Ex vivo expansion of apheresis-derived peripheral blood hematopoietic progenitors

Because the administration of hematopoietic growth factors and the use of stem cell support often fails to alleviate the neutropenic phase induced by cytotoxic drugs, several investigators have attempted to expand ex vivo hematopoietic progenitors for clinical use. These attempts have clearly shown that the cultured cells are functional and can be safely administered to patients, but that the in vivo performance is disappointing and the concept as a whole is not yet clinically useful. The major reasons for these unsuccessful attempts are thought to be cumbersome cell fractionation techniques, contamination, prolonged incubation, and the use of less than ideal cytokine combinations. In response, we have developed a simple procedure for ex vivo expansion of myeloid progenitor cells. In this assay, unfractionated mononuclear cells from apheresis donors are incubated in nonpyrogenic plastic bags for 7 days in the presence of culture medium either containing fetal calf serum or human plasma, granulocyte colony-stimulating factor, and stem cell factor. We have demonstrated that under these conditions the number of colony-forming units (CFU) granulocyte-macrophage (CFU-GM) and of CFU-granulocyte-macrophage-erythroid-megakaryocyte (CFU-GEMM) increased 7- and 9-fold, respectively, by day 7 and the number of burst-forming units-erythroid (BFU-E) increased 2.7-fold by day 5 of culture. Significant increases in the numbers of cells expressing CD34+, CD34+/CD38+, CD34+/CD33+, CD34+/CD15+, and CD34+/CD90+ and significant declines in the numbers of cells expressing CD34+/CD38- and CD19 surface antigens were also observed. The relative numbers of cells expressing T-cell markers and CD56 surface antigen did not change. By using different concentrations of various hematopoietic growth factor combinations, we can increase the number of mature and immature cells of different hematopoietic lineages.     

Ex vivo expansion of human hematopoietic progenitors and cells to support high-dose chemoradiation therapy: five years of clinical experience

The identification of cytokines-soluble or membrane-bound regulators of hematopoietic stem and progenitor cell survival, proliferation, and differentiation - and the definition of culture conditions that enable cell and progenitor expansion, has lead to the first clinical trials using cultured cells in addition to or in place of unmanipulated cells. The use of ex vivo expanded cells can improve several aspects of autologous and allogeneic hematopoietic cell and progenitor transplantation, such as reducing or abolishing the nadir that follows high-dose chemoradiation therapy regimens, or reducing the clinical risks associated with the use of small numbers of progenitors as in cord blood transplantation and in autologous transplantation for poor mobilizers. In addition, biological questions raised by ex vivo expansion are shared by scientists and clinicians interested in gene transfer into hematopoietic stem cells. We here review the biological problems associated with ex vivo expansion: defining efficient culture conditions, considering not only scientific and biological issues but also regulatory and commercial issues, defining appropriate surrogate endpoints that predict engraftment and superior clinical efficacy to that obtained with the use of unmanipulated grafts. We also review the results of the first clinical trials that have demonstrated the feasibilty of this approach, and have shown some of its limitations; demonstration of clinical efficacy will require more preclinical and clinical work.     

Strategies to expand transduced hematopoietic stem cells in vivo.

Data in mice suggest that in vivo selection strategies will expand the numbers of transduced hematopoietic stem cells (HSC) to levels sufficient for clinical therapies, and it is argued that comparable strategies will benefit larger animals and humans. To test this assumption, we performed virtual gene therapy in mouse and cat, species in which the in vivo kinetics of HSC are defined. In the simulated experiments, 10% of HSC and 50% of short-term repopulating cells were transduced with a gene allowing a conditional replication or apoptosis advantage. After transplantation, differentiation proceeded stochastically and contributions of transduced cells were tracked for 2 years. Fifty independent transplantations were simulated per species for each analysis. When transduced HSC had a 2-fold increased chance of replication (self-renewal) extending for 4, 10, or 20 weeks after transplantation, or a 5-fold replication advantage extending for 4 weeks, results in mice were far better than in cat, a larger animal, with slower baseline HSC cell cycle kinetics. Similarly, when transduced HSC had a 2-, 4-, or 10-fold decreased chance of apoptosis, extending for 20 or more weeks after transplantation, the murine studies were poor predictors of feline results. Simulation may allow one to optimize and/or understand the limitations of a gene therapy strategy.     

