Stem Cells of the Skin and Their Lineages
Adult stem cells reside in most tissues of our body. Although more restricted than embryonic stem cells in the types of tissues they make, these stem cells survive long-term, and regenerate both themselves and their resident tissues during normal wear and tear (homeostasis) and following injury. Tissue stem cells typically reside in specialized microenvironments (niches) and are used sparingly, but can be mobilized quickly and expand to repair wounds when the tissue is damaged. During normal homeostasis, tissue stem cells often exist in two distinct states: a quiescent state in which they are not making tissue and a primed state in which they are either actively making tissue or more readily activated when needed. Once activated, tissue stem cells produce shorter-lived progenitors (sometimes called ‘transit-amplifying cells’) that divide rapidly several times, but then progress to terminally differentiate to form their respective tissue.
My laboratory uses mammalian skin as a model system to study adult stem cells. At the surface of our body, skin epidermis is readily accessible, and its stem cells can be cultured and passaged long-term without losing their stemness. Shortly after gastrulation, embryonic skin begins as a single layer of multipotent epithelial cells. These cells not only give rise to the epidermis, as delineated above, but also its appendages, which include hair follicles, sebaceous glands, sweat glands and even mammary glands. In the adult, the stem cells of each of these epithelial tissues all express keratins 5 and 14, are rich in integrins and reside along the basement membrane separating the epithelium from the dermis (Gonzales and Fuchs, 2017; Hsu and Fuchs 2022). However, the stem cells are defined in both their task and their specialized gene expression programs by their local microenvironment (niche), presenting a rich and fascinating diversity for exploring the molecular mechanisms underlying the remarkable ability of stem cells to make and repair tissues throughout the lifetime of the animal.
The mouse hair follicle offers a particularly attractive model for dissecting the mechanisms involved. Like most mammals, mice have a thick hair coat. Each hair follicle has a resident niche (the bulge) of stem cells, responsible for regenerating the follicle below the bulge and producing the hair and its channel. Across the hair coat, follicles undergo synchronized, episodic bouts of activity, where they are either resting in quiescence or actively undergoing tissue (hair) regeneration. This has made the hair follicle the best system to interrogate how stem cells transition from a state where they are not making tissue to one where they actively regenerate a tissue (in this case, hair follicle/hair).
We recently discovered an elaborate network of lymphatic vessels that intimately associate with the stem niche in each hair follicle, and interconnect the follicles to coordinate niche activity (Gur-Cohen et al., 2019). This is not simply a quirk of the hair cycle, as lymphatics also intimately associate with the intestinal stem cell niche (crypt) (Niec et al., 2022). We learned that lymphatics act as a signaling hub to impact stem cell behavior. Whether lymphatics drain immune cells to keep stem cell niches in an immune privileged state, whether they remove toxins to preserve stem cells or whether drainage is needed to control fluid pressure at the niche remain fascinating questions for future study.
Hair follicles stem cells (HFSCs) can rest in quiescence for weeks, even months, adjacent to a specialized mesenchymal niche stimulus, the dermal papilla. During this time, epithelial-mesenchymal crosstalk at the base of the bulge stem cell niche (“the hair germ”) accumulates stimulating factors, and when the threshold is reached, these primed stem cells in the hair germ launch a new hair cycle (Tumbar et al., 2004). We now know that this initial activation step arises from a combination of WNT signaling and inhibition of BMP signaling (Greco et al., 2009). 
Interestingly, once activated by WNT signaling and BMP inhibition, the primed stem cells in the adult hair germ generate short-lived progenitors which start to make SHH (sonic hedgehog). By conditionally inactivating the genes encoding either SHH or its receptor, we discovered that SHH signals in two ways: first, it instructs the dermal papilla to make more BMP inhibitors and induce expression of proactivating FGF7; second, it stimulates the otherwise quiescent bulge to restock the stem cell pool and to make the shaft (outer root sheath or ORS) that pushes the SHH-expressing short lived progenitors away from the bulge niche (Hsu et al., 2014). Distanced from the SHH signal, the stem cells return to quiescence until the next hair cycle. By contrast, the short-lived progenitors continue to interact with the dermal papilla and receive the necessary signals to continue to proliferate and differentiate, producing the hair and its channel, or inner root sheath (IRS). As short-lived progenitors exhaust their proliferative capacity, the follicle degenerates, drawing the dermal papilla up to the base of the stem cell niche. (Hsu et al., 2014).
