Name: derivation and differentiation of canine ovarian mesenchymal stem cells
Uploaded: 2018-12-16T13:00:00.000+00:00
Duration: 11 min 41 s
Description: Watch this Scientific Journal Video about Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells at JoVE.com

Yazarlar köpek sterilizasyon program UNESP-FCAV, lütfen yumurtalık sağlamak için kabul etmiş oluyorsunuz. Bu eser FAPESP (işlem No 2013/14293-0) ve burunları gelen hibe tarafından desteklenmiştir.

Bu deney dört melez kadın köpek bir köpek sterilizasyon programı seçmeli ameliyat sonrası hibe yumurtalık ile gerçekleştirildi. Bu deney UNESP-FCAV (protokol No 026991/13) hayvanların kullanımı Etik Komitesi tarafından kabul edildi.
1. deneysel hazırlık
<ol><li>Hazırlamak veya 500 mL steril Dulbecco'nın fosfat tamponlu tuz (DPBS) kalsiyum ve magnezyum olmadan satın alın.</li>
	<li>Bir collagenase hazırlamak enzim DPBS 1 ml 40 µg karıştırılarak çözüm stok. 0.2 µm şırınga filtre kullanarak filtre uygulama ve 2 mL aliquots-20 ° C'de depolayın</li>
	<li>Dulbecco'nın değiştirilmiş kartal Orta (DMEM) düşük glikoz % 10 fetal serum ve antibiyotiklerin % 1 ile kültür ortamından hazırlayın.</li>
	<li>Steril malzeme toplamak: makas ve forseps, doku kültürü flask, yemekler, tüpler ve 10 mL ve 5 mL pipetler.</li>
	<li>Standart hücre kültür Labaratuar donanımları kullanın: bir biyolojik emanet dolabı (BSC), % 5 CO2 ve 38 ° C ve bir santrifüj ayarlamak bir hücre kültür kuluçka makinesi.</li>
	<li>BSC temiz: eldivenli elleriyle % 70 ile sterilize alkol, UV ışığı kullanarak.</li>
</ol>2. izolasyon yumurtalık mezenkimal kök hücre
<ol><li>Ameliyattan sonra onların varış laboratuarındaki kadar PBS bir konik tüp buzda yumurtalık unutmayın.</li>
	<li>100 mm Petri tabağı PBS 10 mL ile doldurulması.</li>
	<li>Yumurtalık tüpü çıkarması ve PBS ile ilk Petri kabına aktarın. Onları hareketleri karıştırma ile yıkayın.</li>
	<li>Yavaşça yumurtalık almak ve başka bir çanak içine yerleştirin. Steril cımbız ve makas ile çok küçük parçalara, yaklaşık 1 mm boyutunda doku kıyma.</li>
	<li>Yumurtalık dokusu bir 35 mm Petri kabına aktarın ve collagenase 2 mL ekleyin. Doku biraz daha kıyma.</li>
	<li>38 ° c kuluçka 3 h için Petri kabına yerleştirin ve yavaşça dairesel hareketler her 20 min ile Petri kabına sallamak.</li>
	<li>3 saat sonra Petri kabına kuluçka makinesi kaldırın.</li>
	<li>Yemeğin içeriğini 15 mL tüp aktarmak. Genişletme ortam 5 mL ekleyin.</li>
	<li>Santrifüjü önce tüpü yavaşça tersine çevirin. 2100 x g 7 dk. kaldır at tüp süpernatant santrifüj kapasitesi ve Pelet genişleme medya 3 mL ile resuspend.</li>
	<li>Pelet T25 biberondan aktarın. Kuluçka % 5 ile şişe koyun 3 h için 38 ° C'de CO2 . Not: Prosedürün en önemli kısmı kuluçka 3 h sonra medya değiştirmektir.</li>
	<li>Şişe kuluçka makinesi kaldırın ve medya kalan doku ile kaldırın. Taze genişleme medya 3 mL şişe ekleyin.</li>
	<li>Medya her 48 h değiştirmek ve hücresel izdiham için şişe gözlemlemek. Not: Yordamı ana adımları Şekil 1' de görülebilir.</li>
</ol>3. yumurtalık mezenkimal kök hücre genişlemesi
<ol><li>
Hücre geçiş
	<ol>
<li>Hücreleri izdiham ulaştığınızda, medyayı şişeden çıkarın.</li>
		<li>PBS ile 3 mL şişe yıkama. PBS kaldırın.</li>
		<li>Tripsin ve şişe 38 ° C'de kuluçka 3 dakika süreyle hafifçe dokunun ayırmak için hücreleri yardımcı şişe yerine 1,5 mL ekleyin. Not: Hücreleri izdiham yaklaşık 5 - 7 sonra ilk kaplama d ulaştırılması gerekmektedir.</li>
		<li>Genişletme ortam 3 mL ekleyin ve içeriği bir konik tüp aktarın.</li>
		<li>2100 x g 7 dk. kaldır at tüp süpernatant santrifüj kapasitesi ve 1 mL genişleme medya ekleyin.</li>
		<li>Yavaşça tüp içeriğini lunaparkçı.</li>
		<li>10 µL sayma ve hücreleri ile tüp da kuluçka makinesine koy hücre gerçekleştirmek için örnek bir aliquot al.</li>
		<li>Karışımı içine hemasitometre yer 10 µL.</li>
	</ol>
</li>
	<li>
Hücreleri sayma
	<ol>
<li>Hemasitometre kılavuz çizgileri üzerinde odaklanmak ve mikroskop'ın 10 X objektif kullanın.</li>
		<li>Beş küçük kareler (Şekil 2) içinde hücreleri saymak.</li>
		<li>Sayılan sayısı mililitre başına hücre sayısı tahmin etmek için 50.000 tarafından çarpın.</li>
	</ol>
</li>
</ol>4. yumurtalık mezenkimal kök hücre farklılaşma
Not: Hill ve ark. içinde kurulan esaslarına göre farklılaşma deneyleri gerçekleştirilmiştir 9.
<ol><li>Kuyuda nüsha, 4-şey kültür tabak başına tohum 1.0 x 104 hücre.</li>
	<li>Genişletme ortam 1 mL ekleyin.</li>
	<li>Yavaşça dairesel hareketler ile çanak kışkırtmak.</li>
	<li>24 saat sonra arzu edilen farklılaşma medya ile kültür ortamı değiştirin.</li>
	<li>İçin osteojenik, Adipojenik ve chondrogenic farklılaşma, kuluçkaya indüksiyon medya 30 d için hücrelerle, Medya değiştirme ile her 3 ö. belirli farklılaşma kitleri her bu deneyleri için kullanıldı.</li>
	<li>Nörojenik lineage farklılaşma için indüksiyon medya, medya değiştirilmesi her 3 d ile 10 d için hücreleri kuluçkaya. Burada kullanılan ortamı DMEM düşük glikoz, 2 mM valproik asit, 1 µM hidrokortizon, 10 µM forskolin, 5 mM potasyum klorür, 5 µg/mL insülin ve 200 µM butylated hydroxyanisole yer.</li>
	<li>Endoderm medya reaktif üreticinin yönergelerine göre 5 d için endoderm öncüleri, kuluçkaya.</li>
	<li>Primordial germ hücreli lineage farklılaşma için indüksiyon medya medya değiştirilmesi her 3 d ile 14 d için hücrelerde kuluçkaya. Burada kullanılan medya bulunan DMEM, %10 FBS, 1 mM sodyum pyruvate, 10 ng/mL LIF, 1 mM gerekli olmayan amino asitler, 2 mM L-glutamin, 5 µg/mL insülin, 0,1 mM β-mercaptoethanol, 60 µM putrescine, 20 µg/mL transferrin, 10 ng/mL fare epidermal büyüme faktörü, 1 ng/mL insan temel fibroblast büyüme faktörü, 40 ng/mL insan gliyal hücre satırı kaynaklı nörotrofik faktör ve 15 mg/L penisilin.</li>
</ol>

