人脐带血来源造血干细胞体外扩增的研究进展

造血干细胞（HSCs）是血液系统中的一类成体干细胞群,具有自我更新和多谱系分化两个基本特征。造血干细胞移植（HSCT）可以治疗退行性疾病和多种血液系统疾病。脐带血来源造血干细胞（CB HSCs）是降低HLA配型要求的突破点,但单份脐带血中HSCs数量不能满足使用要求,为了获得足够数量的CB HSCs,体外扩增是一种可行的方法。近几年,学者们探索了多种体外扩增方法,包括优化细胞生长因子混合物、与基质细胞共培养及加入小分子化合物（SMCs）激动剂等。目前应用细胞因子联合小分子的扩增方法在多个临床试验中获得成功。本文对目前体外扩增CB HSCs的研究进展做一综述。     

用悬浮搅拌培养体系体外大量扩增人脐血造血干/祖细胞

目的 :建立一种简便、有效的脐血造血干 /祖细胞体外大量扩增培养体系。方法 :淋巴细胞分离液分离的脐血单个核细胞在SCF ,IL - 3,IL - 6三种细胞因子的作用下 ,于悬浮搅拌培养体系中培养 ,分析其总细胞数、CFU -GM、CD34+ 细胞的扩增倍数。结果 :脐血单个核细胞在悬浮搅拌培养体系中培养 12天后 ,其总细胞数、CFU -GM、CD34+ 细胞的扩增倍数分别为 6 .31± 1.5 2 ,2 0 .6 3± 1.5 4和 7.11± 1.12。结论 :悬浮搅拌培养体系是脐血造血干 /祖细胞体外大量扩增的有效培养体系。     

Hematopoietic capacity of preterm cord blood hematopoietic stem/progenitor cells

Full-term cord blood (TCB) hematopoietic stem/progenitor cells (HSC/HPCs) are used for stem cell transplantation and are well characterized. However, the properties of preterm cord blood (PCB) HSC/HPCs remain unclear. In the present study, we compared HSC/HPCs from TCB and PCB with respect to their expression of surface markers, homing capacity and ability to repopulate HSCs in the NOD/Shi-scid mice bone marrow. The proportion of CD34+CD38− cells was significantly higher in PCB. On the other hand, the engraftment rate of TCB CD34+ cells into NOD/Shi-scid mice was significantly higher than PCB CD34+ cells. The expression of VLA4 was stronger among TCB CD34+ cells than PCB CD34+ cells. Moreover, there was a positive correlation between the proportion of CD34+CXCR4+ cells and gestational age. These data suggest that the homing ability of HSCs increases during gestation, so that TCB may be a better source of HSCs for transplantation than PCB.     

Experimental study on ex vivo expanded hematopoietic stem/progenitor in the two step culture from human umbilical cord blood transplanted into NOD/SCID mice

The effects of hematopoietic stem/progenitor cells (HSPCs) expanded in the two step coculture with human bone marrow mesenchymal stem cells (hMSCs) on the hematopoietic reconstruction of irradiated NOD/SCID mice were studied. Mononuclear cells (MNCs) were isolated from human umbilical cord blood (UCB) and cultured in the non-coculture scheme of rhSCF + rhG-CSF + rhMDGF combination and the coculture scheme of rhSCF + rhG-CSF + rhMDGF + hMSCs. Sublethally-irradiated NOD/SCID mice were transplanted with ex vivo expanded HSPCs with the dose of 8.5 × 10⁶ cells per mouse. After transplantation, the dynamics of WBC in the transplanted mice was measured periodically, and the Alu sequence fragment special for human in the transplanted mice was inspected by PCR. Results showed that the coculture scheme increased proliferation of UCB-derived HSPCs. After transplantation with expanded HSPCs, the population of WBC in the transplanted mice increased in 12 d and reached the first peak in 25 d, then showed the second increasing of WBC in 45∼55 d. Expanded cells from the coculture scheme appeared to be favorable for the second increasing of WBC in the transplanted mice. After 85 d, the Alu sequence fragment was detected in the probability of 87.5% (7/8) for the non-coculture scheme and 88.9% (8/9) for the coculture scheme. __________ Translated from Journal of Zhejiang University (Science Edition), 2005, 32 (4) [译自: 浙江大学学报 (理学版), 2005, 32 (4)]     

