Questo lavoro è stato sostenuto da NIH NINDS concedere R01 NS074388 581 (CL Limoli), California Institute for Regenerative Medicine (CIRM) Grant RS1-00413 (LLC), CIRM formazione di Grant TG2-0115 (MMA), e una sovvenzione CIRM a Giove a sostegno della documentazione video.

<ol>
	<li>Acharya, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consequences+of+ionizing+radiation-induced+damage+in+human+neural+stem+cells.">Consequences of ionizing radiation-induced damage in human neural stem cells.</a> Free. Radic. Biol. Med. , (2010).</li><li>Acharya, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+radiation-induced+cognitive+impairment+through+cranial+transplantation+of+human+embryonic+stem+cells.">Rescue of radiation-induced cognitive impairment through cranial transplantation of human embryonic stem cells.</a> Proc. Natl. Acad. Sci. U. S. A. 106, 19150-19155 (2009).</li><li>Boyer, A., Khan, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+11.">Ch. 11.</a> Treatment planning in Radiation Oncology. , (2007).</li><li>Lawson, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+application+of+IMRT+based+radiosurgery+to+treat+multiple+or+large+irregular+lesions+and+verification+of+infra-red+frameless+localization+system.">Intracranial application of IMRT based radiosurgery to treat multiple or large irregular lesions and verification of infra-red frameless localization system.</a> J. Neurooncol. 97, 59-66 (2010).</li><li>Ling, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Commissioning+and+quality+assurance+of+RapidArc+radiotherapy+delivery+system.">Commissioning and quality assurance of RapidArc radiotherapy delivery system.</a> Int. J. Radiat. Oncol. Biol. Phys. 72, 575-581 (2008).</li><li>Otto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volumetric+modulated+arc+therapy:+IMRT+in+a+single+gantry+arc.">Volumetric modulated arc therapy: IMRT in a single gantry arc.</a> Med. Phys. 35, 310-317 (2008).</li><li>Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Rat Brain in stereotaxic coordinates. , (2007).</li><li>Hess, D. C., Borlongan, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-based+therapy+in+ischemic+stroke.">Cell-based therapy in ischemic stroke.</a> Expert. Rev. Neurother. 8, 1193-1201 (2008).</li><li>Hefferan, M. P., Johe, K., Feldman, E. L., Lunn, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+immunosuppressive+therapy+for+spinal+grafting+of+human+spinal+stem+cells+in+a+rat+model+of+ALS.">Optimization of immunosuppressive therapy for spinal grafting of human spinal stem cells in a rat model of ALS.</a> Cell. Transplant. , (2011).</li></ol>

Nessun conflitto di interessi dichiarati.

