We thank Dr. Tadashi Koyama for technical help. This work was supported in part by JSPS KAKENHI Grant Numbers 23249045, 15K09211, 15K19172; a grant to the Respiratory Failure Research Group from the Ministry of Health, Labour and Welfare, Japan; a grant for research on allergic disease and immunology, Japan.

1. Vorbereitung und Kultur von Lungenepithelzellen <ol><li> Kultur A549 humanen Lungenepithelzellen (adhärente Zelllinie) in Dulbeccos modifiziertem Eagle-Medium (DMEM) mit 10% fötalem Rinderserum (FBS) ergänzt, 100 IU / ml Penicillin und 100 ug / ml Streptomycin. </li><li> Entfernen und entsorgen Sie die Zellkulturmedien von Kulturschale und wäscht einmal mit 5: - 10 ml phosphatgepufferter Salzlösung (PBS). Nach dem Waschen aspirieren sofort die PBS. </li><li> 2 ml Trypsin / Ethylendiamintetraessigsäure (EDTA) (0,05%) und inkubiere bei 37 ° C und 5% CO 2 für 3 min. </li><li> Sammle die abgelösten Zellen in Zentrifugenröhrchen, enthaltend Zellkulturmedium, zentrifugieren die Röhrchen bei RT für 4 min bei 150 × g. </li><li> Resuspendieren der Zellen in 2 ml Zellkulturmedium pelletieren und eine Probe für die Zellzählung entfernen. Zählen Sie die Anzahl der Zellen einer Zählkammer mit Trypanblau-Färbung mit der Lebensfähigkeit der Zellen zu überprüfen. </li><li> Seed A549-Zellen auf10 cm Polystyrol - Platten in einer Dichte von 0,5-1,0 x 10 6 Zellen / Schale mit 10 ml Medium für Gelkontraktion Assays. Seed die Zellen auf 6 - Well - Polystyrol - Platten mit einer Dichte von 0,5 bis 1,0 × 10 5 Zellen / Vertiefung mit 2 ml Medium für EMT Bestätigung. Inkubieren der Zellen bei 37 ° C und 5% CO 2 für 24 Std. </li></ol> 2. EMT Verfahren <ol><li> Zugabe von 10 &amp; mgr; l von TGF-β1 (5 ug / ml) und 10 &amp; mgr; l TNF-α (10 ug / ml) auf die Platte für Gelkontraktion Assay ausgesät in Schritt 1.6. Hinzufügen 2 ul TGF-β1 (5 ug / ml) und 2 &amp; mgr; l TNF-α (10 ug / ml) auf die Platte für EMT Bestätigung ausgesät in Schritt 1.6. Inkubieren bei 37 ° C und 5% CO 2 für 48 Stunden. </li></ol> 3. Bestätigung der EMT Verfahren durch PCR und Western Blot <ol><li> Bestätigen Änderung in der Morphologie (von Kopfsteinähnliche Form auf die Spindel) der behandelten Zellen unter Verwendung von Phasenkontrastmikroskopie. Hinweis: Normal A549 Zellen haben Cobble stone förmig und dreieckig geformte Optik , die ein Merkmal von Epithelzellen, aber nach Stimulation mit TGF-β1 und TNF-α, erscheinen die Zellen lang und spindelförmige daß ähnelt Mesenchymzellen 14,15. </li><li> Bewerten Sie die Expression eines epithelialen Marker, wie zum Beispiel E-Cadherin und mesenchymalen Marker, wie zB N-Cadherin, Vimentin und α-Glattmuskelaktin unter Verwendung von PCR oder Western-Blot. <ol><li> Extrahieren der Gesamt - RNA aus den Zellen unter Verwendung von RNA - Extraktions - Kits 16 und synthetisieren cDNA unter Verwendung von reverser Transkriptase 17 gemäß dem Protokoll des Herstellers. </li><li> Messen Sie die Expression von mRNA - Spiegel unter Verwendung von Echtzeit - PCR - System 18 und monomeren Cyaninfarbstoff PCR - Kit nach den Anweisungen des Herstellers. Die spezifischen Primer für GAPDH (Glycerinaldehyd - 3-phosphat - Dehydrogenase), ACTA2 (alpha-Actin-2), CDH1 (Cadherin-1; E-Cadherin) und VIM (Vimentin) sind in Tabelle 1 gezeigt </strong&gt;. </li><li> Lysieren die Zellen eine Lyse - Puffer (Tabelle 2) Lösung , die 1% Protease - Inhibitor - Cocktail verwendet. Messen alle Probenproteinkonzentrationen unter Verwendung eines Protein - Assay - Kit 19,20, und die gleichen Mengen an Protein zu einem Polyacrylamidgel. <ol><li> Führen SDS - Gel-Elektrophorese und halbtrockenen Transfer der Proteine ​​auf eine PVDF - Membran 21. Inkubieren Sie die Membran mit primären Antikörpern in Blocking-Puffer mit 1 - 2 Stunden. Nach der Inkubation waschen zweimal die Membran mit Waschpuffer und Inkubation der Membran 1 Stunde mit dem zweiten Antikörper. Hinweis: Lesen Sie die Materialien / Ausrüstung Tabelle für Antikörper und in diesen