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 border="0" cellpadding="0" cellspacing="0" width="1201">
	<tbody>
		<tr height="60">
			<td height="60" style="height:60px;width:309px;">Matrigel Basement Membrane Matrix</td>
			<td style="width:327px;">BD Bioscience (San Jose, CA-USA)</td>
			<td style="width:149px;">354230</td>
			<td style="width:416px;">Murine ECM proteins 
			Stock Concentration: 100% (9-12 mg/ml) 
			Final Concentration: 100%</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Dulbecco&#39;s Phosphate-Buffered Saline (D-PBS)</td>
			<td style="width:327px;">Sigma (St. Louis, MO, USA)</td>
			<td style="width:149px;">D8537</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">70 &#956;m Filters</td>
			<td style="width:327px;">Miltenyi Biotec (BergischGladbach, Germany)</td>
			<td style="width:149px;">130-095-823</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ficoll-Paque PREMIUM&#160;</td>
			<td>GE Healthcare (Uppsala, Sweden)</td>
			<td style="width:149px;">17-5442-03</td>
			<td>medium for discontinuos density gradient centrifugation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pooled human AB type serum (PhABS)</td>
			<td>LONZA (Walkersville MD-USA)</td>
			<td style="width:149px;">14-490E</td>
			<td style="width:416px;">Final Concentration: 10%</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;">Glutamax-I</td>
			<td style="width:327px;">ThermoFisher (Waltham,&#160; MA USA)</td>
			<td style="width:149px;">35050-038</td>
			<td style="width:416px;">Stabilized L-Glutamine 
			Stock Concentration: 100X 
			Final Concentration: 2 mM</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:327px;">Sigma (St. Louis, MO, USA)</td>
			<td style="width:149px;">A8412</td>
			<td style="width:416px;">Stock Concentration: 7.5% 
			Final Concentration: 0.5%</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Sodium Azide</td>
			<td style="width:327px;">Sigma (St. Louis, MO, USA)</td>
			<td style="width:149px;">S8032</td>
			<td>Final Concentration: 0.02%</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:309px;">Penicillin/Streptomycin (Pen Strep)</td>
			<td style="width:327px;">Gibco (Grand Island, NY, USA)</td>
			<td style="width:149px;">15070-063</td>
			<td style="width:416px;">Antibiotics 
			Stock Concentration: 5,000 UI/mL penicillin, 5,000 ug/mL Streptomycin 
			Final Concentration: 50 UI/mL penicillin, 50 ug/mL Streptomycin</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">T-75 culture flask for suspension cultures</td>
			<td style="width:327px;">Greiner Bio-one&#160; (Frickenhausen, Germany)</td>
			<td style="width:149px;">658 190</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">T-75 culture flask TC treated</td>
			<td style="width:327px;">Greiner Bio-one&#160; (Frickenhausen, Germany)</td>
			<td style="width:149px;">658170</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:309px;">TrypLE Select</td>
			<td style="width:327px;">ThermoFisher (Waltham,&#160; MA USA)</td>
			<td style="width:149px;">12563-011</td>
			<td style="width:416px;">Animal- free proteases detaching solution 
			Stock Concentration: 1X 
			Final Concentration: 1X</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:309px;">Trypsin/EDTA</td>
			<td style="width:327px;">ThermoFisher (Waltham,&#160; MA USA)</td>
			<td style="width:149px;">15400-054</td>
			<td style="width:416px;">Phenol red free 
			Stock Concentration: 0.5% 
			Final Concentration: 0.25%</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">anti-CD90 APC antibody&#160; (CD90)</td>
			<td style="width:327px;">MiltenyiBiotec (BergischGladbach, Germany)</td>
			<td style="width:149px;">130-095-402</td>
			<td>Final Concentration: 1:40</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">anti-CD45 APC-Vio770 antibody (CD45)</td>
			<td>MiltenyiBiotec (BergischGladbach, Germany)</td>
			<td style="width:149px;">130-096-609</td>
			<td>Final Concentration: 1:40</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">anti-CD73 PE antibody (CD73)</td>
			<td>MiltenyiBiotec (BergischGladbach, Germany)</td>
			<td style="width:149px;">130-095-182</td>
			<td>Final Concentration: 1:40</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">anti-CD31 PE Vio-770 antibody (CD31)</td>
			<td style="width:327px;">MiltenyiBiotec (BergischGladbach, Germany)</td>
			<td style="width:149px;">130-105-260</td>
			<td>Final Concentration: 1:40</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Mouse IgG1 APC antibody</td>
			<td style="width:327px;">MiltenyiBiotec (BergischGladbach, Germany)</td>
			<td style="width:149px;">130-098-846</td>
			<td>Final Concentration: 1:40</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Mouse IgG2a APC Vio770 antibody</td>
			<td style="width:327px;">MiltenyiBiotec (BergischGladbach, Germany)</td>
			<td style="width:149px;">130-096-637</td>
			<td>Final Concentration: 1:40</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Mouse IgG1 PE antibody</td>
			<td style="width:327px;">MiltenyiBiotec (BergischGladbach, Germany)</td>
			<td style="width:149px;">130-098-845</td>
			<td>Final Concentration: 1:40</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Mouse IgG1 PE Vio-770 antibody</td>
			<td style="width:327px;">MiltenyiBiotec (BergischGladbach, Germany)</td>
			<td style="width:149px;">130-098-563</td>
