The Potential of Nail Mini-Organ Stem Cells in Skin, Nail and Digit Tips Regeneration.

Nails are highly keratinized skin appendages that exhibit continuous growth under physiological conditions and full regeneration upon removal. These mini-organs are maintained by two autonomous populations of skin stem cells. The fast-cycling, highly proliferative stem cells of the nail matrix (nail stem cells (NSCs)) predominantly replenish the nail plate. Furthermore, the slow-cycling population of the nail proximal fold (nail proximal fold stem cells (NPFSCs)) displays bifunctional properties by contributing to the peri-nail epidermis under the normal homeostasis and the nail structure upon injury. Here, we discuss nail mini-organ stem cells' location and their role in skin and nail homeostasis and regeneration, emphasizing their importance to orchestrate the whole digit tip regeneration. Such endogenous regeneration capabilities are observed in rodents and primates. However, they are limited to the region adjacent to the nail's proximal area, indicating the crucial role of nail mini-organ stem cells in digit restoration. Further, we explore the molecular characteristics of nail mini-organ stem cells and the critical role of the bone morphogenetic protein (BMP) and Wnt signaling pathways in homeostatic nail growth and digit restoration. Finally, we investigate the latest accomplishments in stimulating regenerative responses in regeneration-incompetent injuries. These pioneer results might open up new opportunities to overcome amputated mammalian digits and limbs' regenerative failures in the future.

Are fingernails a key to unlocking the puzzle of mammalian limb regeneration?

Some mammalian digit tips, including those of mice and human children, can regenerate following amputation, whereas mammalian limb regeneration does not occur. One major difference between the digit tip and the rest of the limb is the presence of the nail, which is necessary for this type of regeneration. This couples well with the finding that canonical Wnt signalling and Lgr6, an agonist of Wnt signalling that marks nail stem cells, are necessary for digit tip regeneration. This viewpoint essay discusses the role of the nail in digit tip regeneration and explores whether nail stem cells and their presumptive niche can be solely accountable for why regeneration is possible in the digit tip, but not the rest of the limb.

Extracellular vesicles derived from Msh homeobox 1 (Msx1)-overexpressing mesenchymal stem cells improve digit tip regeneration in an amputee mice model.

In adult mammals, limb regeneration is limited by the absence of blastemal cells (BCs) and the lack of the regenerative signaling cascade. The utilization of transgenic cells circumvents the limitations associated with the absence of BCs. In a previous investigation, we successfully regenerated mouse phalanx amputations using blastema-like cells (BlCs) generated from bone marrow-derived mesenchymal stem cells (mBMSCs) overexpressing Msx1 and Msx2 genes. Recently, extracellular vesicles (EVs) have emerged as potent biological tools, offering a promising alternative to manipulated cells for clinical applications. This research focuses on utilizing BlCs-derived extracellular vesicles (BlCs-EVs) for regenerating mouse digit tips. The BlCs were cultured and expanded, and then EVs were isolated via ultracentrifugation. The size, morphology, and CD81 marker expression of the EVs were confirmed through Dynamic Light Scattering (DLS), Scanning Electron Microscope (SEM), and Western Blot (WB) analyses. Additionally, WB analysis demonstrated the presence of MSX1, MSX2, FGF8, and BMP4 proteins. The uptake of EVs by mBMSCs was shown through immunostaining. Effects on cell proliferation, migration, and osteogenic activity post-treatment with BlCs-EVs were assessed through MTT assay, scratch assay, and Real-time PCR. The regenerative potential of BlCs-EVs was evaluated in a mouse digit tip amputation model using histological assessments. Results indicated that BlCs-EVs enhanced several abilities of mBMSCs, such as migration, proliferation, and osteogenesis in vitro. Notably, BlCs-EVs significantly improved digit tip regeneration in mice, promoting the formation of new bone and nails, which was absent in control groups. In summary, BlCs-EVs are promising tools for digit tip regeneration, avoiding the ethical concerns associated with using genetically modified cells.

Toeing the line between regeneration and fibrosis.

Understanding the remarkable capacity of vertebrates to naturally regenerate injured body parts has great importance for potential translation into human therapeutic applications. As compared to other vertebrates, mammals have low regenerative capacity for composite tissues like the limb. However, some primates and rodents can regenerate the distal tips of their digits following amputation, indicating that at least very distal mammalian limb tissues are competent for innate regeneration. It follows that successful digit tip regenerative outcome is highly dependent on the location of the amputation; those proximal to the position of the nail organ do not regenerate and result in fibrosis. This distal regeneration versus proximal fibrosis duality of the mouse digit tip serves as a powerful model to investigate the driving factors in determining each process. In this review, we present the current understanding of distal digit tip regeneration in the context of cellular heterogeneity and the potential for different cell types to function as progenitor cells, in pro-regenerative signaling, or in moderating fibrosis. We then go on to discuss these themes in the context of what is known about proximal digit fibrosis, towards generating hypotheses for these distinct healing processes in the distal and proximal mouse digit.

