Human embryonic stem-cell derivatives for full reconstruction of the pluristratified epidermis: a preclinical study
Human embryonic stem-cell derivatives for full reconstruction of the pluristratified epidermis: a preclinical study
复制标题
DOI：
10.1016/s0140-6736(09)61496-3
复制
发表时间：
2009-11-21
期刊：
影响因子：
168.9
通讯作者：
Waksman, Gilles
中科院分区：
文献类型：
--
作者：
Guenou, Hind;Nissan, Xavier;Waksman, Gilles
大面积烧伤的细胞治疗依赖于体外重建的自体表皮。然而，目前程序的有效性受到培养患者自身角质形成细胞所需延迟的限制。为了评估人类胚胎干细胞（hESC）的角质形成细胞后代是否可用于形成临时皮肤替代品，用于等待自体移植的患者，方法将来自H9和SA01系的hESC至少一式三份地接种在成纤维细胞饲养细胞上，在补充有骨形态发生蛋白4的培养基中40天，抗坏血酸通过定量PCR、荧光激活细胞分选和免疫细胞化学技术在整个过程中对细胞分化进行分子表征。角质形成细胞的分子分化和功能能力，以构建一个人的表皮进行了评估，在体外和体内。结果从人胚胎干细胞，我们产生了一个同质的人口的细胞，表现出表型特征的基础角质形成细胞。这些细胞中编码角蛋白14、角蛋白5、整合素α 6、整合素β 4、胶原蛋白VII和层粘连蛋白5的基因的表达水平与基底角质形成细胞中的那些相似。在人工基质上接种后，衍生自hESC的角质形成细胞（K-hESC）形成多层表皮。角蛋白-14免疫染色可见于基底室，角蛋白10存在于基底层上的层中。表皮分化的晚期标志物--外膜蛋白和聚丝蛋白仅在最上层检测到。在移植到5只免疫缺陷小鼠上12周后，来自K-hESC的表皮具有与成熟人类皮肤一致的结构。人类外皮蛋白是适当地位于棘层和颗粒层和一些Ki67阳性细胞被检测到在basal layer.Interpretation人胚胎干细胞可以分化成基底角质形成细胞是完全功能性的，即，能够构建一个pluisteriodated表皮。这种资源可以开发为等待自体移植的患者提供临时皮肤替代品。
Background Cell therapy for large burns is dependent upon autologous epidermis reconstructed in vitro. However, the effectiveness of current procedures is limited by the delay needed to culture the patient's own keratinocytes. To assess whether the keratinocyte progeny of human embryonic stem cells (hESCs) could be used to form a temporary skin substitute for use in patients awaiting autologous grafts, we investigated the cells' capability of constructing a pluristratified epidermis.Methods hESCs from lines H9 and SA01 were seeded at least in triplicate on fibroblast feeder cells for 40 days in a medium supplemented with bone morphogenetic protein 4 and ascorbic acid. Molecular characterisation of cell differentiation was done throughout the process by quantitative PCR, fluorescence-activated cell sorting, and immunocytochemical techniques. Keratinocyte molecular differentiation and functional capacity to construct a human epidermis were assessed in vitro and in vivo.Findings From hESCs, we generated a homogeneous population of cells that showed phenotypic characteristics of basal keratinocytes. Expression levels of genes encoding keratin 14, keratin 5, integrin alpha 6, integrin beta 4, Collagen VII, and laminin 5 in these cells were similar to those in basal keratinocytes. After seeding on an artificial matrix, keratinocytes derived from hESCs (K-hESCs) formed a pluristratified epidermis. Keratin-14 immunostaining was seen in the basal compartment, with keratin 10 present in layers overlying the basal layer. Involucrin and filaggrin, late markers of epidermal differentiation, were detected in the uppermost layers only. 12 weeks after grafting onto five immunodeficient mice, epidermis derived from K-hESCs had a structure consistent with that of mature human skin. Human involucrin was appropriately located in spinous and granular layers and few Ki67-positive cells were detected in the basal layer.Interpretation hESCs can be differentiated into basal keratinocytes that are fully functional-ie, able to construct a pluristratified epidermis. This resource could be developed to provide temporary skin substitutes for patients awaiting autologous grafts.