C.D.M. was supported by the American College of Surgeons (ACS) Resident Research Scholarship. M.S.H. was supported by the California Institute for Regenerative Medicine (CIRM) Clinical Fellow training grant TG2-01159. M.S.H., H.P.L., and M.T.L. were supported by the American Society of Maxillofacial Surgeons (ASMS)/Maxillofacial Surgeons Foundation (MSF) Research Grant Award. H.P.L. was supported by NIH grant R01 GM087609 and a gift from Ingrid Lai and Bill Shu in honor of Anthony Shu. H.P.L. and M.T.L. were supported by the Hagey Laboratory for Pediatric Regenerative Medicine and The Oak Foundation. M.T.L. was supported by NIH grants U01 HL099776, R01 DE021683-01, and RC2 DE020771. D.C.W. was supported by NIH grant 1K08DE024269, the Hagey Laboratory for Pediatric Regenerative Medicine, and the Stanford University Child Health Research Institute Faculty Scholar Award.

Not: numuneleri rıza veren hastadan elde edildi. Tüm protokoller gözden geçirilmiş ve uygun Stanford Üniversitesi Kurumsal Değerlendirme Kurulu tarafından onaylanmıştır. insan doku ve hücrelerinin işlerken, her zaman kurum tarafından belirtilen, Biyogüvenlik Seviyesi 2 (BSL2) önlemlere uymak. Reaktiflerin 1. Hazırlık <ol><li> FACS tampon hazırlanması: 10 ml FBS, 188 5 ml Poloxamer 500 ml steril fosfat tamponlu tuz için 5 ml Pen-Strep (PBS) ekleyin. </li><li> Karışımı sindirmek hazırlayın: 0.375 gr C hemolyticum kollajenaz tip II tozu ve 5 mL Poloxamer steril 199 / EBSS ortamı 188 500 ml ekleyin. </li><li> Standart orta hazırlayın: 50 ml FBS ve 500 mL Dulbecco Modifiye Eagle Ortamı için 5 ml Pen-Strep ekleyin. </li></ol> 2. Hasat ve TSK izolasyonu NOT: yeterli kurumsal onaylar insan dokusu kullanarak ve izolasyon saat yerinde olduğundan emin olunuman kök hücreleri. Insan karın elde kuşatan, ya da seçmeli liposuction uygulanan sağlıklı bir donörden cilt altı yağ uyluk. Bir plastik emme kabı içinde yağ tutun. <ol><li> Orijinal plastik kutunun içinde yağ: 1 oranında bir 1, steril PBS ilave edin. (500 mi yağ var ise, 500 ml PBS ilave). 30 saniye için hafif ajitasyon ile karıştırın. Sulu tabaka, emme bağlanmış bir 10 ml plastik pipet ile sulu tabakanın alt (Şekil 1) ve daha sonra aspire yerleşmek ve atın izin verin. </li><li> büyük bir plastik kap içine yağ süzün ve 1 karışımı sindirimi ekleyin: 1 oranında. Kabı kapatın ve% 70 etanol ile dışını temizleyin. parafin film ile kapağı mühür. </li><li> 30 dakika boyunca 37 ° C&#39;de 180 rpm&#39;de yörüngesel karıştırıcıda bu kabı çalkalayın. </li><li> : 1 oranında bir 1, standart orta ekleyerek sindirim nötralize. (1000 ml yağ karışımı varsa, 1.000 ml standart orta ekleyin). </li><li> eşit 25 eşit sayıda içine karışımı dağıtın0 ml plastik konik santrifüj tüpleri ve santrifüj 4 ° C &#39;de 20 dakika boyunca 300 x g&#39;de kanşım </li><li> Süpernatantı aspire ve bozulmamış pelet bırakmak için dikkatli olun. Her konik tüp için 5 ml standart ortamda pelet tekrar. </li><li> Bir 50 ml konik santrifüj tüpüne 100 mikronluk bir filtre boyunca süspansiyon filtre. Daha sonra, 4 ° C&#39;de 15 dakika boyunca 300 x g&#39;de santrifüjleyin. </li><li> Süpernatantı aspire ve 5 ml oda sıcaklığında RBC parçalama tamponunda pelletini. Karışım, daha sonra oda sıcaklığında 15 dakika boyunca (RT) 300 xg&#39;de santrifüj, oda sıcaklığında 5 dakika boyunca bekletin. </li><li> Süpernatantı aspire ve 15 ml standart orta pelletini. Yine, 50 ml konik santrifüj