Denning-Kendall Patricia, Singha Sakon, Bradley Ben, Hows Jill
University of Bristol, Division of Transplantation Sciences, Bristol, United Kingdom.
Stem Cells. 2003;21(6):694-701. doi: 10.1634/stemcells.21-6-694.
The long-term culture-initiating cell (LTC-IC) assay is a physiological approach to the quantitation of primitive human hematopoietic cells. The readout using identification of cobblestone area-forming cells (CAFC) has gained popularity over the LTC-IC readout where cells are subcultured in a colony-forming cell assay. However, comparing the two assays, cord blood (CB) mononuclear cell (MNC) samples were found to contain a higher frequency of CAFC than LTC-IC (126 +/- 83 versus 40 +/- 31 per 10(5) cells, p = 0.0001). Overall, 60% of week-5 cobblestones produced by CB MNC were not functional LTC-IC and were classified as "false." Separation of CB MNC using immunomagnetic columns showed that false cobblestones were CD34(-)/lineage(+). Purified CD34(+) cells, as expected, gave very similar readouts in the two assays, with 4,084 and 3,468/10(5) cells being CAFC and LTC-IC, respectively. CD34(-)/lineage(-) cells did not form cobblestones or become CD34(+) on stroma or in cytokine culture. Human CB MNC contain a population of mature lineage(+) cells, possibly mature T or B cells, which, although producing cobblestone areas (CA), are not functional LTC-IC. The CAFC readout by this method, therefore, is unreliable for estimation of primitive hematopoietic cells by limiting dilution analysis in whole human CB or MNC and also may not detect CD34(-) CA stem cells.
长期培养起始细胞（LTC-IC）测定法是一种定量原始人类造血细胞的生理学方法。通过鉴定鹅卵石区域形成细胞（CAFC）的读数方法，相较于在集落形成细胞测定中对细胞进行传代培养的LTC-IC读数方法，已越来越受到欢迎。然而，比较这两种测定方法时发现，脐带血（CB）单个核细胞（MNC）样本中CAFC的频率高于LTC-IC（每10⁵个细胞中分别为126±83和40±31，p = 0.0001）。总体而言，CB MNC产生的第5周鹅卵石中有60%不是功能性LTC-IC，被归类为“假阳性”。使用免疫磁珠柱分离CB MNC显示，假阳性鹅卵石为CD34⁻/谱系⁺。正如预期的那样，纯化的CD34⁺细胞在两种测定中给出了非常相似的读数，每10⁵个细胞中CAFC和LTC-IC分别为4,084和3,468。CD34⁻/谱系⁻细胞在基质上或细胞因子培养中既不形成鹅卵石，也不会变为CD34⁺。人类CB MNC包含一群成熟的谱系⁺细胞，可能是成熟的T或B细胞，它们虽然能产生鹅卵石区域（CA），但不是功能性LTC-IC。因此，通过这种方法的CAFC读数，对于通过全人类CB或MNC中的有限稀释分析来估计原始造血细胞是不可靠的，并且可能也检测不到CD34⁻ CA干细胞。
