Miller J S, Alley K A, McGlave P
Division of Hematology, University of Minnesota Medical School, Minneapolis 55455.
Blood. 1994 May 1;83(9):2594-601.
We have recently described a marrow stroma-dependent long-term culture system that supports differentiation of CD34+ human marrow primitive progenitors into natural killer (NK) cells. We postulate that CD7 expression may be an early event in commitment of hematopoietic progenitors to the NK lineage. Here we compare the characteristics of CD34+7- and CD34+7+ marrow cells cultivated in the stroma-based NK culture system. These CD34+ populations were further compared with a marrow derived, more committed, CD34-7+ progenitor to emphasize the continuum of NK development and to highlight differences between progenitors in our assays. No progenitor proliferated when plated in media without stroma, underscoring the importance of stroma in NK differentiation. Plating progenitor populations in interleukin-2 containing media directly on preestablished, allogeneic, irradiated marrow stroma for 5 weeks resulted in CD56+CD3- NK cells; however, characteristics of the cultured populations differed. Fold expansion and cloning efficiency of the CD34+7+ population, determined by a functional limiting dilution assay was significantly higher than of the CD34+7- or CD34+7+ populations. This suggests that the CD34+7+ population is highly enriched for an NK progenitor and a possible intermediate in NK lineage differentiation. Further dividing the CD34+7+ population by the relative fluorescence of CD7 into CD34+7+dim and CD34+7+bright populations showed that the CD34+7+bright population exhibited a significantly higher cloning frequency than parallel experiments with CD34+7+dim cells (11.8% +/- 2.4% v 2.4% +/- 0.7%, n = 6; P = .005). Plating of the more primitive CD34+7- population in a transwell system (which separates progenitors from stroma by a microporous membrane) prevents differentiation into NK cells. In contrast, plating of CD34+7+ progenitors in transwells resulted in generation of NK cells. These data suggest that primitive, but not more mature NK progenitors may require direct contact with stroma for the initial differentiation steps. Finally, differentiation of the NK progenitors in this stroma-dependent model results in expression of CD2 not present on any of the starting populations. This observation suggests that marrow stroma can stimulate CD2 expression on NK progenitors in a previously undescribed fashion that may be analogous to the thymic effect on CD2 expression in immature T lymphocytes. These observations identify early steps in the commitment of primitive marrow CD34+ hematopoietic progenitors to a lymphoid lineage and underscore the importance of coexpression of CD7 with CD34 as an early lymphoid commitment characteristic and direct progenitor-stroma interactions in this process.
我们最近描述了一种依赖骨髓基质的长期培养系统，该系统支持CD34⁺人类骨髓原始祖细胞分化为自然杀伤（NK）细胞。我们推测CD7表达可能是造血祖细胞向NK谱系定向分化过程中的早期事件。在此，我们比较了在基于基质的NK培养系统中培养的CD34⁺7⁻和CD34⁺7⁺骨髓细胞的特征。将这些CD34⁺群体与源自骨髓的、分化程度更高的CD34⁻7⁺祖细胞进一步比较，以强调NK细胞发育的连续性，并突出我们实验中祖细胞之间的差异。当接种在无基质的培养基中时，没有祖细胞增殖，这突出了基质在NK细胞分化中的重要性。将祖细胞群体接种在含有白细胞介素-2的培养基中，直接接种在预先建立的、同种异体的、经辐照的骨髓基质上5周，可产生CD56⁺CD3⁻NK细胞；然而，培养群体的特征有所不同。通过功能有限稀释分析确定，CD34⁺7⁺群体的扩增倍数和克隆效率显著高于CD34⁺7⁻或CD34⁺7⁻群体。这表明CD34⁺7⁺群体高度富集了NK祖细胞，并且可能是NK谱系分化中的一个可能的中间阶段。通过CD7的相对荧光将CD34⁺7⁺群体进一步分为CD34⁺7⁺dim和CD34⁺7⁺bright群体，结果显示，与CD34⁺7⁺dim细胞的平行实验相比，CD34⁺7⁺bright群体表现出显著更高的克隆频率（11.8%±2.4%对2.4%±0.7%，n = 6；P = 0.005）。将更原始的CD34⁺7⁻群体接种在transwell系统中（通过微孔膜将祖细胞与基质分离）可阻止其分化为NK细胞。相反，将CD34⁺7⁺祖细胞接种在transwell中可产生NK细胞。这些数据表明，原始的但不是更成熟的NK祖细胞在初始分化步骤中可能需要与基质直接接触。最后，在这个依赖基质的模型中，NK祖细胞的分化导致起始群体中均不存在的CD2表达。这一观察结果表明，骨髓基质可以以一种先前未描述的方式刺激NK祖细胞上的CD2表达，这可能类似于胸腺对未成熟T淋巴细胞上CD2表达的影响。这些观察结果确定了原始骨髓CD34⁺造血祖细胞向淋巴谱系定向分化的早期步骤，并强调了CD7与CD34共表达作为早期淋巴谱系定向特征以及在此过程中祖细胞与基质直接相互作用的重要性。
