Bu çalışma Sağlık Araştırma hibe R01-DE021683-01 ve Sağlık Araştırması hibe R01-DE019434 National Institutes MTL Ulusal Sağlık Enstitüleri tarafından desteklenen; MTCDCW Howard Hughes Tıp Enstitüsü Araştırma Bursu ACS Franklin Martin Fakültesi Araştırma Bursu, Çocuk Rejeneratif Tıp Hagey Laboratuvarı ve Stanford Üniversitesi Çocuk Sağlığı Araştırma Enstitüsü Fakültesi Akademik Ödülü tarafından desteklenmiştir.

NOT: Tüm hasta numuneleri bilgilendirilmiş rıza ile elde edilmiştir ve deneysel protokolleri gözden ve Stanford Üniversitesi Kurumsal Değerlendirme Kurulu (Protokol # 2188 # 9999 ve) tarafından onaylanmıştır. 1. Hücre İzolasyonu ve Kültür: <ol><li> Genel / lokal anestezi altında karın, kanadını, ve / veya uyluk bölgesi seçmeli lipoaspiration geçiren sağlıklı kadın hastalarda insan deri altı yağ dokusu edinin. Kurumsal Değerlendirme Kurulu (KİK) onayı insan dokularından TSK izole protokolü elde edilmiş olduğundan emin olun ve bu tür malzemeler ile çalışırken kurumsal güvenlik önlemlerine uyun. </li><li> Lipoaspirated adipoz dokudan SVF elde edilmesi için, ilk olarak 1 x steril fosfat-tamponlu tuzlu su, eşit hacimlerde (PBS) lipoaspirate üç kez yıkayın. Dikkatle aspire ve alt sulu tabaka atın. </li><li> Ben Hank de Kolajenaz 0.075% Tip: kollajenaz sindirim tampon hazırlayınDengeli Tuz Çözeltisi (HBSS). FACS tamponu hazırlayın: 2% FBS, 1% P188 ve PBS içinde% 1 Pen-Strep. Bir piyasada mevcut 0.22 mikron gözenek boyutu hızlı akış polietersülfon filtre kullanarak her iki çözümleri Filtre. </li><li> Yıkandı, yağ dokusu sindirimi için, 37 ° C (yaklaşık 180 sallar / dk) 60 dakika boyunca çalkalanan bir su banyosu içinde güvenli bir şekilde sindirim gemi kolajenaz sindirimi tampon maddesinin eşit hacmi ekleyin ve koyun. Not: Bu sallama esnasında maksimum sindirim için izin vermesi, gerekli olandan daha büyük bir hacim sindirim kap kullanmak için en iyi (yani, kolajenaz sindirim tamponu 250 mi, 1 litrelik bir steril şişe içine yıkanmış adipoz dokusu 250 ml) eklenmiştir. </li><li> FACS tampon maddesinin eşit hacmi ilave edildikten ve 5 dakika boyunca oda sıcaklığında oturmak için izin enzimatik aktivitesini nötralize. Daha sonra, 4 ° C&#39;de 20 dakika boyunca 233 x g&#39;de santrifüje tabi tutun. </li><li> Dikkatle aspire ve yüksek yoğunluklu SVF pelet rahatsız etmemek için özen, süpernatant atın. </li><li> RT kırmızı 5-10 ml pelletiniHücre topağı boyutuna göre, liziz tamponunda. Oda sıcaklığında 5 dakika boyunca 233 x g&#39;de 5 dakika santrifüj oda sıcaklığında oturup çözeltisi bırakın. </li><li> Aspire yüzer madde ve topak boyutuna göre geleneksel büyüme ortamı 5-10 ml ekleyerek pelletini (Dulbecco Modifiye Eagle Ortamı [DMEM] /% 10 FBS /% 1 penisilin-streptomisin solüsyonu [Pen-strep]). </li><li> Selüler artığın ayrılması için bir 100 um naylon hücre süzgecinden Süspansiyon filtre edilir. </li><li> 4 ° C&#39;de 5 dakika boyunca 233 x g&#39;de santrifüj ve pelet bozmadan süpernatant atılır. </li><li> Topak boyutuna dayalı geleneksel büyüme ortamı 5-10 ml olacak şekilde pelletini. </li><li> 15 cm&#39;lik standart kültür çanak 2 x 10 6 hücre yerleştirin ve 37 ° C /% 21 O 2,% 5 CO 2 birincil kültürleri O / N kurmak. 37 ° C /% 21 O2, büyüme ortamı içinde,% 5 CO2 seviyesinde kendiliğinden farklılaşmasını önlemek için alt konfluent seviyelerde muhafaza edin. NOT: Tam Hücre yoğunluğuKonfluent 15 cm plaka yaklaşık 4-6 x 10 6 hücre olduğunu. </li></ol> 2. Boyama <ol><li> Boyama öncesi 36 saat için 37 ° C /% 21 O2,% 5 CO2, geleneksel bir büyüme ortamı içinde kültür TSK / ayırma. </li><li> (Imalatçının protokolüne göre) Accutase kullanılarak kültür plakalarından Asansör TSK. </li><li> Bir kez (% 2 FBS ve% 1 Pen-strep ile PBS) FACS tamponu ile hücreleri yıkayın. </li><li> 4 ° C&#39;de 5 dakika boyunca 233 x g&#39;de santrifüjleyin. </li><li> Pelet bozmadan süpernatant atın. FACS tampon 0.5-1 ml hücrelerin tekrar (hücre sayısı ve arzu edilen antikor miktarı ile ilgili olarak). NOT: hücre süspansiyonu hacmi, FACS antikor