新一代细胞疗法：造血祖细胞体外扩增与定向诱导分化

第一代细胞疗法是成熟血细胞的全血输注,以后逐渐发展为成分输血。第二代细胞疗法是包括骨髓、动员外周血及脐带血的造血干细胞移植。近年来,体外扩增造血祖细胞的技术方法已逐渐成熟,在液体培养体系中加入FL(Flt-3 ligand),SCF,IL-1,IL-3,GM-CSF,G-CSF,Epo等细胞因子,可使细胞总数及各系祖细胞得以大量扩增,部分技术方法还可明显地扩增原始的早期祖细胞,(LTC-IC,CD34~ CD38~-细胞,CFU-GEMM等),如FL,Epo等细胞因子的组合,CD34~ CD38~-细胞亚群的扩增,负调控因子(如TGF-β,LIF及MIP-1α,IL-8,PF4等趋化因子)的应用,基质细胞的支持和继续灌注培养等。此外,利用上述细胞因子和FL,SCF,G-CSF,Epo,Tpo IL-4,FNF-α,IL-7,IL—15等的不同组合,还可定向地诱导CD34~ 细胞向粒系、红系、巨核系/血小板、NK细胞、树突状细胞(dendritic cells,DC)及淋巴细胞等方向分化与扩增。这些技术方法与方案已开始进入临床试用,在造血调控研究,造血干细胞移殖,大剂量化疗后的造血细胞支持治疗,肿瘤生物免疫治疗,基因治疗等领域将具有广阔的应用前景。     

大鼠骨髓间充质干细胞体内造血分化过程中加入硝普钠的作用

背景:硝普钠作为血管扩张剂加入骨髓间充质干细胞培养过程中,对其分化潜能发生何种影响?目的:观察大鼠骨髓间充质干细胞体内向造血细胞分化过程中加入硝普钠后的变化,并与单纯细胞移植的效果进行比较。设计:随机区组,对照观察。单位:广东医学院微生物免疫学教研室,广州医学院病理生理教研室。材料:实验于2006-08在广州医学院完成。选取清洁级出生七八周balb/c小鼠27只作为受体,随机数字表法分为3组:细胞移植组、硝普钠 细胞移植组、空白对照组,9只/组。另选取4周龄SD大鼠1只作为实验用骨髓间充质干细胞的供体来源。注射用硝普钠(50mg/支,北京双鹤现代医药技术有限责任公司,国药准字H11020907)。方法:①无菌条件下取出SD大鼠的股骨,进行骨髓间充质干细胞的分离培养。传至六七代作为供体细胞,消化离心,调整浓度为1×109L-1。细胞悬液中加入荧光素标记的抗体,流式细胞术检测表型。②取50mg/支的注射用硝普钠1支,加入2.5mL生理盐水混匀,从中取出1.0mL液体加入到100mL的生理盐水中,配成终浓度为200mg/L,配置后4h内使用。③各组小鼠细胞移植前均经5.0GyX射线全身照射4h,吸收剂量率为1.45Gy/min。照射完毕后,细胞移植组直接经尾静脉输注0.3mL骨髓间充质干细胞悬液(含1.5×106个细胞);硝普钠 细胞移植组先注射已配好的200mg/L硝普钠液体0.15mL,1min后立即输注骨髓间充质干细胞悬液0.3mL(含1.5×106个细胞);空白对照组输注等量无血清培养液。④移植后60d,各组存活小鼠眼眶外周取血,处死后常规制备骨髓及脾脏单细胞悬液,流式细胞术检测大鼠源性造血细胞CD11a与CD45的植入水平。主要观察指标:①骨髓间充质干细胞培养扩增情况。②不同组织大鼠源性造血细胞的植入水平检测。结果:作为受体的27只balb/c小鼠均存活至实验结束。①骨髓间充质干细胞培养扩增情况:培养3d后细胞贴壁,形态比较均一,长梭形,至第6天细胞90%融合,无重叠。传代后24h内细胞完全贴壁,长梭形,增殖生长迅速,3d即达到完全融合。②不同组织大鼠源性造血细胞的植入水平检测:细胞移植组、硝普钠 细胞移植组在外周血、骨髓、脾细胞悬液中均可检测到低表达的大鼠源性造血细胞CD11a与CD45,且硝普钠 细胞移植组明显强于细胞移植组(t=2.619,P<0.05);空白对照组CD11a与CD45呈阴性表达。结论:大鼠骨髓间充质干细胞具有向造血细胞分化的潜能,硝普钠可促进其分化。     

