The entire protocol after cell isolation takes approximately 2 hr.
1. Microsurgical Preparation
<ol>
	<li>Perform stem cell isolation from the donor bone marrow and fluorescent labeling of the isolated subpopulation according to standardized protocols9,12 Allow cells to rest during the time the animal operation is performed. For fluorescent labeling we recommend CFDA (Carboxyfluorescein diacetate succinimidyl ester).</li>
	<li>Anesthetize a male mouse weighing 20-25 g with ketamine (75 mg/kg) and xylazine (2.5 mg/kg). Throughout the surgical procedures and experimental protocols, anesthesia should be maintained by supplements (10% of initial injection, i.p.) as needed (usually every 30-60 min).</li>
	<li>Shave gently the anterior aspect of the right scrotum as well as the right thigh and groin. Collect all lose hair with a moistened cotton swab. Place the mouse ventral side up on a plexiglass viewing stage and fix the feet using elastic drape. The stage is custom-made, consisting of a base plate and a second plate at the caudal edge of the base plate for elevation of both legs and the scrotum. Place the stage on a heating pad to maintain body temperature at 37 &#176;C. After fixation on the stage, place the animal under an operation microscope and perform the following operation steps using 10- to 16-fold magnification.</li>
	<li>Make the initial incision using skin scissors at the right thigh, just anterior of the femoral vessels from the knee until to the groin.</li>
	<li>Identify the femoral artery and follow it proximally, mobilizing the artery from surrounding soft tissue. Identify and cut a large branch to the left testicle using thermocautery. Thereafter place a stay suture superficially at the left testicle. Gentle traction will facilitate further proximal mobilization of the femoral artery.</li>
	<li>After completion of the proximal mobilization, identify and cut a large branch running medially at the thigh&#160;by thermocautery. Thereafter ligate the femoral artery in the distal part of the right thigh. Gentle traction on the ligature will aid further preparation.</li>
	<li>Turn attention now to the proximal femoral artery again and place a suture around the artery as proximally as possible. Prepare a knot but do not tie it down yet. Apply gentle traction to the suture and create an arterial kinking.</li>
	<li>After establishing the kinking, incise the artery is carefully using a microscissor. Take special care not to damage the femoral vein.</li>
	<li>Insert a prepared microcatheter (inner diameter 0.28 mm) via the incision. The catheter must be deaired (0.9% sodium chloride, NaCl) and connected to a 1 ml syringe prior insertion.</li>
	<li>After successful insertion of the catheter, carefully loosen the kinking in order to facilitate passage of the catheter. Arterial blood flow should be immediately visible within the catheter. Gently advance the catheter a few millimeters beyond the previous kinking. Thereafter, tie down the prepared ligature for fixation of the catheter.</li>
	<li>Gently flush the catheter (0.9% NaCl) and aspirate to confirm correct position. Use a piece of tape to further secure the catheter to the operation stage.</li>
	<li>After adequate fixation, put the catheter aside for the following preparation steps of the cremaster muscle. Apply regular flushing and aspiration of the catheter every 5-10 min in order to prevent clot formation.</li>
	<li>Turn attention now to the right scrotum and make an incision by cutting the skin and fascia above the ventral aspect of the right scrotum. Take care not to touch the underlying tissue with any instruments.</li>
	<li>Extend the incision up to the inguinal fold and caudally to the distal end of the scrotum.</li>
