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. Hazırlıklar ve Kültür Akciğer Epitel Hücreleri <ol><li> Dulbecco Modifiye Edilmiş Eagle Ortamı (DMEM) içinde kültür A549 insan akciğer epitel hücreleri (yapışık hücre çizgisi), 100 ug / ml streptomisin ve% 10 fetal sığır serumu (FBS), 100 lU / ml penisilin, ve. </li><li> Çıkarın ve Kültür bir tabaktan alınan hücre kültürü ortamı atmak ve 5 ile bir kez yıkayın - fosfat tamponlu tuzlu su, 10 ml (PBS). Yıkandıktan sonra, hemen PBS aspire. </li><li> 3 dakika boyunca CO2 2 ml tripsin / etilendiamintetraasetik asit (EDTA) (% 0.05) ilave edin ve 37 ° C&#39;de inkübe ve% 5. </li><li> Hücre kültürü ortamı, santrifüj 150 x g&#39;de 4 dakika boyunca oda sıcaklığında tüpler ihtiva eden santrifüj tüplerine müstakil hücreleri toplamak. </li><li> hücreler, hücre kültür ortamının 2 ml pelet ve hücre sayımı için bir örnek çıkarın. hücre canlılığı kontrol etmek için Tripan mavi leke hemasitometre kullanarak hücrelerin sayısını. </li><li> Tohum A549 hücreleri0.5 bir yoğunlukta en az 10 cm polistiren plakaları - jel daralma deneyi için ortam, 10 ml 1.0 x 10 6 hücre / çanak. 0.5 yoğunluğunda 6 oyuklu polistiren plâkalar üzerine hücreleri Tohum - 1.0 x 10 5 hücre / göz ile EMT onay orta 2 mi. 24 saat boyunca CO2 37 ° C&#39;de inkübe hücreleri ve% 5. </li></ol> 2. EMT Prosedürü <ol><li> Aşama 1.6 tohumlandı jel daralma deneyi için plakaya TGF-ß1 (5 ug / ml) 10 ul ve TNF-a, 10 ul (10 ug / ml) eklenir. Aşama 1.6 tohumlandı EMT onay plakasına TGF-ß1 (5 ug / ml) 2 ul ve TNF-a 2 ul (10 ug / ml) eklenir. 48 saat boyunca CO2 37 ° C&#39;de inkübe edilir ve% 5. </li></ol> PCR ve Western Blot ile EMT Usul 3. Onay <ol><li> Faz kontrast mikroskopi kullanılarak tedavi hücrelerin (şekil mili için çakıl taşı gibi itibaren) morfoloji değişikliği onaylayın. Not: Normark A549 hücreleri, epitel hücreleri bir özelliğidir çakıl taşı gibi ve üçgen şekilli bir görünüşe sahip, ancak, TGF-ß1 ve TNF-a ile uyarılmasından sonra hücreler uzun görünür ve mil bu mezenkimal hücreler 14,15 benzer şekilli. </li><li> Bu E-kadherin gibi bir epitel işaretleyici ekspresyonunu değerlendirmek ve PCR ve Batı lekeleme ile örneğin N-kaderin, vimentin ve α-düz kas aktin gibi mezenşimal belirteçleri. <ol><li> RNA özütleme kiti 16 ile hücrelerden toplam RNA ekstrakte edin ve üreticinin protokolüne uygun olarak, ters transkriptaz 17 kullanarak cDNA sentezlemek. </li><li> Gerçek zamanlı PCR sistemi 18 ve üreticilerin talimatlarına göre monomerik siyanin boyası PCR kiti kullanılarak mRNA seviyelerinin ifadesini ölçün. GAPDH için spesifik primerler (gliseraldehid 3-fosfat dehidrojenaz), ACTA2 (alfa-aktin-2), CDH1 (kaderin-1; E-kadherin) ve VIM (vimentin), Tablo 1 &#39;de gösterilmiştir </st&gt; Rong. </li><li> Lyse bir parçalama tamponu (Tablo 2)% 1 proteaz önleyici kokteyl içeren çözelti ile hücreleri. Bir protein tahlil kiti 19,20 kullanarak tüm numune protein konsantrasyonlarını ölçmek ve bir poliakrilamid jel protein aynı miktarda uygulanır. <ol><li> SDS jel-elektroforez ve PVDF membranı 21 proteinlerin yarı-kuru transferi gerçekleştirin. 