NOD/SCID小鼠人源化TCR Vβ亚家族T细胞分布与克隆性分析

目的 应用脐血CD34 细胞移植NOD/SCID鼠所建立的人源化SCID模型,分析人源化TCR Vβ亚家族T淋巴细胞分布与克隆性.方法 磁珠分选法分离脐血中CD34 细胞,分别经尾静脉输入亚致死剂量照射的NOD/SCID小鼠.第6周处死小鼠提取外周血、骨髓、胸腺的RNA,RT-PCR扩增人TCR Vβ亚家族基因,并用基因扫描进行T细胞克隆性分析.结果 采用RT-PCR技术在模型小鼠骨髓中检测到部分人TCR Vβ亚家族基因Vβ1、2、9、13、19.经进一步基因扫描分析,发现部分TCR Vβ亚家族基因Vβ9、13、19呈寡克隆表达.结论 在NOD/SCID模型可重建分化成熟的TCR Vβ亚家族T细胞.未能检测到全部人TCR VβT细胞的原因可能与免疫重建不完全或存在移植物抗宿主的反应有关.人源T淋巴细胞在模型鼠骨髓中分化成熟.     

人源化NOD/SCID小鼠的细胞免疫重建

目的探讨人脐带血CD34+细胞移植于非肥胖性糖尿病/重症联合免疫缺陷(NOD/SCID)小鼠后的免疫重建作用。方法免疫磁珠法分选人脐血CD34+细胞,经外侧尾静脉移植于亚致死量照射的NOD/SCID小鼠。移植后4、6、8、10周流式细胞术动态监测小鼠外周血人源CD45+、CD3+、CD56+细胞的数量;10周后PCR检测小鼠骨髓人ALU基因的表达,免疫组织化学染色检测小鼠脾脏人源CD3+、CD56+细胞的表达。结果 NOD/SCID小鼠经照射后骨髓腔内有核细胞及巨细胞数量均明显减少或消失,达到理想的清髓预处理效果;移植后4、6、8、10周,移植组所有存活小鼠外周血均可检测到人源性CD45+、CD3+、CD56+细胞的表达,人源淋巴细胞的数量随时间的延长而变化,8周时达到高峰,10周仍有较高的比例。10周时移植组所有存活小鼠骨髓细胞均检测到人ALU基因的表达,脾脏均检测到人CD3+、CD56+细胞;未移植组小鼠照射后2周内全部死亡。结论经照射后的NOD/SCID小鼠通过移植人脐血CD34+细胞成功地建立了hu-SRC-NOD/SCID模型,并有效地重建了小鼠细胞免疫系统。     

细胞因子介导的人脐血单个核细胞体外扩增并重建NOD/SCID小鼠造血及免疫功能

背景:细胞因子介导的脐血造血细胞体外扩增有望能解决脐血移植数量不足的问题。目的:实验拟确立在无基质培养条件下体外扩增脐血单个核细胞最合适的细胞因子组合及干细胞因子、FLT3配基协同促聚核细胞生成因子对造血及免疫重建功能的影响。方法:将新鲜脐血标本分离的单个核细胞接种于含有不同细胞因子组合的无血清无基质培养体系中培养7d,根据不同细胞因子组合分组,将3因子干细胞因子+FLT3配基+促聚核细胞生成因子组合在无血清无基质条件下扩增培养7d前后的脐血单个核细胞移植给经亚致死量照射的NOD/SCID小鼠。在扩增培养0,7d检测脐血CD34+细胞数及CD34+CXCR4+,CD34+CD62L+,CD34+CD49d+的细胞数。脐血移植6周后通过流式细胞仪,PCR法检测存活小鼠体内的人源性细胞。结果与结论:移植6周后,存活小鼠体内均可检测到人源性CD45+细胞,扩增脐血移植组的NOD/SCID小鼠存活率和人特异性基因捡出率均高于新鲜脐血移植组和高于生理盐水移植组(P0.05)。扩增脐血组存活NOD/SCID小鼠骨髓中可检测到人髓系细胞(CD33+),T淋巴细胞(CD4+),B淋巴细胞(CD19+)和人造血干细胞成分(CD34+)细胞的表达。结果提示干细胞因子+FLT3配基+促聚核细胞生成因子3因子组合脐血造血细胞体外扩增是最合适的细胞因子组合,其扩增的脐血单个核细胞能够植入并重建NOD/SCID小鼠的造血及免疫功能。     

