Diese Arbeit wird durch NIH / NCI gewähren R01CA-118916 gefördert

1. Auszug epithelialen Stammzellen aus menschlicher Haut <ol><li> Vor dem Start des Verfahrens zur Isolierung von epithelialen Stammzellen muss man die jeweiligen Medien und Reagenzien (siehe Tabelle 1) vorzubereiten. </li><li> Frische erwachsenen Menschen Kopfhaut ab Facelift Verfahren oder Stanzbiopsie erfasst werden, dann in DMEM / 10% FBS / Dispase (4 mg / ml) über Nacht bei 4 ° C inkubieren Inkubation für 2-4 h bei 37 ° C ist auch wirksam. Hautstücke sollten eine maximale Breite von 1 cm bis zum Enzym zu durchdringen zu ermöglichen. </li><li> Übertragen Sie die Haut in einem sterilisierten Petrischale, ziehen Sie jedes einzelne Haar von der Haut durch Ergreifen des Haares in der Nähe der Hautoberfläche und zieht fest und glatt. Wählen Follikel Telogen Bühne, auf ihre Morphologie unter einem Binokular auf, schneiden Sie die Ausbuchtung Region (Abbildung 1A) übertragen Follikel in einen 15 mL sterilisiert Röhre. Anagen Follikel kann auch verwendet werden, wenn Schnitt am oberen 1 / 3 der Follikel Haarfollikel &quot;Bulge&quot;-Region (Abbildung 1B) erhalten werden. </li><li> Inkubieren der isolierten Follikel Fragmente in einer Mischung aus 0,05% Trypsin-EDTA (GIBCO) und Versene (0,53 mM EDTA 4NA, GIBCO) (1:1) für 15-20 min bei Raumtemperatur unter Schütteln in regelmäßigen Abständen, 4 ml DMEM + 10 % FBS zu stoppen Reaktion, Spin Down für 5 min bei 800 Umdrehungen pro Minute. Alternativ können Follikelgewebe Fragment Explantate in Kultur ohne Trypsin verdaut werden zur Kultivierung von Zellen aus einzelnen Follikel platziert. </li><li> Überstand vorsichtig (save ~ 0,2-0,5 ml zu verlieren Zellen) und in 1 mL KCM (Keratinozytenmedium), ohne EGF resuspendieren. </li></ol> 2. Kultur Primary epithelialen Stammzellen <ol><li> Bevor die Kultivierung der epithelialen Stammzellen, Mitomycin C-behandelten 3T3-J2-Zellen (Feeder-Zellen) wie folgt zubereitet werden sollte. 3T3-J2-Zellen werden in DMEM mit 10% FBS vor der Behandlung kultiviert. Entfernen Kulturmedien und waschen Zelle zweimal mit PBS, behandeln Zelle mit 15μg / ml Mitomycin C in DMEM ohne Serum für 2 Stunden bei 37 ° C, 5% CO 2. Entfernen Sie die Mitomycin C-haltige DMEM zwei Waschgänge mit PBS werden die Zellen sofort einsatzbereit. (Mitomycin C-behandelten 3T3-J2-Zellen können auch vorher vorbereitet werden und bei -80 ° C auftauen und Saatgut die Zellen einen Tag vor Kultivierung primärer epithelialen Stammzellen, bei 37 ° C, 5% CO 2. 150.000-200.000 3T3-J2 Zellen pro Vertiefung der 6-Well-Platte wird empfohlen. </li><li> Seed isoliert Haarfollikel Stammzellen aus Schritt 1.5 auf Mitomycin 3T3-J2-Zellen in KCM ohne EGF über Nacht bei 37 C-behandelten ° C, 5% CO 2, wechselte er zur EGF-haltigen KCM am nächsten Tag. Die Zellen werden bei 37 ° C in einer feuchten Atmosphäre mit 5% CO 2. Alle Keratinozyten werden mit KCM mit EGF zugeführt alle 2 Tage gewachsen und für 14-20 Tage. Überprüfen Zelle täglich unter dem Mikroskop. Haarfollikelstammzellen bilden Kolonien (Abbildung 2A). Haarfollikel Explantate wird Auswüchse nach 10-14 Tagen (Abbildung 2B) zu bilden. </li></ol> 3. Passage epithelialen Stammzellen <ol><li> Wash-Zellen mit PBS einmal. Entfernen 3T3-J2 Feeder-Zellen durch Versene (auf RT vorgewärmt) Behandlung. Inkubieren Kultur aufgetischt in Versene für 5 Minuten, dann schütteln und absaugen Feeder-Zellen. </li><li> Wash Haarfollikelstammzellen entweder mit Versene oder PBS einmal. </li><li> Add vorgewärmten (37 ° C, kritisch) Trypsin-EDTA (2X) und bei 37 ° C für 7 Minuten oder länger, wenn dies für die Haarfollikel Stammzellen zu lösen. Nicht länger als 15 Minuten am besten ist. </li><li> Add KCM w / o EGF, um das Trypsin zu stoppen, sanft Pipette auf und ab, um Zellen zu zerstreuen, Spin-Down bei 200g, 5 Minuten. </li><li> Re-suspend-Zellen mit KCM ohne EGF, Graf und re-Platte auf Mitomycin C-behandelten 3T3-J2-Zellen-Schicht. </li></ol> 4. Verewigen epithelialen Stammzellen Primäre Zellen, auch adulte Stammzellen, zu erreichen Seneszenz nach serieller Passage und Monate in Kultur. Immortalisierung von primären Stammzellen ist ein effektiver Weg, um dieses Problem zu lindern. <ol><li> Platte