Experimental study on ex vivo expanded hematopoietic stem/progenitor in the two step culture from human umbilical cord blood transplanted into NOD/SCID mice

The effects of hematopoietic stem/progenitor cells(HSPCs)expanded in the two step coculture with human bone marrow mesenchymal stem cells(hMSCs)on the hematopoietic reconstruction of irradiated NOD/SCID mice were studied.Mononuclear cells(MNCs)were isolated from human umbilical cord blood(UCB)and cultured in the non-coculture scheme of rhSCF+rhG-CSF +rhMDGF combination and the coculture scheme of rhSCF+rhG-CSF+rhMDGF+hMSCS.Sublethally-irradiated NOD/SCID mice were transplanted with ex vivo expanded HSPCS with the dose of 8.5×106 cells per mouse.After transplantation.the dynamics of WBC in the transplanted mice was measured periodically,and the Alu sequence fragment special for human in the transplanted mice was inspected by PCR.Results showed that the coculture scheme increased proliferation of UCB-derived HSPCs.After transplantation with expanded HSPCS,the population of WBC in the transplanted mice increased in 12 d and reached the first peak in 25 d,then showed the second increasing of WBC in 45～55 d.Expanded cells from the coculture scheme appeared to be favorable for the second increasing of WBC in the transplanted mice.After 85 d,the Alu sequence fragment was detected in the probability of 87.5%(7/8)for the non-coculture scheme and 88.9%(8/9)for the coculture scheme.     

Human mesenchymal stem cells from the umbilical cord matrix: Successful isolation and ex vivo expansion using serum-/xeno-free culture media

Mesenchymal stem cells (MSC) could potentially be applied in therapeutic settings due to their multilineage differentiation ability, immunomodulatory properties, as well as their trophic activity. The umbilical cord matrix (UCM) represents a promising source of MSC for biomedical applications. The number of cells isloated per umbilical cord (UC) unit is limited and ex vivo expansion is imperative in order to reach clinically meaningful cell numbers. The limitations of poorly defined reagents (e.g. fetal bovine serum, which is commonly used as a supplement for human MSC expansion) make the use of serum-/xeno-free conditions mandatory. We demonstrated the feasibility of isolating UCM-MSC by plastic adherence using serum-/xeno-free culture medium following enzymatic digestion of UCs, with a 100% success rate. 2.6 ± 0.21 × 10⁵ cells were isolated per UC unit, of which 1.9 ± 0.21 × 10⁵ were MSC-like cells expressing CD73, CD90, and CD105. When compared to adult sources (bone marrow-derived MSC and adipose-derived stem/stromal cells), UCM-MSC displayed a similar immunophenotype and similar multilineage differentiation ability, while demonstrating a higher expansion potential (average fold increase of 7.4 for serum-containing culture medium and 11.0 for xeno-free culture medium (P3-P6)). The isolation and expansion of UCM-MSC under defined serum-/xeno-free conditions contributes to safer and more effective MSC cellular products, boosting the usefulness of MSC in cellular therapy and tissue engineering.     

Application of proteoglycan extracted from the nasal cartilage of salmon heads for ex vivo expansion of hematopoietic progenitor cells derived from human umbilical cord blood

Highly purified proteoglycan (PG) extracted from the nasal cartilage of salmon heads was applied to the ex vivo expansion of hematopoietic progenitor cells prepared from human umbilical cord blood in serum-free cultures supplemented with the combination of early-acting cytokines, thrombopoietin (TPO), interleukin-3 (IL-3) and stem cell factor (SCF). PG showed no promoting effects on the cell proliferation rate; however, they promoted the generation of progenitor cells for granulocyte-macrophages, erythrocytes and/or megakaryocytes in culture with TPO alone or SCF plus TPO. However, no promoting effect was observed in a combination of IL-3 plus SCF, which showed the highest cell proliferation rate. PG failed to promote the generation of mixed colony-forming units (i.e. the relatively immature cells in hematopoiesis). These results suggest that PG acts on the relatively mature stem/progenitor cells, and may function as a regulatory factor in the differentiation pathway of hematopoiesis.     