转JAK2基因脐血CD34+细胞体外扩增与生物学特性研究

目的： 探讨转基因JAK2介导的脐血干祖细胞长期扩增调控的可行性和转基因细胞的生物学特征。方法: 构建逆转录病毒载体MGI-F₂JAK2，内含有JAK2基因的功能催化区和2个与小分子靶向基因合成药物(AP20187)结合的位点蛋白(F36v，F₂)。应用MiniMACS磁珠分选系统纯化分离脐血CD34⁺细胞，用含JAK2的逆转录病毒上清转染脐血CD34⁺细胞。转染后的CD34⁺细胞在IMDM培养体系中，将细胞分为AP20187组;FL组；TPO组；AP20187+FL+TPO (AFT) 组。对扩增后的细胞定期检测基因转移后GFP动态变化、细胞免疫标记、造血祖细胞集落培养、染色体核型分析和裸鼠致瘤实验。结果: 分选的CD34⁺细胞纯度>91%，基因转移率为49.32%±6.21%；只有AP20187+FL+TPO组可以使转基因的脐血CD34⁺细胞大量增殖，扩增至第8周时细胞数达10⁹，CD34⁺细胞GFP的阳性率由基线水平逐渐上升并于第8周时达到90%以上；细胞表型为CD33⁺、CD61⁺、Gly-A⁺部分阳性；CD38⁺、HLA-DR⁺强阳性；CD2、CD7、CD19接近阴性。扩增的CD34+细胞可分别形成BFU-E、CFU-GM、CFU-Mix并以CFU-GM集落为主。扩增后CD34⁺细胞检测染色体核型正常，裸鼠实验无致瘤特性。结论: 转染JAK2 基因的人脐血CD34⁺细胞协同FL和TPO细胞因子可以体外长期扩增脐血干祖细胞，对今后研究细胞信号转导、造血调控以及开展干细胞和基因治疗都有潜在的应用价值。     

胸腺共移植对异种骨髓造血细胞移植后T细胞发育的影响

目的　探讨胎儿胸腺小叶共移植对异种骨髓移植后人源性T细胞发育的影响 ,为在体研究人造血细胞向T细胞发育提供实验模型 ,并为异种骨髓移植构建银屑病动物模型奠定基础。方法　将人骨髓单个核细胞及胎儿胸腺小叶共移植于SCID鼠 ,皮下注射骨髓基质细胞 ,并结合IL 2腹腔内注射 ,以未移植胸腺小叶者为对照 ,流式细胞术动态检测移植后不同时间SCID鼠外周血中人源性细胞及CD3 细胞、CD4 细胞、CD8 细胞的比例。结果　实验组移植 4周后 ,SCID鼠外周血中已可检测到人源性细胞 (CD4 5 细胞 ) ,8周出现少量CD3 细胞 ,12周可检测到CD4 细胞、CD8 细胞 ,移植 2 4周内 ,SCID鼠外周血人源性细胞及各亚群T细胞比例逐渐增多 ,2 4周达到最大比例。而未移植胸腺的对照组 ,仅可在外周血检测到人源性CD4 5 细胞 ,而无成熟T细胞存在。结论　胎儿胸腺小叶共移植模式可以促进人源性造血细胞在SCID鼠体内向T细胞发育分化 ,并可使其活性在体内维持较长时间。     

