ROCK Inhibitor Y-27632 Increases Thaw-Survival Rates and Preserves Stemness and Differentiation Potential of Human Wharton's Jelly Stem Cells After Cryopreservation
ROCK Inhibitor Y-27632 Increases Thaw-Survival Rates and Preserves Stemness and Differentiation Potential of Human Wharton's Jelly Stem Cells After Cryopreservation
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DOI：
10.1007/s12015-010-9184-8
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发表时间：
2010-12-01
影响因子：
4.8
通讯作者：
Bongso, Ariff
中科院分区：
文献类型：
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作者：
Gauthaman, Kalamegam;Fong, Chui-Yee;Bongso, Ariff
ROCK抑制剂Y-27632抑制细胞凋亡并增加冻融细胞的增殖。我们研究了Y-27632对人脐带沃顿氏胶质干细胞（hWJSC）的作用，以用于（1）解冻存活（2）增殖和（3）冷冻保存后的干细胞性和分化潜力的保持。将hWJSC分成4组[Gp I：未处理的hWJSC对照; Gp II：冷冻前用Y-27632（10 μ M）预处理24小时; Gp III：冷冻培养基中的Y-27632（10 μ M）和Gp IV：用Y-27632（10 μ M）预处理24小时并包含在冷冻培养基中]。使用快速冷冻方法冷冻所有组，并在评估细胞凋亡、细胞增殖、干细胞性和分化之前在液氮中在-196 ℃下储存90天。解冻后，与对照组相比，第II、III和IV组显示出改善的细胞附着、增加的解冻存活率（活/死细胞计数）和增加的细胞增殖（台盼蓝和MTT测定）。解冻后，处理组中的CD标记物干性特征、形态学和正常核型得以维持，并且没有明显的细胞凋亡证据（膜联蛋白V-FITC和TUNEL测定）。解冻后，qRT-PCR显示处理组中抗凋亡BCL 2基因上调，促凋亡BAX基因和细胞周期调节因子（P53和P21）下调。来自所有组的经处理的冻融hWJSC分化成神经元表型（神经元形态和GFAP、β-3微管蛋白和SOX 2的表达）。冷冻保存后hWJSC的解冻存活率增加以及干细胞性和分化潜力的保留对于它们在脐带血库中的储存是有用的，用于未来的再生医学目的。
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 mu M) for 24 h before freezing; Gp III: Y-27632 (10 mu M) in freezing medium and Gp IV: Pretreatment with Y-27632 (10 mu M) for 24 h and inclusion in freezing medium]. All groups were frozen using a rapid freezing method and stored at -196A degrees 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, beta-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.