Name: generation of human adipose stem cells through dedifferentiation of mature adipocytes in ceiling cultures
Uploaded: 2015-03-07T14:00:00.000+00:00
Duration: 7 min 35 s
Description: Watch this Scientific Journal Video about Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures at JoVE.com

This study was supported by Natural Sciences and Engineering Research Council of Canada Discovery Grant (371697-2011, AT). The authors want to acknowledge the help of bariatric surgeons Drs S. Biron, F-S. Hould, S. Lebel, O. Lescelleur, P. Marceau as well as Christine Racine and Caroline Gagnon from the IUCPQ Tissue Bank. We thank Mr Jacques Cadorette from the IUCPQ&#8217;s audiovisual services for video shooting and editing.

Etik deyimi: Proje öncesinde hasta alımına IUCPQ Araştırma Etik Kurulu tarafından onaylanmıştır. Bu haber / video amaçla, 2 hastada dokuların elde: 1) 57 kg BMI ile 50,7 kg / m 2 ve 2) 35 yaşındaki kadın hasta bir BMI ile 62 yaşındaki erkek hasta, / 2 m. Deneyler hem yağ bölmeleri ile yapılabilir, ancak bu videonun amacı için bir yağ bölmeye sınırlı kalmıştır. Videonun teknik özellikleri, hastanın 1 ile yapıldı ve FABP4 immünofloresan hasta 2 dediferansiye hücreleri ile gerçekleştirildi. 1. Örnek İşleme <ol><li> Laparoskopik obezite cerrahisi sırasında omentum ve deri altı yağ bölmelerden yağ dokusu toplamak için cerrahlara isteyin. </li><li> Çabuk oda sıcaklığında laboratuvara yağ numuneleri getirmek ve hemen işleme. </li><li> Steril olmayan bir atmosferde, laboratuarda sindirim yapın.hücreler daha sonra kültür odasına aktarılır ve, steril koşullar altında kültive edilir. Kontaminasyonu önlemek için, sindirim önce distile ve filtre su ile KRH tampon hazırlamak ve bir filtrasyon (0.22 um filtresi) ile izleyin. Şişeye ve plaka hazırlanması için hücre kültürü kaputu etanol önce transferi ile iyice temizleyin tüpler. </li><li> Önceden ağırlıklı çanak ve kayıt ağırlığı yağ dokusu yerleştirin. Parafine önce en az 24 saat boyunca oda sıcaklığında% 10 formalin tamponu içinde her bir doku numunesi (yaklaşık 1 cm2), bir parça düzeltildi. Immünohistokimyası deneyler için bu gömülü örneği (gösterilmemiştir tekniği) kullanın. </li><li> (- Teknik gösterilmemiştir örneğin, gen ekspresyonu) bütün yağ dokuları üzerinde ileri çalışmalar için -80 ° C&#39;de depolamadan önce sıvı azot içinde bir 50 ml tüp ve flash dondurularak başka bir parça yerleştirin. </li></ol> 2. kollajenaz sindirimi <ol><li> 50 ml&#39;lik tu kalan yağ dokusu parçası yerleştirinsindirim için olabilir. </li><li> Sindirme tüpü numunenin gramı başına kolajenaz (350 U / ml) ile takviye edilmiş KRH-WB 4 ml ilave edilir. </li><li> Makasla yağ dokusu kıyma. </li><li> 45 dakikalık bir kuluçka süresinin (en fazla 1 saat), bir çalkalayıcıda olmak üzere 37 ° C, 90 rpm, en fazla, kıyılmış adipoz doku süspansiyonu yerleştirin. </li></ol> Adipositlerinde ve preadipositlerin 3. saflaştırılması <ol><li> Bir plastik geniş