粒细胞集落刺激因子动员对供者外周血细胞树突状细胞亚群的影响

目的：研究粒细胞集落刺激因子(G-CSF)动员对外周血单个核细胞（PBMNC）中树突状细胞（DC）亚群及其功能的影响。方法：以CD34^-Lin^- HLA-DR⁺细胞群设门4色荧光分析方法检测25例无关供者G-CSF动员前、后外周血细胞DC亚群；ELISA检测其血清相关细胞因子IL-12p40、IL-10、IFN-γ、IL-4活性，并分析DC₁/DC₂（CD11c⁺CD123^- /CD11c^-CD123⁺）比值与CD34⁺细胞含量的相关性。结果：G-CSF动员后, 供者PBMNC 的DC₁/DC₂比值显著低于动员前（P<0.05）；DC₁/DC₂与CD34⁺细胞含量呈负相关（r=-0.438, P<0.05），CD34⁺细胞/MNC≥0.4%时， DC₁/DC₂倒置；而血清相关细胞因子IL-12p40、IL-10、IFN-γ、IL-4水平无显著改变（P>0.05）； DC₂ HLA-DR表达上调而CD83不表达。结论： G-CSF动员后，供者外周血CD34⁺细胞数量增加的同时，DC₂数量也增加，这些DC₂尽管HLA-DR表达上调，但仍处前体细胞状态，不直接分泌或调节Th2细胞分泌免疫抑制因子。     

人源化SCID小鼠模型的建立及其鉴定

目的：探讨在SCID小鼠体内移植人免疫细胞，建立人源化SCID小鼠模型及其特性鉴定。方法：SCID小鼠腹腔注射环磷酰胺（CTX）抑制骨髓造血，连续4天后，通过腹腔注射移植人外周血单个核细胞（PBMC）。4、8和12周后分别取小鼠外周血、脾脏、肝脏。荧光显微镜下观察SCID小鼠外周血中人CD3^＋、CD19^＋细胞；流式细胞仪测定全血中人CD3^＋、CD19^＋细胞百分率；免疫组织化学分析SCID小鼠肝脏和脾脏中人CD3^＋、CD19^＋细胞；ELISA检测SCID小鼠血清中人免疫球蛋白含量。结果：（I）SCID小鼠移植人外周血单个核细胞4、8和12周后在小鼠外周血中通过荧光显微镜下可观察到人CD3^＋、CD19^＋细胞，4周后流式细胞仪测得小鼠外周血单个核细胞中人CD19^＋、CD3^＋细胞百分率分别为10．6％、31．7％；（2）免疫组织化学结果显示在小鼠脾脏中存在人CD3^＋、CD19^＋细胞；（3）移植人外周血单个核细胞4、8和12周后ELISA测得小鼠血清中人免疫球蛋白的含量分别为390、1100和1040μg／ml。结论：成功地在SCID小鼠体内建立了人免疫系统。     

mPEG遮蔽供者淋巴细胞减轻移植物抗宿主病的研究

目的 :探讨向SCID小鼠移植甲氧基聚乙二醇 (mPEG)修饰的人脐血单个核细胞时 ,因供体淋巴细胞表面抗原被遮蔽降低了移植物抗宿主病 (GVHD)而不影响其干祖细胞造血功能重建。方法 :(1)用流式细胞仪检测修饰前后人脐血单个核细胞中CD4 、CD8 T细胞及CD4 /CD8 T细胞比率的变化。 (2 )观察遮蔽前后 ,人脐血干祖细胞在体外培养形成CFU GM的差异。 (3)将修饰前后的单个核细胞移植到SCID小鼠体内 ,观察GVHD出现的时间和小鼠活存状况。 (4)移植后 7wk左右测定小鼠骨髓中人源CD4 5 细胞的含量。结果 :(1)mPEG几乎可完全遮蔽T细胞表面的CD4和CD8抗原。 (2 )修饰前、后的干祖细胞 ,体外培养形成CFU GM的数量没有明显差异。 (3)将修饰的人脐血单个核细胞移植到小鼠体内 ,GVHD出现的时间晚于未修饰组 ,提高了小鼠的活存率。 (4)移植后 4 7d ,活杀小鼠的骨髓细胞中 ,可检测到人的CD4 5 细胞。结论 :用mPEG遮蔽供体T细胞表面的CD4、CD8抗原 ,最终减轻了宿主对移植物的免疫应答 ,而干祖细胞增殖、分化的功能未受到明显影响。     