Effects of human yolk sac endothelial cells on supporting expansion of hematopoietic stem/progenitor cells from cord blood

In order to investigate the effects of human yolk sac-derived endothelial cells (hYSECs) on the expansion of human hematopoietic stem/progenitor cells (HS/PCs) from umbilical cord blood (UCB) in vitro, we purified hYSEC-like cells from 4-5 week human yolk sacs, which were morphologically similar to endothelial cells and expressed CD31, CD144 and vWF characteristics of endothelial cells. Then we isolated CD34(+) cells from UCB in culture under three different conditions: with hematopoietic cytokines (CKs), contact-coculture or noncontact-coculture with hYSECs supplemented with CKs, and found that the contact-coculture system had the strongest expansion efficiency in the total cells' (TCs) ability to form HPP-CFCs. Erythroid burst-forming units (BFU-E) increased 52.35-fold, 20.26-fold and 27.77-fold, respectively, compared with pre-expansion. We detected that the mRNA of Notch ligands such as Jagged1, Delta1 and Delta4 could express in hYSECs after contacted culture with UCB-CD34(+) cells but not the noncontacted cells by RT-PCR analysis. Therefore, we concluded that the contact-coculture system supplemented with CKs could support the expansion of UCB-HS/PCs in vitro, especially high potential proliferative colony-forming cells (HPP-CFC) and BFU-E, perhaps owing to Notch signal pathway.     

脐血造血细胞体外保存及扩增的研究

研究了脐血造血细胞在4℃冰箱保存过程中单核细胞活力、造血性能的变化以及细胞因子种类、组合、不同培养基添加成分对造血细胞扩增的影响。研究表明脐血在4℃冰箱保存应不超过3d;细胞因子组合SCF IL-6 FL TPO扩增CFU—C的效果最佳;培养基体系中添加脐血混合血浆对扩增CFU—C作用明显,脐血混合血浆的最佳浓度为25％。     

UM171和SR1对不同来源造血干/祖细胞体外扩增的作用研究

目的探讨小分子化合物UM171和SR1对脐带血、供者动员外周血和淋巴瘤患者自体动员外周血3种来源的造血干/祖细胞(HSPCs)体外扩增的作用。方法将3种来源的CD34+细胞分别予以UM171、SR1干预后进行体外扩增培养,记为对照组、UM171组、SR1组和UM171+SR1组。通过细胞计数检测各组总有核细胞的数量,流式细胞术检测HSPCs的比例、各谱系分化细胞的比例和HSPCs上归巢相关因子CXCR4的表达水平。多组数据若满足方差齐性,采用单因素方差分析,组间两两比较采用LSD-t检验;若方差不齐,多组间比较以及两两比较均采用Kruskal-Wallis检验。结果与对照组比较,UM171和SR1均能促进3种来源HSPCs的比例升高,同时UM171能够增加3种来源HSPCs的扩增倍数。与对照组比较,UM171处理后脐带血来源的CD33^+(髓系)细胞的比例升高,CD41^+(巨核)细胞的比例降低;SR1处理后3种来源的CD3-CD56^+(自然杀伤)细胞的比例均升高。体外扩增48 h后各组HSPCs上CXCR4的表达较培养前增加。结论UM171能够有效扩增3种来源HSPCs的数量,促进脐带血来源HSPCs分化为髓系细胞并抑制其分化为巨核细胞。SR1能够促进3种来源HSPCs分化为自然杀伤细胞。体外扩增培养可以提高3种来源HSPCs上CXCR4的表达水平。     

Role of the plasma membrane ROS-generating NADPH oxidase in CD34+ progenitor cells preservation by hypoxia

