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31270.9±8640.5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53.3±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50.6±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。      

组蛋白去乙酰化酶3对外周CD4+ T细胞分化的影响

目的 探讨组蛋白去乙酰化酶3（HDAC3）对外周CD4⁺ T细胞分化及功能的调控作用。方法 采用CD4cre酶介导Hdac3杂合基因缺失小鼠（Hdac3^fl/flCD4^cre+/-）及其野生型正常对照小鼠（Hdac3^fl/fl，WT），流式细胞术检测HDAC3缺失对外周CD4⁺和CD8⁺ T细胞比例和数量的影响；在体外佛波酯（PMA）和离子霉素（Ionomycin）刺激条件下，流式细胞术检测HDAC3缺失对CD4⁺ T细胞中IFN-γ、IL-4和IL-17A的表达以及Tfh细胞产生的影响；采用ELISA检测HDAC3缺失对小鼠血清IFN-γ、IL-4和IL-17表达的影响；分选Hdac3^fl/flCD4^cre+/-和WT小鼠外周初始CD4⁺ T细胞，分别在Th1和Th2分化条件下培养，细胞内染色检测HDAC3缺失对Th1、Th2以及Th17相关细胞因子及其特异转录因子表达的影响；采用Microarray检测HDAC3缺失对CD4⁺ T细胞分化亚群相关基因表达的影响；采用链脲佐菌素（STZ）处理小鼠构建I型糖尿病（TIDM）疾病模型，检测HDAC3缺失对T1DM发病的影响。结果 与WT小鼠相比，Hdac3^fl/flCD4^cre+/-小鼠外周CD4⁺和CD8⁺ T细胞的比例和数量显著降低。Hdac3^fl/flCD4^cre+/-小鼠CD4⁺ T细胞及血清中IFN-γ的表达显著降低，而IL-4和IL-17A的表达显著增加，Tfh细胞比例也显著增加；HDAC3缺失抑制体外培养CD4⁺ T细胞向Th1分化但促进其向Th2分化；Microarray检测发现HDAC3缺失导致Th1型细胞谱系基因表达降低，而Th2、Th17以及Tfh细胞谱系基因表达增加；在STZ诱导条件下，HDAC3缺失抑制小鼠T1DM的发生和CD4⁺ T细胞向Th1分化。结论 HDAC3促进外周CD4⁺ T细胞向Th1细胞分化并加重T1DM的发生。     

外周血Treg细胞、T淋巴细胞及其亚群与早期宫颈癌的关系及对淋巴结转移的预测价值研究

摘要 目的：分析外周血Treg细胞、T淋巴细胞及其亚群与早期宫颈癌的关系及对淋巴结转移的预测价值。方法：选择我院自2017年1月至2020年12月接诊的60例接受子宫颈癌根治术及盆腔淋巴清扫术的早期宫颈癌患者作为观察组,另选同期的60例健康体检者作为对照组。比较两组外周血Treg细胞、T淋巴细胞及其亚群水平,使用受试者工作特征曲线（ROC）下面积（AUC）评价外周血Treg细胞、T淋巴细胞及其亚群对淋巴结转移的预测效能。结果：观察组外周血Treg细胞、CD8⁺T细胞水平高于对照组,CD3⁺T细胞、CD4⁺T细胞、CD4⁺/CD8⁺比值均低于对照组（P＜0.05）;观察组术后外周血Treg细胞、CD8⁺T细胞水平较术前降低,CD3⁺T细胞、CD4⁺T细胞、CD4⁺/CD8⁺比值均较术前升高（P＜0.05）;在60例早期宫颈癌患者中,发生淋巴结转移12例;淋巴结转移组术前外周血Treg细胞水平、CD8⁺T细胞高于非淋巴结转移组,CD3⁺T细胞、CD4⁺T细胞、CD4⁺/CD8⁺比值均低于非淋巴结转移组（P＜0.05）;经多因素Logistic回归分析,外周血Treg细胞、CD3⁺T细胞、CD4⁺/CD8⁺比值均是早期宫颈癌患者发生淋巴结转移的独立预测因素（P＜0.05）;经ROC曲线分析,外周血Treg细胞、CD3⁺T细胞联合CD4⁺/CD8⁺比值预测早期宫颈癌患者发生淋巴结转移的AUC为0.910。结论：外周血Treg细胞、T淋巴细胞及其亚群水平与早期宫颈癌的病情演变有关,其中外周血Treg细胞、CD3⁺T细胞联合CD4⁺/CD8⁺比值预测淋巴结转移的效能较好,值得进一步研究应用。     

