Reprogramming of human dermal fibroblasts into inductive dermal papilla cells

在过去的几十年里，毛囊(HF)的再生受到了极大的关注，因为对 有效治疗包括脱发和皮肤损伤在内的许多疾病。当前面临的挑战 药理学和外科手术方法促使人们努力开发更有效的治疗方法。 解决办法。基于细胞的HFs生物工程是一种很有希望的策略，可以克服 再生HFs，实现“毛茸茸”的壮举。主要的障碍是我们无法隔离和繁殖人类 具有诱导能力的毛乳头(DP)成纤维细胞是主要的间充质细胞 角质形成细胞构建HF结构。尽管已知DP成纤维细胞构成真皮小生境， 在HF的基础上，从HFs中分离人DP成纤维细胞被证明是技术困难和低效的。 分离的DP成纤维细胞在正常培养条件下增殖缓慢，原因是缺少关键上皮和 微环境线索。此外，人DP成纤维细胞在体外会迅速丧失毛发诱导性。 扩张，并未能产生HFs。真皮成纤维细胞与真皮成纤维细胞在解剖上的亲缘关系和共同起源 DP成纤维细胞提示真皮成纤维细胞可能是一种可行的选择，可以提供大量 诱导性DP成纤维细胞。然而，真皮成纤维细胞是否可以 重新编程为具有DP毛发诱导性，用于HF生物工程。我们的长期目标是开发小说 为临床程序生产全功能人类HFs的生物工程方法。使用三个- 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活性 改善并导致类HF结构的形成。此外，我们确定了五个主要的转录 对DP表型和诱导性至关重要的因子(TF)。我们的中心假设是DP表型 可以通过激活主DP-TF在人皮肤成纤维细胞(DFS)中驱动，从而重新编程无毛 表皮到毛囊的命运。在第一个目标中，我们将确定人皮肤成纤维细胞是否可以 重新编程以建立DP特征和毛发诱导性。我们将激活五大类的表达 使用CRISPR激活系统的人DFS中与DP诱导性相关的TF。此外，我们将确定哪些 在重新编程人皮肤成纤维细胞以启动DP基因方面，Tf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，我们将确定是否 复合球体中重编程的真皮成纤维细胞可在ESS中诱导HFs。我们将结合使用 3D复合球体和预制的微孔在ESS中创造了一个诱导性的生态位。这项工作将提供一种 对与遗传程序有关的新机制知识的新的深入理解，这些知识对 促进DP特征和HF的形态发生。结果意义重大，因为它们将提供 关于容易获得的真皮成纤维细胞能否被重新编程为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、全功能HFs和皮肤构造方面的进展。

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.