羟基淀粉沉淀对免疫磁珠法分离脐血CD34^＋细胞及造血祖细胞增殖功能的影响

目的：应用羟基淀粉沉淀去除脐血红细胞和免疫磁株（MACS）阳性选择,建立富集,分离脐血CD34^ 细胞的简易实用方法,通过造血祖细胞集落培养分析其在细胞因子作用下的增殖潜能,观察羟基淀粉沉淀对造血细胞增殖功能的影响,方法：采用羟基淀粉沉淀和改进的MACS富集,分离脐血CD^34^ 细胞,以甲纤半固体造血细胞培养法观察其粒－单系细胞（CFU－GM）和红系爆式集落（BFU－E）的数量和特性。结果：脐血分离前,CD34^ 细胞纯度为1．5％－3％,MACS分离后其纯度可达85％,羟基淀粉沉淀和MACS分离可达91％,CD34^ 造血祖细胞培养有明显的增殖,采用IL－3,IL－6,SCF,GM－CSF,EPO等细胞因子组合扩增培养9－14d,CD34^ 细胞在液体培养中有明显扩增,MACS分离后CD34^ 细胞总数增加39倍,羟基淀粉沉淀和MACS分离可增加45倍。结论：应用羟基淀粉沉淀和改进的MACS系统可有效富集,分离肌血CD34^ 细胞,羟基粉沉淀对CD34^ 细胞的富集,分离无影响,脐血细胞分离后作造血祖细胞培养,可产生丰富的CFU－GM和BFU－E,说明羟基淀粉沉淀分离的CD34^ 细胞的增殖功能优于MACS分离CD34^ 细胞的增殖功能。     

分化抑制培养体系体外扩增脐血造血细胞的研究

目的:探讨分化抑制培养体系对脐血造血细胞体外扩增效应。方法:分化抑制培养体系与脐血单个核细胞(MNC)体外共同培养7 d,检测MNC细胞总数、CFC、CD34 细胞反应扩增效果,并检测CD34 细胞表面归巢相关黏附分子VLA-4(CD49d)、VLA-5(CD49e)、LFA-1(CD11a)、HCAM(CD44)、L-selectin(CD62L)的表达率。结果:分化抑制培养体系组明显扩增脐血MNC细胞总数、CFC、CD34 细胞(均P<0．05),对照组培养脐血MNC细胞总数明显下降,CFC和CD34 细胞完全死亡(均P<0．01)。CD34 细胞表面各黏附分子CD49d、CD44和C1362L表达与扩增前相当(均P>0．05),而CD49e和CD11a表达明显高于扩增前(均P<0．05)。结论:分化抑制培养体系体外显著扩增脐血造血细胞,并且扩增后的造血干(祖)细胞总体上保持其表面归巢相关黏附分子的表达,归巢功能不会减低,是一种安全有效的扩增体系。     

原代骨髓基质细胞联合细胞因子体外诱导ES-D3细胞造血分化的研究

目的：研究原代骨髓基质细胞联合细胞因子（VEGF，SCF和TPO）体外诱导ES-D3细胞造血分化的效率，探讨体外高效诱导胚胎干细胞生成造血干／祖细胞的方法。方法：取6～8周昆明鼠骨髓制备小鼠骨髓基质细胞（BMSC）饲养层。采用二步诱导法，先将ES-D3细胞诱导分化为拟胚体（EB），再将EB置于4种体系下诱导分化造血干／祖细胞；第1组：自发分化对照组；第2组：细胞因子组（VEGF加SCF加TPO）；第3组：BMSC饲养层组；第4组：BMSC饲养层加细胞因子组（BMSC加VEGF加SCF加TPO）。诱导分化1周的部分细胞行造血祖细胞集落培养。流式细胞仪检测诱导培养7d和14d的各组细胞中干细胞特异抗原（CD34）、祖细胞特异抗原（c-kit）、粒细胞表面抗原（CD11b）、红系细胞特异抗原（Ter119）阳性表达率。间接免疫荧光染色法显示CD34、c-kit、CD11b、Te〉119阳性细胞。RT-PCR检测造血转录因子GATA-2、FIk-1、SCL、p-H1和p-major珠蛋白的表达。结果：诱导7d生成的细胞均表达造血CD34和Pkit，诱导14d生成的细胞表达单核巨噬细胞、CD11b和Te〉119，且诱导细胞表达造血转录因子（GATA-2、SCL、β-H1、β-major）基因mRNA，培养在特定条件下可形成造血祖细胞集落。从生成造血细胞和造血集落的数量分析，骨髓基质细胞饲养层与细胞因子合用时诱导效率明显高于单用组和对照组（P〈0．05或0．01）。结论：骨髓基质细胞饲养层与细胞因子合用，体外可以促进ES-D3细胞的早期造血分化，产生的造血前体细胞可进一步形成造血集落并分化成熟，表达与造血转录和早期造血分化相关的基因。     

