Diese Studie wurde von der National Institutes of Health Research Stipendium R01-DE021683-01 und National Institutes of Health Forschungsstipendium R01-DE019434 zu MTL unterstützt; Howard Hughes Medical Institute Forschungsstipendium zu MTCDCW wurde von der ACS Franklin Martin Fakultät Research Fellowship, The Hagey Labor für Pädiatrische Regenerative Medizin und der Stanford University Child Health Research Institute Fakultät Scholar Award unterstützt.

HINWEIS: Alle Patientenproben wurden mit Einwilligung erhalten, und experimentelle Protokolle wurden überprüft und von der Stanford University Institutional Review Board (Protokoll # 2188 und # 9999) zugelassen. 1. Zell Isolierung und Kultur: <ol><li> Erhalten menschlichen Hautfettgewebe von gesunden weiblichen Patienten nach elektiven lipoaspiration des Bauches, Flanke, und / oder Oberschenkelbereich unter örtlicher / Vollnarkose. Stellen Sie sicher, dass Institutional Review Board (IRB) Approbation ist für die Protokoll zur Isolierung von ASC aus menschlichen Geweben gewonnen wurden, und folgen institutionelle Schutzmaßnahmen beim Umgang mit derartigen Materialien. </li><li> Um die SVF vom lipoaspirated Fettgewebe zu erhalten, zuerst waschen die lipoaspirate dreimal mit gleichen Volumina 1x sterile phosphatgepufferte Kochsalzlösung (PBS). Vorsichtig absaugen und entsorgen Sie die untere wässrige Schicht. </li><li> Bereiten Sie die Kollagenase-Verdauungspuffer: 0,075% Typ I Kollagenase in HanksBalanced Salt Solution (HBSS). Bereiten FACS-Puffer: 2% FBS, 1% P188 und 1% Pen-Strep in PBS. Filtern beide Lösungen mit Hilfe eines im Handel erhältlichen 0,22 um Porengrße fast flow Polyethersulfon Filter. </li><li> Zu den gewaschenen Fettgewebe verdauen, ein gleiches Volumen von Kollagenase-Verdauungspuffer und legen Digerierbehälter sicher in einem Schüttelwasserbad für 60 min bei 37 ° C (etwa 180 Shakes / min). HINWEIS: Es empfiehlt sich, ein größeres Volumen Aufschlussgefäß als erforderlich verwenden, da dies ermöglicht eine maximale Verdauung beim Schütteln (dh 250 ml Kollagenaseverdau Puffer, 250 ml gewaschen Fettgewebe in einem 1L sterile Flasche). </li><li> Neutralisieren enzymatische Aktivität durch Zugabe eines gleichen Volumens von FACS-Puffer und Stehenlassen bei RT für 5 min stehen. Als nächstes Zentrifuge bei 233 × g für 20 min bei 4 ° C. </li><li> Vorsichtig absaugen und den Überstand verwerfen, kümmert sich nicht um die hochdichte SVF Pellet stören. </li><li> Das Pellet in 5-10 ml RT rotZelllysepuffer, bezogen auf die Pelletgröße. Verlassen Lösung bei RT für 5 Minuten und Zentrifugation bei 233 × g für 5 min bei RT sitzen. </li><li> Überstand wird abgesaugt und das Pellet durch Zugabe von 5-10 ml traditionellen Wachstumsmedien, bezogen auf die Pelletgröße (Dulbeccos modifiziertes Eagle-Medium [DMEM] / 10% FBS / 1% Penicillin-Streptomycin-Lösung [Pen-Strep]). </li><li> Filtern der Suspension durch ein 100 um Nylon Zellsieb um Zelltrümmer zu entfernen. </li><li> Zentrifuge bei 233 xg für 5 Minuten bei 4 ° C, und den Überstand verwerfen, ohne dass der Pellets. </li><li> Das Pellet in 5-10 ml traditionellen Wachstumsmedien, bezogen auf die Pelletgröße. </li><li> Je 2 x 10 6 Zellen in einem 15 cm-Standard Kulturschale und stellen Primärkulturen O / N bei 37 ° C / 21% O 2, 5% CO 2. Zu halten, bei sub-konfluenten Ebenen spontane Differenzierung bei 37 ° C / 21% O 2, 5% CO 2 in Wachstumsmedium zu verhindern. HINWEIS: Die Zelldichte in einem vollkonfluenten 15 cm Platte beträgt ca. 4-6 x 10 6 Zellen. </li></ol> 2. Die Färbung <ol><li> Kultur ASC in herkömmlichen Wachstumsmedium bei 37 ° C / 21% O 2, 5% CO 2, für 36 Stunden vor der Färbung / Sortieranlagen. </li><li> Fahrstuhl ASC von Kulturplatten mit Accutase (nach Herstellerprotokoll). </li><li> Waschen Sie die Zellen einmal mit FACS-Puffer (PBS mit 2% FBS und 1% Pen-Strep). </li><li> Zentrifuge bei 233 × g für 5 min bei 4 ° C. </li><li> Überstand verwerfen, ohne dass der Pellets. Die Zellen in 0,5-1 ml FACS-Puffer (je nach Zellzahl und der Menge des Antikörpers gewünscht). ANMERKUNG: Das Volumen der Zellsuspension, in Übereinstimmung mit den Empfehlungen des FACS Antikörper Herstellers bestimmt Antikörperkonzentration. Jedoch Titration von