üreticinin tavsiyeleri doğrultusunda, antikor konsantrasyonunu belirler. Bununla birlikte, antikor konsantrasyonu titrasyon, 1 için bir başlangıç ​​konsantrasyonu kullanılarak: bir antikor kullanan ilk defa ise, 100, genellikle tavsiye edilir. </li><li> Toplam sayısınıBir hemasitometre kullanarak hücreleri. </li><li> 1,5 ml santrifüj tüplerine rezervasyon 100 ul&#39;lik bir boyanmamış örnek ve (kullanılan floresan antikor sayısına göre) tek renkli kontrol için kullanılır. </li><li> (Üreticinin talimatlarına göre, önceden belirlenmiş konsantrasyonlarda) uygun şekilde floresan ile etiketlenmiş monoklonal antikor ekleyin ve ışıktan korunan, buz üzerinde 20-30 dakika boyunca inkübe edin. <ol><li> Osteojenik alt popülasyonunu etiketlemek için: 01:50 konsantrasyonunda FACS tamponu içerisinde, anti-CD90 (APC, anti-insan CD90 (Thy1)) ya da anti-CD105 (FITC anti-insan CD105 (endoglin)) seyreltin. </li><li> Diğer hücre popülasyonları (örneğin, hematopoietik ve endotelial hücreler), etiketlemek için: Anti-CD45 (anti-insan CD45 Pasifik mavi), anti-CD34 (anti-insan CD34 APC) ve / veya anti-CD31 (anti-insan CD31 PE seyreltin ) FACS 1:50 arasında bir konsantrasyona kadar tampon. </li><li> Doğrudan konjüge edilmiş antikorlar kullanılarak değil, uygun bir streptavidin-konjuge ikinci bir biyotinlenmiş primer antikor kullanımı1 bir seyreltme örneğin streptavidin PE-Cy7 olarak li antikor: 100. </li></ol></li><li> Süpernatan aspire, 4 ° C&#39;de 5 dakika boyunca 233 x g&#39;de FACS tamponu ve santrifüj tüpleri doldurun: inkübasyondan sonra, hücreler iki kez yıkayın. </li><li> Hücre kümeleri kaldırmak için bir 70 mikron naylon hücre süzgecinden 400-500 ul FACS tampon ve filtre örneklerinde hücrelerin tekrar. </li><li> Filtre en FACS tüplerine etiketli hücreler aktarın ve analiz süresince buz üzerinde örnekleri tutmak. </li></ol> 3. floresan aktive edilen hücre sınıflandırma NOT: Aşağıdaki adımlar önceki floresan-aktive hücrede bilgi sıralama (FACS) veya yetenekli bir teknisyen yardımı zorunlu. <ol><li> Ileri dağılım (FSC), yan dağılım (SSC) incelemek için, tasarım analizi araziler uygun FACS yazılımını kullanarak, Fikoeritrin (PE) -Texas Kırmızı, Pasifik Mavi, Fluoresein İzotiosiyanat (FITC), alofikosiyanin (APC) ve diğer fluorofor kullanımıd hücreleri etiketlemek için. </li><li> Hücre sıralayıcı, kısaca girdap hücreleri tekrar süspansiyon her numune içine yerleştirmeden önce. </li><li> Hücre kalıntıları hariç tutmak için ve taban çizgisi otoflüoresan belirlemek için, toplam hücre popülasyonunun kapılarını ayarlamak için lekelenmemiş numune analizi ile başlar. </li><li> Lekesiz numune propidiyum iyodür (PI) ekleyin ve toplam hücre nüfus içindeki canlı hücrelerin nüfusu görselleştirmek için analiz. Bu nüfusun etrafında bir kapı oluşturun. </li><li> Floresan tazminat ayarlamak için her florokrom için tek renkli kontrol örneği analiz. </li><li> Sıralama için kapıları konumunu ayarlamak için lekeli örnek analiz. </li><li> Hücreler ölü hücreleri boyamak için lekeli örnek PI ekleyin. </li><li> Etiketli hücre popülasyonlarının sıralayın. Sıralama 1,5 ml santrifüj tüplerine ya da kültür ortamı içeren konik tüp (15 mi) içine ya. </li><li> El hücre gereken miktarı, elde edildikten sonra sıralama bırakın. </li><li> BizeFACS ing, küçük bir kısmını analiz (örn., 500 hücreler) edinilen hücre popülasyonunun bir saflık kontrolü gerçekleştirecek. NOT: Bu adım sıralanmış hücre popülasyonlarının saflığını onaylar. İdeal olarak, saflık% 90-95 daha büyük olmalıdır. </li><li> 4 ° C&#39;de 5 dakika boyunca 233 x g&#39;de santrifüje hücreler hemen% 10 FBS içeren taze ortam 1 ml sıralama tamamlanmasını ve tekrar süspansiyon, aşağıdaki C. Not: kullanılan ortamın miktarı, hücre peleti büyüklüğüne göre arttırılabilir. </li><li> Jelatin kaplı plakalar üzerinde plaka hücre canlılığını geliştirmek. Nihai hücre verimi, plaka 6-çukurlu plaka içindeki oyuk başına 300,000 hücre, 12 oyuklu bir plaka içerisinde oyuk başına 100,000 hücre bağlı olarak değişir. </li></ol>

