Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 119074, Singapore.
Stem Cell Rev Rep. 2010 Dec;6(4):665-76. doi: 10.1007/s12015-010-9184-8.
The ROCK inhibitor Y-27632 inhibits apoptosis and increases proliferation of frozen-thawed cells. We examined the role of Y-27632 on human umbilical cord Wharton's jelly stem cells (hWJSCs) for (1) thaw-survival (2) proliferation and (3) preservation of stemness and differentiation potential after cryopreservation. hWJSCs were allotted to 4 groups [Gp I: Untreated hWJSC controls; Gp II: Pretreatment with Y-27632 (10 μM) for 24 h before freezing; Gp III: Y-27632 (10 μM) in freezing medium and Gp IV: Pretreatment with Y-27632 (10 μM) for 24 h and inclusion in freezing medium]. All groups were frozen using a rapid freezing method and stored at -196°C in liquid nitrogen for 90 days before evaluation for apoptosis, cell proliferation, stemness and differentiation. After thawing, Groups II, III and IV showed improved cell attachment, increased thaw-survival (live/dead cell counts) and increased cell proliferation (Trypan blue and MTT assay) compared to controls. CD marker stemness profiles, morphology and normal karyotypes were maintained in the treatment groups after thawing and there was no obvious evidence of apoptosis (Annexin V-FITC and TUNEL assays). After thawing, qRT-PCR demonstrated up-regulation of the anti-apoptotic BCL2 gene and down-regulation of the pro-apoptotic BAX gene and cell cycle regulators (P53 and P21) in the treatment groups. Treated frozen-thawed hWJSCs from all groups differentiated into a neuronal phenotype (neuronal morphology and expression of GFAP, β-3 tubulin and SOX2). Increased thaw-survival and retention of stemness and differentiation potential in hWJSCs following cryopreservation is useful for their storage in cord blood banks for future regenerative medicine purposes.
ROCK 抑制剂 Y-27632 抑制细胞凋亡并增加冷冻解冻细胞的增殖。我们研究了 Y-27632 在人脐带华通氏胶间充质干细胞（hWJSCs）中的作用，用于（1）解冻存活，（2）增殖，（3）冷冻保存后保持干细胞特性和分化潜能。hWJSCs 分为 4 组[Gp I：未经处理的 hWJSC 对照；Gp II：冷冻前用 Y-27632（10μM）预处理 24 小时；Gp III：Y-27632（10μM）在冷冻培养基中；Gp IV：冷冻前用 Y-27632（10μM）预处理 24 小时并包含在冷冻培养基中]。所有组均采用快速冷冻法冷冻，并在-196°C 液氮中储存 90 天，然后评估凋亡、细胞增殖、干细胞特性和分化。解冻后，与对照组相比，Gp II、Gp III 和 Gp IV 组显示出更好的细胞附着、增加的解冻存活率（死活细胞计数）和增加的细胞增殖（台盼蓝和 MTT 测定）。解冻后，处理组的 CD 标志物干细胞特征、形态和正常核型得以维持，且无明显的凋亡证据（Annexin V-FITC 和 TUNEL 测定）。解冻后，qRT-PCR 显示，处理组中的抗凋亡 BCL2 基因上调，促凋亡 BAX 基因和细胞周期调节剂（P53 和 P21）下调。来自所有组的经处理的冷冻解冻 hWJSCs 分化为神经元表型（神经元形态和 GFAP、β-3 微管蛋白和 SOX2 的表达）。冷冻保存后 hWJSCs 解冻存活率增加和干细胞特性及分化潜能保留对其在脐带血库中的储存用于未来再生医学目的是有用的。