La ricerca è in corso un notevole esplorando la miriade di modi in cui le cellule staminali possono essere usati clinicamente per ripristinare le normali funzioni a tessuti danneggiati, invecchiato e malato 8. L&#39;eventuale realizzazione di questi sforzi richiederanno una conoscenza dettagliata del comportamento delle cellule trapiantate in microambienti unici che si distinguono dalle intatto tessuto normale. Il nostro lavoro ha dimostrato che all&#39;interno del letto del tessuto irradiato, NSCs cranialmente innestati può ripristinare funzionalmente cognizione, dove sopravvivono, migrano e si differenziano lungo linee neurali e gliali 2. Proprio come queste cellule mediare il recupero della cognizione è incerta al momento, ma non dipende da conducendo una serie di procedure sperimentali attentamente controllata in maniera riproducibile. Abbiamo queste procedure dettagliate critiche qui nel tentativo di accelerare il potenziale traslazionale di terapie con cellule staminali per ameliorating avversi effetti cognitivi associatigestione clinica del cervello e di altre forme di cancro. Ulteriori considerazioni che possono avere un impatto significativo della qualità dei dati sono evidenziati di seguito. Terapie a base di trapianto dipende da cellule staminali come il reagente critico, e di conseguenza, la cura deve essere esercitata per caratterizzare adeguatamente le culture, mantenere la sterilità e di utilizzare i numeri passaggio abbinati per l&#39;affidabilità dei risultati. Trapiantato cellule staminali umane sono stati etichettati con BrdU prima dell&#39;intervento, per fornire un mezzo per il monitoraggio in vivo. Nelle nostre condizioni sperimentali, NSCs trapiantato non ha subito la proliferazione estesa, in modo che la diluizione del marchio BrdU non è stato problematico. In alternativa, trapiantato cellule staminali umane possono essere distinte dalle cellule ospiti mediante immunocolorazione per il consumo umano marcatori specifici come la proteina di matrice nucleare (h-NUC o hNUMA) 9 o umano-specifico antigene nucleare (HuNu) 2. Le cellule staminali umane potrebbe anche essere etichettati conuna serie di marcatori fluorescenti per facilitarne l&#39;identificazione all&#39;interno del cervello ospitante. Attenzione ai parametri di irradiazione definire con precisione le tecniche di erogazione della dose, e quelle qui descritte modello attuali pratiche cliniche in oncologia di radiazione. Trapianto riproducibile di cellule staminali nel cervello è anche critica, ed è realizzato utilizzando uno strumento digitale stereotassico. La capacità di micromanipulate proprio un micro-siringa per l&#39;impianto di cellule staminali in strutture del cervello piccolo come l&#39;ippocampo, riduce notevolmente l&#39;errore umano. Con questo apparato, NSC sono stati trapiantati in quattro siti diversi che attraversa il posteriore, anteriore alle regioni dell&#39;ippocampo di ratto. Dorso-ventrale (DV) coordinate sono state determinate in base all&#39;esperienza atimici nudo (ATN) ratti tale che gli interventi chirurgici non provocherebbe danni alla formazione dell&#39;ippocampo 2. Una sezione coronale attraverso il cervello del ratto rivela strutture chiave della incl formazione dell&#39;ippocampouding il giro dentato (DG), il dentato ilo (DH) e CA1 e CA3 sottocampi (Fig. 8). NSC trapiantati, introdotto dorsalmente dal tratto ago visibile (Nt, Redline), vengono visualizzati come colorazione scura (marrone), le cellule depositate presso il trapianto-release del sito (Tr) proprio sotto il corpo calloso (CC), che successivamente migrare in tutto il setto -temporale asse dell&#39;ippocampo.

<table border="1"><tbody><tr><td> Nome del materiale </td><td> Tipo </td><td> Azienda </td><td> Numero di catalogo </td><td> Commento </td></tr><tr><td> Strumento digitale Stereotassica w/45 ° e 18 ° Earbars </td><td> Cranica trapianto chirurgico di setup </td><td> Leica Microsystems (in precedenza: MyNeuroLab) </td><td> 39463501 </td><td> Utili accessori: Stage con riscaldamento e variabile sorgente di corrente (per mantenere la temperatura corporea durante l&#39;intervento chirurgico) </td></tr></tbody></table>

stem cell transplantation strategies for the restoration of cognitive dysfunction caused by cranial radiotherapy

