Reprogramming of human dermal fibroblasts into inductive dermal papilla cells

毛囊（HF）再生在过去的几十年中受到极大的关注，这是由于对毛囊再生的需求增加。 有效治疗许多疾病，包括脱发和皮肤损伤。当前面临的挑战 药理学和手术方法已经激发了开发更有效的治疗方法的努力， 解决方案基于细胞的HFs生物工程成为克服HFs中的障碍的有希望的策略。 再生高频并实现“多毛”的壮举。主要的障碍是我们无法隔离和传播人类 毛乳头（DP）成纤维细胞具有诱导能力，其是指导分化的主要间充质细胞。 角质形成细胞构建HF结构。虽然DP成纤维细胞是已知的构成一个真皮生态位的权利， 作为HF的基础，从HF中分离人DP成纤维细胞被证明在技术上是困难和低效的。 分离的DP成纤维细胞在正常培养条件下增殖缓慢，这是由于缺少关键的上皮细胞， 微环境线索此外，人DP成纤维细胞在离体培养期间快速丧失毛发诱导性。 膨胀和不能产生HF。真皮成纤维细胞和成纤维细胞之间的解剖学接近性和共同起源， DP成纤维细胞表明，真皮成纤维细胞可能代表了一个可行的选择，提供大量的 诱导性DP成纤维细胞。然而，尚未探索真皮成纤维细胞是否可以被 重新编程以具有用于HF生物工程的DP毛发诱导性。我们的长期目标是开发新颖的 生物工程方法来生产用于临床程序的全功能人HF。使用三- 三维（3D）复合球体模型，我们发现人DP成纤维细胞的毛发诱导性大大增加， 改善并导致形成HF样结构。此外，我们确定了五个主要的转录 因子（TF）是DP表型和诱导性的核心。我们的中心假设是DP表型 可以通过激活主DP-TF在人真皮成纤维细胞（DF）中驱动，从而重新编程非毛细胞 表皮到毛囊的命运。在第一个目标中，我们将确定人真皮成纤维细胞是否可以被 重新编程以建立DP特征和毛发诱导性。我们将激活五个主要的表达 使用CRISPR激活系统的人DF中的DP诱导相关TF。此外，我们将确定哪些 TF在人皮肤成纤维细胞重编程启动DP基因的转录活性最高 表达、DP-角质形成细胞相互作用和DP HF诱导性。在第二个目标中，我们将确定 复合球体中的重编程真皮成纤维细胞可以在ESS中诱导HF。 三维复合球体和预制微孔，以创建一个诱导生态位在ESS。这项工作将提供一个 新的和深入的了解新的机制知识有关的遗传程序，是至关重要的， 促进DP特征和HF形态发生。这些成果意义重大，因为它们将提供 关于容易获得的真皮成纤维细胞是否可以重编程为DP样成纤维细胞的重要数据 用于HF生物工程。这些数据将促进生物工程的进展，全功能的HFs和皮肤构建。

Hair follicle (HF) regeneration has received great attention in the last decades due to increased demands for effective cures for many diseases that involve hair loss and skin injuries. The challenges met in current pharmacological and surgical approaches have provoked the efforts in developing more effective therapeutic solutions. Cell-based bioengineering of HFs emerges as a promising strategy to overcome the hurdles in regenerating HFs and achieve a “hairy” feat. The main obstacle is our inability to isolate and propagate human dermal papilla (DP) fibroblasts with inductive capabilities, which are the major mesenchymal cells instructing keratinocytes to build the HF structure. Although DP fibroblasts are known to constitute a dermal niche right at the base of the HF, isolation of human DP fibroblasts from HFs is proven to be technically difficult and inefficient. Isolated DP fibroblasts proliferate slowly under normal culture conditions due to missing key epithelial and microenvironmental cues. Furthermore, human DP fibroblasts quickly lose hair inductivity during ex vivo expansion and fail to produce HFs. The anatomical proximity and shared origin between dermal fibroblasts and DP fibroblasts suggest that dermal fibroblasts may represent a viable option for supplying a large number of inductive DP fibroblasts. However, dermal fibroblasts have not been explored whether they can be reprogrammed to possess DP hair inductivity for HF bioengineering. Our long-term goal is to develop novel bioengineering approaches to produce fully functional human HFs for clinical procedures. Using a three- dimensional (3D) composite spheroid model, we found that the hair inductivity of human DP fibroblasts is greatly improved and leads to the formation of HF-like structure. Furthermore, we identified five major transcription factors (TFs) that are central to DP phenotype and inductivity. Our central hypothesis is that the DP phenotype can be driven in human dermal fibroblasts (DFs) by activating master DP-TFs, thus reprogramming non‐hairy epidermis to a follicular fate. In the first aim, we will determine whether human dermal fibroblasts can be reprogrammed to establish DP characteristics and hair inductivity. We will activate the expression of five major DP inductivity-related TFs in human DFs using a CRISPR activation system. Furthermore, we will identify which TF has the highest transcriptional activity in reprogramming human dermal fibroblasts to initiate DP gene expression, DP-keratinocyte interaction, and DP HF inductivity. In the second aim, we will determine whether reprogrammed dermal fibroblasts in composite spheroids can induce HFs in ESS. We will use a combination of 3D composite spheroid and premade micropores to create an inductive niche in ESS. This work will provide a new and in-depth understanding of novel mechanistic knowledge regarding genetic programs that are critical to promoting DP characteristics and HF morphogenesis. The outcomes are significant because they will provide important data as to whether easily available dermal fibroblasts can be reprogrammed into DP-like fibroblasts for HF bioengineering. The data will promote progress in bioengineering fully functional HFs and skin constructs.