Intestinal stem cells remain viable after prolonged tissue storage
Intestinal stem cells remain viable after prolonged tissue storage
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DOI：
10.1007/s00441-013-1674-y
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发表时间：
2013-11-01
影响因子：
3.6
通讯作者：
Henning, Susan J.
中科院分区：
文献类型：
--
作者：
Fuller, Megan K.;Faulk, Denver M.;Henning, Susan J.
肠干细胞（ISC）负责正常稳态期间和损伤后上皮的更新。因此，它们具有显着的治疗潜力。然而，ISC 能否在组织储存中存活尚不清楚。我们假设，虽然大多数上皮细胞可能死亡，但 ISC 在 4°C 下仍能存活至少 24 小时。为了探索这一假设，将 C57Bl6/J 或 Lgr5-LacZ 小鼠的空肠取出并立即处理或置于 4°C 下的磷酸盐缓冲盐水中。在储存 24、30 或 48 小时后进行上皮的延迟分离。在光学显微镜水平上，尽管绒毛上皮细胞广泛凋亡，但小肠隐窝在 30 小时内保持形态完整，并且可通过 Lgr5-LacZ 阳性识别 ISC。电子显微镜显示 ISC 在 24 小时内保持高度完整性。通过流式细胞术评估时，ISC 比其他上皮细胞（包括邻近的潘氏细胞）更能抵抗变性，在所有时间点都具有更高的活力。培养的分离隐窝在组织储存 24 小时后，形成复杂肠状体的能力没有丧失，培养 7 天后的效率仍保持在 80% 以上。储存 30 小时后，效率下降，但出芽能力得以保留。我们的结论是，随着隔离的延迟，ISC 仍然具有活力并保留其增殖能力。相反，上皮的其余部分，包括潘氏细胞，表现出变性和程序性细胞死亡。如果这些发现在人体组织中重现，4°C 下的储存可能为收获隐窝或 ISC 用于治疗应用提供宝贵的时间窗口。
Intestinal stem cells (ISCs) are responsible for renewal of the epithelium both during normal homeostasis and following injury. As such, they have significant therapeutic potential. However, whether ISCs can survive tissue storage is unknown. We hypothesize that, although the majority of epithelial cells might die, ISCs would remain viable for at least 24 h at 4 A degrees C. To explore this hypothesis, jejuna of C57Bl6/J or Lgr5-LacZ mice were removed and either processed immediately or placed in phosphate-buffered saline at 4 A degrees C. Delayed isolation of epithelium was performed after 24, 30, or 48 h storage. At the light microscope level, despite extensive apoptosis of villus epithelial cells, small intestinal crypts remained morphologically intact for 30 h and ISCs were identifiable via Lgr5-LacZ positivity. Electron microscopy showed that ISCs retained high integrity for 24 h. When assessed by flow cytometry, ISCs were more resistant to degeneration than the rest of the epithelium, including neighboring Paneth cells, with higher viability across all time points. Cultured isolated crypts showed no loss of capacity to form complex enteroids after 24 h tissue storage, with efficiencies after 7 days of culture remaining above 80 %. By 30 h storage, efficiencies declined but budding capability was retained. We conclude that, with delay in isolation, ISCs remain viable and retain their proliferative capacity. In contrast, the remainder of the epithelium, including the Paneth cells, exhibits degeneration and programmed cell death. If these findings are recapitulated in human tissue, storage at 4 A degrees C might offer a valuable temporal window for the harvesting of crypts or ISCs for therapeutic application.