Craig W, Kay R, Cutler R L, Lansdorp P M
Terry Fox Laboratory, British Columbia Cancer Agency, Departments of Medicine, Pathology, and Medical Genetics, University of British Columbia, Vancouver, Canada.
J Exp Med. 1993 May 1;177(5):1331-42. doi: 10.1084/jem.177.5.1331.
Expression of Thy-1 on hematopoietic cells from human fetal liver (FL), cord blood (CB), and bone marrow (BM) was studied with a novel anti-Thy-1 antibody, 5E10. Specificity of 5E10 for human Thy-1 was demonstrated by immunoprecipitation of a 25-35-kD molecule, and the sequence of a cDNA that was cloned by immunoselection of COS cells transfected with a cDNA library derived from a 5E10+ cell line. Two- and three-color immunofluorescence staining experiments revealed that the Thy-1 expression is restricted to, an average, 1-4% of FL, CB, and BM cells, and binding to these cell types is essentially restricted to a very small subset of lymphoid cells and approximately 25% of CD34+ cells. Thy-1+ CD34+ cells were further characterized as CD38lo/CD45RO+/CD45RA-/CD71lo/c-kit(lo) and rhodamine 123dull. When CD34+ cells were sorted on the basis of Thy-1 expression, the majority of clonogenic cells were recovered in the CD34+Thy-1- fraction, whereas the majority of cells capable of producing myeloid colonies after 5-8 wk of long-term culture (long-term culture initiating cells) were recovered in the Thy-1+CD34+ fraction. In addition to CD34+ cells, Thy-1 was found to be expressed on a variable, very small number (< 1%) of CD34- mononuclear cells in BM, CB, and peripheral blood that were further characterized as CD3+ CD4+ lymphocytes. The restricted expression of Thy-1 on primitive hematopoietic cells is in agreement with a previous report (Baum et al., 1992. Proc. Natl. Acad. Sci. USA. 89:2804) in which Thy-1 expression was used to enrich for primitive hematopoietic cells from fetal tissue. Compared with those previous studies, we found Thy-1 expression on a larger proportion of CD34+ cells (25% in our study vs. 5% in Baum et al.) and furthermore performed studies on Thy-1 expression on CD34+ cells from CB, FL, and BM in relation to markers that are known to be differentially expressed on hematopoietic cells. Taken together our results indicate that Thy-1-specific antibody 5E10 is an attractive tool for further studies on the biology and purification of human stem cells.
利用新型抗Thy-1抗体5E10，研究了人胎肝（FL）、脐血（CB）和骨髓（BM）造血细胞上Thy-1的表达情况。通过对一个25 - 35kD分子进行免疫沉淀，以及对用源自5E10 +细胞系的cDNA文库转染的COS细胞进行免疫筛选所克隆的cDNA序列，证实了5E10对人Thy-1的特异性。双色和三色免疫荧光染色实验表明，Thy-1表达平均局限于FL、CB和BM细胞的1% - 4%，并且与这些细胞类型的结合基本上局限于淋巴细胞的一个非常小的亚群以及大约25%的CD34 +细胞。Thy-1 + CD34 +细胞进一步被鉴定为CD38lo/CD45RO + /CD45RA - /CD71lo/c-kit(lo)和罗丹明123染色暗淡。当根据Thy-1表达对CD34 +细胞进行分选时，大多数集落形成细胞在CD34 + Thy-1 -部分中被回收，而在长期培养5 - 8周后能够产生髓系集落的大多数细胞（长期培养起始细胞）在Thy-1 + CD34 +部分中被回收。除了CD34 +细胞外，还发现Thy-1在BM、CB和外周血中数量可变且极少（<1%）的CD34 -单核细胞上表达，这些细胞进一步被鉴定为CD3 + CD4 +淋巴细胞。Thy-1在原始造血细胞上的限制性表达与之前的一份报告（Baum等人，1992年。美国国家科学院院刊。89:2804）一致，在该报告中Thy-1表达被用于从胎儿组织中富集原始造血细胞。与之前的那些研究相比，我们发现Thy-1在更大比例的CD34 +细胞上表达（我们的研究中为25%，而Baum等人的研究中为5%），并且还对来自CB、FL和BM的CD34 +细胞上Thy-1的表达与已知在造血细胞上差异表达的标志物之间的关系进行了研究。综合我们的结果表明，Thy-1特异性抗体5E10是进一步研究人干细胞生物学和纯化的一个有吸引力的工具。