内皮祖细胞培养鉴定及其体外修复球囊损伤血管的实验研究

目的观察体外培养人脐带静脉血(脐血)内皮祖细胞(EPCs)对球囊损伤血管的修复作用。方法采用贴壁选择法培养人脐血内皮祖细胞,分别采用流式细胞术、DiIacLDL吞噬试验及Ⅷ因子相关抗原免疫组化对培养细胞进行鉴定。球囊损伤法制备去内膜兔腹主动脉段,与EPCs共孵育使再内皮化。结果体外成功培养出人脐血内皮祖细胞,流式细胞术分析显示培养6d后,白细胞分化抗原34(CD34)、血管内皮钙黏素(VECadherin)、血管内皮生长因子受体2(VEGFR2)及白细胞分化抗原133(AC133)阳性细胞比例分别为(65±12)%、(45±10)%、(31±10)%与(20±1)%,CD34VECadherin、VEGFR2VECadherin、CD34AC133及VEGFR2AC133双阳性细胞比例分别为(42±10)%、(28±15)%、(19±1)%与(15±7)%,DiIacLDL吞噬试验及Ⅷ因子相关抗原免疫组化阳性。人脐血EPCs与去内膜兔腹主动脉共培育使之再内皮化,形成新内膜。结论利用脐血可成功培养出内皮祖细胞,体外培养内皮祖细胞可修复内皮损伤血管。     

血管紧张素Ⅱ对定向造血祖细胞BFU-E体外扩增的影响

目的：研究血管紧张素Ⅱ(AngⅡ)参与造血调控的机制及其对造血干／祖细胞BFU-E定向优势分化的作用。方法：将脐血来源的CD34^ 细胞在不同的细胞因子组合中悬浮培养，使它们向红系造血祖细胞优势分化，分别在不同时间对培养细胞进行3种处理：一部分细胞提取：RNA，用RT-PCR方法检测AngⅡ受体：mR-NA的转录；一部分添加AngⅡ和其他细胞因子继续培养3天后转移到半固体培养基中进行集落计数；其余细胞在原来的条件下继续悬浮培养。结果：新分离的CD34^ 细胞表面无AngⅡ受体mRNA的表达，此时添加AngⅡ对造血祖细胞生长无明显作用；在CD34^ 向红系优势分化体系中悬浮培养7天后，细胞中能检测到AngⅡ受体mRNA的表达，此时添加AngⅡ可刺激BFU-E的扩增；在培养第14天，细胞中己无AngⅡ受体表达，此时添加AngⅡ对红系祖细胞的扩增已无明显作用。结论：红系祖细胞在分化过程中有AngⅡ受体mRNA的表达，AngⅡ可刺激早期红系造血细胞的增生，并可提高造血细胞向红系定向优势分化的效率。     

脐血造血干/祖细胞人类端粒酶逆转录酶基因的表达及意义

目的 探讨人类端粒酶逆转录酶（hTERT）基因在脐血造血干/祖细胞中的表达及意义。方法 采用核酸原位杂交法对不同培养时间和不同培养条件下脐血CD34^ 细胞中hTERT基因的表达进行了测定。结果 新鲜分离的脐血中CD34^ 细胞低表达hTERT,阳性率为13%,体外培养5-7d hTERT表达增高,以干细胞因子、白细胞介素-3（IL-3）、IL-6和Flt-3配体组合条件下增高最显著,阳性率为48%,转化生长因子-β1和全反式维甲酸可抑制hTERT表达。结论 hTERT基因在脐血CD34^ 细胞中低水平表达,在体外扩增体系中,优化细胞因子组合能显著上调hTERT基因,负调控因子和诱导分化剂则下调hTERT基因。     

