Niemeyer G P, Hudson J, Bridgman R, Spano J, Nash R A, Lothrop C D
Scott-Ritchey Research Center, College of Veterinary Medicine, Auburn University, Ala., USA
Exp Hematol. 2001 Jun;29(6):686-93. doi: 10.1016/s0301-472x(01)00638-5.
The purpose of this study was to purify and characterize canine hematopoietic progenitor cells for surface antigen phenotype and reconstitution ability. Canine hematopoietic progenitor cells were isolated by density gradient sedimentation, lineage depletion with monoclonal antibodies, and fluorescence-activated cell sorting (FACS) for selection of cells with low-forward and right-angle scatter that were rhodamine 123 (Rh-123)(dull). Isolated cells were characterized for expression of CD34, c-kit, and Flt-3. A canine/murine xenograft model and a mixed-chimerism assay were used to examine the in vivo proliferative potential of isolated cells. The lineage-positive (Lin(+)) cells represented 80 +/- 11% (n = 22) of the input mononuclear cells. Lineage depletion resulted in a fourfold increase in colony-forming unit granulocyte/monocyte (CFU-GM), a 2.5-fold increase in burst-forming unit-erythroid (BFU-E), and a twofold increase in the number of Rh-123(dull) cells over nonlineage-depleted bone marrow mononuclear cells (BMMCs). Lineage depletion led to a 2.7-fold enrichment of CD34 cells, a 10.4-fold enrichment of c-kit cells, and a 10.8-fold enrichment of CD34/c-kit(+1) cells over total BMMCs. Nineteen percent of lineage-negative (Lin(-)) cells were positive for Flt-3. Injection of canine cells into irradiated (400 rads) NOD/SCID mice resulted in the detection of canine CD45(+) cells with BMMCs, Lin(-) cells, or Rh-123(dull) cells. Transplantation of purified Lin(-) cells in dog leukocyte antigen-matched littermates resulted in low-level engraftment for at least 10 weeks. The development of methods for purification and characterization of canine hematopoietic progenitor cells should enhance the utilization of the canine model for a variety of experimental and therapeutic purposes.
本研究的目的是纯化犬造血祖细胞，并对其表面抗原表型和重建能力进行表征。通过密度梯度沉降、用单克隆抗体去除谱系细胞以及荧光激活细胞分选（FACS）来分离犬造血祖细胞，以选择具有低前向散射和直角散射且罗丹明123（Rh-123）染色暗淡的细胞。对分离出的细胞进行CD34、c-kit和Flt-3表达的表征。使用犬/鼠异种移植模型和混合嵌合体分析来检测分离细胞的体内增殖潜力。谱系阳性（Lin(+)）细胞占输入单核细胞的80±11%（n = 22）。去除谱系细胞后，集落形成单位粒细胞/单核细胞（CFU-GM）增加了四倍，爆式红细胞集落形成单位（BFU-E）增加了2.5倍，与未去除谱系细胞的骨髓单核细胞（BMMCs）相比，Rh-123染色暗淡的细胞数量增加了两倍。去除谱系细胞导致CD34细胞富集2.7倍，c-kit细胞富集10.4倍，CD34/c-kit(+1)细胞相对于总BMMCs富集10.8倍。19%的谱系阴性（Lin(-)）细胞Flt-3呈阳性。将犬细胞注射到经辐照（400拉德）的NOD/SCID小鼠体内后，在BMMCs、Lin(-)细胞或Rh-123染色暗淡的细胞中检测到犬CD45(+)细胞。将纯化的Lin(-)细胞移植到与犬白细胞抗原匹配的同窝仔犬中，至少10周内实现了低水平植入。犬造血祖细胞纯化和表征方法的开发应能提高犬模型在各种实验和治疗目的中的利用率。
