Laminin-511-derived recombinant fragment and Rho kinase inhibitor Y-27632 facilitate serial cultivation of keratinocytes differentiated from human embryonic stem cells.
Laminin-511-derived recombinant fragment and Rho kinase inhibitor Y-27632 facilitate serial cultivation of keratinocytes differentiated from human embryonic stem cells.
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层粘连蛋白-511衍生的重组片段和Rho激酶抑制剂Y-27632促进了角质形成细胞与人类胚胎干细胞区分开的系列培养。
DOI：
10.1016/j.reth.2021.07.004
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发表时间：
2021-12
影响因子：
4.3
通讯作者：
Umezawa A
中科院分区：
文献类型：
--
作者：
Takagi R;Tanuma-Takahashi A;Akiyama S;Kaneko W;Miura C;Yamato M;Shimizu T;Umezawa A
衍生自多能干细胞的角质形成细胞在针对角质形成细胞优化的常规培养条件下具有短的增殖寿命。最近，Rho激酶抑制剂Y-27632已被用作培养基的标准补充剂，其中出生后角质形成细胞的增殖寿命显著延长。此外，重组人层粘连蛋白-511被证明是促进培养的表皮角质形成细胞增殖的粘附配体。基于这一认识，评价了Y-27632和层粘连蛋白511衍生的重组片段（称为层粘连蛋白-511 E8片段（LN-511-E8））用于建立从人胚胎干细胞（hESC）分化的角质形成细胞的培养方法的效力。将来自hESC的分化细胞（其在先前的研究中建立为具有临床等级）接种到用LN-511-E8包被的培养皿上，并在补充有Y-27632的无血清培养基中与小鼠饲养层共培养。在连续培养之前，通过胰蛋白酶消化将hESC衍生的角质形成细胞与其他分化的细胞分离。将分离的hESC衍生的角质形成细胞用于评价克隆形成性、角质形成细胞标志物的基因表达分析、通过气升培养的终末分化效力和通过连续培养的长期增殖活性。此外，评价Y-27632、LN-511-E8和小鼠饲养层对hESC衍生的角质形成细胞增殖的功效。通过胰蛋白酶消化成功地分离出具有克隆生长活性的hESC衍生的角质形成细胞，并表现出分化能力，以形成表达表皮角质形成细胞的祖细胞和分化标记的分层表皮等同物。Y-27632和LN-511-E8是维持hESC衍生的角质形成细胞在使用小鼠饲养层的连续培养中的增殖活性所必需的，在对数生长期具有稳定的倍增时间。这些结果表明Y-27632和LN-511-E8用于连续培养hESC衍生的角质形成细胞的效用，其具有在临床情况下制造用于再生分层上皮组织的同种异体细胞产物的潜力。
Keratinocytes derived from pluripotent stem cells have a short proliferative lifespan under conventional culture conditions that are optimized for keratinocytes. Recently, a Rho kinase inhibitor, Y-27632, had been used as a standard supplement for culture medium in which the proliferative lifespan of postnatal keratinocytes was markedly expanded. In addition, recombinant human laminin-511 was demonstrated to be an adhesive ligand for promoting proliferation of cultured epidermal keratinocytes. Based on this knowledge, efficacies of Y-27632 and a laminin511-derived recombinant fragment, known as laminin-511 E8 fragment (LN-511-E8), were evaluated for establishing cultivation methods of keratinocyte differentiated from human embryonic stem cells (hESC). Differentiated cells from hESCs, which were established with clinical grade in previous study, were seeded onto culture dishes coated with LN-511-E8 and co-cultured with a mouse feeder layer in serum-free medium supplemented with Y-27632. Before serial cultivation, hESC-derived keratinocytes were separated from other differentiated cells by trypsinization. The isolated hESC-derived keratinocytes were used for evaluating clonogenicity, gene expression analysis for keratinocyte markers, potency of terminal differentiation by air-lifting culture, and long-term proliferation activity by serial cultivation. Moreover, efficacies of Y-27632, LN-511-E8, and mouse feeder layer were evaluated on proliferation of hESC-derived keratinocytes. hESC-derived keratinocytes with activity of clonal growth were successfully isolated by trypsinization and exhibited potency of differentiation to form stratified epidermal equivalents with expressions of progenitor and differentiation markers of epidermal keratinocyte. Y-27632 and LN-511-E8 were required for maintaining the proliferative activity of the hESC-derived keratinocytes in serially cultivation using mouse feeder layer with stable doubling time during logarithmic growth phase. These results indicate the utility of Y-27632 and LN-511-E8 for serial cultivation of hESC-derived keratinocytes, which have a potential for fabricating allogeneic cellular products in clinical situations for regeneration of stratified epithelial tissues.
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影响因子：
5.2
作者：
Dabelsteen, Sally;Hercule, Paula;Barron, Patricia;Rice, Meghan;Dorsainville, Gregory;Rheinwald, James G.
通讯作者：
Rheinwald, James G.
影响因子：
4.8
作者：
Ido, Hiroyuki;Nakamura, Aya;Sekiguchi, Kiyotoshi
通讯作者：
Sekiguchi, Kiyotoshi
影响因子：
9.2
作者：
McMullan, R;Lax, S;Hotchin, NA
通讯作者：
Hotchin, NA
DOI：
10.1016/s0190-9622(89)70160-2
发表时间：
1989-08-01
影响因子：
13.8
作者：
PHILLIPS, TJ;KEHINDE, O;GILCHREST, BA
通讯作者：
GILCHREST, BA
影响因子：
64.5
作者：
RHEINWALD, JG;GREEN, H
通讯作者：
GREEN, H