体外护增对脐血细胞重建千血功能的影响

目的：探讨体外扩增对人脐血造血细胞重建造血功能的影响。方法：采用祖细胞集落测定、造血细胞体外液体扩增培养和脾结节形成法等技术,研究体外扩增的人脐血细胞和新鲜脐血细胞输注对致死性照射小鼠体内造血重建能力的影响。结果：与新鲜脐血细胞移植相比较,扩增的人脐血细胞同样能够处长小鼠生存时间,更快地加速外周血常规恢复,同时脾脏有多系细胞组成的造血结节（CFU-S）,移植后第30天骨髓中培养出粒单系祖细胞（CFU-GM）和焊增性红系祖细胞（CFU-E）,且CFU-S、CFU-GM和CFU-E计数在两实验组间差异无显著性意义。结论：人脐血扩增的造血细胞同新鲜细胞一样能够在致死性照射小鼠体内重建造血,体外扩增培养并未影响其早期的造血重建能力。     

Caspase-3在脐血CD34+造血干/祖细胞中的表达及意义

目的：探讨脐血CD34^＋干／祖细胞在不同细胞因子支持下的体外扩增过程中Caspase-3表达及意义，方法：采用RT－PCR、Wester blot和流式细胞仪分析技术测定脐血CD34^ 细胞在体外扩增过程中的生物学特性及Caspase-3的表达。结果：Caspase-3 mRNA在新鲜分离的脐血CD34^ 细胞中低水平表达，在细胞因子支持下体外增养3d，扩增的CD34^ 细胞中Caspase-3 mRNA和蛋白质表达上调，但在该两种细胞中仅能检测到分子量为32000的无活性酶原形式的Caspase-3，随着体外培养时间的延长，在IL－3、IL－6和GM－CSF组合条件下，Caspase-3被激活，可检测到分子量为20000的裂解片段。结论：虽然造血干细胞的凋亡是个复杂的过程，但在脐血CD34^ 干／祖细胞体外扩增过程中，Caspase-3参与了凋亡事件并发挥着重要的作用。     

雌二醇对系统性红斑狼疮患者骨髓造血干/祖细胞的影响

目的 体外观察雌二醇（E2）对系统性红斑狼疮（SLE）患者骨髓干／祖细胞的影响。方法 分别用流式细胞仪、半固体休落培养观察17例SLE患者骨髓CD34^ 细胞、CD34^ CD38^－亚群、混合细胞集落（GEMM－CFU）和粒巨噬细胞集落（GM－CFU）的改变。体外观察E2、E2＋其拮抗剂他莫西芬对SLE患者骨髓GM－CFU的作用。结果 ①SLE患者CD34^ 细胞、GEMM－CFU、GM－CFU的形成校正这对照组明显减少；②E2对女性SLE较男性SLE、正常对照组的GM－CFU集落形成有明显的抑制作用；③他莫西芬可逆转E2对女性SLE患者的GM－CFU抑制作用。结论 SLE患者较后期的干细胞、祖细胞存在异常，雌激素可能介导了SLE患者骨髓造血干／祖细胞异常的发生。     

CD34^＋细胞与支气管哮喘

CD34是一种阶段特异性的白细胞分化抗原，选择性地表达于早期造血干／祖细胞表面。近年研究发现CD34阳性（CD34^ ）细胞在哮喘患者骨髓、外周血及肺组织局部明显增多，它可能参与哮喘时特异性嗜酸粒细胞性气道炎症的形成。深入研究CD34^ 细胞在哮喘中的作用有助于阐明该疾病的发生机制，并为其治疗拓展新的思路。     

Evaluation of ex vivo expansion potential of cord blood and bone marrow hematopoietic progenitor cells using cell tracking and limiting dilution analysis