Ex vivo purging of stem cell autografts using cytotoxic cells

Autologous stem cell transplantation (SCT) is the treatment alternative offered to patients that cannot benefit from allogeneic transplantation due to lack of suitable donor or age limitations. However, the outcome of autologous SCT is largely hindered by the high relapse rate. Two major factors can account for relapse after autologous SCT: the persistence of residual malignant cells resistant to chemo/radiotherapy left either in the body or in the autograft. Therefore, the rationale for purging autografts of residual malignant cells comes from the limitations of conventional high-dose chemo/radiotherapy in achieving a complete eradication of residual tumor cells in the marrow. To date, different purging modalities have been exploited. Immunological methods of purging present the advantage of being non-cross-reactive with conventional chemotherapy. These immunologic methods include depletion using antibody targeting of the malignant cells, ex vivo activation/generation of the autologous cytotoxic cells, in particular that of natural killer/lymphokine-activated killer (NK/LAK) and cytokine-induced killer (CIK) cells, and ex vivo purging of autografts using cytotoxic cell lines. The generation of ex vivo-expanded and activated autologous cytotoxic cells (CTL or NK) has generated increasing interest for the treatment of different malignancies. Unfortunately, the isolation and expansion of these cells have proven to be technically difficult. As an alternative, the use of cytotoxic cell lines as immune effectors has been proposed. The two available human cytotoxic cell lines TALL104 and NK-92 are currently in clinical trials and a number of studies have suggested their effectiveness as an immunotherapeutic agent including for ex vivo purging of autografts.     

Controlling culture dynamics for the expansion of hematopoietic stem cells

The ex vivo expansion of hematopoietic stem cells (HSCs) is the subject of intense commercial and academic interest due to the potential of HSCs to be a renewable source of material for cellular therapeutics. Unfortunately, because methodologies have not yet been developed to grow clinically relevant numbers of HSCs (or their derivatives) consistently, the potential of this technology is limited. Manipulation of the in vitro culture microenvironment, primarily through cytokine supplementation, has been the predominant approach in studies attempting to expand primary human HSC numbers in vitro. While promising results have been obtained, it is becoming clear that novel methods must be developed before cellular therapies using these stem cells can become routine. Ideally, bioprocesses must be designed to target specifically the growth of stem cell populations while incorporating positive and negative feedback from potentially dynamic mature and maturing cell populations. The product of these culture systems should consist of not only HSCs, but also of cells that allow the engraftment of HSCs and, ideally, cells responsible for the immediate or accelerated functional support of patients. Development of such "designer transplants" will require combining optimal culture conditions capable of amplifying HSC numbers with novel approaches for finely controlling the number, functional capabilities, and characteristics of potentially therapeutic cells in these very complex cell culture systems.     

Life span of multipotential hematopoietic stem cells in vivo

The findings reported in this study highlight several important features of the development of hematopoietic stem cells after transplantation into irradiated recipients. First, they demonstrate the existence of a class of primitive multipotential stem cells that can function for a significant portion of the lifetime of a mouse (15 mo). In addition, they clearly show that these primitive stem cells can be infected with recombinant retroviruses and thus would be appropriate targets for gene therapy in somatic tissues. Second, our data indicate that the progeny of some, but not all, of the primitive stem cells have fully expanded into the various hematopoietic lineages by 2 mo after reconstitution. Finally, our analysis of the secondary recipients provides strong evidence suggesting that the primitive stem cell population can actually clonally expand. Our current experiments are aimed at determining the extent to which this expansion can occur and whether or not this expansion can be influenced by exogenous factors.     

Ex vivo expansion of early and late megakaryocyte progenitors

Our goal is to produce ex vivo-expanded human megakaryocytes (MK) cells from peripheral blood progenitor cell (PBPC) harvests for use in supplementing conventional autografts. In this paper we show the megakaryocytopoietic productivity of small-scale in vitro serum-free cultures of human CD34+ cells containing MK growth and development factor (MGDF) and stem cell factor (Kit ligand; SCF) +/- granulocyte colony-stimulating factor (G-CSF). Cultures were characterized after 3, 6, 9, and 13 days by flow cytometry and clonogenic assays. CD34+ cells expanded 5.2- and 3.4-fold, and produced 2.2 and 2.4 CD34+/41(+) cells per seeded CD34+ cell after 6 and 9 days in culture, respectively. None were detected at day 13. CD41+ cells expanded exponentially over 13 days. Colony-forming unit-megakaryocyte (CFU-MK) also expanded exponentially, but the proportion of the most primitive CFU-MK dropped from 45% to 1.5% and to <1% after 6 and 9 days, respectively. G-CSF increased total cell expansion, but decreased CD41+ frequency, yielding no gain in MK production. We also found that PB CD34+ cells cultured for 3-6 days are richer in primitive MK progenitors, while those cultured for 9-13 days have greater numbers of more differentiated MKs. Overall, the combination of MGDF+SCF proved sufficient for expanding CD34+/CD41+ cells. As the stage of ex vivo MK differentiation most conducive to optimal platelet production in vivo is not known, we are planning a clinical trial to determine the efficacy of ex vivo-expanded MKs on platelet recovery in relation to MK maturity.     

