Name: isolating mesangiogenic progenitor cells (mpcs) from human bone marrow
Uploaded: 2016-07-15T15:00:00.000+00:00
Duration: 9 min 53 s
Description: Watch this Scientific Journal Video about Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow at JoVE.com

Yazarlar, özellikle kemik iliği örnekleri ve insan osteo-ataları onun uzmanlık sağlamak için, Dr. Paolo Parchi, Cerrahi bölümü, Tıp ve Moleküler Patoloji ve Yoğun Bakım Tıbbı, Pisa Üniversitesi teşekkür etmek istiyorum

NOT: yazılı onayı sonrasında HBM örnekleri kalça protezi için ortopedik cerrahi sırasında elde edilmiştir. Hemen heparin 500 UI içeren 20 ml şırınga oyma femur boynu osteotomisinden sonra ve femoral önce, taze BM aspire etmek için kullanılmıştır. Protokol bir dengeleme kaynağına geniş bir uygulama kabul edilir. İnsan Kemik İliği Mononükleer Hücreleri 1. İzolasyon (HBM-ÇUŞ) <ol><li> Dulbecco fosfat tamponlu tuzlu su çözeltisi (D-PBS) uygulanarak, 50 ml taze BM 10 ml ve çevirerek karıştırın - 5 seyreltilir. Aynı şekilde, iki yeni 50 ml konik tüpler 25 ml dağıtmak her tüpe D-PBS 25 ml ekleyin ve ters çevirerek karıştırın. </li><li> borular çözeltisi mineral kemik parçalarının ayrılması ve yağ, oda sıcaklığında 10 dakika için beklemeye bırakın. </li><li> Dikkatle mineral kemik parçası pelet bozmadan 70 mikron filtreler aracılığıyla steril bir Pasteur pipeti ve filtre hücre süspansiyonu ile yüzen yağ çıkarmak. </li> &lt;li&gt;, aralıklı yoğunluk gradyan santrifüj için ortam, 15 ml (1.077 gr / ml) ile dört adet 50 ml tüpler ayarlayın. Bu ortam, oda sıcaklığında olduğundan emin olun. </li><li> yoğunluk gradyan ortamının üstüne seyreltilmiş BM 25 ml - Yavaşça 20 yatıyordu. Karıştırma katmanları önlemek için tüp duvarları üzerinde hücre süspansiyonu damlama izin dikkatle bu işlemi gerçekleştirmek. </li><li> Fren devre dışı, oda sıcaklığında, 30 dakika boyunca 400 x g&#39;de yoğunluk gradyan santrifüjleme yapın. </li><li> Steril bir Pasteur pipeti kullanarak iki faz arasında yer alan hücrelerin beyazımsı halka toplayın ve taze bir 50 ml tüp aktarın. </li><li> taze kültür ortamı hücreleri yıkayın: fenol kırmızısı içermeyen, düşük glukoz (1,000 mg / L), Dulbecco Modifiye Eagle Ortamı (DMEM),% 10 (h / h) havuzlanmış insan AB tipi serumu (Phab&#39;lar), 2 mM L-glutamin ve antibiyotikler (DMEM /% 10 Phab&#39;lar). 5 dakika boyunca 400 x g&#39;de santrifüjleyin. </li><li> taze DMEM 10 ml /% 10 Phab&#39;ları - Süpernatant aspire ve 5 pelet yeniden askıya. </li><li> Hücre sayımı devam edin.Tripan 1 seyreltme: 1 ile beyaz kan hücrelerinin sayısını belirler. hemasitometre Bu uygulayın ve bir faz kontrast mikroskop altında gözlemlemek. hücre sayısı küçük ve mükemmel yuvarlak eritrosit ve mavi boyalı ölü hücreleri hariç. NOT: Oldukça MPC kurtarma onların performansı için ekran Phab&#39;ları toplu tavsiye edilir. Farklı kökenli en sera MSC-benzeri hücrelerin daha yüksek oranları ile MPC kültürleri ile sonuçlanırken, ABD kaynaklarından Phab&#39;lar iyi sonuçlar vermiştir. </li></ol> HBM-ÇUŞ&#39;ların gelen MPCs 2. izolasyonu <ol><li> Eden taze DMEM /% 10 Phab&#39;lar 15 ml hidrofobik, T-75 balonları ayarlama ve pH ve sıcaklık 30 dakika boyunca% 5 CO2 içinde 37 ° C&#39;de ön-kuluçkalama ile denge sağlar. </li><li> Tohum 4 - şişesi başına 6 x 10 7 HBM-uluslu şirketlerin ve 48 saat boyunca% 5 CO2 içinde 37 ° C&#39;de inkübe edin. </li><li> Aspire edin ve kaplardan olan ve yapışmayan hücreler atılır. eden taze DMEM /% 10 Phab&#39;lar 15 ml ilave edilir ve 37 ° C&#39;de inkübe% 5 CO 2. ORTA her 48 saat değişen 8 gün - 6 kültürleri korumak. İSTEĞE BAĞLI: MPC veriminin arttırılması için, 2.3 yapışmayan hücreler yeni bir kültür şişesi içinde yeniden kaplanmış ve primer kültürlerin için tarif edildiği gibi korunabilir. </li><li> Aspire ve şişeler orta atmak taze DMEM ile yıkayın ve çözüm ayırma hayvan serbest proteaz 2 ml ekleyin. 15 dakika boyunca (uzun kuluçka kaçının) - 5 37 ° C&#39;de inkübe edin. </li><li> 5 dakika boyunca 400 x g&#39;de hücre süspansiyonu ve santrifüj taze DMEM /% 10 Phab&#39;lar 10 ml ilave edilir ve aspire </li><li> Aspire ve supernatant atın ve 1 pelet yeniden askıya - taze DMEM 2 ml /% 10 Phab&#39;ları. Adım 1.10 açıklandığı gibi hücre sayısında geçin. NOT: ayıraç ayırma olarak tripsin / EDTA kullanmayın. PPM&#39;lerin dirençli tripsin. kültürlerin morfolojik tarama hücre hasat öncesi, yüksek iğ şeklinde MSC-benzeri hücrelerin varlığını değerlendirmek için tavsiye edilir. MSC benzeri hücrelerin durumda önemli miktarda d vardıretected seçici kontamine hücreleri yok ederek hücre ürününün saflığını artırmak. Bunu yapmak için, bir şekilde tripsin emdirme 2 dakika boyunca tripsin 2 ml / EDTA,% 0.05 ilave ederek, MPC hasat öncesinde gerçekleştirilebilir. Yıkama kültürler iki kez DMEM /% 10 Phab&#39;lar 5 ml, daha sonra yukarıdaki gibi tedavi proteaz geçin. </li></ol> 3. Hücre Karakterizasyonu <ol><li> Akış sitometrisi <ol><li> Yıkama çözeltisi içinde 10 5, taze müstakil hücrelerin benzer örneklerin ayarlayın: D-PBS% 0.5 (h / h) inek serumu albümini (BSA) ve% 0.02 (ağ / hac) sodyum azid ile takviye edilmiştir. 