	<li>Carefully separate underlying soft and connective tissue above the cremaster sack, without touching it. Identify the distal end of the cremaster, and place a stay suture here to slightly extend the muscle. Resect now remaining soft tissue beneath the muscle sack by gently pulling the muscle caudally and anteriorly.</li>
	<li>After complete separation from connective tissue, open the cremaster sack at one point on the cranial edge using scissors. Extend the incision from this opening down to distal end of the muscle.</li>
	<li>Seal the small vessel connecting the testicle and the cremaster using thermocautery, thereafter cut the remaining connections between the muscle and the testicle using scissors.</li>
	<li>Lay aside the testicle, out of the microscopy field.</li>
	<li>The opened cremaster now lays flat on the stage. Place four 6-0 Prolene stay sutures at the edges of the tissue and fix them with elastic drape in order to spread the cremaster muscle tissue. From now on continuously moisten the tissue with Phosphate-buffered saline (PBS) using a 1 ml syringe (Figure 1).</li>
	<li>Allow now the preparation to rest for 15 min. Thereafter inject 0.05-0.1 ml of 1% Rhodamine-Dextran via the left femoral artery for contrast enhancement of the microvasculature. If any chemokines or inflammatory agents are planned for application, you should now topically moisten the muscle with the respective reagent using a 1 ml syringe. In the case of a control experiment, continue moistening with PBS.</li>
</ol>
2. Intravital Microscopy
<ol>
	<li>Keep the mouse fixed on the operation stage and move under a fluorescence microscope modified for epi-illumination and connected to a CCD (charge-coupled device) video camera. Take special care not to dislocate the femoral artery catheter.</li>
	<li>Conduct experiments using a water immersion objective.</li>
	<li>After adjusting the objective for sufficient visualization of the cremaster microcirculation and before cell injection, stochastically define six postcapillary venules for later analysis of firm stem cell adherence.</li>
	<li>Administer fluorescent-labeled stem cells, dissolved in 200 &#956;l PBS, at 0.4 x 106 cells per injection, with a total of five consecutive injections utilizing the femoral artery catheter and a 1 ml syringe. Carefully shake the cell sample prior to the first injection.</li>
	<li>Record stem cell rolling immediately after cell injection, thereby defining &#34;rolling&#34; as a more than 50% reduction of cell velocity along the endothelial lining in combination with typical cellular &#34;stick and release&#34; movements. Perform counting of rolling stem cells and all stem cells passing a predefined venular distance during 1 min of observation following every single injection. The amount of stem cell rolling is expressed as percentage of all passing cells. Do not count cells exhibiting random brief tethering phenomena as rolling cells.</li>
	<li>Following the last cell injection determine the number of firmly adherent stem cells by counting and express the amount in relation to the calculated endothelial surface of the six predefined venules (diameter x length x &#960; ) as adherent cells/mm2 . Firm adhesion is considered when no cell movement is detectable for 30 sec.</li>
	<li>After completion of the intravital microscopy carefully remove the cremaster muscle and store it in correspondence to later analysis (immunohistochemistry or molecular biology). Sacrifice the animal by cervical dislocation. The analysis of intravital microscopy recordings for quantification of stem cell rolling and firm endothelial adhesion, as well as microvascular hemodynamics,&#160;such as red blood cell velocity (mm/sec), vessel length (&#956;m), vessel diameter (&#956;m), wall share rate (sec-1); should be performed offline by an investigator blinded to the respective experimental tissue treatment utilizing an image processing software.</li>
</ol>