培养前后脐血CD34~＋细胞在NOD/SCID鼠体内造血重建功能的比较

目的:以NOD/SCID小鼠为模型, 经半致死剂量照射后输注新鲜或培养后的造血细胞, 以比较培养前后脐血CD34 细胞的造血重建功能.方法:从新鲜脐血中分离单个核细胞(MNC), 采用干细胞因子(SCF)、血小板生成素(TPO)、Flt3配体(FL)、白细胞介素3(IL-3)和白细胞介素6(IL-6)细胞因子组合体外培养14 d.通过MiniMACS免疫磁性吸附柱从新鲜或培养后的MNC中分离CD34 细胞, 4×105个CD34 细胞和5×106CD34-细胞混合后通过尾静脉输注入NOD/SCID小鼠中.饲养过程中动态观察外周血象恢复情况, 6周后检测小鼠骨髓和脾脏细胞中人源细胞及各系造血细胞的含量.结果:体外培养MNC 14 d后, 总细胞扩增了1.78倍;细胞移植6周后, 输注新鲜和培养后造血细胞的小鼠均存活, 在小鼠骨髓和脾脏中均可检测到人源细胞及各系人源血细胞和人特异ALU基因序列, 小鼠外周血象恢复到辐照前水平.培养后CD34 细胞在小鼠体内的植入水平与新鲜CD34 细胞的相近, 而其各系人源血细胞的含量高于新鲜CD34 细胞. 结论:体外培养14 d后的CD34 细胞仍保持了体内植入和重建造血的能力, 且其多系造血重建能力优于新鲜CD34 细胞.     

绿色荧光蛋白转基因小鼠CD34+/CD45+细胞在脊髓全横断大鼠模型中的存活及迁移

目的:探讨CD34⁺/CD45⁺细胞移植入脊髓全横断大鼠模型后的存活、迁移及分布情况。方法:体外培养绿色荧光蛋白（GFP）转基因小鼠骨髓细胞，经CD34、CD45单克隆抗体鉴定后移植入脊髓全横断大鼠模型脊髓横断处尾侧，分别在术后24 h、48 h、1周、2周、4周和8周行左心腔内灌注，取出脊髓，连续切片（片厚 10 μm），置于荧光显微镜下观察切片中有无绿色荧光细胞，并观察荧光细胞的分布范围；用免疫组化法检测CD34⁺/CD45⁺细胞的存活。 结果:脊髓横断处头尾两侧均可见绿色荧光细胞，且多分布于灰质中，散在或聚集成片；免疫组化法可见切片中有CD34⁺/CD45⁺细胞散在。 结论: CD34⁺/CD45⁺细胞可在脊髓全横断大鼠模型的脊髓中存活，并可迁移至脊髓横断处头侧，且随时间的延长迁移距离有所增加。     

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.     

人骨髓间质干细胞向造血细胞分化潜能的实验研究

目的:在体研究人骨髓间质干细胞(hBMMSCs)向造血细胞分化的潜能。方法:将hBMMSCs经尾静脉注射给环磷酰胺处理的严重联合免疫缺陷(SCID)小鼠,利用流式激活细胞分析系统(FACS)检测hBMMSCs输注后存活35d的SCID小鼠外周血、骨髓和脾脏中人源性造血细胞的表型和水平。结果:hBMMSCs输注组外周血(PB)、骨髓(BM)和脾脏(spleen)中可检测到人CD45⁺/H-2D^d-、CD34⁺/H-2D^d-细胞,而对照组PB、BM和脾脏均未检测到上述表型的人造血细胞。结论:hBMMSCs具有向造血细胞分化的潜能。     

Interleukin-3 promotes proliferation and differentiation of human hematopoietic stem cells but reduces their repopulation potential in NOD/SCID mice