JAK2转基因骨髓造血干/祖细胞体外长期扩增调控及定向分化的研究

目的　探讨酪氨酸激酶JAK2转基因骨髓造血干 祖细胞体外长期扩增调控和定向分化潜能的可行性。方法　克隆了一个MSCV based逆转录病毒载体称作MGI F2Jak2 ,它编码一个绿色荧光蛋白 (GFP)和两个改进的FK5 0 6结合蛋白 (F36v)与JAK2组成的融合蛋白 ,F36v是二聚化化学诱导物AP2 0 187的结合位点。应用该载体转染GpE 86包装细胞株 ,将C5 7BL 6小鼠的骨髓细胞与照射过15 0 0cGy的GpE 86产毒细胞株共同培养实现基因转导。转导后的细胞在X VIVO 15培养体系中扩增 ,分为①空白对照组 ;②AP2 0 187组 ;③干细胞因子 (SCF)组 ;④AP2 0 187 SCF组。扩增的细胞用直接法标记藻红蛋白荧光素 (PE)结合的抗Sca1、c kit、CD34 、Gr1、CD1 1b、TER 119、CD4 1 、B2 2 0和CD3单克隆抗体以流式细胞仪检测 ,并进行定向分化、祖细胞集落培养和脾细胞集落形成单位 (CFU S)的研究。结果　只有AP2 0 187 SCF组可以持续地使转基因的骨髓造血干 祖细胞大量增殖 ,扩增 80d后细胞可达10 1 4倍 ,细胞倍增时间约 30h ,扩增细胞的表型为CD34 、c kit和Sca1等干细胞表面标志强阳性 ,其它细胞表面标志如Gr1、CD1 1b、TER119、CD4 1 、B2 2 0、CD3接近阴性。所扩增的细胞在不同的细胞因子组合下 ,可定向分化为粒细胞、巨噬细胞、红细胞、     

Ex vivo expansion of cord blood haematopoietic stem/progenitor cells under physiological oxygen tensions: clear‐cut effects on cell proliferation,differentiation and metabolism

Physiologically low O₂ tensions are believed to regulate haematopoietic stem cell (HSC) functions in the bone marrow (BM; 0–5%). In turn, placenta and umbilical cord are characterized by slightly higher physiological O₂ tensions (3–10%). We hypothesized that O₂ concentrations within this range may be exploited to augment the ex vivo expansion/maintenance of HSCs from umbilical cord (placental) blood (UCB). The expansion of UCB CD34⁺‐enriched cells was studied in co‐culture with BM mesenchymal stem/stromal cells (MSCs) under 2%, 5%, 10% and 21% O₂. 2% O₂ resulted in a significantly lower CD34⁺ cell expansion (25‐fold vs 60‐, 64‐ and 92‐fold at day 10 for 5%, 21%, 10% O₂, respectively). In turn, 10% O₂ promoted the highest CD34⁺CD90⁺ cell expansion, reaching 22 ± 5.4‐ vs 5.6 ± 2.4‐ and 5.7 ± 2.0‐fold for 2%, 5% and 21% O₂, respectively, after 14 days. Similar differentiation patterns were observed under different O₂ tensions, being primarily shifted towards the neutrophil lineage. Cell division kinetics revealed a higher proliferative status of cells cultured under 10% and 21% vs 2% O₂. Expectedly, higher specific glucose consumption and lactate production rates were determined at 2% O₂ when compared to higher O₂ concentrations (5–21%). Overall, these results suggest that physiological oxygen tensions, in particular 10% O₂, can maximize the ex vivo expansion of UCB stem/progenitor cells in co‐culture with BM MSCs. Importantly, these studies highlight the importance of exploiting knowledge of the intricate microenvironment of the haematopoietic niche towards the definition of efficient and controlled ex vivo culture systems capable of generating large HSCs numbers for clinical applications. Copyright © 2013 John Wiley & Sons, Ltd.     

Methodologies for isolation,expansion and differentiation of somatic stem cells(tissue stem cells)

Recent studies have shown that pluriponten stem cells are also found in adult tissue. These adult stem cells has a great advantage over other stem cells sources, human embryos or human fetal tissue, that trouble many people on ethical grounds. While the adult stem cells have great potential for use in the cell therapy, we must pass numerous technical hurdles to reach a goal. Here we describe a subset of problems encountered in isolation and expansion of somatic stem cells for clinical application. As it is necessary to obtain the enough number of cells to supply the defect tissue and organ, we required purification and ex vivo expansion of stem cells, overcoming senescence. We also refer to the argument about plasticity of adult stem cells.     