tüpüne 100 mikronluk bir filtre boyunca filtre edin. </li><li> Yeni 50 ml konik, yoğunluk tabanlı ayırma (reaktif listesine bakınız) için tasarlanmış 15 mL polisükroz çözüm ekleyin. Sonra, bir 45 ° açıyla tüp tutarak dikkatlice yüzüne hücreleri pipetTüp polisükroz çözeltisi üzerine hücre süspansiyonu hafifçe katmanları (Şekil 2), böylece. NOT: Bu çözeltinin yüzeyine pipet hücreleri için önemlidir. Bu geri dönüşü olmayan bir karışım oluşturmak gibi bir hücre süspansiyonu polisükroz çözüm katmayın. Benzer şekilde, polisükroz çözeltisi merkezine hücreleri pipetle değildir. </li><li> Oda sıcaklığında 10 dakika boyunca 300 x g&#39;de bu karışımın santrifüjleyin. Düşük ivme kullanın (- 2 10 üzerinden 3) ve bu adım için sıfıra santrifüj fren işlevini ayarlayın. NOT: ortasında bulutlu, alt net ve somon renkli üstünde: Üç katmanlar görünür olacaktır. orta tabaka ilgi hücreleri içerir. </li><li> 10 ml pipet kullanarak dikkatlice orta tabakayı kaldırmak ve yeni bir 50 ml konik tüp transfer. NOT: tamamen orta tabakanın bazı geride bırakmak daha üst ve alt katmanları bazılarını almak orta tabaka transferi ve bu süreçte daha iyidir. </li> &lt;li&gt; hemasitometre kullanarak hücre sayımı ve toplam canlı hücre sayısını belirlemek. Not: Genellikle kullanımı tripan mavi hücreleri sayma yardımcı olmak için% 0.8 bir son konsantrasyona kadar, hücre karışımına ilave edildi. Canlı canlı hücreler mavi boya kadar sürmez ve beyaz görünecektir. </li></ol> BMPR IB pozitif hücrelerin ve hücre ihtiva eden ıskelesının Sıralama 3. FACS <ol><li> 4 ° C&#39;de 5 dakika boyunca 300 x g&#39;de hücre süspansiyonu santrifüj. (Adım 2.13 yapılan hücre sayımına bağlı olarak), 100 uL başına 1.000.000 hücre konsantrasyonunda FACS tamponu içerisinde tekrar süspansiyon hücreleri. </li><li> etiketli plastik santrifüj tüpüne en az 10 milyon hücre Transferi &quot;BMPR-IB.&quot; Ayrı, etiketli ayrı bir plastik santrifüj tüpüne 100 uL FACS tampon bir milyon hücreleri transferi &quot;boyanmamış.&quot; &quot;Boyanmamış&quot; tüp 1 ml FACS tampon ekleyin. FACS exper kısmı sıralama buz üzerinde FACS tamponu içerisinde hücrelerin kalan tutuniment yapılmaktadır. Bu hücreleri etiketlemek &quot;Unsorted.&quot; kontroller daha sonra deneyde olarak kullanın. </li><li> üreticinin talimatlarına göre &quot;BMPR IB&quot; tüp bir flüoresan anti-insan BMPR IB antikorun uygun miktarda. antikor dağıtmak için hafifçe yukarı ve aşağı karışımı pipetle. NOT: 1:10 seyreltme bir insan BMPR-IB / ALK-6 APC-konjuge antikor kullanmak (detaylar için reaktif listesine bakınız). Ancak, farklı üreticilerin antikorlar farklı tavsiye edilen konsantrasyonlarda olacaktır. </li><li> ışığı tutan bir şekilde buz kovası örtün. Hücre / antikor karışımı, 30 dakika boyunca (ya da antikor üretici talimatlarına göre) yapılmaktadır izin verin. Not: Anti-BMPR IB antikor, bir ikincil antikor gerektiren birincil konjuge olmayan antikor olarak gelir, üreticinin talimatlarına uygun olarak ikincil antikor ile buz üzerinde ayrı bir boyama adımı gerçekleştirmek. </li><li> Her iki tüpleri santrifüj4 ° C&#39;de 5 dakika boyunca 300 x g. pelet aspire için dikkatli olmak, süpernatant aspire. 