Name: stimulation of stem cell niches and tissue regeneration in mouse skin by switchable protoporphyrin ix-dependent photogeneration of reactive oxygen species in situ
Uploaded: 2020-05-08T12:30:00.000+00:00
Duration: 10 min 5 s
Description: Watch this Scientific Journal Video about Stimulation of Stem Cell Niches and Tissue Regeneration in Mouse Skin by Switchable Protoporphyrin IX-Dependent Photogeneration of Reactive Oxygen Species In

This work has been supported by grants from Ministerio de Econom&#237;a y Competitividad (RTC-2014-2626-1 to JE) and Instituto de Salud Carlos III (PI15/01458 to JE) of Spain. EC has been supported by the Atracci&#243;n de Talento Investigador grant 2017-T2/BMD-5766 (Comunidad de Madrid and UAM).

All mouse husbandry and experimental procedures must be conducted in compliance with local, national, international legislation and guidelines on animal experimentation.
1. Induction of hair growth, burn induction and identification of long-term BrdU LRCs in the tail skin epithelium wholemounts
NOTE: Use 10-day or 7-week old C57BL/6 mice, preferably littermates, for the experimental designs described below. In all the experimental procedures, animals will be anesthetized by 3% isoflurane inhalation or euthanized by cervical dislocation as indicated.
<ol>
	<li>Induction of hair growth in the back skin of mice in the second telogen (resting) phase (about day 50 post-natal)
	<ol>
		<li>Anesthetize mice with inhalation of 3% isoflurane. Confirm full deep anaesthesia by lack of a pedal reflex (firm toe pinch). Shave two independent side by side regions of the back skin in each single mouse using hair clippers and depilatory cream (Table of Materials). Use the left side for the control and the right side for treatment. 
		NOTE: Check that, after shaving, the subjacent back skin is pinkish and not grey/black, an indicator of melanogenesis and entrance into the anagen (growing) phase in this mouse strain.</li>
		<li>Wash thoroughly with PBS to remove all cream remains and proceed to induction of transient in situ production of nonlethal ROS levels as described in section 2.1.</li>
		<li>Record hair follicle growth through daily acquisition of high-resolution images of the control and treated back skin areas in each animal (e.g., using an HD camera coupled to a 5&#8722;20x binocular lens).</li>
	</ol>
	</li>
	<li>Induction of 2nd degree burn lesions in the back skin of mice in the second telogen (resting) phase (about day 50 post-natal)
	<ol>
		<li>Anesthetize mice. Shave the whole back skin region in each single mouse using hair clippers and depilatory cream and wash thoroughly with PBS to remove all cream remains. 
		NOTE:&#160;Administer in situ subcutaneous injections of lidocaine 0.5% (2 mg/ml) in sterile saline, not exceeding 7 mg/ml, just before the burning procedure.</li>
		<li>Preheat a brass bar (1 cm in cross-section) at 95 &#176;C by immersing in boiling water and then apply on the central region of the dorsal back skin surface of each mouse for 5 s.</li>
		<li>Just after burn generation, intraperitoneally inject the animals with 1 mL of physiological solution (0.9% NaCl) on an electric blanket to prevent dehydration. Let the animals recover for 24 h and proceed to induction of transient in situ production of nonlethal ROS levels as described in section 2.3.</li>
		<li>Record burn wound progression through daily acquisition of high-resolution images of the control and treated back skin areas in each animal (e.g., using an HD camera coupled to a 5&#8722;20x binocular lens).</li>
	</ol>
	</li>
	<li>Generation and identification of long-term BrdU LRCs in the tail skin epithelium
	<ol>
		<li>Inject 10/14-day old littermates intraperitoneally (no anaesthesia) once a day for 4 consecutive days with 50 mg/kg bodyweight BrdU dissolved in PBS. After the labelling phase, allow mice to grow for 50&#8722;60 days before any treatment. 
		NOTE:&#160;Use a fresh needle for each injection.&#160;</li>
		<li>Proceed as described in section 2.3 for the induction of transient in situ nonlethal ROS production in the tail skin at different times before the preparation of tissue wholemounts.</li>
		<li>To prepare wholemounts of tail epidermis, euthanize mice by cervical dislocation and clip the tails with surgical scissors.
		<ol>
			<li>Use a scalpel to make a straight longitudinal incision all along the tail and peel the whole skin as a single piece from the backbone. Incubate the peeled skin in 5 mM EDTA in PBS in 5 mL tubes for 4 h at 37&#160;&#176;C and carefully separate intact sheets of epidermis from the dermis using forceps.</li>
			<li>Fix the tissue in 4% formaldehyde in PBS for at least 72 h at room temperature (RT) and proceed for BrdU detection using appropriate antibodies.</li>
			<li>Use fluorescence/confocal microscopy to identify and quantify LRCs in each experimental condition, including light controls and photodynamic treatments at different times before the preparation of tissue wholemounts, as previously described in detail19,20. 
			NOTE: Fixed epidermal sheets may be stored in PBS containing 0.02% sodium azide at 4&#160;&#176;C for up to three months. Fixed epidermal sheets may be used for immunolocalization of required proteins following standard histological sections procedures.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Induction of transient production of nonlethal ROS levels in mouse skin
NOTE: To induce transient production of nonlethal ROS levels in mouse skin, a photodynamic treatment using a precursor of the endogenous photosensitizer PpIX, in this case, methyl-aminolevulinate (mALA), and red light will be used.
<ol>
	<li>To switch on transient ROS production for the induction of hair growth in the back skin, prepare the animals as indicated in section 1.1.
	<ol>