In the absence of conclusive assays capable of determining the functionality of ex vivo expanded human hematopoietic progenitor cells, we combined cell tracking with the membrane dye PKH2, immunostaining for CD34, and limiting dilution analysis to estimate the frequency of long-term hematopoietic culture-initiating cells (LTHC-ICs) among de novo-generated CD34+ cells. Umbilical cord blood (CB) and bone marrow (BM) CD34+ cells were stained with PKH2 on day 0 and cultured with stem cell factor (SCF) and interleukin-3 (IL-3) in short-term stromal cell- free suspension cultures. Proliferation of CD34+ cells in culture was tracked through their PKH2 fluorescence relative to day 0 and the continued expression of CD34. As such, it was possible to identify cells that had divided while maintaining the expression of CD34 (CD34+PKH2dim) and others that expressed CD34 but had not divided (CD34+PKH2bright). In all such cultures, a fraction of both BM and CB CD34+ cells failed to divide in response to cytokines and persisted in culture for up to 10 days as CD34+PKH2bright cells. Between days 5 and 7 of culture, CD34+PKH2bright and CD34+PKH2dim cells were sorted in a limiting dilution scheme into 96-well plates prepared with medium, SCF, IL-3, IL-6, granulocyte-macrophage colony-stimulating factor, and erythropoietin. Cells proliferating in individual wells were assayed 2 weeks later for their content of clonogenic progenitors and the percentage of negative wells was used to calculate the frequency of LTHC-ICs in each population. Among fresh isolated BM and CB CD34+ cells, the frequencies of LTHC-ICs were 2.01% +/- 0.98% (mean +/- SEM) and 7.56% +/- 2.48%, respectively. After 5 to 7 days in culture, 3.00% +/- 0.56% of ex vivo-expanded BM CD34+PKH2bright cells and 4.46% +/- 1.10% of CD34+PKH2dim cells were LTHC-ICs. In contrast, the frequency of LTHC-IC in ex vivo expanded CB CD34+ cells declined drastically, such that only 3.87% +/- 2.06% of PKH2bright and 2.29% +/- 1.75% of PKH2dim cells were determined to be initiating cells after 5 to 7 days in culture. However, when combined with a calculation of the net change in the number of CD34+ cells in culture, the sum total of LTHC-ICs in both BM and CB cells declined in comparison to fresh isolated cells, albeit to a different degree between the two tissues.(ABSTRACT TRUNCATED AT 400 WORDS)     

Umbilical cord blood from preterm human fetuses is rich in committed and primitive hematopoietic progenitors with high proliferative and self-renewal capacity.

Human umbilical cord blood (CB) has been recognized as a source of hematopoietic stem cells for transplantation. While hematopoietic properties of neonatal CB from full-term pregnancies have been well characterized, little is known about CB from early gestational ages. We analyzed the content and the growth properties of primitive and committed hematopoietic progenitors in preterm CB from second trimester (week 16-28; n = 17) and early third trimester (week 29-34; n = 17) in comparison with term CB (n = 18). The frequency of CD34+ and CD34+CD38- cells was significantly higher in preterm than in term CB (mean, 2.51% and 0.56% vs 0.88% and 0.13%;p < 0.002). The number of colony forming units (CFU) in preterm CB was about twofold higher (230 +/- 6 vs 133 +/- 14/ 10(5) mononuclear cells; p < 0.05) and correlated with the content of CD34+ progenitors (r = 0.73). Long-term culture initiating cells (LTC-IC) were enriched about 2.5-fold (6.7 +/- 2.9 vs 2.6 +/- 1.2/10(5) cells; p < 0.05). Progenitors from preterm CB could be expanded in stroma-free liquid cultures supplemented with hematopoietic growth factors as efficiently as progenitors from term neonates. In short-term cultures containing erythropoietin (Epo), interleukin (IL)-1, IL-3, and IL-6, or granulocyte- (G-) and granulocyte-macrophage colony-stimulating factor (GM-CSF) together with stem cell factor (SCF) or Flt3 ligand (FL), expansion of CFUs was six- to eightfold at week 1. In long-term cultures containing thrombopoietin (TPO) and FL, an approximately 1000-fold expansion of multilineage progenitors was observed at week 10. In summary, we show that preterm CB compared with term CB is richer in hematopoietic progenitors, and that precursors from preterm CB can be extensively expanded ex vivo. This may have implications for the development of transplantation and gene transfer strategies targeting circulating fetal stem cells.     