şişenin içine örgü 400 uM naylondan yağ birkaç parçaları ile saydam çözelti dökülür. </li><li> Cımbız ile, naylon örgü hücre hazırlama ovmak ve KRH-WB 5 ml suyla yıkayın. </li><li> Incelikle içinde plastik boru ve boru ucunda bağlı bir 60cc şırınga ile bir 50 ml tüp içine süzüldü hücre süspansiyonu aktarın. </li><li> Olgun adipositlerle süspansiyon hücreleri yüzdürme ile tampon üst ulaşmasını sağlayan, yaklaşık 10 dakika bekletin. </li><li> Yavaş yavaş 60cc enjektörler kullanılarak tüpün altındaki tampon aspirege emme. </li><li> KRH-WB 20 ml yıkama ekleyin. 2 ek yıkama için adım 3.4 tekrarlayın. </li><li> Hücre miktarına bağlı olarak, 5 ya da 10 ml&#39;lik bir son hacme adiposit süspansiyon getirmek için tampon toplayın. 5. bölümünde adımlarla takibi. </li><li> Arzu edildiği takdirde ayrıca, birinci hücre kültürü santrifüj ile 60cc şırınga (3000 rpm, RT, 5 dk) ile toplanmış tampon stromal vasküler kısmını kurtarma (tekniği gösterilmemiştir). </li></ol> 4. Olgun Adiposit Hücre Sayısı <ol><li> Yük bir sayım odasında (hemositometre) hafifçe sarsıldı adiposit süspansiyonu 10 ul. Dört nüsha olarak hücre sayısını yapın. </li><li> Izole olgun hücrelerin sayısını hesaplayın. </li></ol> T-25 şişesi içine 5. Olgun Adiposit farksızlaşma <ol><li> DMEM / F12-20% buzağı serumu ile hacminin ¾ 25 cm 2 doku kültürü şişesi doldurun. </li><li> Hücre sayımı göre, balona 500.000 olgun hücreleri dökün. </li&gt; <li> Tam ortam ile 50 ml tüp kullanarak balon doldurun ve mümkün olduğu kadar çok kabarcıklarını çıkarmak. </li><li> Şişenin üzerindeki unvented kapağı vidalayın. </li><li> Kontaminasyonu önlemek için inkübasyon öncesinde etanol ile şişeyi temizleyin. </li><li> Hücresel yapışmayı bozabilir kültür hareketi önlemek için dokunmadan baş aşağı bir hafta boyunca şişeyi inkübe. </li><li> Ters kültür 7 gün balon tersine önce, hafifçe ani hareketlerinden kaçınmak, şişeyi işlemek ve aspirasyon şişeye tüm orta kaldırmak. </li><li> DMEM-F12-20% buzağı serumu 12 ml ekleyin ve standart tekniklerle hücreleri yetiştirmek. Filtre edilmiş, havalandırmalı kapak şişeye ilave edilebilir. </li></ol> 6 yuvalı plaka içine 6. Olgun Adiposit farksızlaşma <ol><li> 6 gözlü bir plakanın her bir alt kısmında bir lamel yerleştirin </li><li> Her lamel üstüne ½ &quot;plastik burcu ekleyin. </li><li> % 20 dana serumu-DMEM ortamı 8 ml ile kuyu doldurun. </li><li> Her plastik burç üzerinde bir lamel koyun. </li><li> Slayt ve slayt altında hücresinin enjekte edilmesi için bir tüp (oyuk başına 50,000 hücre) ile pipet ucu yerleştirin. </li><li> Bir hafta boyunca% 5 CO2 ile 37 ° C &#39;de bir standart hücre kültürü inkübatöründe inkübe edin. </li><li> Her bir göze% 20 dana serumu ile takviye edilmiş bir ortam 2 ml ihtiva eden ve takip kültüre ilişik hücrelerle Arka lamel. </li><li> Hücreler immunfloresan dahil olmak üzere birçok amaçlar için dediferansiyonunu geçiren ile lamel kullanın (tekniği gösterilmemiştir). </li></ol>

