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 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 differentiation of natural killer cells from human umbilical cord blood CD34+ progenitor cells

Abstract

Natural Killer (NK)-cells are peripheral blood lymphocytes that represent an important arm of the innate immune system. NK-cells play a critical role in the immune surveillance against tumors and virally infected cells in a major histocompatibiliy complex (MHC)-unrestricted fashion. We have explored such capacities of NK-cells after differentiation from hematopoietic stem and progenitor cells derived from human umbilical cord blood. Several culture conditions have been established supporting proliferation and subsequent differentiation of these cells in terms of receptor expression and specific lysis depending on the growth conditions in the presence and absence of supportive stromal feeders. We show that acquisition of Killer Immunoglobulin Receptor (KIR) as well as NK Cytotoxicity Receptor expressions is independent of culture condition whereas absence of stromal feeders did not support acquisition of CD94/NKG2A expression. Such KIR-positive/NKG2A-negative cells generated under different culture conditions showed strong and specific cytolytic activity which could have impact on further immunotherapeutic strategies.     

Impairment in the telocyte/CD34+ stromal cell network in human rheumatoid arthritis synovium

Telocytes (TCs)/CD34⁺ stromal cells have recently emerged as peculiar interstitial cells detectable in a variety of organs throughout the human body. TCs are typically arranged in networks establishing unique spatial relationships with neighbour cells and likely contributing to the maintenance of tissue homeostasis by both cell-to-cell contacts and releasing extracellular vesicles. Hence, TC defects are being increasingly reported in different pathologies, such as chronic inflammatory and fibrotic conditions. In this regard, TCs/CD34⁺ stromal cells have been shown to constitute an intricate interstitial network in the subintimal area of the normal human synovial membrane, but whether they are altered in chronic synovitis has yet to be explored. We therefore undertook a morphologic study to compare the distribution of TCs/CD34⁺ stromal cells between normal synovium and chronically inflamed synovium from patients with rheumatoid arthritis (RA) by using CD34 immunohistochemistry and CD31/CD34 double immunofluorescence. CD34 immunostaining revealed that, at variance with normal synovium, the inflamed and hyperplastic RA synovial tissue was nearly or even completely devoid of TCs/CD34⁺ stromal cells. Double immunofluorescence confirmed that almost all CD34⁺ tissue components detectable in RA synovium were blood vessels coexpressing CD31, while a widespread network of CD31⁻/CD34⁺ TCs was clearly evident in the whole sublining layer of normal synovium. In the context of the emerging diverse roles of TCs/CD34⁺ stromal cells in the regulation of tissue homeostasis and structure, the remarkable impairment in their networks herein uncovered in RA synovium may suggest important pathophysiologic implications that will be worth investigating further.     

Support of hMSCs transduced with TPO/FL genes to expansion of umbilical cord CD34+ cells in indirect co-culture

A novel indirect co-culture system was established to support ex vivo expansion of hematopoietic progenitors in umbilical cord blood (UCB) by using thrombopoietin (TPO)/Flt-3 ligand (FL)-transduced human-marrow-derived mesenchymal stem cells (tfhMSCs) as a feeder. UCB CD34⁺ cells were isolated and cultured by using five culture systems in serum-containing or serum-free medium. Suitable aliquots of cultured cells were taken to monitor cell production, clonogenic activity, and long-term culture-initiating culture (LTC-IC) output. Finally, the severe-combined immunodeficient mouse (SCID) repopulating cell (SRC) assay was performed to confirm the ability of the indirect co-cultured cells from the tfhMSCs system to reconstitute long-term hematopoiesis. Results showed significant differences in the number of total nucleated cells (TNCs) among the culture systems with respect to serum-containing medium or serum-free medium during 14-day culture. In addition, on day 14, the outputs of CD34⁺ cells, the colony-forming units (CFUs) in culture, and the CFUs in mixed colonies containing erythroid and myeloid cells and megakaryocytes in the tfhMSC indirect co-culture system were significantly enhanced. The LTC-IC assay demonstrated that the tfhMSCs indirect co-culture system had the strongest activity. The SCID-SRC assay confirmed the extensive ability of the expanded cells from the tfhMSCs indirect co-culture systems to reconstitute long-term hematopoiesis. Furthermore, polymerase chain reaction analysis demonstrated the presence of human hematopoietic cells in the bone marrow and peripheral blood cells of non-obese diabetic/SCID mice. Thus, hMSCs transduced with TPO/FL, in combination with additive cytokines, can effectively expand hematopoietic progenitors from UCB in vitro. The tfhMSC indirect co-culture system may therefore be a suitable system for ex vivo manipulation of primitive progenitor cells under non-contact culture conditions.This work was supported by the Zhejiang Scientific Foundation (no. 2003C23015).     