NLR、25-（OH）D3、IL-6、PCT与重症肺炎支原体肺炎患儿免疫功能和预后不良的关系研究

摘要 目的：探讨中性粒细胞与淋巴细胞比值（NLR）、25-羟维生素D3 [25-（OH）D3]、白细胞介素-6（IL-6）、降钙素原（PCT）与重症肺炎支原体肺炎（MMP）患儿免疫功能和预后不良的关系。方法：选取2019年2月至2021年12月我院收治的106例重症MMP患儿作为重症组,同期收治的101例轻症MMP患儿（轻症组）作为对照。检测外周血中性粒细胞计数、淋巴细胞计数、T淋巴细胞亚群以及血清25-（OH）D3、IL-6、PCT水平,计算NLR。分析NLR、25-（OH）D3、IL-6、PCT与T淋巴细胞亚群的相关性。重症MMP患儿治疗后随访半年,根据重症MMP患儿的预后情况分为预后良好组（75例）和预后不良组（31例）,多因素Logistic回归分析影响重症MMP患儿预后的因素。结果：重症组NLR、IL-6、PCT水平,CD8⁺高于轻症组（P＜0.05）,25-（OH）D3水平、CD3⁺、CD4⁺、CD4⁺/CD8⁺低于轻症组（P＜0.05）。NLR、IL-6、PCT水平与CD3⁺、CD4⁺、CD4⁺/CD8⁺呈负相关（P＜0.05）,与CD8⁺呈正相关（P＜0.05）;25-（OH）D3与CD3⁺、CD4⁺、CD4⁺/CD8⁺呈正相关（P＜0.05）,与CD8⁺呈负相关（P＜0.05）。多因素Logistic回归分析显示：肺大片实变影、NLR（较高）、IL-6（较高）、PCT（较高）是重症MPP患儿预后不良的危险因素（P＜0.05）,25-（OH）D3（较高）是保护因素（P＜0.05）。结论：重症MMP患儿NLR、IL-6、PCT水平升高,25-（OH）D3水平降低,且与细胞免疫功能低下以及预后不良有关,检测NLR、IL-6、PCT、25-（OH）D3有助于评估重症MMP患儿的预后。     

YKL-40、CD4+/CD8+比值与腺病毒肺炎患儿炎性因子和并发喘息的关系研究

摘要 目的：探讨血清壳多糖酶3样蛋白1（YKL-40）、外周血CD4⁺/CD8⁺比值与腺病毒肺炎（AP）患儿炎性因子和并发喘息的关系。方法：选取2019年10月～2022年10月湖北省妇幼保健院收治的97例AP患儿为AP组,根据是否并发喘息分别为喘息组和无喘息组,另选取同期50例体检健康儿童为对照组。收集AP患儿的临床资料,采用酶联免疫吸附法检测血清YKL-40和炎性因子[白细胞介素（IL）-6、IL-8、肿瘤坏死因子-α（TNFα）]水平,流式细胞术检测外周血CD4⁺、CD8⁺比例并计算CD4⁺/CD8⁺比值。采用Spearman相关性分析AP患儿血清YKL-40、CD4⁺/CD8⁺比值与炎性因子水平的相关性,多因素Logistic回归分析AP患儿并发喘息的影响因素。结果：与对照组比较,AP组血清YKL-40、外周血CD8⁺比例升高,CD4⁺比例、CD4⁺/CD8⁺比值降低（P＜0.05）。AP组血清IL-6、IL-8、TNF-α水平高于对照组（P＜0.05）。Spearman相关性分析显示,AP患儿血清YKL-40与IL-6、IL-8、TNF-α水平呈正相关,外周血CD4⁺/CD8⁺比值与IL-6、IL-8、TNF-α水平呈负相关（P＜0.05）。97例AP患儿住院期间喘息发生率为50.52%（49/97）。多因素Logistic回归分析显示,呼吸衰竭、小气道病变、特应性体质和血清IL-6、IL-8、TNF-α、YKL-40升高为AP患儿并发喘息的独立危险因素,外周血CD4⁺/CD8⁺比值升高为独立保护因素（P＜0.05）。结论：AP患儿血清YKL-40水平升高和外周血CD4⁺/CD8⁺比值降低,与炎性因子水平升高和并发喘息密切相关。     

