Sensory nerves in the spotlight of the stem cell niche

Abstract Niches are specialized tissue microenvironments that control stem cells functioning. The bone marrow mesenchymal stem cell niche defines a location within the marrow in which mesenchymal stem cells are retained and produce new cells throughout life. Deciphering the signaling mechanisms by which the niche regulates stem cell fate will facilitate the use of these cells for therapy. Recent studies, by using state‐of‐the‐art methodologies, including sophisticated in vivo inducible genetic techniques, such as lineage‐tracing Cre/loxP mediated systems, in combination with pharmacological inhibition, provide evidence that sensory neuron is an important component of the bone marrow mesenchymal stem cell niche. Strikingly, knockout of a specific receptor in sensory neurons blocked stem cell function in the bone marrow. The knowledge arising from these discoveries will be crucial for stem cell manipulation in the future. Here, we review recent progress in our understanding of sensory nerves biology in the stem cell niche.


| Bone marrow mesenchymal stem cells (BMSCs)
Adult endogenous stem cells are fundamental for maintaining tissue homeostasis due to their extraordinary capacity to form specialized cell populations in a coordinated way according to the needs of the organism. 1 Mesenchymal stem cells were first discovered within the bone marrow. 2 Subsequent studies have identified mesenchymal stem cells in various other adult tissues. 3 BMSCs are characterized as postnatal selfrenewing multipotent stem cells, forming all skeletal tissues. 2,4 In culture, these cells can form a clonal progeny of transplantable cells, equal to the one that generated them. 5 After transplanted in vivo, BMSCs can form bone organoids. 6 A single BMSC is a bona fide stem cell as it can initiate a clonal population in vitro, which then may create a full organoid in vivo with transplantable BMSCs, being self-renewing and multipotent. 2,7 BMSCs are also defined as skeletal stem cells as they can be located within the skeleton, able to give rise to various skeletal tissues, and have an innate capacity to start a recapitulation of bone organogenesis in vivo. [8][9][10][11] BMSCs are essential for the development, lifelong turnover, and regenerative ability of bones in our organism. 12,13 The ability of mesenchymal stem cells derived from variable sources to repair tissues placed them in the center of attention of numerous groups due to their promising potential in regenerative medicine for multiple disorders. 14,15 Therefore, in the last two decades, it became clear that understanding the biology of these cells may lead to the treatment of several diseases.

| Stem cells and their niches
Accurate regulation over stem cell differentiation is crucial for appropriate tissue homeostasis and organogenesis. 16 Stem cells occupy particular microenvironments, also termed niches, 17 which keep them in an undifferentiated and self-renewing state. Defining and understanding the mechanisms that restrict niche signaling exclusively to stem cells is crucial to determine how stem cells undergo self-renewal while their progeny differentiate. Extensive studies in a variety of tissues have highlighted the importance of the microenvironment in modulating stem cell behavior, including skin, 18 intestine, 19 stomach, 20 skeletal muscle, 21 bone marrow, 22 liver, 23 brain, 24,25 and others. 26,27 Despite significant progress made in our knowledge of which signals foster stem cell quiescence or activation, some constituents of stem cells niches remain unrevealed to date. This is due to the complexity of tissue microenvironment content and its dynamics. Understanding the role of niche components in stem cell behavior is vital for our knowledge of organ homeostasis and disease, and to fully exploit stem cell therapeutic potential.

| BMSC niche
Although the precise location of the BMSC niche has not been determined so far, several studies suggest that MSCs reside in perivascular sites, associated with blood vessels. 28 Therefore, MSCs have been compared to pericytes. [29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45] Nevertheless, whether these two cell types correspond to the same cell is not clear yet. 11,46 BMSCs maintenance within the adult bone is essential for skeletal homeostasis and reconstruction after damage. 47 BMSCs are often in a quiescent state, and extrinsic factors from their medullar niche can activate their self-renewal, proliferation, or differentiation. Examination of the BMSC microenvironment in the bone marrow revealed that BMSC behavior is markedly affected by interactions with cellular components of this local niche, both directly, by physical contact, or indirectly, by ligation of secreted soluble molecules. 48 Multiple local signaling cues can influence BMSCs fate, such as interleukins, 49,50 chemokines, 51 Wnt ligands, 52,53 FGF2, 54 and others. 48,55 Oxygen also may be a key regulatory factor in the BMSC niche. 56 Perturbations in the architecture of extracellular matrix constituents in the bone marrow influence BMSC behavior. 57