<ol>
	<li>Zhang, H. H., Kumar, S., Barnett, A. H., Eggo, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ceiling+culture+of+mature+human+adipocytes:+use+in+studies+of+adipocyte+functions.">Ceiling culture of mature human adipocytes: use in studies of adipocyte functions.</a> J Endocrinol. 164 (2), 119-128 (2000).</li><li>Matsumoto, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mature+adipocyte-derived+dedifferentiated+fat+cells+exhibit+multilineage+potential.">Mature adipocyte-derived dedifferentiated fat cells exhibit multilineage potential.</a> J Cell Physiol. 215 (1), 210-222 (2008).</li><li>Poloni, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+dedifferentiated+adipocytes+show+similar+properties+to+bone+marrow-derived+mesenchymal+stem+cells.">Human dedifferentiated adipocytes show similar properties to bone marrow-derived mesenchymal stem cells.</a> Stem Cells. 30 (5), 965-974 (2012).</li><li>Perrini, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+gene+expression+and+cytokine+release+profiles+highlight+the+heterogeneity+of+distinct+subsets+of+adipose+tissue-derived+stem+cells+in+the+subcutaneous+and+visceral+adipose+tissue+in+humans.">Differences in gene expression and cytokine release profiles highlight the heterogeneity of distinct subsets of adipose tissue-derived stem cells in the subcutaneous and visceral adipose tissue in humans.</a> PLoS One. 8 (3), e57892 (2013).</li><li>Kishimoto, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+osteoblastic+differentiation+ability+of+human+dedifferentiated+fat+cells+is+higher+than+that+of+adipose+stem+cells+from+the+buccal+fat+pad.">The osteoblastic differentiation ability of human dedifferentiated fat cells is higher than that of adipose stem cells from the buccal fat pad.</a> Clin Oral Investig. , (2013).</li><li>Kou, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+phenotype+and+tissue-specific+nature+of+multipotent+cells+derived+from+human+mature+adipocytes.">The phenotype and tissue-specific nature of multipotent cells derived from human mature adipocytes.</a> Biochem Biophys Res Commun. 444 (4), 543-548 (2014).</li><li>Jumabay, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotent+stem+cells+derived+from+mouse+and+human+white+mature+adipocytes.">Pluripotent stem cells derived from mouse and human white mature adipocytes.</a> Stem Cells Transl Med. 3 (2), 161-171 (2014).</li><li>Sugawara, A., Sato, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+dedifferentiated+fat+cells+for+periodontal+tissue+regeneration.">Application of dedifferentiated fat cells for periodontal tissue regeneration.</a> Hum Cell. 27 (1), 12-21 (2014).</li><li>Kikuta, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteogenic+effects+of+dedifferentiated+fat+cell+transplantation+in+rabbit+models+of+bone+defect+and+ovariectomy-induced+osteoporosis.">Osteogenic effects of dedifferentiated fat cell transplantation in rabbit models of bone defect and ovariectomy-induced osteoporosis.</a> Tissue Eng Part A. 19 (15-16), 1792-1802 (2013).</li><li>Obinata, D., et al. <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+mature+adipocyte-derived+dedifferentiated+fat+(DFAT)+cells+improves+urethral+sphincter+contractility+in+a+rat+model.">Transplantation of mature adipocyte-derived dedifferentiated fat (DFAT) cells improves urethral sphincter contractility in a rat model.</a> Int J Urol. 18 (12), 827-834 (2011).</li><li>Jumabay, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dedifferentiated+fat+cells+convert+to+cardiomyocyte+phenotype+and+repair+infarcted+cardiac+tissue+in+rats.">Dedifferentiated fat cells convert to cardiomyocyte phenotype and repair infarcted cardiac tissue in rats.</a> J Mol Cell Cardiol. 47 (5), 565-575 (2009).</li><li>Ohta, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mature+adipocyte-derived+cells,+dedifferentiated+fat+cells+(DFAT),+promoted+functional+recovery+from+spinal+cord+injury-induced+motor+dysfunction+in+rats.">Mature adipocyte-derived cells, dedifferentiated fat cells (DFAT), promoted functional recovery from spinal cord injury-induced motor dysfunction in rats.</a> Cell Transplant. 17 (8), 877-886 (2008).</li><li>Moreno-Navarrete, J. M. F. -. r., Symonds, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+2.">Ch. 2.</a> Adipose Tissue Biology. , 17-38 (2012).</li><li>Poulos, S. P., Dodson, M. V., Hausman, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+line+models+for+differentiation:+preadipocytes+and+adipocytes.">Cell line models for differentiation: preadipocytes and adipocytes.</a> Exp Biol Med (Maywood. 235 (10), 1185-1193 (2010).</li><li>Casteilla, L., Penicaud, L., Cousin, B., Calise, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choosing+an+adipose+tissue+depot+for+sampling:+factors+in+selection+and+depot+specificity).">Choosing an adipose tissue depot for sampling: factors in selection and depot specificity).</a> Methods Mol Biol. 456, 23-38 (2008).</li><li>Tchernof, A., Despres, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+human+visceral+obesity:+an+update.">Pathophysiology of human visceral obesity: an update.</a> Physiol Rev. 93 (1), 359-404 (2013).</li><li>Rodbell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+of+Isolated+Fat+Cells.+I.+Effects+of+Hormones+on+Glucose+Metabolism+and+Lipolysis.">Metabolism of Isolated Fat Cells. I. Effects of Hormones on Glucose Metabolism and Lipolysis.</a> J Biol Chem. 239, 375-380 (1964).</li><li>Watson, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Markers+and+Functional+Attributes+of+Human+Adipose-Derived+Stem+Cells+and+Dedifferentiated+Adipocyte+Cells+from+Subcutaneous+Fat+of+an+Obese+Diabetic+Donor.">Comparison of Markers and Functional Attributes of Human Adipose-Derived Stem Cells and Dedifferentiated Adipocyte Cells from Subcutaneous Fat of an Obese Diabetic Donor.</a> Adv Wound Care. 3 (3), 219-228 (2014).</li></ol>

