Goff J P, Shields D S, Greenberger J S
Department of Radiation Oncology, University of Pittsburgh and University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA.
Blood. 1998 Dec 1;92(11):4098-107.
There is a need to determine whether culture conditions may exist for ex vivo expansion of hematopoeitic stem cells (HSC), which favor solely proliferative self-renewal of HSC as opposed to proliferation with differentiation. Using single cells, we studied the effects of individual and combinations of cytokines in serum-free medium on the kinetics of the first cell doubling and the resulting phenotype of each of individual daughter cell. CD34(+)Thy-1(+)lin- cells were plated 1 cell per well in Terasaki plates in serum-free medium containing cytokines. Each well containing a single cell was monitored daily over 7 days for maintenance, division, or death. When division occurred in an individual well, the phenotype of the daughter cells was determined by staining with anti-CD34 fluorescein isothiocyanate (FITC)- and phycoerythrin (PE)-conjugated lineage specific antibodies. The cumulative percent of wells with an undivided single cell, wells in which the cell had divided, and wells in which the cell had died were scored. The number of doublets with conserved phenotype (CD34(+)lin-) was compared to those wells with one or more differentiated daughter cells (CD34(+)lin+). Over 7 days, cells cultured in single factors showed that between 13% (interleukin-6 [IL-6]) and 29% (thrombopoietin [TPO]) of the cells were undivided, between 13% (IL-1) and 35% (TPO) of the cells doubled, and between 35% (TPO) and greater than 60% (IL-11, IL-1, or hepatocyte growth factor [HGF]) died. When combinations of cytokines were used over 7 days, between 5% (FLT-3 ligand [FLT-3L], stem cell factor [SCF], IL-3, IL-6, granulocyte colony-stimulating factor [G-CSF], beta nerve growth factor [betaNGF]) and 22% (FLT-3L + HGF) of the cells remained undivided, between 15% (HGF, IL-1, IL-11, G-CSF) and 68% (SCF + TPO) of the cells had doubled and between 27% (FLT-3L + TPO) and 70% (HGF, IL-1, IL-11, G-CSF) died. The combination of FLT-3L + TPO induced the highest total percent (64. 6%) of cells with conserved phenotype (percent conserved doublets + percent with 1 cell conserved), followed by SCF + TPO, (50%) and the combination of FLT-3L, SCF, IL-3, IL-6, G-CSF, betaNGF (53%). These combinations also produced the highest yield of cells with conserved phenotype after one division (FLT-3L + TPO - 81 cells/100 initial cells, SCF + TPO - 68 cells/100 initial cells) (P =.01). Observation of the time of the initial cell division and phenotype of the daughter cells allowed us to identify candidate combinations of cytokines that promote maintenance of lin- cells (TPO), or recruit the primitive cells to divide and undergo phenotypic self-renewal (FLT-3L + TPO, SCF + TPO).
有必要确定是否存在有利于造血干细胞（HSC）体外扩增的培养条件，这种条件应仅支持HSC的增殖性自我更新，而非伴有分化的增殖。我们使用单细胞研究了无血清培养基中细胞因子单独及组合对首次细胞加倍动力学以及每个子代细胞所产生表型的影响。将CD34(+)Thy-1(+)lin-细胞以每孔1个细胞的密度接种于含有细胞因子的无血清培养基的特拉斯基培养板中。每天监测每个含有单个细胞的孔，持续7天，观察细胞的维持、分裂或死亡情况。当单个孔中发生细胞分裂时，通过用抗CD34异硫氰酸荧光素（FITC）和藻红蛋白（PE）偶联的谱系特异性抗体染色来确定子代细胞的表型。对未分裂的单个细胞孔、细胞已分裂的孔以及细胞已死亡的孔的累积百分比进行评分。将具有保守表型（CD34(+)lin-）的双细胞数量与具有一个或多个分化子代细胞（CD34(+)lin+）的孔进行比较。在7天内，在单一因子培养的细胞中，13%（白细胞介素-6 [IL-6]）至29%（血小板生成素 [TPO]）的细胞未分裂，13%（IL-1）至35%（TPO）的细胞发生了加倍，35%（TPO）至超过60%（IL-11、IL-1或肝细胞生长因子 [HGF]）的细胞死亡。当在7天内使用细胞因子组合时，5%（Flt3配体 [FLT-3L]、干细胞因子 [SCF]、IL-3、IL-6、粒细胞集落刺激因子 [G-CSF]、β神经生长因子 [βNGF]）至22%（FLT-3L + HGF）的细胞未分裂，15%（HGF、IL-1、IL-11、G-CSF）至68%（SCF + TPO）的细胞发生了加倍，27%（FLT-3L + TPO）至70%（HGF、IL-1、IL-11、G-CSF）的细胞死亡。FLT-3L + TPO组合诱导具有保守表型的细胞的总百分比最高（64.6%）（保守双细胞百分比 + 1个细胞保守的百分比），其次是SCF + TPO（50%）以及FLT-3L、SCF、IL-3、IL-6、G-CSF、βNGF组合（53%）。这些组合在一次分裂后也产生了具有保守表型的细胞的最高产量（FLT-3L + TPO - 81个细胞/100个初始细胞，SCF + TPO - 68个细胞/100个初始细胞）（P =.01）。对初始细胞分裂时间和子代细胞表型的观察使我们能够确定促进lin-细胞维持（TPO）或促使原始细胞分裂并进行表型自我更新（FLT-3L + TPO、SCF + TPO）的细胞因子候选组合。