2 saat 1 - blokaj tamponunda primer antikorlar ile membran inkübe edin. İnkübasyondan sonra yıkama tamponu ile iki defa zarın yıkanması ve ikinci antikor ile membran 1 saat inkübe edilir. Not: Bu çalışmalarda kullanılan antikorlar ve seyreltileri malzemeleri / donatım tabloya bakınız. Bloke edici tampon bileşenleri için Tablo 2&#39;ye bakınız. </li><li> iki kez yıkama tamponu ile membran yıkayın. Soğuk CCD kamera 11,23 kullanarak Western lekeleme tespit kiti 22 ile membran fotoğraf çekmek. </li></ol></li></ol></li></ol> 4. Değerlendirilmesi EMT için Jel Daralma Deneyi <ol><li> Hücre kültürü kabından koşullu ortam aspire ve 5 ile iyice yıkayın - ölü hücreleri çıkarmak için 10 ml PBS. Yıkandıktan sonra, hemen PBS aspire. </li><li> 3 dakika boyunca CO2,% 0.05 tripsin / EDTA 2 ml ilave edilir ve 37 ° C&#39;de inkübe ve% 5. </li><li> DMEM 150 x g&#39;de 4 dakika boyunca oda sıcaklığında tripsin inhibitörü (1 mg / ml), santrifüj tüpleri ile takviye içeren santrifüj tüplerine müstakil hücreleri toplamak. </li><li> damıtılmış su olan tip 1 kollajen jel karıştırın ve DMEM konsantre edildi 4 x 1.75 mg / ml&#39;lik bir konsantrasyon elde etmek için kolajen seviyesini ayarlamak için ve 1 x DMEM konsantrasyonu. Bu adımı sırasında buz üzerinde jel ortamı tutmak için emin olun. Not: Jel ortamı 6 mi yapmak için, iyi 3.5 mi tip 1 kollajen jel (3 mg / ml) 1.5 ila 4 x konsantre edildi mL DMEM ve 1 ml damıtılmış su karıştırılır. </li><li> 500 ul PBS hücre pelletini ve hücre sayımı için bir örnek çıkarın. OrtakTripan mavi boyası ile bir hemositometre kullanılarak hücre sayısı, hücre canlılığı ile kontrol edin. </li><li> Yavaşça, bir hücre 3.0 x 10 5 hücre yoğunluğu / oyuk (6.0 x 10 5 hücre / ml) ve elde ancak hızla jelleşme olmadan pipetleme ile karıştırarak seviyesini ayarlamak için jel orta ekleyin. </li><li> düzgün silindir biçiminde yapmak için hızlı ve dikkatli bir şekilde 24 oyuklu işlenmemiş plakanın her oyuğuna 0.5 ml koyun. Herhangi bir hava kabarcıkları jeller (Şekil 1A) kirletmesine izin vermemek için dikkatli olun. hücreleri içeren jel orta viskoz ve kolayca kuyuda jel ve form hilal şekli olabilir: Not. </li><li> 37 ° C&#39;de bir hücre kuluçka makinesi içinde, 15 dakika boyunca inkübasyona bırakılır,% 5 CO2 ve% 95 nem, tamamen jel. </li><li> Bir yönde bir çevresi çekmek için bir şekilde sterilize edilmiş bir spatula taşıyarak bozmadan plaka jeller ayırın. Bir spatula kullanarak, yavaşça 60 mm doku kültürü yemekleri jeller aktarmakya da TGF-ß1 (5 ng / ml) ve TNF-a (10 ng / ml) olmadan DMEM /% 1 FBS, 5 ml ihtiva etmektedir. </li><li> Yavaşça jeller ortamda yüzen sağlamak için yemekler sallayın. 37 ° C&#39;de,% 5 CO2 ve% 95 nemde bir hücre kuluçka makinesi içinde inkübe edin. </li></ol> Jel Boyutu 5. Ölçüm <ol><li> bir görüntü analiz sistemi kullanılarak, 0, 24, 48, ve 72 saat sonra kolajen jel boyutu ölçülür. <ol><li> Jel dokümantasyon sistemi (Şekil 2A) açın ve ışık geçirmeyen bir kabinin içine yemekler koymak. Sonra kabine yemeklerin kapağını çıkarmak. </li><li> İlgili jel analiz yazılımı açın. kabine jellerin görüntü göstermek için menü çubuğunda &quot;görüntü elde etme lütfen&quot; tıklayın. Sonra, jeller (Şekil 2B) fotoğraf çekmek için menü çubuğunda &quot;acquire&quot; tıklayın. </li><li> Ölçüm bölgesini menü çubuğunda (Şekil 2C) &quot;algılama düğmesini&quot; tıklayın ve ayarlamak (sarı daire ifareyi sürükleyerek n Şekil 2C). Ardından, menü çubuğunda &quot;otomatik algılama düğmesi&quot; (Şekil 2B düğmesini bakınız). Yazılım otomatik olarak bir ısı haritası (Şekil 2B) gösteren jeller algılar. </li><li> Görüntü işleme ile jellerin taslağını çıkarmak ve anahat (Şekil 2E) ile çevrili alanlar hesaplamak için &quot;Tamam&quot; a tıklayın. Alan hesaplandıktan sonra, hesaplanan alan ayrı bir pop-up penceresi görüntülenir. Not: jeller görüntüleme sırasında birbirleriyle örtüşen, hafifçe steril bir pipet kullanarak jeller taşıyın. </li></ol></li></ol>