扶正方对Lewis肺癌小鼠免疫功能、PI3K/AKT信号通路和外周血IL-2、IL-6、INF-γ的影响

摘要 目的：观察扶正方对Lewis肺癌小鼠免疫功能、磷脂酰肌醇3激酶（PI3K）/蛋白激酶B（AKT）信号通路和外周血白细胞介素（IL）-2、IL-6、γ干扰素（INF-γ）的影响。方法：将40只Lewis肺癌小鼠随机分为模型组（M组）、扶正方低剂量组（A组）、扶正方高剂量组（B组）、顺铂组（S组）,每组10只,A组、B组分别给予扶正方0.4 mL/20 g、0.8 mL/20 g灌胃,M组给予生理盐水0.4 mL/20 g灌胃,S组给予顺铂1 mg/mL,0.4 mL灌胃,连续14d,比较各组小鼠一般情况、肿瘤重量,胸腺指数、脾脏指数、脾脏CD3⁺细胞、CD4⁺细胞、CD8⁺细胞比例细胞百分比,鼠肿瘤组织PI3K/AKT信号通路蛋白表达水平及外周血IL-2、IL-6、INF-γ水平。结果：A组、B组、S组小鼠肿瘤重量低于M组,S组小鼠肿瘤重量低于A组、B组（P<0.05）,治疗前各组小鼠体重比较无统计学差异（P>0.05）,治疗后A组、B组小鼠体重高于S组、M组（P<0.05）。A组、B组小鼠胸腺指数显著高于M组、S组（P<0.05）。A组、B组CD3⁺、CD4⁺、CD4⁺/CD8⁺显著高于M组、S组,CD8⁺显著低于M组、S组（P<0.05）,B组CD3⁺、CD4⁺、CD4⁺/CD8⁺显著高于A组,CD8⁺低于A组（P<0.05）。A组、B组、S组小鼠肿瘤组织PI3K蛋白、AKT蛋白表达水平显著低于M组（P<0.05）。A组、B组、S组小鼠外周血IL-2、INF-γ水平显著高于M组,IL-6水平显著低于M组（P<0.05）。结论：扶正方可以提升Lewis肺癌小鼠免疫功能,调节IL-2、IL-6、INF-γ细胞因子水平,抑制PI3K/AKT信号通路起到抗肺癌的作用。     

IL-7和胸腺基质细胞系(MTEC5)诱导胸腺CD3- CD4- CD8- 细胞分化

胸腺细胞发育是细胞因子及胸腺细胞与胸腺基质细胞相互作用的结果.观察了IL-7及小鼠胸腺基质上皮细胞系MTEC5对成年小鼠CD3^- CD4^- CD8^- 胸腺细胞发育的影响.IL-7能促进TN细胞增殖.TN细胞经在IL-7条件下培养后表达CD3-TCR分子,其中20％～40％为TCRγδ⁺,60％～80％为TCRαβ⁺,但保持CD4^- CD8^- .该细胞获得对 Con A、抗CD3mAb及抗TCRmAb进行增殖应答的能力,抗CD28mAb能促进这种增殖应答,证明表达的CD3-TCR分子功能成熟.当在该系统中加入MTEC5,部分TN细胞转变为CD3⁺TCRβ⁺CD4⁺CD8^- 和CD3⁺TCRβ⁺CD4^- CD8⁺SP细胞,表明MTEC5可诱导TN细胞分化为表现型成熟的胸腺细胞.     