Another remarkable feature of bulge stem cells is that they normally only function in hair regeneration, but they harbor the potential to also make epidermis and sebaceous glands. We first learned of this plasticity of bulge stem cells when we fluorescently marked and purified the stem cells from their native niche, cultured them and then engrafted the progeny from individual stem cell-derived clones back onto the skin of a hairless mouse. The graft produced hundreds of fluorescent hair follicles, sebaceous glands and epidermis (Blanpain et al., 2004). This experiment not only illustrated the enormous tissue-regenerating potential of these stem cells, but also showed that these stem cells possess multipotency. It also told us that the niche microenvironment imposes restraints upon what these stem cells do. Indeed, following a shallow injury in which the skin is denuded of its epidermis, hair follicle stem cells exit their niche, migrate up into the wound bed, undergo a fate switch and thereafter act as epidermal stem cells to make the skin’s barrier and fend off infection (Gonzales et al., 2021).(Blanpain et al., 2004).
We observed similar stem cell plasticity when we purified and tested the myoepithelial stem cells from sweat glands (Lu et al., 2012; Blanpain and Fuchs, 2014). Similar to myoepithelial stem cells of mammary glands, these stem cells normally act unipotently and only replenish dying myoepithelial cells of the gland. However, when purified by fluorescence activated cell sorting (FACS) and transplanted directly into a mammary fat pad, the stem cells can regenerate the complete bi-layered gland, and the new luminal cells secrete sweat. Moreover, when engrafted to the skin, these stem cells can make epidermis. An area of interest in my lab is to understand the environmental cues that dictate the fascinating plasticity of epithelial stem cells, and to elucidate the chromatin remodeling that leads to the changes in gene expression necessary to generate different tissues from a common progenitor.
To understand how a stem cell chooses its differentiation pathway, we have taken several approaches. The beauty of the hair follicle as a model is that it is one of the few systems where sufficient quantities of stem cells can be isolated directly from their native niche in order to carry out high throughput analyses in vivo. Adding to the advantages are the synchrony of hair follicles and the spatial and temporal landscape of the stem cells and their lineages. We initially used fluorescence activated cell sorting (FACS) to purify cells directly from skin at different time points along the lineages and generate a battery of lineage-specific profiles at the mRNA (RNA-seq) and chromatin level (transcription factor, histone modification and accessibility landscaping). In combination with single cell RNA-seq (scRNA-seq) and spatial transcriptomics, we’ve now been able to temporally and spatially map how stem cells change their program of gene expression as they become activated and differentiate (Yang et al., Cell 2017; Niec et al., 2022). Overall, these approaches have allowed us to exploit this information to understand how stem cells and/or their progeny receive signals, change their program of gene expression and progress along a particular lineage.
(Tumbar et al., 2004; Blanpain et al., 2004; Keyes et al., 2013). For the hair follicle, >350 mRNAs are selectively upregulated in the bulge stem cells relative to their short-lived progeny (Tumbar et al., 2004; Blanpain et al., 2004;Yang et al., 2017). A number of these changes are in transcription factors and epigenetic regulators. In vivo ChIP-seq on HFSCs and their short-lived progeny and bioinformatic analyses have unveiled which genes bind these transcription factors, what types of histone modifications occur within the locus, and how this changes as the stem cells progress to form transiently dividing cells that then terminally differentiate along one of the 7 distinct concentric cell layers that constitute the hair and its channel (Lien et al., 2011; 2014; Adam et al., 2015; 2018; 2020). By engineering inducible-conditional knockouts to selectively remove these transcription factors and/or histone modifications in the stem cells, we’ve learned their physiological relevance.
Based upon these analyses, many of the transcription factors are essential for maintaining the HFSCs in their native niche. Without LHX2, the stem cell niche becomes a sebaceous gland (Folgueras et al., 2013). Without SOX9, the niche becomes an epidermal cyst (Kadaja et al., 2014). TCF3 and TCF4 antagonize WNT signaling. Without them, quiescent HFSCs precociously activate a new hair cycle (Lien et al., 2014). Conversely, without β-catenin, the DNA effector of WNT signaling, quiescent HFSCs never reenter a new hair cycle (Lien et al., 2014). The precise mechanisms underlying the antagonistic phenotypic behavior between TCF3/4 and β-catenin still remain to be elucidated, although in the natural hair cycle, WNT-signaling is accompanied by a switch from TCF3/4 to TCF1/LEF1, which work together with β-catenin to drive new hair growth (Adam et al., 2018).
NFATc1 and FOXC1 are downstream of BMP signaling and are required to maintain HFSC quiescence. In their absence, HFs cycle continuously (Horsley et al., 2008; Lay et al., 2016). During aging, BMP levels become elevated in the skin, and the resting intervals between hair cycling become increasingly longer. Although we can stimulate old follicles by lowering BMPs, inhibiting NFATc1 and/or knocking out NFATc1 or FOXC1, continuous hair regeneration results in precocious hair greying and baldness (Keyes et al., 2013; Lay et al., 2016). Since HFSCs can be cultured long-term without losing their stemness, this precocious exhaustion of stem cells in vivo suggests that surrounding niche cells or other changes in the skin are responsible for the failure of the stem cells to keep up with increased demands on tissue regeneration. More studies are still needed in the quest for the Fountain of Youth!