1 ml FACS tampon maddesi içinde tekrar süspansiyon pelet hücreleri. Yine 5 dakika boyunca 300 xg&#39;de tüpler santrifüj. Süpernatantı dikkatlice aspire 1 ml başına 1.000.000 hücre konsantrasyonunda FACS tamponu içerisinde tekrar süspansiyon hücreleri. </li><li> Yeni etiketli cam FACS tüpler içine 40 mikron hücre süzgeçler aracılığıyla &quot;BMPR-IB&quot;, &quot;unstained&quot; ve &quot;Unsorted&quot; hücreleri Filtre. Hücreler filtre geride değil emin olmak için 500 uL FACS tamponu ile filtre durulayın. Ayrıca &quot;BMPR-IB-pozitif&quot; ve etiketli iki ayrı plastik santrifüj tüplerine 2 ml standart orta ekleyin &quot;BMPR-IB-negatif.&quot; FACS makine sıralanır hücreleri toplamak için bu ikisini kullanın. </li><li> FACS makinede, floresan işaretleyici için negatif bir kapı tanımlamak için lekesiz hücreleri kullanın. </li><li> 100 mikron memesi, ilgili tüplere con içine sıralama BMPR-IB pozitif ve negatif hücreler kullanılarakStandart ortam taining. FACS makine bu yeteneği varsa sıralama sırasında 4 ° C&#39;de soğutulmuş bu tüpler tutun. NOT: Sharon ve arkadaşları tarafından JOVE 13. Maddenin bakın. FACS sıralama için mükemmel bir protokol için. </li><li> Bir hemasitometre kullanarak, her bir tüp içinde hücreleri sayın. 4 ° C&#39;de 5 dakika boyunca 300 xg&#39;de &quot;&quot;, BMPR-IB-pozitif &quot;BMPR-IB-negatif&quot; ve &quot;Unsorted&quot; tüpleri Santrifüj ve hücre pelet rahatsız etmemek için dikkatli olmak, süpernatant aspire. Daha sonra 250 uL başına 1.000.000 hücre konsantrasyonunda standart ortam içinde tekrar süspansiyon hücreleri. </li><li> Ön-kesim, 4 mm PLGA (poli [laktik-ko-glikolik asit]) hidroksiapatit ile kaplanmış iskeleleri elde edilir. 24-iyi plaka iyi her bir iskele yerleştirin. BMPR-IB-pozitif Unsorted, ve BMPR-IB-negatif (Şekil 3): (. I .e, 200.000 hücreleri) üç gruptan birine hücre süspansiyonu 50 ul her iskele örtün. NOT: LütfenLo ve arkadaşları tarafından JOVE madde 14 başvurun. PLGA iskelelerinin yapımı ile ilgili detaylar için. </li><li> Kısmen, (iskelelerinin kuruma önlemek için), kapağı plakası kapağı ve 30 dakika boyunca 37 ° C &#39;de, bir hücre kültürü inkübatöründe inkübe standart ortam ile çevre boş kuyu doldurun. </li></ol> Kalvaryum Kusur ve ACS içeren Scaffold Uygulama 4. oluşturulması NOT: yeterli kurumsal onaylar canlı farelerde kalvaryal kusurların oluşturulması için yerinde olduğundan emin olun. Bu protokol Stanford Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından onaylanmıştır. <ol><li> inhale izofluran gaz veya enjekte uzun etkili anestezik kokteyl kullanarak CD-1 çıplak fare anestezisi. <ol><li> Tek başına gaz kullanmak, bir anestezi odasına fare koyun ve 2 oksijen akışını başlatmak - izofluran% 2 konsantrasyonda 3 L / dk. fare F sonraully bilinçsiz, bir emici ped ile sıcak su sirkülasyon battaniye üzerine o eğilimli yerleştirin ve oksijen ve izofluran aynı konsantrasyon ile bir anestezi burun konisi içine burun yerleştirin. Dikkatle solunum hızını izlemek ve gerektiğinde izofluran konsantrasyonu ayarlayın. </li><li> yukarıda tarif edildiği gibi anestezi bölmesi fare anestezi, enjekte uzun etkili anestezik kokteyl kullanmak ve daha sonra anestezi kokteyl enjekte edilmesiyle gerçekleştirilebilmektedir. Anestezi odasından fareyi çıkarın ve davranışlarını gözlemlemek. NOT: Fare kısaca anestezi ancak birkaç dakika içinde ortaya çıkabilir yine tam enjekte anestezi etkisi altında anestezi gerekmektedir. bir emici ped ile kaplı bir sıcak su sirkülasyon battaniye fare aktarın. Not: bir anestetik kokteyl sağlamak için, normal tuzlu su içinde ketamin 10 mg / mL ve ksilazin, 1 mg / ml ihtiva eden bir karışım, intraperitoneal