Described herein are embodiments of (i) methods of treating hair loss, (ii) methods for obtaining and delivering senescent cells into the skin for the purposes of hair growth stimulation, and (iii) methods for producing and delivering senescent cell associated secretory phenotype (SASP) molecules and their combinations into the skin for the purposes of hair growth stimulation.
Hair loss (alopecia) results from (i) shortening of the growth phase of the hair regeneration cycle (aka anagen phase) so that progressively shorter hairs are produced and (ii) lengthening the rest phase of the cycle (aka telogen phase) so that hair follicles stop producing new hairs or (iii) the combination of the above two mechanisms.
Current available treatments for hair loss involve approaches for extending the duration of the growth phase. There is a deficiency in the art for hair loss treatments that involve shortening the rest phase and for treatments that effectively cause a transition of dormant hair follicles from the telogen to anagen phase.
In some embodiments, a method for enhancing or inducing hair growth in a subject at an area affected by hair loss is provided. In some embodiments, the methods include delivering at least one senescence associated secretory phenotype (SASP) factor, or at least one senescent cell or cell type that secretes said at least one SASP factor, to the subject at the area affected by hair loss. In some embodiments, the at least one senescent cell or cell type comprises at least one cell type that is non-replicative or exhibits a non-replicative phenotype. In some embodiments, the delivery of the at least one senescent cell or cell type or of the at least one SASP factor induces one or more of lengthening an anagen phase and shortening a telogen phase of a hair follicle in the area affected by hair loss. In some embodiments, the lengthening of the anagen phase and/or shortening of the telogen phase of the hair follicle enhances or induces hair growth in the subject at the area affected by hair loss.
In some embodiments, the at least one senescent cell is a melanocyte. In some embodiments, the melanocyte is derived from a nevus skin. In some embodiments, the nevus is a hairy nevus. In some embodiments, the SASP factor is an osteopontin polypeptide. In some embodiments, the osteopontin polypeptide recruits myeloid cells to the area affected by hair loss. In some embodiments, the myeloid cells secrete additional osteopontin polypeptides or other SASP factors to further enhance or induce hair growth in the subject at the area affected by hair loss. In some embodiments, the delivering comprises topical delivery of the at least one senescent cell or cell type. In some embodiments, the topical delivery is performed following application of a microneedle device. In some embodiments, the topical delivery is performed following application of a fractional laser treatment. In some embodiments, the delivering comprises topical delivery of the at least one SASP factor.
In some embodiments, the topical delivery is performed following application of a microneedle device. In some embodiments, the topical delivery is performed following application of a fractional laser treatment.
In some embodiments, a method is provided that comprises delivering at least one type of senescent cell or cell type into a hair loss affected area of the skin.
In some embodiments, a method is provided that comprises injecting at least one factor secreted by senescent cells and that is/are a senescent cell associated secretory phenotype (SASP) molecule into a hair loss affected area of the skin. In some embodiments, a method is provided that comprises exposing at least one type of normal cell to at least one oncogenic factor to induce their senescence.
In some embodiments, a method is provided that comprises exposing at least one normal cell to at least one type of senescence-inducing factor.
In some embodiments, a method is provided that comprises delivering a composition, wherein the composition is made up at least in part, substantially, or completely of factors derived from SASP.
In some embodiments, a method is provided that comprises delivering at least one factor derived from SASP and at least one senescent cell or cell type into a hair loss affected area.
In some embodiments, a method is provided comprising delivering at least one factor derived from SASP and at least one factor derived from an immune cell into a hair loss affected area.
In some embodiments, a method is provided comprising delivering a cocktail of factors produced by co-culturing at least one senescent cell with at least one immune cell or cell type.
In some embodiments, a composition is provided comprising at least one factor derived from SASP.
In some embodiments, the SASP factors include: Angptl4, Axl, Bmp4, Clqtnf2, Clqtnf5, Clqtnf7, Ccl17, Ccl4, Ccl5, Ccl6, Ccl9, Ctsb, Cxcl12, Cxcl9, Dhh, Dkk3, Fgf7, Frzb, Fstl1, Gdfl0, Igfbp2, Igfbp4, Igfbp7, Il10, Il1a, Il1f9, Inhba, Insl6, Mif, Mmp11, Mmpl2, Mmp14, Mmp2, Mmp23, Mmp3, Nrg2, Ogn, Omd, Pdgfa, Plat, Postn, Retnla, Sct, Sparc, Spp1, Timp1, Tnf, Tnfaip6, Wif1, and Wisp1. In some embodiments, the SASP factors specific to human nevus skin: ANGPTL7, BAMBI, CCL18, DKKL1, FGFBP2, FRZB, GDF1, GDF10, GDF11, GDF15, IL17D, MMP17, PDGFD, SPP1, TNFSF12, C1QTNF5, NRG3, PLAT, and TIMP2.
In some embodiments, a composition is provided comprising at least one factor derived from senescent cells cultured together with at least one type of immune cell or cell type.
In some embodiments, a composition is provided comprising some or all factors derived from SASP.
In some embodiments, a composition is provided comprising all factors listed in Table 1.1.