Antikörper-Konzentration, unter Verwendung einer Ausgangskonzentration von 1: 100 wird in der Regel empfohlen, wenn es das erste Mal ist, einen Antikörper zu verwenden. </li><li> Zählen der Gesamtzahl derZellen unter Verwendung eines Hämocytometers. </li><li> Reserve 100 ul Aliquots in 1,5-ml-Zentrifugenröhrchen für eine ungefärbte Probe und einfarbige Kontrollen (entsprechend der Anzahl von fluoreszierenden Antikörpern verwendet wird) verwendet werden. </li><li> Fügen Sie die entsprechenden fluoreszenzmarkierten monoklonalen Antikörpern (in vorgegebenen Konzentrationen, den Anweisungen des Herstellers) und Inkubation für 20 bis 30 Minuten auf Eis, vor Licht geschützt. <ol><li> Osteogene Subpopulation beschriften: Verdünnen anti-CD90 (APC-anti-Mensch CD90 (Thy1)) bzw. anti-CD105 (FITC-Anti-Human CD105 (Endoglin)) in FACS-Puffer auf eine Konzentration von 1:50. </li><li> Um andere Zellpopulationen (zB hämatopoetische und Endothelzellen) zu kennzeichnen: Verdünnen anti-CD45 (Anti-Human CD45 Pacific Blue), Anti-CD34 (Anti-Human CD34 APC), und / oder anti-CD31 (Anti-Human CD31 PE ) in FACS-Puffer auf eine Konzentration von 1:50. </li><li> Wenn nicht mit direkt konjugierten Antikörper, mit einem biotinylierten primären Antikörpers mit einem geeigneten Streptavidin-konjugierten zweitenary-Antikörper, wie beispielsweise Streptavidin PE-Cy7, bei einer Verdünnung von 1: 100. </li></ol></li><li> Nach der Inkubation wurden die Zellen werden zweimal: Füllrohre mit FACS-Puffer und Zentrifugieren bei 233 × g für 5 min bei 4 ° C, Absaugen Stand. </li><li> Die Zellen in 400 bis 500 &amp; mgr; l FACS-Puffer und Filterproben durch einen 70 &amp; mgr; m Nylon Zellsieb um Zellklumpen zu entfernen. </li><li> Übertragen markierten Zellen in FACS-Röhrchen mit Filter oben und halten Proben auf Eis für die Dauer der Analyse. </li></ol> 3. Fluoreszenz-aktivierte Zellsortierung Hinweis: Die folgenden Schritte Mandat Vorkenntnisse in fluoreszenzaktivierte Zellsortierung (FACS) oder die Hilfe von einem Fachmann. <ol><li> Mit der entsprechenden Software FACS, Konstruktionsanalyse Grundstücke zum Vorwärtsstreuung (FSC), Seitenstreuung (SSC) zu untersuchen, Phycoerythrin (PE) -Texas Red, Pacific Blue, Fluoresceinisothiocyanat (FITC), Allophycocyanin (APC) und andere Fluorophor Verwendungd, um Zellen zu markieren. </li><li> Vor dem Laden in den Zellsortierer, Vortexen jede Probe, um die Zellen zu resuspendieren. </li><li> Beginnen Sie mit der Analyse der ungefärbten Probe, um Tore zu der gesamten Zellpopulation gesetzt, um Zelltrümmer ausschließen und die Baseline Autofluoreszenz zu bestimmen. </li><li> Hinzufügen Propidiumiodid (PI) auf die ungefärbten Probe und zu analysieren, um die Population von lebenden Zellen in der Gesamtzellpopulation zu visualisieren. Konstruieren Sie ein Tor um diese Bevölkerung. </li><li> Analysieren Sie einen einfarbigen Kontrollprobe für jedes Fluorochrom um Fluoreszenzkompensation eingestellt. </li><li> Analysieren Sie die gefärbten Probe, um die Position der Tore für die Sortierung festlegen. </li><li> Fügen PI auf die gefärbte Probe, um abgestorbene Zellen zu färben. </li><li> Die markierten Zellpopulationen zu sortieren. Sortieren entweder in 1,5 ml Zentrifugenröhrchen oder konische Röhren (15 ml) enthaltenden Kulturmedium. </li><li> Manuell einzustellen Sortier, wenn die erforderliche Menge von Zellen erhalten worden ist. </li><li> Unsing FACS, führen eine Überprüfung der Reinheit durch Analyse eines kleinen Bruchteil (z. B. 500 Zellen) der erfassten Zellpopulation. HINWEIS: Dieser Schritt bestätigt die Reinheit der sortierten Zellpopulationen. Idealerweise sollte die Reinheit größer als 90-95% betragen. </li><li> Zentrifuge Zellen bei 233 g für 5 min bei 4 ° C unmittelbar nach der Beendigung des Sortierens und Resuspension in 1 ml frischem Medium, enthaltend 10% FBS. HINWEIS: Die Menge des verwendeten Mediums kann auf der Grundlage der Größe der Zellpellet erhöht werden. </li><li> Platte auf mit Gelatine beschichtete Platten Lebensfähigkeit der Zellen zu verbessern. Abhängig von dem jeweiligen Zellausbeute, Platte 300.000 Zellen pro Vertiefung in einer Platte mit 6 Vertiefungen, 100.000 Zellen pro Vertiefung in einer Platte mit 12 Vertiefungen. </li></ol>