<ol>
	<li>Gazit, Z., Pelled, G., Sheyn, D., Kimelman, N., Gazit, D., Atala, A., Lanza, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells.">Mesenchymal stem cells.</a> Handbook of Stem Cells (2nd Edition). , 513-527 (2013).</li><li>Zipori, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nature+of+stem+cells:+state+rather+than+entity.">The nature of stem cells: state rather than entity.</a> Nature Reviews Genetics. 5 (11), 873-878 (2004).</li><li>Auersperg, N., Wong, A. S., Choi, K. C., Kang, S. K., Leung, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+surface+epithelium:+biology,+endocrinology,+and+pathology.">Ovarian surface epithelium: biology, endocrinology, and pathology.</a> Endocrine Reviews. 22 (2), 255-288 (2001).</li><li>Ahmed, N., Thompson, E. W., Quinn, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-mesenchymal+interconversions+in+normal+ovarian+surface+epithelium+and+ovarian+carcinomas:+an+exception+to+the+norm.">Epithelial-mesenchymal interconversions in normal ovarian surface epithelium and ovarian carcinomas: an exception to the norm.</a> Journal of Cellular Physiology. 213 (3), 581-588 (2007).</li><li>Bukovsky, A., Svetlikova, M., Caudle, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oogenesis+in+cultures+derived+from+adult+human+ovaries.">Oogenesis in cultures derived from adult human ovaries.</a> Reproductive Biology and Endocrinology. 3 (1), 17 (2005).</li><li>Gong, S. P., 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=Embryonic+stem+cell-like+cells+established+by+culture+of+adult+ovarian+cells+in+mice.">Embryonic stem cell-like cells established by culture of adult ovarian cells in mice.</a> Fertility and Sterility. 93 (8), 2594-2601 (2010).</li><li>Johnson, J., Canning, J., Kaneko, T., Pru, J. K., Tilly, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+stem+cells+and+follicular+renewal+in+the+postnatal+mammalian+ovary.">Germline stem cells and follicular renewal in the postnatal mammalian ovary.</a> Nature. 428 (6979), 145 (2004).</li><li>Deans, R. J., Moseley, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells:+biology+and+potential+clinical+uses.">Mesenchymal stem cells: biology and potential clinical uses.</a> Experimental Hematology. 28 (8), 875-884 (2000).</li><li>Trinda Hill, A. B. T., Therrien, J., Garcia, J. M., Smith, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal-like+stem+cells+in+canine+ovary+show+high+differentiation+potential.">Mesenchymal-like stem cells in canine ovary show high differentiation potential.</a> Cell Proliferation. 50 (6), 12391 (2017).</li><li>Dominici, M. L. B. 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=Minimal+criteria+for+defining+multipotent+mesenchymal+stromal+cells.+The+International+Society+for+Cellular+Therapy+position+statement.">Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.</a> Cytotherapy. 8, 4315-4317 (2006).</li><li>Friedenstein, A. J., Piatetzky-Shapiro, I. I., Petrakova, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteogenesis+in+transplants+of+bone+marrow+cells.">Osteogenesis in transplants of bone marrow cells.</a> Development. 16 (3), 381-390 (1966).</li><li>Kuznetsov, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-colony+derived+strains+of+human+marrow+stromal+fibroblasts+form+bone+after+transplantation+in+vivo.">Single-colony derived strains of human marrow stromal fibroblasts form bone after transplantation in vivo.</a> Journal of Bone and Mineral Research. 12 (9), 1335-1347 (1997).</li></ol>

