This research was supported in part by Program for Creating Start-ups from Advanced Research and Technology (START Program) from the Japan Society for the Promotion of Science (ST261006IP, TM) and by Program for the Strategic Research Foundation at Private Universities (2014-2019) (S1411018, TM) from the Ministry of Education, Sports, Science and Technology.

Proben von menschlichem subkutanen Fettgewebes wurden von Patienten in der Chirurgie in der Abteilung für Plastische Chirurgie, Urologie, Kinderchirurgie und Orthopädische Chirurgie der Nihon Universität Itabashi Hospital (Tokyo, Japan) erhalten. Die Patienten gaben informierte Zustimmung geschrieben, und die Ethikkommission der Nihon University School of Medicine der Studie zugelassen. 1. Gewebepräparation <ol><li> Bringen Sie die Gewebeprobe aus dem Operationssaal zu Labor. </li><li> Wash 1-2 g Fettgewebe mit 5 ml PBS (-) bei Raumtemperatur (RT) einmal, in einem 50-ml-Röhrchen und dann die Probe in einem 10 cm Plastikschale. </li></ol> 2. Collagenaseverdau <ol><li> Entfernen der Probe aus der PBS (-), dann werden 10 ml steriler 0,1% (w / v) Lösung Kollagenase (Kollagenase Typ II) in die Schale der Gewebeprobe enthält. </li><li> Mince das Gewebe mit einer chirurgischen Schere für etwa 15 Minuten, bis er eine pürierte erreicht bestehenparenz. </li><li> Anschließend verdauen das zerkleinerte Gewebe in dem 0,1% (w / v) Kollagenase-Lösung bei 37 ° C für 30 min (60 rpm) in einem 50 ml Röhrchen unter leichtem Schütteln auf einem Schüttler. </li><li> Filtern Sie die aufgeschlossene Probe durch ein 100 um-Filter in ein 50 ml Röhrchen. Als nächstes wurden 10 ml DCM passieren mit 2% FBS durch den Filter ergänzt (siehe Tabelle der Materialien). </li></ol> 3. Zell Isolation <ol><li> Zentrifugieren der filtrierten Zellen bei 100 · g für 1 min bei RT. </li><li> An dieser Stelle vorbereiten 5 ml DCM mit 2% FBS in einer 50-ml-Tube ergänzt. </li><li> die schwimmenden Fettzellen unter Verwendung einer 1000 ul Pipette und fügen Sie die Zellen in ein 50 ml Röhrchen, das Medium Als nächstes erstellen. </li><li> Anschließend schütteln Sie die 50-ml-Röhrchen vorsichtig das DCM-Medium mit den Zellen zu mischen. </li><li> Zentrifuge dann das Rohr bei 100 × g für 1 min bei RT. </li><li> Verwerfen das Medium an der Unterseite des Rohrs eine 18 G-Nadel und einer 20 ml Spritze. </li><li> Als nächstes werden 10 ml DCM supplemented mit 2% FBS zu den gesammelten Zellen. </li><li> Mischen Sie die DCM mit den Zellen durch Schütteln des 50-ml-Röhrchen sanft. </li><li> Zentrifugieren Sie die Röhrchen bei 100 g für 1 min bei RT. </li></ol> 4. Plating Cells <ol><li> Hinzufügen 41 ml DCM oder DMEM, supplementiert mit 20% FBS auf eine 12,5 cm-Kolben. </li><li> Als nächstes wird unter Verwendung einer 200 ul Pipette 40 ul Fettzellen in den Kolben hinzufügen. </li><li> Dann füllen Sie den Kolben mit DCM oder DMEM mit 20% FBS ergänzt. Kritischste Schritt: An dieser Stelle sicher, dass die Zellen im gesamten Medium verteilt sind. Eine Sichtprüfung der abgelösten Zellen und danach den Kolben eines runden Petrischale auf dem Deckel halten es flach zu halten. </li><li> Schließlich wird der Kolben im Inkubator und lassen Sie es für 7 Tage bei 37 ° C in einem 5% CO 2 Inkubator ungestört. </li><li> Nach einer Woche Inkubation kehren die Kolben und prüfen, ob DFAT Zellen. Als nächstes beurteilen Tüchtigkeit DFAT Zellgeneration wie seit fibrobl an dieser Stelle 10 beschriebenast-ähnlichen Zellen (DFAT Zellen) werden in den Regionen unterscheiden sich von Fettzellen. </li><li> 5 ml DCM oder DMEM, supplementiert mit 20% FBS in den Kolben der früheren Zellkulturmedium nach der Entfernung. Lassen Sie die DFAT Zellen für eine weitere Woche zu wachsen, nachdem das Medium verändert. </li><li> Mit Hilfe eines Zellzählers, zählen die Anzahl der DFAT unter verschiedenen Zellkulturbedingungen (DCM gegen DMEM) erzeugten Zellen. </li></ol>