口服维生素D对实验性脑疟小鼠树突状细胞的影响

维生素D（VD）为固醇类衍生物,除发挥抗佝偻病作用,还具有一定的免疫调节功能。主要研究VD对实验性脑疟小鼠树突状细胞的影响。结果显示,与对照组相比,在伯氏疟原虫（P.bANKA）感染前给予VD,可降低C57BL/6小鼠发生脑型疟疾的风险,存活时间明显延长。VD预处理后,脾脏中髓样树突状细胞（CD11c⁺CD11b⁺）和浆样树突状细胞（CD11c⁺B220⁺）百分率明显降低,CD11c⁺MHCII⁺细胞、CD11c⁺TLR4⁺细胞和CD11c⁺TLR9⁺细胞的百分含量也显著减少。由此提示,预防性口服VD可抑制感染小鼠树突状细胞的数量和功能,对脑型疟疾的发生具有一定预防作用,为新型抗疟药物的研发提供参考。     

肝硬化患者的免疫功能障碍与血清壳多糖酶3样蛋白1表达的关系

摘要 目的：探究肝硬化患者的免疫功能障碍与血清壳多糖酶3样蛋白1（CHI3L1）表达关系。方法：招募2018年2月至2020年8月在我院就诊的160例肝硬化患者。通过Child-Pugh评分对肝病的严重程度进行分组,轻度组（n=73）和重度组（n=87）。在本院健康检查中心招募60例年龄相仿且无任何明显疾病或感染的人员作为对照组。并检测CHI3L1蛋白表达、CHI3L1浓度、MR浓度、CD3⁺T、CD25⁺T和CD69⁺T水平以及血浆细胞因子的浓度。结果：对照组较轻度和重度组AST、ALT浓度升高,且轻度组较重度组升高（P<0.05）。Cr血清浓度各组比较无差异（P>0.05）。轻度组中93.15 %患者为Child-Pugh A,重度组中全部为Child-Pugh B/A。重度组较轻度组肝硬化程度严重（P<0.05）。重度组和轻度组较对照组CHI3L1蛋白表达升高,重度组较轻度组升高（P<0.05）。重度组和轻度组血清CHI3L1水平较对照组升高（P<0.05）,重度组较轻度组升高（P<0.05）。重度组和轻度组血清CD163和甘露糖受体（MR）水平较对照组升高（P<0.05）,重度组较轻度组升高（P<0.05）。重度组和轻度组血清CD3⁺T、CD25⁺T和CD69⁺T水平较对照组升高,重度组血清较轻度组升高（P<0.05）。重度组和轻度组白细胞介素（IL-1β）、IL-6、肿瘤坏死因子α（TNF-α）和干扰素（IFN-γ）浓度较对照组升高,重度组较轻度组升高（P<0.05）。Logistic回归分析,提示患者血清CHI3L1、MR、CD3⁺T、CD25⁺T、CD69⁺T与患者肝硬化程度相关（P<0.05）。结论：肝硬化患者中血清CHI3L1与肝硬化严重程度呈正相关,CHI3L1促进T细胞活化、增殖和炎性细胞因子的分泌,这导致加重了免疫介导的肝损伤和纤维化的发展。     

布地奈德联合特布他林雾化吸入治疗对毛细支气管炎患儿潮气呼吸肺功能、T细胞亚群及血清炎性因子的影响

摘要 目的：探讨布地奈德联合特布他林雾化吸入治疗对毛细支气管炎患儿潮气呼吸肺功能、T细胞亚群及血清炎性因子的影响。方法：选取2019年1月~2019年12月期间我院收治的毛细支气管炎患儿98例,采用随机数字表法分为对照组（常规方案治疗）和观察组（对照组基础上加用布地奈德联合特布他林雾化吸入治疗）,各49例。记录两组治疗1周后总有效率。对比两组治疗前、治疗1周后的潮气呼吸肺功能指标[吸气/呼气时间比（Ti/Te）、达峰时间比（TPTEF/TE）、达峰容积比（VPEF/VE）、每千克潮气量（Vt/kg）]、T细胞亚群[CD3⁺、CD4⁺、CD8⁺及CD4⁺/CD8⁺]、血清炎性因子[降钙素原（PCT）、C反应蛋白（CRP）]。比较两组不良反应发生率。结果：+、CD4⁺、CD4⁺/CD8⁺均高于治疗前,且观察组高于对照组（P<0.05）,PCT、CRP、CD8+均低于治疗前,且观察组低于对照组（P<0.05）。两组患儿不良反应发生率对比无统计学差异（P>0.05）。结论：雾化吸入特布他林联合布地奈德治疗毛细支气管炎患儿疗效显著,可有效改善患儿T细胞亚群、潮气呼吸肺功能,减轻患儿炎性反应,且安全性好。     