<ol>
	<li>Madlambayan, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=marrow+stem+and+progenitor+cell+contribution+to+neovasculogenesis+is+dependent+on+model+system+with+SDF-1+as+a+permissive+trigger.">marrow stem and progenitor cell contribution to neovasculogenesis is dependent on model system with SDF-1 as a permissive trigger.</a> Blood. 114, 4310-4319 (2009).</li><li>Lapidot, T., Dar, A., Kollet, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+do+stem+cells+find+their+way+home?.">How do stem cells find their way home?.</a> Blood. 106, 1901-1910 (2005).</li><li>Lehr, H. A., Vollmar, B., Vajkoczy, P., Menger, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+fluorescence+microscopy+for+the+study+of+leukocyte+interaction+with+platelets+and+endothelial+cells.">Intravital fluorescence microscopy for the study of leukocyte interaction with platelets and endothelial cells.</a> Methods Enzymol. 300, 462-481 (1999).</li><li>Gavins, F. N., Chatterjee, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+for+the+study+of+mouse+microcirculation+in+anti-inflammatory+drug+research:+focus+on+the+mesentery+and+cremaster+preparations.">Intravital microscopy for the study of mouse microcirculation in anti-inflammatory drug research: focus on the mesentery and cremaster preparations.</a> J. Pharmacol. Toxicol. Methods. 49, 1-14 (2004).</li><li>Leister, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+peritoneal+cavity+chamber+for+intravital+microscopy+of+the+liver+under+conditions+of+pneumoperitoneum.">A peritoneal cavity chamber for intravital microscopy of the liver under conditions of pneumoperitoneum.</a> Surg. Endosc. 17, 939-942 (2003).</li><li>Sriramarao, P., Anderson, W., Wolitzky, B. A., Broide, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+bone+marrow-derived+mast+cells+roll+on+P-selectin+under+conditions+of+flow+in+vivo.">Mouse bone marrow-derived mast cells roll on P-selectin under conditions of flow in vivo.</a> Lab Invest. 74, 634-643 (1996).</li><li>Baez, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+open+cremaster+muscle+preparation+for+the+study+of+blood+vessels+by+in+vivo+microscopy.">An open cremaster muscle preparation for the study of blood vessels by in vivo microscopy.</a> Microvasc. Res. 5, 384-394 (1973).</li><li>Bagher, P., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+cremaster+muscle+preparation+for+intravital+imaging+of+the+microcirculation.">The mouse cremaster muscle preparation for intravital imaging of the microcirculation.</a> J. Vis. Exp. , e2874 (2011).</li><li>Kaminski, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+NOS+is+required+for+SDF-1alpha/CXCR4-mediated+peripheral+endothelial+adhesion+of+c-kit++bone+marrow+stem+cells.">Endothelial NOS is required for SDF-1alpha/CXCR4-mediated peripheral endothelial adhesion of c-kit+ bone marrow stem cells.</a> Lab Invest. 88, 58-69 (2008).</li><li>Furlani, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HMGB-1+induces+c-kit++cell+microvascular+rolling+and+adhesion+via+both+toll-like+receptor-2+and+toll-like+receptor-4+of+endothelial+cells.">HMGB-1 induces c-kit+ cell microvascular rolling and adhesion via both toll-like receptor-2 and toll-like receptor-4 of endothelial cells.</a> J. Cell Mol. Med. 16, 1094-1105 (2012).</li><li>Furlani, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+the+intravascular+administration+of+mesenchymal+stem+cells+safe?+Mesenchymal+stem+cells+and+intravital+microscopy.">Is the intravascular administration of mesenchymal stem cells safe? Mesenchymal stem cells and intravital microscopy.</a> Microvasc. Res. 77, 370-376 (2009).</li><li>Donndorf, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysing+migratory+properties+of+human+CD+133(+)+stem+cells+in+vivo+after+intraoperative+sternal+bone+marrow+isolation.">Analysing migratory properties of human CD 133(+) stem cells in vivo after intraoperative sternal bone marrow isolation.</a> Cell Transplant. , (2012).</li></ol>