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Yazarlar ifşa etmek herhangi bir çıkar çatışmaları var.

Bu çalışmada geliştirilen protokol iki adımları içerir. İlk adım, ikinci bir basamaklı bir jel tahlili daralma ise, EMT indüklemek için gerçekleştirilir. hücrelerin EMT uygulanan onaylamak için önemli olduğundan, adım 2 morfolojik ve gen ekspresyon değişiklikleri için mükemmel bir tamamlayıcı sağlar. Daha önceki çalışmalar, A549 hücrelerinin EMT, TGF-ß1 sadece 24 tarafından indüklendiğini göstermiştir; Daha önce 10 rapor ettiği gibi, ancak, TNF-α tedavisi EMT...

Fibroz, interstisyel akciğer hastalığı, kardiyak fibroz, karaciğer sirozu, böbrek yetmezliği, terminal, sistemik skleroz, otoimmün hastalık 1 gibi çeşitli kronik progresif hastalıkların patogenezinde rol oynamaktadır. interstisyel akciğer hastalıkları arasında, idiyopatik pulmoner fibrozis (IPF) kronik ilerleyici bir hastalıktır ve kötü prognozu gösterir. IPF patolojik bulgusudur prognoz ile ilişkili olduğu aktive fibroblast ve miyofibroblastlar oluşan fibroblastik odaklar geliştirilmesidir. Bu tür akciğer fibroblast kökenleri aslen ikamet akciğer fibroblastlar dahil olmak üzere ve kemik iliği fibrositlerden dolaşan birkaç mezenkimal hücrelerden, elde edilecek önerilmektedir. Son zamanlarda, epitelyal mezenkimal geçişi (EMT) mezenkimal hücreler 2 oluşumu ile ilişkili olduğu öne sürülmüştür ve fibrotik hastalıkların patogenezine katkıda bulunmak. Bu düşünülmektedir EMT tümör invazyonu ve metastaz 3 olmak üzere fetal gelişim sürecinde önemli roller, yara iyileşmesi ve kanserin ilerlemesini, oynar. EMT süreci takiben, epitel hücreler, E-kadherin gibi epitelyal belirteçleri kaybıyla mezenkimal hücrelerin yeteneği elde etmek ve örneğin vimentin gibi mezenşimal belirteçleri ve α-düz kas aktini (SMA) 4,5 ekspresyonuyla. Önceki çalışmalar EMT süreç böbrek 6 ve akciğer 7 doku fibrozis gelişimi ile ilişkili olduğunu kanıt gösterdi. Ayrıca, kronik inflamasyon fibrotik hastalığı 8 teşvik; Bundan başka, tümör nekroz faktörü üst aile üyesini 14 (TNFSF14; LIGHT) gibi enflamatuar sitokinler, tümör nekroz faktörü (TNF) -a ve interlökin-1, EMT 9-12 geliştirmek için gösterilmiştir. Kollajen jel daralma tahlili, fibroblastlar tip I gömülü olduğu bir kollajen bazlı hücre kasılması deneyikolajen jeli üç boyutlu, kontraktilite değerlendirilmesi için in vitro model bir idealdir. Kontraktilite fibroblast karakteristik işlevlerinden biridir ve normal yara onarımı ve fibrozis 13 katkıda bulunur. Bu deneyde, fibroblast ek bazı koşullar altında, mekanik gerilim üretilmesi için gereken I integrin bağlantılı mekanizmalar vasıtasıyla kolajen tip, ve bunun sonucu olarak doku daralmaya yol düşünülmektedir. Burada, jel daralma deneyinin geliştirilmesi EMT yapılan hücrelerde kontraktil fonksiyonun satın değerlendirmek üzere uyarlanmış olduğu bildirilmektedir. Bu raporda, bu modifiye edilmiş deney EMT uygulanan mezenkimal hücreler kontraktiliteııin değerlendirilmesi için uygun olduğunu göstermektedir.