önerilir. Bu karışımın standart doz300 uL 100 mg / kg ve ksilizin 10 mg / kg ketamin eşittir 30 g fare için. Enjekte anestezi ile ilgili daha fazla bilgi için tartışma bölümüne bakın. </li><li> Anestezi derinliğini belirlemek için bir ayak tutam manevrası kullanın. hafifçe cerrah tarafından sıkışmak zaman, yeterince anestezi fare pençesini geri olmaz. </li><li> Korneanın kuruma önlemek için göz merhemi sürün. </li><li> 1 mg / kg buprenorfin SR subkutan yönetme. NOT: Üstün ağrının cerrahi sonuçlarına önce analjezi sağlanması. </li><li> tüm prosedürü sırasında, fare solunum hızını izlemek. NOT: - / dk 100 nefesler düzgün izofluran altında anestezi bir fare 50 bir solunum oranına sahip olmalıdır. Artmış solunum hızı azalmış solunum hızı anestezi çok derin olduğunu gösteriyor olabilir iken anestezi, çok hafif olduğunu gösterir. </li></ol></li><li> povidon-iyot ile kafatası dorsal deri hazırlıkine çözeltisi% 70 etanol ile üç kez, ardından. maruz cerrahi siteden ayrılmadan, steril örtüler ile örtün. NOT: steril cerrahi eldiven giyin ve işlem sırasında steril tekniği korumak. Ancak böyle steril örtü ve otoklava araçlar olarak steril yüzeyleri ve nesneler dokunun. Asistan aydınlatma, açık dikiş paketleri, vb ayarlamak mı </li><li> 15 bıçak neşter kullanılarak, dorsal kafatası çoğunluğu boyunca uzanan bir sagital orta hat kesi yapmak. ince dişli forseps kullanarak, sağ parietal kemik açığa kesi sağ tarafındaki deriyi geri çekin. </li><li> otoklavlanıp 4 mm elmas kaplı trefin matkap ile bir matkap kullanarak, paryetal kemik yoluyla kusur matkap. dura mater tabakası içine kemik geçmiş kusur uzatmayın. (Şekil 4) </li><li> defekt içine bir iskele yerleştirin ve sonra naylon dikiş çalışan cilt kesisi kapatın. </li><li> fare izleyin ve standart postoperatif bakım a sağlamakkurumsal kurallarına şeklinizi ona göre. <ol><li> o kurtarır iken temiz bir kafes içinde temiz bir kağıt havlu üzerinde fare tutun. kafes bir yarısı, bir ılık su çevrimli battaniyesi üzerine yerleştirilmelidir ve fare başlangıçta bu devre, yerleştirilmelidir. </li><li> o ambulate için yeterli bilinci yerine kadar bir fare gözetimsiz bırakmayın. tamamen iyileşti kadar diğer fareler ile bir kafes için ameliyat geçirmiş bir fare iade etmeyin. </li></ol></li><li> test edilecek olan her iskele için yeni bir fare ile yukarıdaki adımları tekrarlayın. sıcak bir boncuk sterilizatör veya diğer uygun yöntem kullanılarak ameliyatlar arasında cerrahi aletler sterilize. </li><li> Kısa bir süre işlemden sonra ve daha sonra 2, 4, 6, ve 8 hafta sonra, kalvaryal kusur, kapağın oranının analizi için bir mikro CT tarama kullanımı. NOT: Levi ve ark makalesine bakın. Kalvarial kusurların mikro BT&#39;de açıklaması için 6. </li><li> Ne zaman deney komple birTe, temiz bir ötanazi bölme içine yerleştirilmesi ve 2 L / dakikalık bir hızda bölme içine, karbon dioksit gazı akışı başlayarak fareler euthanize. solunuma tamamen (sonra yaklaşık 10 dakika) durdurdu sonra ötenazi onaylamak için servikal dislokasyon gerçekleştirin. </li><li> Deney tamamlandıktan sonra isteğe bağlı olarak, kullanım kesit ve histolojik boyama defekti içinde kemik oluşumunu değerlendirmek. NOT: McArdle ve arkadaşları tarafından makalesine bakın. Kemik hazırlama ayrıntılar için 12 histoloji için numune. Histolojik ve boyama teknikleri gözden geçirmek için, bu tür Cerrahi Patoloji 15 Manual olarak histolojik el kitabına bakın. </li></ol>