The compositions and related methods summarized above and set forth in further detail below describe certain actions taken by a practitioner; however, it should be understood that they can also include the instruction of those actions by another party. Thus, actions such as “transplanting at least one senescent cell type” includes “instructing the transplantation of at least one senescent cell type.”
Hair loss (aka alopecia) in humans results from two changes in the so-called hair growth cycle, the physiological cyclic process of hair synthesis by the hair follicle: (a) shortening of the growth phase (aka anagen), so that progressively shorter and shorter hairs are being produced; and (b) lengthening of the rest phase (aka telogen), so that hair follicles stop making new hairs all together for a prolonged period of time.
Pharmacological solutions to hair loss involve modulating signaling pathways that normally induce longer growth phase and shorter rest phase with either systemically, locally or topically delivered pharmaceuticals. To-date, the most prominent anti-hair loss effect was recorded for the agents that in one way or another reduce androgen signaling, and their effect is primarily directed toward lengthening the growth phase. The effect of reduced androgen signaling on telogen-to-anagen transition is not significant. For this reason, the anti-hair loss effect of Finasteride, for example, is very gradual and takes several years to fully show. Specifically, dormant hair follicles need to spontaneously enter a new anagen phase first for the anagen phase lengthening effect of Finasteride to be observable.
In mice, many other signaling pathways have been identified whose activation or suppression can promote transition of hair follicles from telogen to new anagen phase. However, for the most part, their effects on growth phase activation in humans have not been studied. Furthermore, some of the key signaling molecules that can stimulate new growth phase are also potent growth factors that have many other, often undesirable off-target side effects. For example, WNT signaling, which can active hair growth in mice, can also signal to promote growth of cancer cells. Therefore, use of WNT molecules for treating hair loss might result in higher risk of skin tumorigenesis.
To-date, no therapeutic solution exists in humans for (i) efficiently activating new hair growth phase, and (ii) simultaneously increasing duration of the growth phase, and thus length of hairs.
“Hairy nevus” is an under-studied and very poorly understood phenomenon. Nevus is a type of benign skin lesion that is pigmented and contains increased number of specialized melanocytes. Unlike normal skin, hairy nevus skin lesions contain many so-called senescent melanocytes that become senescent as the result of acquiring an oncogenic mutation. An example of human hair nevus with enhanced hair growth as compared to surrounding non-nevus skin is shown in
Normal body hairs, called vellus hairs, are typically very short, thin and often non-pigmented and, thus, barely visible. However, these hairs transform into prominent scalp-like hairs that are long, thick and pigmented (aka terminal hairs) once inside of the nevus boundaries. Clinically, vellus-to-terminal hair transformation is highly desirable and forms basis for treating hair loss, when achieving many terminal hairs is the ultimate goal.
Studies using several mouse models tested the hypothesis of whether specialized senescent melanocytes in the nevus skin can drive activation of hair growth. These studies showed that senescent melanocytes indeed prominently enhance hair growth. The studies also showed that senescent melanocytes achieve this effect via SASP factors that they secrete. Generally, SASP represents a set of secreted signaling molecules, enriched in members of inflammatory signaling pathways that are produced by all types of senescent cells, including senescent melanocytes.
SASP Profile in Senescent Melanocytes Derived from “Hairy Nevus”
The SASP profile of senescent melanocytes derived from hairy nevus skin was evaluated and established by RNA-sequencing on sorted cells. From this analysis, multiple candidate molecular players have been identified that appear to be responsible for promoting hair growth in the nevus, either as individual molecules, or in combination. Taken together, based on this data it was determined that senescent-cell derived SASP factors are the primary drivers of enhanced hair growth. This indicates that exposing dormant (telogen) hair follicles to either senescent cells or senescent cell-derived SASP or components of SASP, as in accordance with several embodiments disclosed herein, induces their activation and enhance hair growth.
Moreover, the data shows that senescent melanocyte-produced SASP also induces recruitment into the skin and activation of immune cells, specifically macrophages. RNA-sequencing studies on sorted nevus skin macrophages showed that they also secrete many of the same SASP factors and other additional inflammatory cytokines. Thus, in effect macrophages and their secreted molecules amplify and potentiate hair growth-inducing effect of senescent cell-derived SASP factors. Thus, SASP or components of SASP with macrophage-derived signaling factors may result in potentiation of the hair growth inducing effect.
In some embodiments described herein, SASP factors are collected and purified for skin injection from cultured senescent melanocytes or any other type of senescent cell (fibroblasts, keratinocytes, etc.). Advantageously, it has been determined that different senescent cells can be used because different varieties of senescent cells share large portions of their SASP molecular profiles. In some embodiments described herein, secreted factors are collected and purified from the co-culture of senescent cells with the immune cells, such as macrophages. Once collected, these “bioactive factor cocktails” can be delivered into skin via a number of ways, including but not limited to direct intra-dermal injection, topical delivery following application of a micro-needle device or fractional laser treatment.
In some embodiments described herein hair growth is stimulated by (i) senescent cells or (ii) senescent cell derived bioactive SASP cocktail of signaling molecules, or (iii) signaling molecule cocktails produced by a combination of senescent cells and macrophages.