Co-culture of human CD34+ cells with mesenchymal stem cells increases the survival of CD34+ cells against the 5-aza-deoxycytidine- or trichostatin A-induced cell death

It has been suggested that epigenetic regulation plays an important role in maintaining the stemness and lineage differentiation of hematopoietic stem cells (HSCs), 5-aza-deoxycytidine (aza-D) and Trichostatin A (TSA) being candidate additives for HSC ex vivo expansion. Although they have potent activity to maintain the stemness, they can also cause serious cell death. This study examined the effects of mesenchymal stem cells (MSCs) on the maintenance of CD34+ cells driven by aza-D and TSA in culture with the combined cytokines of thrombopoietin, flt-3 ligand, stem cell factor, interleukin-3, and interleukin-6. In cultures without MSCs, although aza-D and TSA retained the CD34 frequency 4 to 8 times more than in the cytokines alone, a large portion of cells underwent apoptotic cell death. Consequently, CD34+ cell expansion could not be achieved in any condition without MSCs. In cultures with MSCs, the total cell number was higher in aza-D or TSA than in any conditions in the cultures without MSCs. The CD34 frequency was also similar to the level in the cultures in aza-D or TSA without the MSCs. These results suggest that a co-culture of CD34+ cells with the MSCs might not simply deliver the proliferation signals but also stemness and survival signals, and overlap the action of epigenetic regulators.     

Factors affecting retrovirus-mediated gene transfer to human CD34+ cells

BACKGROUND: Retrovirus-mediated gene transfer is a useful technology in studying the biology of hematopoietic stem cells (HSCs) as well as in developing gene therapy products for a variety of human diseases. One of the most important factors determining the success of these studies is the number of HSCs receiving the gene of interest. METHODS: We tested various parameters for their influences on gene transfer efficiency to CD34+ cells derived from bone marrow. Based on a literature survey, three medium formulations of CD34+ cells have been compared for their effects on gene delivery efficiency and differentiation of them. We also tested whether FBS, used in the medium formulation, could be replaced with human serum or synthetic material. RESULTS: Formulation A, consisting of stem cell factor, Flt-3 ligand, thrombopoietin, and IL-3, provided optimum results in that it maintained the highest percentage of CD34+ cells during the culture as well as produced the highest gene delivery efficiency. It was found that the synthetic serum substitute containing bovine serum albumin, insulin and human transferrin could replace the fetal bovine serum present in the original formulation A without compromising gene transfer efficiency. When the transduction procedure was repeated three times, the gene could be delivered in up to 60% of the cell population. Gene delivery efficiency was comparable between CD34+ cells derived from bone marrow and mobilized peripheral blood. CONCLUSIONS: Our data could be useful in designing a procedure for stem cell gene therapy and providing a basis for further improving the conditions for gene transfer to various HSCs.     

Uptake and intracytoplasmic storage of pigmented particles by human CD34+ stromal cells/telocytes: endocytic property of telocytes

We studied the phagocytic‐like capacity of human CD34+ stromal cells/telocytes (TCs). For this, we examined segments of the colon after injection of India ink to help surgeons localize lesions identified at endoscopy. Our results demonstrate that CD34+ TCs have endocytic properties (phagocytic‐like TCs: phTCs), with the capacity to uptake and store India ink particles. phTCs conserve the characteristics of TCs (long, thin, bipolar or multipolar, moniliform cytoplasmic processes/telopodes, with linear distribution of the pigment) and maintain their typical distribution. Likewise, they are easily distinguished from pigment‐loaded macrophages (CD68+ macrophages, with oval morphology and coarse granules of pigment clustered in their cytoplasm). A few c‐kit/CD117+ interstitial cells of Cajal also incorporate pigment and may conserve the phagocytic‐like property of their probable TC precursors. CD34+ stromal cells in other locations (skin and periodontal tissues) also have the phagocytic‐like capacity to uptake and store pigments (hemosiderin, some components of dental amalgam and melanin). This suggests a function of TCs in general, which may be related to the transfer of macromolecules in these cells. Our ultrastructural observation of melanin‐storing stromal cells with characteristics of TCs (telopodes with dichotomous branching pattern) favours this possibility. In conclusion, intestinal TCs have a phagocytic‐like property, a function that may be generalized to TCs in other locations. This function (the ability to internalize small particles), together with the capacity of these cells to release extracellular vesicles with macromolecules, could close the cellular bidirectional cooperative circle of informative exchange and intercellular interactions.     