primären epithelialen Stammzellen (~ 350.000 Zellen / Well von 6-Well-Platte) bei Passage 1 auf Feeder-Schicht (3T3-J2) und Kultur für 2 Tage. </li><li> Kultur PA317 LXSN 16E6E7 Zelllinie einen Tag nach der Aussaat der primären epithelialen Stammzellen mit DMEM mit 10% FBS. (Diese Linie stellt die amphotrope Retrovirus LXSN16E6E7 die HPV16 E6 und E7 offene Leserahmen kodiert, und die kann verwendet werden, um stabil zu infizieren und zu verewigen vielen Zelltypen werden). </li><li> Gönnen PA317 LXSN 16E6E7 Zelllinie mit Mitomycin C für 2 Stunden (Same-Protokoll mit 3T3-J2-Zellen). Add Mitomycin C behandelt PA317 LXSN 16E6E7 Zelllinie zur primären epithelialen Stammzellen, Co-Kultur für 6 Tage in KCM mit EGF. Renew frischen KCM mit EGF Medien alle 2 Tage. </li><li> Entfernen 3T3-J2 und PA317 LXSN 16E6E7 Zelle mit Versene. Add Mitomycin-C behandelt 3T3-J2 NHP-Zellen (Neomycin, Hygromycin, Puromycin resistenter, ~ 200.000 Zellen pro Well), die epithelialen Stammzellen. Die Zellen werden unter 0,2 mg / ml G418 (Gibco) für zusätzliche ausgewähltitional 6 Tage. Renew frischen G418 mit KCM mit EGF Medien alle 2 Tage. Surviving epithelialen Stammzellen Stammzellen verewigt werden. Ein gut von primären epithelialen Stammzellen, die nicht mit PA317 LXSN 16E6E7 Zelle co-kultiviert als Kontrolle für G418 Auswahl sollte eingestellt werden, sollten alle Zellen in diesem gut G418 nach 6 Tagen Auswahl getötet werden. </li></ol> 5. Repräsentative Ergebnisse Frühe Passage der Haut epithelialen Stammzellen und verewigt epithelialen Stammzellen bilden dichte Kolonien, bestehend aus kleinen Keratinozyten (Abbildung 2A) von Feeder-Zellen umgeben. Wenn in serumfreiem Medium mit definierten Ergänzungen kultiviert, ohne Feeder-Schicht werden die Stammzellen zerstreuen und bilden keine dichte Kolonien (sie wachsen als einzelne Zellen und kleine Cluster). Immortalized epithelialen Stammzellen eine stabile Phänotyp für&gt; 12 Monate kontinuierlicher Übergang, sondern eher enger Kolonien als die primären Zellen zu bilden. Sie bilden keine Verankerung unabhängigen Kolonien in Soft-Agar-Assays, was darauf hinweist, dass diese immortalisierten Zellen besitzen nicht die Eigenschaften der Krebszellen. Beide primären und immortalisierten epithelialen Stammzellen ausdrückliche Haarfollikel Stammzellen Cytokeratin 15 (3A), und können in epidermale, Haarfollikel und Talgdrüsen Linien (Abb. 3B-D) zu unterscheiden. <img src="/files/ftp_upload/2561/2561fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Gerupft Haare. Nach dem Rupfen aus Dispase-behandelte Haut erscheinen Haarfollikel entweder als Telogen Club Haaren (A) mit einer Kugel von Zellen umgebenden Boden der Haare oder Anagenhaare (B) mit einer Hülse aus Epithel um die Länge der Haare. Das Telogen Wulstregion bildet eine deutliche morphologische Region zu Mikro-sezieren, während der Anagen Ausbuchtung ist weniger ausgeprägt. Die Anagenphase Birne enthält Matrixkeratinozyten, was Transit-Verstärkung Zellen, die auch isoliert und kultiviert werden können. <img src="/files/ftp_upload/2561/2561fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Stammzellen Kulturen. (A) Epitheliale Stammzellen aus der Haut bilden feste epithelialen Kolonien (durch E), wenn in KCM mit einem Feeder-Schicht von Zellen (gekennzeichnet durch F) kultiviert. (B) Haarfollikel Explantate Anlass zu epithelialen Auswüchse. Das Telogen Wulstregion (gekennzeichnet durch T) ist es, die Schüssel angeschlossen und umgeben Epithelzelle Kolonie zusammen mit Feeder-Zellen. <img src="/files/ftp_upload/2561/2561fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Stammzell-Differenzierung. (A) Stammzellen Kolonien zu halten Expression von epithelialen Markern wie Zytokeratin 15. (B) Skin epithelialen Stammzellen können entlang der Haarfollikel Linie durch K6Hf Ausdruck bestimmt unterschieden werden. (C) Die Zellen können auf de-epidermalized Dermis oder anderen Matrices an der Luft-Flüssigkeit-Grenzfläche kultiviert werden und wird eine geschichtete Epidermis mit Hornschicht, körnige Schicht und Dorn-Schicht zu bilden. (D) Stammzellen können entlang der Talgdrüsen Linie mit Oil Red positive Globuli induziert werden.