The development of macrophages from human CD34+ haematopoietic stem cells in serum-free cultures is optimized by IL-3 and SCF

The derivation of human macrophages from peripheral blood monocytes remains a convenient method for the study of macrophage biology. However, for macrophage differentiation, a significant proportion of development has occurred prior to the monocyte stage; monocyte subsets also have varying potential for differentiation. Differentiation of macrophages from a less mature precursor, such as CD34⁺ haematopoietic stem cells, can further inform with regard to the development of macrophage-lineage cells. CD34⁺ cells were cultured in serum-free medium containing Flt3L, SCF, IL-3, IL-6 and M-CSF. Using differing combinations of growth factors, the effect on cell proliferation and differentiation to adherent macrophage-like cells was determined. The proliferative response of CD34⁺ cells to M-CSF was determined during the initial phase of cell culture. Thirteen combinations of SCF, IL-3, IL-6 and M-CSF were then compared to determine the optimum combination for proliferation. Adherence was used to isolate mature macrophages, and the macrophage-like phenotype was confirmed by analyses of surface markers, histo-morphology and phagocytosis. This study refines the means by which large numbers of macrophages are obtained under serum-free conditions from haematopoietic precursors.     

Individual and synergistic cytokine effects controlling the expansion of cord blood CD34(+) cells and megakaryocyte progenitors in culture

Background aimsExpansion of hematopoietic progenitors ex vivo is currently investigated as a means of reducing cytopenia following stem cell transplantation. The principal objective of this study was to develop a new cytokine cocktail that would maximize the expansion of megakaryocyte (Mk) progenitors that could be used to reduce periods of thrombocytopenia.MethodsWe measured the individual and synergistic effects of six cytokines [stem cell factor (SCF), FLT-3 ligand (FL), interleukin (IL)-3, IL-6, IL-9 and IL-11] commonly used to expand cord blood (CB) CD34⁺ cells on the expansion of CB Mk progenitors and major myeloid populations by factorial design.ResultsThese results revealed an elaborate array of cytokine individual effects complemented by a large number of synergistic and antagonistic interaction effects. Notably, strong interactions with SCF were observed with most cytokines and its concentration level was the most influential factor for the expansion and differentiation kinetics of CB CD34⁺ cells. A response surface methodology was then applied to optimize the concentrations of the selected cytokines. The newly developed cocktail composed of SCF, thrombopoietin (TPO) and FL increased the expansion of Mk progenitors and maintained efficient expansion of clonogenic progenitors and CD34⁺ cells. CB cells expanded with the new cocktail were shown to provide good short- and long-term human platelet recovery and lymphomyeloid reconstitution in NOD/SCID mice.ConclusionsCollectively, these results define a complex cytokine network that regulates the growth and differentiation of immature and committed hematopoietic cells in culture, and confirm that cytokine interactions have major influences on the fate of hematopoietic cells.     

Expansion of CD34+ cells from human umbilical cord blood by FL and/or TPO gene transfected human marrow stromal cell lines