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The authors have no disclosures to make.

Protokol çerçevesinde kritik adımlar TSK Hasat sırasında, kritik bir adım kollajenaz ile yağ yeterli sindirim olduğunu. Yetersiz sindirim TSK düşük bir verimle sonuçlanır. BMPR-IB + hücrelerinin FACS sıralama sırasında, dikkatli pozitifliği açısından kapısı tanımlamak önemlidir. çok gevşek kapıları tanımlanması saf değildir sıralı nüfus neden olabilir. kalvarial defekt oluşturulması sırasında, kafatası kemik yoluyla kusur delmek için ancak dura mater iç...

yaralanma, enfeksiyon veya invaziv kanser kaynaklanan başlıca kemik defektleri hastanın iyileşmesi ve yaşam kalitesi üzerinde önemli bir etkiye sahip. Teknikleri yerde hastanın kendi vücudunda sağlıklı kemik ile bu kusurları doldurmak için var, ama bu transfer, kendi morbidite ve komplikasyonları 1, 2, 3 riskini taşır. Ayrıca, bazı kusurları yeterli donör kemik defekti doldurmak için mevcut değildir o kadar büyük veya karmaşık. Protez cihazlar kemikli defektleri doldurmak için potansiyel bir seçenek ancak bu enfeksiyon riski, donanım arızası ve yabancı cisim reaksiyonu 4 dahil olmak üzere çeşitli dezavantajları ile ilişkilidir. Bu nedenlerden dolayı hastanın kendi hücrelerini 5 kullanan biyolojik kemik yerine geçen mühendislik olasılığı büyük ilgi var. Yağ-kaynaklı stromal hücreler (TSK)onlar hastanın kendi yağ dokusunda bolca mevcuttur ve yeni kemik dokusu 6, 7 üreterek kemik defektleri iyileşmek için yeteneğini göstermiştir çünkü bu uygulama için potansiyel var. TSK hücreler ve çeşitli çalışmalar çeşitli nüfus spesifik hücre yüzey belirteçleri için seçilerek geliştirilmiş osteojenik aktivitesi 8, 9 hücre popülasyonları göstermiştir vardır. En yüksek osteojenik potansiyele sahip TSK seçilmesi, bu hücreleri ile seribaşı bir iskele büyük bir kemik defekti yeniden olabilir olasılığını artırmak olacaktır. Kemik morfogenetik proteini (BMP) sinyal kemik farklılaşmasını ve oluşumu 10 ve TSK 11 osteogenesis için önemli olduğu bilinen BMP reseptörü tip lb (BMPR-IB) düzenlenmesi için çok önemlidir. Son zamanlarda, biz olabilir b BMPR-IB o ifadeyi göstermiştirE geliştirilmiş osteojenik aktivite 12 TSK için seçmek için kullanılır. Burada, bir in vivo kalvaryal hata modeli kullanarak osteojenik aktivitesi deneyi takiben, insan yağ TSK BMPR-IB-sentezleyen izolasyonu için protokol göstermektedir.

