Onyia J E, Clapp D W, Long H, Hock J M
Endocrine Division, Lilly Research Labs, Indiana University School of Dentistry, Indianapolis 46285, USA.
J Bone Miner Res. 1998 Jan;13(1):20-30. doi: 10.1359/jbmr.1998.13.1.20.
We transduced osteoprogenitor cells with recombinant retrovirus and analyzed proviral integration patterns into chromosomal DNA to detect for the first time the clonal and cellular fate of osteoprogenitor-derived progeny cells. Metaphyseal bone cells and diaphyseal stromal cells were isolated from the distal femurs of young rats, transduced with the vM5neolacZ recombinant retrovirus, and selected in the neomycin analog, G418. Following surgical marrow ablation of a femur in one leg of mature rats, retroviral-transduced metaphyseal or diaphyseal cells were injected into the ablated site. These rats were killed 5-6 days later. Metaphyseal and diaphyseal cells were isolated from distal femurs, selected in G418, and stained for beta-galactosidase (beta-gal+). The number and clonal origin of transduced progenitor cells were determined. High numbers of beta-galactosidase colonies with an osteoblast phenotype were obtained following metaphyseal transplants and detected in 100% of metaphyseal and none of diaphyseal specimens. In contrast, beta-galactosidase colonies derived from diaphyseal transplants were detected in 50% of specimens in both the metaphysis and diaphysis, and the absolute number of progenitor cell colonies was 60-fold less than metaphyseal transplants. Provirus was only detected in the ablated bones and not in the contralateral bone or other tissues. Proviral integration fragment analysis showed a single integration site for recovered metaphyseal cell clones, consistent with their origination from a common single progenitor. This is one of the first demonstrations of successful transplantation of clonal osteoprogenitors to their site of origin in bone. It may be possible to use these cells to target genes to bone for therapeutic use in skeletal and hematopoietic diseases.
我们用重组逆转录病毒转导骨祖细胞，并分析原病毒整合到染色体DNA中的模式，首次检测骨祖细胞来源的子代细胞的克隆情况和细胞命运。从幼年大鼠的股骨远端分离干骺端骨细胞和骨干基质细胞，用vM5neolacZ重组逆转录病毒转导，并在新霉素类似物G418中进行筛选。在成年大鼠的一条腿的股骨进行手术骨髓消融后，将逆转录病毒转导的干骺端或骨干细胞注射到消融部位。5-6天后处死这些大鼠。从股骨远端分离干骺端和骨干细胞，在G418中筛选，并进行β-半乳糖苷酶染色（β-gal+）。确定转导祖细胞的数量和克隆起源。干骺端移植后获得了大量具有成骨细胞表型的β-半乳糖苷酶菌落，在100%的干骺端标本中检测到，而骨干标本中均未检测到。相比之下，在干骺端和骨干的50%的标本中检测到源自骨干移植的β-半乳糖苷酶菌落，祖细胞菌落的绝对数量比干骺端移植少60倍。仅在消融的骨中检测到原病毒，在对侧骨或其他组织中未检测到。原病毒整合片段分析显示，回收的干骺端细胞克隆有一个单一的整合位点，这与其源自共同的单个祖细胞一致。这是克隆性骨祖细胞成功移植到其在骨中的起源部位的首批证明之一。有可能利用这些细胞将基因靶向到骨，用于骨骼和造血疾病的治疗。