		<li>Apply ~25 mg of mALA in the form of topical cream (Table of Materials) on the right region, keeping the left side as an internal control avoiding inter-individual differences. Incubate for 2.5 h in the dark, wash off the excess cream thoroughly with PBS. To confirm anesthesia depth, monitor the animals&#160;every 10 mins until fully recovered. 
		NOTE: The production of PpIX in the back skin should be tested in situ by its red fluorescence under blue light (407 nm) excitation.</li>
		<li>Anesthetize the animals.</li>
		<li>Irradiate the whole back skin with an adequate red-light source (Table of Materials) for a total dose of 2.5&#8722;4 J/cm2. Keep mice on an electric blanket until their complete recovery and proceed as described in step 1.1.3. 
		NOTE: Irradiance should be adjusted by manipulating the distance between the light source and the tissue and measured using a power energy meter (Table of Materials). The experiment is considered finished when full hair growth is observed in any of the independent shaved regions of each animal. The whole procedure involves just a single photo treatment.</li>
	</ol>
	</li>
	<li>To switch on transient ROS production for healing improvement of 2nd degree burn lesions, prepare the animals as indicated in section 1.2.
	<ol>
		<li>Apply ~25 mg of mALA in the form of topical cream all along the burned surface, encompassing about 4 mm of adjacent tissue. Incubate for 2.5 h in the dark, wash off the excess cream thoroughly with PBS.&#160;To confirm anesthesia depth, monitor the animals&#160;every 10 mins until fully recovered.</li>
		<li>Anesthetize the animals.</li>
		<li>Irradiate the whole back skin with an adequate red-light source for a total dose of 2.5&#8722;4 J/cm2. Keep the mice on an electric blanket until their complete recovery and proceed as described in step 1.2.4. 
		NOTE: The experimental procedure for each animal is considered finished when full burn healing is observed. The whole procedure involves just a single photo treatment.</li>
	</ol>
	</li>
	<li>To switch on transient ROS production in tail skin, prepare mice as indicated in section 1.3, apply ~25 mg of mALA in the form of topical cream all along the dorsal tissue area and proceed as described in section 2.1 for back skin. Perform photo treatments and correspondent light controls 24 h, 48 h, or 72 h before animal euthanasia and further extraction of tail skin whole mounts. 
	NOTE: In all experimental designs, assay the ROS-dependence of the process by using antioxidant ROS scavengers (e.g., daily inoculation of 100 mg/kg bodyweight N-acetyl-cysteine by intraperitoneal injection of a 20 mg/mL solution in PBS, pH 7.2, starting 5 days before mALA treatments or, alternatively, two doses of 100 mg/mL ascorbic acid in 50% ethanol, spaced 30 min, topically applied on the skin in the time interval between the mALA treatments and red-light irradiation).</li>
</ol>
3. ROS detection in the skin
<ol>
	<li>Ex vivo evaluation of ROS production in tail skin after photodynamic treatment using hydroethidine 
	NOTE: Hydroethidine is a non-fluorescent molecule that reacts specifically with ROS to produce fluorescent dye 2-hydroxyethidium (hET).
	<ol>
		<li>Incubate whole tail skins, obtained as described in section 1.3.3, for 3 h at 37&#160;&#176;C in 5 mM EDTA in PBS solution (control samples) or additionally containing 2 mM mALA (photodynamic treatment samples).</li>
		<li>In all cases, add hydroethidine to a final concentration of 3.2 &#956;M from a 25 mg/mL stock in dimethyl sulfoxide (DMSO) and incubate for 1 h in the dark at RT.</li>
		<li>Stretch the tail skin samples over a glass surface using fingertips and irradiate with 636 nm red light at a 10 J/cm2 fluence.</li>
		<li>Proceed immediately to separate the epidermis from the dermis and to fix the epidermal sheets as described in section 1.3.3.</li>
		<li>Evaluate the hET red emission under a fluorescence/confocal microscope using green exciting light, capture high quality images and proceed with further analysis. 
		NOTE: Use hET staining of tissue samples in the absence of photodynamic treatment as a negative control for hET autoxidation.</li>
	</ol>
	</li>
	<li>In vivo detection of ROS production in the back skin after induction of hair growth and burn healing followed by photodynamic treatment 
	NOTE: This step is performed using 2&#8242;,7&#8242;-dichlorodihydrofluorescein diacetate (DHF-DA), a cell permeant non-fluorescent compound that, after cleavage by intracellular esterase enzymes, specifically reacts with ROS giving the 2&#8242;,7&#8242;-dichlorofluorescein (DCF) fluorescent dye.
	<ol>
		<li>Use animals prepared for induction of hair growth (section 1.1) or burn healing (section 1.2). Just before topical mALA cream treatments (see sections 2.1 and 2.2), topically dispense 100 &#956;L of 1 mg/mL in 50% ethanol of DHF-DA on all target control/treated skin areas, let the skin fully absorb the material and proceed forward with topical mALA cream application.</li>
		<li>Incubate treated animals for 4 h in the dark, wash the topical mALA cream thoroughly off the skin with PBS and dispense a second dose of 100 &#956;L of DHF-DA solution on the skin.&#160;To confirm anesthesia depth, monitor the animals&#160;every 10 mins until fully recovered.</li>
		<li>Incubate treated animals for 50 min in the dark, anesthetize and irradiate the whole back skin with a fluence ranging from 2.5 to 4 J/cm2 of 636 nm red light using a LED lamp.</li>
		<li>Immediately after irradiation, evaluate ROS levels generated in the skin using an in vivo imaging system (Table of Materials). Set the filter box for 445&#8722;490 nm excitation and 515&#8722;575 nm emission, capture high quality images and proceed with further analysis. 
		NOTE: Use DHF-DA staining of tissue samples in the absence of photodynamic treatment as negative control for DHF-DA autoxidation.</li>
	</ol>
	</li>
</ol>

