Bruno B, Goerner M A, Nash R A, Storb R, Kiem H P, McSweeney P A
Clinical Research Division, Fred Hutchinson Cancer Research Center, University of Washington Medical School, Seattle, USA.
Biol Blood Marrow Transplant. 2001;7(10):543-51. doi: 10.1016/s1083-8791(01)70020-1.
Human CD34+ cells have been shown to retain long-term hematopoietic engrafting potential in preclinical and clinical studies. However, recent studies of human and murine CD34- stem cells suggest that these are functionally important early progenitors. Using autologous transplantation, we investigated whether canine CD34 and CD34- marrow cells could be transduced and give rise to long-term hematopoiesis. CD34+Lin- and CD34-Lin- cell populations purified by fluorescence-activated cell sorting were separately cocultivated with retroviral vectors LN (CD34+Lin-) and LNY (CD34-Lin-), which carry the neomycin (neo) gene. After myeloablative total body irradiation (920 cGy), 3 dogs received transplants of both CD34+Lin- cells and CD34-Lin- cells and 2 dogs received only CD34-Lin- cells. Untransduced autologous marrow cells were given to ensure hematopoietic recovery. Using CFU-C assays, transduction efficiencies of CD34+Lin- cells ranged from 6% to 18% with no CFU-C formation from CD34-Lin- cells. PCR-based detection of the neo gene from WBCs was used to detect transduced cells weekly after transplantation. Additional PCR studies in 3 dogs given both CD34+Lin- and CD34-Lin- cells were performed on monocytes, granulocytes, and T cells (2 dogs, one at 7.5 months and the other at 9 months) and granulocytes (1 dog at 12 months). LN was detected up to 12 months posttransplantation in WBCs and mono-myeloid and lymphoid populations from 3 dogs receiving transplants of transduced CD34+Lin- cells. LNY was not detected at any time after transplantation in 5 dogs that received transduced CD34-Lin- cells. Whereas canine CD34+Lin- marrow cells contributed to long-term multilineage hematopoiesis, progeny of CD34-Lin- progenitor cells were not detected after transplantation in these experiments.
在临床前和临床研究中，已证明人类CD34+细胞具有长期造血植入潜力。然而，最近对人类和小鼠CD34-干细胞的研究表明，这些细胞是功能重要的早期祖细胞。我们采用自体移植方法，研究犬CD34和CD34-骨髓细胞是否能够被转导并产生长期造血。通过荧光激活细胞分选纯化的CD34+Lin-和CD34-Lin-细胞群体分别与携带新霉素（neo）基因的逆转录病毒载体LN（CD34+Lin-）和LNY（CD34-Lin-）共培养。在进行920 cGy的清髓性全身照射后，3只犬接受了CD34+Lin-细胞和CD34-Lin-细胞的移植，2只犬仅接受了CD34-Lin-细胞移植。给予未转导的自体骨髓细胞以确保造血恢复。使用集落形成单位-细胞（CFU-C）测定法，CD34+Lin-细胞的转导效率为6%至18%，而CD34-Lin-细胞未形成CFU-C。移植后每周使用基于PCR的白细胞中neo基因检测来检测转导细胞。对3只同时接受CD34+Lin-和CD34-Lin-细胞移植的犬，在单核细胞、粒细胞和T细胞（2只犬，一只在7.5个月，另一只在9个月）以及粒细胞（1只犬在12个月）上进行了额外的PCR研究。在接受转导的CD34+Lin-细胞移植的3只犬的白细胞、单核-髓系和淋巴系群体中，移植后12个月仍可检测到LN。在接受转导的CD34-Lin-细胞移植的5只犬中，移植后任何时间均未检测到LNY。虽然犬CD34+Lin-骨髓细胞有助于长期多系造血，但在这些实验中，移植后未检测到CD34-Lin-祖细胞的后代。