S-亚硝基谷胱甘肽诱导脐带血CD34+细胞来源的巨核细胞产生血小板

为了探讨一氧化氮供体S-亚硝基谷胱甘肽（S—nitrosoglutathione,GSNO）对脐带血CD34^＋细胞分化来源的巨三核细胞产生血小板的可能作用,我们采用免疫磁珠法从8例健康产妇足月顺产的胎儿脐带血中分选CD34^＋细胞,并在含血小板生成素（thrombopoietin,TPO,50ng／ml）、白细胞介素-3（IL-3,10ng／ml）、干细胞因子（stem cell factor,SCF,50ng／ml）和重组人粒-巨噬细胞刺激因子（rHu GM—CSF,20ng／ml）的无血清培养基中培养14d。随后,用免疫磁珠法分选CD61^＋细胞。CD61^＋细胞在含有（实验组）和缺乏（对照组）GSNO（20mg／ml）的无血清培养基[含TPO（50ng／ml）、IL-3（10ng／ml）、SCF（50ng／ml）]中培养不同时间（30min、2h）。采用流式细胞仪检测培养体系中的血小板数;电子显微镜观察巨核细胞的形态学;倒置显微镜和流式细胞仪观察凝血酶诱导的血小板凝集;ELISA方法检测巨核细胞中cGMP的含量。结果显示,与对照组比较,实验组血小板数量明显增加（P〈0．05）;电子显微镜下可见巨核细胞有明显伪足形成和突出;凝血酶诱导后在倒置显微镜和流式细胞仪上均可观察到血小板凝集现象;GSNO作用后巨核细胞中的cGMP明显升高（P〈0．05）。这些结果提示,GSNO可以促进脐带血CD34^＋细胞来源的巨核细胞产生具有一定功能的血小板,其产生的机制可能部分与cGMP途径有关。     

Resveratrol improves ex vivo expansion of CB-CD34+ cells via downregulating intracellular reactive oxygen species level

Intracellular reactive oxygen species (ROS) play important roles in the ex vivo expansion of hematopoietic stem and progenitor cells (HSPCs). In this study, the effects of resveratrol (RES), on the ex vivo expansion of HSPCs were investigated by analyzing CD34⁺ cells expansion and biological functions, with the objective to optimize ex vivo culture conditions for CD34 ⁺ cells. Among the five tested doses (0, 0.1, 1, 10, 20, and 50 μM), 10 μM RES was demonstrated to be the most favorable for ex vivo CD34 ⁺ cells expansion. In the primary cultures, 10 μM RES favored higher expansion folds of CD34 ⁺ cells, CD34 ⁺CD38 ⁻ cells, and colony-forming units (CFUs) ( P < 0.05). It was found that the percentages of primitive HSPCs (CD34 ⁺CD38 ⁻CD45R ⁻CD49f ⁺CD90 ⁺ cells) in 10 μM RES cultures were higher than those without RES. Further, in the secondary cultures, expanded CD34 ⁺ cells derived from primary cultures with 10 μM RES exhibited significantly higher total cells and CD34 ⁺ cells expansion ( P < 0.05). In the semisolid cultures, the frequency of CFU-GM and total CFUs of 10 μM RES group were both higher than those of without RES group, demonstrating that CD34 ⁺ cells expanded with 10 μM RES possessed better biological function. Furthermore, the addition of 10 μM RES downregulated the intracellular ROS level via strengthening the scavenging capability of ROS, and meanwhile reducing the percentages of apoptotic cells in cultures. Collectively, RES could stimulate the ex vivo expansion of CD34 ⁺ cells, preserved more primitive HSPCs and maintain better biological function by alleviating intracellular ROS level and cell apoptosis in cultures.     

Initial CD34+ cell-enrichment of cord blood determines hematopoietic stem/progenitor cell yield upon ex vivo expansion

Since umbilical cord blood (UCB), contains a limited hematopoietic stem/progenitor cells (HSC) number, successful expansion protocols are needed to overcome the hurdles associated with inadequate numbers of HSC collected for transplantation. UCB cultures were performed using a human stromal‐based serum‐free culture system to evaluate the effect of different initial CD34⁺ cell enrichments (Low: 24 ± 1.8%, Medium: 46 ± 2.6%, and High: 91 ± 1.5%) on the culture dynamics and outcome of HSC expansion. By combining PKH tracking dye with CD34⁺ and CD34⁺CD90⁺ expression, we have identified early activation of CD34 expression on CD34^? cells in Low and Medium conditions, prior to cell division (35 ± 4.7% and 55 ± 4.1% CD34⁺ cells at day 1, respectively), affecting proliferation/cell cycle status and ultimately determining CD34⁺/CD34⁺CD90⁺ cell yield (High: 14 ± 1.0/3.5 ± 1.4‐fold; Medium:22 ± 2.0/3.4 ± 1,0‐fold; Low:31 ± 3.0/4.4 ± 1.5‐fold) after a 7‐day expansion. Considering the potential benefits of using expanded UCB HSC in transplantation, here we quantified in single UCB units, the impact of using one/two immunomagnetic sorting cycles (corresponding to Medium and High initial progenitor content), and the average CD34⁺ cell recovery for each strategy, on overall CD34⁺ cell expansion. The higher cell recovery upon one sorting cycle lead to higher CD34⁺ cell numbers after 7 days of expansion (30 ± 2.0 vs. 13 ± 1.0 × 10⁶ cells). In particular, a high (>90%) initial progenitor content was not mandatory to successfully expand HSC, since cell populations with moderate levels of enrichment readily increased CD34 expression ex‐vivo, generating higher stem/progenitor cell yields. Overall, our findings stress the importance of establishing a balance between the cell proliferative potential and cell recovery upon purification, towards the efficient and cost‐effective expansion of HSC for cellular therapy. J. Cell. Biochem. 112: 1822–1831, 2011. © 2011 Wiley‐Liss, Inc.     

