Diese Arbeit wurde durch ein Stipendium des National Institute of Arthritis and Muskel-Skelett-Skin Diseases, NIH RO1AR054458 unterstützt.

1. Kennzeichnung von hMSCs mit Endorem <ol><li> Die Zellen sind zu 80% Konfluenz mindestens 18 Stunden vor der Etikettierung gewachsen. </li><li> Während dieser Zeit ist die Kennzeichnung Medien, indem Endorem, um die Probe in einer Dosis von 100 ug Fe / ml Serum-freien Medien vorbereitet. </li><li> Nachdem sich die Zellen bis zur Konfluenz gezüchtet werden, ist Kulturmedien abgesaugt und die Zellen werden mit PBS gewaschen oder Serum-freien Medien 1x. </li><li> Als nächstes wird die Spüllösung abgesaugt und die vorbereitete Beschriftung Medien aufgenommen. Die Zellen werden dann bei 37 ° C, 5% CO 2 für 4 Stunden. </li><li> Nach 4 Stunden Inkubation wird die Kennzeichnung Medien abgesaugt und die Zellen werden mit PBS gespült und mit Trypsin behandelt wie gewohnt. </li><li> Sobald trypsiniert werden die Zellen mit PBS gewaschen 3x und zentrifugiert bei 400rcf für 5 Minuten. </li><li> Nach diesem Waschschritt werden die Zellen resuspendiert, gezählt, geprüft für die Lebensfähigkeit, und als für Experimente benötigt. </li></ol> 2. Vorbereitung Agarose <ol><li> Nachdem die Zellen markiert wurden, ist es Zeit, um die Agarose-Lösung vorzubereiten. </li><li> An einem schönen Skala werden 80 mg Agarose Pulver gewogen und in 2 ml PBS. </li><li> Diese Lösung wird dann sanft geschüttelt und autoklaviert. </li><li> Nach ca. 40 min, kann die Flüssigkeit Agarose-Lösung aus dem Autoklaven entnommen werden. Das Volumen sollte gemessen und dementsprechend mit vorgewärmter PBS eingestellt. </li><li> Dann wird die Lösung auf 42 ° C abgekühlt </li></ol> 3. Erstellen MASI <ol><li> Wenn die Agarose-Lösung hat die gewünschte Temperatur erreicht ist, werden die Zellen gezählt und zentrifugiert zum letzten Mal in einem 1,5 ml Eppendorf-Röhrchen. </li><li> Nach der Zentrifugation wird der Überstand verworfen und die Zellen werden mit dem DMEM gemischt, um eine Konzentration von 30 x 10 6 Zellen / ml erreichen. </li><li> Nun werden die Zellen mit dem 42 ° C warme Agarose gemischt. </li><li> Ein kalter Pipettenspitze kann Agarose auf in der Spitze zu verfestigen, daher ist es zur Vorwärmung der Spitze durch Pipettieren heißen sterile PBS nach oben und unten empfohlen </li><li> Nun, das gleiche Volumen an Agarose als Medien aufnimmt und gründlich mit der Zelle-media-Suspension gemischt, so dass eine homogene Suspension entsteht. </li><li> Halten Sie die Eppendorf-Röhrchen in einem vorgeheizten Wasserbad beim Mischen, so dass die Agarose nicht abkühlen genug, um zu erstarren. </li><li> Wenn die Suspension homogen ist, sind die Zellen in den Defekt implantiert. </li></ol><img src="/files/ftp_upload/1793/1793fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Koronare T2-gewichteten SE-Bildern (TR 4000 ms / TE 18,27 ms) einer Patella Probe mit implantierten hMSCs in Knorpeldefekte.