All animal experiments, conducted for this manuscript have been approved by the local animal care and use committee.
	The authors declare that they have no competing financial interests.

Intravital fluorescence microscopy allows for direct visualization and quantitative analysis of stem cell-endothelium interactions. The cremaster muscle model is particularly useful, since microsurgical exposure and techniques intravital visualization have been well established during the research on leucocyte-endothelium interactions and various chemical components can be selectively applied to the target tissue by simple superfusion technique. Due to the absence of movement artifacts and severe image noise, this partic...

The purpose of the present article is to describe the technique of intravital microscopy (IVM) applied on the mouse cremaster microcirculation for direct observation and analysis of peripheral bone marrow stem cell migration.
The current concept of stem cell based tissue and organ regeneration involves the homing of bone marrow derived stem cells to the injured tissue1. A crucial step for successful stem cell migration includes stem cell interaction with the local endothelium within the injured organ followed by transendothelial migration and eventually organ engraftment2. Intravital analysis of these stem cell - endothelial cell interactions forms an ideal parameter for the quantification of a stem cell based regenerative response in different pathophysiological settings in vivo. Furthermore, it seems conceivable, that, in the future, intravital analysis of the migratory capacity of specific stem cell populations within standardized microcirculatory environments, might be applied prior advancing these populations to further clinical testing.
Intravital microscopy has been initially developed for the observation and quantification of leucocyte-endothelial cell interaction in vivo in the field of inflammatory research3. First successful recordings of intravital microscopy studies were reported by Cohnheim already in the 19th century, studying frogs&#39; tongues and mesenteries under a light microscope4. Since its first use IVM has undergone a tremendous technical development and today certain essential components form prerequisites in order to perform quantitative IVM: (i) tissue preparations that permit optical access, (ii) molecular probes that can be detected by a microscope, (iii) a microscope connected to a detection system and (iv) computer based analysis systems that can extract parameters of interest from the image data set4.
A variety of tissue preparations has been introduced for IVM studies including the mesenteries and liver of the mouse and rat5, the dorsal skinfold chambers of mouse 6 and hamster, the rabbit ear and the hamster cheek pouch to name a few.
However, in the following we will focus on the mouse cremaster muscle, representing an ideal tissue for intravital observations, as preparation and visualization are possible by a well-standardized surgical procedure, and in general no problems of movement artifacts occur. The open cremaster muscle preparation was carried out for the first time in the 1970s by Baez and colleagues 7. Originally described for rats, it has been adopted successfully also to the mouse8. After previous studies had mainly focused on leucocyte interactions with the vessel wall, our own group recently introduced the mouse cremaster muscle preparation as a valuable tool for direct visualization and quantitative analysis of stem cell-endothelium interactions within a defined microenvironment9. Various stem cell subpopulations have been studied utilizing this model, including murine c-kit+ bone marrow stem cell and mesenchymal stem cells, as well as human CD 133+ bone marrow stem cells10-12. Following cell isolation from donor bone marrow and fluorescent labeling for visualization, the stem cells are selectively applied into the cremaster microcirculation utilizing an arterial injection via the femoral artery, thereby avoiding any cell entrapment within remote organs. Furthermore, the cremaster muscle model is particularly useful since various chemokines potentially mediating local stem cell migration within the respective settings, can be topically applied to the target tissue by simple superfusion technique.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent</td>
			<td>Company</td>
			<td>Catalog Number</td>
		</tr>
		<tr>
			<td>Carboxy-fluorescein diacetate succinimidylester (CFDA)</td>
			<td>Invitrogen, Carlsbad, CA, USA</td>
			<td>C1157</td>
		</tr>
		<tr>
			<td>Rhodamine 6 G (1%)</td>
			<td>Sigma-Aldrich, Munich, Germany</td>
			<td>83697</td>
		</tr>
		<tr>
			<td>Ketamin (Ketanest)</td>
			<td>Pfizer, Berlin, Germany</td>
			<td>not available</td>
		</tr>
		<tr>
			<td>Xylacin (Rompun)</td>
			<td>Bayer, Leverkusen, Germany</td>
			<td>not available</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate - buffered saline (PBS)</td>
			<td>PAN Biotech, Aidenbach, Germany</td>
			<td>P04-36500</td>
		</tr>
	</tbody>
</table>

intravital microscopy of the microcirculation in the mouse cremaster muscle for the analysis of peripheral stem cell migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

In general, interaction of directly injected stem or progenitor cells with the vascular endothelium within the cremaster muscle microcirculation is a rare event and occurs exclusively in postcapillary venules (diameter: 30-80 &#956;m). Due to fluorescence labeling rolling and firmly adherent stem cells can be clearly quantified in separation from circulating endogenous leucocytes in the observed venules (Figure 2).
The microcirculatory conditions represented by the blood flow ...

Watch this Scientific Journal Video about Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration at JoVE.com

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

intravital-microscopy-microcirculation-mouse-cremaster-muscle-for

Research

JoVE Journal

Biology

15.2K Views.  University Rostock.  Прижизненные микроскопия кремастер микроциркуляции мыши М. предлагает уникальное и хорошо стандартизированы в естественных условиях модели для анализа периферической костного мозга миграции стволовых клеток. 