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All commercial applications of the procedures described in this work are protected by a CSIC-UAM patent (EP2932967A1) authored by EC, MIC and JE and licensed to Derma Innovate SL for commercial exploitation. JE and JJM have an advisory position in Derma Innovate SL.

Here, we present a methodology that allows a transient activation of endogenous ROS production in vivo in mouse skin with physiological effects. The methodology is based on a photodynamic procedure to induce a controlled and local stimulation of the endogenous photosensitizer PpIX (Figure 1B). This experimental approach is an interesting tool to study ROS biology in in vivo experimental systems constituting a significant advance over methodologies using external ROS sources (usually hydrogen peroxide) and allowing controlled and local production of ROS in the tissue/sample.
Given that aminolevulinate-based precursors are administered in excess to promote the accumulation of PpIX inside the cells, a critical step in this methodology is the establishment of an adequate light dose to induce transient production of ROS levels in the tissue below the damage threshold but showing a strong stimulatory effect. Currently there are no available technologies to directly quantify the exact amount of any type of ROS that is produced in cells and tissues. In our methodology, it is still not possible to establish a direct correlation between a given light dose, the exact amount of ROS produced, and a given biological effect (e.g., cell death or cell proliferation). For this reason, the light dose (fluence) for any particular experimental model should be established empirically by the researcher using qualitative or semiqualitative parameters of choice for each situation. In the case of mouse skin, we choose an easily measurable transition between cell death and tissue damage and the induction of a significant and transient proliferative wave.
The methodology presented here has proven to be very effective in the improvement of skin regeneration in different processes, including burn healing and hair follicle growth. These observations pave the way for the implementation of therapeutic applications of this technology in clinics for the treatment of incidental or chronic burns and wounds or for different pathologies skin and, particularly, of the hair follicle involving a defective stem cell functioning.