In the present study we explored systematically the influence of human interleukin-3 (IL-3) on the cord blood (CB) cell-derived production of human hematopoietic cells in the bone marrow, blood, and spleen of chimeric nonobese/severe combined immunodeficient mice ((NOD/SCID) mice. CB mononuclear cells and MACS-enriched CB CD34(+) cells were injected into irradiated NOD/SCID mice. The mice were additionally transplanted with a stably transfected rat fibroblast cell line expressing the human IL-3 gene (Rat-IL-3) constitutively, or with the nontransfected rat fibroblast cell line as a control (Rat-1). Rat-IL-3 mice displayed a higher engraftment of human hematopoietic cells in bone marrow, spleen, and peripheral blood compared with mice with Rat-1 cotransplantation. When we transplanted their total bone marrow cell population into secondary mice, surprisingly, mice transplanted with bone marrow cells from Rat-1 mice displayed a higher proportion of human hematopoietic cells compared with Rat-IL-3 mice. As expected, bone marrow cultures (BMCs) from Rat-IL-3 mice contained a higher proportion of human cells than Rat-1 bone marrow cells. However, when BMCs were passaged to new flasks, we observed a higher proportion of human cells in BMCs from Rat-1 mice compared with BMCs from Rat-IL-3 mice. IL-3 promotes the proliferation and differentiation of hematopoietic stem cells in chimeric bone marrow. In addition, IL-3 may play a role in the depletion of hematopoietic stem cells in chimeric bone marrow. In the absence of IL-3, the hematopoietic stem cells may remain in a quiescent state and proliferation can be induced by stimuli, including secondary transplantation or cell passage.     

Functional characterization of TPO-expanded CD34+ cord blood cells identifies CD34- CD61- cells as platelet-producing cells early after transplantation in NOD/SCID mice and rCD34+ cells as CAFC colony-forming cells

Transplantation of thrombopoietin (TPO)-expanded cord blood CD34(+) cells accelerates human platelet recovery in NOD/SCID mice. It is unknown which subpopulations of the TPO-expanded cells mediate accelerated platelet recovery and bone marrow (BM) engraftment. In this study, the contribution of these subpopulations to human platelet appearance in the blood and BM engraftment was studied in NOD/SCID mice. Following transplantation of CD34(-) /CD61(-)/lineage(-) cells (Lin(-)), human platelets were detected in the blood of recipient mice from day 4. Both time to platelet recovery and blood platelet counts at 6 weeks after transplantation showed Lin(-) dose dependence. The Lin(-) population was virtually negative for lineage marker expression and lacked CD42b expression but was heterogeneous with regard to CD36 and CD38 expression, reflecting a population in transit but not fully committed toward the megakaryocyte (MK) lineage. Although no definitive phenotype could be established of the cells generating prompt platelet production and cells generating platelets 6 weeks after transplantation, this relatively heterogeneous Lin(-) population is prerequisite to accelerate platelet recovery in vivo. The interval to platelet recovery after transplantation of the CD34(+) cells remaining after expansion (rCD34(+)) was similar to mice transplanted with nonexpanded CD34(+) cells, although the total platelet counts and the engraftment levels in the BM were lower. Cobblestone area-forming cell colony-forming cells resided mostly in the rCD34(+) population. The pro-MK CD61(+) cells did not contribute to human platelet recovery or engraftment in the BM. Our study shows that not all expanded cells appear critical for transplantation. These data support that functional characterization of the expanded cell populations is warranted to make future expansion protocols suitable for clinical application.     

Transduction of umbilical cord blood CD34+ NOD/SCID-repopulating cells by simian foamy virus type 1 (SFV-1) vector