5 dakika boyunca 400 x g&#39;de santrifüjleyin. DİKKAT: Sodyum azid toksiktir. </li><li> Yıkama çözeltisi 200 ul pelet yeniden askıya ve anti-CD90, anti-CD45, anti-CD73 ve floresan boyalar ( &quot;test&quot;) ile konjuge edilmiş anti-CD31 antikor ekleyin; paralel izotip kontrolleri ( &quot;CTRL&quot;) kurdu. NOT: antikorları boyama miktarları titrasyon veya edi tarafından belirlenmelidirüreticinin talimatlarına artırılabilir. </li><li> 30 dakika boyunca 4 ° C&#39;de inkübe edin. </li><li> 5 dakika boyunca 400 x g&#39;de santrifüjleyin. Yıkama solüsyonu 500 ul hücreleri yeniden askıya ve 7 9 10 sitometresinde renkli akış en az 5 x 10 4 olayları kazanır. </li><li> nokta-araziler ile sonuçlarını analiz ve kadran ayarlamak için &quot;CTRL&quot; kaydedilmiş olayları kullanın. NOT: MPC kültür olarak kültür tanımlamak amacıyla CD73 negatif CD90 negatif CD45 + CD31 + hücrelerin yüzdesi% 95 üzerinde olmalıdır. % 98 - bazı özel uygulamalar için, örneğin, gen ekspresyon analizi, geri kesme 97 arttırılmalıdır. </li></ol></li><li> Nestin algılama ve F-aktin organizasyon analizi <ol><li> Kültür oda slaytlar levhalar taze izole edilmiş MPCs (20,000 / cm2). Hücreler,% 5 CO2 içinde 37 ° C&#39;de gece boyunca inkübe etmek suretiyle uygun izin verin. </li><li> yıkama solüsyonu ve fi hücreleri yıkayın% 4 x 15 dakika için oda sıcaklığında (ağırlık / hacim), para-formaldehid. sabitleştirici, yıkama solüsyonu ilave 2 dakika boyunca inkübe ve dökün kaldırmak için. </li><li> iki kez yıkama tekrarlayın. </li><li> D-PBS içinde geçirgenliği hücreler oda sıcaklığında 15 dakika boyunca% 0.05 (h / h) Triton X-100 ile takviye edilmiştir. </li><li> proteinsiz sinyal arttırıcı (oda sıcaklığında 30 dakika) ya da standart engelleme çözeltisi ile reaksiyonu durdurun (D-PBS% 3 ile desteklenmiş (w / v) BSA). </li><li> çözümü engelleme / sinyal arttırıcı çıkarın. </li><li> 7 ug / ml ilave anti-insan Nestin birincil antikor (a / h) ile nemlendirilmiş bir odada gece boyunca 4 ° C&#39;de inkübe edin. Buna paralel olarak, değil özel floresan sinyalleri değerlendirmek için izotipik kontrol antikorları kullanın. </li><li> D-PBS ekleyerek slaytlar yıkayın, 2 dakika bekletin ve dökün. İki kez tekrarlayın. </li><li> 2 ug / ml ilave fluoresan boya konjuge sekonder antikor (a / h) ve karanlıkta 1 saat süreyle 4 ° C&#39;de inkübe edin. </li><li> Yıkama yukarıda kayar. </li><li> Floresan phalloidin (5 UI / ml) ilave, 30 m, oda sıcaklığında bırakınve karanlıkta D-PBS içinde 3 kere yıkayın. </li><li> odası duvarları kaldırmak ve çekirdekler tespiti için antifade reaktif ve 4 &#39;, 6-diamidino-2-fenilindol (DAPI) ile desteklenmiş, sulu yapıştırma vasatı içerisinde montaj slaydları. 7 9 10 görüntüleme geçin. </li></ol></li></ol> MPCs 4. Mesengenic Farklılaşma <ol><li> Levha 2 x 10 4 / cm2 TC-muamele T75 kültür şişelerinde taze izole edilmiş MPCs ve hücreler,% 5 CO2 içinde 37 ° C de DMEM /% 10 Phab&#39;lar gece boyunca uygun bildirin. </li><li> Mezenkimal stromal hücre genişlemesi (MSC-SC ortamı) için tasarlanmış, standart düşük serum ortamı, yaklaşık 200 ul / cm2, DMEM /% 10 Phab&#39;lar değiştirin. indüksiyon 7 ila 10 gün arasında, tipik olarak, (P1-mezenkimal) konflüansa hücreleri büyütün. 2 günde orta yenileyin. </li><li> Aspire ve şişeler orta atmak taze MSC-RS ortamı ile yıkayın ve çözüm ayırma hayvan serbest proteaz 2 ml ekleyin. 37 ° C&#39;de inkübe edin76, 5 C - 15 dakika (uzun kuluçka kaçının). </li><li> 5 dakika boyunca 400 x g&#39;de hücre süspansiyonu ve santrifüj taze MSC-SC ortamı 10 ml aspire </li><li> Taze MSC-RS orta 2 ml - Süpernatant aspire ve 1 pelet yeniden askıya. Adım 1.10 açıklandığı gibi hücre sayısında geçin </li><li> 5 x 10 3 hücre / cm 2-3 ekim onlara alt kültüre devam edin. (P2-MKH&#39;lerin) izdiham hücreleri büyür. </li><li> 4.5 adım ve bölüm 3&#39;te tarif edildiği gibi karakterize devam etmek için aşama 4.3 altında tarif edildiği gibi bir proteaz sindirimi ile Hasat hücreleri. Not: CD73 + CD90 + CD45 negatif CD31 negatif hücrelerin yüzdesi daha düşük% 95, sadece kısmi türev gösterir ve ayrıca bir kültür geçişi gerektirecektir. </li><li> Levha P2-MSC&#39;ler 2 x