Reactive oxygen species (ROS) are the result of the chemical reduction of molecular oxygen to form water, and include singlet oxygen, superoxide anion, hydrogen peroxide and the hydroxyl radical1,2,3. ROS have a very short lifespan due to their extremely chemical reactive nature. In aerobic organisms, ROS are incidentally formed inside the cells as a major leaky by-product of aerobic respiration (electron transport chain) in the mitochondria. Transient accumulation of high levels of ROS in the cell results in an oxidative stress condition that may provoke the irreversible inactivation of proteins, lipids and sugars and the introduction of mutations in the DNA molecule2,3,4,5. The gradual accumulation of oxidative damage in cells, tissues and whole organisms steadily increases with time and has been associated with the induction of cell death programs, several pathologies, and the ageing process2,3,4,6.
Aerobic organisms have steadily evolved efficient molecular mechanisms to tackle excess ROS accumulation in cells and tissues. These mechanisms include members of the superoxide dismutase (SOD) protein family, which catalyze superoxide radical dismutation into molecular oxygen and hydrogen peroxide, as well as different catalases and peroxidases which use the antioxidant pool (glutathione, NADPH, peroxiredoxin, thioredoxin7,8) to catalyze the subsequent conversion of hydrogen peroxide to water and molecular oxygen.
However, several reports support the role of ROS as key components of molecular circuits that regulate critical cell functions, including proliferation, differentiation and mobility2,3,4. This concept is further supported by the initial identification and characterization of dedicated ROS-producing mechanisms in aerobic organisms, including lipoxygenases cyclooxygenases and NADPH oxidases9,10. In this sense, ROS exhibit an active role during vertebrate embryo development11,12,13 and key roles for these molecules in the regulation of specific in vivo physiological functions have been reported in different experimental systems, including the differentiation program of hematopoietic progenitors in Drosophila14, healing induction in zebrafish, or tail regeneration in Xenopus tadpoles15. In mammals, ROS have been involved in the self-renewal/differentiation potential of neural stem cells in a neurosphere model16 and in the deregulation of intestinal stem cell function during colorectal cancer initiation17. In the skin, ROS signalling has been associated with epidermal differentiation and the regulation of the skin stem cell niche and the hair follicle growth cycle18,19.
In this perspective, a major experimental limitation to determine the physiological roles of ROS in biological systems, both in normal or pathological conditions, is the lack of adequate experimental tools to induce controlled production of these molecules in cells and tissues, accurately resembling their physiological production as second signalling messengers. At present, most experimental approaches involve the administration of exogenous ROS, mostly in the form of hydrogen peroxide. We have recently implemented an experimental approach to switch on a transient, nonlethal in vivo production of endogenous ROS in the mouse skin, based on the administration of precursors of the endogenous photosensitizer protoporphyrin IX (PpIX; e.g., aminolaevulinic acid or its methyl derivative methylaminolevulinate) and further irradiation of the sample with red light to induce the in situ formation of ROS from intracellular molecular oxygen (Figure 1). This photodynamic procedure may be efficiently used to stimulate resident stem cell niches, thus activating the regenerative programs of the tissue19,20 and opening the way for new therapeutic modalities in skin regenerative medicine. Here, we present a detailed description of the protocol, showing representative examples of stimulation of stem cell niches, measured as an increase in the number of long-term 5-bromo-2&#8217;-deoxyuridine (BrdU) label retaining cells (LRCs) in the bulge region of the hair follicle19,21, and subsequent activation of regeneration programs (acceleration of hair growth and burn healing processes) induced by transient, nonlethal ROS production in the skin of C57Bl6 mouse strain.

<table><tbody><tr><td>2&amp;prime;,7&amp;prime;-Dichlorofluorescin diacetate</td><td>Sigma Aldrich</td><td>D6883-50MG</td><td></td></tr><tr><td>5&#039;-bromo-2&#039;-deoxiuridine</td><td>Sigma Aldrich</td><td>B5002-500MG</td><td></td></tr><tr><td>Anti-Bromodeoxyuridine-Fluorescein</td><td>Roche</td><td>11202693001</td><td></td></tr><tr><td>Depilatory cream (e.g., Veet)</td><td>Veet</td><td></td><td></td></tr><tr><td>Dihydroethidium</td><td>Sigma Aldrich</td><td>37291-25MG</td><td></td></tr><tr><td>In Vivo imaging system, e.g., IVIS Lumina 2</td><td>Perkin Elmer</td><td></td><td></td></tr><tr><td>mALA in the form of topical cream, e.g.,METVIX Crema 160 mg/g</td><td>Galderma</td><td></td><td></td></tr><tr><td>Power energy meter (e.g., ThorLabs Model PM100D)</td><td>ThorLabs</td><td></td><td></td></tr><tr><td>Red light source, e.g., 636 nm Aktilite LED lamp</td><td>Photocure ASA</td><td></td><td></td></tr></tbody></table>

stimulation of stem cell niches and tissue regeneration in mouse skin by switchable protoporphyrin ix-dependent photogeneration of reactive oxygen species in situ

