עבודה זו מומנה על ידי מענקים SMM: GIA0855953G AHA, NIH 1R01 HL091105-01. תמיכה נוספת סופק על ידי: DW: NIH RO1DK075013, DDK וקרן Kleberg; תזרים UCCC cytometry Core (NIH 5 P30 CA 46934-15), את UCCC microarray הליבה (NCI P30 CA 46934-14).

<ol>
	<li>Goodell, M., Brose, K., Paradis, G., Conner, A. S., Mulligan, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+functional+properties+of+murine+hematopoietic+stem+cells+that+are+replicating+in+vivo.">Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo.</a> J. Exp. Med. 183, 1797-1806 (1996).</li><li>Jun, 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=The+Pathology+of+Bleomycin+Induced+Fibrosis+is+Associated+with+Loss+of+Resident+Lung+Mesenchymal+Stem+Cells+Which+Regulate+Effector+T-Cell+Proliferation.">The Pathology of Bleomycin Induced Fibrosis is Associated with Loss of Resident Lung Mesenchymal Stem Cells Which Regulate Effector T-Cell Proliferation.</a> STEM CELLS. , (2011).</li><li>Martin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+lung+side+population+cells+have+mesenchymal+stem+cell+potential.">Adult lung side population cells have mesenchymal stem cell potential.</a> Cytotherapy. 10, 140-151 (2008).</li><li>Majka, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+progenitor+populations+in+skeletal+muscle+are+bone+marrow+derived+and+exhibit+different+cell+fates+during+vascular+regeneration.">Distinct progenitor populations in skeletal muscle are bone marrow derived and exhibit different cell fates during vascular regeneration.</a> The Journal of Clinical Investigation. 111, 71-79 (2003).</li><li>Goodell, M. A., McKinney-Freeman, S., Camargo, F. D., Helgason, D. C., Miller, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+in+Molecular+Biology.">Methods in Molecular Biology.</a> Basic Cell Culture Protocols. 290, 343-352 (2005).</li><li>Tadjali, M., Zhou, S., Rehg, J., Sorrentino, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+Isolation+of+Murine+Hematopoietic+Stem+Cells+by+Expression+of+an.">Prospective Isolation of Murine Hematopoietic Stem Cells by Expression of an.</a> Abcg2/GFP Allele. Stem Cells. 24, 1556-1563 (2006).</li><li>Zhou, S., Schuetz, J. D., Bunting, K. D., Colapietro, A. -. M., Sampath, J., Morris, J. J., Lagutina, I., Grosveld, G. C., Osawa, M., Nakauchi, H., Sorrentino, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ABC+transporter+Bcrp1/ABCG2+is+expressed+in+a+wide+variety+of+stem+cells+and+is+a+molecular+determinant+of+the+side-population+phenotype.">The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype.</a> Nature Medicine. 7, 1028-1034 (2001).</li><li>Chateauvieux, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+profile+of+mouse+stromal+mesenchymal+stem+cells.">Molecular profile of mouse stromal mesenchymal stem cells.</a> Physiological Genomics. 29, 128-138 (2007).</li><li>Gregory, C. A., Prockop, D. J., Spees, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-hematopoietic+bone+marrow+stem+cells:+Molecular+control+of+expansion+and+differentiation.">Non-hematopoietic bone marrow stem cells: Molecular control of expansion and differentiation.</a> Experimental Cell Research. 306, 330-335 (2005).</li><li>Menicanin, D., Bartold, P., Zannettino, A., Gronthos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+Profiling+of+Mesenchymal+Stem+Cells.">Genomic Profiling of Mesenchymal Stem Cells.</a> Stem Cell Reviews and Reports. 5, 36-50 (2009).</li><li>Summer, R., Fitzsimmons, K., Dwyer, D., Murphy, J., Fine, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+an+Adult+Mouse+Lung+Mesenchymal+Progenitor+Cell+Population.">Isolation of an Adult Mouse Lung Mesenchymal Progenitor Cell Population.</a> Am. J. Respir. Cell Mol. Biol. 37, 152-159 (2007).</li><li>Waid, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+T+cell+subset+described+as+CD4loCD40++T+cells+(TCD40)+in+human+type+1+diabetes.">A unique T cell subset described as CD4loCD40+ T cells (TCD40) in human type 1 diabetes.</a> Clinical Immunology. 124, 138-148 (2007).</li></ol>

אין ניגודי אינטרסים הכריז.