Human stem-progenitor cells from neonatal cord blood have greater hematopoietic expansion capacity than those from mobilized adult blood

OBJECTIVE: In this study we compared the hematopoietic capacity of CD34+ cell preparations from neonatal cord blood (CB) vs adult mobilized peripheral blood (PBSC) before and after ex vivo culture. METHODS: CD34+ cell preparations purified from CB or PBSC were cultured in serum-free medium containing FKT: FLT-3 ligand (FL), KIT ligand (KL), and thrombopoietin (TPO). RESULTS: After 1-4 weeks ex vivo culture, CB CD34+ cell preparations had greatly increased numbers of total cells, CD34+ cells, and colony-forming cells (CFC). In contrast, ex vivo-cultured PBSC CD34+ cell preparations generated far less in vitro assessed hematopoietic capacity. Nonobese diabetic severe combined immunodeficient mouse (NOD/SCID) engrafting potential (SEP) was maintained in ex vivo-cultured CB CD34+ cell preparations, whereas ex vivo-cultured PBSC lost SEP. CB CD34+ cells continued to proliferate throughout 3 weeks ex vivo, whereas after 1 week, no additional cell divisions were detected in PBSC CD34+ cells. After 3 weeks in culture, the average CB CD34+ cell had divided more than 5 times, as compared to only 2 times for the average PBSC CD34+ cell. CONCLUSION: CB CD34+ cell preparations generated massively increased in vitro assessed hematopoietic capacity and maintained SEP during 1- to 4-week ex vivo cultures. In contrast, ex vivo-cultured PBSC CD34+ cell preparations generated far less in vitro assessed hematopoietic capacity and decreased SEP. The differences in the in vitro proliferative indices of membrane dye-labeled CD34+ cells from CB vs PBSC correlated with these functional differences.     

Differences in the frequency of normal and clonal precursors of colony-forming cells in chronic myelogenous leukemia and acute myelogenous leukemia.

Acute myelogenous leukemia (AML) is a clonal disease that is heterogeneous with respect to the pattern of differentiative expression of the leukemic progenitors. In some patients, the involved stem cells manifest pluripotent differentiative expression, whereas in others, the involved progenitors manifest differentiative expression mainly restricted to the granulocytic pathway. This is in contrast to chronic myelogenous leukemia (CML) which is a clonal disease known to arise in a pluripotent stem cell. Therefore, we tested whether these leukemias could be distinguished with respect to their involvement of immature precursors by studying colony-forming cells (CFC) and their precursors from four glucose-6-phosphate dehydrogenase (G6PD) heterozygous patients with AML and five patients with CML. CFC were separated from their precursors by FACS for expression of CD33 and CD34 followed by growth in a long-term culture (LTC) system. The vast majority of CFC express both the CD33 and CD34 antigens, but their less mature precursors, detected by their ability to give rise to CFC in LTC, express only CD34. In three of the four patients with AML, the CD33-CD34+ cells produced CFC in LTC that appeared to be predominantly or completely normal (ie, nonclonal) in origin. In the fourth patient, a significant enrichment of nonclonal progenitors was obtained in the CD33-CD34+ population, but these cells may also have included significant numbers of clonal cells. In contrast, in four of five patients with CML, cultures of both the CD33-CD34+ and CD33+CD34+ populations produced CFC in LTC that were almost entirely clonal in origin, whereas in the fifth patient a substantial number originated from nonclonal stem cells. These data indicate that granulocyte/monocyte progenitors are predominantly clonally derived in CML and AML. In CML, their precursors are also predominantly clonal, but in some cases of AML they are not. These findings may have implications for understanding the success or failure of current therapies of AML and CML.     