Hypoxia favored the preservation of progenitor characteristics of hematopoietic stem and progenitor cells (HSPCs) in bone marrow. This work aimed at studying the role of reactive oxygen species (ROS)-generating NADPH oxidase system regulated by hypoxia in ex vivo cultures of cord blood CD34⁺ cells. The results showed that NADPH oxidase activity and ROS generation were reduced in hypoxia with respect to normal oxygen tension. Meanwhile the ROS generation was found to be inhibited by diphenyleneiodonium (the NADPH oxidase inhibitor), or N-acetylcysteine (the ROS scavenger). Accordingly NADPH oxidase mRNA and p67 protein levels decreased in hypoxia. The analysis of progenitor characteristics, including the proportion of cultured cells expressing the HSPCs marker CD34⁺CD38⁻, colony production ability of the colony-forming cells (CFCs), and the re-expansion capability of the cultured CD34⁺ cells, showed that either 5% pO₂ or reduced ROS favored preserving the characteristics of CD34⁺ progenitors, and promoted the expansion of CD34⁺CD38⁻ cells as well. The above results demonstrated that hypoxia effectively maintained biological characteristics of CD34⁺ cells through keeping lower intracellular ROS levels by regulating NADPH oxidase.     

HIV-gp120 induced cell death in hematopoietic progenitor CD34+ cells

Haematologic abnormalities accompany the majority of HIV-1 infections. At present it is unclear whether this is due directly to HIV infection of hematopoietic progenitor cells, or whether this results from an indirect mechanism secondary to HIV infection. Here we provide evidence for an indirect mechanism, whereby hematopoietic progenitor cells undergo HIV gp120-induced apoptosis (programmed cell death) even in the absence of HIV infection. Freshly isolated, purified human hematopoietic progenitor CD34+ cells, derived from both umbilical cord blood and bone marrow, co-expressed the CD4 marker at low density on their surface. Although these CD34+CD4+ cells theoretically should be capable of productive infection by HIV, we found that HIV-IIIB could not establish productive infection in these cells. Nonetheless, gp120 from IIIB could bind the cells. Thus, binding of gp120 did not correlate with infectivity. Furthermore, binding of gp120 was a specific event, leading to apoptosis upon crosslinking with anti-gp120 through a fas-dependent mechanism. If apoptosis is also observed in vivo even in uninfected hematopoietic cells, this could contribute significantly to the impairment in hematopoietic cell number and function. Our data suggest a novel indirect mechanism for depletion of CD34+ and CD34+-derived cells even in the absence of productive viral infection of these cells.     

一种新型生物反应器在造血干/祖细胞体外扩增中的应用

针对造血干/祖细胞体外扩增对培养环境的需求, 结合静/动态培养的特点, 开发了一种新型的生物反应器用于造血干/祖细胞的体外扩增.在该生物反应器内, 采用SCF TPO Flt-3细胞因子组合, 比较了静态和循环培养两种方式体外扩增脐血CD34 细胞的效果.培养7 d后, 总细胞分别扩增了(13.86 ± 4.26)和(7.23 ± 2.67)倍, 显示静态培养有利于总细胞的扩增; CD34 细胞扩增倍数、培养物中CD34 细胞含量均相近, 无显著性差异; 而CD34 CD38-细胞扩增倍数以及培养物中CD34 CD38-细胞的百分含量分别为(1.82 ± 0.58)和(3.90 ± 0.85)倍以及(9.45 ± 4.85)和(37.47 ± 14.06)%, 循环培养明显高于静态培养.可见, 在该生物反应器内, 采用静态和循环两种培养方式, 均能实现造血干/祖细胞的体外扩增, 但静态培养促使造血干细胞向定向祖细胞分化, 而循环培养则更有利于早期造血干细胞的扩增.     