אנחנו צריכים להתאים שיטה המשמשת בתחילה כדי לזהות תאים hematopoietic BM לבודד אוכלוסייה ספציפית של MSC ריאות תושב. בשל שחזור של בידוד תאים אלה היו אז מאופיין גם MSC. המוצא שלהם הוגדר תושב בריאה עכבר בוגר (להבדיל BM נגזרת) ופרופיל פנוטיפי מולקולרית מתועד 2. היכולת שוב ושוב לבודד אוכלוסיה זו מתאפיינת מאפשר מחקר נוסף של חשיבות ביולוגית תפקיד של MSC הריאה במהלך הומאוסטזיס רקמה ומחלות. ההגדרה האחרונה של אוכלוסייה זו in vivo ברקמת הריאה הן Murine האדם ומקל על הפיתוח של אסטרטגיה טיפולית המכוונת להציל את התאים אנדוגני כדי להקל לתקן ריאות במהלך פציעה ומחלה.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><th> שם מגיב </th><th> חברה </th><th> מספר קטלוגי </th><th> הערות (אופציונלי) </th></tr><tr><td> פוספט בופר סליין (PBS) </td><td> סיגמא </td><td> P-5368 </td><td></td></tr><tr><td> הנקס שנאגרו תמיסת מלח (HBSS) </td><td> Thermo Scientific </td><td> SH30588.01 </td><td></td
></tr><tr><td> 0.2% וורטינגטון סוג 2 collagenase </td><td> וורטינגטון ביוכימיקלים </td><td> LS004202 </td><td></td
></tr><tr><td> תא דם אדום תמוגה חיץ </td><td> eBiosciences </td><td> 00-4333-57 </td><td></td
></tr><tr><td> DMEM </td><td> Invitrogen Gibco </td><td> 11965-092 </td><td></td
></tr><tr><td> Hoechst 33342 צבע </td><td> סיגמא </td><td> B2261 </td><td></td
></tr><tr><td> CD45-APC </td><td> BD Pharmingen </td><td> 559864 </td><td></td
></tr><tr><td> Propidium יודיד </td><td> סיגמא </td><td> 81845 </td><td></td
></tr><tr><td> , ממ </td><td> Thermo Scientific </td><td> SH30265.01 </td><td>; </td
></tr><tr><td> FBS </td><td> Invitrogen </td><td> 16000-069 </td><td></td
></tr><tr><td> 0.5% טריפסין / EDTA </td><td> Cellgro </td><td> 25-053-Cl </td><td></td
></tr><tr><td> MesenCult השלם בינוני </td><td> תא גזע טכנולוגיות </td><td> 05511 </td><td></td
></tr><tr><td> 0.4% w / v Giemsa פתרון מכתים </td><td> סיגמא </td><td> GS1L </td><td></td
></tr><tr><td> 4% paraformaldehyde </td><td> מיקרוסקופית אלקטרונים שירותים </td><td> 15710 </td><td> Paraformeldehyde 16% הוא מדולל ל -4% באמצעות PBS </td
></tr><tr><td> Carboxyfluorescein succinimidyl אסתר (CFSE) </td><td> סיגמא </td><td> 21888 </td><td></td
></tr></tbody></table>

isolation &#38; characterization of hoechstlow cd45negative mouse lung mesenchymal stem cells

תושב רקמות mesenchymal בתאי גזע (MSC) הם הרגולטורים חשוב לתיקון רקמות או התחדשות, סיסטיק דלקת, אנגיוגנזה היווצרות הגידול. יחדיו מחקרים אלה מראים כי MSC ריאות תושב לשחק תפקיד במהלך הומאוסטזיס פגיעה ברקמת הריאה, ולתקן במהלך מחלות כגון פיברוזיס ריאתי (PF) ויתר לחץ דם עורקי (PAH). כאן אנו מתארים בטכנולוגיה כדי להגדיר אוכלוסייה של MSC ריאות תושב. ההגדרה של אוכלוסייה זו ברקמת הריאה vivo באמצעות להגדיר סט של סמנים מאפשר בידוד חוזרות ונשנות של אוכלוסייה היטב מאופיינת בתאי גזע על ידי זרימת cytometry ולימוד סוג תא תפקוד ספציפי.