| Nerves in the bone marrow
Nerves penetrating the bone marrow were described for the first time more than 50 years ago. 80 As the bone marrow occupies spaces deep within our body and is encased by an outer hard compact bone, experimental assessments were initially difficult. Later studies confirmed these initial discoveries demonstrating that medullar innervations can signal and communicate with cells in the bone marrow. 81 Innervations in the bone marrow are mostly associated with blood vessels. 80,82 The pattern of similar wiring of nerves and blood vessels is well established in other organs, and suggests that they support each other. 83 Accordingly, it is possible that vascular and neuronal networks also have a functional connection within the bone marrow.
Because of the perivascular location of BMSCs, the interaction of BMSCs with nerve projections is also likely.
So far, the sympathetic nerves were the most explored nerve fibers in the bone marrow. Innervations expressing tyrosine-hydroxylase 79 and Neuropeptide Y, 84   in vivo, as well as their ability to differentiate into osteoblasts in vitro, 92 suggesting that sensory innervations are essential for BMSCs maintenance in their niche.

| Sensory nerves in the bone marrow
It is well established that prostaglandins play essential roles in bone metabolism. 93 Amid prostaglandins, prostaglandin E 2 (PGE 2 ) induces bone formation. 94 Strikingly, genetic deletion of prostaglandin E 2 receptor (EP4) specifically from sensory nerve fibers, by using Advillin-Cre/ EP4-floxed mice, inhibited osteogenesis and induced adipogenesis. 92 Moreover, similarly to what was observed after sensory nerves depletion, BMSCs number declined in the bone marrow, along with their capacity to self-renew and to differentiate into the osteogenic lineage 92 ( Figure 1). Genetic ablation of the prostaglandin receptor from other cells present in the bone marrow microenvironment (osteoblasts or BMSCs) did not present alterations in the bone or in BMSC behavior. 92 Overall, these findings imply that EP4 receptor in sensory nerves is crucial for BMSC behavior control in the bone marrow.
Hu and colleagues also showed, by using Ocn-Cre/COX2-floxed mice and pharmacological inhibition, that osteoblasts are the source of PGE 2 which acts on sensory nerves that consequently affect BMSC behavior. 92 Remarkably, Hu 92 Some caveats need to be given due consideration when using such systems, including insufficient gene knockdown and compensatory pathway upregulation.
Thus, the analysis of the expression level of the gene being deleted as well as of other genes that may be affected will clarify this point.
Moreover, gene ablation that occurs in the germline can culminate in developmental compensatory mechanisms. [95][96][97] Models with inducible time and tissue-specific gene ablation would overcome potential physiological compensatory processes that can alter the true function of a specific gene. 98 Such strategies are being used to study the role of individual proteins in distinct pathophysiologic conditions. [99][100][101][102][103][104][105][106] Thus, this may be addressed by analyzing bone marrow from Advillin-CreER/EP4-floxed mice in which EP4 deletion in sensory nerve fibers can be temporally controlled.
The main findings from this work are based on the data obtained from Advillin-Cre mice. 92 Note, however, that expression of advillin is not restricted to peripheral sensory neurons that innervate the bone marrow. Thus, in Advillin-Cre/EP4-floxed mice, EP4 is also eliminated from sensory nerves in several other tissues, besides the bone. Therefore, it remains to be explored whether BMSC phenotype in this mouse model is due to EP4 deletion that happens specifically in the bone. Moreover, it was recently discovered that advillin is also expressed in the peripheral neuronal projections innervating the vasculature coming from sympathetic, parasympathetic, and enteric neurons. 107 Interestingly, even some non-neuronal cells may be targeted in these mice, that is, Merkel 107,108 and Tuft 109 cells. Hence, it is possible that some of the effects observed in BMSCs in Advillin-Cre/ EP4-floxed mice are not due to sensory neurons exclusively. To clarify whether EP4 is eliminated from any other medullar components, the bone marrow from Advillin-Cre/EP4-floxed/TdTomato mice should be examined in which all components from which EP4 is being deleted will be labeled. Furthermore, mouse models more specific to sensory nerves can be used for comparison, such as Nav1.8-Cre mice.