<ol>
	<li>Lanzoni, G., 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=Concise+review:+clinical+programs+of+stem+cell+therapies+for+liver+and+pancreas.">Concise review: clinical programs of stem cell therapies for liver and pancreas.</a> Stem Cells. 31 (10), 2047-2060 (2013).</li><li>de Girolamo, L., 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=Mesenchymal+stem/stromal+cells:+a+new+"cells+as+drugs"+paradigm.+Efficacy+and+critical+aspects+in+cell+therapy.">Mesenchymal stem/stromal cells: a new "cells as drugs" paradigm. Efficacy and critical aspects in cell therapy.</a> Curr. Pharm. Des. 19 (13), 2459-2473 (2013).</li><li>Lindroos, B., Suuronen, R., Miettinen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+adipose+stem+cells+in+regenerative+medicine.">The potential of adipose stem cells in regenerative medicine.</a> Stem Cell. Rev. 7 (2), 269-291 (2011).</li><li>Yan, J., Tie, G., Xu, T. Y., Cecchini, K., Messina, L. M. <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+as+a+treatment+for+peripheral+arterial+disease:+current+status+and+potential+impact+of+type+II+diabetes+on+their+therapeutic+efficacy.">Mesenchymal stem cells as a treatment for peripheral arterial disease: current status and potential impact of type II diabetes on their therapeutic efficacy.</a> Stem Cell. Rev. 9 (3), 360-372 (2013).</li><li>Ringden, O., Keating, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stromal+cells+as+treatment+for+chronic+GVHD.">Mesenchymal stromal cells as treatment for chronic GVHD.</a> Bone Marrow Transplant. 46 (2), 163-164 (2011).</li><li>Matsumoto, 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=Mature+adipocyte-derived+dedifferentiated+fat+cells+exhibit+multilineage+potential.">Mature adipocyte-derived dedifferentiated fat cells exhibit multilineage potential.</a> J. Cell. Physiol. 215 (1), 210-222 (2008).</li><li>Lessard, J., 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=Generation+of+human+adipose+stem+cells+through+dedifferentiation+of+mature+adipocytes+in+ceiling+cultures.">Generation of human adipose stem cells through dedifferentiation of mature adipocytes in ceiling cultures.</a> J. Vis. Exp. (97), (2015).</li><li>Lessard, J., 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=Characterization+of+dedifferentiating+human+mature+adipocytes+from+the+visceral+and+subcutaneous+fat+compartments:+fibroblast-activation+protein+alpha+and+dipeptidyl+peptidase+4+as+major+components+of+matrix+remodeling.">Characterization of dedifferentiating human mature adipocytes from the visceral and subcutaneous fat compartments: fibroblast-activation protein alpha and dipeptidyl peptidase 4 as major components of matrix remodeling.</a> PLoS One. 10 (3), 0122065 (2015).</li><li>Peng, X., 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=Phenotypic+and+Functional+Properties+of+Porcine+Dedifferentiated+Fat+Cells+during+the+Long-Term+Culture+In+Vitro.">Phenotypic and Functional Properties of Porcine Dedifferentiated Fat Cells during the Long-Term Culture In Vitro.</a> Biomed. Res. Int. 2015, 673651 (2015).</li><li>Kono, S., Kazama, T., Kano, K., Harada, K., Uechi, M., Matsumoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+and+functional+properties+of+feline+dedifferentiated+fat+cells+and+adipose-derived+stem+cells.">Phenotypic and functional properties of feline dedifferentiated fat cells and adipose-derived stem cells.</a> Vet. J. 199 (1), 88-96 (2014).</li><li>Bellin, M., Marchetto, M. C., Gage, F. H., Mummery, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cells:+the+new+patient.">Induced pluripotent stem cells: the new patient.</a> Nat. Rev. Mol. Cell Biol. 13 (11), 713-726 (2012).</li></ol>

The authors have nothing to disclose.

Ältere Adipozyten , die in vitro Dedifferenzierung, ein Verfahren bekannt , als Deckenkultur unterzogen werden , können zu einer primitiveren Phänotyp zurückkehren und proliferativen Fähigkeiten gewinnen. Diese Zellen werden als dedifferenzierte Fett (DFAT) -Zellen bezeichnet. Die multilineage Differenzierungspotential von DFAT Zellen wurde bewertet. Durchflusszytometrie Analyse und Genexpressionsanalyse ergab , dass DFAT Zellen im Vergleich zu ASCS 6 sehr homogen waren. In der Tat ist das Zello...