Yazarlar ifşa gerek yok.

Burada kanıt MSCs ovariectomy sonra biyolojik atık olarak kabul edilir köpek yumurtalık dokusu yalıtılmış olmasını sağlamak. Birçok hücre tipleri-ebilmek bulunmak içinde Yumurtalık nedeniyle gerçeğini, başarılı bir şekilde büyüdü hücreleri bir monolayer fibroblast benzeri görünümdeki seçili plastik hızlı onların bağlılığı dayalı MSCs seçmek için bir protokol evlenme teklif etti.
MSCs türetmeye kemik iliği ilk rapor MSCs plastik yapışma kapasitesini k...

Kök hücreler üzerinde odak önemli ölçüde güçlü rejeneratif tıp terapiler keşfetmek toplu amacı tarafından yakıt oldu bir araştırma çaba son on yıl içinde artmıştır yayımlanmış çalışmaları sayısı. Kök hücreler var iki birincil tanımlama işaretleri: öz - yenileme ve farklılaşma. Mezenkimal kök hücre normal doku ciro için sorumlu ve embriyonik kök hücre1' e göre farklılaşma daha sınırlı bir kapasiteye sahip. Son zamanlarda, birçok çalışma geniş bir MSCs farklılaşma göstermiştir ve olup farklar embriyonik ve yetişkin kök hücreleri arasındaki tüm2adlı bir konudur tartışma altında.
Ovarium yüzey epitel hücreleri yanıt olarak farklı türdeki ayırt etmek için kapasite istinat hem epitelyal ve mezenkimal işaretçileri3, ifade farklılaşmış, bir kaydedilmemiş katman hücre, nispeten daha az olduğunu çevre sinyalleri4. Yumurtalık kök hücre tam konumunu iyi bilinen değildir; Ancak, tunica albuginea bipotential ataları germ hücreleri5' e çıkmasına önerilmiştir. İmmünolojik araştırmalar bu hücreler stromal kökenli6 veya içinde bulunduğu veya proksimal Over yüzey7onaylanmadığına karar. Mezenkimal kök hücre hücre adezyon8' önemli bir rol oynamaktadır çok sayıda reseptörleri hızlı beri bir deney bir nüfus hızlı adezyon içeren hücrelerin seçerek hücreleri nüfusu açıkça ayırmak hipotezi test etmek için tasarlanmıştır doğada mezenkimal olarak characterizable. Son zamanlarda, bizim grup MSCs türetmeye yumurtalık dokusu hücreleri hızlı yapışma9sergilenmesi arıtılmış nüfusu elde etmek için onların ilk 3 h kültür, kültür tabakta plastik yüzeyine yapışma üzerine kurulu kapasiteli bildirdi. Burada, yumurtalık dokusu mezenkimal kök hücre izolasyon için geliştirilen yöntem açıklanmaktadır.

