Early apoptosis largely accounts for functional impairment of CD34+ cells in frozen-thawed stem cell grafts
Early apoptosis largely accounts for functional impairment of CD34+ cells in frozen-thawed stem cell grafts
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DOI：
10.1089/152581602321080619
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发表时间：
2002-12-01
期刊：
影响因子：
--
通讯作者：
Schuurhuis, GJ
中科院分区：
文献类型：
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作者：
De Boer, F;Dräger, AM;Schuurhuis, GJ
干细胞移植物的质量评估通常通过流式细胞术CD34（+）计数或新鲜材料克隆输出评估来进行。在此之前，我们使用Syto16染色法在纯化的冻融CD34（+）细胞中发现了早期凋亡的发生，而通透性标记物7-氨基放线菌素D （7-AAD）无法检测到。Syto（高）/7-AAD（-）细胞为活细胞，Syto16（低）/7-AAD（-）细胞为早期凋亡细胞，Syto16（低）/7-AAD（+）细胞为死亡细胞。这在随后使用淋巴瘤患者冻融移植的研究中得到证实。在本研究中，我们研究了多发性骨髓瘤和淋巴瘤患者的移植细胞早期凋亡过程的功能后果。两个移植物组syto16定义的平均存活率分别为41%和42%，而仅使用7-AAD的移植物组分别为78%和72%。已建立的早期凋亡标志物膜联蛋白V在Syto16检测到的早期凋亡中约有50%的缺失。相比之下，CD34（+）细胞在匹配组淋巴瘤的非操纵全血移植中的活力。在4℃下保存72小时后，即使采用Syto16检测，也超过90%。冷冻保存后的CFU恢复（中位数26-33%）与Syto16后的CD34（+）恢复相匹配，但与7-AAD校正不匹配。相比之下，全血移植的菌落形成单位（CFU）恢复接近100%。此外，早期凋亡的CD34（+）细胞失去了向基质细胞衍生因子-1 α (sdf -1 α)迁移的能力。CD34（+）细胞中Syto16（高）/7-AAD（-）比例的建立为更准确地确定干细胞移植物中存活的非凋亡CD34（+）细胞的数量。该检测的进一步发展应允许将其纳入临床流式细胞术实验室解冻后样品的常规CD34（+）评估。
Quality assessment of stem cell grafts is usually performed by flow cytometric CD34(+) enumeration or assessment of clonogenic output of fresh material. Previously, we identified the occurrence of early apoptosis, not detectable with the permeability marker 7-amino actinomycin D (7-AAD), in purified frozen-thawed CD34(+) cells, using the vital stain Syto16. Syto(high)/7-AAD(-) cells were defined as viable, Syto16(low)/7-AAD(-) cells as early apoptotic and Syto16(low)/7-AAD(+) as dead. This was confirmed in a subsequent study using frozen-thawed transplants of lymphoma patients. In the present study on grafts from multiple myeloma and lymphoma patients, we investigated the functional consequences of the early apoptotic process. The mean Syto16-defined viability was 41 and 42%, respectively, for both graft groups, compared to 78% and 72%, respectively, using 7-AAD only. The established early apoptosis marker annexin V missed roughly 50% of the early apoptosis detected with Syto16. In contrast, viability of CD34(+) cells in nonmanipulated whole blood transplants from a matched group of lymphoma. patients, after 72 h of storage at 4degreesC, was more than 90%, even with the Syto16 assay. CFU recovery (median 26-33%) after cryopreservation matched CD34(+) recovery after Syto16, but not 7-AAD correction. In contrast, colony-forming unit (CFU) recovery in the whole blood transplant was close to 100%. Furthermore, early apoptotic CD34(+) cells had lost migratory ability toward stromal cell derived factor-1alpha (SDF-1alpha). The establishment of a Syto16(high)/7-AAD(-) proportion of CD34(+) cells offers a new approach for. a more correct determination of the number of viable nonapoptotic CD34(+) cells in stem cell grafts. Further development of this assay should allow its incorporation into the routine CD34(+) assessment of post-thawed samples in clinical flow cytometry laboratories.