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Die Zelle Extraktion und Kultur Methoden beschrieben sind überraschend leicht und reproduzierbar. Wir haben epithelialen Stammzellen in Zellkulturen von Dutzenden von Menschen in einer breiten Altersspanne, einschließlich Patienten mit angeborenen Hautdefekte 18 erzeugt wird. Am besten ist es, den Prozess auf den Tag des Gewebes Ernte zu beginnen, jedoch Zellen bleiben in den Medien auf Eis tragfähig für mehrere Tage, die Erleichterung versand, wenn nötig. Ausrangierte Facelift Haut liefert Hunderte von ...

Keratinocyte Medien (KCM) [DMEM und Ham s F12 (GIBCO, 3:1), Adenin (Sigma, 180 mM), 10% fötalem Rinderserum (GIBCO), Cholera-Toxin (ICN, 0,1 nM), Penicillin / Streptomycin (GIBCO, 100 U / ml und 100 mg / ml), Hydrocortison (Sigma, 0,4 mg / ml, 1,1 mm), T/T3 (Transferrin, GIBCO, 5 g / ml, 649 nM, und Triiod-l-thyronine, Sigma, 2 nM) , Insulin (Sigma, 5 mg / ml, 862 nM) und EGF (Sigma, 10 ng / ml, 1,6 nM), pH 7,2]

