Dos Santos Jeniffer Farias, Freitas-Marchi Bruna Letícia, Reigado Gustavo Roncoli, de Assis Silvia Romano, Maria Engler Silvya Stuchi, Chambergo Alcalde Felipe Santiago, Nunes Viviane Abreu
Laboratory of Skin Physiology and Tissue Bioengineering, School of Arts, Sciences and Humanities, University of Sao Paulo (EACH-USP), São Paulo, São Paulo, Brazil.
Skin Biology Group, iNOVA Pele, School of Pharmaceutical Sciences (FCF), University of São Paulo, São Paulo, São Paulo, Brazil.
Front Cell Dev Biol. 2023 Jan 12;10:1012637. doi: 10.3389/fcell.2022.1012637. eCollection 2022.
In skin traumas, such as burns, epidermal homeostasis is affected, often requiring clinical approaches. Different therapeutic strategies can be used including transplantation, besides the use of synthetic or natural materials with allogeneic cells. In this context, tissue engineering is an essential tool for skin regeneration, and using mesenchymal stem cells (MSC) from the umbilical cord appears to be a promising strategy in regenerative medicine due to its renewal and differentiation potential and hypo immunogenicity. We evaluated the transdifferentiation of MSC from umbilical cord into keratinocytes in three-dimensional (3D) skin models, using dermal equivalents composed by type I collagen with dermal fibroblasts and a commercial porcine skin decellularized matrix, both cultured at air-liquid interface (ALI). The expression of epidermal proteins cytokeratins (CK) 5, 14 and 10, involucrin and filaggrin was investigated by real-time PCR and immunofluorescence, in addition to the activity of epidermal kallikreins (KLK) on the hydrolysis of fluorogenic substrates. The cultivation of MSCs with differentiation medium on these dermal supports resulted in organotypic cultures characterized by the expression of the epidermal markers CK5, CK14, CK10 and involucrin, mainly on the 7 day of culture, and filaggrin at 10 day in ALI. Also, there was a 3-fold increase in the KLK activity in the epidermal equivalents composed by MSC induced to differentiate into keratinocytes compared to the control (MSC cultivated in the proliferation medium). Specifically, the use of collagen and fibroblasts resulted in a more organized MSC-based organotypic culture in comparison to the decellularized matrix. Despite the non-typical epithelium structure formed by MSC onto dermal equivalents, the expression of important epidermal markers in addition to the paracrine effects of these cells in skin may indicate its potential use to produce skin-based substitutes.
在皮肤创伤（如烧伤）中，表皮稳态会受到影响，通常需要临床治疗方法。除了使用含有同种异体细胞的合成或天然材料外，还可采用包括移植在内的不同治疗策略。在此背景下，组织工程是皮肤再生的重要工具，由于脐带间充质干细胞（MSC）具有更新和分化潜力以及低免疫原性，将其用于再生医学似乎是一种很有前景的策略。我们在三维（3D）皮肤模型中评估了脐带间充质干细胞向角质形成细胞的转分化，该模型使用由I型胶原蛋白与真皮成纤维细胞组成的真皮替代物以及商业猪皮肤脱细胞基质，二者均在气液界面（ALI）培养。通过实时聚合酶链反应和免疫荧光研究了表皮蛋白细胞角蛋白（CK）5、14和10、内披蛋白和丝聚蛋白的表达，此外还研究了表皮激肽释放酶（KLK）对荧光底物水解的活性。在这些真皮支架上用分化培养基培养间充质干细胞，形成了器官样培养物，其特征在于表皮标志物CK5、CK14、CK10和内披蛋白的表达，主要在培养第7天出现，而丝聚蛋白在ALI培养10天时出现。此外，与对照组（在增殖培养基中培养的间充质干细胞）相比，诱导分化为角质形成细胞的间充质干细胞组成的表皮替代物中的KLK活性增加了3倍。具体而言，与脱细胞基质相比，使用胶原蛋白和成纤维细胞可形成更有组织的基于间充质干细胞的器官样培养物。尽管间充质干细胞在真皮替代物上形成的上皮结构不典型，但这些细胞在皮肤中的重要表皮标志物表达以及旁分泌作用可能表明其在生产皮肤替代物方面具有潜在用途。
