Lemieux M E, Rebel V I, Lansdorp P M, Eaves C J
Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, Canada.
Blood. 1995 Aug 15;86(4):1339-47.
In this report, we describe a modification of the assay for long-term culture-initiating cells (LTC-IC) that allows a subset of murine LTC-IC (designated as LTC-ICML) to express both their myeloid (M) and lymphoid (L) differentiative potentials in vitro. The modified assay involves culturing test cells at limiting dilutions on irradiated mouse marrow feeder layers for an initial 4 weeks under conditions that support myelopoiesis and then for an additional week under conditions permissive for B-lymphopoiesis. All of the clonogenic pre-B progenitors (colony-forming unit [CFU] pre-B) detected in such postswitch LTC appear to be the progeny of uncommitted cells present in the original cell suspension because exposure of lymphoid-restricted progenitors to myeloid LTC conditions for > or = 7 days was found to irreversibly terminate CFU-pre-B production and, in cultures initiated with limiting numbers of input cells (no progenitors of any type detected in > 70% of cultures 1 week after the switch), the presence of CFU-pre-B was tightly associated with the presence of myeloid clonogenic cells, regardless of the purity of the input population. Limiting dilution analysis of the proportion of negative cultures measured for different numbers of input cells showed the frequency of LTC-ICML in normal adult mouse marrow to be 1 per 5 x 10(5) cells with an enrichment of approximately 500-fold in the Sca-1+ Lin-WGA+ fraction, as was also found for competitive in vivo repopulating units (CRU) and conventionally defined LTC-IC. LTC-ICML also exhibited the same resistance to treatment in vivo with 5-fluorouracil (5-FU) as CRU and LTC-IC, thereby distinguishing these three populations from the great majority of both in vitro clonogenic cells and day 12 CFU-S. The ability to quantitate cells with dual lymphoid and myeloid differentiation potentials in vitro, without the need for their prior purification, should facilitate studies of totipotent hematopoietic stem cell regulation.
在本报告中，我们描述了一种对长期培养起始细胞（LTC-IC）检测方法的改进，该方法能使一部分小鼠LTC-IC（命名为LTC-ICML）在体外同时表达其髓系（M）和淋巴系（L）分化潜能。改进后的检测方法包括将测试细胞以有限稀释度接种在经照射的小鼠骨髓饲养层上，在支持髓系造血的条件下初始培养4周，然后在允许B淋巴细胞生成的条件下再培养1周。在这种转换后的LTC中检测到的所有克隆性前B祖细胞（集落形成单位[CFU]前B）似乎都是原始细胞悬液中未定向分化细胞的后代，因为发现淋巴系受限祖细胞暴露于髓系LTC条件下≥7天会不可逆地终止CFU-前B的产生，并且在以有限数量的输入细胞起始的培养物中（转换后1周，>70%的培养物中未检测到任何类型的祖细胞），CFU-前B的存在与髓系克隆性细胞的存在紧密相关，而与输入群体的纯度无关。对不同数量输入细胞的阴性培养物比例进行的有限稀释分析表明，正常成年小鼠骨髓中LTC-ICML的频率为每5×10⁵个细胞中有1个，在Sca-1⁺Lin⁻WGA⁺组分中富集约500倍，竞争性体内再植单位（CRU）和传统定义的LTC-IC也是如此。LTC-ICML在体内对5-氟尿嘧啶（5-FU）治疗的抗性也与CRU和LTC-IC相同，从而将这三个群体与绝大多数体外克隆性细胞和第12天的CFU-S区分开来。能够在体外定量具有双淋巴系和髓系分化潜能的细胞，而无需事先对其进行纯化，这将有助于对全能造血干细胞调节的研究。