To elucidate the effect of gene transfected marrow stromal cell on expansion of human cord blood CD34⁺ cells, a culture system was established in which FL and TPO genes were transfected into human stromal cell line HFCL. To establish gene transfected stromal cells co-culture system, cord blood CD34⁺ cells were purified by using a magnetic beads sorting system. The number of all cells and the number of CD34⁺ cells and CFC (CFU-GM and BFU-E) were counted in different culture systems. The results showed that in all 8 culture systems, SCF+IL-3+HFT manifested the most potent combination, with the number of total nucleated cells increasing by (893.3 ±52.1)-fold, total progenitor cells (CFC) by (74.5 ±5.2)-fold and CD34⁺ cells by 15.7-fold. Maximal expansions of CFC and CD34⁺ cells were observed at the end of the second week of culture. Within 14 days of culture, (78.1 ± 5.5)-fold and (57.0 ± 19.7)-fold increases in CFU-GM and BFU-E were obtained. Moreover, generation of LTC-IC from amplified CD34⁺ cells within 28 days was found only in two combinations, i.e. SCF+IL-3+FL+TPO and SCF+IL-3+HFT, and there was no significant difference between these two groups statistically. These results suggest that human umbilical cord blood CD34⁺ cells can be extensively expandedex vivo by using gene transfected stromal cells along with cytokines.     

Ex vivo expansion of hematopoietic stem cells in a proliferation chamber with external stirred conditioning tank: Sequential optimization of growth factors

The insufficient number of hematopoietic stem cells (HSCs) extracted from various cell sources creates the need for the use of growth factors in the culture systems. Numerous types and concentrations of growth factors have been reported in the literature. In this study, we investigated the effect of three important hematopoietic growth factors thrombopoietin (TPO), stem cell factor (SCF), and Fms‐like tyrosine kinase‐3 ligand (Flt‐3) as well as cell‐seeding density on ex vivo expansion of human HSCs using a factorial design. Sequential optimization was then followed by methods of steepest ascent and central composite design. The optimum concentrations of growth factors were 50, 90, and 34 ng/mL for SCF, TPO, and Flt‐3, respectively, at an initial cell density of 2.5 × 10⁵ cells/mL. Effective expansion factor value (EEF) of HSCs increased considerably and revealed almost similar results when the cells were cultured in a 24‐well plate (EEF = 4.54 ± 0.43) and a proliferation chamber with an external stirred conditioning tank (PC‐ESCT; EEF = 5.1 ± 0.35) at seeding density of 2.5 × 10⁵ cells/mL after 7 days. The cells did not show considerable changes in proliferation when they were cultured in medium containing serum or in a commercial serum‐free medium at the optimum concentrations of the growth factors. The present study demonstrated the optimum condition of hematopoietic growth factors as well as the potential of PC‐ESCT for ex vivo expansion of HSCs.     

Metallothionein in human immunomagnetically selected CD34+ haematopoietic progenitor cells

Human umbilical CD34⁺ immature haematopoietic cells were rapidly and efficiently obtained from light density MNC (mononuclear cells) by MACS (magnetic cell sorting). An ex vivo expanded population of CD34⁺ was cultured in serum‐free medium supplemented with cytokines FL (flt3 ligand), SCF (stem cell factor) and TPO (thrombopoietin) in order to obtain a sufficient number of CD34⁺ cells. CD34⁺ cells expanded from cord blood for 7 days were demonstrated to increase in the absolute number of CD34⁺ cells by 5.12±2.47‐fold (mean±S.D., n=3). Flow cytometric analysis demonstrated that the percentage of CD34 antigen expression after expansion of the culture was 97.81±1.07%, whereas it was 69.39±10.37% in none‐expanded CD34⁺ cells (mean±S.D., n=3), thus defining a system that allowed extensive amplification accompanied by no maturation. MTs (metallothioneins), low molecular weight, cysteine‐rich metal‐binding proteins, exhibit various functions, including metal detoxification and homoeostasis. We here examined the expression pattern of functional members of the MT gene family in immature CD34⁺ cells and compared it with more mature CD34^? cells in order to strengthen the proposed function of MT in differentiation. Cells were cultured in RPMI 1640 medium, with or without different zinc supplements for 24 h. Relative quantitative expression of MT isogenes in the mature CD34^? cells was higher than in the immature CD34⁺ cells. IHC (immunohistochemical staining) revealed an increased MT protein biosynthesis in CD34^? cells, greater than in CD34⁺ cells. Therefore, the role of MT in differentiation of human haematopoietic progenitor cells from human cord blood is reported for the first time.     

