Prosper F, Vanoverbeke K, Stroncek D, Verfaillie C M
Department of Medicine, University of Minnesota, Minneapolis 55455, USA.
Blood. 1997 Jun 1;89(11):3991-7.
We have recently shown that more than 90% of long-term culture initiating cells (LTC-IC) mobilized in the peripheral blood (PB) of normal individuals express HLA-DR and CD38 antigens and can sustain hematopoiesis for only 5 weeks. However, 10% of LTC-IC in mobilized PB are CD34+ HLA-DR- and CD34+ CD38- and can sustain hematopoiesis for at least 8 weeks. We now examine the ex vivo expansion potential of CD34+ HLA-DR+ cells (rich in mature LTC-IC) and CD34+ HLA-DR- cells (rich in primitive LTC-IC) in granulocyte colony-stimulating factor (G-CSF) mobilized PB progenitor cells (PBPC). Cells were cultured in contact with M2-10B4 cells (contact) or in transwells above M2-10B4 (noncontact) without and with interleukin-3 (IL-3) and macrophage inflammatory protein (MIP-1alpha) for 2 and 5 weeks. Progeny were evaluated for the presence of colony-forming cells (CFC) and LTC-IC. When CD34+ HLA-DR+ PB cells were cultured in contact cultures without cytokines, a threefold expansion of CFC was seen at 2 weeks, but an 80% decrease in CFC was seen at week 5. Further, the recovery of LTC-IC at week 2 was only 17% and 1% at week 5. This confirms our previous observation that although CD34+ HLA-DR+ mobilized PB cells can initiate long-term cultures, they are relatively mature and cannot sustain long-term hematopoiesis. In contrast, when CD34+ HLA-DR- mobilized PB cells were cultured in contact cultures without cytokines, CFC expansion persisted until week 5 and 49% and 11% of LTC-IC were recovered at week 2 and 5, respectively. As we have shown for steady state bone marrow (BM) progenitors, recovery of LTC-IC was threefold higher when CD34+ HLA-DR- PBPC were cultured in noncontact rather than contact cultures, and improved further when IL-3 and MIP-1alpha were added to noncontact cultures (96 +/- 2% maintained at week 5). We conclude that although G-CSF mobilizes a large population of "mature" CD34+ HLA-DR+ LTC-IC with a limited proliferative capacity, primitive CD34+ HLA-DR- LTC-IC present in mobilized PB have similar characteristics as LTC-IC from steady state BM: (1) they can be maintained in noncontact cultures containing IL-3 and MIP-1alpha for at least 5 weeks; (2) they are subject to the same proliferation inhibitory influences of contact with stroma. Since the absolute number of primitive LTC-IC (week 8 LTC-IC) per mL of G-CSF mobilized PB is similar to that per mL of steady state BM, these studies further confirm that G-CSF mobilized PBPC may have similar long-term repopulating abilities as steady state BM.
我们最近发现，正常个体外周血（PB）中动员的长期培养起始细胞（LTC-IC）超过90%表达HLA-DR和CD38抗原，且只能维持造血5周。然而，动员的PB中10%的LTC-IC是CD34+HLA-DR-和CD34+CD38-，并能维持造血至少8周。我们现在研究粒细胞集落刺激因子（G-CSF）动员的PB祖细胞（PBPC）中CD34+HLA-DR+细胞（富含成熟LTC-IC）和CD34+HLA-DR-细胞（富含原始LTC-IC）的体外扩增潜力。细胞与M2-10B4细胞接触培养（接触培养）或在M2-10B4上方的Transwell中培养（非接触培养），分别在无细胞因子、添加白细胞介素-3（IL-3）和巨噬细胞炎性蛋白（MIP-1α）的条件下培养2周和5周。评估子代中集落形成细胞（CFC）和LTC-IC的存在情况。当CD34+HLA-DR+PB细胞在无细胞因子的接触培养中培养时，2周时CFC出现三倍扩增，但在第5周时CFC减少80%。此外，第2周时LTC-IC的恢复率仅为17%，第5周时为1%。这证实了我们之前的观察结果，即尽管CD34+HLA-DR+动员的PB细胞可以启动长期培养，但它们相对成熟，无法维持长期造血。相反，当CD34+HLA-DR-动员的PB细胞在无细胞因子的接触培养中培养时，CFC扩增持续到第5周，第2周和第5周时LTC-IC的恢复率分别为49%和11%。正如我们对稳态骨髓（BM）祖细胞所显示的那样，当CD34+HLA-DR-PBPC在非接触培养而非接触培养中培养时，LTC-IC的恢复率高出三倍，当在非接触培养中添加IL-3和MIP-1α时进一步提高（第5周时维持在96±2%）。我们得出结论，尽管G-CSF动员了大量增殖能力有限的“成熟”CD34+HLA-DR+LTC-IC，但动员的PB中存在的原始CD34+HLA-DR-LTC-IC具有与稳态BM中的LTC-IC相似的特征：（1）它们可以在含有IL-3和MIP-1α的非接触培养中维持至少5周；（2）它们受到与基质接触的相同增殖抑制影响。由于每毫升G-CSF动员的PB中原始LTC-IC（第8周LTC-IC）的绝对数量与每毫升稳态BM中的相似，这些研究进一步证实G-CSF动员的PBPC可能具有与稳态BM相似的长期重建能力。