Transplantation of ex vivo expanded cord blood cells using the copper chelator tetraethylenepentamine: a phase I/II clinical trial

The copper chelator tetraethylenepentamine (TEPA; StemEx) was shown to attenuate the differentiation of ex vivo cultured hematopoietic cells resulting in preferential expansion of early progenitors. A phase I/II trial was performed to test the feasibility and safety of transplantation of CD133+ cord blood (CB) hematopoietic progenitors cultured in media containing stem cell factor, FLT-3 ligand, interleukin-6, thrombopoietin and TEPA. Ten patients with advanced hematological malignancies were transplanted with a CB unit originally frozen in two fractions. The smaller fraction was cultured ex vivo for 21 days and transplanted 24 h after infusion of the larger unmanipulated fraction. All but two units contained <2 x 10(7) total nucleated cells (TNCs) per kilogram pre-expansion. All donor-recipient pairs were mismatched for one or two HLA loci. Nine patients were beyond first remission; median age and weight were 21 years and 68.5 kg. The average TNCs fold expansion was 219 (range, 2-620). Mean increase of CD34+ cell count was 6 (over the CD34+ cell content in the entire unit). Despite the low TNCs per kilogram infused (median=1.8 x 10(7)/kg), nine patients engrafted. Median time to neutrophil and platelet engraftment was 30 (range, 16-46) and 48 (range, 35-105) days. There were no cases of grades 3-4 acute graft-versus-host disease (GVHD) and 100-day survival was 90%. This strategy is feasible.     

Evaluation of the effect of cryopreservation on ex vivo expansion of hematopoietic progenitors from cord blood.

In previous studies, we identified a cytokine cocktail including thrombopoietin, Flt-3 ligand, interleukin (IL)-6 and IL-11 in serum-free medium, suitable to induce significant and sustained ex vivo expansion of primitive hematopoietic stem cells (HSCs) from cord blood (CB) for up to 10 weeks. The aim of the present study was to evaluate the effects of cryopreservation on ex vivo expansion of HSCs and their committed progenitors. CD34+ cells were purified from CB units, each of which was processed in part as such and in part as cryopreserved and thawed, then expanded for 5 weeks in serum-free medium with the cytokine cocktail described above. We determined the number of nucleated cells (NC), CD34+, CD34+/38(-)/33(-), CD34+/61+, CD61+ cells and the clonogenic potential. After 2 weeks the median fold expansion of NC, CD34+ and CD34+/38(-)/33(-) cells was around two log both with fresh and cryopreserved CB and the expansion continued similarly until week 5. Our data suggest that this serum free protocol induces similar ex vivo expansion of HSCs and their committed progenitors from both fresh and cryopreserved CB. Our findings can be useful in view of clinical applications, since CB used for transplantation is stored in the cryopreserved state.     

Normal bone marrow hematopoietic stem cell reserves and normal stromal cell function support the use of autologous stem cell transplantation in patients with multiple sclerosis

Bone marrow (BM) stem cell reserves and function and stromal cell hematopoiesis supporting capacity were evaluated in 15 patients with multiple sclerosis (MS) and 61 normal controls using flow cytometry, clonogenic assays, long-term BM cultures (LTBMCs) and enzyme-linked immunosorbent assays. MS patients displayed normal CD34+ cell numbers but a low frequency of colony-forming cells (CFCs) in both BM mononuclear and purified CD34+ cell fractions, compared to controls. Patients had increased proportions of activated BM CD3+/HLA-DR+ and CD3+/CD38+ T cells that correlated inversely with CFC numbers. Patient BM CD3+ T cells inhibited colony formation by normal CD34+ cells and patient CFC numbers increased significantly following immunomagnetic removal of T cells from BMMCs, suggesting that activated T cells may be involved in the defective clonogenic potential of hematopoietic progenitors. Patient BM stromal cells displayed normal hematopoiesis supporting capacity indicated by the CFC number in the nonadherent cell fraction of LTBMCs recharged with normal CD34+ cells. Culture supernatants displayed normal stromal derived factor-1 and stem cell factor/kit ligand but increased flt-3 ligand levels. These findings provide support for the use of autologous stem cell transplantation in MS patients. The low clonogenic potential of BM hematopoietic progenitors probably reflects the presence of activated T cells rather than an intrinsic defect.     

