This work was supported by OTKA (Hungarian Scientific Research Fund) D45933, T049621, T&Eacute;T (Hungarian Science and Technology Foundation) A4/04, Arg-17/2006 and SIN, Bolyai, &Ouml;veges Fellowships and T&Aacute;MOP 4.2.2-08/1/KMR-2008-0004 and 4.2.1/B 09/1/KMR-2010-0001. OTKA 83803. We would like to thank William Gesztes for providing the voice-over.

1. Preparing H9c2 cardiomyoblast cells
H9c2 rat cardiomyoblasts were obtained from ATCC (Wesel, Germany) and expanded in high glucose (4.5 g/L) DMEM containing 10% fetal bovine serum, 4 mM L-glutamine, 100 U/ml penicillin and 100 &mu;g/ml streptomycin. The H9c2 myoblast cell line is derived from embryonic rat heart, it is used as an in vitro model for both skeletal and cardiac muscle 8,9.
Prepare 12 well plate of H9c2 cells:
<ol>
	<li>Remove culture medium from Petri dish of H9c2 cells.</li>
	<li>Wash cells twice with 5 ml PBS and digest with 2-3 ml 0.05% trypsin/EDTA.</li>
	<li>Place Petri dish for 5 minutes in the 37&deg;C incubator.</li>
	<li>Inhibit trypsin with 10 ml DMEM culture medium.</li>
	<li>Spin down the cells at 1200 RPM for 8 minutes.</li>
	<li>After removing the supernatant, resuspend cells in 1 ml DMEM culture medium and count cells with hemocytometer using Trypan Blue staining.</li>
	<li>Seed 30,000 H9c2 cells in 2 ml DMEM per well in a 12 well plate.</li>
	<li>Place the plate into the 37&deg;C CO2 incubator for 24 hours.</li>
</ol>
2. Simulated ischemia/reperfusion
Ischemia/reperfusion was simulated in vitro by performing oxygen glucose deprivation (OGD) on H9c2 cell cultures. This procedure was performed on the stage of the Zeiss confocal microscope with the PeCon cell incubation system (Erbach, Germany) allowing the observation of the cells during OGD. 
<ol>
	<li>Remove medium by aspiration from 12 well plate of H9c2 cells.</li>
	<li>Wash cells twice with PBS.</li>
	<li>Add 3 ml glucose free medium per well.</li>
	<li>Place the plate into the cell incubation system.</li>
	<li>Start nitrogen gas to purge the oxygen out of the incubation system.</li>
	<li>Wait until the level of oxygen reaches 0.5% then start the timer. 0.5% oxygen means approximately 3 mmHg partial tension. Subject cells to 150 minutes of simulated ischemia.</li>
	<li>At the end of simulated ischemia, remove the glucose free medium from the cells.</li>
	<li>Pipette 2 ml normal culture medium to the wells.</li>
	<li>Place the 12 well plate for 30 minutes in the 37&deg;C CO2 incubator; this is the "reperfusion period".</li>
</ol>
3. Preparing rescue mesenchymal stem cells
Bone marrow of human origin was taken into T75 flasks and diluted with Dulbecco's Modified Eagle's Medium (DMEM) culture medium containing 10% fetal calf serum (FCS), 100 U/ml penicillin and 100 &mu;g/ml streptomycin, 4 mM L-glutamine and 1 g/l glucose. The flasks were incubated at 37&deg;C in a fully humidified atmosphere of 5% CO2 for 3 days. After the incubation period the BMSCs adhered to the surface of the flasks and the remaining components of bone marrow were eliminated by washing with PBS. The used BMSCs were between 1 and 5 passages in the experiments. The identity of cells was confirmed by the presence of lineage-specific cell surface markers with flow cytometry. Hematopoietic linage-specific surface markers (CD34, CD45) and mesenchymal surface markers (CD73, CD90, CD105 and CD166) were investigated.
During simulated ischemia, prepare rescue mesenchymal stem cells for transplantation.
<ol>
	<li>Dilute the fluorescent dye Vybrant DiD in 1:200 ratio in DMEM culture medium.</li>
	<li>Aspirate medium from the rescue cells and add 300 &mu;l DMEM with Vybrant DiD.</li>
	<li>Place Petri dish for 30 minutes in the 37&deg;C CO2 incubator.</li>
	<li>Remove culture medium from Petri dish of mesenchymal stem cells.</li>
	<li>Wash cells twice with 5 ml PBS and digest with 2-3 ml 0.05% trypsin/EDTA.</li>
	<li>Place Petri dish for 5 minutes in the 37&deg;C incubator.</li>
	<li>Inhibit trypsin with 10 ml DMEM culture medium.</li>
	<li>Spin down the cells at 1200 RPM for 8 minutes.</li>
	<li>After removing the supernatant, resuspend cells in 1 ml DMEM culture medium and count cells with hemocytometer using Trypan Blue staining.</li>
	<li>After 30 minutes at the end of the reperfusion add 20,000 mesenchymal stem cells per well to the postischemic H9c2 cardiac myoblasts.</li>
	<li>Place co-culture of cells into the 37&deg;C CO2 incubator for 24 hours.</li>
</ol>
4. Analysis with confocal microscopy
After the simulated ischemia/reperfusion injury, cells are incubated in normal culture medium for 24 hours.
<ol>
	<li>After 24 hours wash cells twice with PBS.</li>
	<li>Stain the cells in 500 &mu;l live/dead dye solution for 30 minutes (5 nM calcein and 400 nM Ethidium-homodimer-2 in PBS) (5x104 cell/ml).</li>