altı kuyucuklu TC ile tedavi edilmiş plakalarda, 10 4 / cm2 ve büyümesi de MSC-SC ortamı içinde birikene kadar. </li><li> Mark &quot;Hayır Diff&quot; olarak iki kuyu ve MSC-RS orta yenileyin. </li><li> Mark iki iyi&quot;Osteo&quot; ve özellikle MSC farklılaşması için tasarlanmış 200 ul / standart osteojenik ortamın cm 2 ile orta yerine kadar s. </li><li> Mark iki &quot;Adipo&quot; olarak kuyu ve özellikle MSC farklılaşması için tasarlanmış 200 ul / standart adipogenic ortamın cm 2 ile orta yerine. </li><li> Bütün ortam maddesi her 48 saat değiştirerek% 5 CO2 içinde 37 ° C&#39;de kültürler koruyun. NOT: Bu son derece Test tekrarlanabilir standart piyasada mevcut ayırt medyayı kullanılması tavsiye edilir. hücre içi lipid damlacıkları birikimi adipogenic kaynaklı hücrelerde belirgin iken ayırt şartlarda 2/3 hafta sonra, kireçlenme, osteojenik kaynaklı kültürlerde görülür. </li><li> Aspire kültür ortamı daha sonra D-PBS ile yıkayın atmak ve. </li><li> Oda sıcaklığında 15 dakika için para-formaldehid (ağ / hac)% 4, 1 ml ekleyerek kültürleri sabitleyin. </li><li> sabitleştirici, D-PBS ekleyin 2 dakika boyunca inkübe ve dökün kaldırmak için. Tekrarlama yıkamaiki kez. </li><li> İki &quot;Adipo&quot; 200 nM Nil Kırmızı çözelti kuyular işaretli iki &quot;Osteo&quot; birlikte hidroksiapatit özel floresan çözüm ve bir &quot;Hayır Diff&quot; kuyular işaretli birlikte bir &quot;Hayır Diff&quot; Leke. Oda sıcaklığında 11,12, 30 dakika süreyle inkübe edilir. </li><li> çözümler boyama çıkarın ve iki kez D-PBS içinde yıkayın. </li><li> Ekleme, D-PBS çıkarın D-PBS% 50 (h / h) gliserin ile takviye edilmiş ve görüntüleme 7 9 10 geçin. </li></ol> 5. MPC Sfero çimlenme Deneyi <ol><li> 3D parçacıklarının üretilmesi için bir petri çanağı kapağın iç yüzeyi üzerinde yeni izole edilmiş MPC süspansiyonu (1.5 x 10 4 hücre / damla) 20 ul damla yatmaktadır. NOT: taşıma damla kendi yırtılmasına yol açabilir gibi, fazla bırakmaya son derece tavsiye ediyoruz. </li><li> Dikkatle D-PBS asılı buharlaşmasını damla önlemek için içeren bir Petri kabı tekrarlamak için kapağı kullanın. CO2,% 5 37 ° C&#39;de inkübe edinbir gecede hücreler 3D küresellerde agrega için izin vermek. </li><li> önceden soğutulmuş 24 oyuklu bir kültür plakasına standart ECM proteinlerinin 300 ul hacimde eklenmesiyle murin hücre dışı matris (ECM) bir protein kalın bir jel ayarlama, ve 30 dakika süreyle 37 ° C&#39;de inkübe edin. </li><li> Dikkatle Petri kabı kapağı devrilebilir ve yavaşça steril bir Pasteur pipeti kullanarak parçacıklarının pick up. </li><li> ECM proteini jel üzerine parçacıklarının Lay standart VEGF zengin endotelyal hücre büyüme ortamı 700 ul alikotları ekleyin ve% 5 CO2 içinde 37 ° C&#39;de inkübe edin. </li><li> 24 saat sonra ve kültür 7 gün, 4X büyütme gücü 3D kültürlerin fotoğraf çekmek. Son istilacı hücre ve sfero kenarı arasındaki radyal mesafe ölçülerek görüntü analiz yazılımı uygulayan sferoidler çimlenme değerlendirin. en az 20 farklı yönleri boyunca tekrar ölçer. Ortalama mesafe ne zaman 50 mikron ya da üzerinde olumlu olarak kabul edilir. </li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+hitting+the+right+spot+with+mesenchymal+stromal+cells.">Concise review: hitting the right spot with mesenchymal stromal cells.</a> Stem Cells. 28 (8), 1446-1455 (2010).</li><li>Corselli, 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=The+tunica+adventitia+of+human+arteries+and+veins+as+a+source+of+mesenchymal+stem+cells.">The tunica adventitia of human arteries and veins as a source of mesenchymal stem cells.</a> Stem Cells Dev. 21 (8), 1299-1308 (2012).</li><li>Bieback, K., 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+alternatives+to+fetal+bovine+serum+for+the+expansion+of+mesenchymal+stromal+cells+from+bone+marrow.">Human alternatives to fetal bovine serum for the expansion of mesenchymal stromal cells from bone marrow.</a> Stem Cells. 27 (9), 2331-2341 (2009).</li><li>Watson, L., Elliman, S. J., Coleman, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+isolation+to+implantation:+a+concise+review+of+mesenchymal+stem+cell+therapy+in+bone+fracture+repair.">From isolation to implantation: a concise review of mesenchymal stem cell therapy in bone fracture repair.</a> Stem Cell Res Ther. 5 (2), 51 (2014).</li><li>Pacini, S., Petrini, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+MSCs+angiogenic+cells?+New+insights+on+human+nestin-positive+bone+marrow-derived+multipotent+cells.">Are MSCs angiogenic cells? New insights on human nestin-positive bone marrow-derived multipotent cells.</a> Front Cell Dev Biol. 2, 20 (2014).</li></ol>