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			<td height="21" style="height:21px;width:216px;">Gel Documentation System&#160;</td>
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			<td>Gel imager</td>
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			<td height="42" style="height:42px;width:216px;">gel analyzing software</td>
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			<td style="width:119px;">Densitograph, ver. 3.00</td>
			<td>analysing software bundled with AE-6911FXN</td>
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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.

EMT sırasında, epitel hücreleri E-kaderin gibi epitel belirteçleri, kaybetmek ve bu vimentin ve α-düz kas 4,5 aktin olarak mezenkimal belirteçler, ifadesini kazanır. TGF-ß1 ve TNF-a ile A549 insan akciğer epitel hücrelerine inkübasyonu EMT indükler. Normal A549 hücrelerinin görünümü epitel hücreleri (Şekil 3A) bir özelliğidir şekil ve üçgen şekli gibi çakıl taşı, ancak, TGF-ß1 ve TNF-a ile uyarılmış sonra görünümü mezenşi...

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.

Bir Gelişimi<em&gt; İn Vitro</em&gt; Deney Mezenkimal Hücreleri Uyguladığımız Epitel-Mezenkimal Geçiş kasılma Fonksiyonu değerlendirin

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 Görüntüleme.  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.

Video: 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.

Bir Enzimatik Yöntem Kaymaklı Kemik İliği Örneklerinden Mezenkimal Kök Hücreler Kurtarma

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

Gelişim Biyolojisi

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.

Vitro ve in vivo Modelleri Kornea Endotel-mezenkimal Geçiş Çalışması için

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.

Fare Endometrial stromal ve epitelyal hücrelerin izolasyonu için<em&gt; İn Vitro</em&gt; sidualizasyonu

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.

Vitro Simüle mikrogravite altında fare Preantral follikül gelişimi

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.

Vitro İnsan Mezenkimal Kök hücre farklılaşma fonksiyonel Cardiomyocyte benzeri hücre içine

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.

Aort yüzük ortak kültür tahlil: Mezenkimal Stromal hücre Vitro anjiogenik potansiyelini değerlendirmek için uygun bir araç

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.

Zebra balığı embriyo geliştirilmesinde bir Vitro tahlil hematopoetik kök ve yaratıcı Quantitate gelişimi (HSPCs) hücreleri

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.

Çekirdeklerin dağıtım, proteinler ve sitoiskeleti çalışmaya Caenorhabditis elegans Germline sayısal analizi

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.

Somite türevleri indüklenen insan nesil Vitro Pluripotent kök hücreler

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.

Ultraviyole-C (UV-C) Hasarına Yanıt Olarak Birincil Fare Oküler Yüzey Hücrelerinde/Kök Hücrelerinde Oksidatif Hasarın Değerlendirilmesi

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