Die Zelltherapie und Tissue Engineering sind heiße Themen auf dem Gebiet der regenerativen Medizin 1-5. Während diese therapeutischen Modalitäten großes Versprechen halten, die derzeit einige technische Probleme, ihre klinische Anwendung behindern. In diesem Zusammenhang ist, wie bei der Erzeugung von iPS-Zellen, alle Therapien Tissue Engineering müssen Zellen frei von äußeren Gen Transduktionen produzieren, um die Sicherheit der Patienten zu gewährleisten. Dementsprechend waren wir die erste Gruppe erfolgreich menschliche DFAT Zellen produzieren 6. Mehrere andere Arbeitsgruppen haben unsere Methode , da angenommen DFAT Zellen von Säugetieren 7-9, weiter hervorheben , die Nützlichkeit unseres Modells zu generieren. Im Laufe von mehreren Studien haben wir festgestellt, dass die Qualität der Zellkulturumgebung durch Einstellen des Inhalts des Zellmediums verändert werden. Diese Erkenntnis hat zu einer Erhöhung der Erfolgsrate von DFAT Zellproduktion geführt und Zellqualität verbessert; beide kritischen Faktoren ineffizient Zellen für zukünftige klinische Studien zu generieren. In dieser Hinsicht verbesserte einer DFAT Kulturmedium (DCM, ein Medium ähnliche Zellmedium stammen mesenchymalen, die rekombinantes humanes Insulin, Serumalbumin, L-Glutaminsäure, verschiedene Fettsäuren und Cholesterin enthält) und ein Verfahren zur DFAT Zellgeneration und Proliferation wurde entwickelt (weitere Informationen über den Inhalt von DCM ist auf Anfrage erhältlich). Mit dieser Methode hohe Qualität DFAT Zellen wurden mit der Fähigkeit, erzeugt in verschiedene Zelltypen, einschließlich adipogenen, osteogenen und chondrogenen Zellen zu differenzieren. Insgesamt verbessert diese validierten Zellkultur Protokoll, um die Qualität der DFAT Zellen und kann zur Verbesserung der klinischen Anwendung der Zelltherapie und Tissue Engineering sehr nützlich sein.

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			<td height="21" style="height:21px;width:243px;">CSTI303-MSC medium&#160;</td>
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			<td style="width:143px;">Takasago Medical Industry Co., Ltd</td>
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			<td height="42" style="height:42px;width:243px;">Falcon Cell Strainer 100um Yellow</td>
			<td style="width:143px;">CORNING LIFE SCIENCES&#160;</td>
			<td style="width:155px;">DL 352360</td>
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			<td height="63" style="height:63px;width:243px;">Falcon 12.5cm&#178; Rectangular Canted Neck Cell Culture Flask with Blue Vented Screw Cap</td>
			<td style="width:143px;">CORNING LIFE SCIENCES&#160;</td>
			<td style="width:155px;">353107</td>
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			<td height="21" style="height:21px;width:243px;">18G needle</td>
			<td style="width:143px;">NIPRO</td>
			<td style="width:155px;">02-002</td>
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			<td height="21" style="height:21px;width:243px;">20ml Syringe&#160;</td>
			<td style="width:143px;">NIPRO</td>
			<td style="width:155px;">08-753</td>
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			<td height="21" style="height:21px;width:243px;">Z Series Coulter Counter</td>
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an efficient method to obtain dedifferentiated fat cells

Tissue Engineering und Zelltherapie vielversprechend klinisch. In dieser Hinsicht multipotenten Zellen wie mesenchymalen Stammzellen (MSCs), können therapeutisch, in naher Zukunft wieder herzustellen Funktion geschädigte Organe verwendet. Dennoch einige technische Probleme, einschließlich der hochinvasiven Verfahren von MSCs und die Ineffizienz rund um ihre Verstärkung zu isolieren, behindern gegenwärtig die möglichen klinischen Verwendung dieser therapeutischen Modalitäten. Hierin stellen wir eine hocheffiziente Methode zur Erzeugung von dedifferenzierten Fettzellen (DFAT), MSC-ähnlichen Zellen. Interessanterweise kann DFAT Zellen in verschiedene Zelltypen, einschließlich adipogenen, osteogenen, chondrogenen Zellen und differenzieren. Obwohl andere Gruppen zuvor verschiedene Verfahren zur Erzeugung von DFAT Zellen aus reifen Fettgewebe präsentiert haben, unsere Methode ermöglicht es uns, mehr DFAT Zellen effizient zu produzieren. In dieser Hinsicht zeigen wir, dass DFAT Kulturmedium (DCM), ergänzt mit 20% FBS,ist wirksamer DFAT Zellen bei der Erzeugung als DMEM, ergänzt mit 20% FBS. Zusätzlich können die Zellen durch DFAT unserem Zellkulturverfahren hergestellt in verschiedene Gewebetypen redifferenziert werden. Als solche ein sehr interessantes und nützliches Modell für die Untersuchung von Gewebe Dedifferenzierung dargestellt.