The authors have no competing financial interests or other conflicts of interest.

In the last decades, MSCs have been extensively researched and pre-clinically evaluated for possible application in the treatment of various bone/articular, immunological, neurological, cardiovascular, gastrointestinal and hematological disorders14,15. The easy and inexpensive isolation of multipotent MSCs, from many different tissues, together with their lack of significant immunogenicity16, contribute to make these cells one of the most interesting cell population to be applied in cell based therapies. Nonetheless, the very low frequency in the tissue of origin represents a great limitation to the MSCs application in clinics, forcing the expansion of these cells, in vitro, before the infusion or transplantation. 
Expanded MSC cultures have revealed high grades of heterogeneity and variability17-19 making it difficult to reach a consensus about MSC production and characterization protocols. Moreover, recent investigations suggested the presence of multiple in vivo MSC ancestors in a wide range of tissues, which contribute to culture heterogeneity10,20. In fact, it has been proposed that particular culture conditions possibly select or simply promote specific sub-populations of MSCs progenitors present, in various percentages, in "crude" and unprocessed samples like bone marrow (hBM-MNCs) or adipose tissues (stromal vascular fraction)2. Thus, the variability in MSC-initiating cell populations together with the great number of different enrichment/isolation and culture protocols applied, represent a great obstacle to the definition of feasible MSC-based therapies.
A crucial factor affecting heterogeneity of MSC cultures is serum supplementation21. In our hands replacement of FBS with PhABS in primary cultures from hBM-MNCs, combined with high density seeding on hydrophobic plastics, led to the isolation of a novel highly adherent cell population with distinct biological features named MPCs5,6. We observed that the addition of small percentages of PhABS to FBS primary cultures also allowed MPC isolation, suggesting the presence of MPC inducing agents in the human serum6. At the moment, the MPC isolation/characterization protocol is a unique method available to obtain almost pure MPCs. The protocol has been carefully adjusted and it is highly reproducible for quality screening of MPC preparations before further applications.
MPCs could be used as a source for MSC production, thus limiting the variability introduced by use of unfractionated starting material. The precise definition of the multiple steps characterizing MPC mesengenic differentiation reported9 would allow synchronized mesenchymal cell expansion. Nonetheless, this latest condition could be realized exclusively applying highly purified MPC population, as a consequence the characterization of the cell products obtained by the protocol described here, results of crucial importance. This isolating method has been reported allowing MPC recovery with purity generally around 95%. However, donor/patient variability together with the variability related to the different batches of human pooled serum applied, could lead to a significant percentage of MSC-like cells co-isolated together with MPCs, under selective conditions. 
It is not clear if these "contaminating" MSC-like cells could arise from the other different in vivo progenitors described in bone marrow22 or from uncontrolled and spontaneous MPC differentiation. In any case, a consistent percentage of MSC-like cells in the MPC products nullify the possibility to applying these cells as homogeneous starting material for the MSC expansion. Thus, here it has been suggested a simple and inexpensive method, based on the MPC resistance to trypsin digestion, increasing the purity of the MPC products. Similar or even better results in purifying MPC cultures could be achieved by fluorescent or magnetic cell sorting performing CD73 and/or CD90 depletion, but significantly prolonging the process time and increasing the costs.
Moreover, MPCs showed expression of pluripotency-associated markers and Nestin, all rapidly lost during mesengenic differentiation7. Sprouting assay revealed MPC ability to invade murine ECM protein gel. Taken together these results indicate that MPCs have to be considered a more immature progenitor, retaining angiogenic potential. Nonetheless, the initial enthusiasm about mesodermal differentiation potential of MPCs is actually waning. In fact, after more than 7 years of studies on MPCs, mesengenic and angiogenic potential have been extensively described5-9, but differentiation toward any other cells of mesodermal origin is still lacking. Thus, here we propose a new, and more rigorous, definition of these cells as "Mesangiogenic Progenitor Cells", maintaining the acronym MPCs.
We also believe that most controversies about MSC angiogenic potential could be related to the heterogeneous composition of expanded cultures consisting of sub-populations of MPCs and MSCs in variable percentages23.
Finally, MPCs could also play a crucial role for the implementation of CBMPs applicable for tissue reconstruction, as these cells could also support the neo-vascularization. In fact, future studies on regeneration should take in consideration that the newly formed tissue growth should be supported by concomitant neo-angiogenesis. The co-existence of mesengenic and angiogenic potential in MPCs could significantly improve the regeneration potential of new therapeutic approaches that involve these interesting cells.

Mezenşimal stromal hücreler (MSC&#39;ler) bunların çok soyundan farklılaşma kapasitesi ve büyüme faktörleri / sitokinler salgılama hem de bağışıklık sistemi 1 bir rol hemopoiesis destekleme kapasiteleri için ilgili klinik değeri vardır. MSC-temelli tedaviler, hücre üretimi ve uygulaması tanımı tıbbi ürün (CBMP) tedavileri 3 tabanlı hücrenin güvenlik ve etkinlik için özel uluslararası düzenlemeye özellikle dikkat, geniş klinik ve klinik öncesi araştırma 2 nesne olmuştur. İnsan MSC&#39;ler örneğin fetal sığır serumu (FBS) ve sığır tripsin gibi ek ve hayvan kökenli, reaktifler içeren ortamda yoğun kültürlenir. Bu nedenle, hücre manipülasyonu ile ilişkili enfeksiyon riskleri, hastalar prion maruz gibi proteinler, peptidler ya da hayvan kökenli diğer biyomoleküllerin bağlantılı immünolojik riski ile karşı karşıya birlikte hücre hasat ve Transplan sonra devam olabilirtation 4. sorunu aşmak için, havuzlanmış insan AB tipi serumu (Phab&#39;lar) FBS yerine, insan kemik iliği (SM), hayvan içermeyen ortam içinde türevi MKH&#39;lerin kültürlenmiştir. Bu koşullar altında büyüyen MSC ile birlikte bir yeni hücre popülasyonunu tespit edilmiştir. Bu hücreler morfolojik ve MKH gelen fenotipik farklı ve kendine özgü bir gen tanımlama profili yanı sıra karakteristik büyüyen / yapışma özellikleri göstermiştir. Bu muhafaza hem mesengenic ve potansiyel anjiyojenik ve bu nedenle adlandırılmıştır Mezodermal Ata Hücreleri (MPCs) 5. Daha sonra, biz saflık 6 yüksek sınıfta MPCs üretmek için seçici ve tekrarlanabilir kültür koşulları tanımlamak başardık. Biz daha MPCs morfolojik ve biyolojik özellikleri incelenmiştir. PPM&#39;ler bisiklet, olumlu-Nestin yavaş gösterdi, Ki-67-negatif ve uzun telomerlerin 5 ile karakterize kromozomlar ile. Onlar plur dileipotency ilişkili transkripsiyon faktörleri Ekim-4 ve Nanog ziyade MSC ana düzenleyicileri Runx2 ve SOX9 7. mezenkimal belirteçler CD73, CD90, CD166 eksik ise fenotipik, PPM&#39;ler MSC daha düşük seviyede endoglin (CD105) olarak ifade edilmiştir. PPM&#39;lerin de özellikle podosome benzeri yapılar 8 ayakta yapışma PECAM (CD31) tutarlı ekspresyonu ile karakterize edilen molekül, integrinler αL (CD11a) Branşman (CD11b) αX (CD11c) ve integrin β2 (CD18) farklı bir modelini göstermektedir . Standart MSC genişleme medyada, PPM&#39;ler derhal 9 sinyalizasyon Wnt5 / Calmodulin hücre aktivasyonunu içeren bir ara aşamasında MKH ayrışmıştır. Murin hücre dışı matris (ECM) proteini 3D kültürlerinde sferoidler filiz kabiliyetleri ile gösterildiği gibi MPCs da anjiyojenik özellikleri muhafaza. anjiyojenik potansiyeli hızla mesengenic soy boyunca MPC farklılaşma sonra kayboldu. Burada yanlısı mevcutprotokollere‖ izole etmek ve HBM kan numunelerinden yüksek ölçüde saflaştırılmış MPCs karakterize etmek için optimize edilmiştir. MPC mesengenic ve anjiyojenik farklılaşması için tekrarlanabilir protokoller de tarif edilmiştir.