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Keiner der Autoren ein finanzielles Interesse in irgendwelchen der Produkte, Geräte oder in diesem Manuskript erwähnt Drogen. Keiner der Autoren haben eine konkurrierende finanzielles Interesse zu berichten.

Derzeit bleibt die Isolierung homogene Subpopulationen von ASC vom SVF menschlichen Fettgewebe eine anspruchsvolle aber wünschenswertes Ziel. Isolation von Pro-osteogenen ASC Subpopulationen ist besonders wünschenswert, da solche Zellen verwendet werden, um die Bildung und die Homöostase von Skelettgewebe zu untersuchen. Die SVF von Fettgewebe birgt jedoch erhebliche Heterogenität bezüglich Stammzellenkapazität und Differenzierungspotential. 11 wird die molekulare Grundlage dieser Heterogenität nicht a...

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.

<table border="0" cellpadding="0" cellspacing="0" width="618">
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		<tr height="42">
			<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>
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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.

Verwendung CD90 als ein Marker für Zellen mit erhöhter Osteogenese ergibt Isolierung eines hochangereicherten Populationen menschlicher ASCs (1A, 1B). ASC wurden mit Pacific Blue-konjugierten Anti-Human-CD45 FITC-konjugiertem Anti-Human CD105 und APC-konjugiertem Anti-Human-CD90 gefärbt. Nach dem Sortieren der Reinheitsgrad betrug mehr als 98%, wie durch die Post-Sortieranalyse quantifiziert. Definieren von Gruppen von Zellen auf Basis von Transkriptionsprofilen für ange...

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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.

Isolierung und Anreicherung von menschlichen Fettgewebe gewonnene Stromazellen für verbesserte Osteogenesis

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 Aufrufe.  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.

Serie: 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.

Isolierung und Differenzierung von Fettgewebe gewonnenen Stammzellen aus Porcine Subkutan Fettgewebes

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

Forschung

Entwicklungsbiologie

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.

Immunomagnetische Trennung von Fett Depot-spezifische Sca1<sup&gt; Hoch</sup&gt; Fettgewebe gewonnene Stammzellen (ASC)

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.

Isolierung und Expansion von mesenchymalen Stamm- / Stroma-Zellen aus humanen Placenta Gewebe gewonnen

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.

Chondrogene Differenzierung Induktion von Fettgewebe gewonnene Stammzellen durch Zentrifugalkraft Gravity

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.

Schnelle Isolierung von BMPR-IB + Fettgewebe gewonnene Stromazellen für die Verwendung in einem Schädeldachdefektheilung Modell

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.

Isolierung von Maus Endometrial Epithelial und Stromazellen für<em&gt; In Vitro</em&gt; decidualization

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.

Isolierung und Charakterisierung von mesenchymalen Stromazellen aus Nabelschnur und Fetal Placenta

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

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

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

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

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

In Vivo Tracking der menschlichen Fettgewebe mesenchymalen Stammzellen in einem Rattenmodell Knie Arthrose mit fluoreszierenden lipophile Membran Farbstoff

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

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.

Isolation, Kultur und Differenzierung von Stromazellen Knochenmarkzellen und Osteoklasten-Vorläufer von Mäusen

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.

Isolierung und Charakterisierung von mesenchymaler Stromazellen Herzzellen von Endomyocardial bioptischen Proben von Arrhythmogenic Kardiomyopathie Patienten

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