Studien verwendeten Verdünnungen. Siehe Tabelle 2 für die Komponenten der Blocking - Puffer. </li><li> Waschen Sie die Membran mit Waschpuffer zweimal. Machen Sie Fotos von der Membran mit der Western - Blot - Nachweis - Kit 22 eine kalte CCD - Kamera 11,23 verwenden. </li></ol></li></ol></li></ol> 4. Gelkontraktion Assay zur Beurteilung von EMT <ol><li> Aspirieren den konditionierten Medien aus dem Zellkulturgefäß, und wäscht gut mit von 5 bis 10 ml PBS abgestorbene Zellen zu entfernen. Nach dem Waschen aspirieren sofort die PBS. </li><li> 2 ml 0,05% Trypsin / EDTA und Inkubation bei 37 ° C und 5% CO 2 für 3 min. </li><li> Sammeln Sie die abgelösten Zellen in Zentrifugenröhrchen, das DMEM mit Trypsin-Inhibitor ergänzt (1 mg / ml), Zentrifuge die Röhrchen bei RT für 4 min bei 150 × g. </li><li> Mix Typ-1-Kollagen-Gel mit destilliertem Wasser, und 4 × konzentriert DMEM anpassen, um die Volumina einer Kollagenkonzentration von 1,75 mg zu erreichen / ml und 1 × DMEM Konzentration. Achten Sie darauf, während dieser Schritt das Gel-Medium auf Eis zu halten. Hinweis: Um 6 ml Gel-Medium zu machen, gut mischen 3,5 ml Typ-1-Kollagen-Gel (3 mg / ml), 1,5 ml 4 × konzentriert DMEM, und 1 ml destilliertem Wasser. </li><li> eine Probe für die Zellzählung Resuspendieren des Zellpellets in 500 &amp; mgr; l PBS und zu entfernen. Grafdie Anzahl der Zellen, die ein Hämozytometer mit Trypanblau-Färbung mit der Lebensfähigkeit der Zellen zu überprüfen. </li><li> Füge das Gelmedium das Volumen einzustellen , um eine Zelldichte von 3,0 × 10 5 Zellen zu erreichen / Vertiefung (6,0 × 10 5 Zellen / ml) und sanft , aber es schnell und ohne Gelbildung durch Pipettieren gemischt. </li><li> Dispense 0,5 ml des Gemisches in jede Vertiefung einer 24-Well nicht behandelten Platte schnell und sorgfältig gepflegte zylindrische Form zu machen. Achten Sie darauf , keine Luftblasen zu ermöglichen , die Gele (1A) zu verunreinigen. Hinweis: Das Gelmedium, die Zellen enthält, viskos ist und kann leicht gelieren und Form sichelförmige Form in dem Bohrloch. </li><li> Inkubiere die Platte für 15 min in einem Zellinkubator bei 37 ° C, 5% CO 2 und 95% Feuchtigkeit vollständig zu gelieren. </li><li> Lösen sich von der Platte Gelen ohne Brechen durch einen sterilisierten Spatel in einer Weise bewegt einen Umfang in eine Richtung zu ziehen. Mit einem Spatel übertragen sanft die Gele bis 60 mm Gewebekulturschalendie 5 ml DMEM / 1% FBS mit oder ohne TGF-β1 (5 ng / ml) und TNF-α (10 ng / ml). </li><li> Sie vorsichtig die Gerichte schütteln Gele auf dem Medium schweben zu gewährleisten. Inkubieren in einem Zellinkubator bei 37 ° C, 5% CO 2 und 95% Luftfeuchtigkeit. </li></ol> 5. Messung der Gel-Größe <ol><li> Messen der Größe Kollagengel nach 0, 24, 48 und 72 h ein Bildanalysesystem verwendet wird. <ol><li> Schalten Sie das Geldokumentationssystem (2A), und stellen Geschirr in die Lichtabschirmungs Schrank. Dann nehmen Sie den Deckel von Geschirr im Schrank. </li><li> Öffnen Sie die zugehörige Gel Analysesoftware. Klicken Sie auf die &quot;Bildaufnahme-Taste&quot; in der Menüleiste, um das Bild der Gele im Schrank zu zeigen. Klicken Sie dann auf &quot;Acquire&quot; in der Menüleiste Bilder der Gele (2B) zu nehmen. </li><li> Klicken Sie auf die &quot;Erkennung Taste&quot; in der Menüleiste (Abbildung 2C) und stellen Sie den Messbereich (gelber Kreis in 2C) durch Ziehen der Maus. Klicken Sie dann auf die &quot;Auto-detect - Taste&quot; in der Menüleiste (siehe Taste in 2D). Die Software erkennt automatisch die Gele zeigt eine Heatmap (2D). </li><li> &quot;OK&quot; klicken , zu extrahieren , die Umrisse der Gele durch Bildverarbeitung und auf Gebieten , die von der Gliederung (Abbildung 2E) umgeben berechnen. Nachdem die Fläche berechnet wird, wird die berechnete Fläche in separaten Popup-Fenster angezeigt. Hinweis: Wenn die Gele einander während der Bildgebung überlappen, Gele bewegen vorsichtig mit einer sterilen Pipettenspitze. </li></ol></li></ol>