<table><tbody><tr><td>Matrigel Basement Membrane Matrix</td><td>BD Bioscience (San Jose, CA-USA)</td><td>354230</td><td>Murine ECM proteins&lt;br/&gt; Stock Concentration: 100% (9 - 12 mg/ml)&lt;br/&gt; Final Concentration: 100%</td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline (D-PBS)</td><td>Sigma (St. Louis, MO, USA)</td><td>D8537</td><td></td></tr><tr><td>70 &amp;mu;m Filters</td><td>Miltenyi Biotec (BergischGladbach, Germany)</td><td>130-095-823</td><td></td></tr><tr><td>Ficoll-Paque PREMIUM&amp;nbsp;</td><td>GE Healthcare (Uppsala, Sweden)</td><td>17-5442-03</td><td>medium for discontinuos density gradient centrifugation</td></tr><tr><td>Pooled human AB type serum (PhABS)</td><td>LONZA (Walkersville MD-USA)</td><td>14-490E</td><td>Final Concentration: 10%</td></tr><tr><td>Glutamax-I</td><td>ThermoFisher (Waltham,&amp;nbsp; MA USA)</td><td>35050-038</td><td>Stabilized L-Glutamine&lt;br/&gt; Stock Concentration: 100x&lt;br/&gt; Final Concentration: 2 mM</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma (St. Louis, MO, USA)</td><td>A8412</td><td>Stock Concentration: 7.5%&lt;br/&gt; Final Concentration: 0.5%</td></tr><tr><td>Sodium Azide</td><td>Sigma (St. Louis, MO, USA)</td><td>S8032</td><td>Final Concentration: 0.02%</td></tr><tr><td>Penicillin/Streptomycin (Pen Strep)</td><td>Gibco (Grand Island, NY, USA)</td><td>15070-063</td><td>Antibiotics&lt;br/&gt; Stock Concentration: 5,000 UI/ml penicillin, 5,000 &amp;#956;g/ml Streptomycin&lt;br/&gt; Final Concentration: 50 UI/ml penicillin, 50 &amp;#956;g/ml Streptomycin</td></tr><tr><td>T-75 culture flask for suspension cultures</td><td>Greiner Bio-one&amp;nbsp; (Frickenhausen, Germany)</td><td>658 190</td><td></td></tr><tr><td>T-75 culture flask TC treated</td><td>Greiner Bio-one&amp;nbsp; (Frickenhausen, Germany)</td><td>658170</td><td></td></tr><tr><td>TrypLE Select</td><td>ThermoFisher (Waltham,&amp;nbsp; MA USA)</td><td>12563-011</td><td>Animal-free proteases detaching solution&lt;br/&gt; Stock Concentration: 1x&lt;br/&gt; Final Concentration: 1x</td></tr><tr><td>Trypsin/EDTA</td><td>ThermoFisher (Waltham,&amp;nbsp; MA USA)</td><td>15400-054</td><td>Phenol red free&lt;br/&gt; Stock Concentration: 0.5%&lt;br/&gt; Final Concentration: 0.25%</td></tr><tr><td>anti-CD90 APC antibody&amp;nbsp; (CD90)</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-095-402</td><td>Final Concentration: 1:40</td></tr><tr><td>anti-CD45 APC-Vio770 antibody (CD45)</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-096-609</td><td>Final Concentration: 1:40</td></tr><tr><td>anti-CD73 PE antibody (CD73)</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-095-182</td><td>Final Concentration: 1:40</td></tr><tr><td>anti-CD31 PE Vio-770 antibody (CD31)</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-105-260</td><td>Final Concentration: 1:40</td></tr><tr><td>Mouse IgG1 APC antibody</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-098-846</td><td>Final Concentration: 1:40</td></tr><tr><td>Mouse IgG2a APC Vio770 antibody</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-096-637</td><td>Final Concentration: 1:40</td></tr><tr><td>Mouse IgG1 PE antibody</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-098-845</td><td>Final Concentration: 1:40</td></tr><tr><td>Mouse IgG1 PE Vio-770 antibody</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-098-563</td><td>Final