			<td>Final Concentration: 1:40</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:309px;">Low Glucose Dulbecco&#39;s Modified Eagle Medium&#160; (DMEM)</td>
			<td style="width:327px;">ThermoFisher (Waltham,&#160; MA USA)</td>
			<td style="width:149px;">13-1331-82</td>
			<td style="width:416px;">Phenol red-free minimal essential medium 
			Stock Concentration: 1&#39;000 mg/l glucose</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Fetal Bovine Serum&#160; (FBS)</td>
			<td style="width:327px;">ThermoFisher (Waltham,&#160; MA USA)</td>
			<td style="width:149px;">10500</td>
			<td style="width:416px;">Stock Concentration:0.2 mg/mL 
			Final Concentration: 2 &#956;g/mL</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Prolong Gold antifade reagent with 4&#8217;,6-diamidino-2-phenylindole</td>
			<td style="width:327px;">Invitrogen (Waltham, MA,&#160; USA)</td>
			<td style="width:149px;">P-36931</td>
			<td style="width:416px;">Aqueous mounting medium + DAPI 
			Final Concentration: 1X</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Paraformaldehyde</td>
			<td style="width:327px;">Sigma (St. Louis, MO, USA)</td>
			<td style="width:149px;">P6148</td>
			<td style="width:416px;">Fixative 
			Final Concentration: 4%</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">LAB-TEK two-well chamber slides</td>
			<td style="width:327px;">Sigma (St. Louis, MO, USA)</td>
			<td style="width:149px;">C6682</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Anti-Nestin antibody [clone 10C2]</td>
			<td style="width:327px;">Abcam (Cambridge, UK)</td>
			<td style="width:149px;">ab2035</td>
			<td style="width:416px;">Stock Concentration: 1 mg/ml 
			Final Concentration: 7 &#956;g/ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Alexa Fluor 555 Phalloidin</td>
			<td style="width:327px;">ThermoFisher (Waltham,&#160; MA USA)</td>
			<td style="width:149px;">A34055</td>
			<td style="width:416px;">Stock Concentration: 200 UI/ml 
			Final Concentration: 5 UI/ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">Triton X-100</td>
			<td style="width:327px;">Euroclone (Milan, Italy)</td>
			<td style="width:149px;">EMR237500</td>
			<td style="width:416px;">Final Concentration: &#39;0,05%</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">MesenPRO RS Medium&#160; (MSC-RS medium)</td>
			<td style="width:327px;">ThermoFisher (Waltham,&#160; MA USA)</td>
			<td>12746-012</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:309px;">Alexa Fluor 488 anti-mouse SFX kit</td>
			<td style="width:327px;">ThermoFisher (Waltham,&#160; MA USA)</td>
			<td style="width:149px;">A31619</td>
			<td style="width:416px;">Goat anti-mouse secondary antibody + Signal enhancer 
			Stock Concentration: 2 mg/ml 
			Final Concentration: 2 &#956;g/ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Pasteur Pipette</td>
			<td style="width:327px;">Kartell Labware&#160; (Noviglio (MI), ITALY )</td>
			<td>329</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">StemMACS AdipoDiff&#160; Media</td>
			<td style="width:327px;">MiltenyiBiotec (BergischGladbach, Germany)</td>
			<td style="width:149px;">130-091-679</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">StemMACS OsteoDiff&#160; Media</td>
			<td style="width:327px;">MiltenyiBiotec (BergischGladbach, Germany)</td>
			<td style="width:149px;">130-091-678</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Osteoimage Bone mineralization Assay</td>
			<td style="width:327px;">LONZA (Walkersville MD-USA)</td>
			<td style="width:149px;">PA-1503</td>
			<td>Hydroxyapatite specific fluorescent staining solution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">50mL Polystyrene conical tube</td>
			<td style="width:327px;">Greiner bio-one (Kremsm&#252;nster 
			Austria)</td>
			<td style="width:149px;">227261</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:309px;">Nile Red</td>
			<td style="width:327px;">ThermoFisher (Waltham,&#160; MA USA)</td>
			<td style="width:149px;">N1142</td>
			<td style="width:416px;">Fluorescent staining solution for lipids 
			Stock Concentration: 100 mM 
			Final Concentration: 200 Nm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Glycerin</td>
			<td style="width:327px;">Sigma (St. Louis, MO, USA)</td>
			<td style="width:149px;">G2289</td>
			<td>Final Concentration: &#39;50%</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Polistirene Petri dishes</td>
			<td style="width:327px;">Sigma (St. Louis, MO, USA)</td>
			<td style="width:149px;">P5606</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">24-well plates TC-treated</td>
			<td style="width:327px;">Greiner Bio-one GmbH (Frickenhausen, Germany)</td>
			<td style="width:149px;">662160</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Endothelial Growth Medium, EGM-2 BulletKit&#160; (EGM-2)</td>
			<td style="width:327px;">LONZA (Walkersville MD-USA)</td>
			<td style="width:149px;">CC-3162</td>
			<td>VEGF-rich endothelial cell growth medium</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Leica Qwin Image Analisys Software</td>
			<td style="width:327px;">Leica (Wetzlar, Germany)</td>
			<td style="width:149px;"></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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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 ...