Video: Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic by-products. This balance is achieved by the movement of blood through the microcirculation, which encompasses the smallest branches of the vascular supply throughout all tissues and organs. Arterioles branch from arteries to form networks that control the distribution and magnitude of oxygenated blood flowing into the multitude of capillaries intimately associated with parenchymal cells. Capillaries provide a large surface area for diffusional exchange between tissue cells and the blood supply. Venules collect capillary effluent and converge as they return deoxygenated blood towards the heart. To observe these processes in real time requires an experimental approach for visualizing and manipulating the living microcirculation.
The cremaster muscle of rats was first used as a model for studying inflammation using histology and electron microscopy post mortem1,2. 
The first in vivo report of the exposed intact rat cremaster muscle investigated microvascular responses to vasoactive drugs using reflected light3. However 
curvature of the muscle and lack of focused illumination limited the usefulness of this preparation. The major breakthrough entailed opening the muscle, 
detaching it from the testicle and spreading it radially as a flat sheet for transillumination under a compound microscope4. While shown to be a valuable 
preparation to study the physiology of the microcirculation in rats5 and hamsters6, the cremaster muscle in mice7 has proven particularly useful in dissecting 
cellular pathways involved in regulating microvascular function8-11 and real-time imaging of intercellular signaling12. 
The cremaster muscle is derived from the internal oblique and transverse abdominus muscles as the testes descend through the inguinal canal13. 
It serves to support (Greek: cremaster = suspender) and maintain temperature of the testes. As described here, the cremaster muscle is prepared as a thin flat sheet 
for outstanding optical resolution. With the mouse maintained at a stable body temperature and plane of anesthesia, surgical preparation involves freeing the muscle 
from surrounding tissue and the testes, spreading it onto transparent pedestal of silastic rubber and securing the edges with insect pins while irrigating it 
continuously with physiological salt solution. The present protocol utilizes transgenic mice expressing GCaMP2 in arteriolar endothelial cells. GCaMP2 is a 
genetically encoded fluorescent calcium indicator molecule12. Widefield imaging and an intensified charge-coupled device camera enable in vivo study of 
calcium signaling in the arteriolar endothelium.

A tissue preparation is described for visualization and experimental manipulation of the living microcirculation. In anesthetized male mice, the thin, highly vascularized cremaster muscle is prepared for intravital microscopy to study microvascular networks including arterioles, capillaries and venules. This preparation is readily adapted for rats and hamsters.

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic ...

Microiontophoresis and Micromanipulation for Intravital Fluorescence Imaging of the Microcirculation

Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental preparation. Microiontophoresis can simulate synaptic transmission1 by delivering neurotransmitters and neuropeptides onto neurons reproducibly2. Negligible volume (fluid) displacement avoids mechanical disturbance to the experimental preparation. Adapting these techniques to the microcirculation3 has enabled mechanisms of vasodilation and vasoconstriction to be studied at the microscopic level in vivo4,5. A key advantage of such localized delivery is enabling vasomotor responses to be studied at defined sites within a microvascular network without evoking systemic or reflexive changes in blood pressure and tissue blood flow, thereby revealing intrinsic properties of microvessels.
A limitation of microiontophoresis is that the precise concentration of agent delivered to the site of interest is difficult to ascertain6. Nevertheless, its release from the micropipette tip is proportional to the intensity and duration of the ejection current2,7, such that reproducible stimulus-response relationships can be readily determined under defined experimental conditions (described below). Additional factors affecting microiontophoretic delivery include solute concentration and its ionization in solution. The internal diameter of the micropipette tip should be &tilde; 1 &mu;m or less to minimize diffusional 'leak', which can be counteracted with a retaining current. Thus an outward (positive) current is used to eject a cation and a negative current used to retain it within the micropipette.
Fabrication of micropipettes is facilitated with sophisticated electronic pullers8. Micropipettes are pulled from glass capillary tubes containing a filament that 'wicks' solution into the tip of the micropipette when filled from the back end ("backfilled"). This is done by inserting a microcapillary tube connected to a syringe containing the solution of interest and ejecting the solution into the lumen of the micropipette. Micromanipulators enable desired placement of micropipettes within the experimental preparation. Micromanipulators mounted on a movable base can be positioned around the preparation according to the topography of microvascular networks (developed below). 
The present protocol demonstrates microiontophoresis of acetylcholine (ACh+ Cl-) onto an arteriole of the mouse cremaster muscle preparation (See associated protocol: JoVE ID#2874) to produce endothelium-dependent vasodilation. Stimulus delivery is synchronized with digitized image acquisition using an electronic trigger. The use of Cx40BAC-GCaMP2 transgenic mice9 enables visualization of intracellular calcium responses underlying vasodilation in arteriolar endothelial cells in the living microcirculation.