NFiB is a transcription factor which is specific to the HFSCs, but functions by repressing the expression of genes that are essential for the differentiation of the melanocyte stem cells, which reside within the same stem cell niche (Chang et al., 2013; Adam et al., 2020). These two stem cell populations must be activated at the same time so that differentiating melanocytes can transfer pigment to the differentiating hair cells to provide the natural coloring to our hair. Loss of NFiB uncouples this crosstalk and leads to the precocious activation of a key NFiB target gene that encodes a secreted melanocyte differentiation factor (Chang et al., 2013).(Chang et al., 2013).
There are a number of additional transcription factors and epigenetic regulators which are enhanced in the complex milieu of HFSC chromatin, and there is still much to be learned. Of the epigenetic regulators, we’ve thus far examined only the role of polycomb chromatin repressor complexes and their targeted methylation of H3K27, which we’ve shown function critically in controlling the fate switch from a stem cell to a committed, transit-amplifying state (Ezhkova et al., 2009; 2011; Lien et al., 2011). Our in vivo ChIP-seq studies with mutually exclusive H3K27ac unearthed an additional component of this fascinating switch: a small cohort (~5%) of the total genes expressed by HFSCs are regulated by large open chromatin domains, called ‘super-enhancers’. These enhancers are typified by the presence of ‘epicenters’, which are strong peaks of H3K27ac associated with clusters of densely packed binding motifs for the key stemness genes noted above (Adam et al., 2015). In a paradigm first set for cultured embryonic stem cells, the genes encoding the key stemness transcription factors of in vivo tissue stem cells are themselves regulated by super-enhancers, as are BMPs and their receptors and WNT-inhibitors and WNT receptors.
Probing more deeply, we’ve learned that epicenters are essentially molecular “zipcodes” that tells the transcriptional machinery “this is who I am (e.g. a HFSC) and where I am (e.g. in a quiescent niche). For the HFSC, the epicenter binds not only HFSC transcription factors, but also the DNA effectors for BMP signaling (pSMAD1/SMAD4) and WNT signaling antagonists (TCF3/4). In so doing, the expression of genes regulated by super-enhancers are highly sensitive to the microenvironment of the stem cell. We can demonstrate this functionally by excising an epicenter and using it as an enhancer to drive eGFP in mice: an epicenter mapped from ChIP-seq analysis of in vivo HFSCs is only active in the quiescent HFSC niche; eGFP expression is silenced when the HFSCs are cultured, and reactivated when the HFSCs are engrafted and HFs are re-established in vivo (Adam et al., 2015). By contrast, an epicenter mapped from similar analysis of cultured HFSCs is only active in vivo when the skin is wounded (Adam et al., 2015). Indeed, rich in serum and growth factors, the culture environment mimics a wound state!
In performing functional analyses to interrogate the physiological relevance of our chromatin landscaping, we have learned that super-enhancers are at the roots of stem cell plasticity (Ge et al., 2017). We are aided in our strategy by the power of our in utero lentiviral delivery method, which enables us to perform rapid functional analyses and reap the benefits of high throughput technology.
By dissecting the regulatory elements that drive gene expression in a particular cell type and state, we are also generating tailor-made enhancers to drive Cre-recombinases and/or transgene expression with greater precision than before. Finally, the short-list of super-enhancer regulated genes is rich in stemness genes and human skin disease genes, offering a plethora of new, unexplored genes on this list for future study. Systematically working our way through the functional significance and mechanism of action of epigenetic and transcriptional controls on stem cells can now proceed at a rate that is on par with worm or fly genetics.
Stem Cells: Coping With Stress and Inflammation.
At the interface between the environment and the body, the skin epithelium is subjected to a vast array of stresses, ranging from wounding/injury to mechanical stresses to microbial stresses to climate changes and oncogenic stresses. To survive long-term, stem cells must be able to cope with stress. At all costs, the stem cells must be ready to rapidly respond and repair a breach in the skin barrier should it arise. Increasingly, a major focus of the laboratory is how stem cells equip themselves to with the ability to cope and withstand these traumas. Integral to this talent is their ability to communicate with resident immune cells and to recruit and coordinate responses with inflammatory cells.