isolating-mesangiogenic-progenitor-cells-mpcs-from-human-bone-marrow

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

8.0K 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

Isolation of Human Mesenchymal Stem Cells and their Cultivation on the Porous Bone Matrix

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs bovine bone matrix Nukbone (NKB) as a scaffold. Thus, the application of MSCs in repair and tissue regeneration processes depends principally on the efficient implementation of the techniques for placing these cells in a host tissue. For this reason, the design of biomaterials and cellular scaffolds has gained importance in recent years because the topographical characteristics of the selected scaffold must ensure adhesion, proliferation and differentiation into the desired cell lineage in the microenvironment of the injured tissue. This option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) employs bovine bone matrix as a cellular scaffold and is an efficient culture technique because the cells respond to the topographic characteristics of the bovine bone matrix Nukbone (NKB), i.e., spreading on the surface, macroporous covering and colonizing the depth of the biomaterial, after the cell isolation process. We present the procedure for isolating and culturing MSCs on a bovine matrix.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs the bovine bone matrix Nukbone (NKB) as a scaffold.

İnsan Mezenkimal Kök Hücre İzolasyonu ve Gözenekli Kemik Matrix kendi Yetiştirme

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific ...

Gelişim Biyolojisi

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

Embriyonik Kök Hücreleri Kardiyak Progenitör Hücrelerinin türetilmesi

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

Periferik kan Hematopoetik Progenitör Hücreleri İnsan Sinir Kök Hücre Doğrudan İndüksiyon

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

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 ...

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

Periventriküler Yetişkin Rat Bölgesinde ve İnsan Omurilik gelen Nöral Kök / Progenitör Hücreler izolasyonu

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

İnsan uyarılmış pluripotent kök hücrelerin türetilmiş Piramidal Nöronlar gelen Dendritik Spine Üç boyutlu Niceleme

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by distinct pools of SCs, which are located in the permanent portion of the HF, a region known as the bulge. These multipotent bulge SCs were initially identified as slow cycling label retaining cells; however, their isolation has been made feasible after identification of specific cell markers, such as CD34 and keratin 15 (K15). Here, we describe a robust method for isolating bulge SCs and epidermal keratinocytes from mouse HFs utilizing fluorescence activated cell-sorting (FACS) technology. Isolated hair follicle SCs (HFSCs) can be utilized in various in vivo grafting models and are a valuable in vitro model for studying the mechanisms that govern multipotency, quiescence and activation.

An ideal model for studying adult stem cell biology is the mouse hair follicle. Here we present a protocol for isolating different populations of hair follicles stem cells and epidermal keratinocytes, employing enzymatic digestion of mouse dorsal skin followed by FACS analysis.

Dorsal Fare Cilt Saç Folikül Kök Hücreler ve epidermal keratinositler Ayırma

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by ...