In some embodiments, a method is provided that comprises transplanting at least one senescent cell type into a hair loss affected area. In some embodiments, a method is provided that comprises transplanting a population of senescent cells into a hair loss affected area.
In some embodiments, the population of senescent cells is a pure population of senescent cells. For example, the method comprises transplanting a population of senescent cells that are greater than 70% pure, greater than 80% pure, greater than 90% pure, or greater than 95% pure.
In some embodiments, a method is provided that comprises delivering at least one senescent cell and at least one factor derived from SASP to a hair loss affected area.
Any of the senescent cells described herein can be derived from any organism. In some embodiments, the senescent cells are human senescent cells. In some embodiments, the senescent cells are any one or more of senescent melanocytes, senescent fibroblasts, senescent keratinocytes, or senescent adipocytes. In some embodiments, the senescent cell is any cell type that is senescent or has entered a senescent phenotype. In some embodiments, a senescent phenotype includes a non-replicative phenotype.
In some embodiments, a method is provided that comprises transplanting at least one factor derived from SASP into a hair loss affected area. In some embodiments, a method is provided that comprises adding at least one factor produced by immune cells into a hair loss affected area. In some embodiments, a method is provided that comprises adding at least one factor from SASP and at least one factor from immune cells into a hair loss affected area.
In some embodiments, a method is provided that comprises delivering a composition into a hair loss affected area, wherein the composition is made up at least in part, substantially or completely of factors derived from SASP. In some embodiments, a method is provided that comprises delivering at least one factor derived from SASP and at least one factor derived from at least one immune cell into a hair loss affected area.
In some embodiments, a method is provided comprising delivering at least one factor derived from culturing senescent cells with at least one type of immune cell into a hair loss affected area. The senescent cells are any senescent cells in the skin. In some embodiments, the at least one senescent cell comprises any one or more of senescent melanocytes, senescent fibroblasts, senescent keratinocytes, or senescent adipocytes. The immune cells are any immune cells. In some embodiments, the immune comprises any one or more of neutrophils, eosinophils, basophils, lymphocytes, monocytes, and macrophages.
In some embodiments, a method is provided comprising delivering a cocktail of factors produced by co-culturing at least one senescent cell with at least one immune cell. The at least one senescent cell is any senescent in cell found in the skin. In some embodiments, the at least one senescent cell is any one or more of senescent melanocytes, senescent fibroblasts, senescent keratinocytes, or senescent adipocytes. The at least one immune cell is any immune cell type. In some embodiments, the at least one immune cell is any one or more of neutrophils, eosinophils, basophils, lymphocytes and monocytes. In some embodiments, the immune cell is a macrophage.
In some embodiments, a method is provided comprising delivering at least one senescent cell and at least one factor derived from senescent cells into a hair loss affected area. The at least one senescent cell is any senescent cell. In some embodiments, the senescent cell is any one or more of senescent melanocytes, senescent fibroblasts, senescent keratinocytes, and senescent adipocytes.
In some embodiments, in any of the methods described herein that comprise delivering one or more factors derived from SASP, including but not limited to any one or more of the factors listed in Table 1.
In some embodiments, one or more SASP factors are produced by one or more cells. In some embodiments, the one or more cells include at least one of a mammalian cell, a human cell, a mouse cell, a rat cell, a bacterial cell, a yeast cell, and/or any other type of cell capable of producing the one or more SASP factors. In some embodiments, one or more SASP factors are secreted from a cell. In some embodiments, the one or more SASP factors are secreted into a medium. In some embodiments, one or more SASP factors are isolated and/or purified after being secreted. For example, the one or more SASP factors may be isolated and/or purified from a supernatant after centrifuging cells and media associated with the cells. In some embodiments, one or more SASP factors are isolated and/or purified without being secreted from cells. In some embodiments, one or more SASP factors are produced recombinantly in a cell, such as through the use of standard molecular biology techniques. In some embodiments, one or more SASP factors are produced synthetically. In some embodiments, one or more SASP factors are purchased commercially.
In some embodiments, the SASP factors include mouse SASP factors such as Angptl4, Axl, Bmp4, Clqtnf2, Clqtnf5, Clqtnf7, Ccl17, Ccl4, Ccl5, Ccl6, Ccl9, Ctsb, Cxcl12, Cxcl9, Dhh, Dkk3, Fgf7, Frzb, Fstl1, Gdfl0, Igfbp2, Igfbp4, Igfbp7, Il10, Il1a, Il1f9, Inhba, Insl6, Mif, Mmp11, Mmp12, Mmp14, Mmp2, Mmp23, Mmp3, Nrg2, Ogn, Omd, Pdgfa, Plat, Postn, Retnla, Sct, Sparc, Spp1, Timp1, Tnf, Tnfaip6, Wif1, and/or Wisp1. In some embodiments, the SASP factors include human nevus skin SASP factors such as ANGPTL7, BAMBI, CCL18, DKKL1, FGFBP2, FRZB, GDF1, GDF10, GDF11, GDF15, IL17D, MMP17, PDGFD, SPP1, TNFSF12, C1QTNF5, NRG3, PLAT, and/or TIMP2. Some embodiments, include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, any number therebetween, or more, of the SASP factors described or identified herein.
