McNiece Ian K, Almeida-Porada Graça, Shpall Elizabeth J, Zanjani Esmail
Experimental Hematology Laboratory, University of Colorado Health Sciences Center, Denver 80262, USA.
Exp Hematol. 2002 Jun;30(6):612-6. doi: 10.1016/s0301-472x(02)00805-6.
Cord blood (CB) products are becoming routinely used in unrelated allogeneic transplantation for smaller pediatric patients. Because of the low numbers of cells in CB compared to bone marrow or peripheral blood progenitor cells, their use is more limited in larger adults. Therefore, we developed ex vivo expansion conditions for CB and currently are transplanting ex vivo expanded CB products to patients receiving high-dose chemotherapy. As there is concern that ex vivo expansion may exhaust long-term engrafting cells, the current clinical protocols consist of both an expanded fraction and an unexpanded fraction. To determine the effect of expansion culture on long-term engrafting cells, we evaluated the short- and long-term engrafting potential of ex vivo expanded CB using a fetal sheep xenogeneic transplant model.
CD 34(+) cells were selected from CB products and cultured in a two-step procedure in the presence of stem cell factor, megakaryocyte growth and differentiation factor, and granulocyte colony-stimulating factor for 14 days. Starting cells (CD34(+) cells), and cultured cells (day 7 and day 14 cells) were transplanted in 60-day-old fetal sheep and evaluated at various time points post transplant for the presence of human cells. Long-term engrafting cells were assessed by serial passage into secondary and tertiary recipients.
Day 14 expanded CB cells provided more rapid engraftment than either the day 7 expanded cells or the day 0 cells; however, this engraftment was transient, and no human cells were detectable at 16 months post transplant in the animals that received the day 14 expanded cells. Day 0 cells had engrafted animals at 2 months post transplant and both the day 0 and day 7 cells persisted to 16 months or longer. In the secondary animals, the day 0 and day 7 cells engrafted equivalently at 3 months post transplant; however, no secondary engraftment resulted from the day 14 cells. The levels of engraftment in secondary animals receiving day 7 cells decreased with time to barely detectable levels at 12 months post transplant.
Ex vivo expansion of CB CD34(+) cells under the conditions described results in the generation of increased mature cells and progenitors that are capable of more rapid engraftment in fetal sheep compared to unexpanded CB CD34(+) cells. The expanded cells engrafted primary sheep but lacked secondary and tertiary engrafting potential. These studies demonstrate that although ex vivo expanded cells may be able to provide rapid short-term engraftment, the long-term potential of expanded grafts may be compromised. Therefore, clinical protocols may require transplantation of two fractions of cells, an expanded CB graft to provide rapid short-term engraftment and an unmanipulated fraction of CB graft to provide stem cells for long-term engraftment.
脐血（CB）产品正常规用于较小儿科患者的非亲缘异基因移植。由于与骨髓或外周血祖细胞相比，脐血中的细胞数量较少，其在较大成年患者中的应用更为受限。因此，我们开发了脐血的体外扩增条件，目前正在将体外扩增的脐血产品移植给接受大剂量化疗的患者。鉴于有人担心体外扩增可能会耗尽长期植入细胞，当前的临床方案包括一个扩增部分和一个未扩增部分。为了确定扩增培养对长期植入细胞的影响，我们使用胎羊异种移植模型评估了体外扩增脐血的短期和长期植入潜力。
从脐血产品中筛选出CD34(+)细胞，并在干细胞因子、巨核细胞生长和分化因子以及粒细胞集落刺激因子存在的情况下，分两步培养14天。将起始细胞（CD34(+)细胞）以及培养细胞（第7天和第14天的细胞）移植到60日龄的胎羊体内，并在移植后的不同时间点评估人细胞的存在情况。通过连续传代至二级和三级受体来评估长期植入细胞。
第14天扩增的脐血细胞比第7天扩增的细胞或第0天的细胞植入更快；然而，这种植入是短暂的，在接受第14天扩增细胞的动物中，移植后16个月未检测到人类细胞。第0天的细胞在移植后2个月植入动物体内，第0天和第7天的细胞均持续存在至16个月或更长时间。在二级动物中，第0天和第7天的细胞在移植后3个月植入情况相同；然而，第14天的细胞未产生二级植入。接受第7天细胞的二级动物中的植入水平随时间下降，在移植后12个月降至几乎检测不到的水平。
在所描述的条件下对脐血CD34(+)细胞进行体外扩增，会产生比未扩增的脐血CD34(+)细胞更多的成熟细胞和祖细胞，这些细胞在胎羊中能够更快地植入。扩增的细胞植入了一级绵羊，但缺乏二级和三级植入潜力。这些研究表明，尽管体外扩增细胞可能能够提供快速的短期植入，但扩增移植物的长期潜力可能会受到损害。因此，临床方案可能需要移植两部分细胞，一部分扩增的脐血移植物用于提供快速的短期植入，另一部分未处理的脐血移植物用于提供长期植入的干细胞。
