Inducing hair follicle neogenesis with secreted proteins enriched in embryonic skin
Introduction
Compared with the profound spontaneous restoration of lost tissues and organs in lower vertebrates [[1], [2], [3]], mammals show limited regeneration after injury and damaged tissues are often replaced by morphologically and functionally debilitating scars [4]. To promote regeneration, attempts have been made by transplantation of stem cells, organoids or bioartificial organs [[5], [6], [7]], yet in most cases functional incorporation and maintenance of transplanted cells remains challenging [8]. In skin, it usually heals by fibrosis rather than regeneration after injury, leading to scar formation and permanent loss of skin appendages, prominently hair follicles (HFs) [9]. Although promotion of skin healing has been attempted by transplanting cultured keratinocytes or skin equivalents [10,11], regeneration of HFs is still an unmet clinical need.
The HF is a mini-organ composed of an epithelial cylinder and specialized dermal papilla (DP) fibroblasts [12]. Similar to other ectodermal mini-organs, HF development depends on well-choreographed epithelial-mesenchymal interactions initiated by the crosstalk between epithelial and dermal embryonic skin progenitors [12]. During HF morphogenesis, epithelial progenitors gradually adopt a follicular fate, whereas dermal progenitors differentiate into DP fibroblasts. In normal adult skin, new HFs generally can not form [12]. As an exception, HFs can regenerate spontaneously in the center of very large wounds and with low efficiency in humans via incompletely understood mechanisms [[13], [14], [15]]. Such examples indicate that, in principle, the potential for HF neogenesis is preserved and can possibly be unleashed.
To induce new HFs postnatally, Oliver et al. demonstrated that microdissected DP could induce new HFs from epidermis when transplanted into the subepidermal space [16]. Following this seminal work, HF neogenesis in adults has largely relied on culture-expanded DP fibroblasts, which are capable of reinitiating developmental epithelial-mesenchymal interactions with competent keratinocytes [[17], [18], [19], [20]]. The HF-inducing ability of cultured DP fibroblasts is easily lost during culture and this limits their use for large-scale HF regeneration [17]. In addition to adult DP cells, freshly isolated newborn murine dermal fibroblasts have also been demonstrated to be capable of inducing HF neogenesis [7,19,[21], [22], [23]], but such HF inductivity of dermal fibroblasts are quickly lost in postnatal life.
An alternative strategy for inducing new HFs is through the use of embryonic skin [24]. In contrast to adult skin, wounded embryonic skin heals without scarring and with extensive HF neogenesis [25]. When embryonic dermal tissues are combined with postnatal epithelium, they are able to induce new HFs [24]. What are the key factors that define the HF-inducing ability of embryonic skin? It was demonstrated that isolated embryonic dermal cells are able to induce new HFs when combined with keratinocytes [26]. On the other hand, it has also been suggested that embryonic tissues create unique extracellular environments conducive to regeneration [25,27]. The cell-free extracellular matrix from adult tissue has been shown to enhance tissue regeneration [6,[27], [28], [29], [30]]. Isolation of defined factors present in the extracellular matrix that can elicit neogenesis of an organ can open novel, therapeutically amenable organ-specific regeneration protocols. However, induction of the neogenesis of a specific organ by defined extracellular factors has not been achieved. Here, we demonstrate that a cocktail of 3 secreted protein factors enriched in the developmental skin can induce neogenesis of the HF miniorgan through reactivating the epithelial-mesenchymal crosstalk between adult skin cells.
Section snippets
Animals
All animal experiments were approved by the Institutional Animal Care and Use Committee at National Taiwan University. Animals were housed in animal facilities of National Taiwan University. Wistar rats were purchased from BioLASCO Taiwan Co. and C57BL/6 mice and nude mice (BALB/cAnN-Foxn1nu/CrlNarl) were from Taiwan National Laboratory Animal Center. Z/AP mice expressing the lacZ reporter gene to overexpress β-galactosidase were from Jackson Laboratory [31]. For invasive experiments, animals
Induction of HF neogenesis by cell-free extract from E15.5-E17.5 embryonic rat skin
We first determined whether cell-free extracts from embryonic skin can induce HF neogenesis. In the HF neogenesis assay, the contamination of preformed HFs in keratinocytes can lead to HF formation without co-administration of inductive mesenchymal cells [21]. We carefully removed preformed HFs from epidermal keratinocytes to avoid this and the isolated keratinocytes alone did not grow any new HFs in patch assays for HF neogenesis (Fig. S1b, and S1b’). Formation of new HFs in the HF neogenesis
Discussion
We show that the three core proteins, Apoa1, Lgals1, and Lum are necessary and sufficient to induce HF neogenesis from adult cells, and that additional factors can further enhance the efficiency. ApoA1 is the major protein component of plasma high-density lipoprotein particles [55]. Due to their function in lipid metabolism and transport, apolipoproteins are actively involved in tissue healing and regeneration where newly formed lipid membranes are required [[56], [57], [58]]. In addition to
Conclusion
Regeneration of lost tissues in adults depends on the recapitulation of the developmental process of the respective organs. Using the HF regeneration as a model, we unveiled a novel method to re-elicit the developmental scheme for organ neogenesis. We demonstrated that defined extracellular proteins enriched in the embryonic developmental environment can reactivate organogenesis from adult cells. Three core proteins, Apoa1, Lgals1 and Lum, are necessary and sufficient for this regenerative
Acknowledgements
We thank the staff of the 8th Core Lab, Department of Medical Research, National Taiwan University Hospital for technical support and lab members in the S.J.L. lab for discussion. The mass spectrometry analyses using the Orbitrap Fusion mass spectrometers were performed by the Common Mass Spectrometry Facilities, Institute of Biological Chemistry, Academia Sinica. Analysis of RNA-sequencing data was performed by the Bioinformatics Core, Center of Genomic Medicine, National Taiwan University.
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