In any of the methods described herein that comprise delivering at least one or more factors derived from immune cells, the factors are derived from any white blood cell type. In some embodiments, the factors derived from any white blood cell type are produced from hematopoietic stem cells. In some embodiments, the factors derived from white blood cells are derived from any one or more of neutrophils, eosinophils, basophils, lymphocytes and monocytes. In some embodiments, the immune cell from which the one or more factors derive is a macrophage.
In some embodiments, delivering comprises at least one intradermal injection. In some embodiments, delivering comprises multiple repetitive intradermal injections. In some embodiments, the delivering comprises topical delivery. In some embodiments, the topical delivery follows application of a microneedle device or a fractional laser treatment.
Any of the compositions disclosed herein can be delivered into a hair loss affected area through any of the methods disclosed herein.
In some embodiments, a method of producing senescent cells is provided that comprises exposing one or more normal cells to one or more oncogenic factors. In some embodiments, a method of producing senescent cells is provided that comprises exposing one or more normal cells to one or more senescent inducing factors. In some embodiments, a method of producing senescent cells is provided that involves repetitive passaging of cells to achieve replicative senescence.
In some embodiments, a composition is provided that comprises at least one factor derived from SASP. In some embodiments, a composition is provided that comprises at least one factor derived from SASP and at least one factor derived from one immune cell type. In some embodiments, at least one factor derived from SASP is any one or more of the factors listed in Table 1.
In compositions that comprise factors derived from immune cells, the factors are derived from any one or more white blood cells or any combination of white blood cells. In some embodiments, the factors derived from any one or more of white blood cells that are produced from hematopoietic stem cells. In some embodiments, the factors from white blood cells are derived from any one or more of neutrophils, eosinophils, basophils, lymphocytes and monocytes. In some embodiments, the immune cells from which the one or more factors derive, are macrophages.
In some embodiments, a composition is provided that comprises at least one factor derived from senescent cells that are cultured with at least one type of immune cell. The senescent cells can comprise any senescent cell found in the skin. In some embodiments, the senescent cells comprise any one or more of the following: senescent melanocytes, senescent fibroblasts, senescent keratinocytes, and senescent adipocytes. In some embodiments, the at least one factor derived from senescent cells include but are not limited any one or more of the factors listed in Table 1.1.
In some embodiments, a composition is provided that comprises all factors derived from SASP. In some embodiments, the composition is provided comprising each of the factors listed in Table 1.1.
In some embodiments, a composition is provided that includes one or more SASP factors. In some embodiments, the composition includes a medium or supernatant containing one or more SASP factors. In some embodiments, the SASP factors that are included in the medium or supernatant, are secreted by a cell into the medium or supernatant.
Some aspects of the embodiments discussed above are disclosed in further detail in the following examples, which are not in any way intended to limit the scope of the present disclosure
Several transgenic mice were used as models for the oncogene-induced senescence in melanocyte cell lineage to verify that they display overactive hair growth, replicating human hairy nevus lesions. One such mouse model is Tyr-NrasQ61K. Similar to human hairy nevus, skin of these mice showed large number of ectopic senescent melanocytes (
The so-called pulse-chase technique showed that hair follicle stem cells (aka bulge stem cells) are less quiescent and more active in the skin of Tyr-NrasQ61K mice as compared to control mice (
Injection of fluorescently labeled senescent melanocytes in the skin of mice resulted in ectopic activation of hair growth (
In certain embodiments as shown by FACS analysis in
The present example shows that hair follicle stem cells can exist in quiescence, and change their transcriptome and composition, and that hair regeneration dramatically enhances in the presence of senescent melanocytes. It was shown here that the latter activate a senescence-associated secretory phenotype (SASP), containing pro-inflammatory factors. Osteopontin is a new SASP factor involved in hair regeneration. Osteopontin injection was shown to be sufficient to induce new hair growth, and to recruit myeloid cells which amplify osteopontin levels and enhance SASP effect on hair regeneration. Deletion of osteopontin, its receptor, Cd44, or depletion of myeloid cells all markedly reversed enhanced hair regeneration by senescent melanocytes. While conventionally senescent cells are viewed as being detrimental for tissue regeneration potential, it is here shown that they can enhance regeneration by enriching stem cell niche for SASP signaling and immune cell modulation.
It was shown here that senescent cells can induce dramatic loss of quiescence by tissue stem cells (SCs) and this is enabled by their unique secretome, the SASP. It is here shown that senescent melanocytes in pigmented nevus skin (aka mole) signal via SASP to hyper-activate hair SCs, leading to prominently enhanced hair regeneration. SASP recruits myeloid cells which, in turn, amplify and enrich it for novel pro-regenerative signaling factors. Osteopontin is herein identified as a novel SASP factor, responsible for enhanced hair regeneration. Activation of tissue progenitors by aged tissue cells provides a novel paradigm in SC biology.