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T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD90+(Thy-1)+Positive+Selection+Enhances+Osteogenic+Capacity+of+Human+Adipose-Derived+Stromal+Cells.">CD90 (Thy-1) Positive Selection Enhances Osteogenic Capacity of Human Adipose-Derived Stromal Cells.</a> Tissue Eng Part A. , (2012).</li></ol>

Yazarların hiçbiri bu metinde belirtilen ürünler, cihazlar veya uyuşturucu herhangi bir mali ilgi var. Yazarların hiçbiri rapor için herhangi bir rakip mali bir ilgi var.

Şu anda, insan adipoz dokusu SVF TSK homojen alt popülasyonlarının izolasyonu arzu hedefi olsa zorlu kalır. Bu tür hücreler, kemik doku oluşumu ve homeostazı incelemek için kullanılabilir gibi pro-osteojenik ASC alt popülasyonlarının izole edilmesi özellikle tercih edilir. Ancak, adipoz doku SVF. Hücre kapasitesi ve farklılaşma potansiyelinin kök açısından bu heterojenite için moleküler temeli hücrelerinin toplanmış nüfus anlaşılmaktadır olamaz 11 önemli heterojenitesini barın...

The heterogeneous nature of stem cell populations is not yet fully understood and remains a major impediment to the development of clinically effective stem cell-based therapeutic applications. One of the most common ways to characterize a heterogeneous population of stem cells is to employ a cell sorting method, such as fluorescence-activated cell sorting (FACS), to separate cells based on their surface marker expression profiles. As sorting methods become more complex, it becomes possible to identify more distinct functional subpopulations of cells. Microfluidic-based technologies are becoming more and more frequently utilized in analysis of gene expression at the single cell level. Multiplexed quantitative polymerase chain reaction (qPCR) within a microfluidic chip allows for effective and reliable high-resolution, single cell transcriptional analysis.1-5
In a previous study using single cell transcriptional profiling of 48 genes, considerable transcriptional heterogeneity was observed among ASCs.6 However, the distribution of genes MSX2, BMP-5, BMP-7, ALP, OCN, RUNX2 exhibited a strong association with a cluster of cells possessing highly osteogenic transcriptional profiles. To isolate cells according to this osteogenic gene expression profile, surface antigen expression patterns were correlated with transcription patterns and surface marker expression of endoglin (CD105) was subsequently discovered to closely correlate with enhanced osteogenic differentiation potential of ASCs. Independent of CD105 expression, expression of surface receptor Thy-1 (CD90), a glycosyl-phosphatidylinositol-linked membrane protein previously shown by Chen et al. to be associated with osteoprogenitor cells, was also correlated with osteogenic gene expression.6,7 These findings provide the opportunity to prospectively isolate subpopulations within the larger heterogeneous pool of ASCs with increased osteogenic capacity for cell-based bone tissue engineering applications.