<table><tbody><tr><td>DPBS&amp;nbsp;</td><td>Thermo Fisher</td><td>14190144</td><td></td></tr><tr><td>Collagenase I</td><td>Thermo Fisher</td><td>17100017</td><td></td></tr><tr><td>Tissue flask</td><td>Corning</td><td>CLS3056</td><td></td></tr><tr><td>DMEM low glucose</td><td>Thermo Fisher</td><td>11054020</td><td></td></tr><tr><td>FBS</td><td>Thermo Fisher</td><td>12484-010</td><td></td></tr><tr><td>TrypLE express</td><td>Thermo Fisher</td><td>12604021</td><td></td></tr><tr><td>StemPro Adipogenesis Differentiation Kit</td><td>Thermo Fisher</td><td>A1007001</td><td></td></tr><tr><td>StemPro Chondrogenesis Differentiation Kit</td><td>Thermo Fisher</td><td>A1007101</td><td></td></tr><tr><td>StemPro Osteogenesis Differentiation Kit</td><td>Thermo Fisher</td><td>A1007201</td><td></td></tr><tr><td>STEMdiff Definitive Endoderm Kit</td><td>StemCell</td><td>5110</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Thermo Fisher</td><td>15070063</td><td></td></tr><tr><td>CD45</td><td>AbD Serotec</td><td>MCA 2035S</td><td></td></tr><tr><td>CD34</td><td>AbD Serotec</td><td>MCA 2411GA</td><td></td></tr><tr><td>CD90</td><td>AbD Serotec</td><td>MCA 1036G</td><td></td></tr><tr><td>CD44</td><td>AbD Serotec</td><td>MCA 1041</td><td></td></tr><tr><td>Nestin</td><td>Milipore</td><td>MAB353</td><td></td></tr><tr><td>&amp;beta;-Tubulin&amp;nbsp;</td><td>Milipore</td><td>MAB1637</td><td></td></tr><tr><td>DDX4</td><td>Invitrogen</td><td>PA5 -23378</td><td></td></tr><tr><td>IgG- FITC</td><td>AbD Serotec</td><td>STAR80F</td><td></td></tr><tr><td>IgG- FITC</td><td>AbD Serotec</td><td>STAR120F</td><td></td></tr></tbody></table>

derivation and differentiation of canine ovarian mesenchymal stem cells

Mezenkimal kök hücre (MSCs) ilgi yalıtım, genişleme ve kültür kolaylığı nedeniyle son on yıl içinde artmıştır. Son zamanlarda, çalışmalar bu hücreler sahip geniş farklılaşma kapasite göstermiştir. Yumurtalık hücre tabanlı terapiler için umut verici bir aday MSCs içinde zengin ve sık sık biyolojik atık olarak ovariectomy ameliyat sonra atılan nedeniyle gerçeğini temsil eder. Bu makalede, yalıtım, genişleme, için yordamlar açıklanır ve MSCs farklılaşma hücre sıralama gerekliliği olmadan köpek yumurtalık türetilmiştir teknikleri. Bu iletişim kuralı rejeneratif tıp için önemli bir araç nedeniyle klinik çalışmalar son derece türevlenebilir bu hücrelerde geniş uygulanabilirliği temsil eder ve terapötik kullanır.