Cyclic hair regeneration is tightly controlled at the level of stem cell quiescence (Yi, 2017), and naturally occurring conditions of excessive hair growth are rare. A hairy pigmented nevus is a type of benign skin lesion in humans with prominently enhanced hair growth (
Several inflammatory cytokines and growth factors are part of SASP, and their signaling roles are being rapidly recognized in modulating biological processes, including normal embryonic development (Storer et al., 2013), cellular plasticity and reprogramming (Mosteiro et al., 2016; Ritschka et al., 2017), injury repair (Chiche et al., 2017; Demaria et al., 2014), and cancer progression (Capell et al., 2016; Herranz et al., 2015; Laberge et al., 2015; Ruhland et al., 2016; Yoshimoto et al., 2013).
It was considered how senescent melanocytes affect HF cell populations with active roles in hair regeneration: bulge SCs, hair germ (HG) progenitors and DP fibroblasts. Their transcriptomes were profiled by RNA-sequencing (RNA-seq) following cell sorting. Bulge and HG cells were isolated from Tyr-NrasQ61K;K14-H2B-GFP mutant and K14-H2B-GFP control mice as GFP+/CD34+/Pcadlo and GFP+/CD34neg/Pcadhi populations, respectively (Greco et al., 2009). Subset of DP fibroblasts was isolated from Tyr-NrasQ61K;Sox2-GFP mutant and Sox2-GFP control mice as GFP+;CD133+ population (Driskell et al., 2009). Bulge and DP cells were profiled at P30 and P56, when dorsal HFs in control, wildtype (WT) mice are in second anagen and telogen, respectively. HG progenitors were profiled at P56, since they exist only during telogen phase.
RNA-seq analysis on bulge SCs revealed prominent differences between Tyr-NrasQ61K and WT mice at both time points (
To address these possibilities, bulge SCs were compared between P56 Tyr-NrasQ61K and WT mice on single-cell RNA-seq. Analysis shows that WT bulge SCs consist of two distinct types (top-right and bottom-right clusters in
Next, loss of quiescence was confirmed in functional assays. A pulse-chase experiment was performed on bulge SCs. Mice were pulsed with EdU between days P27-P34, when WT HFs are in early anagen and their SCs are proliferative (Hsu et al., 2011), and then chased till day P92. On cytometric analysis, the total number of bulge SCs in Tyr-NrasQ61K mice did not significantly differ from WT, however, there was prominent loss of EdU-retaining SCs (n=4) (
It was then asked what signaling changes accompany loss of quiescence by bulge SCs in Tyr-NrasQ61K mice. To address this, bulk RNA-seq data was analyzed, which has higher depth of coverage compared to single-cell data. First, it was confirmed that quiescence markers from single-cell analysis, Nfatc1 and Hopx, were also downregulated in P56 NrasQ61K vs. WT SCs on bulk analysis. Other quiescence markers were also downregulated in NrasQ61K SCs: Axin2 (Lim et al., 2016), Col17a1 (Matsumura et al., 2016), Fgf18 (Kimura-Ueki et al., 2012) and Foxc1 (Lay et al., 2016; Wang et al., 2016) (
Compared to WT control, P56 NrasQ61K HG progenitors upregulate several transcriptional factors, including Grhl3, Meis2, Ovol1, Sox6/15, canonical (Wnt3/3a/7b/10a) and non-canonical WNT ligands (Wnt5a/5b/11/16), multiple cytokines Ccl1/2/7/20/27a/27b, interleukins Il1a/1b/1f5/1f9/6/24/34 and chemokines Cxcl9/16, as well as Fst and Ngf. They downregulate transcriptional factors Id1/2/4, Lhx2, Sox5/13, Tbx1, multiple WNT pathway components: Dkk3, Fzd2/3/7/8, Lgr4/5, Lef1, Lrp6, Tcf7l1/12, Wnt7a/10b, Wif1, and Hedgehog pathway members Gli1/2, Ptch1/2 (
A melanocyte lineage was isolated as tdTomato+ cells from Tyr-NrasQ61K;Tyr-CreERT2;tdTomato mutant and Tyr-CreERT2;tdTomato control mouse skin. Transcriptome of P56 mutant samples was then compared to both P30 anagen and P56 telogen WT samples (
Considering a rapidly emerging role for immune cell signaling in hair cycle activation (Ali et al., 2017; Amberg et al., 2016; Castellana et al., 2014; Chen et al., 2015; Gay et al., 2013; Lee et al., 2017; Wang et al., 2017b), experiments were conducted to see if and how immune cell secretome is altered in the nevus skin. Transcriptomes were profiled of all skin-resident CD45-expresing hematopoietic cells (
Taken together, the combined secretome of senescent melanocytes and myeloid cells distinctly enriches signaling environment of nevus skin for multiple inflammatory pathway ligands (
A transcript for osteopontin was one of the most prominently upregulated signaling factors in multiple nevus skin cell types (
Next, it was considered whether osteopontin plays functional role in hair growth phenotype of Tyr-NrasQ61K mice and if it is sufficient to induce new hair cycle in WT mice. Tyr-NrasQ61K;Spp1−/− mice were generated to test if osteopontin deletion rescues hair cycle quiescence in nevus skin. Indeed, compared to Tyr-NrasQ61K mice, whose HFs start cycling ectopically already at P23 (