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	<tbody>
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			<td height="42" style="height:42px;width:275px;">Name of Reagent/Material</td>
			<td style="width:152px;">Company</td>
			<td style="width:112px;">Catalog Number</td>
			<td style="width:79px;">Comments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Disposable 250 mL Conical Tubes</td>
			<td style="width:152px;">Corning (Thomas Scientific)</td>
			<td style="width:112px;">2602A43</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:152px;">Gibco</td>
			<td style="width:112px;">15140-122</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">DMEM, high glucose, GlutaMAX Supplement</td>
			<td style="width:152px;">Gibco</td>
			<td style="width:112px;">10566-016</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS, pH 7.4</td>
			<td style="width:152px;">Gibco</td>
			<td>10010-023</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Betadine - Antiseptic Povidone/Iodine Solution</td>
			<td style="width:152px;">Purdue&#160;</td>
			<td>PFC-67618015017</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hank&#39;s Balanced Salt Solution, 1X</td>
			<td style="width:152px;">Cellgro</td>
			<td>21-023-CV</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum, Certified, US Origin</td>
			<td style="width:152px;">Gibco</td>
			<td>16000-044</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase from&#160;Clostridium histolyticum</td>
			<td style="width:152px;">Sigma-Aldrich</td>
			<td>C0130-5G</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">ACCUTASE Cell Detachment Solution</td>
			<td style="width:152px;">Stem Cell Technologies</td>
			<td>7920</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">APC Mouse Anti-Human CD90</td>
			<td style="width:152px;">BD Pharmingen</td>
			<td style="width:112px;">559869</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">FITC Mouse anti-Human CD105 (Endoglin)</td>
			<td style="width:152px;">BD Pharmingen</td>
			<td style="width:112px;">561443</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Anti-Human CD45 eFluor 450 (Pacific Blue replacement)&#160;</td>
			<td style="width:152px;">eBioscience</td>
			<td style="width:112px;">48-9459-41</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Anti-Human CD34 APC</td>
			<td style="width:152px;">eBioscience</td>
			<td style="width:112px;">17-0349-41</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Anti-Human CD31 (PECAM-1) PE</td>
			<td style="width:152px;">eBioscience</td>
			<td style="width:112px;">12-0319-41</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Streptavidin PE-Cyanine7</td>
			<td style="width:152px;">eBioscience</td>
			<td style="width:112px;">25-4317-82</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">BD FACS Aria II instrument</td>
			<td style="width:152px;">BD Biosciences</td>
			<td style="width:112px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">BD FACSDiva Software</td>
			<td style="width:152px;">BD Biosciences</td>
			<td style="width:112px;"></td>
			<td style="width:79px;"></td>
		</tr>
	</tbody>
</table>

isolation and enrichment of human adipose-derived stromal cells for enhanced osteogenesis

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

İnsan TSK (Şekil 1A, 1B) bir yüksek oranda zenginleştirilmiş popülasyonlarının izole edilmesi geliştirilmiş osteogenez sonuçlar hücreleri için bir marker olarak CD90 kullanılması. TSK Pasifik Mavi-konjüge anti-insan CD45 FITC-konjüge anti-insan CD105 ve APC-konjuge edilmiş anti-insan CD90 ile lekelenmiştir. Sonrası sıralama analizi ile nicelleştirilmiştir olarak sıralama sonra, saflık derecesi, daha büyük% 98 idi. İki yeni alt popülasyonlar muhte...