En1 and Lmx1b do not recapitulate embryonic dorsal-ventral limb patterning functions during mouse digit tip regeneration.

The mouse digit tip regenerates following amputation. How the regenerate is patterned is unknown, but a long-standing hypothesis proposes developmental patterning mechanisms are re-used during regeneration. The digit tip bone exhibits dorsal-ventral (DV) polarity, so we focus on En1 and Lmx1b, two factors necessary for DV patterning during limb development. We investigate whether they are re-expressed during regeneration in a developmental-like pattern and whether they direct DV morphology of the regenerate. We find that both En1 and Lmx1b are expressed in the regenerating digit tip epithelium and mesenchyme, respectively, but without DV polarity. Conditional genetics and quantitative analysis of digit tip bone morphology determine that genetic deletion of En1 or Lmx1b in adult digit tip regeneration modestly reduces bone regeneration but does not affect DV patterning. Collectively, our data suggest that, while En1 and Lmx1b are re-expressed during mouse digit tip regeneration, they do not define the DV axis during regeneration.

Nail-associated mesenchymal cells contribute to and are essential for dorsal digit tip regeneration.

Here, we ask why the nail base is essential for mammalian digit tip regeneration, focusing on the inductive nail mesenchyme. We identify a transcriptional signature for these cells that includes Lmx1b and show that the Lmx1b-expressing nail mesenchyme is essential for blastema formation. We use a combination of Lmx1bCreERT2-based lineage-tracing and single-cell transcriptional analyses to show that the nail mesenchyme contributes cells for two pro-regenerative mechanisms. One group of cells maintains their identity and regenerates the new nail mesenchyme. A second group contributes specifically to the dorsal blastema, loses their nail mesenchyme phenotype, acquires a blastema transcriptional state that is highly similar to blastema cells of other origins, and ultimately contributes to regeneration of the dorsal but not ventral dermis and bone. Thus, the regenerative necessity for an intact nail base is explained, at least in part, by a requirement for the inductive nail mesenchyme.

Cellular Heterogeneity and Lineage Restriction during Mouse Digit Tip Regeneration at Single-Cell Resolution.

Innate regeneration following digit tip amputation is one of the few examples of epimorphic regeneration in mammals. Digit tip regeneration is mediated by the blastema, the same structure invoked during limb regeneration in some lower vertebrates. By genetic lineage analyses, the digit tip blastema has been defined as a population of heterogeneous, lineage-restricted progenitor cells. These previous studies, however, do not comprehensively evaluate blastema heterogeneity or address lineage restriction of closely related cell types. In this report, we present single-cell RNA sequencing of over 38,000 cells from mouse digit tip blastemas and unamputated control digit tips and generate an atlas of the cell types participating in digit tip regeneration. We computationally define differentiation trajectories of vascular, monocytic, and fibroblastic lineages over regeneration, and while our data confirm broad lineage restriction of progenitors, our analysis reveals 67 genes enriched in blastema fibroblasts including a novel regeneration-specific gene, Mest.

Mesenchymal Precursor Cells in Adult Nerves Contribute to Mammalian Tissue Repair and Regeneration.

Peripheral innervation plays an important role in regulating tissue repair and regeneration. Here we provide evidence that injured peripheral nerves provide a reservoir of mesenchymal precursor cells that can directly contribute to murine digit tip regeneration and skin repair. In particular, using single-cell RNA sequencing and lineage tracing, we identify transcriptionally distinct mesenchymal cell populations within the control and injured adult nerve, including neural crest-derived cells in the endoneurium with characteristics of mesenchymal precursor cells. Culture and transplantation studies show that these nerve-derived mesenchymal cells have the potential to differentiate into non-nerve lineages. Moreover, following digit tip amputation, neural crest-derived nerve mesenchymal cells contribute to the regenerative blastema and, ultimately, to the regenerated bone. Similarly, neural crest-derived nerve mesenchymal cells contribute to the dermis during skin wound healing. These findings support a model where peripheral nerves directly contribute mesenchymal precursor cells to promote repair and regeneration of injured mammalian tissues.