Microiontophoresis entails movement of ions from a micropipette in response to a difference in electrical potential between the inside and outside of the micropipette. Biologically active molecules are thereby delivered in proportion to electrical current. We illustrate acetylcholine microiontophoresis in conjunction with micromanipulation to study endothelium-dependent vasodilation in the microcirculation.

Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental  preparation. ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study.
RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34- cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34- cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment.
In summary, this paper offers a method of using RNA-Seq technology to identify potential regulators of self-renewal and differentiation in EML cells. The key factors identified are subjected to downstream functional analysis in vitro and in vivo.

RNA-sequencing and bioinformatics analyses were used to identify significantly and differentially expressed transcription factors in Lin-CD34+ and Lin-CD34- subpopulations of mouse EMLcells. These transcription factors might play important roles in determining the switch between self-renewing Lin-CD34+ and partially differentiated Lin-CD34- cells.

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as ...

Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo cell-free layer width and its spatiotemporal variations can provide a comprehensive understanding of hemodynamics in microcirculation. In this study, we used an intravital microscopic system coupled with a high-speed video camera to quantify the cell-free layer widths in arterioles in vivo. The cremaster muscle of Sprague-Dawley rats was surgically exteriorized to visualize the blood flow. A custom-built imaging script was also developed to automate the image processing and analysis of the cell-free layer width. This approach enables the quantification of spatiotemporal variations more consistently than previous manual measurements. The accuracy of the measurement, however, partly depends on the use of a blue filter and the selection of an appropriate thresholding algorithm. Specifically, we evaluated the contrast and quality of images acquired with and without the use of a blue filter. In addition, we compared five different image histogram-based thresholding algorithms (Otsu, minimum, intermode, iterative selection, and fuzzy entropic thresholding) and illustrated the differences in their determination of the cell-free layer width.

This study demonstrates the surgical preparation of the rat cremaster muscle for the visualization of the in vivo cell-free layer. Considerable factors affecting the accuracy of the cell-free layer width measurement are discussed in this study.

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

Intravital Subcellular Microscopy of the Mammary Gland

The mammary gland constitutes a model par excellence for investigating epithelial functions, including tissue remodeling, cell polarity, and secretory mechanisms. During pregnancy, the gland expands from a primitive ductal tree embedded in a fat pad to a highly branched alveolar network primed for the formation and secretion of colostrum and milk. Post-partum, the gland supplies all the nutrients required for neonatal survival, including membrane-coated lipid droplets (LDs), proteins, carbohydrates, ions, and water. Various milk components, including lactose, casein micelles, and skim-milk proteins, are synthesized within the alveolar cells and secreted from vesicles by exocytosis at the apical surface. LDs are transported from sites of synthesis in the rough endoplasmic reticulum to the cell apex, coated with cellular membranes, and secreted by a unique apocrine mechanism. Other preformed constituents, including antibodies and hormones, are transported from the serosal side of the epithelium into milk by transcytosis. These processes are amenable to intravital microscopy because the mammary gland is a skin gland and, therefore, directly accessible to experimental manipulation. In this paper, a facile procedure is described to investigate the kinetics of LD secretion in situ, in real-time, in live anesthetized mice. Boron-dipyrromethene (BODIPY)665/676&#160;or monodansylpentane are used to label the neutral lipid fraction of transgenic mice, which either express soluble EGFP (enhanced green fluorescent protein) in the cytoplasm, or a membrane-targeted peptide fused to either EGFP or tdTomato. The membrane-tagged fusion proteins serve as markers of cell surfaces, and the lipid dyes resolve LDs &#8805; 0.7 &#181;m. Time-lapse images can be recorded by standard laser scanning confocal microscopy down to a depth of 15-25 &#181;m or by multiphoton microscopy for imaging deeper in the tissue. The mammary gland may be bathed with pharmacological agents or fluorescent dyes throughout the surgery, providing a platform for acute experimental manipulations as required.