Considering that osteopontin becomes prominently upregulated in immune cells and fibroblasts at the edge of healing skin wounds (Liaw et al., 1998; Mori et al., 2008), it was asked if it might mediate wound-induced hair cycle activation phenomenon, when HFs in WT mice enter ectopic anagen at the wound margin. A previous study implicated Tnf as one of the signaling mediators of this phenomenon (Wang et al., 2017b). It is shown here that compared to WT mice, Spp1−/− mice show significantly fewer ectopic anagen HFs at the margin of 7 mm full-thickness wounds 11 days post-wounding (
Myeloid cells are a source and target for osteopontin signaling in the context of various inflammatory conditions, including in skin (Buback et al., 2009; Giachelli et al., 1998; Liaw et al., 1998; Mori et al., 2008). Considering this and prominent increase in Spp1 levels in Tyr-NrasQ61K myeloid cells on bulk (
Next, it was examined whether Cd44, a receptor for osteopontin (Weber et al., 1996), mediates its effect in nevus skin. On RNA-seq Cd44 was highly expressed in multiple skin cell types, including bulge and HG progenitors, both in Tyr-NrasQ61K and control mice (
Hyper-activated hair cycling in nevus skin resembles the phenotype of K14-Wnt7a mice that overexpress canonical WNT ligand (Plikus et al., 2011). WNT signaling plays a role in physiological hair cycle activation (Choi et al., 2013; Greco et al., 2009; Kandyba et al., 2013; Lien et al., 2014; Lowry et al., 2005) and it is elevated and drives early stages of melanocytic nevus formation (Pawlikowski et al., 2013). Foci of WNT reporter-active cells were consistently found in the dermis of Tyr-NrasQ61K;TOPGAL mice (
Signaling aspects of hairy pigmented nevi in humans were also examined. Whole-tissue RNA-seq revealed prominent transcriptome differences between hairy nevi and adjacent normal facial skin, as well as patient-to-patient variability (
Genome-wide cross comparison of purified melanocytes, bulge, DP, sHG, and myeloid cells via FACS sorting, was used to identify osteopontin as a new SASP molecule for hair stem cell activation in the senescent melanocytic nevi skin. Osteopontin is expressed in the mouse uterus (Qi et al., 2014), but its function seems redundant because osteopontin knockout (Spp1−/−) mice are viable and normal grossly (Liaw et al., 1998). In the adult, the level of osteopontin expression is upregulated following injury or under other pathological circumstances, such as cell transformation (Mori et al., 2008; Zhou et al., 2005). The expression pattern of osteopontin reflects its multifunctional feature in response to diverse stimuli (Cooper et al., 2005; Liu et al., 2004). In the normal skin, it is absent in the bulge and sHG, suggesting a non-permissive role of osteopontin in the SC compartments. Although it is expressed in melanocytes, DP, and dermal myeloid cells in a hair cycle-dependent manner, osteopontin is dispensable for normal HF SC regeneration, as normal cyclic hair growth was observed in Spp1−/− mice. However, it was involved in precocious hair growth in the nevus skin, evidenced by loss of ability to regenerate HF SC when osteopontin is deleted in Spp1−/− mice. This finding highlights its general role as a stress sensor that was demonstrated previously in connection with altered wound healing in Spp1−/− mice (Liaw et al., 1998).
Skin operates as a complex organ consisting of different structures with multiple cell types. Their interaction with each contributing to specific function is involved in SC regeneration. Osteopontin was upregulated in melanocytes, bulge, DP, and myeloid population in the nevus skin. Normal HF SC regeneration is controlled by both its immediate niche cell DP (Rendl et al., 2008) and other cell types in the skin such as adipocytes (Festa et al., 2011; Plikus et al., 2008). On one hand, upregulation of osteopontin in HF niche cell DP (9.8-fold) and bulge SC (73.9-fold) suggests that the mode of osteopontin action on HF SC regeneration can be direct through modulation of niche and SC themselves. On the other hand, the high osteopontin expressing senescent melanocytes (11.8-fold) can recruit myeloid cells and increase osteopontin expression on those cells (30.2-fold), suggesting a positive feedback loop to sensitize other cell types, and to produce osteopontin in the nevus skin. However, this premature anagen hair phenotype was lost when myeloid cells are depleted, suggesting an important role of myeloid cells in promoting hair growth in the nevus skin. Although SCs are regulated by their niches which are usually nearby and are able to produce rapid-response paracrine factors, extra-niches in the skin which contain of various cell types with distinct functions also participate normal SC regeneration. In this regard, our finding supports the notion of extra-niche cell interaction with SC to regulate hair cycle in the presence of senescent cells.