<ol>
	<li>Mauck, R. L., Yuan, X., Tuan, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondrogenic+differentiation+and+functional+maturation+of+bovine+mesenchymal+stem+cells+in+long-term+agarose+culture.">Chondrogenic differentiation and functional maturation of bovine mesenchymal stem cells in long-term agarose culture.</a> Osteoarthritis Cartilage. 14, 179-189 (2006).</li><li>Pittenger, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilineage+potential+of+adult+human+mesenchymal+stem+cells.">Multilineage potential of adult human mesenchymal stem cells.</a> Science. 284, 143-147 (1999).</li><li>Rahfoth, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+allograft+chondrocytes+embedded+in+agarose+gel+into+cartilage+defects+of+rabbits.">Transplantation of allograft chondrocytes embedded in agarose gel into cartilage defects of rabbits.</a> Osteoarthritis Cartilage. 6, 50-65 (1998).</li><li>Henning, T. D., Boddington, S., Daldrup-Link, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labeling+hESCs+and+hMSCs+with+iron+oxide+nanoparticles+for+non-invasive+in+vivo+tracking+with+MR+imaging.">Labeling hESCs and hMSCs with iron oxide nanoparticles for non-invasive in vivo tracking with MR imaging.</a> J Vis Exp. , (2008).</li></ol>

Das beschriebene Protokoll bietet eine reproduzierbare Möglichkeit, MSCs mit Eisenoxid-Nanopartikel-Label und Implantat dieser markierten MSCs in Knorpeldefekte. Diese Technik ermöglicht nicht-invasive Darstellung von Stammzell-Transplantationen in Knorpeldefekte mit der MR-Bildgebung, die eine frühzeitige Erkennung von MASI Fehler erlaubt. Eine Dislokation oder Efflux der markierten Zellen können basierend auf einem Verschwinden des Etiketts von der Transplantation vor Ort auf MR-Bildern diagnostiziert werden. Eine...

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		<th>Material Name</th>
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		<th>Catalogue Number</th>
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<td>D-MEM High Glucose</td>
<td>Reagent</td>
<td>Sigma</td>
<td>D5648</td>
<td>&nbsp;</td>
<tr>
<td>PBS (Ca++, Mg++ free)</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>14190-144</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin-EDTA 0.05%</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25399-120</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30071.03</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>15140-122</td>
<td>&nbsp;</td>
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<td>Ferumoxides (Endorem)</td>
<td>
</td>
<td>Guerbet</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Agarose Typ VII</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A9045</td>
<td>Dilute Agarose in PBS to final concentration of 4%</td>
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implantation of ferumoxides labeled human mesenchymal stem cells in cartilage defects

Das Gebiet des Tissue Engineering integriert die Prinzipien der Ingenieur-, Zell-Biologie und der Medizin auf die Regeneration von bestimmten Zellen und funktionelle Gewebe. Matrix verbunden Stammzellen Implantate (MASI) sollen Knorpeldefekte durch arthritischen oder traumatische Gelenkverletzungen zu regenerieren. Adulten mesenchymalen Stammzellen (MSCs) haben die Fähigkeit, in Zellen des chondrogene Linie zu unterscheiden und haben vielversprechende Ergebnisse für zellbasierte Gelenkknorpel Reparatur-Technologien. Autologe MSCs können aus einer Vielzahl von Geweben isoliert werden, können in Zellkulturen, ohne ihre Differenzierungspotenzial erweitert werden, und haben gezeigt, chondrogene Differenzierung in vitro und in vivo 1, 2. Um lokale Speicherung und Lebensfähigkeit der transplantierten MSCs in Knorpeldefekte bieten, ist ein Gerüst benötigt, das auch spätere Differenzierung und Proliferation. Die Architektur des Gerüsts führt Gewebebildung und ermöglicht die extrazelluläre Matrix, die von den Stammzellen produzierte, zu erweitern. Frühere Untersuchungen haben gezeigt, dass ein 2% iges Gerüst kann die Entwicklung von stabilen hyalinen Knorpel zu unterstützen und nicht induziert Immunreaktionen drei. Langfristige Aufbewahrung von transplantierten Stammzellen in MASI ist entscheidend für die Knorpelregeneration. Kennzeichnung von MSCs mit Eisenoxid-Nanopartikel können für langfristige In-vivo-Tracking mit nicht-invasiven MR Bildgebung 4. Diese Präsentation wird zeigen, Techniken zur Kennzeichnung MSCs mit Eisenoxid-Nanopartikel, die Erzeugung von Zell-Agarose-Konstrukte und die Implantation dieser Konstrukte in Knorpeldefekte. Die markierten Konstrukte können nicht-invasiv mit MR-Imaging verfolgt werden.