Köpek yumurtalık mezenkimal kök hücre izolasyon:
Yumurtalık MSC yalıtım yordamı Şekil 1' de özetlenmiştir. Cerrahi, doku kıyma, collagenase sindirim ve medya Değiştir 3 h başında kültür sonra sonra hızlı plastik yapışkanlı özellikleri sözde MSC nüfusuyla başarıyla köpek yumurtalık dokusundan izole edildi. Hasat hücreleri hızla kültü...

Watch this Scientific Journal Video about Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells at JoVE.com

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

Burada, yalıtım, genişleme ve köpek yumurtalık dokusundan mezenkimal kök hücre farklılaşma için bir yöntem açıklanmaktadır.

Türetme ve köpek yumurtalık mezenkimal kök hücre farklılaşma

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies ...

Bu işlemin genel amacı, izole etmek, genişletmek ve mezenkimal sapları ayırt etmek tir canine yumurtalık dokusundan elde edilen satıyor. Bu deney, köpek sterilizasyon programında elektif cerrahi sonrası bağışlanan dört melez dişi köpeğin yumurtalıkları ile gerçekleştirilmiştir. Bu deney Sao Paulo State University, FVAC hayvanların kullanımı etik komitesi tarafından onaylanmıştır.Temsili sonuçlar. Tartışma.Sonuç.

derivation-differentiation-canine-ovarian-mesenchymal-stem

Research

JoVE Journal

Developmental Biology

5.6K Görüntüleme.  São Paulo State University (UNESP). Bu işlemin genel amacı, izole etmek, genişletmek ve mezenkimal sapları ayırt etmek tir canine yumurtalık dokusundan elde edilen satıyor. Bu deney, köpek sterilizasyon programında elektif cerrahi sonrası bağışlanan dört melez dişi köpeğin yumurtalıkları ile gerçekleştirilmiştir. Bu deney Sao Paulo State University, FVAC hayvanların kullanımı etik komitesi tarafından onaylanmıştır.Temsili sonuçlar. Tartışma.Sonuç.

Video: Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

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 ...

Gelişim Biyolojisi

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 ...

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Immunosurgery İnsan Embriyonik Kök Hücreleri türetilmesi

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Biyoloji

An Orthotopic Mouse Model of Ovarian Cancer using Human Stroma to Promote Metastasis

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant transgenic mouse models of ovarian cancer exist, these mice are expensive and take a long time to develop tumors. Intraperitoneal injection xenograft models lack human stroma and do not accurately model ovarian cancer metastasis. Even patient derived xenografts (PDX) do not fully recapitulate the human stromal microenvironment as serial PDX passages demonstrate significant loss of human stroma. The ability to easily model human ovarian cancer within a physiologically relevant stromal microenvironment is an unmet need. Here, the protocol presents an orthotopic ovarian cancer mouse model using human ovarian cancer cells combined with patient-derived carcinoma-associated mesenchymal stem cells (CA-MSCs). CA-MSCs are stromal progenitor cells, which drive the formation of the stromal microenvironment and support ovarian cancer growth and metastasis. This model develops early and diffuses metastasis mimicking clinical presentation. In this model, luciferase expressing ovarian cancer cells are mixed in a 1:1 ratio with CA-MSCs and injected into the ovarian bursa of NSG mice. Tumor growth and metastasis are followed serially over time using bioluminescence imaging. The resulting tumors grow aggressively and form abdominal metastases by 14 days post injection. Mice experienced significant decreases in body weight as a marker of systemic illness and increased disease burden. By day 30 post injection, mice met endpoint criteria of &gt;10% body weight loss and necropsy confirmed intra-abdominal metastasis in 100% of mice and 60%-80% lung and parenchymal liver metastasis. Collectively, orthotopic engraftment of ovarian cancer cells and stroma cells generates tumors that closely mimic the early and diffuse metastatic behavior of human ovarian cancer. Furthermore, this model provides a tool to study the role of ovarian cancer cell: stroma cell interactions in metastatic progression.

Orthotopic engraftment of ovarian cancer cells mixed with human stromal cells provides a mouse model that exhibits rapid, diffuse metastatic behavior that is characteristic of human ovarian cancer. This model also allows for the study of tumor cell and stromal cell interactions, as well as their role in tumor progression and metastasis.

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant ...