Foamy viruses are nonpathogenic retroviruses that offer unique opportunities for gene transfer into various cell types including hematopoietic stem cells. We used a simian foamy virus type 1 vector (SFV-1) containing a LacZ reporter gene with a titer of 1-5 x 10(6) viral particles/ml that was free of replication-competent retrovirus to transduce human umbilical cord blood CD34+ cells. Transduced CD34+ cord blood cells were transplanted into NOD/SCID mice and plated in serum-free methylcellulose culture to determine the transduction efficiency of human hematopoietic progenitor cells. A transduction efficiency of about 20% was obtained. At 6-10 weeks posttransplantation, human hematopoietic cell engraftment and marking were determined. Marrow from transplanted mice demonstrated human cell engraftment by the presence of human (CD45+) cells containing both CD19+ lymphoid and CD33+ myeloid cells. Serial sampling of NOD/SCID bone marrow revealed the presence of 6.7-14.0% CD45+ cells at 6 weeks posttransplant as compared to 3.6-27.2% CD45+ cells at 9-10 weeks posttransplant. Human progenitors examined from NOD/SCID bone marrow cells 9 weeks posttransplant revealed from 7.4 to 25.9% of the colonies exhibiting X-gal staining. Our study demonstrates the ability of a simian foamy virus vector to transduce the SCID-repopulating cell and offers a promising new gene delivery system for use in hematopoietic stem cell gene therapy.     

Hematopoietic repopulating ability of cord blood CD34(+) cells in NOD/Shi-scid mice

Although umbilical cord blood (CB) is increasingly being used as an alternative to bone marrow (BM) as a source of transplantable hematopoietic stem cells (HSC), information on the hematopoietic repopulating ability of CB HSC is still limited. We recently established a xenotransplantation system in NOD/Shi-scid mice to evaluate human stem cell activity. In the present study, we transplanted 5 to 10 x 10(4) CB CD34(+) cells into six NOD/Shi-scid mice treated with anti-asialo GM1 antiserum to investigate the hematopoietic repopulating ability of CB. The BM of all recipients contained human CD45(+) cells 10 to 12 weeks after the transplantation (43.8 +/- 17.7%). Clonal culture of the recipient BM cells revealed the formation of various types of human hematopoietic colonies, including myelocytic, erythroid, megakaryocytic, and multilineage colonies, indicating that CB HSC can differentiate into hematopoietic progenitors of various lineages. However, the extent of the differentiation and maturation differed with each lineage. CD13(+)/CD14(+)/CD33(+) myelocytic cells were mainly repopulated in BM and peripheral blood (PB). While CD41(+) megakaryocytic cells and platelets were present, few glycophorin A(+)CD71(+) or hemoglobin alpha-containing erythroid cells were detected. CD19(+) B cells were the most abundantly repopulated in NOD/Shi-scid mice, but their maturational stage differed among the hematopoietic organs. Most of the BM CD19(+) cells were immature B cells expressing CD10 but not surface immunoglobulin (Ig) M, whereas more mature CD19(+)CD10(-) surface IgM(+) B cells were predominantly present in spleen and PB. CD3(+) T cells were not detected even in the recipient thymus. The transplantation to the NOD/Shi-scid mouse may provide a useful tool for evaluating the repopulating ability of transplantable human HSC.     

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.     

Inhibition of CD26 in human cord blood CD34+ cells enhances their engraftment of nonobese diabetic/severe combined immunodeficiency mice

CD26, a surface serine dipeptidylpeptidase IV (DPPIV) expressed on different cell types, cleaves the amino-terminal dipeptide from some chemokines, including stromal-derived factor-1 (SDF-1/CXCL12). SDF-1/CXCL12 plays important roles in hematopoietic stem cell (HSC) homing, engraftment, and mobilization. Inhibition of CD26 peptidase activity enhances homing, engraftment, and competitive repopulation in congenic mouse bone marrow cell transplants. Our studies evaluated a role for CD26 in in vivo engraftment of HSCs from human umbilical cord blood (CB) into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Pretreating purified CD34(+) human CB cells with Diprotin A, a DPPIV inhibitor, for 15 min significantly enhanced engraftment. Treatment did not affect differentiation of CD34(+) cells in vivo, as measured phenotypically by human markers CD33, CD38, CD19, and CD34. We found that the percentage of CD26(+) cells within the more immature cells (CD34(+)CD38()) was significantly higher than in the more mature population (CD34(+)CD38(+)). These results suggest that inhibition of CD26 may be one way to enhance engraftment of limiting numbers of stem cells during CB transplantation.     

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.