Watch this Scientific Journal Video about Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects at JoVE.com

Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

Ziel der Präsentation ist es, eine hohe Reproduzierbarkeit der Methode zur Matrix verbunden Stammzellen Implantate in Knorpeldefekte, die visualisiert werden MR-Bildgebung erzeugen kann demonstrieren. Stammzellen sind mit von der FDA zugelassene Ferumoxides mit Agarose, implantiert in Knorpeldefekte und abgebildet mit einem 7T MR-Scanner bezeichnet.

Die Implantation von Ferumoxides Labeled humanen mesenchymalen Stammzellen in Knorpeldefekte

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and ...

implantation-ferumoxides-labeled-human-mesenchymal-stem-cells

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JoVE Journal

Biology

10.5K Aufrufe.  Medical Center, University of California San Francisco. Ziel der Präsentation ist es, eine hohe Reproduzierbarkeit der Methode zur Matrix verbunden Stammzellen Implantate in Knorpeldefekte, die visualisiert werden MR-Bildgebung erzeugen kann demonstrieren. Stammzellen sind mit von der FDA zugelassene Ferumoxides mit Agarose, implantiert in Knorpeldefekte und abgebildet mit einem 7T MR-Scanner bezeichnet.

Serie: Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

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.

Isolation und tierisches Serum Freie Expansion der menschlichen Nabelschnur Derived mesenchymale Stromazellen (MSCs) und Endothelial Colony Forming Progenitorzellen (ECFCs)

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

Forschung

Biologie

Peptides from Phage Display Library Modulate Gene Expression in Mesenchymal Cells and Potentiate Osteogenesis in Unicortical Bone Defects

Two novel synthetic peptides accelerate bone formation and can be delivered using a collagen matrix. The aim of this study was to investigate the effects on bone repair in a unicortical defect model. Treatment of mesenchymal cells produced an increase in alkaline phosphatase activity, showed nodule formation by the cells, and increased the expression of genes for runx2, osterix, bone sialoprotein, and osteocalcin. A collagen sponge soaked with peptide promoted repair of bone defects, whereas the control was less effective. The results from this study demonstrated that mesenchymal cells treated with peptide in vitro differentiate towards osteogenesis, and, that peptides delivered in vivo using a collagen sponge promote the repair of unicortical defects.

A phage display library was used to identify peptide sequences that target bone. The objective was to investigate the effect of these peptides on mesenchymal cell differentiation and to determine their effect on bone regeneration.

Peptide aus Phage Display Bibliothek Genexpression modulieren in mesenchymalen Zellen und potenzieren Osteogenesis in unikortikale Knochendefekten

Two novel synthetic peptides accelerate bone formation and can be delivered using a collagen matrix. The aim of this study was to investigate the effects ...

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

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

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

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

Reprogrammierung menschlichen Körperzellen in induzierte pluripotente Stammzellen (IPSCs) mit retroviralen Vektoren mit GFP

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

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.

Induktion und Analyse von epithelialen zu mesenchymalen Transition

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.

Isolation und Kultur der Dental epithelialen Stammzellen aus dem erwachsenen Maus Schneidezahn

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

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

Effiziente Erzeugung Menschen induzierten pluripotenten Stammzellen aus menschlichen Körperzellen mit Sendai-Virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

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

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

Profiling Individuelle humanen embryonalen Stammzellen durch quantitative RT-PCR

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

A Simple Benchtop Filtration Method to Isolate Small Extracellular Vesicles from Human Mesenchymal Stem Cells

