Final Report Summary - CLASSIC (deviCes for Light impact on Skin Stem Cells)
CLaSSiC is an interdisciplinary, intersectoral research project, leveraging insights on molecular, cellular and biophysics impact of light on skin stem cells towards novel devices for regenerative therapy. The aging demographic creates a need for effective therapeutic solutions addressing age-related and age-independent skin disorders, including failure of wound healing, hair loss or unwanted hair growth. All have negative psychological impact affecting millions and present significant personal and clinical cost burdens to society. Light therapy, or photobiomodulation, have been showing encouraging clinical efficacy. Photobiomodulation is a type of light-tissue interaction that relies on photobiological and photochemical effects, triggered by relatively low irradiance (in W/cm2) and products employing low level light treatment have been reported to lead to encouraging clinical efficacy.
Translation to commercial devices had already started, however, the field has developed rather empirically, lacking basic mechanistic research.
CLaSSiC aims were to unlock the potential for new light-based devices for management of skin and hair disorders, while working synergistically with complimentary collaborators in a two-partner network comprising PHILIPS (Netherlands) and the Centre for Skin Sciences (University of Bradford, UK). A board of 8 very experienced supervisors working across disciplines and sectors trained and mentored 3 early stage researchers who had the opportunity to work at superb research facilities at both locations.
Specifically, CLaSSiC’s objectives were to define molecular mechanisms controlling the activity of skin and hair follicle ‘stem’ cells mediated by visible light applicable for the development of novel devices and to translate the obtained knowledge into the therapeutically and commercially promising areas of focus (wound healing, hair regeneration and/or removal). An invited Viewpoint article was published in Experimental Dermatology describing the ‘state of the art’ in relation to experiments exposing cells to light. This Viewpoint article highlighted inconsistencies and knowledge gaps in the published research on photobiomodulation and generated high interest in the field and demonstrated the lack of consistency on approach. Once mapped out, this facilitated rational design of experiments in the project as well as the most appropriate design of the device built to expose cells to light probing a wide range of parameters in a systematic way.
The integrated CLaSSiC PhD projects revealed for the first time that a variety of skin and hair follicle cells; including stem cells, express several light sensitive receptors, that intrinsically could be sensitive to light as contain binding sites for light absorbing chromophore, covering UV-blue-green spectral. These included Cryptochromes 1 and 2 (CRY1, 2) and Opsins (OPN1-SW, OPN2, OPN3 and OPN5). Of great interest is that in contrast to the dogma about the benefits of red wavelengths of light these light receptors absorb in the blue to green-region of the visible spectrum. Next to discovery of putative receptors of light their relevance to function of skin and hair follicle cells was investigated. It was determined that CRY1 exerts positive effects on hair growth and as such this receptor has functionality in human hair biology. The stabilization of CRY1 protein with a pharmacological component (KL001) helped maintain healthy hair growth in an ex vivo human hair follicle model while ‘silencing’ of CRY1 induced premature catagen development, meaning that without CRY1 hair growth is not maintained. Research to find out why CRY1 was important for hair growth by ‘silencing’ of CRY1 gene expression in the outer root sheath (stem cell progenies) cells in vitro caused downregulation of genes involved in the control of proliferation, including the cyclin dependent kinase 6 (CDK6). This showed that CRY1 was linked to the cell-cycle. In epidermal keratinocytes, silencing CRY1 promoted cell differentiation, again suggesting that CRY1 was needed to maintain cell proliferation in epidermis and hair follicle. The project also investigated how CRY1 was regulated and discovered that the opsin, OPN3 was involved. OPN3 has a positive effect on hair follicle cells, including their metabolic activity and proliferation. OPN3 silencing in the outer root sheath cells (stem cell progenies) resulted in the altered expression of genes involved in the control of proliferation and apoptosis, processes crucial to normal hair growth and the hair cycle as well as wound healing.
Having found expression of opsins and cryptochromes in skin and hair follicles, the project team next investigated whether they respond to visible light in ways that would be likely to impact skin and hair function. Because the investigated CRY1, OPN2 and 3 receptors greatly absorb in the blue to green-region of the visible spectrum, the effect of blue light (447-453 nm, 3.2 J/cm2, 16 nm full width half maximum) on the behaviour of epidermal keratinocytes, distinct populations of fibroblasts and hair follicle in vitro/ex vivo was evaluated. In hair follicles, daily treatment with blue light significantly prolonged anagen phase ex vivo that was associated with sustained proliferation in the hair follicle cells. In addition, blue light stimulated proliferation of the outer root sheath cells in vitro, which was abrogated by silencing of OPN3. Low levels of blue light stimulated early differentiation of epidermal keratinocytes, which was shown to be mediated by OPN3 and circadian clock mechanisms. However, low levels of blue light inhibited keratinocyte proliferation and this was shown to be mediated by the circadian clock independently of OPN3. The low levels of blue light were able to help heal wounds and this seemed to be due to up-regulation, by blue (but not red) light, of both CRY1 and OPN3.
Fibroblast cells were also investigated for their light responses. Additionally, critical culture and treatment conditions that have dramatic impact on the outcome of specific light treatment of these human skin dermal cells were identified. In particular, the impact of environmental oxygen concentration, cell confluency and serum all influenced how light impacted fibroblast cell populations. Oxidative stress is implicated in skin ageing and the induction of reactive oxygen species (ROS) by short visible wavelengths was observed with ROS species created in or close to mitochondria, which is significant given the central role of mitochondria in cellular metabolism. ROS at low levels is important to cell function; too much ROS is, however, harmful and this was shown when higher doses of blue light increased extracellular matrix turnover; something linked to ageing.