The nature of an injury or stress instructs the epithelium how quickly to respond to the potential threat and establishes the communication network involved in recruiting immune cells and collaborating with them to patch a breach in the barrier. Recently, we discovered that a breach in the terminally differentiated cells that make the skin’s barrier can be sensed directly by the underlying stem cells, through the shared, aberrant intercellular interface (Lay et al., 2018). Independent of the resulting bacterial infiltration, the stem cells sense the breach and transmit chemokine and cytokine distress signals to recruit and galvanize dendritic cells and regulatory T cells within the skin. Turnabout is fair play: the activated Tregs in turn appear to stimulate the stem cells to proliferate and patch the barrier breach (Lay et al., 2018). Increasing evidence suggests that establishing communication lines with these special T cells may be of general importance for tissue stem cells to efficiently repair their wounds.
As we age, our wounds heal more slowly. We’ve found that at least in part, the poor performance emanates from failed conversation between resident immune cells and aged skin stem cells. In mice, the epidermis houses a unique population of γδ T cells, called dendritic epidermal T cells (DETCs), that orchestrate wound repair. Upon wounding, epidermal progenitors upregulate expression of Skint genes that are essential for alerting DETCs, which in turn produce growth factors that accelerate wound-repair (Keyes et al., 2016). In aging skin, this communication network breaks down, and as a result, epidermal progenitors are left on their own to re-epithelialize the wound bed, without the help of DETCs(Keyes et al., 2016). Although human epidermis lacks DETCs, similar impairments in the dialogue between other tissue resident T cells and stem cells could underlie chronic wounds in aged individuals.
As the complex crosstalk between immune cells and skin stem cells continues to unfold, we’ve learned that the stem cells markedly change their chromatin landscape and program of gene expression in response to these various encounters. In wondering how long these epigenetic changes last, we discovered that while most chromatin changes resolve with the pathology of inflammation, some changes linger and can still be detected in chromatin 6 months after the inflammatory assault (Naik et al., 2017). These epigenetic scars of their inflammatory experiences heighten the stem cell’s sensitivity to subsequent exposures, and equips them with enhanced tissue repair capacity. Despite the advantage to fixing barrier breaches, inflammatory memory is likely to be at the roots of a host of human inflammatory disorders of epithelial tissues, including psoriasis and atopic dermatitis.
One of the hallmarks of inflammatory disorders such as psoriasis, atopic dermatitis, asthma and inflammatory bowel disease is that their epithelial cells all hyperproliferate in response to a particular inflammatory stimulus. Following the irritant, the pathology returns back to normal, but then it returns, often in the same spot and with increasing severity. Of additional intrigue is that the secondary trigger need not be the same as the initial stimulus, setting this kind of memory apart from that of adaptive immunity (B and T cells). As we delved deeper, we learned that tucked inside the nucleus of an epidermal stem cell was an epigenetic memory of the initial inflammatory experience. The memory persists independent of lymphocytes or macrophages. We showed that these epigenetic memory marks act as inflammation sensors and are typically found in enhancers associated with genes that need to be rapidly activated in wound repair (Naik et al., 2017). Working out the mechanism underlying inflammatory memory, we showed that establishing memory requires both an inflammatory transcription factor to open the chromatin and the general stress factor c-fos/jun to remodel it and transcribe the associated gene. During this process, stem cell factors now gain accessibility to these enhancers such that following inflammation, when the inflammatory and stress factors are no longer there, the stem cell factors keep the chromatin accessible. As the chromatin is now open, all that is needed is general stress to induce c-fos and “recall” the memory (Larsen et al., 2021).
Our bioinformatics have suggested that the mechanism we’ve unearthed is broadly applicable to other types of inflammatory memory. In addition, we now know that many other types of stem cells harbor similar types of inflammatory memory. While evolutionarily beneficial (the ability to repair subsequent wounds faster, broader resistance to pathogens), there are also downsides to memory (chronic inflammation, increased susceptibility to cancer). The mechanisms of how stem cells retain and recall memories not only of inflammation but also other types of tissue experiences have remarkable similarities to memory in brain cells, and our work suggests that many if not most long-lived cells of our body (like stem cells and neurons) may harbor memories of their experiences (Gonzales et al., 2021; Niec et al., Cell 2021; Naik and Fuchs, 2022). With its profound importance to body and tissue fitness, this arena has developed into a major focus of my group, with a strong bent towards elucidating the interactions between stem cells and their niches in times of health and stressful encounters.
An endless sea of questions remain. What happens to tissue memories as we age? Can we acquire memories when we’re older? Are they the same as when we’re younger? How many kinds of memories are there and what are the consequences? Do our memories keep endowing us with heightened sensitivity or do we become tolerant to repetitive bouts of the same memory? Are memories kept in the skin dependent upon overall organismal fitness? If so how? Dissecting the underlying mechanisms involved will be a first step in identifying new strategies to “erase memory” and treat such disorders.