The authors declare no conflict of interest.

Tavan kültür tekniği ile olgun adipositlerin farksızlaşma yerli yağ dokusunun küçük bir örnek adipoz kök hücreleri elde etmek yeni bir yaklaşımdır. Deneyimlerimize ve diğerleri 2 bu göre, bir gram doku 25 cm2 bir balon plaka ve homojenliği Poloni ve arkadaşları 3 ile gösterilmiştir olan DFAT hücre popülasyonu elde etmek için yeterlidir. Adiposit farksızlaşma bağımsız olarak yaş, cinsiyet ve diğer özellikleri, herhangi bir donörden alınan hücrelerle mümkün görünüyor. DFAT elde çıkan nüfus arasında, bir kaç yuvarlak veya tamamen farksızlaştığı vermedi kısmen uzatılmış hücreler kalır. Onlar tripsin-medya karışımı yüzer gibi bu hücreler genellikle kültür geçitten atılır. Bu hücrelerin multipotency kurulmuş ve hücre tedavisi 2,3 için onların kullanımını destekler. Bunların yüksek çoğalma kapasitesi de rapor edilmiştir ki bu ikök hücre uygulamaları 2 için hücre kültürü sa değerli yönü. İnsan DFAT hücreleri ile yapılan çalışmalar, bu da tekrar eden ve farklılaşma kapasitesi 18 göre aynı donörden ASC daha etkili olabileceğini göstermiştir. Son zamanlarda vaka çalışması DFAT hücreleri adipositler ve osteoblast içine ayırt etmek daha verimli ve aynı birey, obezite ve diyabet 18 ile bir donörden ASC daha yüksek telomeraz düzeyleri olduğunu desteklemektedir. Böylece, tavan kültürünün kullanımı zaten kullanılan ASC daha verimli yağ kök hücreleri sağlayabilir. Bununla birlikte, ilave deneyler açıkça bu noktayı belirlemek için önemlidir. Bizim 6-yuvalı plaka tavan kültürü tekniği farksızlaşma prosesinin bir çalışma sağlar. Hücrelerin en az sayıda kaplanmış ve spesifik zaman noktalarında çalışma sağlar edilebilir. Örneğin, adipositler underg gelen immünofloresan gerçekleştirmek için bir 6-yuvalı plaka mikroskop lamı toplananmeler farksızlaşma (Şekil 2). Veya floresan olmayan, miscroscopy Sahne, dediferansiyasyon çeşitli yönlerini değerlendirmek için büyük önem taşımaktadır. Kök hücre uygulamaları yanı sıra, DFAT hücreler fizyolojik çalışmalar için ilginç bir modelini temsil eder. Sadece bir kaç çalışmada, gen ekspresyonu ve her iki hücre tipinde işlevlerini incelenmiştir. Aynı yağ bölmeden kısa, ASC ve DFAT gen ifadesi ve salgılanması 4 benzerlikler gösterdi. Aynı vericiden alınan ASC ve DFAT arasında daha fazla karşılaştırmalar gereklidir. Sonuç olarak, biz adipoz doku kollajenaz sindirim köklü teknik ve tavan kültür tekniği kullanılarak insan adipoz dokudan DFAT hücreleri elde etmek için nasıl bu teknik raporda göstermek. Bizim orijinal 6-plaka formatı daha yaygın kullanılan şişe yöntemi büyük populasyona üretimi için izin verir ise farksızlaşma süreci hakkında bilgi artırmaya yardımcı olabilirDFAT hücre saplamalar. Bu protokolün en önemli kısıtlılığı, hasta yazılı ve bilgilendirilmiş onam elde etmek için bir ameliyat ekibi ve etik yönetimi ile işbirliği dayanan insan yağ dokusu erişim olduğunu. Olgun hücreler manipülasyon önlemleri gerektiren son derece duyarlıdır. Bu en iyi sonuçları elde etmek için, protokol, özellikle T-25flask olarak kaplama için adipositlerin sayısı ve 6-yuvalı plaka formatı optimize edilmiş ve önemli bir ek modifikasyonlar ya da sorun beklenebilir.