一种新型生物反应器在造血干/祖细胞体外扩增中的应用

针对造血干/祖细胞体外扩增对培养环境的需求, 结合静/动态培养的特点, 开发了一种新型的生物反应器用于造血干/祖细胞的体外扩增。在该生物反应器内, 采用SCF+TPO+Flt-3细胞因子组合, 比较了静态和循环培养两种方式体外扩增脐血CD34+细胞的效果。培养7 d后, 总细胞分别扩增了(13.86 ± 4.26)和(7.23 ± 2.67)倍, 显示静态培养有利于总细胞的扩增; CD34+细胞扩增倍数、培养物中CD34+细胞含量均相近, 无显著性差异; 而CD34+CD38-细胞扩增倍数以及培养物中CD34+CD38?细胞的百分含量分别为(1.82 ± 0.58)和(3.90 ± 0.85)倍以及(9.45 ± 4.85)和(37.47 ± 14.06)%, 循环培养明显高于静态培养。可见, 在该生物反应器内, 采用静态和循环两种培养方式, 均能实现造血干/祖细胞的体外扩增, 但静态培养促使造血干细胞向定向祖细胞分化, 而循环培养则更有利于早期造血干细胞的扩增。     

用差异显示法分析不同生长环境中的造血干细胞/祖细胞基因表达的初步研究

对比分析不同生长环境中的脐血CD34+造血干/祖细胞基因表达变化。方法: 采用静态和动态培养系统培养脐血单个核细胞,1周后收获CD34+造血干/祖细胞, 提取总RNA, 用差异显示法对比分析在不同生长环境中造血干/祖细胞基因表达的差异。 结果: 在所使用的差异显示条件下,得到30个差异表达基因片段,其中一个差异表达片段为RAN基因,该基因属于RAS癌基因家族,可能与造血细胞增殖有关。结论: 不同生长环境影响CD34+造血干/祖细胞的基因表达,这些差异表达的基因可为优化体外培养环境,扩增造血细胞提供分子基础。     

Quality evaluation of umbilical cord blood progenitor cells cryopreserved with a small-scale automated liquid nitrogen system

The performance of a small-scale automated cryopreservation and storage system (Mini-BioArchive system) used in the banking of umbilical cord blood (UCB) units was evaluated. After thawing the units, the viability and recovery of cells, as well as the recovery rate of hematopoietic progenitor cells (HPCs) such as CD34⁺ cells, colony-forming unit-granulocyte-macrophage (CFU-GM), and total CFU were analyzed. Twenty UCB units cryopreserved using the automated system and stored for a median of 34 days were analyzed. Mean CD34⁺ cell viabilities before freezing were 99.8 ± 0.5% and after thawing were 99.8 ± 0.4% in the large bag compartments and 99.7 ± 0.5% in the small compartments. The mean recovery values for total nucleated cells, CD34⁺ cells, CFU-GM, and total CFU were 94.8 ± 16.0%, 99.3 ± 18.6%, 103.9 ± 20.6%, and 94.3 ± 12.5%, respectively in the large compartments, and 95.8 ± 25.9%, 106.8 ± 23.9%, 101.3 ± 23.3%, and 93.8 ± 19.2%, respectively in the small compartments. A small-scale automated cryopreservation and storage system did not impair the clonogenic capacity of UCB HPCs. This cryopreservation system could provide cellular products adequate for UCB banking and HPC transplantation.     

Cytokine effect on ex vivo expansion of haemopoietic stem cells from different human sources

Human pluripotential stem cells (PSC) are currently the target for transplantation attempts and genetic manipulation. We have therefore investigated the frequency and the expansion potential of PSC’s in different types of blood samples. CD 34⁺ cells were thus obtained from human bone marrow (BM), as well as from peripheral blood (PB) and cord blood (CB) samples. After immuno-magnetic separation the highest yields of CD 34⁺ cells were from BM (1.08–2.25%) and CB (0.42–1.32%) while PB samples gave much lower values. Suspension cultures of PSC’s from the three sources were then set up, in the presence of combinations of haemopoietic growth factors. A remarkable amplification of the nucleated cell pool was observed reaching a maximum between 10 and 15 days of culture; earliest and maximum expansion (up to 220-fold) was achieved when Erythropoietin (Epo) was added to the culture medium, but this resulted in reduction of colony-forming cells and differentiation into erythroid progenitors. Clonogenic tests for BFU-E’s derived colonies showed a peak value at 5 days of liquid culture. Further studies are advisable to establish the best cytokine combination for a valuableex vivo expansion, coupled with preservation of stem cell properties.