid in the development of a novel cell therapy strategy to overcome the quantitative and qualitative limitations of EPC therapy.     

A Simple Model System Enabling Human CD34+ Cells to Undertake Differentiation Towards T Cells

Background

Channelling the development of haematopoietic progenitor cells into T lymphocytes is dependent upon a series of extrinsic prompts whose temporal and spatial sequence is critical for a productive outcome. Simple models of human progenitor cells development depend in the main on the use of xenogeneic systems which may provide some limitations to development.

Methods and Findings

Here we provide evidence that a simple model system which utilises both human keratinocyte and fibroblast cell lines arrayed on a synthetic tantalum coated matrix provides a permissive environment for the development of human CD34⁺ haematopoietic cells into mature CD4⁺ or CD8⁺ T lymphocytes in the presence of Interleukin 7 (IL-7), Interleukin 15 (IL-15) and the Fms-like tyrosine kinase 3 ligand (Flt-3L). This system was used to compare the ability of CD34⁺ cells to produce mature thymocytes and showed that whilst these cells derived from cord blood were able to productively differentiate into thymocytes the system was not permissive for the development of CD34⁺ cells from adult peripheral blood.

Conclusions/Significance

Our study provides direct evidence for the capacity of human cord blood CD34⁺ cells to differentiate along the T lineage in a simple human model system. Productive commitment of the CD34⁺ cells to generate T cells was found to be dependent on a three-dimensional matrix which induced the up-regulation of the Notch delta-like ligand 4 (Dll-4) by epithelial cells.