In vitro dedifferentiation of mature adipocytes is achieved through a technique called ceiling culture1. Because of their natural tendency to float in aqueous solutions, isolated mature adipocytes adhere to the surface of an inverted flask fully filled with culture medium. Over a few days, cells modify their spherical morphology and become fibroblast-like cells. The resulting cells, called dedifferentiated fat (DFAT) cells, are multipotent2. Research articles on adipocyte dedifferentiation, especially on human cells, are limited. However, they have already provided interesting information regarding multipotency, cell phenotype and replicative capacity of DFAT cells2. Mature adipocytes originating from various fat compartments have been successfully dedifferentiated including those originating from human visceral and subcutaneous adipose tissues2-4. In addition to these depots, Kishimoto and collaborators sampled adipose tissue from the buccal fat pads and dedifferentiated adipocytes into DFAT cells5. Matsumoto and collaborators successfully generated subcutaneous DFAT cells from patients covering a wide range of ages, and the majority of cells had a high proliferative rate and less than 6% of senescence even after 10 passages in culture2.
DFAT cells have been successfully re-differentiated into several lineages, including adipogenic, osteogenic, chondrogenic and neurogenic lineages2,3,6. These cells express several embryonic stem cell markers such as Nanog and the four identified pluripotent factors Oct4, c-myc, Klf4 and Sox23. They also express markers specific to each of the three germ layers7. In addition, DFAT cells are similar to Bone Marrow-derived Mesenchymal Stem Cells (BM-derived MSC) based on their epigenetic signature3. Exploiting the stem cell capacity of DFAT cells, many groups have investigated their potential to treat or improve various diseases8,9. Improvements of pathologic conditions, such as infracted cardiac tissue, spinal cord injury and urethral sphincter dysfunction, have been observed with DFAT cell injections in rat models of disease10-12. 
In addition to the stem cell properties of DFAT cells, they may represent a new cellular model for adipocyte physiology studies. The 3T3-L1 cell line is often used for this purpose as these cells differentiate into adherent, lipid-storing adipocytes under adipogenic stimulation13. However, these cells originate from mouse embryo tissue13. Also, depot-specificity cannot be investigated with this model and it may not fully reflect human adipocyte physiology14. Other laboratories work with isolated adipose cells from murine fat depots, but fat distribution is not dimorphic in mice and anatomical configuration of the rodent's abdominal cavity prevents from extrapolating directly to humans15. In order to study adipocytes in the context of the physiopathology of human obesity, consideration of body fat distribution and fat depot-specific differences has become essential16. Some limitations of primary preadipocyte cultures, including cell quantities obtained from adipose tissue biopsy samples and their senescence after a few passages in culture, created the need for alternate models. Perrini and collaborators investigated depot-specificity in gene expression of DFAT cells originating from visceral and subcutaneous fat and compared them to adipose-derived stem cells (ASC) from the same fat depot. They demonstrated that differences in gene expression and function where mainly found between depots than between cell types, suggesting that DFAT cells are physiologically close to ASC from the same depot. DFAT cells may represent an interesting alternative to available models for studies on fat distribution in the pathophysiology of human obesity. Moreover, ceiling culture is a promising method to obtain adult stem cells for tissue engineering purposes. 
Here, we describe collagenase digestion, a widely-used technique to isolate mature adipocytes from the subcutaneous and/or visceral fat samples17, and the subsequent steps to perform ceiling culture and dedifferentiate these cells into multipotent, fibroblast-like cells.