While the origin of osteopontin is complex (it could be derived from senescent melanocytes or myeloid cells), it appears to have an active role in regeneration of HF SC in senescent skin. A feature of SASP is to attract immune cells. Senescent cells in tumors can recruit immune cells through the SASP and allow tumor clearance (Xue et al., 2007), whereas prolonged SASP can enhance tumor proliferation, migration, and invasion (Bavik et al., 2006), demonstrating distinct functions of the immune cells in the senescent environment. SASP factors are mostly characterized in culture and found in senescent cells (Capell et al., 2016; Coppe et al., 2008; Pawlikowski et al., 2013), they have an altered expression profile enriched in growth factors, chemokines, and ECM remodeling enzymes. The list of nevus-derived factors is extensive. An array of SASP factors (cytokines and chemokines) such as CCL17, CXCL9, CXCL3, and IL1b were found in the myeloid cells but not in the senescent melanocytes, whereas matrix metalloproteinases (MMP3, 12, and 14 in myeloid and MMP11 and 23 in senescent melanocytes, respectively) were altered in both cell types. The expression of osteopontin has an effect on the behavior of neighboring cells in transducing paracrine/autocrine signaling.
Experimental Procedures used in Example 5
All experiments were performed in accordance with University of California Irvine's Animal Care and Use Committee guidelines. B6.129S6(Cg)-Spp1tm1Blh/J (Liaw et al., 1998), B6.129(Cg)-Cd44tm1Hbg/J (Protin et al., 1999), B6(Cg)-Tyrc-2J/J (Townsend et al., 1981), B6.CB17-Prkdcscid/SzJ (Blunt et al., 1995), B6;129S-Sox2tm2Hoch/J (Arnold et al., 2011), Tyr-NrasQ61K (Ackermann et al., 2005) were purchased from The Jackson Laboratory. Tetracycline controlled triple mutant mice of myeloid lineage specific depletion were created by crossing LysM-Cre, Rosa-reTA and TetODTA (Chen et al., 2015).
One-month-old mice were injected with EdU (5 μg/g body weight) via i.p. daily for seven consecutive days, followed by a chase period of 8 weeks. Mouse dorsal skin was harvested; half was fixed in 4% PFA, embedded in paraffin and examined by immunohistochemistry (IHC) using EdU imaging kit (Molecular Probe). The other half was used for flow cytometry quantification using EdU flow kit (Molecular Probe). Both IHC showing Edu positive cells among total numbers of follicles and FACS analyzing triple positive CD34+CD49f+ Edu+ cells were used to quantify EdU positive cells. At least two sections per animal and three to five animals per group were used for analysis.
Intradermal delivery of protein-soaked agarose beads was performed according to Plikus 2008. Briefly, recombinant mouse SPP1 protein (R&D) was reconstituted in 0.1% BSA at a final concentration of 1.3 mg/ml. Affi-gel blue gel beads (Bio-Rad) were washed three times in sterile PBS and then resuspended with recombinant protein (vol/vol) in 0.1% BSA on ice for 1 hr before injection. For both recombinant SPP1 protein and BSA control, four consecutive daily injections, including 24, 48, and 72 hrs after first bead implantation, were performed to the same skin region using a 26G needle to create a pouch, then delivered by a glass micropipette, introducing about 100 beads/20 μl bead solution using a microinjector under the back skin from p51 to p53.
For paraffin-embedded sections, back skins were fixed with 4% (vol/vol) paraformaldehyde (PFA) overnight at 4° C., followed by dehydration with 20%, 50%, and 70% of ethanol. Sections were permeabilized for 15 min in PBS+0.1% Triton X-100 (PBST) and blocked for at least 1 hr at room temperature using PBST+3% BSA. Mouse Abs were blocked with M.O.M. block kit according to manufacturer's instructions. Primary antibodies (Abs) were incubated overnight at 4° C. and secondary Abs were incubated 1 hr at RT. Frozen sections were cryopreserved in Optimal Cutting Temperature compound (OCT). The following antibodies and dilutions were used. Spp1 (1:20, goat, R&D), CD45 (1:100, rabbit, BD Biosciences), F4/80 (1:100, rabbit, BD Biosciences). Nuclei were stained with 4060-diamidino-2-phenylindole (DAPI). For j-gal staining, think sections (20 μm) were incubated in 1 mg/ml X-gal substrate in PBS with 1.3 mM MgCl2, 3 mM K3Fe(CN)6, and 3 mM K4Fe(CN)6 at 37° C. overnight. Hematoxylin and Eosin staining was performed using standard methods. Percent positive area was calculated using ImageJ. All images were captured with a Nikon dissecting or Nikon Ti-E Upright microscope.
Single cells from mouse whole back skin were lysed in RIPA buffer containing a cocktail of protease inhibitors (Roche). 25 μg of each cell lysates (n=3 samples per group) were loaded onto a 12% separating Bis-Tris gel. The proteins were transferred to a nitrocellulose membrane. The membrane was incubated with the Spp1 primary antibody or anti-GAPDH at a concentration of 2.5 μg ml-1. The blot was developed with Enhanced Chemiluminescence Plus Developer.
Total RNA from FACS sorted cells was extracted using RNeasy Mini Kit (QIAGEN) coupled with its on-column DNase digestion protocol. This total RNA was then reverse-transcribed by Superscript III (Life Technologies) in the presence of Oligo-dT. The Full length cDNA was normalized to equal amount using house keep genes GAPDH or 18s.
