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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. Adult stem cells typically reside in specialized microenvironments (niches) and are used sparingly, but self-renew long-term. They often exist in two distinct states: a quiescent state in which they are not making tissue and a primed or active state in which they are actively making tissue. When activated, they produce shorter-lived progenitors (transit-amplifying cells) that divide rapidly and then progress to terminally differentiate to form their respective tissue cells. 

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 will give rise to the epidermis, and its appendages, which include hair follicles, sebaceous glands, sweat glands and even mammary glands. In the adult, each of these epithelial tissues has its own niche(s) of stem cells, presenting a rich and fascinating diversity for exploring the molecular mechanisms underlying the “fountain of youth” properties of stem cells and their remarkable ability to make and repair tissues.

The mouse hair follicle offers a particularly attractive model for dissecting the mechanisms involved, and for elucidating how stem cells transition from a quiescent state where they are not making tissue to an active state, where they churn out tissue, in this case hair. 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, the follicles undergo synchronized bouts of activity. The bulge stem cells can rest in quiescence for weeks, even months, adjacent to a specialized dermal cell cluster, the dermal papilla. During this time, epithelial-mesenchymal crosstalk at the base of the stem cell niche (“the hair germ”) accumulates stimulating factors, and when the threshold is reached, 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). Soon thereafter, these activated stem cells generate the short-lived progenitors which start to make SHH (sonic hedgehog).

To elucidate the role for this new stimulus, we used mouse genetics and inactivated the genes encoding either SHH or its receptor and specifically in the stem cells or other niche progeny. We discovered that SHH signals in two ways: first, it stimulates the otherwise quiescent bulge to restock the stem cell pool; second, it stimulates the dermal papilla, instructing it to make more BMP inhibitors and induce expression of FGF7 (Hsu et al., 2014). As the new hair follicle grows downward, the SHH-expressing, short-lived progenitors and the dermal papilla are pushed downward, away from the bulge niche. Too distant to receive the SHH signal, the stem cells return to quiescence. Still receiving the BMP inhibitory and FGF7 signals, the short-lived progenitors proliferate, enabling the hair to grow. When the short-lived progenitors exhaust their proliferative capacity, the follicle degenerates and this draws the dermal papilla upward again to the stem cell niche. A new resting phase is launched and persists until the crosstalk is sufficient to activate a new cycle. With each hair cycle, our hair follicles spend longer periods at rest. By investigating why this happens, we hope to address whether this knowledge might be useful in restoring the youthful activity of our skin stem cells as we age (Keyes et al., 2013).

A remarkable feature of bulge stem cells is that they normally only function in hair regeneration, but they possess 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 tells us that the niche microenvironment imposes restraints upon what these stem cells do.

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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. An ongoing approach of the lab is to express different fluorescent proteins under the control of various skin promoters, active at different stages in stem cells and their lineages. Through FACS, we've purified cells at different time points along the lineages and generated a battery of lineage-specific profiles, enabling us to define at an mRNA (RNA-seq) and chromatin (ChIP-seq) level how stem cells change as they transition from quiescence to activation to lineage determination. Our global objective is to exploit this information to understand how stem cells receive signals, change their program of gene expression and select a lineage.  We also want to understand the functional significance of these changes. The beauty of the hair follicle as a model is that it is currently the only system where sufficient quantities of stem cells can be isolated directly from their native niche in order to carry out whole-genome wide analyses in vivo. This eliminates the caveats arising from culturing cells, namely induction of a stress response and large-scale epigenetic changes in gene expression.

For the hair follicle, >150 mRNAs are selectively upregulated in the bulge stem cells relative to their short-lived progeny (Tumbar et al., 2004; Blanpain et al., 2004; Keyes et al., 2013). A number of these changes are in transcription factors and epigenetic regulators. We’ve conducted in vivo chromatin immunoprecipitation and high throughput sequencing (ChIP-seq) on chromatin from hair follicle stem cells (HFSCs) and their short-lived progeny. Bioinformatics reveals which genes bind these transcription factors, 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. By conducting high throughput RNA sequencing (RNA-seq) on HFSCs lacking each of these genes, we’ve learned which target genes depend upon binding these transcription factors. Finally, by engineering inducible-conditional knockouts to selectively remove these transcription factors in the stem cells, we’ve learned the physiological relevance of these factors.

Based upon these analyses, TCF3/TCF4, LHX2 and SOX9 are all essential for maintaining the hair follicle stem cells in their native niche (Nguyen et al., 2006; 2009; Rhee et al., 2006; Folgueras et al., 2013Lien et al., 2011; 2014; Nowak et al., 2008; Kadaja et al., 2014). In addition, LHX2 represses sebaceous gland differentiation: following its loss, the stem cell niche soon becomes a sebaceous gland (Folgueras et al., 2013). SOX9 represses epidermal differentiation: following its loss, the niche becomes an epidermal cyst (Kadaja et al., 2014). TCF3 and TCF4 repress HF differentiation: following their loss, quiescent HFSCs precociously activate a new hair cycle (Lien et al., 2014). TCF3 and TCF4 can partner with β-catenin, which is stabilized and becomes nuclear upon Wnt signaling: if β-catenin is silenced in the quiescent HFSCs, they never reenter a new hair cycle. In their native niche, quiescent HFSCs express a transcriptional repressor TLE4 which binds to TCF3 and TCF4: our findings are consistent with the view that Wnt signaling functions by relieving TCF3/4/TLE4-mediated repression (Lien et al., 2014).

NFATc1 is required for maintaining HFSC quiescence, and in its absence, HFs cycle precociously (Horsley et al., 2008). Additionally, NFATc1 is downstream of BMP signaling, offering a potential explanation as to why BMP signaling must be lowered to activate hair cycling. A major feature of the aging HFSC signature is elevated NFATc1 target genes, and we can stimulate old follicles by inhibiting NFATc1 (Keyes et al., 2013). A major question still to be answered is whether HFSCs have an endless capacity for hair cycling and whether this same phenomenon operates in aging scalp hairs in humans. If so, these findings may open new doors for future therapeutics.

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). 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).

There are a number of additional transcription factors and epigenetic regulators which are enhanced in the complex milieu of HF stem cell 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, 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). In coming years, we will continue to systematically work our way through the functional significance and mechanism of action of epigenetic and transcriptional controls on stem cells as they transit from a quiescent to activated to committed state. When coupled with our recent ability to efficiently knockdown genes in a few days using lentiviral-mediated shRNA delivery (Beronja et al., 2010), this now becomes a powerful tool for exploiting bioinformatics analyses to gain biological insights.

Our ultimate goal is to understand how external signals from the surrounding niche microenvironment impact chromatin dynamics to achieve tissue production. Equally important will be the expression of specific genes that enables them to remodel their cytoskeleton and adhesive contacts and either form a stratified epidermis or an epithelial bud that can then develop into a hair follicle (Perez-Moreno et al., 2003; Blanpain and Fuchs, 2009; Hsu et al., 2014). While our model is the skin, the problem is a general one of how a single epithelial stem cell gives rise to a spatially organized, functional tissue. It is also integrally linked to understanding the basis of cancer progression.

 

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 t