Here, we describe a protocol to induce switchable in vivo photogeneration of endogenous reactive oxygen species (ROS) in mouse skin. This transient production of ROS in situ efficiently activates cell proliferation in stem cell niches and stimulates tissue regeneration as strongly manifested through the acceleration of burn healing and hair follicle growth processes. The protocol is based on a regulatable photodynamic treatment that treats the tissue with precursors of the endogenous photosensitizer protoporphyrin IX and further irradiates the tissue with red light under tightly controlled physicochemical parameters. Overall, this protocol constitutes an interesting experimental tool to analyze ROS biology.

The topical administration of the mALA precursor in the mouse back and tail skin results in a significant accumulation of PpIX in the whole tissue and, noticeably, in the hair follicle, as demonstrated by the reddish-pink fluorescence of this compound under blue light (407 nm) excitation (Figure 2A,C). Subsequent irradiation of treated tissue with red light (636 nm) at a fluence of 2.5&#8722;4 J/cm2 promotes transient production of ROS in the tissue, particularly in the bulge region of the hair follicle (Figure 2B,D).
Switching on nonlethal ROS production in mouse skin in vivo promotes a significant increase in the number of LRCs, categorized as somatic stem cells, in the bulge region of the hair follicle two days after photo treatments (Figure 3, left panels). Notably, the increase in the number of LRCs is transient, restoring to normal levels 6 days after treatments (Figure 3, right panel). As this region is one of the main stem cell niches in mouse skin, a transient induction of cell proliferation in this region mainly reflects the functional activation of the bulge niche and of the resident stem cell programs of proliferation and differentiation22,23.
The ROS-dependent activation of the bulge hair follicle niche is further associated with physiological responses in the skin. Thus, transient ROS production notably accelerates the skin healing process after a 2nd degree burn (Figure 4A,B). Quantification of the gradual reduction of the damaged/scab skin area demonstrates the robustness and statistical significance of the wound healing acceleration process induced by PpIX-dependent transient ROS production in the tissue (Figure 4C). In the same way, nonlethal ROS levels strongly promote hair growth after shaving during the second coordinated telogen (Figure 5A), a phase during which the hair follicle is refractory to respond to growth stimuli22,23, constituting an adequate way to evaluate the potential of new compounds and/or processes to stimulate hair growth. Notably, the use of antioxidant compounds like ascorbic acid (AA) results in a statistically significant reduction in the number of animals showing accelerated hair growth (Figure 5B). In addition, ROS production in the skin after photo treatments, quantified by the fluorescent emission of DHF in the skin, is also significantly reduced by antioxidant compounds (Figure 5C). Together, these results demonstrate that ROS production after PpIX-based photo treatments is strictly required to induce a physiological response in the tissue.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60859/60859fig1.jpg" /> 
Figure 1: Theoretical background for the controlled switching on of endogenous photodynamic production of ROS in situ in cells and tissues using the heme biosynthetic pathway. (A) Schematic representation of the basic photochemical reactions resulting in molecular oxygen excitation during photodynamic treatments. Upon absorption of light with the appropriate &#955;, a photosensitizer molecule (PS) in the ground state S0 undergoes a transition to an excited singlet state S1. Since any excited state is energetically less preferable than the ground state, the molecule returns to S0 after a short period of time. Most PS have a high quantum efficiency for the transition from S1 to the triplet state T1, generally characterized by a relative long lifetime. Activated PS in the excited triplet state can react with other molecules via two different pathways. A type I photochemical reaction is the transfer of electrons to adjacent molecules to form radical species; these radicals are likely to react with molecular oxygen to produce ROS, including superoxide anion (&#8226;O2-), hydrogen peroxide (H2O2) and hydroxyl radical (&#8226;OH). A type II photochemical reaction represents the dominant process for most PS employed in PDT. During this reaction, the transfer of energy (not electrons) to molecular oxygen (whose configuration in the ground state is the triplet, 3O2) drives the formation of the non-radical but highly reactive singlet oxygen (1O2). The photoproducts formed during these reactions trigger a cascade of biochemical events resulting in an oxidative stress that finally causes cell death or that can potentially stimulate cell growth. (B) 5-aminolevulinic acid (ALA) is a natural precursor in the heme biosynthetic pathway, which involves both mitochondrial and cytosolic cellular compartments. The ALA synthase enzyme activity is regulated by a negative feedback control whereby free heme, the final product of this pathway, inhibits the synthesis of ALA from glycine and succinyl CoA. The administration of exogenous ALA or its derivative methyl aminolevulinate (mALA) bypasses the regulatory feedback system, so that downstream metabolites, especially protoporphyrin IX (PpIX), are accumulated in the cell inducing photosensitization. The rate-limiting characteristics of ferrochelatase, catalyst enzyme of the iron insertion in PpIX, promote the accumulation of this endogenous PS compound. PBG = porphobilinogen. This figure has been modified from Carrasco et al.19. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60859/60859fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60859/60859fig2.jpg" /> 
Figure 2: Photodynamic treatment with mALA and red light induces transient production of ROS in the skin. (A) Accumulation of endogenous PpIX after mALA topic treatment in back skin. The left side in the same animal was used as control. (B) Left panel: PpIX-dependent ROS (mALA+Light) production monitored by DHF-DA. Right panel: time-course analysis of relative ROS production in back skin; the relative integrated density of DHF-DA fluorescent emission of mALA+Light versus Light regions in each animal was quantified at different times after irradiation and normalized as described in methodology. The mean &#177; SE was represented (n = 4 for each time point). (C) Localization of PpIX in tail skin (fluorescence microscopy images). (D) ROS production in tail skin after mALA+Light as revealed by hET showing an increased and sustained accumulation in the bulge region of the hair follicle. Representative confocal microscopy images (maximum projections) are shown. Scale bar = 100 &#181;m. This figure has been modified from Carrasco et al.19. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60859/60859fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60859/60859fig3.jpg" /> 
Figure 3: Switching on in situ ROS production in the skin promotes a significant increase of stem cells in the bulge region of the hair follicle niche. Left panels: representative confocal microscopy images (maximum projections) showing the localization of BrdU label retaining cells (LRC) in mouse tail skin whole mounts and the evident increase of LRC in the bulge region of hair follicles 2 days after PpIX-based phototreatments. Right panel: quantification of the number of LRC in the hair follicle bulge region. The mean + SE (n = 4) is represented. Scale bar = 50 &#181;m. This figure has been modified from Carrasco et al.19. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60859/60859fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60859/60859fig4.jpg" /> 
Figure 4: Switching on in situ ROS production in the skin accelerates burn healing. (A) PpIX production induced by mALA in burn injured regions in treated animals as compared to control samples. (B) Burn healing evolution in mALA+Light treated and control animals. (C) Time-course quantification of burned areas (left panel) showing accelerated burn healing in mALA+Light treated animals; the mean + SE (n = 4) of unhealed area is represented. Area-under-the-curve analysis (right panel) demonstrating statistical differences between both time-course curves (p &#8804; 0.06). This figure has been modified from Carrasco et al.19. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60859/60859fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60859/60859fig5.jpg" /> 
Figure 5: Switching on in situ ROS production in the skin stimulates hair growth. (A) Top row: Induction of hair growth during the refractory telogen phase by mALA+Light (right side of dorsal skin) as compared to light control region (left side). Bottom row: Both ROS production in the skin and the acceleration of hair growth induced by mALA+Light are inhibited by ascorbic acid (AA) antioxidant treatment. (B) Quantification of the % of animals showing accelerated hair growth in mALA-PT as compared to control region in the absence or presence of the antioxidant AA (n = 4 in 3 independent experiments). (C) Quantification of the ROS production inhibition in dorsal skin induced by AA during mALA-PT (n = 4). In all cases, bars represent mean + SE. This figure has been modified from Carrasco et al.19. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60859/60859fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Stimulation of Stem Cell Niches and Tissue Regeneration in Mouse Skin by Switchable Protoporphyrin IX-Dependent Photogeneration of Reactive Oxygen Species In 

Stimulation of Stem Cell Niches and Tissue Regeneration in Mouse Skin by Switchable Protoporphyrin IX-Dependent Photogeneration of Reactive Oxygen Species In Situ

The aim of this protocol is to induce transient in vivo production of nonlethal levels of reactive oxygen species (ROS) in mouse skin, further promoting physiological responses in the tissue.