血小板生成素诱导胎肝CD34~ 造血干/祖细胞增殖分化与周期蛋白表达的关系

为了解胚胎时期巨核细胞增殖分化特有的内在机制 ,本研究观察了在体外培养体系中 ,胎肝源CD3 4 造血干 /祖细胞在血小板生成素 (thrombopoietin ,TPO)作用下增殖分化特征与相关周期蛋白B1、D1和D3表达及细胞内水平变化的关系。结果发现 :( 1)经 12d培养后 ,TPO使胎肝源CD3 4 细胞数从 1× 0 5个细胞 /ml增加到 13 12± 4 0 6× 10 5个细胞 /ml,CD4 1 细胞增加到了 95 % ,CD3 4 细胞下降到了 3 % ,大部分细胞的DNA倍性为 2N ,少数为 4N ,无大于 4N巨核细胞 ,TPO对MegaCultTm C胶原半固体培养体系中胎肝源CD3 4 细胞形成CFU Mk集落产率的影响呈明显的剂量效应关系 ;( 2 )在整个培养期间 ,周期蛋白B1表达逐渐增加 ,并保持在一个高水平上 ,培养后期 ,高水平的周期蛋白B1出现在G1期细胞上 ;( 3 )周期蛋白D1和D3表达先增加 ,培养后期细胞内水平下降 ,且以G2期细胞为主。该结果提示 :( 1)TPO通过上调周期蛋白B1和在所有细胞周期时限上调周期蛋白D1和D3表达 ,促进巨核细胞祖细胞的增殖分化 ;( 2 )周期蛋白B1在G2 M期的持续高水平和周期蛋白D1和D3在G2 M期的水平下降 ,可能导致胎肝源巨核细胞核内有丝分裂延迟或阻滞。     

FLT3配基在人骨髓基质细胞系中的基因转移与表达

目的：研究逆转录病毒介导的FL在骨髓基质细胞系HFCL中的表达。方法：采用脂质体法将重组质粒pLF-SN/HFCL和空载体pLXSN/HFCL转染包装细胞PA317,G418筛选抗性克隆,用抗性克隆上清液感染HFCL。RT－PCR和基因组DNA－PCR检测外源基因mRNA水平的表达及染色体的整合,小鼠CFU－GM集落法检测FL生物学活性。结果：在mRNA水平上有FL的表达,染色体基因组中整合有标记neo基因和FL基因。活性测试结果显示转染的骨髓基质细胞分泌FL。结论：提示骨髓基质细胞可作为基因治疗的靶细胞。     

Exposure to a MRI‐type high‐strength static magnetic field stimulates megakaryocytic/erythroid hematopoiesis in CD34+ cells from human placental and umbilical cord blood

The biological response after exposure to a high‐strength static magnetic field (SMF) has recently been widely discussed from the perspective of possible health benefits as well as potential adverse effects. To clarify this issue, CD34⁺ cells from human placental and umbilical cord blood were exposed under conditions of high‐strength SMF in vitro. The high‐strength SMF exposure system was comprised of a magnetic field generator with a helium‐free superconducting magnet with built‐in CO₂ incubator. Freshly prepared CD34⁺ cells were exposed to a 5 tesla (T) SMF with the strongest magnetic field gradient (41.7 T/m) or a 10 T SMF without magnetic field gradient for 4 or 16 h. In the harvested cells after exposure to 10 T SMF for 16 h, a significant increase of hematopoietic progenitors in the total burst‐forming unit erythroid‐ and megakaryocytic progenitor cells‐derived colony formation was observed, thus producing 1.72‐ and 1.77‐fold higher than the control, respectively. Furthermore, early hematopoiesis‐related and cell cycle‐related genes were found to be significantly up‐regulated by exposure to SMF. These results suggest that the 10 T SMF exposure may change gene expressions and result in the specific enhancement of megakaryocytic/erythroid progenitor (MEP) differentiation from pluripotent hematopoietic stem cells and/or the proliferation of bipotent MEP. Bioelectromagnetics 30:280–285, 2009. © 2009 Wiley‐Liss, Inc.     

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.