Schuurhuis G J, Muijen M M, Oberink J W, de Boer F, Ossenkoppele G J, Broxterman H J
Department of Haematology, University Hospital Vrije Universiteit, Amsterdam, The Netherlands.
Bone Marrow Transplant. 2001 Mar;27(5):487-98. doi: 10.1038/sj.bmt.1702809.
Apoptosis is the common cell death pathway which is initiated by a variety of different stimuli. The recognition of early apoptotic events would markedly improve reliability and convenience of apoptosis assays. In the present study the vital stain SytoR 16 in combination with the permeability marker 7-amino actinomycin D, (7-AAD) has been used to identify an early stage of apoptosis, not detected with trypan blue or 7-AAD alone or with conventional apoptosis tests and not consistently and only partly detected by the early apoptosis marker annexin V. The method was established using solid tumour cell lines treated with TNF. Subsequently we applied it to determine apoptotic populations in CD34(+) peripheral blood progenitor cells obtained from growth factor and/or chemotherapy mobilised patients and frozen/thawed according to standard stem cell transplantation protocols. In a cell line model as well as CD34(+) progenitor cells, different subpopulations with decreased SytoR 16 fluorescence (SytoR 16int or SytoR 16low, compared with the normal SytoR 16high) appeared which are not, or only partly, apoptotic using conventional techniques including morphology or 7-AAD staining: eg percentages of SytoR 16(int)/7-AAD(-) and SytoR 16(low)/7-AAD(-) may amount to the majority of cells present in a particular CD34(+) sample. Second, upon further incubation these subpopulations become late apoptotic/secondary necrotic much faster than the unmodified SytoR 16high population, as determined with 7-AAD staining and morphology. Third, these cells have strongly or completely reduced clonogenic capacity for committed (CFU-GM) and early (LTC-IC, determined only for CD34(+) cells) progenitors. This technique needs the inclusion of a blocker of P-glycoprotein, which is highly active in CD34(+) progenitor cells. This prevents the interference of the detection of SytoR16(low) apoptotic cells by SytoR 16low cells resulting from P-glycoprotein activity. By comparison with other apoptosis markers we found that early apoptotic subpopulations were detected in the order SytoR 16 > annexin V > 7-AAD. In conclusion, the combination of SytoR 16 and 7-AAD detects apoptotic events earlier than conventional apoptosis techniques or annexin V. Compared to the presently available viability tests, it allows a much better estimation of the number of viable clonogenic CD34(+) cells after freeze/thawing.
凋亡是由多种不同刺激引发的常见细胞死亡途径。识别早期凋亡事件将显著提高凋亡检测的可靠性和便利性。在本研究中，活性染料SytoR 16与通透性标记物7-氨基放线菌素D（7-AAD）联合使用，以识别凋亡的早期阶段，而单独使用台盼蓝或7-AAD、传统凋亡检测方法均无法检测到该阶段，早期凋亡标记物膜联蛋白V也不能始终如一且仅部分检测到该阶段。该方法是通过用肿瘤坏死因子处理实体瘤细胞系建立的。随后，我们将其应用于确定从生长因子和/或化疗动员的患者获得并按照标准干细胞移植方案进行冷冻/解冻的CD34(+)外周血祖细胞中的凋亡群体。在细胞系模型以及CD34(+)祖细胞中，出现了不同的亚群，其SytoR 16荧光降低（与正常的SytoR 16高相比，为SytoR 16int或SytoR 16low），使用包括形态学或7-AAD染色在内的传统技术检测，这些亚群并非凋亡细胞，或仅部分为凋亡细胞：例如，SytoR 16(int)/7-AAD(-)和SytoR 16(low)/7-AAD(-)的细胞百分比可能占特定CD34(+)样本中细胞的大多数。其次，进一步孵育后，这些亚群比未修饰的SytoR 16高群体更快地进入晚期凋亡/继发性坏死，这通过7-AAD染色和形态学确定。第三，这些细胞对定向祖细胞（CFU-GM）和早期祖细胞（仅针对CD34(+)细胞测定的LTC-IC）的克隆形成能力已大幅降低或完全丧失。该技术需要加入P-糖蛋白阻滞剂，其在CD34(+)祖细胞中具有高活性。这可防止因P-糖蛋白活性导致的SytoR 16low细胞干扰对SytoR16(low)凋亡细胞的检测。通过与其他凋亡标记物比较，我们发现早期凋亡亚群的检测顺序为SytoR 16 > 膜联蛋白V > 7-AAD。总之，SytoR 16和7-AAD联合使用比传统凋亡技术或膜联蛋白V能更早地检测到凋亡事件。与目前可用的活力检测方法相比，它能更好地估计冷冻/解冻后存活的具有克隆形成能力的CD34(+)细胞数量。
