Characterization and partial purification of human marrow cells capable of initiating long-term hematopoiesis in vitro.
Characterization and partial purification of human marrow cells capable of initiating long-term hematopoiesis in vitro.
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DOI：
10.1182/blood.v74.5.1563.1563
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发表时间：
1989-10
期刊：
影响因子：
20.3
通讯作者：
Heather J. Sutherland;C. Eaves;A. Eaves;W. Dragowska;Peter M. Lansdorp
中科院分区：
文献类型：
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作者：
Heather J. Sutherland;C. Eaves;A. Eaves;W. Dragowska;Peter M. Lansdorp
为了开发用于分离存在于正常人骨髓中的最原始的造血干细胞的纯化策略，我们将细胞分离技术与在照射的人骨髓贴壁细胞存在下体外启动持续造血的细胞的测定相结合。这些“饲养细胞”是通过以每平方厘米3 × 10（4）个辐照细胞的密度传代培养2- 6周龄的原代长期骨髓培养贴壁层而建立的。将试验“长期培养（LTC）-起始细胞”接种在饲养层顶部，然后将共培养物作为标准长期骨髓培养物维持，每周更换一半培养基并去除一半非贴壁细胞。使用5周后存在的髓系、红系和多系克隆生成祖细胞的总数来提供最初添加的LTC起始细胞的数量的定量评估。使用该测定，LTC起始细胞的密度、光散射和两种细胞表面抗原特性已被定义，并与能够在甲基纤维素中直接形成集落的细胞进行比较。虽然大多数的克隆形成细胞被发现在高前向光散射（FLS）的“原始”窗口，LTC启动细胞有显着较低的FLS属性，在这方面更类似于淋巴细胞。LTC起始细胞也比克隆形成细胞表达更少的HLA-DR抗原。大多数LTC起始细胞位于CD 34（My 10）荧光谱的前2%，而克隆形成细胞位于CD 34荧光谱的前5%。通过组合低FLS、低正交光散射（OLS）、低HLA-DR表达和高CD 34表达，可以获得相对于未分离的骨髓富集约800倍的LTC起始细胞的群体。该群体仅含有0.06%的骨髓细胞和2%的总克隆形成细胞，但保留了原始骨髓中存在的50%至60%的LTC起始细胞。独立纯化这两个群体的能力表明，LTC和克隆形成试验鉴定了不同的，但不一定是不重叠的人骨髓细胞类型。由于克隆形成细胞来源于LTC起始细胞，因此LTC测定清楚地检测到更原始的群体。一个简单的方法，允许纯化这些细胞的三个数量级的高产率的可用性应该是有用的造血早期事件的调查，以及为确定性分离的人造血干细胞与长期在体内重建的潜力。
To develop a purification strategy for isolating the most primitive hematopoietic stem cells present in normal human marrow we have combined cell separation techniques with an assay for cells that initiate sustained hematopoiesis in vitro in the presence of irradiated human marrow adherent cells. These "feeders" were established by subculturing 2- to 6-week-old primary long-term marrow culture adherent layers at a density of 3 x 10(4) irradiated cells per square centimeter. Test "long-term culture (LTC)-initiating cells" were plated on top of the feeders and the cocultures then maintained as standard long-term marrow cultures with half-media changes and removal of half of the nonadherent cells each week. The total number of myeloid, erythroid, and multilineage clonogenic progenitors present after 5 weeks was used to provide a quantitative assessment of the number of LTC-initiating cells originally added. Using this assay, the density, light scatter, and two cell surface antigen properties of LTC-initiating cells have been defined and compared with cells capable of directly forming colonies in methylcellulose. While the majority of the clonogenic cells were found in the high forward light scatter (FLS) "blast" window, LTC-initiating cells had significantly lower FLS properties and in this respect were more similar to lymphocytes. LTC-initiating cells also expressed less HLA-DR antigen than clonogenic cells. The majority of LTC-initiating cells were found in the top 2% of the CD34 (My10) fluorescence profile, whereas clonogenic cells were found throughout the top 5% of the CD34 fluorescence profile. By combining low FLS, low orthogonal light scatter (OLS), low HLA-DR expression, and high CD34 expression, a population could be obtained that was enriched for LTC-initiating cells approximately 800-fold over unseparated marrow. This population contains only 0.06% of the marrow cells and 2% of the total clonogenic cells, but retains 50% to 60% of the LTC-initiating cells present in the original marrow. The ability to purify these two populations independently shows that the LTC and clonogenic assays identify distinct, although not necessarily nonoverlapping cell types in human marrow. Since clonogenic cells are derived from LTC-initiating cells, the LTC assay clearly detects a more primitive population. The availability of a simple approach that allows the purification of such cells by three orders of magnitude in high yield should be useful for the investigation of early events in hematopoiesis as well as for the definitive isolation of human hematopoietic stem cells with long-term in vivo repopulating potential.