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...

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 border="0" cellpadding="0" cellspacing="0" style="width:812px;" width="811">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">2&#8242;,7&#8242;-Dichlorofluorescin diacetate</td>
			<td style="width:104px;">Sigma Aldrich</td>
			<td style="width:115px;">D6883-50MG</td>
			<td style="width:161px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">5&#39;-bromo-2&#39;-deoxiuridine</td>
			<td style="width:104px;">Sigma Aldrich</td>
			<td style="width:115px;">B5002-500MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">Anti-Bromodeoxyuridine-Fluorescein</td>
			<td style="width:104px;">Roche</td>
			<td>11202693001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Depilatory cream (e.g., Veet)</td>
			<td style="width:104px;">Veet</td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">Dihydroethidium</td>
			<td style="width:104px;">Sigma Aldrich</td>
			<td>37291-25MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">In Vivo imaging system, e.g., IVIS Lumina 2</td>
			<td style="width:104px;">Perkin Elmer</td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:432px;">mALA in the form of topical cream, e.g.,METVIX Crema 160 mg/g</td>
			<td style="width:104px;">Galderma</td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">Power energy meter (e.g., ThorLabs Model PM100D)</td>
			<td style="width:104px;">ThorLabs</td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">Red light source, e.g., 636 nm Aktilite LED lamp</td>
			<td style="width:104px;">Photocure ASA</td>
			<td style="width:115px;"></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 ...

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 ...

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Biology

1.9K Views.  Ramón y Cajal University Hospital. 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.

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

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism&prime;s program for development1, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution2-4. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation5,6. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics. 
Reporter transgenes provide a powerful method to study the in vivo function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE&prime;s activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster7 has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster to insert transgenes into specific genome landing sites8-10. This capability has made the quantitative measurement of gene and, relevant here, CRE activity11-13 feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at specialized transgene injection protocols. 
Here, we present a general protocol to quantitatively evaluate a CRE&prime;s activity, and show how this approach can be used to measure the effects of an introduced mutation on a CRE&prime;s activity and to compare the activities of orthologous CREs. Although the examples given are for a CRE active during fruit fly metamorphosis, the approach can be applied to other developmental stages, fruit fly species, or model organisms. Ultimately, a more widespread use of this approach to study CREs should advance an understanding of regulatory logic and how logic can vary and evolve.

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

Phenotypic variation for traits can result from mutations in cis-regulatory element (CRE) sequences that control gene expression patterns. Methods derived for use in Drosophila melanogaster can quantitatively compare the levels of spatial and temporal patterns of gene expression mediated by modified or naturally occurring CRE variants.

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or eliminated either catalytically or spontaneously. Due to the short life spans of most ROS and the diversity of their sources and subcellular localizations, a complete picture can be obtained only by careful measurements using a combination of protocols. Here, we present a set of three different protocols using OxyBurst Green (OBG)-coated beads, or dihydroethidium (DHE) and Amplex UltraRed (AUR), to monitor qualitatively and quantitatively various ROS in professional phagocytes such as Dictyostelium. We optimised the beads coating procedures and used OBG-coated beads and live microscopy to dynamically visualize intraphagosomal ROS generation at the single cell level. We identified lipopolysaccharide (LPS) from E. coli as a potent stimulator for ROS generation in Dictyostelium. In addition, we developed real time, medium-throughput assays using DHE and AUR to quantitatively measure intracellular superoxide and extracellular H2O2 production, respectively.

We adapted a set of protocols for the measurement of reactive oxygen species (ROS) that can be applied in various amoeba and mammalian cellular models for qualitative and quantitative studies.

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical applications. Isolating primary melanocytes from skin tissues by the conventional method usually takes about 3 to 4 weeks to passage sufficiently. More importantly, the tissues used are usually newborn foreskins and it is still a challenge to efficiently isolate primary melanocytes from adult tissues. We recently developed a new isolation method for melanocytes that adds Y-27632, a Rho kinase inhibitor, to the initial culture medium for 48 h. Compared with the conventional protocol, this new method dramatically increases the yield of melanocytes and shortens the time required to isolate melanocytes from foreskin tissues. We now describe this new method in more detail using adult epidermis to efficiently culture primary melanocytes. Importantly, we show that melanocytes obtained from adult tissues prepared by this new method can function normally. This new protocol will significantly benefit studies of pigmentation defects and melanomas using primary melanocytes prepared from easily accessed adult skin tissues.

This paper reports that the addition of Y-27632 to TIVA medium can significantly increase the yield of melanocytes from adult skin tissues.

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical ...

Inhibition of Wound Epidermis Formation via Full Skin Flap Surgery During Axolotl Limb Regeneration

Classical experiments in salamander regenerative biology over the last century have long established that the wound epidermis is a crucial signaling structure that forms rapidly post-amputation and is required for limb regeneration. However, methods to study its precise function at the molecular level over the last decades have been limited due to a paucity of precise functional techniques and genomic information available in salamander model systems. Excitingly, the recent plethora of sequencing technologies coupled with the release of various salamander genomes and the advent of functional genetic testing methods, including CRISPR, makes it possible to re-visit these foundational experiments at unprecedented molecular resolution. Here, I describe how to perform the classically developed full skin flap (FSF) surgery in adult axolotls in order to inhibit wound epidermis formation immediately following amputation. The wound epidermis normally forms via distal migration of epithelial cells in the skin proximal to the amputation plane to seal off the wound from the outside environment. The surgery entails immediately suturing full thickness skin (which includes both epidermal and dermal layers) over the amputation plane to hinder epithelial cell migration and contact with the underlying damaged mesenchymal tissues. Successful surgeries result in the inhibition of blastema formation and limb regeneration. By combining this surgery method with contemporary downstream molecular and functional analyses, researchers can begin to uncover the molecular underpinnings of wound epidermis function and biology during limb regeneration.

This article describes how to perform a surgical method to inhibit wound epidermis formation during axolotl limb regeneration by immediately suturing full thickness skin over the amputation plane. This method allows researchers to investigate the functional roles of the wound epidermis during the early stages of limb regeneration.

Classical experiments in salamander regenerative biology over the last century have long established that the wound epidermis is a crucial signaling ...

Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe

Reactive oxygen species (ROS) play essential roles in intestinal homeostasis. ROS are natural by-products of cell metabolism. They are produced in response to infection or injury at the mucosal level as they are involved in antimicrobial responses and wound healing. They are also critical secondary messengers, regulating several pathways, including cell growth and differentiation. On the other hand, excessive ROS levels lead to oxidative stress, which can be deleterious for cells and favor intestinal diseases like chronic inflammation or cancer. This work provides a straightforward method to detect ROS in the intestinal murine organoids by live imaging and flow cytometry, using a commercially available fluorogenic probe. Here the protocol describes assaying the effect of compounds that modulate the redox balance in intestinal organoids and detect ROS levels in specific intestinal cell types, exemplified here by the analysis of the intestinal stem cells genetically labeled with GFP. This protocol may be used with other fluorescent probes.

The present protocol describes a method to detect reactive oxygen species (ROS) in the intestinal murine organoids using qualitative imaging and quantitative cytometry assays. This work can be potentially extended to other fluorescent probes to test the effect of selected compounds on ROS.