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Die Autoren haben keine Interessenkonflikte offen zu legen.

Das Protokoll in dieser Studie entwickelt umfasst zwei Schritte. Der erste Schritt wird durchgeführt, EMT zu induzieren, während der zweite Schritt ist die Gelkontraktion Assays. Da es wichtig ist, um zu bestätigen, dass die Zellen EMT unterzog, Stufe 2 bietet eine hervorragende Ergänzung zu den morphologischen und Veränderungen der Genexpression. Frühere Studien zeigten , dass EMT von A549 - Zellen durch TGF-β1 induzierten wurde nur 24; aber, wie wir vorher 10, TNF-α - Behandlung berichtet...

Fibrose ist in der Pathogenese verschiedener chronisch progressive Erkrankungen, wie interstitielle Lungenerkrankung, Herzfibrose, Leberzirrhose, terminalem Nierenversagen, systemischer Sklerose, Autoimmunerkrankung und 1 beteiligt. Unter interstitiellen Lungenerkrankungen, ist idiopathischer pulmonaler Fibrose (IPF) ist eine chronische progressive Krankheit und zeigt eine schlechte Prognose. Pathologisches Merkmal der IPF ist die Entwicklung von fibroblastischen Foci von aktivierten Fibroblasten und Myofibroblasten besteht, die mit der Prognose assoziiert sind. Die Ursprünge solcher Lungenfibroblasten vorgeschlagen von mehreren mesenchymalen Zellen abgeleitet werden, einschließlich ursprünglich resident Lungen Fibroblasten und Fibrozyten aus Knochenmark zirkuliert. Vor kurzem epithelial-mesenchymalen Übergang (EMT) wurde mit der Bildung von mesenchymalen Zellen 2, in Verbindung gebracht werden vorgeschlagen und zur Pathogenese von fibrotischen Erkrankungen beizutragen. Es wird vermutet, dass EMT spielt eine wichtige Rolle im Prozess der fötalen Entwicklung, Wundheilung, und das Fortschreiten von Krebs, einschließlich der Tumorinvasion und Metastasierung 3. Nach dem Prozess des EMT, erhalten Epithelzellen die Fähigkeit der mesenchymalen Zellen , die durch Verlust von epithelialen Marker, wie E-Cadherin, und durch Expression von mesenchymalen Marker, wie Vimentin und α-smooth muscle actin (SMA) 4,5. Frühere Studien zeigten die Hinweise , dass EMT Verfahren hat sich mit der Entwicklung von Gewebefibrose in der Niere und der Lunge 6 7 verbunden. Darüber hinaus fördert die chronische Entzündung fibrotischen Erkrankung 8; Ferner, wie inflammatorische Zytokine wie Tumor - Nekrose - Faktor -Superfamilienmitglied 14 (TNFSF14; LIGHT), Tumor - Nekrose - Faktor (TNF) -α, und Interleukin-1β wurden EMT 9-12 gezeigt , zu verbessern. Collagen-Gel Kontraktion Assay, ein Kollagen-basierte Zellkontraktion Test, bei dem Fibroblasten in Typ I eingebettet sindKollagengel dreidimensional, ist ein ideales in vitro - Modell für die Bewertung der Kontraktilität. Kontraktionsfähigkeit ist eines der charakteristischen Funktionen von Fibroblasten und trägt zu einer normalen Wundheilung und Fibrose 13. In diesem Test wird angenommen, dass die Bindung von Fibroblasten Integrin-abhängige Mechanismen Kollagen durch sollen Typ I mechanische Spannung unter bestimmten Bedingungen zu erzeugen, und damit zur Gewebekontraktion führen. Hierbei wird die Entwicklung der Gelkontraktion Assays berichtet angepasst werden, um die Übernahme der kontraktilen Funktion in den Zellen zu bewerten, die EMT unterzog. Dieser Bericht zeigt, dass sich diese modifizierte Test zur Bewertung der Kontraktilität in mesenchymalen Zellen geeignet ist, die EMT unterzog.