Combining Intravital Fluorescent Microscopy (IVFM) with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular niches modulating critical hematopoietic stem cell (HSC) functions1,2. Indeed, a more detailed picture of the hematopoietic microenvironment is now emerging, in which the endosteal and the endothelial niches form functional units for the regulation of normal HSC and their progeny3,4,5. New studies have revealed the importance of perivascular cells, adipocytes and neuronal cells in maintaining and regulating HSC function6,7,8. Furthermore, there is evidence that cells from different lineages, i.e. myeloid and lymphoid cells, home and reside in specific niches within the BM microenvironment. However, a complete mapping of the BM microenvironment and its occupants is still in progress.
Transgenic mouse strains expressing lineage specific fluorescent markers or mice genetically engineered to lack selected molecules in specific cells of the BM niche are now available. Knock-out and lineage tracking models, in combination with transplantation approaches, provide the opportunity to refine the knowledge on the role of specific &#34;niche&#34; cells for defined hematopoietic populations, such as HSC, B-cells, T-cells, myeloid cells and erythroid cells. This strategy can be further potentiated by merging the use of two-photon microscopy of the calvarium. By providing in vivo high resolution imaging and 3-D rendering of the BM calvarium, we can now determine precisely the location where specific hematopoietic subsets home in the BM and evaluate the kinetics of their expansion over time. Here, Lys-GFP transgenic mice (marking myeloid cells)9 and RBPJ knock-out mice (lacking canonical Notch signaling)10 are used in combination with IVFM to determine the engraftment of myeloid cells to a Notch defective BM microenvironment.

Combining Intravital Fluorescent Microscopy IVFM with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Intravital fluorescence microscopy (IVFM) of the calvarium is applied in combination with genetic animal models to study the homing and engraftment of hematopoietic cells into bone marrow (BM) niches.

Intravital floresan mikroskobu (IVFM) kemik iliği nişler hematopoetik hücrelerin Engraftment Dynamics çalışmaya genetik modelleri ile birleştirerek

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular ...

Preparation of Plasma Membrane Vesicles from Bone Marrow Mesenchymal Stem Cells for Potential Cytoplasm Replacement Therapy

We have previously reported on the generation of plasma membrane vesicles (PMVs) through the mechanical extrusion of mammalian cells. The fusion of PMVs with mitochondrial deficient Rho0 cells restored mitotic activity under normal culture conditions. Atherosclerosis, type 2 diabetes, Alzheimer&#39;s disease, and cancer are age-related diseases that have been reported to be associated with multiple mechanical and functional defects in the cytosol and organelles of a variety of cell types. Bone marrow mesenchymal stem cells (BMSCs) represent a unique cell population from the bone marrow that possess self-renewal capabilities while maintaining their multipotency. The supplementation of senescence cells with young cytoplasm from autologous BMSCs via the fusion of PMVs provides a promising approach to ameliorate or even reverse age-associated phenotypes. This protocol describes how to prepare PMVs from BMSCs via extrusion through a polycarbonate membrane with 3 &#181;m pores, determine the existence of mitochondria and examine the maintenance of membrane potential within PMVs using a confocal microscope, concentrate PMVs by centrifugation, and carry out the in vivo injection of PMVs into the gastrocnemius muscle of mice.

Age-related diseases are associated with multiple defects in components of the cytoplasm. Here, we present a protocol to prepare plasma membrane vesicles from bone marrow mesenchymal stem cells. This technique could potentially be used as a means of cytoplasm replacement therapy to ameliorate or even reverse age-associated phenotypes.

Potansiyel Sitoplazma Replasman Tedavisi için Kemik İliği Mezenkimal Kök Hücrelerinden Plazma Membran Vesiküllerinin Hazırlanması

We have previously reported on the generation of plasma membrane vesicles (PMVs) through the mechanical extrusion of mammalian cells. The fusion of PMVs ...

Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the mature Schwann cell fate. We subjected rat and human MSCs to transient hypoxic conditions (1% oxygen for 16 h) followed by expansion as neurospheres upon low-attachment substratum with epidermal growth factor (EGF)/basic fibroblast growth factor (bFGF) supplementation. Neurospheres were seeded onto poly-D-lysine/laminin-coated tissue culture plastic and cultured in a gliogenic cocktail containing &#946;-Heregulin, bFGF, and platelet-derived growth factor (PDGF) to generate Schwann cell-like cells (SCLCs). SCLCs were directed to fate commitment via coculture for 2 weeks with purified dorsal root ganglia (DRG) neurons obtained from E14-15 pregnant Sprague Dawley rats. Mature Schwann cells demonstrate persistence in S100&#946;/p75 expression and can form myelin segments. Cells generated in this manner have potential applications in autologous cell transplantation following spinal cord injury, as well as in disease modeling.

Marrow stromal cells (MSCs) with neural potential exist within the bone marrow. Our protocol enriches this population of cells via hypoxic preconditioning and thereafter directs them to become mature Schwann cells.

Olgun Schwann Hücrelerinin Üretimi İçin Bir Kaynak Olarak Kemik İçi Türe Göre Progenitör Hücrelerin Hipoksik Ön Koşullandırma

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the ...