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rapid isolation of bmpr-ib+ adipose-derived stromal cells for use in a calvarial defect healing model

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

Mikro BT açıkça kafatası defekti gösterecektir ameliyat günü yapılan tarama. Şu anda 4 mm defekt içine hiçbir büyümesi olacak. Daha sonraki taramaları başlangıca kıyasla zamanla kusurun büyüklüğünü ölçmek için zamanla elde edilir. BMPR-IB- ve tasnif hücreleri (Şekil 5) ile karşılaştırıldığında BMPR IB + hücreleri ile tohumlanmış Kusur kusurun daha hızlı kapanmasına göstermelidir. Buna ek olarak, kusur içeren kafatasının kısm...

Watch this Scientific Journal Video about Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model at JoVE.com

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

BMPR-IB + hızlı İzolasyon Kalvaryum Hata Şifa Modeli Kullanılan Stromal Hücreleri yağ kaynaklı

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

rapid-isolation-bmpr-ib-adipose-derived-stromal-cells-for-use

Research

JoVE Journal

Developmental Biology

6.6K Görüntüleme.  Stanford University School of Medicine. Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

Video: Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

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

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

Hematopoetik Hücre Otomatik Ölçümü - undekalsifiye Kemik histolojik Görüntüler stromal hücre etkileşimleri

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

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. ...

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells (ASCs)

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix (ECM) remodeling. In vivo, ECM-rich environment consisting of fibrillar collagens provides a structural support to adipose tissues during the progression and regression of obesity. Physiological ECM remodeling mediated by matrix metalloproteinases (MMPs) plays a major role in regulating adipose tissue size and function1,2. The loss of physiological collagenolytic ECM remodeling may lead to excessive collagen accumulation (tissue fibrosis), macrophage infiltration, and ultimately, a loss of metabolic homeostasis including insulin resistance3,4. When a phenotypic change of the adipose tissue is observed in gene-targeted mouse models, isolating primary ASCs from fat depots for in vitro studies is an effective approach to define the role of the specific gene in regulating the function of ASCs. In the following, we define an immunomagnetic separation of Sca1high ASCs.

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs

We present the techniques required to isolate the stromal vascular fraction (SVF) from mouse inguinal (subcutaneous) and perigonadal (visceral) adipose tissue depots to assess their gene expression and collagenolytic activity. This method includes the enrichment of Sca1high adipose-derived stem cells (ASCs) using immunomagnetic cell separation.

Yağ Deposu özgü SCA1 ve İmmunomanyetik Ayırma<sup&gt; yüksek</sup&gt; Yağ türetilmiş Kök Hücreler (TSK)

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

İnsan Plasenta Doku türetilen İzolasyon ve mezenkimal kök Genişleme / Stromal hücreler