The training elements of CLaSSiC combined scientific training in skin biology, biophysics of light interaction with skin stem cells, and device design and transferable life-skills training opportunities afforded through the combined academic and industry working environments. All three early stage researchers participated in boot camps where they combined business activities with project reviews and had the opportunity to meet leaders in the field as well as Philips business personnel. All ESRs actively participated in conferences key international conference relevant to the field of biomedical optics, dermatology, skin and hair biology giving talks, e.poster presentations and writing full publications. All three ESRs have completed a doctoral degree.
Translation to commercial devices had already started, however, the field has developed rather empirically, lacking basic mechanistic research.
CLaSSiC aims were to unlock the potential for new light-based devices for management of skin and hair disorders, while working synergistically with complimentary collaborators in a two-partner network comprising PHILIPS (Netherlands) and the Centre for Skin Sciences (University of Bradford, UK). A board of 8 very experienced supervisors working across disciplines and sectors trained and mentored 3 early stage researchers who had the opportunity to work at superb research facilities at both locations.
Specifically, CLaSSiC’s objectives were to define molecular mechanisms controlling the activity of skin and hair follicle ‘stem’ cells mediated by visible light applicable for the development of novel devices and to translate the obtained knowledge into the therapeutically and commercially promising areas of focus (wound healing, hair regeneration and/or removal). An invited Viewpoint article was published in Experimental Dermatology describing the ‘state of the art’ in relation to experiments exposing cells to light. This Viewpoint article highlighted inconsistencies and knowledge gaps in the published research on photobiomodulation and generated high interest in the field and demonstrated the lack of consistency on approach. Once mapped out, this facilitated rational design of experiments in the project as well as the most appropriate design of the device built to expose cells to light probing a wide range of parameters in a systematic way.
The integrated CLaSSiC PhD projects revealed for the first time that a variety of skin and hair follicle cells; including stem cells, express several light sensitive receptors, that intrinsically could be sensitive to light as contain binding sites for light absorbing chromophore, covering UV-blue-green spectral. These included Cryptochromes 1 and 2 (CRY1, 2) and Opsins (OPN1-SW, OPN2, OPN3 and OPN5). Of great interest is that in contrast to the dogma about the benefits of red wavelengths of light these light receptors absorb in the blue to green-region of the visible spectrum. Next to discovery of putative receptors of light their relevance to function of skin and hair follicle cells was investigated. It was determined that CRY1 exerts positive effects on hair growth and as such this receptor has functionality in human hair biology. The stabilization of CRY1 protein with a pharmacological component (KL001) helped maintain healthy hair growth in an ex vivo human hair follicle model while ‘silencing’ of CRY1 induced premature catagen development, meaning that without CRY1 hair growth is not maintained. Research to find out why CRY1 was important for hair growth by ‘silencing’ of CRY1 gene expression in the outer root sheath (stem cell progenies) cells in vitro caused downregulation of genes involved in the control of proliferation, including the cyclin dependent kinase 6 (CDK6). This showed that CRY1 was linked to the cell-cycle. In epidermal keratinocytes, silencing CRY1 promoted cell differentiation, again suggesting that CRY1 was needed to maintain cell proliferation in epidermis and hair follicle. The project also investigated how CRY1 was regulated and discovered that the opsin, OPN3 was involved. OPN3 has a positive effect on hair follicle cells, including their metabolic activity and proliferation. OPN3 silencing in the outer root sheath cells (stem cell progenies) resulted in the altered expression of genes involved in the control of proliferation and apoptosis, processes crucial to normal hair growth and the hair cycle as well as wound healing.
Having found expression of opsins and cryptochromes in skin and hair follicles, the project team next investigated whether they respond to visible light in ways that would be likely to impact skin and hair function. Because the investigated CRY1, OPN2 and 3 receptors greatly absorb in the blue to green-region of the visible spectrum, the effect of blue light (447-453 nm, 3.2 J/cm2, 16 nm full width half maximum) on the behaviour of epidermal keratinocytes, distinct populations of fibroblasts and hair follicle in vitro/ex vivo was evaluated. In hair follicles, daily treatment with blue light significantly prolonged anagen phase ex vivo that was associated with sustained proliferation in the hair follicle cells. In addition, blue light stimulated proliferation of the outer root sheath cells in vitro, which was abrogated by silencing of OPN3. Low levels of blue light stimulated early differentiation of epidermal keratinocytes, which was shown to be mediated by OPN3 and circadian clock mechanisms. However, low levels of blue light inhibited keratinocyte proliferation and this was shown to be mediated by the circadian clock independently of OPN3. The low levels of blue light were able to help heal wounds and this seemed to be due to up-regulation, by blue (but not red) light, of both CRY1 and OPN3.
Fibroblast cells were also investigated for their light responses. Additionally, critical culture and treatment conditions that have dramatic impact on the outcome of specific light treatment of these human skin dermal cells were identified. In particular, the impact of environmental oxygen concentration, cell confluency and serum all influenced how light impacted fibroblast cell populations. Oxidative stress is implicated in skin ageing and the induction of reactive oxygen species (ROS) by short visible wavelengths was observed with ROS species created in or close to mitochondria, which is significant given the central role of mitochondria in cellular metabolism. ROS at low levels is important to cell function; too much ROS is, however, harmful and this was shown when higher doses of blue light increased extracellular matrix turnover; something linked to ageing.
The training elements of CLaSSiC combined scientific training in skin biology, biophysics of light interaction with skin stem cells, and device design and transferable life-skills training opportunities afforded through the combined academic and industry working environments. All three early stage researchers participated in boot camps where they combined business activities with project reviews and had the opportunity to meet leaders in the field as well as Philips business personnel. All ESRs actively participated in conferences key international conference relevant to the field of biomedical optics, dermatology, skin and hair biology giving talks, e.poster presentations and writing full publications. All three ESRs have completed a doctoral degree.