<html> In dieser Studie wurde das Verfahren und das Toolkit für DFAT Zellgeneration verbessert (Abbildung 1). Unser Verfahren ermöglicht es uns , DFAT Zellen sowohl mit DCM und DMEM - Medium , das 20% FBS (2A) zu erzeugen. Als solche verglichen wir die Effizienz von DCM und DMEM DFAT Zellen zu erzeugen. In dieser Hinsicht verbesserte DCM DFAT Zellproliferation durch drei Mal , wenn auf DMEM verglichen, unabhängig von der Anzahl von Adipozyten (...

Watch this Scientific Journal Video about An Efficient Method to Obtain Dedifferentiated Fat Cells at JoVE.com

An Efficient Method to Obtain Dedifferentiated Fat Cells

We have modified the conditions for DFAT cell generation and provide herein information regarding the use of an improved growth medium for the production of these cells.

Eine effiziente Methode erhalten Sie Dedifferenzierte Fettzellen

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used ...

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Developmental Biology

7.9K Aufrufe.  Nihon University. We have modified the conditions for DFAT cell generation and provide herein information regarding the use of an improved growth medium for the production of these cells.

Serie: An Efficient Method to Obtain Dedifferentiated Fat Cells

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

Effiziente Ableitung von Retinapigmentepithel-Zellen aus Stammzellen

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

Forschung

Entwicklungsbiologie

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.

Ein enzymatisches Verfahren zur mesenchymalen Stammzellen aus Knochenmarkproben Clotted Rettung

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

A Quick and Efficient Method for the Purification of Endoderm Cells Generated from Human Embryonic Stem Cells

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell replacement therapies. Terminally differentiated cell types could be used for the treatment of various degenerative diseases. In vitro differentiation of these cells towards tissues of the lung, liver and pancreas requires as a first step the generation of definitive endodermal cells. This step is rate-limiting for further differentiation towards terminally matured cell types such as insulin-producing beta cells, hepatocytes or other endoderm-derived cell types. Cells that are committed towards the endoderm lineage highly express a multitude of transcription factors such as FOXA2, SOX17, HNF1B, members of the GATA family, and the surface receptor CXCR4. However, differentiation protocols are rarely 100% efficient. Here, we describe a method for the purification of a CXCR4+ cell population after differentiation into the DE by using magnetic microbeads. This purification additionally removes cells of unwanted lineages. The gentle purification method is quick and reliable and might be used to improve downstream applications and differentiations.

Here, we describe a method for the purification of differentiated human embryonic stem cells that are committed towards the definitive endoderm for the improvement of downstream applications and further differentiations.

Eine schnelle und effiziente Methode zur Reinigung von Entodermzellen generiert aus humanen embryonalen Stammzellen

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell ...

Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such cells are not readily available, they can be obtained using different approaches that include, among the many, reprogramming and trans-differentiation, with advantages and limitations that are specific of the different techniques. Here a new strategy for the conversion of an adult mature fibroblast into an insulin-secreting cell, arbitrarily designated as epigenetic converted cells (EpiCC), is described. The method has been developed, based on the increasing understanding of the mechanisms controlling epigenetic regulation of cell fate and differentiation. In particular, the first step uses an epigenetic modifier, namely 5-aza-cytidine, to drive adult cells into a "highly permissive" state. It then takes advantage of this brief and reversible window of epigenetic plasticity, to re-address cells toward a different lineage. The approach is designated "epigenetic cell conversion". It is a simple and robust way to obtain an efficient, controlled and stable cellular inter-lineage switch. Since the protocol does not involve the use of any gene transfection, it is free of viral vectors and does not involve a stable pluripotent state, it is highly promising for translational medicine applications.

Here, a new method that allows the conversion of adult skin fibroblasts into insulin-secreting cells is presented. This technique is based on epigenetic conversion, does not involve the use of retroviral vectors nor the acquisition of a stable pluripotent state. It is therefore highly promising for translational medicine applications.