Ex vivo expansion CD34+/AC133+ - selected autologous peripheral blood progenitor cells (PBPC) in high-risk breast cancer patients receiving intensive chemotherapy

AC133 antibody provides an alternative to CD34 for the selection and characterization of cells necessary for engraftment in transplant situations. We studied the effect of stem cell factor (SCF), interleukin 3 (IL-3) and interleukin 11 (IL-11) on the ex vivo expansion of human CD34⁺/AC133⁺ progenitors isolated from leukapheresis products from chemotherapy plus granulocyte-colony-stimulating factor (G-CSF) -mobilized adult donors. MiniMACS AC133⁺ isolated cells contained a mean of 85% (80-90) AC133⁺ cells. Enriched AC133⁺ cells co-expressed CD34⁺ 80%, CD71^low 36.6 % and CD33⁺ 6.6 %. After a seven-day ex vivo expansion in the presence of SCF + IL-3 + IL-11, the number of cells increased 19 times. These cells expressed a mean 12% CD34⁺ and 74% CD71⁺ (23% CD 71^high) and 59% CD33⁺. This means that the absolute number of CD34⁺ cells increased slightly, the number of CD33⁺ increased 100 times and cells with high density CD71^high (23%) appeared. These cells represent the cells committed to erythroid differentiation. The increase in the number of CFU-GM with various combinations of cytokines SCF + Il-3 + IL-11 will be discussed. The number of CFU-GM in culture after a seven-day ex vivo expansion in the presence of SCF + IL-3 + IL-11 increased 45 times.     

Flow cytometric and functional characterization of AC133+ cells from human umbilical cord blood

AC133+ cells may represent an alternative source of transplantable haemopoietic progenitor cells to CD34+ cells. Here, we have addressed the characterization of umbilical cord blood (UCB) AC133+ cells and compared their immunophenotypic and functional features with those of UCB CD34+ cells. UCB AC133+ and CD34+ cell fractions were purified by magnetic cell sorting, analysed by flow cytometry, tested for their content in blast cell colony-forming units (CFU-Bl), erythroid and granulocyte-macrophage colony-forming units before and after expansion in the presence of various haemopoietic growth factor combinations. Median AC133+ cell yield was 62.3%, and median AC133+ population purity was 97.9%. AC133+ cells were found to contain significantly more CFU-Bl than CD34+ cells; furthermore, the replating efficiency, i.e. the number of CFU-Bl capable of generating secondary colonies, was higher in the former than in the latter cells. Both AC133+ and CD34+ cells displayed an increased ability to give rise to committed progenitors after 7-day expansion in liquid cultures. These data suggest that the AC133+ cell subset is a heterogeneous pool of immature and more differentiated cells that can be maintained and expanded in well-defined culture conditions. In comparison with CD34+ cells, UCB AC133+ cells appear to contain a higher number of early haemopoietic progenitors.     

Hematopoietic capability of CD34+ cord blood cells: a comparison with CD34+ adult bone marrow cells

The characteristics of hematopoietic progenitor and stem cell (HPC/HSC) populations in mammals vary according to their ontogenic stage. In humans, HPC/HSCs from umbilical cord blood (CB) are increasingly used as an alternative to HPC/HSCs from adult bone marrow (BM) for the treatment of various hematologic disorders. How the hematopoietic activity of progenitor and stem cells in CB differs from that in adult BM remains unclear, however. We compared CD34+ cells, a hematopoietic cell population, in CB with those in adult BM using phenotypic subpopulations analyzed by flow cytometry, the colony-forming activity in methylcellulose clonal cultures, and the repopulating ability of these cells in NOD/Shi-scid (NOD/SCID) mice. Although the proportion of CD34+ cells was higher in adult BM than in CB mononuclear cells, the more immature subpopulations, CD34+ CD33- and CD34+ CD38- cells, were present in higher proportions in CD34+ CB cells. Clonal culture assay showed that more multipotential progenitors were present in CD34+ CB cells. When transplanted into NOD/SCID mice. CD34+ adult BM cells could not reconstitute human hematopoiesis in recipient BM, but CD34+ CB cells achieved a high level of engraftment, indicating that CD34+ CB cells possess a greater repopulating ability. These results demonstrated that human hematopoiesis changes with development from fetus to adult. Furthermore, CD34+ CB cells contained a greater number of primitive hematopoietic cells, including HSCs, than did adult BM, suggesting the usefulness of CD34+ CB cells not only as a graft for therapeutic HSC transplantation but also as a target cell population for ex vivo expansion of transplantable HSCs and for gene transfer in gene therapy.