For single cell suspension, the back skin was incubated in Dispase II solution (Roche) to separate epidermis from dermis. Dermis and/or epidermis was digested into single cells with Collagenase I (Life Technologies) at 37° C. These skin single cells were filtered with strainers (70 μM, followed by 40 μM). Viability dye was used to exclude dead cells. Gated live cells were sorted on FACSAria II sorters (BD Biosciences). FACS acquisition was performed on LSRII flow cytometer (BD Biosciences) and then analyzed with FlowJo software (FlowJo).
Total RNAs from FACS sorted cells including three biological triplicates with RNA integrity number (RIN) >9.1 determined by Agilent 2100 Bioanalyzer Pico chip were selected for cDNA synthesis and amplification. 1 ng of mRNA was used for full length cDNA synthesis, followed by PCR amplification according to Smart-seq2 standard protocol. cDNA libraries were constructed using the Nextera DNA Sample Preparation Kit (Illumina). The libraries were sequenced on the Illumina Next-Seq500 system to an average depth of 10-30 million reads per library using paired 43 bp reads.
For single cell RNA-seq (scRNA-seq) sample preparations on the C1 platform, sorted cells from mouse back skins were captured using the Fluidigm C1 chips according to Fluidigm C1 protocol. A concentration of 200,000-350,000 cells per ml was used for chip loading. After cell capture, chips were examined visually under the microscope to identify the capture rate and empty chambers or chambers with multiple cells were excluded from later analysis. cDNAs were synthesized and amplified on Fluidigm C1 Single-Cell Auto Prep System with Clontech SMARTer Ultra Low RNA kit and ADVANTAGE-2 PCR kit (Clontech). scRNA-seq libraries were constructed in 96-well plates using the Illumina Nextera XT DNA Sample Preparation kit according to Fluidigm C1 manual. Multiplexed libraries were analyzed on Agilent 2100 Bioanalyzer for fragment distribution and quantified using Kapa Biosystem's universal library quantification kit. Libraries were sequenced as 75 bp paired-end reads on the Illumina Next-Seq500 platform. RNA-seq reads were first aligned using STAR v.2.4.2a (Dobin et al., 2013) with parameters ‘--outFilterMismatchNmax 10 --outFilterMismatchNoverReadLmax 0.07 -- outFilterMultimapNmax 10’ to the reference mouse genome (mm10/genocode,vM8) Gene expression level was quantified using RSEM v.1.2.25 (Li and Dewey, 2011) with expression values normalized into Fragments Per Kilobase of transcript per Million mapped reads (FPKM). Samples displaying >9,000,000 uniquely mapped reads and >60% uniquely mapping efficiency were considered for downstream analyses. Differential expression analysis was performed using edgeR v.3.2.2 (Robinson et al., 2010) on protein-coding genes and lncRNAs. Differentially expressed genes were selected by using fold change (FC)>2, false discovery rate (FDR)<0.05 and counts per million reads (CPM)>2.
It is contemplated that various combinations or sub-combinations of the specific features and aspects of the embodiments disclosed above may be made and still fall within one or more of the inventions. Further, the disclosure herein of any particular feature, aspect, method, property, characteristic, quality, attribute, element, or the like in connection with an embodiment can be used in all other embodiments set forth herein. Accordingly, it should be understood that various features and aspects of the disclosed embodiments can be combined with or substituted for one another in order to form varying modes of the disclosed inventions. Thus, it is intended that the scope of the present inventions herein disclosed should not be limited by the particular disclosed embodiments described above. Moreover, while the invention is susceptible to various modifications, and alternative forms, specific examples thereof have been shown in the drawings and are herein described in detail. It should be understood, however, that the invention is not to be limited to the particular forms or methods disclosed, but to the contrary, the invention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the various embodiments described and the appended claims. Any methods disclosed herein need not be performed in the order recited. The methods disclosed herein include certain actions taken by a practitioner; however, they can also include any third-party instruction of those actions, either expressly or by implication. For example, actions such as “transplanting at least one senescent cell type” includes “instructing the transplanting of at least one senescent cell type” In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.
The ranges disclosed herein also encompass any and all overlap, sub-ranges, and combinations thereof. Language such as “up to,” “at least,” “greater than,” “less than,” “between,” and the like includes the number recited. Numbers preceded by a term such as “about” or “approximately” include the recited numbers. For example, “at least one factor . . . ” includes “one factor.”
This application is a Continuation application of U.S. patent application Ser. No. 16/496,383, filed Sep. 20, 2019, which is the U.S. National Phase application under 35 U.S.C. § 371 of International Application No. PCT/US2018/023624, filed Mar. 21, 2018, designating the U.S. and published in English as WO 2018/175630 A1 on Sep. 27, 2018, which claims the benefit of U.S. Provisional Application No. 62/475,688, filed Mar. 23, 2017. The contents of the aforementioned applications are expressly incorporated herein by reference in their entirety.
Number | Date | Country | |
---|---|---|---|
62475688 | Mar 2017 | US |
Number | Date | Country | |
---|---|---|---|
Parent | 16496383 | Sep 2019 | US |
Child | 18309721 | US |