| Mechanism by which sensory nerves affect BMSCs
Cao's group data revealed that sensory nerves regulate BMSC behavior in the bone marrow. 92 Yet it remains uncertain whether this happens by an indirect mechanism via BMSC niche components or by directly acting on BMSCs. The authors suggest that this regulation is via sympathetic nervous system, based on pharmacological inhibition with β adrenergic antagonists. 92 As beta-blockers may have off-target side effects, 110  Interestingly, in another recent work from the same group, Chen and colleagues suggest that sensory nerves' effect on bone formation is through the hypothalamus. 112 The authors show that, in mice stimulated by PGE2, CREB is phosphorylated in the hypothalamus, and this is inhibited by knockout of EP4 receptors in sensory nerves.
Albeit CREB phosphorylation in the hypothalamus may affect sympathetic nerves, 113  One such organ is the skin, largely innervated by sensory nerve fibers. 125 Interestingly, a recent study showed that hyperactivation of the sympathetic nervous system leads to reduced number of melanocyte stem cells in their dermal niche 126 ( Figure 2). These stem cells reside in the hair follicle microenvironment. 127  Interestingly, Cao's group showed that mice with sensory nerves genetic depletion present also impaired hepatic regeneration after hepatectomy, suggesting that sensory nerves compose the liver stem cell niche as well. 112 Notably, the mechanism by which sensory nerves are activated in the liver is different from what happens in the bone marrow, as deletion of EP4 receptor in sensory nerves did not alter hepatic regeneration. 112 During fetal development, hematopoietic stem cells are located in the liver from where they later migrate through the bloodstream to the bone marrow preceding birth, where they remain throughout the adult life. [132][133][134][135] Their expansion in the fetal liver is dependent of portal vessel-associated hepatic mesenchymal stem cells. 132 It will be interesting to explore whether sensory nerves also regulate hepatic mesenchymal stem cells in the fetal liver, and consequently hematopoietic stem cells.

| Sensory nerve involvement in BMSCs' other functions
The most well-established functions of BMSCs, based on which they were named, are related to their capacity to differentiate in multiple cell types, replacing damaged cells. 136 Notably, in addition to their regenerative activities, BMSCs have also been described to present immunomodulatory, immunosuppressive, and anti-inflammatory characteristics. [137][138][139] These capabilities are the basis for the medical exploration by numerous clinical trials of BMSCs in the therapy for inflammatory and immune F I G U R E 2 Sensory nerves role in the hair follicle melanocyte stem cell (MeSC) niche. Two main stem cell populations are present in the hair follicle bulge: MeSCs and hair follicle stem cells (HFSCs). MeSCs generate new pigmented hair. Zhang and colleagues revealed that hyperactivation of sympathetic nerves leads to MeSCs activation, proliferation, and consequently elimination from their niche, leading to hair discoloration. 126 Interestingly, resinoferatoxin (RTX), which causes sensory nerve denervation as well, was used to induce stress. Adrb2, β2-adrenergic receptor; NE, norepinephrine disorders. [140][141][142] BMSCs can affect the behavior of various immune cells, such as T cells, macrophages, and others. [143][144][145] These interactions may grant homeostasis within the tissue. 146,147 In the last decade, several groups have focused their attention on exploring the immune-modulating capacity of the peripheral nervous system. Recent studies have shown that sensory neurons are critical mediators of inflammatory processes in diverse tissues. 128 Importantly, BMSCs are also heterogeneous in their morphology, distribution, anatomical location, origin, molecular markers, and function. [155][156][157][158][159] At least two subpopulations have been described in the bone marrow. 157 Thus, whether only a fraction of BMSCs respond to sensory nerves in response to mechanical loading still needs to be elucidated. It would be important to evaluate whether distinct BMSCs' subsets behave differently during sensory nerves activation.

| Sensory nerves in the cancer microenvironment
Nerves have been reported to promote tumor growth and spread. [160][161][162] Classically, sensory nerves have been associated with tumor-associated pain. 163,164 Interestingly, a new study shows that sensory nerve fibers are involved in tumor progression in vivo. 124 (Figure 3).

| CONCLUSION
In conclusion, the study by Hu and colleagues reveals a novel important role in BMSCs regulation of sensory neurons which innervate the bone marrow. 92 However, our understanding of the neural regulation in the stem cell niche in the bone marrow and other organs remains limited, and the complexity of these interactions in different microenvironments should be elucidated in future studies. A big challenge for the future will be to translate these findings to human patients.
Whether sensory nerves are essential components in the human bone marrow stem cell niche remains to be determined. Improving the availability of human bone marrow samples will be crucial to reach this goal. Future developments in this research are promising.

CONFLICT OF INTEREST
The authors declared no potential conflicts of interest.

AUTHOR CONTRIBUTIONS
All authors wrote and commented on the manuscript.

DATA AVAILABILITY STATEMENT
Data sharing is not applicable to this article as no new data were created or analyzed in this study.