Kusadasi N, van Soest P L, Mayen A E, Koevoet J L, Ploemacher R E
Institute of Hematology, Erasmus University Rotterdam, The Netherlands.
Leukemia. 2000 Nov;14(11):1944-53. doi: 10.1038/sj.leu.2401917.
In view of the limited potential for rapid hematological recovery after transplantation of umbilical cord blood cells (UCB) in adults, we have attempted to expand CD34+ selected hemopoietic stem cells (HSC) and progenitors in 2-week cultures of whole graft pools in the presence or absence of serum and stromal layers, and with various cytokine combinations including (1) FL + TPO; (2) FL + TPO plus SCF and/or IL6; or (3) SCF + IL6. Both in the input material and cultured grafts we determined the number of colony-forming cells (CFC), cobblestone area forming cells (CAFC), the NOD/SCID repopulating ability (SRA), and CD34+ CD38- subset by phenotyping. The highest fold-increase obtained for the number of nucleated cells (nc), CD34+, CD34+ CD38 cell numbers and CFC content was, respectively, 102 +/- 76, 24 +/- 19, 190 +/- 202 and 53 +/- 37 for stroma-free and 315 +/- 110, 25 +/- 3, 346 +/- 410 and 53 +/- 43 for stroma-supported cultures. CAFC week type 6 was maximally 11-fold expanded both under stroma-free and stroma-supported conditions. The FBMD-1 stromal cells supported a modest expansion of CD34+ CD38- cells (27 +/- 18-fold) and nc (6 +/- 4-fold), while a loss of CFC and CAFC subsets was observed. The stromal cells synergized with FL + TPO to give the highest expansion of hemopoietic progenitors. Stromal support could be fully replaced by complementing the FL + TPO stimulated cultures with SCF + IL6. FL + TPO were required and sufficient to give a 10- to 20-fold expansion of the ability of CD34+ UCB cells in 2-week cultures to engraft the BM of NOD/SCID mice. Stromal support, or complementation of the medium with SCF + IL6, did not significantly improve the in vivo engraftment potential. If the SRA and CAFC week 6 assays are accepted as tentative estimates of in vivo engrafting stem cells in humans, our findings may assist in the preparation of UCB grafts to meet the requirements for improved repopulation in the clinical setting.
鉴于成人脐血细胞（UCB）移植后血液学快速恢复的潜力有限，我们尝试在有或无血清及基质层的情况下，用不同细胞因子组合，对全移植物池进行为期2周的培养，以扩增经CD34 + 选择的造血干细胞（HSC）和祖细胞，这些组合包括：（1）FL + TPO；（2）FL + TPO加SCF和/或IL6；或（3）SCF + IL6。我们通过表型分析确定了输入材料和培养移植物中的集落形成细胞（CFC）数量、鹅卵石区域形成细胞（CAFC）数量、非肥胖型糖尿病/严重联合免疫缺陷小鼠（NOD/SCID）的再增殖能力（SRA）以及CD34 + CD38 - 亚群。对于有核细胞（nc）、CD34 + 、CD34 + CD38细胞数量和CFC含量，无基质培养获得的最高扩增倍数分别为102±76、24±19、190±202和53±37，有基质支持培养的分别为315±110、25±3、346±410和53±43。在无基质和有基质支持条件下，第6周的CAFC最大扩增了11倍。FBMD - 1基质细胞支持CD34 + CD38 - 细胞适度扩增（27±18倍）和nc适度扩增（6±4倍），但观察到CFC和CAFC亚群减少。基质细胞与FL + TPO协同作用，使造血祖细胞得到最高程度的扩增。用SCF + IL6补充FL + TPO刺激的培养物可完全替代基质支持。在为期2周的培养中，FL + TPO是使CD34 + UCB细胞植入NOD/SCID小鼠骨髓的能力扩增10至20倍所必需且足够的。基质支持或用SCF + IL6补充培养基并不能显著提高体内植入潜力。如果将第6周的SRA和CAFC检测结果作为对人体内植入性干细胞的初步估计，我们的研究结果可能有助于制备UCB移植物，以满足临床环境中改善再增殖的要求。