</ol>
The evaluation of confocal images for live and dead cells selected by morphology and fluorescence can be performed with the ImageJ software (National Institutes of Health, USA). In case of co-cultures, MSCs can be distinguished from H9c2 cells due to their Vybrant DiD cell labeling. The ratio of dead cells can be evaluated in 4 independent fields of view (objective 10x) for each culture in a blind fashion10.
H9c2 cardiomyoblasts cultured on 42 mm glass coverslips can also be investigated with time lapse video microscopy during and after ischemia/reperfusion injury using the cell incubation system of the confocal microscope. Cell-to-cell interactions such as cell fusion and formation of intercellular nanotubes can be studied over time.
5. Analysis with flow cytometry
After the simulated ischemia/reperfusion injury, cells are incubated in normal culture medium for 24 hours.
<ol>
	<li>After 24 hours, harvest the culture media. It is very important to collect the culture medium also because some dead cells could have detached from the culture dish surface during the 24 hour incubation.</li>
	<li>Wash cells twice with PBS and digest with 0.05% trypsin-EDTA.</li>
	<li>Inhibit trypsin with DMEM culture medium.</li>
	<li>Spin down the cells and the harvested culture medium at 1200 RPM (approximately 150-200g) for 8 minutes.</li>
	<li>Discard the supernatant.</li>
	<li>Suspend the cells in 500 &mu;L live/dead dye solution for 30 minutes (5 nM calcein and 400 nM Ethidium-homodimer-2 in PBS) (5x104 cell/ml).</li>
	<li>Transfer cells to flow cytometry tubes.</li>
	<li>Start the CellQuest Pro software.</li>
	<li>To set up the flow cytometer for measurement, prepare control samples of live and dead cells.
		<ol class="lalpha">
			<li>Plate H9c2 cells at a density of 3x104 in 12-well plates as described in point 1.</li>
			<li>Prepare live cell controls with single trypsinisation as described in point 1.</li>
			<li>Prepare dead cell controls by treatment with 10 &mu;M H2O2 for 1 hour. </li>
			<li>After trypsinisation, resuspend live and dead cell controls in 500 &mu;l live/dead dye solution (5 nM calcein-AM and 400 nM Ethidium-homodimer-2 in PBS).</li>
		</ol>
	</li>
	<li>The instrument settings should be adjusted so the living cells are calcein positive and ethidium homodimer negative, while the dead cells are calcein negative and ethidium homodimer positive.</li>
	<li>The percentage of these populations can be expressed as bars on a graph and statistical analysis can be performed on these values.</li>
	<li>Measure the different experimental groups.</li>
</ol>
6. Representative results:
Our results showed that added mesenchymal stem cells could improve the survival of postischemic cardiac cells. Confocal microscopy images revealed direct cell-to-cell interactions, such as partial membrane connection or intercellular nanotubes, which probably play an important role in the observed protection10. These nanotubes span over distances of several cell diameters, and their diameters were between 200 and 500 nm (white arrows).
<img src="/files/ftp_upload/3575/3575fig1.jpg" alt="Figure 1" /> 
Figure 1. Nanotube formation between cells. Nanotube formation (white arrow) was observed between Vybrant DiO labeled cardiomyoblasts and Vybrant DiD labeled mesenchymal stem cells.
Flow cytometry analysis showed the populations of surviving and necrotic cardiomyoblasts and mesenchymal stem cells. Interestingly, we also observed a third, &#x2018;injured&#x2019; cardiomyoblast cell population which contained calcein but also contained the indicator of the dead cells, ethidium homodimer (Figure 2). 
<img src="/files/ftp_upload/3575/3575fig2.jpg" alt="Figure 2" /> 
Figure 2. Representative dot-plot of the different cell populations after simulated ischemia.
The evaluation of several experiments showed that addition of mesenchymal stem cell to postischemic H9c2 cardiomyoblast increased significantly the percentage of living cells compared to the cardiac myoblasts cultured alone after ischemia (Figure 3).
<img src="/files/ftp_upload/3575/3575fig3.jpg" alt="Figure 3" /> 
Figure 3. Effect of stem cell treatment on postischemic cardiomyoblasts. Percentage of living cells was significantly reduced after simulated ischemia compared to control (control vs. I-R model: 90.36&plusmn;2.60 vs. 10.52&plusmn;0.48, *** p&gt;0.001), which increased after addition of mesenchymal stem cells (I-R model vs. I-R + BMSC: 10.52&plusmn;0.48 vs. 25.21&plusmn;2.13, *** p&gt;0.001). Data represent mean&plusmn;SEM. Statistical analysis of data was carried out using one-way analysis of variance with Tukey's multiple comparison post hoc test.