Watch this Scientific Journal Video about Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis at JoVE.com

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

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

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

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

isolation-enrichment-human-adipose-derived-stromal-cells-for-enhanced

Research

JoVE Journal

Developmental Biology

11.4K Görüntüleme.  Stanford University School of Medicine. The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

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

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

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

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

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

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

Gelişim Biyolojisi

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Santrifüj Yerçekimi ile adipoz kaynaklı Kök Hücrelerin kondrojenik Farklılaşma İndüksiyon

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

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

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

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

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

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

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. Although many efforts have been made to reveal the underlying mechanisms of decidualization, the exact signaling between the epithelial cells that are in contact with the embryo and the underlying stromal cells remains poorly understood. Therefore, studying decidualization in a way that takes both the epithelial and stromal cells into account could improve our knowledge about the molecular details of decidualization. For this purpose, in vivo models of artificial decidualization are physiologically the most relevant; however, manipulation of intercellular communication is limited. Currently, in vitro cultures of endometrial stromal cells are being used to investigate the modulation of decidualization by several signaling molecules. Conventionally, human or mouse endometrial stromal cells are used. However, the availability of human samples is very often limited. Furthermore, the use of murine tissues is accompanied with variety in the method of culturing. This study presents a validated and standardized method to obtain pure Endometrial Epithelial Cell (EEC) and Stromal Cell (ESC) cultures using adult intact mice treated with estrogen for three consecutive days. The protocol is optimized to improve the yield, viability, and purity of the cells and was further extended in order to study decidualization in a coculture of EEC and ESC. This model may be suitable to exploit the importance of both cell types in decidualization and to evaluate the contribution of significant signaling molecules secreted by EEC or ESC during the intercellular communication.

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

This study presents a standardized and validated method for the isolation and culture of primary mouse endometrial stromal and epithelial cells, which can be used in a coculture system to study in vitro decidualization.

Fare Endometrial stromal ve epitelyal hücrelerin izolasyonu için<em&gt; İn Vitro</em&gt; sidualizasyonu

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. ...

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

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

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

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

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

Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular infiltrate and epithelial cell activation. The progenitor cell population during such inflammatory reactions has mostly been investigated using single surface markers, either by histological analysis or by flow cytometry-based techniques. However, novel surface markers identified various functionally distinct subsets within the liver progenitor/stem cell compartment. The method presented here describes the isolation and detailed flow cytometry analysis of progenitor subsets using novel surface marker combinations. Moreover, it demonstrates how the various progenitor cell subsets can be isolated with high purity using automated magnetic and FACS sorting-based methods. Importantly, novel and simplified enzymatic dissociation of the liver allows for the isolation of these rare cell populations with a high viability that is superior in comparison to other existing methods. This is especially relevant for further studying progenitor cells in vitro or for isolating high-quality RNA to analyze the gene expression profile.

Liver injuries are accompanied by progenitor cell expansion that represents a heterogeneous cell population. Novel classification of this cellular compartment allows for the distinguishing of multiple subsets. The method described here illustrates the flow cytometry analysis and high purity isolation of various subsets that can be used for further assays.

İzolasyon ve Roman Yüzey Etiket Kombinasyon ile tanımlanır Karaciğer progenitör Alt Grupları zenginleştirilmesi

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular ...

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

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

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

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

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

Isolation and Characterization of Cardiac Mesenchymal Stromal Cells from Endomyocardial Bioptic Samples of Arrhythmogenic Cardiomyopathy Patients

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The isolation of these cells offers the possibility of studying their involvement in cardiac diseases, and, in addition, provides a useful primary cell model to investigate biological mechanisms.
Here, the method for the isolation of C-MSC from arrhythmogenic cardiomyopathy patients&#39; bioptic samples is described. The endomyocardial biopsy sampling is guided in the right ventricular areas adjacent to the scar visualized by electro-anatomical mapping. The digestion of the biopsies in collagenase and their plating on a plastic dish in culture medium to allow C-MSC growth is described. The isolated cells can be expanded in culture for several passages. To confirm their mesenchymal phenotype, the description of immuno-phenotypical characterization is provided. C-MSC are able to differentiate into several cell types like adipocytes, chondrocytes, and osteoblasts: in the context of ACM, characterized by adipocyte deposits in patients&#39; hearts, the protocols for the adipogenic differentiation of C-MSC and the characterization of lipid droplet accumulation are described.

In this article, a method to isolate cardiac mesenchymal stromal cells from endomyocardial bioptic samples of arrhythmogenic cardiomyopathy patients is provided. Their characterization and the protocol to boost their adipogenic differentiation are described.

Yalıtım ve Endomyocardial Bioptic örnekleri Aritmojenik kardiyomiyopati hasta kardiyak Mezenkimal Stromal hücrelerden karakterizasyonu

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The ...