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development of an in vitro assay to evaluate contractile function of mesenchymal cells that underwent epithelial-mesenchymal transition

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor &#946;, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor &#945;. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.

Während EMT, verlieren Epithelzellen epithelialen Marker, wie E-Cadherin, und gewinnen die Expression von mesenchymalen Marker, wie Vimentin und α-smooth muscle 4,5 Aktin. Inkubation von menschlichen A549 Lungenepithelzellen mit TGF-β1 und TNF-α induziert EMT. Das Erscheinungsbild der normalen A549 - Zellen sind Cobble Stein wie Form und Dreiecksform , die ein Merkmal von Epithelzellen (3A), aber nachdem sie mit TGF-β1 und TNF-α stimuliert, änderte sich...

Watch this Scientific Journal Video about Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition at JoVE.com

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

Entwicklung eines<em&gt; In Vitro</em&gt; Assay Kontraktionsfunktion von mesenchymalen Zellen, die Unterzog Epithelial-mesenchymale Transition zur Bewertung

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

development-an-vitro-assay-to-evaluate-contractile-function

Research

JoVE Journal

Developmental Biology

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

12.1K Aufrufe.  The University of Tokyo Hospital. Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

Serie: Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug &#8211; clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.

Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

Ein enzymatisches Verfahren zur mesenchymalen Stammzellen aus Knochenmarkproben Clotted Rettung

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

Forschung

Entwicklungsbiologie

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema and diminished visual acuity. However, human CECs are prone to compromised proliferative potential. Furthermore, stimulation of cell growth is often complicated by gradual endothelial-mesenchymal transition (EnMT). Therefore, understanding the mechanism of EnMT is necessary for facilitating the regeneration of CECs with competent function. In this study, we prepared a primary culture of bovine CECs by peeling the CECs with Descemet's membrane from the corneal button and demonstrated that bovine CECs exhibited the EnMT process, including phenotypic change, nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, in the in vitro culture. Furthermore, we used a rat corneal endothelium cryoinjury model to demonstrate the EnMT process in vivo. Collectively, the in vitro primary culture of bovine CECs and in vivo rat corneal endothelium cryoinjury models offers useful platforms for investigating the mechanism of EnMT.

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

A primary culture of bovine corneal endothelial cells was used to investigate the mechanism of corneal endothelial-mesenchymal transition. Furthermore, a rat corneal endothelium cryoinjury model was used to demonstrate corneal endothelial-mesenchymal transition in vivo.

In vitro und in vivo Modelle Corneal Endothelial-mesenchymale Transition zu studieren

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema ...

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. Although many efforts have been made to reveal the underlying mechanisms of decidualization, the exact signaling between the epithelial cells that are in contact with the embryo and the underlying stromal cells remains poorly understood. Therefore, studying decidualization in a way that takes both the epithelial and stromal cells into account could improve our knowledge about the molecular details of decidualization. For this purpose, in vivo models of artificial decidualization are physiologically the most relevant; however, manipulation of intercellular communication is limited. Currently, in vitro cultures of endometrial stromal cells are being used to investigate the modulation of decidualization by several signaling molecules. Conventionally, human or mouse endometrial stromal cells are used. However, the availability of human samples is very often limited. Furthermore, the use of murine tissues is accompanied with variety in the method of culturing. This study presents a validated and standardized method to obtain pure Endometrial Epithelial Cell (EEC) and Stromal Cell (ESC) cultures using adult intact mice treated with estrogen for three consecutive days. The protocol is optimized to improve the yield, viability, and purity of the cells and was further extended in order to study decidualization in a coculture of EEC and ESC. This model may be suitable to exploit the importance of both cell types in decidualization and to evaluate the contribution of significant signaling molecules secreted by EEC or ESC during the intercellular communication.

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

This study presents a standardized and validated method for the isolation and culture of primary mouse endometrial stromal and epithelial cells, which can be used in a coculture system to study in vitro decidualization.

Isolierung von Maus Endometrial Epithelial und Stromazellen für<em&gt; In Vitro</em&gt; decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. ...