isolation and culture of adult epithelial stem cells from human skin

Die Homöostase aller selbst erneuernden Geweben ist abhängig von adulten Stammzellen. Als undifferenzierte Stammzellen asymmetrische Teilungen durchlaufen, erzeugen sie Tochterzellen, dass die Stammzellen Phänotyp und Transit-Verstärkung Zellen (TA-Zellen), die aus der Stammzell-Nische zu migrieren, zu unterziehen schnelle Verbreitung und terminal zu differenzieren, um das Gewebe besiedeln zu behalten. Epitheliale Stammzellen in der Epidermis, Haarfollikel und Darm wurden als Zellen mit einem hohen In-vitro-proliferative Potential und so langsam-cycling-Label-Beibehaltung Zellen in vivo 1-3;. Adult-, Gewebe-spezifische Stammzellen sind verantwortlich für die Regeneration der Gewebe, in denen sie während des normalen physiologischen Umsatz als auch in Zeiten von Stress 4-5 befinden. Darüber hinaus sind Stammzellen allgemein als multi-potent, besitzen die Fähigkeit, die zu verschiedenen Zelltypen geben innerhalb der Gewebe 6. Zum Beispiel kann Nagetier Haarfollikelstammzellen generieren Epidermis, Talgdrüsen und Haarfollikel 7-9. Wir haben gezeigt, dass Stammzellen aus dem menschlichen Haarfollikel Wulstregion multi-Potential 10 weisen. Stammzellen haben sich zu einem wertvollen Werkzeug in der biomedizinischen Forschung, die aufgrund ihrer Nützlichkeit als in vitro-System für das Studium der Entwicklungsbiologie, Differenzierung, Tumorentstehung und ihre möglichen therapeutischen Nutzen. Es ist wahrscheinlich, dass adulte epithelialen Stammzellen werden in der Behandlung von Krankheiten wie ektodermale Dysplasien, Monilethrix, Netherton-Syndrom, Menkes Krankheit erblich Epidermolysis bullosa und Alopezien 11-13 nützlich. Darüber hinaus werden andere Hautprobleme wie Brandwunden, chronische Wunden und Geschwüre aus Stammzellen verwandte Therapien 14,15 profitieren. Angesichts des Potenzials für Reprogrammierung von adulten Zellen in einen pluripotenten Zustand (iPS-Zellen) 16,17, kann die leicht zugänglich und erweiterbar adulten Stammzellen in der menschlichen Haut eine wertvolle Quelle von Zellen für die Induktion und Downstream-Therapie für ein breites Spektrum von Krankheiten, einschließlich bieten Diabetes und Parkinson-Krankheit.

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Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

Eine schnelle und robuste Lösung zur Isolierung von lebensfähigen Erwachsenen epithelialen Stammzellen aus menschlicher Haut beschrieben wird. Das Verfahren nutzt enzymatische Verdauung der Haut Kollagen-Matrix, durch Zupfen der Haarfollikel und Isolierung von einzelnen Zellsuspensionen oder Gewebestücke für die Zellkultur gefolgt.

Isolierung und Kultivierung von Adult epithelialen Stammzellen aus menschlicher Haut

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

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28.2K Aufrufe.  University of Massachusetts Medical School. Eine schnelle und robuste Lösung zur Isolierung von lebensfähigen Erwachsenen epithelialen Stammzellen aus menschlicher Haut beschrieben wird. Das Verfahren nutzt enzymatische Verdauung der Haut Kollagen-Matrix, durch Zupfen der Haarfollikel und Isolierung von einzelnen Zellsuspensionen oder Gewebestücke für die Zellkultur gefolgt.

Serie: Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

Isolation und großräumige Ausdehnung der erwachsenen Menschen Endothelial Colony Forming Progenitorzellen

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Forschung

Biologie

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Adulten und embryonalen Skelettmuskel Microexplant Kultur und Isolation der Skelettmuskulatur Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

Isolierung von Stammzellen aus menschlichen Bauchspeicheldrüsenkrebs Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Isolation und Kultur der einzelnen Muskelfasern und ihre Satelliten aus adulten Skelettmuskel

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Naive Adult Stem Cells Isolation von primären humanen Fibroblasten Kulturen

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Induktion und Analyse von epithelialen zu mesenchymalen Transition

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

Manuelle Isolierung von Fettgewebe gewonnene Stammzellen aus menschlichen Lipoaspirates

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

Isolierung, Kultur und Transplantation von Muskel-Satelliten-Zellen

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Isolation und Kultur der Dental epithelialen Stammzellen aus dem erwachsenen Maus Schneidezahn

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Isolierung und Kultur der primären Maus Keratinozyten aus neonatalen und erwachsenen Maus Haut

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...