<table><tbody><tr><td>Bovine serum albumine</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>Adenosine</td><td>Sigma</td><td>A4036</td><td></td></tr><tr><td>Ascorbic acid</td><td>Sigma</td><td>A0278</td><td></td></tr><tr><td>NaCl</td><td></td><td></td><td>Any brand can be used</td></tr><tr><td>KCl</td><td></td><td></td><td>Any brand can be used</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td>Any brand can be used</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td>Any brand can be used</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td></td><td></td><td>Any brand can be used</td></tr><tr><td>HEPES</td><td></td><td></td><td>Any brand can be used</td></tr><tr><td>Glucose</td><td></td><td></td><td>Any brand can be used</td></tr><tr><td>Type I collagenase</td><td>Worthington Biochemical Corp</td><td>LS-004196</td><td></td></tr><tr><td>DMEM/F-12, HEPES, no phenol red</td><td>Gibco-Life Technologies</td><td>11039-021</td><td>Add to medium : 20% calf serum, gentamicin (50 &amp;micro;g/ml) and fungizone (2.5 &amp;micro;g/ml)</td></tr><tr><td>Calf Serum, iron supplemented, from formula-fed calves</td><td>Sigma</td><td>C8056-500 ml</td><td></td></tr><tr><td>1/2 In plastic bushing</td><td>Iberville</td><td>2704-CP</td><td>SKU:1000120918 (Home Depot)</td></tr><tr><td>Liquid nitrogen</td><td>Linde</td><td></td><td></td></tr><tr><td>Formalin soluton, neutral buffered, 10%</td><td>SIGMA</td><td>HT501128</td><td></td></tr><tr><td>Sterile tweezers</td><td></td><td></td><td></td></tr><tr><td>Sterile scissors</td><td></td><td></td><td></td></tr><tr><td>60 cc syringes</td><td>BD Syringe</td><td></td><td></td></tr><tr><td>Plastic tubing</td><td></td><td></td><td></td></tr><tr><td>Krebs-Ringer-Henseleit stock buffer (KRH)</td><td></td><td></td><td>Prepare stock buffer as following: 25 mM HEPES pH 7.6, 125 mM NaCl, 3.73 mM KCl, 5 mM CaCl&lt;sub&gt;2&lt;/sub&gt;.2H&lt;sub&gt;2&lt;/sub&gt;O, 2.5 mM MgCl&lt;sub&gt;2&lt;/sub&gt;.6H&lt;sub&gt;2&lt;/sub&gt;O, 1 mM K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;. Adjust pH to 7.4.</td></tr><tr><td>Krebs-Ringer-Henseleit-Working Buffer (KRH-WB)</td><td></td><td></td><td>Add the following components freshly to KRH buffer: 4% bovine serum albumin, 5 mM glucose, 0.1 &amp;micro;M adenosine, 560 &amp;micro;M ascorbic acid</td></tr><tr><td>KRH-WB supplemented with Type I collagenase</td><td></td><td></td><td>Add 350 U/ml of Type I collagenase</td></tr><tr><td>T25 unvented cap tissue culture flask</td><td>Sarsted or other brand</td><td></td><td></td></tr><tr><td>6-well tissue culture plate</td><td>BD Falcon or other brand</td><td></td><td></td></tr><tr><td>Microscope cover glass 22x22</td><td>Fisherbrand</td><td>12-542-B</td><td></td></tr><tr><td>Sterile beakers</td><td></td><td></td><td></td></tr></tbody></table>

generation of human adipose stem cells through dedifferentiation of mature adipocytes in ceiling cultures

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

Başlıca morfolojik değişimler dediferansiyasyon (Şekil 1) sırasında adipositleri olgun oluşur. Şekil 2&#39;de gösterildiği gibi, farklılaşma bozukluğunun geçiren hücreler, floresan analizi için bir anti-FABP4 antikor ile boyandı. Fibroblast-benzeri hücrelerin bir çoğunluğu bu görülmemiştir yuvarlak morfoloji olan hücreler FABP4 proteini ifade etmiştir. Dediferansiyasyon sonra, DFAT hücreleri birkaç geçişleri için standart prosedürler ile yetiştirilebilir. İnsan omental ve deri altına DFAT hücre çizgileri için 15 kanallarına ulaşmak mümkün olmuştur (veriler gösterilmemiştir). <img alt="Şekil 1," src="/files/ftp_upload/52485/52485fig1.jpg" /> 4 gün sonra (B), 7 gün kültür. Resimleri 12 günde (C) &#39;de bir süre (A) boyunca, olgun adipositlerin dedifferentiating Şekil 1. morfolojisi, bir faz-kontrast mikroskobunun ile inkübasyon sırasında farklı zaman noktalarında alınmıştıre. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52485/52485fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 2," src="/files/ftp_upload/52485/52485fig2.jpg" /> Adipositlerde FABP4 protein Şekil 2. Algılama dediferansiyonunu geçiren. Hücreler, farklılaşmanın giderilmesi için 13 gün sonra sabit ve immünofloresans anti-FABP4 antikor ile boyandı. Çekirdekler DAPI boyama ile görüntülendi. Sol: immünoflüoresans karşılık gelen aydınlık görüntü. Sağ: Birleştirilmiş görüntü gösterilir (FABP4-kırmızı, çekirdekler-mavi-10X). Uzatılmış hücreler ise yuvarlak morfoloji ekspres FABP4, olgun bir adiposit işaretleyici ile adipozitler artık bunu ifade. Ölçek çubuğu: 1 adet = 0.25mm <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52485/52485fig2large.jpg" target="_blank">bu rakamın daha büyük bir versiyonunu görmek için burayı tıklayınız.</a>

Watch this Scientific Journal Video about Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures at JoVE.com

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

Tavan Kültürlerde Olgun adipositlerinde ve dediferansiyonunu ile İnsan adipoz Kök Hücre Üretimi

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

generation-human-adipose-stem-cells-through-dedifferentiation-mature

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

11.7K Görüntüleme.  IUCPQ Research Center. Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

Video: Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

İzolasyon ve İnsan zenginleştirilmesi Gelişmiş Osteogenezi için Stromal Hücreleri adipoz-türevi