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

In-vitro- Wachstum der Maus Preantral Follikel in simulierter Schwerelosigkeit

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

In-vitro- Differenzierung humaner mesenchymaler Stammzellen in funktionellen Cardiomyocyte-ähnlichen Zellen

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature maintenance and pathological malformations can result in severe ischemic diseases, while overly abundant vascular development is associated with cancer and inflammatory disorders. A promising form of pro-angiogenic therapy is the use of angiogenic cell sources, which can provide regulatory factors as well as physical support for newly developing vasculature.
Mesenchymal Stromal Cells (MSCs) are extensively investigated candidates for vascular regeneration due to their paracrine effects and their ability to detect and home to ischemic or inflamed tissues. In particular, first trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a highly promising candidate due to their pericyte-like properties, high proliferative and multilineage potential, immune-privileged properties, and robust paracrine profile. To effectively evaluate potentially angiogenic regenerative cells, it is a requisite to test them in reliable and &#34;translatable&#34; pre-clinical assays. The aortic ring assay is an ex vivo angiogenesis model that allows for easy quantification of tubular endothelial structures, provides accessory supportive cells and extracellular matrix (ECM) from the host, excludes inflammatory components, and is fast and inexpensive to set up. This is advantageous when compared to in vivo models (e.g., corneal assay, Matrigel plug assay); the aortic ring assay can track the administered cells and observe intercellular interactions while avoiding xeno-immune rejection.
We present a protocol for a novel application of the aortic ring assay, which includes human MSCs in co-cultures with developing rat aortic endothelial networks. This assay allows for the analysis of the MSC contribution to tube formation and development through physical pericyte-like interactions and of their potency for actively migrating to sites of angiogenesis, and for evaluating their ability to perform and mediate ECM processing. This protocol provides further information on changes in MSC phenotype and gene expression following co-culture.

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Here, we present a novel application of the aortic ring assay where prelabelled mesenchymal cells are co-cultured with rat aorta-derived endothelial networks. This novel method allows visualization of Mesenchymal Stromal Cells (MSCs) homing and integration with endothelial networks, quantification of network properties, and evaluation of MSC immunophenotypes and gene expression.

Die Aorta Ring Kokultur Assay: Ein komfortables Werkzeug, um angiogenen Potenzial des mesenchymaler Stromazellen Zellen In Vitro zu bewerten

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell lineages that comprise mature blood. Isolation and identification of these HSPCs is difficult because they are defined ex post facto; they can only be defined after their differentiation into specific cell lineages. Over the past few decades, the zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop ex utero, and by 48 h post-fertilization (hpf) have generated definitive HSPCs. Assays to assess HSPC differentiation and proliferation capabilities have been developed, utilizing transplantation and subsequent reconstitution of the hematopoietic system in addition to visualizing specialized transgenic lines with confocal microscopy. However, these assays are cost prohibitive, technically difficult, and time consuming for many laboratories. Development of an in vitro model to assess HSPCs would be cost effective, quicker, and present fewer difficulties compared to previously described methods, allowing laboratories to quickly assess mutagenesis and drug screens that affect HSPC biology. This novel in vitro assay to assess HSPCs is performed by plating dissociated whole zebrafish embryos and adding exogenous factors that promote only HSPC differentiation and proliferation. Embryos are dissociated into single cells and plated with HSPC-supportive colony stimulating factors that cause them to generate colony forming units (CFUs) that arise from a single progenitor cell. These assays should allow more careful examination of the molecular pathways responsible for HSPC proliferation, differentiation, and regulation, which will allow researchers to understand the underpinnings of vertebrate hematopoiesis and its dysregulation during disease.

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

Here, we present a simple method to quantitate hematopoietic stem and progenitor cells (HSPCs) in embryonic zebrafish. HSPCs from dissociated zebrafish are plated in methylcellulose with supportive factors, differentiating into mature blood. This allows the detection of blood defects and allows drug screening to be easily conducted.

Entwicklung eines In-vitro- Assay, hämatopoetischen Stammzellen und Vorläuferzellen Quantitate Zellen (HSPCs) bei der Entwicklung von Zebrafish Embryos

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

Computergestützte Analyse der Keimbahn Caenorhabditis Elegans , die Verteilung der Kerne, Proteine und das Zytoskelett zu studieren

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In vitro-Generation der Somiten Derivate aus menschlichen induzierten Zellen pluripotenten Stammzellen

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

Bewertung von oxidativen Schäden in den primären Maus-Augen-Oberflächenzellen/Stammzellen als Reaktion auf Uv-C-Schäden (UV-C)

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...