Radioterapia fornisce spesso l&#39;unica ricorso clinica per malati di tumori cerebrali primari o metastatici. Mentre benefici, irradiazione cranica può indurre un declino progressivo e invalidante nella cognizione che possono, in parte, essere causati dalla deplezione delle cellule staminali neurali. Dato l&#39;aumento della sopravvivenza dei pazienti con diagnosi di tumore al cervello, qualità della vita in termini di salute cognitiva è diventata una crescente preoccupazione, soprattutto in assenza di una soddisfacente trattamenti a lungo termine. Per risolvere questo serio problema di salute che abbiamo usato cellule staminali sostituzione come strategia per combattere la radiazione indotta declino cognitivo. Il nostro modello utilizza atimici ratti nudi sottoposti a irradiazione cranica. La radiazione ionizzante viene consegnato sia come intero cervello o come un fascio altamente focalizzato l&#39;ippocampo attraverso l&#39;orecchio di un lin (LINAC) ccelerator radiochirurgia stereotassica base. Due giorni dopo irradiazione, huLe cellule staminali neurali dell&#39;uomo (hNSCs) sono stati trapiantati in stereotaxically l&#39;ippocampo. I ratti sono stati poi valutati per cambiamenti cognitivi, la sopravvivenza delle cellule innestate e per l&#39;espressione di specifici marcatori di differenziazione-1 e 4 mesi dopo l&#39;irradiazione. I nostri paradigmi test cognitivi hanno dimostrato che gli animali innestati con hNSCs mostrano significativi miglioramenti delle funzioni cognitive. Stereologia Tutti rivela sopravvivenza significativa (10-40%) delle cellule trapiantate a 1 e 4 mesi dopo il trapianto, dipende dalla quantità e dal tipo di cellule innestate. Cellule engrafted migrare, differenziare lungo linee gliali e neuroni, ed esprimere una gamma di marcatori fenotipici immaturi e maturi. I nostri dati dimostrano direct benefici cognitivi derivanti da innestate cellule staminali umane, suggerisce che questa procedura potrebbe un giorno permettere una strategia promettente per il lungo periodo di restauro funzionale della cognizione negli individui sottoposti a radiot cranialherapy. Per promuovere la diffusione delle procedure critica necessaria per replicare ed estendere i nostri studi, abbiamo fornito una documentazione scritta e visiva di alcuni passaggi chiave del nostro piano sperimentale, con particolare attenzione alla stereotaxic radiosurgey e il trapianto.

Watch this Scientific Journal Video about Stem Cell Transplantation Strategies for the Restoration of Cognitive Dysfunction Caused by Cranial Radiotherapy at JoVE.com

Stem Cell Transplantation Strategies for the Restoration of Cognitive Dysfunction Caused by Cranial Radiotherapy

Pazienti con tumore al cervello sottoposti a radioterapia cranica di routine, e per quanto utili, questo trattamento si traduce spesso in debilitanti disfunzione cognitiva. Questo grave problema irrisolto è allo stato attuale, alcun ricorso clinica, e ha spinto i nostri sforzi per ideare terapie con cellule staminali base per il recupero della radiazione indotta decrementa cognitive.

Strategie di trapianto di cellule staminali per il Restauro di disfunzione cognitiva causata da radioterapia craniale

Radiotherapy often provides the only clinical recourse for those afflicted with primary or metastatic brain tumors. While beneficial, cranial irradiation ...

stem-cell-transplantation-strategies-for-restoration-cognitive

Nanyang Technological University

Research

JoVE Journal

Medicine

14.4K Views.  University of California Irvine. Pazienti con tumore al cervello sottoposti a radioterapia cranica di routine, e per quanto utili, questo trattamento si traduce spesso in debilitanti disfunzione cognitiva. Questo grave problema irrisolto è allo stato attuale, alcun ricorso clinica, e ha spinto i nostri sforzi per ideare terapie con cellule staminali base per il recupero della radiazione indotta decrementa cognitive.

Video: Stem Cell Transplantation Strategies for the Restoration of Cognitive Dysfunction Caused by Cranial Radiotherapy

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

Trapianto ortotopico aortica: un modello di ratto per studiare lo sviluppo di vasculopatia cronica