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No conflicts of interest declared.

The demonstrated protocol is an in vitro approach to the much more complex issue of stem cell therapy in myocardial infarct, with all the advantages and disadvantages of such a model. Obviously it cannot reflect the complex (e.g. immunological) events taking place during and after myocardial infarct but can focus on the direct effects of the added cells on the postischemic cells. The effects of simulated ischemia on H9c2 cardiomyoblasts highly depend on the time of OGD, on the passage number of the used cells an...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>calcein-AM</td>
			<td>Molecular Probes</td>
			<td>L3224, C3099</td>
			<td><a href="http://www.invitrogen.com">http://www.invitrogen.com</a></td>
		</tr>
		<tr>
			<td>ethidium homodimer-2</td>
			<td>Molecular Probes</td>
			<td>L3224, E3599</td>
			<td><a href="http://www.invitrogen.com">http://www.invitrogen.com</a></td>
		</tr>
		<tr>
			<td>Vybrant DiD</td>
			<td>Molecular Probes</td>
			<td>V22887</td>
			<td><a href="http://www.invitrogen.com">http://www.invitrogen.com</a></td>
		</tr>
	</tbody>
</table>
Table 1. Reagents.
<table border="1">
	<tbody>
		<tr>
			<td>Name of the equipment</td>
			<td>Company</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>PeCon cell incubation system for Zeiss microscopes</td>
			<td>PeCon GmbH</td>
			<td><a href="http://www.pecon.biz/">www.pecon.biz/</a></td>
		</tr>
	</tbody>
</table>
Table 2. Equipment.

stem cell transplantation in an in vitro simulated ischemia/reperfusion model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Watch this Scientific Journal Video about Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model at JoVE.com

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

stem-cell-transplantation-an-vitro-simulated-ischemiareperfusion

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Medicine

14.0K ビュー.  Semmelweis University. 我々は、セットアップする方法を示しています in vitroで虚血/再灌流モデルとどのように虚血後の心筋細胞の幹細胞治療の効果を評価する。

ビデオ: Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse model is useful for investigating the pathophysiology of cerebral ischemia-reperfusion and determining potential therapeutic strategies for patients with stroke. Variations in the anatomy of the circle of Willis of C57BL/6 mice affects their infarct volume after cerebral-ischemia-induced injury. Studies have indicated that distal MCA occlusion (MCAO) can overcome this problem and result in a stable infarct size. In this study, we establish a two-vessel occlusion mouse model of cerebral ischemia-reperfusion through the interruption of the blood flow to the right MCA. We distally ligate the right MCA and right common carotid artery (CCA) and restore blood flow after a certain period of ischemia. This ischemia-reperfusion injury induces an infarct of stable size and a behavioral deficit. Peripheral immune cells infiltrate the ischemic brain within the 24 h infiltration period. Additionally, the neuronal loss in the cortical area is less for a longer reperfusion duration. Therefore, this two-vessel occlusion model is suitable for investigating the immune response and neuronal recovery during the reperfusion period after cerebral ischemia.

A mouse model of cerebral ischemia-reperfusion is established to investigate the pathophysiology of stroke. We distally ligate the right middle cerebral artery and right common carotid artery and restore blood flow after 10 or 40 min of ischemia.

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...