<Emphasis Type="Italic">Ex vivo</Emphasis> expansion of hematopoietic cells from CD34<Superscript>+</Superscript> cord blood cells in various culture conditions

This study compared the cell expansion and colony-forming ability of human cord blood stem cells cultured ex vivo with 2 types of cytokine combinations, and 2 types of media characterized by the presence or absence of fetal bovine serum (FBS) in 2 or 3 dimensional (2D or 3D) culture environments. Purified CD34⁺ cells derived from different donors were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) and Ultraculture serum-free medium (SFM) containing the cytokine cocktail-I (coc-I) (EPO, GM-CSF, SCF, and IL-3) or cytokine cocktail-II (coc-II) (TPO, G-CSF, SCF, IL-6, and Flt3/Flk-2 ligand) with or without FBS. Generally, higher CFU-GM values were observed in the IMDM compared to the SFM. In the coc-I conditions, the ‘IMDM + coc-I’ and ‘IMDM + coc-I + FBS’ conditions gave the greatest cell (1,667 ± 274 and 1,600 ± 140-fold, respectively) and colony-forming units (CFU) expansions (BFU-E: 21 ± 3, 36 ± 5; CFU-GM: 95 ± 19, 81 ± 17; and CFU-GEMM: 2 ± 1, 3 ± 1-fold, respectively) in 26 day culture, respectively. In the coc-II conditions, the ‘SFM + coc-II’ condition gave the greatest cell expansion (2,143 ± 134-fold), but the ‘IMDM + coc-II’ condition gave the best CFU-GM expansion (924 ± 110-fold) in 26 day culture. In conclusion, ‘IMDM + coc-I’ and ‘IMDM + coc-II’ were the most accessible conditions for CFU expansion for all culture cases. The 2D stationary culture had affirmative effect on CFU expansion compared to the 3D culture using semisolid Methocult™. These results are believed to be significant in the ex vivo expansion of hematopoietic stem cells.     

Different growth factor requirements for the ex vivo amplification of transplantable human cord blood cells in a NOD/SCID mouse model

The growth factor combination containing early acting cytokines FLT-3 ligand (FL), Stem Cell Factor (SCF) and thrombopoietin (TPO) is able to maintain, for an extended culture period, early stem cells, defined as long-term repopulating NOD/SCID mice (Scid Repopulating Cell-SRC) contained in cord blood (CB). In this culture system, the role of IL-6 and IL-3 has not been clearly established. Using a combination of FL+TPO+SCF with or without IL-6, we were able to form CB CD34+ cells for 30 weeks. The CB CD34+ cells cultured in this system engrafted NOD/SCID mice after 6 weeks of culture; the cells from primary recipients were also able to engraft secondary NOD/SCID mice. When CB CD34+ cells were cultured in the presence of IL-3 in the place of IL-6 we observed an even better expansion of cells and a similar clonogenic progenitor output in the first 8 weeks of culture. However, more primitive LTC-IC output increased up to week 6 with the growth factor combination containing IL-3 and then decreased and disappeared, while with the growth factor combination with or without IL-6 increased up to week 23. Cells cultured for 4 weeks with the 4-factor combination containing IL-3 engrafted NOD/SCID mice less efficiently. Repopulation of NOD/SCID mice was no longer observed when ex vivo expansion was performed for 6 weeks. This study provides some evidence that no differences could be detected in long-term maintenance and even expansion of human primitive cord blood cells cultured with FL+TPO+SCF in the presence or absence of IL-6. Under the culture conditions employed in this study, the presence of IL-3 reduced the repopulating potential of expanded CB CD34+ cells.     

In vitro propagated dendritic cells from patients with human-papilloma virus-associated preneoplastic lesions of the uterine cervix: use of Flt3 ligand