Concentration: 1:40</td></tr><tr><td>Low Glucose Dulbecco&#039;s Modified Eagle Medium&amp;nbsp; (DMEM)</td><td>ThermoFisher (Waltham,&amp;nbsp; MA USA)</td><td>13-1331-82</td><td>Phenol red-free minimal essential medium&lt;br/&gt; Stock Concentration: 1,000 mg/L glucose</td></tr><tr><td>Fetal Bovine Serum&amp;nbsp; (FBS)</td><td>ThermoFisher (Waltham,&amp;nbsp; MA USA)</td><td>10500</td><td>Stock Concentration:0.2 mg/ml&lt;br/&gt; Final Concentration: 2 &amp;mu;g/ml</td></tr><tr><td>Prolong Gold antifade reagent with 4&amp;rsquo;,6-diamidino-2-phenylindole</td><td>Invitrogen (Waltham, MA,&amp;nbsp; USA)</td><td>P-36931</td><td>Aqueous mounting medium + DAPI&lt;br/&gt; Final Concentration: 1x</td></tr><tr><td>Paraformaldehyde</td><td>Sigma (St. Louis, MO, USA)</td><td>P6148</td><td>Fixative&lt;br/&gt; Final Concentration: 4%</td></tr><tr><td>LAB-TEK two-well chamber slides</td><td>Sigma (St. Louis, MO, USA)</td><td>C6682</td><td></td></tr><tr><td>Anti-Nestin antibody [clone 10C2]</td><td>Abcam (Cambridge, UK)</td><td>ab2035</td><td>Stock Concentration: 1 mg/ml&lt;br/&gt; Final Concentration: 7 &amp;mu;g/ml</td></tr><tr><td>Alexa Fluor 555 Phalloidin</td><td>ThermoFisher (Waltham,&amp;nbsp; MA USA)</td><td>A34055</td><td>Stock Concentration: 200 UI/ml&lt;br/&gt; Final Concentration: 5 UI/ml</td></tr><tr><td>Triton X-100</td><td>Euroclone (Milan, Italy)</td><td>EMR237500</td><td>Final Concentration: 0.05%</td></tr><tr><td>MesenPRO RS Medium&amp;nbsp; (MSC-RS medium)</td><td>ThermoFisher (Waltham,&amp;nbsp; MA USA)</td><td>12746-012</td><td></td></tr><tr><td>Alexa Fluor 488 anti-mouse SFX kit</td><td>ThermoFisher (Waltham,&amp;nbsp; MA USA)</td><td>A31619</td><td>Goat anti-mouse secondary antibody + Signal enhancer&lt;br/&gt; Stock Concentration: 2 mg/ml&lt;br/&gt; Final Concentration: 2 &amp;mu;g/ml</td></tr><tr><td>Pasteur Pipette</td><td>Kartell Labware&amp;nbsp; (Noviglio (MI), ITALY )</td><td>329</td><td></td></tr><tr><td>StemMACS AdipoDiff&amp;nbsp; Media</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-091-679</td><td></td></tr><tr><td>StemMACS OsteoDiff&amp;nbsp; Media</td><td>MiltenyiBiotec (BergischGladbach, Germany)</td><td>130-091-678</td><td></td></tr><tr><td>Osteoimage Bone mineralization Assay</td><td>LONZA (Walkersville MD-USA)</td><td>PA-1503</td><td>Hydroxyapatite specific fluorescent staining solution</td></tr><tr><td>50 ml Polystyrene conical tube</td><td>Greiner bio-one (Kremsm&amp;uuml;nster&lt;br/&gt; Austria)</td><td>227261</td><td></td></tr><tr><td>Nile Red</td><td>ThermoFisher (Waltham,&amp;nbsp; MA USA)</td><td>N1142</td><td>Fluorescent staining solution for lipids&lt;br/&gt; Stock Concentration: 100 mM&lt;br/&gt; Final Concentration: 200 Nm</td></tr><tr><td>Glycerin</td><td>Sigma (St. Louis, MO, USA)</td><td>G2289</td><td>Final Concentration: 50%</td></tr><tr><td>Polistirene Petri dishes</td><td>Sigma (St. Louis, MO, USA)</td><td>P5606</td><td></td></tr><tr><td>24-well plates TC-treated</td><td>Greiner Bio-one GmbH (Frickenhausen, Germany)</td><td>662160</td><td></td></tr><tr><td>Endothelial Growth Medium, EGM-2 BulletKit&amp;nbsp; (EGM-2)</td><td>LONZA (Walkersville MD-USA)</td><td>CC-3162</td><td>VEGF-rich endothelial cell growth medium</td></tr><tr><td>Leica Qwin Image Analisys Software</td><td>Leica (Wetzlar, Germany)</td><td></td><td>Image analysis software</td></tr></tbody></table>

