Giampaolo A, Pelosi E, Valtieri M, Montesoro E, Sterpetti P, Samoggia P, Camagna A, Mastroberardino G, Gabbianelli M, Testa U
Department of Hematology-Oncology, Istituto Superiore di Sanità, Rome, Italy.
Stem Cells. 1995 May;13 Suppl 1:90-105.
Intensive efforts have led to the development of methods for stringent purification of adult hematopoietic progenitor cells (HPCs), particularly from peripheral blood (PB). The purification procedure previously reported by our group (Science, 1990) provided a high HPC frequency, but yielded a low HPC recovery (< or = 5-10%). We therefore developed an improved purification methodology based on "potentiated" negative immunobead selection (Step IIIP) by addition of anti-CD45, -11a and -71 monoclonal antibodies (mAbs) to the previously utilized panel of mAbs. This simplified procedure consistently allows not only high level purification but also abundant recovery of early HPCs: the final Step IIIP cell population (0.95 x 10(6) cells/4 PB donors, mean value) features an 81% HPC frequency and a recovery of 45% of the initial HPCs. The purified HPCs bear the primitive HPC phenotype, i.e., they are consistently CD34+, largely CD33-/45RA-, and in part HLA-DR-/low/CD38-/low/Thy-1+. In optimized semi-solid culture, the purified erythroid/multipotent HPCs give rise to macroscopic colonies (10,000-150,000 cells/clone, > 0.5 mm size colonies). This purification methodology compares favorably with previously reported procedures in terms of combined HPC frequency and recovery: availability of a large number of highly purified, early HPCs will provide an experimental tool for analysis of the molecular/cellular basis of early hematopoiesis. We have investigated by reverse transcription-polymerase chain reaction (RT-PCR) the mRNA expression of homeobox B (HOXB) cluster genes in purified HPCs induced in liquid suspension culture to gradual erythroid or granulopoietic (largely eosinophilic) differentiation and maturation by differential growth factor (GF) stimulus. Only B3 is expressed in quiescent HPCs. After GF treatment B3 expression is enhanced in the initial 24 h and then through erythroid and granulopoietic differentiation and maturation. HOXB4 and B5 are induced at slightly later times and expressed through maturation in both lineages, while B6 is selectively induced in granulocytic differentiation. B2 is transiently expressed at low level in the granulopoietic pathway, while it is detected only in advanced stages of erythropoiesis; B7, B8 and B9 are essentially not detected. Functional studies were performed with antisense phosphorothioate oligomers to HOX mRNAs including: 1) anti-B3 oligomer (alpha-B3) treatment of purified HPCs induces a striking blockade of both erythroid and granulomonocytic colony formation, 2) alpha-B6 selectively and markedly inhibits granulomonocytic colony formation, 3) alpha-B4 and alpha-B5 cause a significant, less pronounced decrease of both colony types and finally, 4) alpha-B2 and alpha-B7, alpha-B9 exert little and no effect respectively.
经过大量努力，已开发出用于严格纯化成人造血祖细胞（HPCs）的方法，特别是从外周血（PB）中纯化。我们小组先前报道的纯化程序（《科学》，1990年）可提供高HPC频率，但HPC回收率较低（≤5 - 10%）。因此，我们通过在先前使用的单克隆抗体（mAb）组中添加抗CD45、-11a和-71单克隆抗体，开发了一种基于“增强型”阴性免疫磁珠选择（步骤IIIP）的改进纯化方法。这种简化的程序不仅始终能实现高水平的纯化，还能大量回收早期HPCs：最终的步骤IIIP细胞群体（4名PB供体的0.95×10⁶个细胞，平均值）具有81%的HPC频率和45%的初始HPCs回收率。纯化的HPCs具有原始HPC表型，即它们始终是CD34⁺，大部分是CD33⁻/45RA⁻，部分是HLA-DR⁻/低/CD38⁻/低/Thy-1⁺。在优化的半固体培养中，纯化的红系/多能HPCs可形成肉眼可见的集落（10,000 - 150,000个细胞/克隆，集落大小>0.5毫米）。就HPC频率和回收率而言，这种纯化方法优于先前报道的程序：大量高度纯化的早期HPCs的可用性将为分析早期造血的分子/细胞基础提供实验工具。我们通过逆转录-聚合酶链反应（RT-PCR）研究了在液体悬浮培养中通过不同生长因子（GF）刺激诱导纯化的HPCs向逐渐红系或粒系（主要是嗜酸性粒细胞）分化和成熟过程中同源盒B（HOXB）簇基因的mRNA表达。只有B3在静止的HPCs中表达。GF处理后，B3表达在最初24小时内增强，然后在红系和粒系分化及成熟过程中持续增强。HOXB4和B5在稍晚时候被诱导，并在两个谱系的成熟过程中表达，而B6在粒细胞分化中被选择性诱导。B2在粒系分化途径中短暂低水平表达，而仅在红细胞生成的晚期阶段被检测到；B7、B8和B9基本未被检测到。使用针对HOX mRNA的反义硫代磷酸酯寡聚物进行了功能研究，包括：1）用抗B3寡聚物（α-B3）处理纯化的HPCs可显著阻断红系和粒单核细胞集落形成，2）α-B6选择性且显著抑制粒单核细胞集落形成，3）α-B4和α-B5导致两种集落类型显著但不太明显的减少，最后，4）α-B2和α-B7、α-B9分别几乎没有作用和没有作用。