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

Ricerca

Medicina

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

Un modello murino di Nell&#39;utero Trapianto

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

Quantifying Cognitive Decrements Caused by Cranial Radiotherapy

With the exception of survival, cognitive impairment stemming from the clinical management of cancer is a major factor dictating therapeutic outcome. For many patients afflicted with CNS and non-CNS malignancies, radiotherapy and chemotherapy offer the best options for disease control. These treatments however come at a cost, and nearly all cancer survivors (~11 million in the US alone as of 2006) incur some risk for developing cognitive dysfunction, with the most severe cases found in patients subjected to cranial radiotherapy (~200,000/yr) for the control of primary and metastatic brain tumors1. Particularly problematic are pediatric cases, whose long-term survival plagued with marked cognitive decrements results in significant socioeconomic burdens2. To date, there are still no satisfactory solutions to this significant clinical problem.
We have addressed this serious health concern using transplanted stem cells to combat radiation-induced cognitive decline in athymic rats subjected to cranial irradiation3. Details of the stereotaxic irradiation and the in vitro culturing and transplantation of human neural stem cells (hNSCs) can be found in our companion paper (Acharya et al., JoVE reference). Following irradiation and transplantation surgery, rats are then assessed for changes in cognition, grafted cell survival and expression of differentiation-specific markers 1 and 4-months after irradiation. To critically evaluate the success or failure of any potential intervention designed to ameliorate radiation-induced cognitive sequelae, a rigorous series of quantitative cognitive tasks must be performed. To accomplish this, we subject our animals to a suite of cognitive testing paradigms including novel place recognition, water maze, elevated plus maze and fear conditioning, in order to quantify hippocampal and non-hippocampal learning and memory. We have demonstrated the utility of these tests for quantifying specific types of cognitive decrements in irradiated animals, and used them to show that animals engrafted with hNSCs exhibit significant improvements in cognitive function3.
The cognitive benefits derived from engrafted human stem cells suggest that similar strategies may one day provide much needed clinical recourse to cancer survivors suffering from impaired cognition. Accordingly, we have provided written and visual documentation of the critical steps used in our cognitive testing paradigms to facilitate the translation of our promising results into the clinic.

Cognitive impairment resulting from the radiotherapeutic management of brain tumors represents a clinically intractable condition that adversely impacts quality of life. The capability to critically evaluate potential interventions for ameliorating radiation-induced cognitive decrements ultimately depends on the capability to undertake rigorous quantitative assessments of cognition.

Quantificare Decrementa cognitivi causati da radioterapia craniale

With the exception of survival, cognitive impairment stemming from the clinical management of cancer is a major factor dictating therapeutic outcome. For ...

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.

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.

Trapianto di cellule staminali in un In vitro Simulato ischemia / riperfusione Modello

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

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Differenziazione Isolamento, caratterizzazione e comparata delle cellule umane Dental Pulp staminali derivate da denti permanenti mediante due diversi metodi

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

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.

Neural Stem Cell Transplantation in Sperimentale Contusivi 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 ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

Mouse trapianto renale: Modelli di Allograft Rifiuto

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). 
Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. 
Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system.
Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. 
This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

High Throughput Caratterizzazione di cellule staminali adulte Engineered per consegna di fattori terapeutici per le strategie neuroprotettive

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

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.

Studiare cancro del pancreas staminali caratteristiche cellulari per lo sviluppo di nuove strategie di trattamento

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

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a serious problem. Therefore, the acceptance criteria for organ donors have been extended leading to the usage of marginal kidney grafts. These marginal organs tolerate cold storage poorly resulting in increased preservation injury and higher rates of delayed graft function. To overcome the limitations of cold storage, extensive research is focused on alternative normothermic preservation methods.
Ex vivo normothermic organ perfusion is an innovative preservation technique. The first experimental and clinical trials for ex vivo lung, liver, and kidney perfusions demonstrated favorable outcomes.
In addition to the reduction of cold ischemic injury, the method of normothermic kidney storage offers the opportunity for organ assessment and repair. This manuscript provides information about kidney retrieval, organ preservation techniques, and isolated ex vivo normothermic kidney perfusion (NEVKP) in a porcine model. Surgical techniques, set up for the perfusion solution and the circuit, potential assessment options, and representative results are demonstrated.

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

The severe organ shortage has resulted in increased use of marginal kidney grafts for transplantation. This has triggered interest in alternative storage methods, since marginal grafts especially tolerate cold storage poorly. The technique of normothermic ex vivo kidney perfusion (NEVKP) represents a novel preservation method for kidney grafts prior to &#160;transplantation.

Normotermica<em&gt; Ex Vivo</em&gt; Rene perfusione per la Preservazione della Kidney Innesti prima del trapianto

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a ...