Pawliuk R, Eaves C, Humphries R K
Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, Canada.
Blood. 1996 Oct 15;88(8):2852-8.
Recent assessment of the long-term repopulating activity of defined subsets of hematopoietic cells has offered new insights into the characteristics of the transplantable stem cells of this system; however, as yet, there is very little known about mechanisms that regulate their self-renewal in vivo. We have now exploited the ability to quantitate these cells using the competitive repopulating unit (CRU) assay to identify the role of both intrinsic (ontological) and extrinsic (transplanted dose-related) variables that may contribute to the regulation of CRU recovery in vivo. Ly5.1 donor cells derived from day-14.5 fetal liver (FL) or the bone marrow (BM) of adult mice injected 4 days previously with 5-fluorouracil were transplanted at doses estimated to contain 10, 100, or 1,000 long-term CRU into irradiated congenic Ly5.2 adult recipient mice. Eight to 12 months after transplantation, there was a complete recovery of BM cellularity and in vitro clonogenic progenitor numbers and a nearly full recovery of day-12 colony-forming unit-spleen numbers irrespective of the number or origin of cells initially transplanted. In contrast, regeneration of Ly5.1+ donor-derived CRU was incomplete in all cases and was dependent on both the origin and dose of the transplant, with FL being markedly superior to that of adult BM. As a result, the final recovery of the adult marrow CRU compartment ranged from 15% to 62% and from 1% to 18% of the normal value in recipients of FL and adult BM transplantation, respectively, with an accompanying maximum CRU amplification of 150-fold for recipients of FL cells and 15-fold for recipients of adult BM cells. Interestingly, the extent of CRU expansion from either source was inversely related to the number of CRU transplanted. These data suggest that recovery of mature blood cell production in vivo may activate negative feedback regulatory mechanisms to prematurely limit stem cell self-renewal ability. Proviral integration analysis of mice receiving retrovirally transduced BM cells confirmed regeneration of totipotent lymphomyeloid repopulating cells and provided evidence for a greater than 300-fold clonal amplification of a single transduced stem cell. These results highlight the differential regenerative capacities of CRU from fetal and adult sources that likely reflect intrinsic, genetically defined determinants of CRU expansion but whose contribution to the magnitude of stem cell amplification ultimately obtained in vivo is also strongly influenced by the initial number of CRU transplanted. Such findings set the stage for attempts to enhance CRU regeneration by administration of agents that may enable full expression of regenerative potential or through the expression of intracellular gene products that may alter intrinsic regenerative capacity.
最近对造血细胞特定亚群的长期再增殖活性的评估，为该系统可移植干细胞的特性提供了新的见解；然而，迄今为止，对于体内调节其自我更新的机制仍知之甚少。我们现在利用竞争性再增殖单位（CRU）分析来定量这些细胞的能力，以确定可能有助于体内CRU恢复调节的内在（本体）和外在（移植剂量相关）变量的作用。将来自第14.5天胎肝（FL）或4天前注射5-氟尿嘧啶的成年小鼠骨髓（BM）的Ly5.1供体细胞，以估计含有10、100或1000个长期CRU的剂量，移植到经照射的同基因Ly5.2成年受体小鼠中。移植后8至12个月，无论最初移植的细胞数量或来源如何，BM细胞数量和体外克隆祖细胞数量完全恢复，第12天脾集落形成单位数量几乎完全恢复。相比之下，Ly5.1+供体来源的CRU在所有情况下的再生都是不完全的，并且取决于移植的来源和剂量，FL明显优于成年BM。结果，成年骨髓CRU区室的最终恢复分别为FL和成年BM移植受体正常价值的15%至62%和1%至18%，FL细胞受体伴随的最大CRU扩增为150倍，成年BM细胞受体为15倍。有趣的是，来自任何一种来源的CRU扩增程度与移植的CRU数量呈负相关。这些数据表明，体内成熟血细胞生成的恢复可能会激活负反馈调节机制，过早限制干细胞的自我更新能力。对接受逆转录病毒转导BM细胞的小鼠进行的原病毒整合分析证实了全能淋巴髓系再增殖细胞的再生，并为单个转导干细胞的300倍以上克隆扩增提供了证据。这些结果突出了来自胎儿和成年来源的CRU的不同再生能力，这可能反映了CRU扩增的内在、基因定义的决定因素，但其对体内最终获得的干细胞扩增幅度的贡献也受到最初移植的CRU数量的强烈影响。这些发现为尝试通过给予可能使再生潜力充分表达的药物或通过表达可能改变内在再生能力的细胞内基因产物来增强CRU再生奠定了基础。