isolating mesangiogenic progenitor cells (mpcs) from human bone marrow

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel cell population specifically selected for growth in human serum supplemented medium. These cells are characterized by morphological, phenotypic, and molecular features distinct from MSCs and we named them Mesodermal Progenitor Cells (MPCs). MPCs are round, with a thick highly refringent core region; they show strong, trypsin resistant adherence to plastic. Failure to expand MPCs directly revealed that they are slow in cycling. This is as also suggested by Ki-67 negativity. On the other hand, culturing MPCs in standard medium designed for MSC expansion, gave rise to a population of exponentially growing MSC-like cells. Besides showing mesenchymal differentiation capacity MPCs retained angiogenic potential, confirming their multiple lineage progenitor nature. Here we describe an optimized highly reproducible protocol to isolate and characterize hBM-MPCs by flow cytometry (CD73, CD90, CD31, and CD45), nestin expression, and F-actin organization. Protocols for mesengenic and angiogenic differentiation of MPCs are also provided. Here we also suggest a more appropriate nomenclature for these cells, which has been re-named as "Mesangiogenic Progenitor Cells".

Burada tarif edilen seçici kültür koşulları HBM-çok uluslu% 1.0 bir roman yapışık ve hemen hemen monomorfik hücre popülasyonu izole izin (0,5-2,0 x 10 5 6 HBM-uluslu şirketlerin - taze BM numuneleri, 10 ml) 5,6 . MPCs 5 gibi, hareketsiz, Ki-67-pozitif hücrelerin bulunduğu yuvarlak, - bu büyük (60 um çapında 40) tespit edilmiştir. Morfolojik olarak, bunlar (Şekil 1 A.1 beyaz oklar) daha yüksek bir büyütme güçte filopodia çok gösteren düz, ince çevre ile çevrelenmiş kalın çekirdek bölge ile ayırt edici bir kızarmış yumurta-şeklinde karakterize edilir. Dış hücre sınır Polar uzama genellikle (Şekil 1 A.1 siyah oklar) görülmektedir. Bu morfoloji, standart MSC kültürlerinde rapor tipik iğ şekilli mezenkimal stromal hücre görünüşünden açıkça farklıdır. Akım sitometri m iken CD31 ve CD45 ifade taze izole MPCs% 95 in üzerinde gösterdiesenchymal ilişkili markörler CD90 ve CD73 13 tespit edilemeyen (Şekil 1 A.2) idi. Biz MPCs için dört antijenlerin bu sınırlı kümesi olarak gösterge görüyoruz. MPCs daha ayırt edici özellikleri hücrelerde algılanmaz (Şekil 1 A.3&#39;te kırmızı) bir podosome benzeri yapıların sayısını ortaya noktalı F-aktin dağıtım ve Nestin yoğun anlatım (Şekil 1 A.3&#39;te yeşil) vardır izotipik kontrol antikoru ile lekelenmiş (veriler gösterilmemiştir). Standart RS Kültürleme MPCs orta katlanarak büyüyen MSC-benzeri hücreler (Şekil 1 B.1) içine hızlı farklılaşma MSC genişleme sonuçları için tasarlanmış. Iki geçit sonra hücreler nihayet CD73 + CD90 + CD45 negatif CD31 neg (Şekil 1 B.2) için CD73 negatif CD90 negatif CD45 + CD31 + &#39;dan kendi fenotip geçiş. Bu süreç içinde, PPM&#39;ler yeniden azotaNestin ifade Birkaç nadir hücreleri (Şekil 1 B.3) sınırlı olur ise nize stres liflerinin içine F-aktin. kesin MSC gibi fenotip içine MPC mesengenic farklılaşma farklı hücre morfolojileri ortaya iki farklı adımlar sayesinde gerçekleşir. MSC SC ortamı içinde bir hafta MPC-benzeri hücrelerin bir artık doldurma işleminden sonra halen (Şekil 2, bir P1-MSC), düz, poligonal, çok dallı hücrelerinin konfluent tabaka içinde tespit edilebilir. Başka bir kanala (Şekil 2, bir P2-MSC) iğsi MSC-benzeri hücrelerin neredeyse monomorfik kültür elde etmek için gereklidir. böylece MSC doğasını teyit en az 2 hafta boyunca seçici ortama aktarıldığında bu katlanarak büyüyen hücreler kolayca osteoblast veya adipositlere ayırt edebilirsiniz. Osteojenik kaynaklı kültürlerde, kireçlenme, ya kolorimetrik Alizarin-S leke ya da belirli bir floresan boyalar (Şekil 2 B yeşil) ile tespit edilebilir. adipojenik endüksiyon sonra hücreler(Şekil 2 B kırmızı) ya kolorimetrik Yağ Kırmızı veya floresan Nil Kırmızı leke ortaya lipid damlacığı birikimi gösterir. MPC yazarak anjiyojenez deneyi filizlenme ile teyit edilmiştir. PPM&#39;ler 24 saat VEGF-uyarıcı (Şekil 2 C) sonra 3D sferoidlerde gelen fare ECM protein jel istila (mikron 50) yeteneğini gösterdi. 600 mikron mesafe - Bir hafta hücreleri istila 300 tespit edildikten sonra. Tersine, işgal kapasitesi mesengenic farklılaşma (Şekil 2 D) sonra P2-MKH kayboldu. <img alt="Şekil 1" src="/files/ftp_upload/54225/54225fig1.jpg" /> Şekil 1. Taze İzole MPCs Belirgin Özellikler var. DMEM /% 10 Phab&#39;ları içinde Kültüre HBM-ÇUŞ&#39;larına yedi gün boyunca MKH kolayca ayırt sakin MPCs (A) nüfusu (B) yol açar </strong&gt; morfoloji açısından (A.1, B.1, ölçek çubukları = 100 mikron), fenotip (A.2, B.2), F-aktin dağılımı (A.3&#39;te kırmızı, B.3), ve Nestin ifade (A.3, B.3&#39;te yeşil, ölçek çubuklar 20 mikron =). <a href="https://www.jove.com/files/ftp_upload/54225/54225fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="şekil 2" src="/files/ftp_upload/54225/54225fig2.jpg" /> 2. MPCs orta MSC genişlemesi için tasarlanmış ticari olarak mevcut RS DMEM / Phab&#39;ları in vitro. Değiştirme Standart MKH ve Show çimlenme Angiogenezis içine ayırt Şekil MPCs mesengenic indüksiyon tetikler. Kültür için tek bir hafta sonra bir kaç artık MPCs de MSC-SC ortamı içinde bir başka geçiş birleşik MSC-benzeri hücre popülasyonu (P2-MSC&#39;ler, bir ölçek bar = 100 um) neden olurken, saptanabilir (P1-MSC&#39;ler) vardır. PKalsiyum birikimi (B yeşil) ve lipid damlacığı birikimi (B kırmızı, ölçek çubukları = 200 mikron) gösterdiğine göre 2-MKH ölümcül sırasıyla, uygun uyaranların altında osteosit veya adipositler içine ayırt. MPCs P2-MSC (D, ölçek çubukları = 1,0 mm) bir farkı fare ECM proteini jel (C) sferoidler tutarlı filizlenme gösteriyor. <a href="https://www.jove.com/files/ftp_upload/54225/54225fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a>

Watch this Scientific Journal Video about Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow at JoVE.com

Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

Here we describe an optimized, highly reproducible protocol to isolate Mesodermal Progenitor Cells (MPCs) from human bone marrow (hBM). MPCs were characterized by flow cytometry and nestin expression. They showed the ability to give rise to exponentially growing MSC-like cell cultures while retaining their angiogenic potential.

İnsan Kemik İliği gelen Yalıtımlı Mesangiogenic Ata Hücreleri (MPCs)

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel ...

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Research

JoVE Journal

Developmental Biology

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

8.1K Görüntüleme.  University of Pisa. Here we describe an optimized, highly reproducible protocol to isolate Mesodermal Progenitor Cells (MPCs) from human bone marrow (hBM). MPCs were characterized by flow cytometry and nestin expression. They showed the ability to give rise to exponentially growing MSC-like cell cultures while retaining their angiogenic potential.