The present protocol describes a facile technique for the intravital imaging of the lactating mouse mammary gland by laser scanning confocal and multiphoton microscopy.

The mammary gland constitutes a model par excellence for investigating epithelial functions, including tissue remodeling, cell polarity, and secretory ...

Live Imaging of Microtubule Dynamics in Glioblastoma Cells Invading the Zebrafish Brain

With a dismal median survival time in real populations-between 6 to 15 months-glioblastoma (GBM) is the most devastating malignant brain tumor. Treatment failure is mainly due to the invasiveness of GBM cells, which speaks for the need for a better understanding of GBM motile properties. To investigate the molecular mechanism supporting GBM invasion, new physiological models enabling in-depth characterization of protein dynamics during invasion are required. These observations would pave the way to the discovery of novel targets to block tumor infiltration and improve patient outcomes. This paper reports how an orthotopic xenograft of GBM cells in the zebrafish brain permits subcellular intravital live imaging. Focusing on microtubules (MTs), we describe a procedure for MT labeling in GBM cells, microinjecting GBM cells in the transparent brain of 3 days post fertilization (dpf) zebrafish larvae, intravital imaging of MTs in the disseminating xenografts, altering MT dynamics to assess their role during GBM invasion, and analyzing the acquired data.

We report a technique permitting live imaging of microtubule dynamics in glioblastoma (GBM) cells invading a vertebrate brain tissue. Coupling the orthotopic injection of fluorescently tagged GBM cells into a transparent zebrafish brain with high-resolution intravital imaging allows the measurement of cytoskeleton dynamics during in situ cancer invasion.

With a dismal median survival time in real populations-between 6 to 15 months-glioblastoma (GBM) is the most devastating malignant brain tumor. Treatment ...

Fluorescence Lifetime Macro Imager for Biomedical Applications

This paper presents a new photoluminescence lifetime imager designed to map the molecular oxygen (O2) concentration in different phosphorescent samples ranging from solid-state, O2-sensitive coatings to live animal tissue samples stained with soluble O2-sensitive probes. In particular, the nanoparticle-based near-infrared probe NanO2-IR, which is excitable with a 625 nm light-emitting diode (LED) and emits at 760 nm, was used. The imaging system is based on the Timepix3 camera (Tpx3Cam) and the opto-mechanical adaptor, which also houses an image intensifier. O2 phosphorescence lifetime imaging microscopy (PLIM) is commonly required for various studies, but current platforms have limitations in their accuracy, general flexibility, and usability.
The system presented here is a fast and highly sensitive imager, which is built on an integrated optical sensor and readout chip module, Tpx3Cam. It is shown to produce high-intensity phosphorescence signals and stable lifetime values from surface-stained intestinal tissue samples or intraluminally stained fragments of the large intestine and allows the detailed mapping of tissue O2 levels in about 20 s or less. Initial experiments on the imaging of hypoxia in grafted tumors in unconscious animals are also presented. We also describe how the imager can be re-configured for use with O2-sensitive materials based on Pt-porphyrin dyes using a 390 nm LED for the excitation and a bandpass 650 nm filter for emission. Overall, the PLIM imager was found to produce accurate quantitative measurements of lifetime values for the probes used and respective two-dimensional maps of the O2 concentration. It is also useful for the metabolic imaging of ex vivo tissue models and live animals.

This paper describes the use of a new, fast optical imager for the macroscopic photoluminescence lifetime imaging of long decay emitting samples. The integration, image acquisition, and analysis procedures are described, along with the preparation and characterization of the sensor materials for the imaging and the application of the imager in studying biological samples.

This paper presents a new photoluminescence lifetime imager designed to map the molecular oxygen (O2) concentration in different phosphorescent samples ...