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

Gelişim Biyolojisi

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

Domuz Subkutan yağ dokularındaki İzolasyonu ve Yağ-Türetilmiş Kök Hücre Farklılaşması

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

An Efficient Method to Obtain Dedifferentiated Fat Cells

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used therapeutically, in the near future, to restore function to damaged organs. Nevertheless, several technical issues, including the highly invasive procedure of isolating MSCs and the inefficiency surrounding their amplification, currently hamper the potential clinical use of these therapeutic modalities. Herein, we introduce a highly efficient method for the generation of dedifferentiated fat cells (DFAT), MSC-like cells. Interestingly, DFAT cells can be differentiated into several cell types including adipogenic, osteogenic, and chondrogenic cells. Although other groups have previously presented various methods for generating DFAT cells from mature adipose tissue, our method allows us to produce DFAT cells more efficiently. In this regard, we demonstrate that DFAT culture medium (DCM), supplemented with 20% FBS, is more effective in generating DFAT cells than DMEM, supplemented with 20% FBS. Additionally, the DFAT cells produced by our cell culture method can be redifferentiated into several tissue types. As such, a very interesting and useful model for the study of tissue dedifferentiation is presented.

We have modified the conditions for DFAT cell generation and provide herein information regarding the use of an improved growth medium for the production of these cells.

Etkin Bir Yöntem Dediferansiye Yağ Hücreleri Elde

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used ...

Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. Adipose-derived stromal cells (ASCs) have proven to be an abundant source of multipotent stem cells capable of undergoing osteogenic, chondrogenic, adipogenic, and myogenic differentiation. Many studies have explored the osteogenic potential of these cells in vivo with the use of various scaffolding biomaterials for cellular delivery. It has been demonstrated that by utilizing an osteoconductive, hydroxyapatite-coated poly(lactic-co-glycolic acid) (HA-PLGA) scaffold seeded with ASCs, a critical-sized calvarial defect, a defect that is defined by its inability to undergo spontaneous healing over the lifetime of the animal, can be effectively show robust osseous regeneration. This in vivo model demonstrates the basis of translational approaches aimed to regenerate the bone tissue - the cellular component and biological matrix. This method serves as a model for the ultimate clinical application of a progenitor cell towards the repair of a specific tissue defect.

This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration.

Lipoaspirate itibaren hasat Yağ kaynaklı stromal hücreler kullanarak Kritik ölçekli Kalvaryum Defekt Model tamiri

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. ...

Tıp

Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of cardiovascular disease. ADSCs derived from different adipose tissues show distinct features, and those from the PVAT have not been well characterized. In a recent study, we reported that some ADSCs in the periaortic arch adipose tissue (PAAT) descend from the neural crest cells (NCCs), a transient population of migratory cells originating from the ectoderm.
In this paper, we describe a protocol for isolating red fluorescent protein (RFP)-labeled NCCs from the PAAT of Wnt-1 Cre+/-;Rosa26RFP/+ mice and inducing their adipogenic differentiation in vitro. Briefly, the stromal vascular fraction (SVF) is enzymatically dissociated from the PAAT, and the RFP+ neural crest derived ADSCs (NCADSCs) are isolated by fluorescence activated cell sorting (FACS). The NCADSCs differentiate into both brown and white adipocytes, can be cryopreserved, and retain their adipogenic potential for ~3&#8211;5 passages. Our protocol can generate abundant ADSCs from the PVAT for modeling PVAT adipogenesis or lipogenesis in vitro. Thus, these NCADSCs can provide a valuable system for studying the molecular switches involved in PVAT differentiation.

We present a protocol for the isolation, culture, and adipogenic induction of neural crest derived adipose-derived stem cells (NCADSCs) from the periaortic adipose tissue of Wnt-1 Cre+/-;Rosa26RFP/+ mice. The NCADSCs can be an easily accessible source of ADSCs for modeling adipogenesis or lipogenesis in vitro.

Periaortik Yağ Dokusundan Nöral Adipoz-Türetilmiş Kök Hücrelerin İzolasyon, Kültür ve Adipojenik İndüksiyonu

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of ...

Robust Differentiation of Human iPSCs into a Pure Population of Adipocytes to Study Adipocyte-Associated Disorders

Recent advances in induced pluripotent stem cell (iPSC) technology have allowed the generation of different cell types, including adipocytes. However, the current differentiation methods have low efficiency and do not produce a homogenous population of adipocytes. Here, we circumvent this problem by using an all-trans retinoic-based method to produce mesenchymal stem cells (MSCs) in high yield. By regulating pathways governing cell proliferation, survival, and adhesion, our differentiation strategy allows the efficient generation of embryonic bodies (EBs) that differentiate into a pure population of multipotent MSCs. The high number of MSCs generated by this method provides an ideal source for generating adipocytes. However, sample heterogeneity resulting from adipocyte differentiation remains a challenge. Therefore, we used a Nile red-based method for purifying lipid-bearing mature adipocytes using FACS. This sorting strategy allowed us to establish a reliable way to model adipocyte-associated metabolic disorders using a pool of adipocytes with reduced sample heterogeneity and enhanced cell functionality.