Here, we describe a protocol to induce switchable in vivo photogeneration of endogenous reactive oxygen species (ROS) in mouse skin. This transient ...

stimulation-stem-cell-niches-tissue-regeneration-mouse-skin

Nanyang Technological University

Research

JoVE Journal

Biology

1.9K Views.  Ramón y Cajal University Hospital. Het doel van dit protocol is om voorbijgaande in vivo productie van niet-dodelijke niveaus van reactieve zuurstofsoorten (ROS) in de huid van muizen te induceren, waardoor fysiologische reacties in het weefsel verder worden bevorderd.

Video: Stimulation of Stem Cell Niches and Tissue Regeneration in Mouse Skin by Switchable Protoporphyrin IX-Dependent Photogeneration of Reactive Oxygen Species In Situ

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Developmental Biology

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated &#946;-galactosidase (SA-&#946;-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal &#946;-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry&#160;(IHC). The major limitation of the SA-&#946;-Gal assay is the requirement of fresh or frozen samples.
Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-&#946;-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use&#160;IHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming.

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Here we present a detailed protocol to detect both senescent and pluripotent stem cells in the skeletal muscle upon injury while inducing in vivo reprogramming. This method is suitable for evaluating the role of cellular senescence during tissue regeneration and reprogramming in vivo.

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

Direct Reprogramming of Mouse Fibroblasts into Melanocytes

The loss of function of melanocytes leads to vitiligo, which seriously affects the physical and mental health of the affected individuals. Presently, there is no effective long-term treatment for vitiligo. Therefore, it is imperative to develop a convenient and effective treatment for vitiligo. Regenerative medicine technology for direct reprogramming of skin cells into melanocytes seems to be a promising novel treatment of vitiligo. This involves the direct reprogramming of the patient's skin cells into functional melanocytes to help ameliorate the loss of melanocytes in patients with vitiligo. However, this method needs to be first tested on mice. Although direct reprogramming is widely used, there is no clear protocol for direct reprogramming into melanocytes. Moreover, the number of available transcription factors is overwhelming. 
Here, a concentrated lentivirus packaging system protocol is presented to produce transcription factors selected for reprogramming skin cells to melanocytes, including Sox10, Mitf, Pax3, Sox2, Sox9, and Snai2. Mouse embryonic fibroblasts (MEFs) were infected with the concentrated lentivirus for all these transcription factors for the direct reprogramming of the MEFs into induced melanocytes (iMels) in vitro. Furthermore, these transcription factors were screened, and the system was optimized for direct reprogramming to melanocytes. The expression of the characteristic markers of melanin in iMels at the gene or protein level was significantly increased. These results suggest that direct reprogramming of fibroblasts to melanocytes could be a successful new therapeutic strategy for vitiligo and confirm the mechanism of melanocyte development, which will provide the basis for further direct reprogramming of fibroblasts into melanocytes in vivo.

Here, we describe an optimized direct reprogramming system for melanocytes and a high-efficiency, concentrated virus packaging system that ensures smooth direct reprogramming.

The loss of function of melanocytes leads to vitiligo, which seriously affects the physical and mental health of the affected individuals. Presently, ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various pathologies such as cancer, atherosclerosis and diabetes. We have previously shown that dysfunction of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/ Kelch-like erythroid cell-derived protein 1 (Keap1) signaling pathway leads to extreme ROS imbalance during cutaneous wound healing in diabetes. Since ROS levels are an important indicator of progression of wound healing, specific and accurate quantification techniques are valuable. Several in vitro assays to measure ROS in cells and tissues have been described; however, they only provide a single cumulative measurement per sample. More recently, the development of protein-based indicators and imaging modalities have allowed for unique spatiotemporal analyses. L-012 (C13H8ClN4NaO2) is a luminol derivative that can be used for both in vivo and in vitro chemiluminescent detection of ROS generated by NAPDH oxidase. L-012 emits a stronger signal than other fluorescent probes and has been shown to be both sensitive and reliable for detecting ROS. The time lapse applicability of L-012-facilitated imaging provides valuable information about inflammatory processes while reducing the need for sacrifice and overall reducing the number of study animals. Here, we describe a protocol utilizing L-012-facilitated in vivo imaging to quantify oxidative stress in a model of excisional wound healing using diabetic mice with locally dysfunctional Nrf2/Keap1.