Dendritic cells (DC) are the most efficient antigen presenting cells. The clinical use of DC as vectors for antitumor and anti-infectious disease immunotherapy has been limited by their low level and accessibility in normal tissue. Substantial numbers of DC can be generated from peripheral blood cultured in the presence of interleukin-4 (IL-4) and granulocyte/macrophage-colony-stimulating factor (GM-CSF). We showed in this study that substantial numbers of DC can be obtained from the peripheral blood of patients with (pre)neoplastic lesions of the uterine cervix. The procedure required relatively small blood samples (10 ml) and the presence of 100 U/ml IL-4 and 800 U/ml GM-CSF in the culture medium. There was no significant difference in the morphology, yield, phenotype and function of generated DC between patients with cervical (pre)neoplastic lesions and healthy individuals. When the hematopoietic factor Flt3 ligand (Flt3L, 40 ng/ml) was added, there was an average increase in the DC population of 26% compared to cultures with GM-CSF and IL-4 alone. Approximately 1.2 × 10⁶ cells with the characteristics of dendritic cells could be obtained when Flt3L was included in the medium. The addition of Flt3L did not modify the phenotypic profile of DC (HLA-DR⁺, CD1a⁺, CD4⁺, CD54⁺, CD80⁺, CD86⁺, CD40⁺, CD3⁻ and CD14⁻). In addition, Flt3L generated functional DC capable of stimulating the proliferation of alloreactive T cells. These results suggest that Flt3L, in association with GM-CSF and IL-4, provides an advantageous tool for the large-scale generation of DC and that an immunotherapy based on the use of DC generated in vitro is possible in patients with (pre)neoplastic lesions of the uterine cervix. Received: 8 January 1998 / Accepted: 30 April 1998     

Bone marrow niche-inspired,multiphase expansion of megakaryocytic progenitors with high polyploidization potential

Background aimsMegakaryopoiesis encompasses hematopoietic stem and progenitor cell (HSPC) commitment to the megakaryocytic cell (Mk) lineage, expansion of Mk progenitors and mature Mks, polyploidization and platelet release. pH and pO₂ increase from the endosteum to sinuses, and different cytokines are important for various stages of differentiation. We hypothesized that mimicking the changing conditions during Mk differentiation in the bone marrow would facilitate expansion of progenitors that could generate many high-ploidy Mks.MethodsCD34⁺ HSPCs were cultured at pH 7.2 and 5% O₂ with stem cell factor (SCF), thrombopoietin (Tpo) and all combinations of Interleukin (IL)-3, IL-6, IL-11 and Flt-3 ligand to promote Mk progenitor expansion. Cells cultured with selected cytokines were shifted to pH 7.4 and 20% O₂ to generate mature Mks, and treated with nicotinamide (NIC) to enhance polyploidization.ResultsUsing Tpo + SCF + IL-3 + IL-11, we obtained 3.5 CD34⁺ CD41⁺ Mk progenitors per input HSPC, while increasing purity from 1% to 17%. Cytokine cocktails with IL-3 yielded more progenitors and mature Mks, although the purities were lower. Mk production was much greater at higher pH and pO₂. Although fewer progenitors were present, shifting to 20% O₂/pH 7.4 at day 5 (versus days 7 or 9) yielded the greatest mature Mk production, 14 per input HSPC. NIC more than doubled the percentage of high-ploidy Mks to 40%.ConclusionsWe obtained extensive Mk progenitor expansion, while ensuring that the progenitors could produce high-ploidy Mks. We anticipate that subsequent optimization of cytokines for mature Mk production and delayed NIC addition will greatly increase high-ploidy Mk production.     

Large generation of megakaryocytes from serum-free expanded human CD34 cells

Ex vivo generation of megakaryocytes from hematopoietic stem cells (HSCs) is crucial to HSC research and has important clinical potential for thrombocytopenia patients to rapid platelet reconstruction. In this study, factorial design and steepest ascent method were used to screen and optimize the effective cytokines (10.2 ng/ml TPO, 4.3 ng/ml IL-3, 15.0 ng/ml SCF, 5.6 ng/ml IL-6, 2.8 ng/ml FL, 2.8 ng/ml IL-9, and 2.8 ng/ml GM-CSF) in megakaryocyte induction medium that facilitate ex vivo megakaryopoiesis from CD34⁺ cells. After induction, the maximum fold expansion for accumulated megakaryocytes was almost 5000-fold, and the induced megakaryocytes were characterized by analysis of gene expression, polyploidy and platelet activation ability. Furthermore, the combination of megakaryocyte induction medium and HSC expansion medium can induce and expand a large amount of functional megakaryocytes efficiently, and might be a promising source of megakaryocytes and platelets for cell therapy in the future.