The protocol allows the generation of a pure adipocyte population from induced pluripotent stem cells (iPSCs). Retinoic acid is used to differentiate iPSCs into mesenchymal stem cells (MSCs) which are used for producing adipocytes. Then, a sorting approach based on Nile red staining is used to obtain pure adipocytes.

Adipositle İlişkili Bozuklukları İncelemek için İnsan iPSC'lerinin Saf Bir Adiposit Popülasyonuna Sağlam Farklılaşması

Recent advances in induced pluripotent stem cell (iPSC) technology have allowed the generation of different cell types, including adipocytes. However, the ...

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease.
This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) &#62;35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2&#8211;3 g of fat. These cells can be expanded in culture over 10&#8211;14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5&#8211;6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPAR&#947; agonist-rosiglitazone.
This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

Yalıtım, genişleme ve Adipojenik indüksiyon CD34 + CD31 + insan Omental ve deri altı yağ dokusu endotel hücrelerden

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...

İmmünoloji ve Enfeksiyon

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

Bir İnsan 3D Ekstrasellüler Matrix-Adiposit Kültür Modeli Matrix-Hücre Metabolik Crosstalk Çalışma için

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

Biyomühendislik

Differentiation Capacity of Human Aortic Perivascular Adipose Progenitor Cells

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic lineages. Adipogenic differentiation of progenitor cells is a major mechanism driving adipose tissue expansion and dysfunction in response to obesity. Understanding changes to perivascular adipose tissue (PVAT) is thus clinically relevant in metabolic disease. However, previous studies have been predominately performed in the mouse and other animal models. This protocol uses human thoracic PVAT samples collected from patients undergoing coronary artery bypass graft surgery. Adipose tissue from the ascending aorta was collected and used for explantation of the stromal vascular fraction. We previously confirmed the presence of adipose progenitor cells in human PVAT with the capacity to differentiate into lipid-containing adipocytes. In this study, we further analyzed the differentiation potential of cells from the stromal vascular fraction, presumably containing multi-potent progenitor cells. We compared PVAT-derived cells to human bone marrow MSC for differentiation into adipogenic, osteogenic, and chondrogenic lineages. Following 14 days of differentiation, specific stains were utilized to detect lipid accumulation in adipocytes (Oil red O), calcific deposits in osteogenic cells (Alizarin Red), or glycosaminoglycans and collagen in chondrogenic cells (Masson&#8217;s Trichrome). While bone marrow MSC efficiently differentiated into all three lineages, PVAT-derived cells had adipogenic and chondrogenic potential, but lacked robust osteogenic potential.

The goal of this protocol is to test the ability of progenitor cells derived from human perivascular adipose tissue to differentiate into multiple cell lineages. Differentiation was compared to mesenchymal stem cells derived from human bone marrow, which is known to differentiate into adipocyte, osteocyte, and chondrocyte lineages.

İnsan aort Perivasküler yağ progenitör hücre farklılaşması kapasitesi

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic ...

Biyoloji

Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures (MAAC)

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments for T2D, more physiologically relevant in vitro adipocyte models are required. This study describes a new technique to isolate and culture mature human adipocytes. This method is entitled MAAC (membrane mature adipocyte aggregate culture), and compared to other adipocyte in vitro models, MAAC possesses an adipogenic gene signature that is the closest to freshly isolated mature adipocytes. Using MAAC, adipocytes can be cultured from lean and obese patients, different adipose depots, co-cultured with different cell types, and importantly, can be kept in culture for 2 weeks. Functional experiments can also be performed on MAAC including glucose uptake, lipogenesis, and lipolysis. Moreover, MAAC responds robustly to diverse pharmacological agonism and can be used to study adipocyte phenotypic changes, including the transdifferentiation of white adipocytes into brown-like fat cells.

Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures MAAC

Membrane mature adipocyte aggregate culture (MAAC) is a new method to culture mature human adipocytes. Here we detail how to isolate adipocytes from human adipose and how to set up MAAC.

Membran Olgun Adiposit Agrega Kültürleri (MAAC) Kullanarak İnsan Olgun Adipositlerin İzolasyon ve Kültürü

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments ...