Koizumi K, Sawada K, Yamaguchi M, Notoya A, Tarumi T, Takano H, Fukada Y, Nishio M, Katagiri E, Yasukouchi T, Sato N, Sekiguchi S, Koike T
Department of Internal Medicine II, Hokkaido University School of Medicine, Sapporo, Japan.
Exp Hematol. 1998 Nov;26(12):1140-7.
The aim of this study is to clarify the transitional change of the proliferation and differentiation of human peripheral blood CD34+ cells to megakaryocytic lineage, focusing on its clinical application. We developed a rapid system to purify human peripheral blood CD34+ cells from healthy volunteers, which produced CD34+ cells with a 90% purity. The purified CD34+ cells predominantly consisted of CD41- cells, and the rate of coexpression of CD41 was 0.6% +/- 0.5%. When the purified cells were cultured in liquid phase for 10 days in the presence of recombinant human stem cell factor (rSCF: a ligand for c-kit), interleukin-3 (rIL-3), and thrombopoietin (rTPO: a ligand for Mpl), the number of CD34+/CD41+ cells increased to 19% +/- 7% of total expanded cells on day 4 (4 days of liquid culture) and then gradually decreased to 2.2% +/- 0.6% on day 10. The absolute number of CD34+/CD41+ cells increased and reached a plateau on day 6, and 1.7 +/- 0.6 x 10(5) CD34+/CD41+ cells were produced by 1 x 10(5) CD34+/CD41- day 0 cells. The CD34-/CD41+ cells appeared on day 6, continuously increased in number until day 10, and constituted the main population of expanded cells on day 10, with a value of 38% +/- 18%. On day 10, 19.5 +/- 10.6 x 10(5) of CD34-/CD41+ cells were produced by 1 x 10(5) CD34+/CD41- day 0 cells. The deletion of rTPO from this cytokine combination decreased the number of CD34+/CD41+ and CD34-/CD41+ cells, after days 6 and 8, respectively. Day 0 cells required rIL-3 for promoting colonies containing megakaryocytes, whereas rTPO alone promoted almost no megakaryocytic colonies from day 0 cells. Thus, a combination of IL-3 and SCF expands CD34+/CD41+ cells from CD34+/CD41- cells, and TPO mainly acts to increase CD34-/CD41+ cells. This study suggests that if the expansion of CD34+/CD41+ is performed in vitro, the 6 days' culture of peripheral blood CD34+/CD41- cells with a combination of IL-3 and SCF with TPO provides the most rapid and stable products of CD34+/CD41+ cells for the rapid recovery of platelets in patients with peripheral blood stem cell transplantation.
本研究的目的是阐明人外周血CD34+细胞向巨核细胞系增殖和分化的转变过程，重点关注其临床应用。我们开发了一种从健康志愿者中纯化人外周血CD34+细胞的快速系统，该系统产生的CD34+细胞纯度达90%。纯化的CD34+细胞主要由CD41-细胞组成，CD41的共表达率为0.6%±0.5%。当纯化的细胞在重组人干细胞因子（rSCF：c-kit的配体）、白细胞介素-3（rIL-3）和血小板生成素（rTPO：Mpl的配体）存在的情况下在液相中培养10天时，CD34+/CD41+细胞的数量在第4天（液体培养4天）增加到总扩增细胞的19%±7%，然后在第10天逐渐降至2.2%±0.6%。CD34+/CD41+细胞的绝对数量增加并在第6天达到平台期，1×10⁵个第0天的CD34+/CD41-细胞产生了1.7±0.6×10⁵个CD34+/CD41+细胞。CD34-/CD41+细胞在第6天出现，数量持续增加直至第10天，并在第10天构成扩增细胞的主要群体，比例为38%±18%。在第10天，1×10⁵个第0天的CD34+/CD41-细胞产生了19.5±10.6×10⁵个CD34-/CD41+细胞。从这种细胞因子组合中去除rTPO后，分别在第6天和第8天后，CD34+/CD41+和CD34-/CD41+细胞的数量减少。第0天的细胞需要rIL-3来促进含有巨核细胞的集落形成，而单独的rTPO几乎不能促进第0天细胞形成巨核细胞集落。因此，IL-3和SCF的组合可使CD34+/CD41-细胞扩增为CD34+/CD41+细胞，而TPO主要作用是增加CD34-/CD41+细胞。本研究表明，如果在体外扩增CD34+/CD41+细胞，将外周血CD34+/CD41-细胞与IL-3、SCF和TPO组合培养6天，可为外周血干细胞移植患者血小板的快速恢复提供最快速、稳定的CD34+/CD41+细胞产物。
