Increased megakaryopoiesis in cultures of CD34-enriched cord blood cells maintained at 39 degrees C

Based on previous evidence suggesting positive effects of fever on in vivo hematopoiesis, we tested the effect of hyperthermia on megakaryopoiesis (MK) in ex vivo cultures of CD34-enriched cord blood (CB) cells. The cells were cultured at 37 degrees C or 39 degrees C for 14 days in cytokine conditions optimized for megakaryocyte development and analyzed periodically. Compared to 37 degrees C, the cultures maintained at 39 degrees C produced significantly more (up to 10-fold) total cells, myeloid and MK progenitors, and total MKs, and showed accelerated and enhanced MK maturation with increased yields of proplatelets and platelets. This observation could facilitate clinical applications requiring ex vivo expansion of hematopoietic cells.     

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％。     

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.     

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.     

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 expanded ex vivo by using gene transfected stromal cells along with cytokines.     

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 2     

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。     

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.     

Presence of osteoclast precursor cells during ex vivo expansion of bone marrow-derived mesenchymal stem cells for autologous use in cell therapy

Background aimsTo obtain a cell product competent for clinical use in terms of cell dose and biologic properties, bone marrow-derived mesenchymal stem cells (MSCs) must be expanded ex vivo.MethodsA retrospective analysis was performed of records of 76 autologous MSC products used in phase I or II clinical studies performed in a cohort of cardiovascular patients. In all cases, native MSCs present in patient bone marrow aspirates were separated and expanded ex vivo.ResultsThe cell products were classified in two groups (A and B), according to biologic properties and expansion time (ex vivo passages) to reach the protocol-established cell dose. In group A, the population of adherent cells obtained during the expansion period (2 ± 1 passages) was composed entirely of MSCs and met the requirements of cell number and biologic features as established in the respective clinical protocol. In group B, in addition to MSCs, we observed during expansion a high proportion of ancillary cells, characterized as osteoclast precursor cells. In this case, although the biologic properties of the resulting MSC product were not affected, the yield of MSCs was significantly lower. The expansion cycles had to be increased (3 ± 1 passages).ConclusionsThese results suggest that the presence of osteoclast precursor cells in bone marrow aspirates may impose a limit for the proper clinical use of ex vivo expanded autologous bone marrow-derived MSCs.