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 ...

isolation-and-culture-of-adult-epithelial-stem-cells-from-human-skin

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28.3K 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

Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by distinct pools of SCs, which are located in the permanent portion of the HF, a region known as the bulge. These multipotent bulge SCs were initially identified as slow cycling label retaining cells; however, their isolation has been made feasible after identification of specific cell markers, such as CD34 and keratin 15 (K15). Here, we describe a robust method for isolating bulge SCs and epidermal keratinocytes from mouse HFs utilizing fluorescence activated cell-sorting (FACS) technology. Isolated hair follicle SCs (HFSCs) can be utilized in various in vivo grafting models and are a valuable in vitro model for studying the mechanisms that govern multipotency, quiescence and activation.

An ideal model for studying adult stem cell biology is the mouse hair follicle. Here we present a protocol for isolating different populations of hair follicles stem cells and epidermal keratinocytes, employing enzymatic digestion of mouse dorsal skin followed by FACS analysis.

Isolieren von Haarfollikelstammzellen und epidermalen Keratinozyten von Dorsal Mäusehaut

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by ...

Forschung

Entwicklungsbiologie

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

Isolation und Kultur von Neural Crest Stem Cells aus menschlichen Haarfollikeln

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

Neurowissenschaften

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. In addition, keratinocytes play an essential role in the initiation, maintenance, and regulation of epidermal immune responses by being part of the innate immune system responding to antigenic stimuli in a fast, nonspecific manner. Here, we describe a protocol for isolation of primary human keratinocytes from adult skin, and demonstrate that these cells respond to calcium-induced terminal differentiation, as measured by an increased expression of the differentiation marker involucrin. In addition, we show that the isolated keratinocytes are responsive to IL-1&#946;-induced activation of intracellular signaling pathways as measured by the activation of the p38 MAPK pathway. Taken together, we describe a method for isolation and culturing of primary human keratinocytes from adult skin. Because the keratinocytes are the predominant cell type in the epidermis, this method is useful to study molecular mechanisms in cutaneous biology in vitro.

The human skin acts as a first line of defense against the external environment. We present a method for isolating primary human keratinocytes from adult skin. These isolated keratinocytes are useful in numerous experimental setups, and are a highly suitable model for studying molecular mechanisms in cutaneous biology in vitro.

Generation und Kultivierung der primären menschlichen Keratinozyten von Erwachsenen

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. ...

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 ...

Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human cancers. As a lining tissue, the skin epithelium is often subject to injury and has evolutionarily adapted by acquiring the cellular plasticity necessary to repair damaged tissue. Over the years, several efforts have been made to study epithelial plasticity using in&#160;vitro and ex vivo cell-based models. However, these efforts have been limited in their capacity to recapitulate the various phases of epithelial cell plasticity. We describe here a protocol for generating 3D epidermal spheroids and epidermal spheroid-derived cells from primary neonatal human keratinocytes. This protocol outlines the capacity of epidermal spheroid cultures to functionally model distinct stages of keratinocyte generative plasticity and demonstrates that epidermal spheroid re-plating can enrich heterogenous normal human keratinocytes (NHKc) cultures for integrin&#945;6hi/EGFRlo keratinocyte subpopulations with enhanced stem-like characteristics. Our report describes the development and maintenance of a high throughput system for the study of skin keratinocyte plasticity and epidermal regeneration.

Here we describe a protocol for the systematic cultivation of epidermal spheroids in 3D suspension culture. This protocol has wide-ranging applications for use in a variety of epithelial tissue types and for the modeling of several human diseases and conditions.

Etablierung eines epidermalen Sphäroidkultursystems mit hohem Durchsatz zur Modellierung der Plastizität von Keratinozyten-Stammzellen

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

Isolierung von adulten menschlichen Dermal-Fibroblasten aus der Bauchhaut und Generierung induzierter pluripotenter Stammzellen mit einer nicht-integrierenden Methode

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Biotechnik

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&#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 ...

Biologie

Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid enzymatic dissociation of skin. Primary keratinocytes, fibroblasts, and Schwann cells are all harvested from the human newborn foreskin, which is available following standard of care procedures. The removed skin is disinfected, and the subcutaneous fat and muscle are removed using a scalpel. The method consists of enzymatic and mechanical separation of epidermal and dermal layers, followed by additional enzymatic digestion to obtain single-cell suspensions from each of these skin layers. Finally, single cells are grown in appropriate cell culture media following standard cell culture protocols to maintain growth and viability over weeks. Together, this simple protocol allows isolation, culturing, and characterization of all three cell types from a single piece of skin for in vitro evaluation of skin-nerve models. Additionally, these cells can be used together in co-cultures to gauge their effects on each other and their responses to in vitro trauma in the form of scratches performed robotically in the culture associated with wound healing.

The study of wound healing associated with musculoskeletal injury often requires the assessment of in vitro interactions between Schwann cells (SCs), keratinocytes, and fibroblasts. This protocol describes the isolation, culturing, and characterization of these primary cells from the human foreskin.

Isolierung, Kultur und Charakterisierung von primären Schwann-Zellen, Keratinozyten und Fibroblasten aus der menschlichen Vorhaut

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid ...

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 ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

Isolierung und Kultur von primären oralen Keratinozyten aus dem erwachsenen Mausgaumen

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...