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

We describe a non-invasive in vivo imaging protocol that is streamlined and cost-effective, utilizing L-012, a chemiluminescent luminol-analog, to visualize and quantify reactive oxygen species (ROS) generated in a mouse excisional wound model.

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various ...

Immunology and Infection

Interrogating Individual Autoreactive Germinal Centers by Photoactivation in a Mixed Chimeric Model of Autoimmunity

Autoimmune diseases present a significant health burden. Fundamental questions regarding the development and progression of autoimmune disease remain unanswered. One requirement for advancements in our understanding of the underlying disease mechanisms and cellular dynamics is the precise coupling of the microanatomical location of cell subsets with downstream molecular or functional analyses; a goal that has traditionally been difficult to achieve. The development of stable photoactivatable biological fluorophores and their integration into reporter strains has recently enabled precise microanatomical labeling and tracking of cellular subsets in murine models. Here, we describe how the ability to analyze autoreactive lymphocytes from single germinal centers may help to provide novel insights into autoimmunity, using the combination of a novel chimeric model of autoimmunity with a photoactivatable reporter as an example. We demonstrate a procedure for generating mixed chimeras with spontaneous autoreactive germinal centers populated by lymphocytes carrying a photoactivatable green fluorescent protein reporter. Using in vivo labeling strategies, single germinal centers can be visualized in explanted lymphoid tissues and their cellular constituents photoactivated by two-photon microscopy. Photoactivated lymphocytes from single germinal centers can then be analyzed or sorted flow cytometrically, as single cells or in bulk, and may be subjected to additional downstream molecular and functional analyses. This approach may directly be applied to provide renewed insights in the field of autoimmunity, but the procedure for generating bone marrow chimeras and the photoactivation procedure may additionally find broad application in studies of infectious diseases and tumor metastases.

This protocol describes the generation of mixed murine bone marrow chimeras with spontaneous autoimmune germinal centers, in which autoreactive lymphocytes carry a photoactivatable green fluorescent protein (PA-GFP) reporter. This provides the ability to link cellular location in tissues with downstream molecular and functional analyses.

Autoimmune diseases present a significant health burden. Fundamental questions regarding the development and progression of autoimmune disease remain ...

Generation of Multicue Cellular Microenvironments by UV-Photopatterning of Three-Dimensional Cell Culture Substrates

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand distribution and tissue geometry, have been increasingly shown to play critical roles in governing cell phenotype and behavior. However, these environmental cues and their effects on cells are often studied separately using in vitro platforms that isolate individual cues, a strategy that heavily oversimplifies the complex in vivo situation of multiple cues. Engineering approaches can be particularly useful to bridge this gap, by developing experimental setups that capture the complexity of the in vivo microenvironment, yet retain the degree of precision and manipulability of in vitro systems.
This study highlights an approach combining ultraviolet (UV)-based protein patterning and lithography-based substrate microfabrication, which together enable high-throughput investigation into cell behaviors in multicue environments. By means of maskless UV-photopatterning, it is possible to create complex, adhesive protein distributions on three-dimensional (3D) cell culture substrates on chips that contain a variety of well-defined geometrical cues. The proposed technique can be employed for culture substrates made from different polymeric materials and combined with adhesive patterned areas of a broad range of proteins. With this approach, single cells, as well as monolayers, can be subjected to combinations of geometrical cues and contact guidance cues presented by the patterned substrates. Systematic research using combinations of chip materials, protein patterns, and cell types can thus provide fundamental insights into cellular responses to multicue environments.

Traditionally, cell culture is performed on planar substrates that poorly mimic the natural environment of cells in vivo. Here we describe a method to produce cell culture substrates with physiologically relevant curved geometries and micropatterned extracellular proteins, allowing systematic investigations into cellular sensing of these extracellular cues.

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand ...

Bioengineering

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

We present a protocol for the development and use ofan oxidative stress-model by treating retinal pigment epithelial cells with H2O2, analyzing cell morphology, viability, density, glutathione, and UCP-2 level. It is a useful model to investigate the antioxidant effect of proteins secreted by transposon-transfected cells to treat neuroretinal degeneration.

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...

Stimulation of Stem Cell Niches and Tissue Regeneration in Mouse Skin by Switchable Protoporphyrin IX-Dependent Photogeneration of Reactive Oxygen Species In Situ

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