脐血间充质干细胞的生物学特征及其对造血干/祖细胞体外扩增的支持作用

目的探讨脐血间充质干细胞(MSC)的生物学特征及其对造血干/祖细胞体外扩增的支持作用。方法用液体培养法分离脐血贴壁细胞,采用ELISA方法检测贴壁细胞条件培养液中细胞因子的表达;用流式细胞术分析其免疫表型特征;在成软骨细胞诱导培养条件下诱导细胞分化,并用RTPCR方法检测分化后细胞原胶原Ⅱ型基因的表达。采用分阶段共培养方法观察脐血贴壁细胞对CD34+细胞体外扩增的支持作用。结果脐血单个核细胞纤维样细胞集落形成率为(3.5±0.7)/106。脐血MSC体外至少可以扩增15代。没有分化的脐血MSC表型为CD13、CD29、CD90、CD105、CD166、SH2、SH3和SH4阳性,CD45、CD34和CD14阴性;脐血MSC培养上清中干细胞因子、IL6和肿瘤坏死因子α检测阳性。在成软骨细胞诱导培养基培养条件下,脐血MSC原胶原Ⅱ型基因mRNA表达阳性。脐血MSC与CD34+细胞共掊养14d,CD34+细胞扩增率高于未共培养组4倍。结论脐血MSC具有类似于成体骨髓MSC的特征,对造血干细胞增殖有明显的支持作用。     

体外扩增小鼠造血干/祖细胞及重建造血功能的研究

目的 探讨体内中性粒细胞的快速植入及长期重建造血的能力。方法 分离经氟尿嘧啶处理的雄性BDF1小鼠骨髓单个核细胞，以MoFlo细胞分选系统分离纯化CD34^ /c-kit^ 造血干，祖细胞，体外应用骨髓间充质干细胞共培养和阶段扩增结合的方法分别对分离的单个核细胞和CD34^ /c-kit^ 细胞进行体外扩增，并将扩增的有核细胞移植给经亚致死剂量照射的雌性小鼠。结果 以骨髓间充质干细胞为培养基质细胞，结合分阶段扩增方法，有效提高了各种细胞的扩增倍数，其中单个核细胞扩增的总有核细胞、CD34^ 细胞、GM-CFC和HPP-CFC扩增倍数分别为10．8，4．8，65．9和38．8，CD34^ /c-kit^ 细胞扩增的以上4种细胞扩增倍数分别为76．1，2．9，71．7和51．8；扩增细胞移植后可快速产生体内中性粒细胞的植入，并在2个月后的移植小鼠中仍可检测到移植的造血细胞。结论 与骨髓间充质干细胞的共培养，为造血干，祖细胞的有效扩增提供了良好的环境，分阶段扩增法加快了体外造血干，祖细胞的扩增和成熟，为移植后体内中性粒细胞的快速植入创造了条件。     

人主动脉-性腺-中肾区基质细胞诱导胚胎干细胞分化为造血干细胞及体内造血重建

背景：研究证实多种造血生长因子、基质细胞饲养层及其条件培养液可促进胚胎干细胞向造血干细胞分化。目的：以人主动脉-性腺-中肾（aorta-gonad-mesonephros,AGM）区基质细胞为饲养层体外诱导小鼠胚胎干细胞分化为造血干细胞,并比较不同移植途径对造血干细胞体内造血重建能力的影响。方法：将小鼠E14胚胎干细胞诱导为拟胚体,采用Transwell非接触共培养体系在人AGM区基质细胞饲养层上诱导6d,接种NOD-SCID小鼠检测体内致瘤性。再将诱导后的拟胚体细胞移植经致死量60Coγ射线辐照的BALB/C雌鼠,受鼠随机分为静脉移植组、骨髓腔移植组、照射对照组及正常对照组。结果与结论：拟胚体细胞经人AGM区基质细胞诱导后Sca-1＋c-Kit＋细胞占（13.12±1.30）%。NOD-SCID小鼠皮下接种经人AGM区基质细胞诱导的拟胚体细胞可出现畸胎瘤,经骨髓腔接种未见肿瘤形成。静脉移植组动物全部死亡,骨髓腔移植组生存率为55.6%,移植后21d外周血象基本恢复,存活受鼠检测到供体来源Sry基因。提示小鼠胚胎干细胞经人AGM区基质细胞诱导分化的造血干细胞通过骨髓腔移植安全并具有一定的造血重建能力。