Davis T A, Robinson D H, Lee K P, Kessler S W
Immune Cell Biology Program, Naval Medical Research Institute, Bethesda, MD 20889-5055, USA.
Blood. 1995 Apr 1;85(7):1751-61.
Primary autologous as well as allogeneic and xenogeneic stroma will support human stem cell proliferation and differentiation for several months. In the present study, we investigated the capacity of porcine microvascular endothelial cells (PMVECs) together with combinations of cytokines (granulocyte-macrophage colony-stimulating factor [GM-CSF] + stem factor [SCF], interleukin-3 [IL-3] + SCF + IL-6, and GM-CSF + IL-3 + SCF + IL-6) to support the expansion and development of purified human CD34+ bone marrow cells. In short-term cultures (7 days), the greatest expansion of nonadherent hematopoietic cells and clonogenic progenitors was seen with CD34+ cells in direct contact with PMVEC monolayers (PMVEC contact), followed by PMVEC noncontact and liquid suspension cultures, respectively. Maximal expansion of nonadherent cells (42-fold) and total CD34+ cells (12.6-fold) occurred in PMVEC contact cultures treated with GM-CSF + IL-3 + SCF + IL-6, with similar increases in the number of granulocyte-macrophage colony-forming units (CFU-GM), CFU-mix, erythroid burst-forming units (BFU-E), CFU-blast and CFU-megakaryocyte (CFU-Mk) progenitor cells. Moreover, the number of CD34+ CD38- and CD34+ CD38+ cells increased 148.1-fold and 8.0-fold, respectively. Replating studies show that cells from day 7 dispersed blast cell colonies generated on cytokine-treated PMVEC monolayers have a high replating potential for multilineage progenitor cells. In long-term PMVEC contact cultures, CD34+ cells seeded onto PMVEC monolayers with GM-CSF + IL-3 + SCF + IL-6 showed a total calculated expansion of over 5,000,000-fold of nonadherent cells over 35 days in culture. Maximal clonogenic cell production was observed at day 28, with 6,353-fold for total CFC and comparable increases for CFU-GM, CFU-mix, CFU-blast, BFU-E, and CFU-Mk. The total number of CD34+ cells increased 2,584-fold at day 28. Furthermore, the extended growth kinetics of these cultures indicates that these phenotypically primitive progenitor cells are also functionally expanded on PMVEC monolayers. These results support the hypothesis that direct contact with a PMVEC monolayer supports the initial expansion of hematopoietic progenitor cells with a high replating potential and, possibly, a more primitive phenotype (CD34+, CD34+/CD38-).
原代自体以及异体和异种基质可支持人类干细胞增殖和分化数月。在本研究中，我们研究了猪微血管内皮细胞（PMVECs）与细胞因子组合（粒细胞 - 巨噬细胞集落刺激因子[GM - CSF] + 干细胞因子[SCF]、白细胞介素 - 3[IL - 3] + SCF + IL - 6以及GM - CSF + IL - 3 + SCF + IL - 6）支持纯化的人类CD34 + 骨髓细胞扩增和发育的能力。在短期培养（7天）中，与PMVEC单层直接接触（PMVEC接触）的CD34 + 细胞产生的非贴壁造血细胞和克隆形成祖细胞扩增最多，其次分别是PMVEC非接触培养和液体悬浮培养。在用GM - CSF + IL - 3 + SCF + IL - 6处理的PMVEC接触培养中，非贴壁细胞（42倍）和总CD34 + 细胞（12.6倍）出现最大扩增，粒细胞 - 巨噬细胞集落形成单位（CFU - GM）、CFU - mix、红系爆式集落形成单位（BFU - E）、CFU - blast和巨核细胞集落形成单位（CFU - Mk）祖细胞数量也有类似增加。此外，CD34 + CD38 - 和CD34 + CD38 + 细胞数量分别增加了148.1倍和8.0倍。传代培养研究表明，来自细胞因子处理的PMVEC单层上第7天分散的原始细胞集落的细胞对多谱系祖细胞具有高传代潜力。在长期PMVEC接触培养中，接种到用GM - CSF + IL - 3 + SCF + IL - 6处理过的PMVEC单层上的CD34 + 细胞在培养35天内非贴壁细胞的总计算扩增超过5,000,000倍。在第28天观察到最大克隆形成细胞产生，总集落形成细胞（CFC）为6,353倍，CFU - GM、CFU - mix CFU - blast、BFU - E和CFU - Mk有类似增加。第28天CD34 + 细胞总数增加了2,584倍。此外，这些培养物延长的生长动力学表明这些表型原始的祖细胞在PMVEC单层上也在功能上得到了扩增。这些结果支持这样的假设，即与PMVEC单层直接接触支持具有高传代潜力且可能具有更原始表型（CD34 + CD34 + /CD38 - ）的造血祖细胞的初始扩增。
