利用特定環境蛋白誘導毛囊新生

Abstract

器官發育是一個自體組織生成的複雜過程。毛囊新生是透過角質細胞與真皮細胞間的所展開及持續的交互作用所達成。在成體狀態下誘導毛囊新生需要活化角質細並且誘導上皮-間質交互作用。後者可藉由毛囊真皮乳突細胞誘導，但因細胞來源有限且體外培養過程中容易喪失所需之特性。儘管現階段已有許多研究在探討相關的胚胎發育機制，但如何在成體誘發此發育機制而誘導成體器官再生仍尚未釐清。 本研究在探討可否可利用真皮細胞以誘導出生後的上皮-間質交互作用，而進一步誘導毛囊新生。本研究發現利用特定發育階段的胚胎皮膚所取得的無細胞萃取物，能夠在沒有毛囊真皮乳突細胞或新生鼠真皮細胞的幫助下，在全層皮膚傷口模型或修改的小片毛囊再生模型中誘導毛囊新生。我們發現在此毛囊新生的過程中主要是藉由影響纖維細胞而達成。成體的纖維細胞培養於胚胎皮膚萃取物後，可讓纖維細胞展現誘導毛囊新生的能力；但角質細胞利用此刺激後，並無法誘導毛囊新生。我們透過分析接觸此萃取物的纖維母細胞之磷酸化蛋白質體發現胰島素/類胰島素生長因子訊息傳遞被活化，並在實驗中確認此訊息傳遞路徑在成體纖維細胞誘導毛囊新生的過程是必需的。此外透過分析此胚胎皮膚萃取物的蛋白質體，我們尋找到三個會大量表現在胚胎皮膚的分泌型胞外蛋白，包括脫輔基蛋白質-1，半乳凝素-1和基膜聚醣。動物實驗實驗證實利用此三個蛋白質可與表皮細胞混合後後，可在活體中修改的小片毛囊再生模型中誘導毛囊新生。因此經過特定細胞外蛋白混合物短時間刺激後，成體細胞可以被誘導成具有再生能力的細胞。 器官再生可以透過施與特定的細胞外因子來建構促發組織再生反應的環境。故藉由特定器官的胚胎組織來尋找誘發此器官再生特定蛋白質組合，可以作為促進成體器官再生的重要策略。

Organ development is a sophisticated process of self-organization. Hair follicle neogenesis depends on the initiation and perpetuation of crosstalk between keratinocytes and dermal cells. Regenerating new hair follicles in adults requires reactivation of epithelial-mesenchymal interactions between competent keratinocytes and inductive mesenchymal cells. The latter can be expanded from the preexisting dermal papilla fibroblasts of mature hair follicles, yet this source is limited and cells quickly lose inductive properties in culture. However, despite a growing understanding of the developmental mechanisms involved, little is known about how to reactivate them for adult organ regeneration. We ask whether this epithelial-mesenchymal interaction for hair follicle neogenesis can be initiated without introducing inductive dermal cells in postnatal life. Here we demonstrate a new regenerative approach that does not require dermal papilla fibroblasts. We found that cell-free extract from embryonic skin of specific developmental stages was able to induce hair follicle neogenesis both in a full-thickness wound and in a modified patch assay in mice without the help of inductive dermal papilla cells or newborn dermal cells. Hair follicle neogenesis here was mediated mainly through the effect on fibroblasts. When adult fibroblasts, but not keratinocytes, were cultured with the cell-free extract, they were conferred on the ability to induce new hair follicles. In search of the molecular mechanisms involved through phosphoproteomic analysis, we found that insulin/IGF signaling was activated and required for the hair follicle inductivity in adult fibroblasts. Finally, through proteomics analysis, we identified 3 extracellular/ secreted proteins, including apoliproprotein, galectin-1 and lumican, enriched in embryonic skin. These three proteins together were required and sufficient to induce hair follicle neogenesis in vivo. Thus, a cocktail of organ-specific extracellular proteins from the embryonic environment can render adult cells competent to re-initiate developmental interactions for regeneration. Therefore, short-term exposure to and organ-specific extracellular protein cocktail can render adult cells competent for regeneration through conversion toward a state in which their fate can be further stably committed via re-engagement in developmental interactions. These 3 proteins show a stage-specific co-enrichment in the embryonic dermis during the peri-folliculogenetic period. Mechanistically, exposure to embryonic skin extract or a combination of the 3 proteins altered the gene expression to a hair follicle dermal papilla fibroblast-like profile and activated Igf and Wnt signaling, crucial for the regeneration process. Organ regeneration can be initiated by creating a pro-regeneration environment with defined extracellular factors. Identification of factors to recreate tissue-specific embryonic extracellular context could become an important strategy for promoting regeneration in various adult organs.