Video: Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

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

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

İnsan Kardiyak Doku perivasküler Multipotent Öncü hücre popülasyonlarının izolasyonu

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

Yalıtım endotelyal Progenitor Hücre insan göbek kordon kanı

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

Yalıtım, kültür ve kemik iliği Stromal hücreler ve Osteoklast ataları fareler gelen farklılaşma

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

İnsan Göbek kordonu İzolasyon ve Hayvan Serum Genişleme Mezenkimal stromal hücreler (MKH) ve endotel Koloni Oluşturan Progenitör Hücreler (ECFCs) Türetilmiş

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

Biyoloji

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

Kimlik ve yalıtım Oligopotent ve Lineage için kabul edilen miyeloid ataları fare kemik iliği dan

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

İmmünoloji ve Enfeksiyon

Isolation and Culture of Bone Marrow Mesenchymal Stem Cells from the Human Mandible

Human mesenchymal stem cells (hMSCs) have shown great potential in bone regeneration, immune modulation, and treating refractory chronic diseases. Various origins have been found to obtain hMSCs recently, while bone marrow was still considered the main source. Bone marrow-derived MSCs (BMSCs) from different donor bone sites have distinct characteristics due to microenvironmental factors. Studies have shown that BMSCs from maxillofacial bone may have greater proliferative and osteogenic capacities than BMSCs from long bones or the iliac crest. And maxillofacial BMSCs were considered more suitable for stem cell therapy in the maxillofacial tissues. The mandible, especially the ascending ramal area with sufficient marrow, was a feasible donor site for harvesting BMSCs. This study described a protocol for harvesting, isolating, and culturing human mandibular bone marrow-derived MSCs (hmBMSCs). Furthermore, immunophenotyping of hmBMSCs, proliferation assays, and in vitro induction of osteogenic, adipogenic, and chondrogenic differentiation was performed to identify the cultured cells. Applying this protocol can help the researchers successfully obtain enough high-quality hmBMSCs, which is necessary for further studies of the biological function, microenvironmental effects, and clinical applications.

The present protocol describes an efficient procedure for isolating and culturing of human mandibular bone marrow-derived mesenchymal stem cells using the whole bone marrow adherence method. The cultured cells were identified by cell proliferation assays, flow cytometry, and multilineage differentiation induction.

Human mesenchymal stem cells (hMSCs) have shown great potential in bone regeneration, immune modulation, and treating refractory chronic diseases. Various ...

Biyomühendislik

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

İnsan İskelet Kası Kan-damar-türevli Multipotent Öncüleri İzolasyonu

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Isolation and Identification of Porcine Bone Marrow Mesenchymal Stem Cells and their Derived Extracellular Vesicles

With the development of stem cell therapy in translational research and regenerative medicine, bone marrow mesenchymal stem cells (BM-MSCs), as a kind of pluripotent stem cells, are favored for their instant availability and proven safety. It has been reported that transplantation of BM-MSCs is of great benefit to repairing injured tissues in various diseases, which might be related to modulating the immune and inflammatory responses via paracrine mechanisms. Extracellular vesicles (EVs), featuring a double-layer lipid membrane structure, are considered to be the main mediators of the paracrine effects of stem cells. Recognized for their crucial roles in cell communication and epigenetic regulation, EVs have already been applied in vivo for immunotherapy. However, similar to its maternal cells, most of the studies on the efficacy of transplantation of EVs still remain at the level of small animals, which is not enough to provide essential evidence for clinical translation. Here, we use density-gradient centrifugation to isolate bone marrow cells (BMC) from porcine bone marrow at first, and get porcine BM-MSCs (pBM-MSCs) by cell culture subsequently, identified by the results of observation under the microscope, induced differentiation assay, and flow cytometry. Furthermore, we isolate EVs derived from pBM-MSCs in cell supernatant by ultracentrifugation, proved by the techniques of transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting successfully. Overall, pBM-MSCs and their derived EVs can be isolated and identified effectively by the following protocols, which might be widely used in pre-clinical studies on the transplantation efficacy of BM-MSCs and their derived EVs.

This article elaborates a method to isolate and identify porcine bone marrow mesenchymal stem cells (pBM-MSCs) and extracellular vesicles (EVs) derived from them, providing a methodological basis for the pre-clinical evaluation of transplantation efficacy of BM-MSCs and their derived EVs.

With the development of stem cell therapy in translational research and regenerative medicine, bone marrow mesenchymal stem cells (BM-MSCs), as a kind of ...

Tıp

Sıçan Çene Kemik İliği Mezenkimal Kök Hücrelerinin Niş Bazlı İzolasyonu

Technique for Isolation and Culture of Rat Jaw Bone Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from appendicular bones, BMMSCs derived from the jaw have greater proliferative and osteogenic differentiation ability, gradually becoming important seed cells for jaw defect repair. However, the mandible has a complex bony structure and less cancellous content than appendicular bones. It is difficult to acquire a large number of high-quality jaw-derived marrow mesenchymal stem cells using traditional methods. This study presents a &#39;niche-based approach on stemness&#39; for isolating and culturing rat jaw bone marrow mesenchymal stem cells (JBMMSCs). Primary rat JBMMSCs were isolated and cultured using the whole bone marrow adherent method combined with the bone slice digestion method. The isolated cells were identified as JBMMSCs through cell morphology observation, detection of cell surface markers, and multi-directional differentiation induction. The cells extracted by this method exhibit a &#39;fibroblast-like&#39; spindle shape. The cells are long, spindle-shaped and fibroblast-like. The flow cytometry analysis shows these cells are positive for CD29, CD44, and CD90 but negative for CD11b/c, CD34, and CD45, which is congruent with BMMSCs characteristics. The cells show strong proliferation capacity and can undergo osteogenic, adipogenic, and chondrogenic differentiation. This study provides an effective and stable method for obtaining enough high-quality JBMMSCs with strong differentiation ability in a short time, which could facilitate further studies of the exploration of biological function, regenerative medicine, and related clinical applications.

Yazar Spotlight: Yüksek Saflıkta Çene Kemiği İliği Mezenkimal Kök Hücrelerinin Doku Mühendisliğinde Uygulanması

Mesenchymal stem cells from the jaw bone marrow have significant functions in diverse differentiation, self-renewal, and immune modulation. They have emerged as a crucial reservoir of precursor cells in gene therapy, tissue engineering, and regenerative medicine. Here, we present a unique method for isolating jaw bone marrow mesenchymal stem cells in rats.

Sıçan Çene Kemik İliği Mezenkimal Kök Hücrelerinin İzolasyonu ve Kültürü Tekniği

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from ...