Kanser Araştırmaları

Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem cells&#8221; (CSCs) responsible for tumor initiation, maintenance and relapse following conventional chemotherapies. Identification of ovarian CSCs is therefore an important goal. A commonly used method to assess CSC potential in vitro is the spheres assay in which cells are plated under non-adherent culture conditions in serum-free medium supplemented with growth factors and sphere formation is scored after a few days. Here, we review currently available protocols for human ovarian cancer spheres assays and perform a side-by-side analysis between commonly used multi cell-based assays and a more accurate system based on single cell plating. Our results indicate that both multi cell-based as well as single cell-based spheres assays can be used to investigate sphere formation in vitro. The more laborious and expensive single cell-based assays are more suitable for functional assessment of individual cells and lead to overall more accurate results while multi cell-based assays can be strongly influenced by the density of plated cells and require titration experiments upfront. Methylcellulose supplementation to multi cell-based assays can be effectively used to reduce mechanical artifacts.

In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.

Çoklu ve tek hücre bazlı küreler tahliller kullanılarak, insan yumurtalık karsinoma hücrelerinde Kök Hücre Özelliklerinin Değerlendirilmesi

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem ...

Tıp

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Embriyonik Kök Hücreler farklılaşma oligodendrosit Öncüleri

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

Blastosist Fare trofoblast Kök Hücre türetilmesi

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

Differentiation and Characterization of Osteoclasts from Human Induced Pluripotent Stem Cells

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a single-cell suspension for further use in embryoid body induction. Following mesodermal induction, embryoid bodies undergo hematopoietic differentiation, producing a floating hematopoietic cell population. Subsequently, the harvested hematopoietic cells undergo a macrophage colony-stimulating factor maturation step and, finally, osteoclast differentiation. After osteoclast differentiation, osteoclasts are characterized by staining for TRAP in conjunction with a methyl green nuclear stain. Osteoclasts are observed as multinucleated, TRAP+ polykaryons. Their identification can be further supported by Cathepsin K staining. Bone and mineral resorption assays allow for functional characterization, confirming the identity of bona fide osteoclasts. This protocol demonstrates a robust and versatile method to differentiate human osteoclasts from iPSCs and allows for easy adoption in applications requiring large quantities of functional human osteoclasts. Applications in the areas of bone research, cancer research, tissue engineering, and endoprosthesis research could be envisioned.

This protocol presents the differentiation of human osteoclasts from induced pluripotent stem cells (iPSCs) and describes methods for the characterization of osteoclasts and osteoclast precursors.

İnsan Kaynaklı Pluripotent Kök Hücrelerden Osteoklastların Farklılaşması ve Karakterizasyonu

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a ...

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

Germ benzeri hücreler içine Yenidoğan Fare Cilt Türetilmiş Kök Hücre Farklılaşma<em&gt; In vitro</em

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...

Yağ Dokusu Kaynaklı Kök Hücre İzolasyonu ve Karakterizasyonu

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in addition to their great capacity for self-renewal, migration, and proliferation. Adipose tissue is one of the least invasive and most accessible sources of mesenchymal cells. It has also been reported to have higher yields compared to other sources, as well as superior immunomodulatory properties. Recently, different procedures for obtaining adult mesenchymal cells from different tissue sources and animal species have been published. After evaluating the criteria of some authors, we standardized a methodology that is applicable to different purposes and easily reproducible. A pool of stromal vascular fraction (SVF) from perirenal and epididymal adipose tissue allowed us to develop primary cultures with optimal morphology and functionality. The cells were observed adhered to the plastic surface for 24 h, and exhibited a fibroblast-like morphology, with prolongations and a tendency to form colonies. Flow cytometry (FC) and immunofluorescence (IF) techniques were used to assess the expression of the membrane markers CD105, CD9, CD63, CD31, and CD34. The ability of adipose-derived stem cells (ASCs) to differentiate into the adipogenic lineage was also assessed using a cocktail of factors (4 &#181;M insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 &#181;M dexamethasone). After 48 h, a gradual loss of fibroblastoid morphology was observed, and at 12 days, the presence of lipid droplets positive to oil red staining was confirmed. In summary, a procedure is proposed to obtain optimal and functional ASC cultures for application in regenerative medicine.

Yazar Spotlight: Sprague Dawley Sıçanlarının Yağ Dokusundan Türetilen Mezenkimal Kök Hücrelerin İzolasyonu ve Tanımlanması  

This protocol describes a methodology for isolating and identifying adipose tissue-derived mesenchymal stem cells (MSCs) from Sprague Dawley rats.

Sprague Dawley Sıçanlarının Yağ Dokusundan Elde Edilen Mezenkimal Kök Hücrelerin İzolasyonu ve Tanımlanması

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...

Türetme ve köpek yumurtalık mezenkimal kök hücre farklılaşma

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