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this isolation method is relatively lower, and these methods are inefficient in separating sEV subtypes. This study demonstrates a simple benchtop filtration method to isolate human umbilical cord-derived MSC small extracellular vesicles (hUC-MSC-sEVs), successfully separated by ultrafiltration from the conditioned medium of hUC-MSCs. The size distribution, protein concentration, exosomal markers (CD9, CD81, TSG101), and morphology of the isolated hUC-MSC-sEVs were characterized with nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively. The isolated hUC-MSC-sEVs&#39; size was 30-200 nm, with a particle concentration of 7.75 &#215; 1010 particles/mL and a protein concentration of 80 &#181;g/mL. Positive bands for exosomal markers CD9, CD81, and TSG101 were observed. This study showed that hUC-MSC-sEVs were successfully isolated from hUC-MSCs conditioned medium, and characterization showed that the isolated product fulfilled the criteria mentioned by Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV 2018).

This protocol demonstrates how to isolate human umbilical cord-derived mesenchymal stem cells&#39; small extracellular vesicles (hUC-MSC-sEVs) with a simple lab-scale benchtop setting. The size distribution, protein concentration, sEVs markers, and morphology of isolated hUC-MSC-sEVs are characterized by nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively.

Eine einfache Tischfiltrationsmethode zur Isolierung kleiner extrazellulärer Vesikel aus humanen mesenchymalen Stammzellen

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this ...

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological regulators of organismal homeostasis and important indicators of pathologies; in the meantime, their immense potential to establish accessible and controllable disease therapeutics is emerging. Mesenchymal stem cells (MSCs) can release large amounts of EVs in culture, which have shown promise to jumpstart effective tissue regeneration and facilitate extensive therapeutic applications with good scalability and reproducibility. There is a growing demand for simple and effective protocols for collecting and applying MSC-EVs. Here, a detailed protocol is provided based on differential centrifugation to isolate and characterize representative EVs from cultured human MSCs, exosomes, and microvesicles for further applications. The adaptability of this method is shown for a series of downstream approaches, such as labeling, local transplantation, and systemic injection. The implementation of this procedure will address the need for simple and reliable MSC-EVs collection and application in translational research.

The present protocol describes the differential centrifugation for isolating and characterizing representative EVs (exosomes and microvesicles) from cultured human MSCs. Further applications of these EVs are also explained in this article.

Isolierung, Charakterisierung und therapeutische Anwendung extrazellulärer Vesikel aus kultivierten humanen mesenchymalen Stammzellen

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological ...

Isolierung und Charakterisierung von Stammzellen aus Fettgewebe

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in addition to their great capacity for self-renewal, migration, and proliferation. Adipose tissue is one of the least invasive and most accessible sources of mesenchymal cells. It has also been reported to have higher yields compared to other sources, as well as superior immunomodulatory properties. Recently, different procedures for obtaining adult mesenchymal cells from different tissue sources and animal species have been published. After evaluating the criteria of some authors, we standardized a methodology that is applicable to different purposes and easily reproducible. A pool of stromal vascular fraction (SVF) from perirenal and epididymal adipose tissue allowed us to develop primary cultures with optimal morphology and functionality. The cells were observed adhered to the plastic surface for 24 h, and exhibited a fibroblast-like morphology, with prolongations and a tendency to form colonies. Flow cytometry (FC) and immunofluorescence (IF) techniques were used to assess the expression of the membrane markers CD105, CD9, CD63, CD31, and CD34. The ability of adipose-derived stem cells (ASCs) to differentiate into the adipogenic lineage was also assessed using a cocktail of factors (4 &#181;M insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 &#181;M dexamethasone). After 48 h, a gradual loss of fibroblastoid morphology was observed, and at 12 days, the presence of lipid droplets positive to oil red staining was confirmed. In summary, a procedure is proposed to obtain optimal and functional ASC cultures for application in regenerative medicine.

Autor im Rampenlicht: Isolierung und Identifizierung mesenchymaler Stammzellen aus Fettgewebe von Sprague-Dawley-Ratten  

This protocol describes a methodology for isolating and identifying adipose tissue-derived mesenchymal stem cells (MSCs) from Sprague Dawley rats.

Isolierung und Identifizierung von mesenchymalen Stammzellen aus dem Fettgewebe von Sprague-Dawley-Ratten

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...