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes differentiated from stem cells using cytokines. Unfortunately, cytokine-induced chondrogenic differentiation is costly, time-consuming, and associated with a high risk of contamination during in vitro differentiation. However, biomechanical stimuli also serve as crucial regulatory factors for chondrogenesis. For example, mechanical stress can induce chondrogenic differentiation of stem cells, suggesting a potential therapeutic approach for the repair of impaired cartilage. In this study, we demonstrated that centrifugal gravity (CG, 2,400 &#215; g), a mechanical stress easily applied by centrifugation, induced the upregulation of sex determining region Y (SRY)-box 9 (SOX9) in adipose-derived stem cells (ASCs), causing them to express chondrogenic phenotypes. The centrifuged ASCs expressed higher levels of chondrogenic differentiation markers, such as aggrecan (ACAN), collagen type 2 alpha 1 (COL2A1), and collagen type 1 (COL1), but lower levels of collagen type 10 (COL10), a marker of hypertrophic chondrocytes. In addition, chondrogenic aggregate formation, a prerequisite for chondrogenesis, was observed in centrifuged ASCs.

Mechanical stress can induce the chondrogenic differentiation of stem cells, providing a potential therapeutic approach for the repair of impaired cartilage. We present a protocol to induce the chondrogenic differentiation of adipose-derived stem cells (ASCs) using centrifugal gravity (CG). CG-induced upregulation of SOX9 results in the development of chondrogenic phenotypes.

Santrifüj Yerçekimi ile adipoz kaynaklı Kök Hücrelerin kondrojenik Farklılaşma İndüksiyon

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes ...

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. ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

İnsan göbek bağı ve fetal plasentadan Mezenkimal Stromal Hücre İzolasyonu ve Vasıflandırılması

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the efficacy of cell persistence and biodistribution of haMSCs in animal models. We demonstrated a method to label the cell membrane of haMSCs with lipophilic fluorescent dye. Subsequently, intra-articular injection of the labeled cells in rats with surgically induced KOA was monitored dynamically by an in vivo imaging system. We employed the lipophilic carbocyanines DiD (DilC18 (5)), a far-red fluorescent Dil (dialkylcarbocyanines) analog, which utilized a red laser to avoid excitation of the natural green autofluorescence from surrounding tissues. Furthermore, the red-shifted emission spectra of DiD allowed deep-tissue imaging in live animals and the labeling procedure caused no cytotoxic effects or functional damage to haMSCs. This approach has been shown to be an efficient tracking method for haMSCs in a rat KOA model. The application of this method could also be used to determine the optimal administration route and dosage of MSCs from other sources in pre-clinical studies.

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

This protocol describes an efficient way to monitor the cell persistence and biodistribution of human adipose-derived mesenchymal stem cells (haMSCs) by far-red fluorescence labeling in a rat knee osteoarthritis (KOA) model via intra-articular (IA) injection.

Vivo İnsan yağ elde edilen Mezenkimal kök hücrelerin sıçan diz osteoartrit modelinde floresan lipofilik membran boya ile izleme

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the ...

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for embryo implantation into the uterine lining of the maternal womb. Improper decidualization has been established as a root cause for implantation failure and subsequent early embryo miscarriage. Therefore, understanding the molecular mechanisms underlying decidualization is advantageous to improving the rate of successful births. In vivo based studies of artificial decidualization are often limiting due to ethical dilemmas associated with human research, as well as translational complications within animal models. As a result, in vitro assays through primary cell culture are often utilized to explore the modulation of decidualization via hormones. This study provides a detailed protocol for the isolation of HESC and subsequent artificial decidualization via the supplementation of hormones to the culturing medium. Further, this study provides a well-designed method to knockdown any gene of interest by utilizing lipid-based siRNA transfections. This protocol permits the optimization of culture purity as well as product yield, thereby maximizing the ability to utilize this model as a reliable method to understand the molecular mechanisms underlying decidualization, and the subsequent quantification of secreted agents by decidualized endometrial stromal cells.

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

This study presents a validated and optimized procedure for the isolation and culture of human endometrial stromal cells to conduct in vitro decidualization assay. Further, this study provides a detailed method to efficiently knockdown a specific gene using siRNAs in human endometrial stromal cells.

Vitro Decidualization için insan endometrium Stromal hücre izolasyon

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for ...