Epigenetische Umwandlung als eine sichere und einfache Methode, Insulin absondernden Zellen aus adulten Fibroblasten der Haut zu erhalten

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such ...

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 ...

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of pancreatic progenitor cells that can be used for the study of and future treatment of diabetes. This article outlines a four-stage differentiation protocol designed to generate pancreatic progenitor cells from human embryonic stem cells (hESCs). This protocol can be applied to a number of human pluripotent stem cell (hPSC) lines. The approach taken to generate pancreatic progenitor cells is to differentiate hESCs to accurately model key stages of pancreatic development. This begins with the induction of the definitive endoderm, which is achieved by culturing the cells in the presence of Activin A, basic Fibroblast Growth Factor (bFGF) and CHIR990210. Further differentiation and patterning with Fibroblast Growth Factor 10 (FGF10) and Dorsomorphin generates cells resembling the posterior foregut. The addition of Retinoic Acid, NOGGIN, SANT-1 and FGF10 differentiates posterior foregut cells into cells characteristic of pancreatic endoderm. Finally, the combination of Epidermal Growth Factor (EGF), Nicotinamide and NOGGIN leads to the efficient generation of PDX1+/NKX6-1+ cells. Flow cytometry is performed to confirm the expression of specific markers at key stages of pancreatic development. The PDX1+/NKX6-1+ pancreatic progenitors at the end of stage 4 are capable of generating mature &#946; cells upon transplantation into immunodeficient mice and can be further differentiated to generate insulin-producing cells in vitro. Thus, the efficient generation of PDX1+/NKX6-1+ pancreatic progenitors, as demonstrated in this protocol, is of great importance as it provides a platform to study human pancreatic development in vitro and provides a source of cells with the potential of differentiating to &#946; cells that could eventually be used for the treatment of diabetes.

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Here we describe a 4-stage protocol to differentiate human embryonic stem cells to NKX6-1+ pancreatic progenitors in vitro. This protocol can be applied to a variety of human pluripotent stem cell lines.

Effiziente Differenzierung von pluripotenten Stammzellen zu NKX6-1<sup&gt; +</sup&gt; Pancreatic Vorläufern

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Eine neuartige Mamma-Fat-Pad-Transplantationstechnik zur Visualisierung der Gefäßerzeugung von vaskulären endothelialen Stammzellen

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

Wing development in Drosophila melanogaster is an ideal model for studying morphogenesis at tissue level. These appendages develop from a group of cells named wing imaginal discs formed during embryonic development. In the larval stages the imaginal discs grow, increasing its number of cells and forming monolayered epithelial&#160;structures. Inside the pupal case, the imaginal discs bud out and fold into bilayers along a line that becomes the future margin of the wing. During this process, the longitudinal primodia veins originate vein cells on the prospective dorsal and ventral surfaces of the wing. During the pupal stage the stripes of vein cells of each surface communicate in order to generate tight tubes; at the same time, the cross-veins begin their formation.
With the help of appropriate molecular markers, it is possible to identify the major elements composing the wing during its development. For this reason, the ability to accurately detect transcripts or proteins in this structure is critical for studying their abundance and localization related to the development process of the wing.
The procedure described here focuses on manipulating pupal wings, providing detailed instructions on how to dissect the wing during the pupal stage. The dissection of pupal tissue is more difficult to perform than their counterparts in third instar larvae. This is why this approach was developed, to obtain rapid and efficient high quality samples. Details of how to immunostain and mount these wing samples, to allow the visualization of proteins or cell components, are provided in the protocol. With little expertise it is possible to collect 8-10 high quality pupal wings in a short amount of time.

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

The ability to accurately detect transcripts or proteins in Drosophila tissues is critical for studying their abundance and localization related to the development process. Here is the description of a straightforward procedure to dissect pupal wings. These wings can be used as samples in several techniques (immunohistochemistry, PCR assay, etc.).

Eine schnelle und effiziente Methode, um Pupal Flügel von Drosophila geeignet für Immunodetections oder PCR-Assays sezieren

Wing development in Drosophila melanogaster is an ideal model for studying morphogenesis at tissue level. These appendages develop from a group of cells ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

Computergestützte Analyse der Keimbahn Caenorhabditis Elegans , die Verteilung der Kerne, Proteine und das Zytoskelett zu studieren

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

Eine vereinfachte und effiziente Methode zur primären menschlichen Keratinozyten aus Erwachsenen Hautgewebe zu isolieren

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Eine effiziente Methode erhalten Sie Dedifferenzierte Fettzellen

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