Watch this Scientific Journal Video about Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells at JoVE.com

Isolation &amp; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

במאמר זה אנו מדגימים את הבידוד של Murine ריאות תושב בתאי גזע mesenchymal (ריאות MSC), הרחבה שלהם, אפיון וניתוח של מאפייני המערכת החיסונית.

בידוד &amp; אפיון Hoechst נמוך CD45 שלילי עכבר mesenchymal ריאות בתאי גזע

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

isolation-characterization-hoechstlow-cd45negative-mouse-lung

Research

JoVE Journal

Biology

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

20.3K Views.  University of Colorado Denver. במאמר זה אנו מדגימים את הבידוד של Murine ריאות תושב בתאי גזע mesenchymal (ריאות MSC), הרחבה שלהם, אפיון וניתוח של מאפייני המערכת החיסונית.

Video: Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

בידוד בעלי חיים הרחבת חינם סרום של טבורי האדם נגזר תאים סטרומה mesenchymal (MSCs) ו האנדותל יצירת המושבה ובתאים (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

בידוד גזירת תאים עובריים בעכבר ז&#39;רמינל

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

בידוד של בלוטת הרוק עכבר תאים גזע

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

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.

דור של עכברים שמקורם בתאי גזע pluripotent מושרים

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

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

אינדוקציה וניתוח של אפיתל לmesenchymal מעבר

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

בידוד והתרבות של תאי גזע השיניים אפיתל ממבוגרי העכבר הארקטיק

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O2, 5% CO2 conditions. To deplete fibroblasts (CD146-) and to ensure a population of only L-MSCs (CD146+), positive selection for CD146+ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

בידוד של CD146<sup&gt; +</sup&gt; תאי סטרומה mesenchymal ריאות התושב מריאות העכברוש

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

תא הטרנס-הפנימית המוני הזרקה של תאי גזע עובריים מוביל שיעור גבוה יותר של Chimerism

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Isolation, Characterization, And High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). Therefore, assessing mitochondrial function in primary TECs may provide valuable insight into the bioenergetic status of the cells, providing insight into the pathophysiology of CKD. While there are a number of complex protocols available for the isolation and purification of proximal tubules in different species, the field lacks a cost-effective method optimized for&#160;tubular cell isolation without the need for purification. Here, we provide an isolation protocol that allows for studies focusing on both primary mouse proximal and distal renal TECs. In addition to cost-effective reagents and minimal animal procedures required in this protocol, the isolated cells maintain high energy levels after isolation and can be sub-cultured up to four&#160;passages, allowing for continuous studies. Furthermore, using a high throughput extracellular flux analyzer, we assess the mitochondrial respiration directly in the isolated TECs in a 96-well plate for which we provide recommendations for the optimization of cell density and compound concentration. These observations suggest that this protocol can be used for renal tubular ex vivo studies with a consistent, well-standardized production of renal TECs. This protocol may have broader future applications to study mitochondrial dysfunction associated with renal disorders for drug discovery or drug characterization purposes.

This protocol provides a cost-effective approach to isolate and characterize mouse primary renal tubular cells that can subsequently be sub-cultured to assess renal biological functions ex vivo, including mitochondrial bioenergetics.

ניתוח השטף חוץ-תאי בידוד, אפיון, תפוקה גבוהה של העכבר הראשי כליות צינורי בתאי אפיתל

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

בידוד, אפיון, התמיינות של תאי גזע הלב מהלב העכבר למבוגרים

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...