Il supporto e la consulenza di Markus Pajtler e Robert Bohrer sono stati preziosi per la visualizzazione di questo protocollo.

Gli autori affermano che gli esperimenti sugli animali sono stati eseguiti in conformità con le Comunità europee direttiva del Consiglio (86/609/CEE), seguendo le linee guida del NIH per quanto riguarda la cura e l&#39;uso di animali nelle procedure sperimentali e le norme stabilite dalla Animal Istituzionale Cura e uso Comitato (IACUC) presso l&#39;Università di Duisburg-Essen (Germania). Parte 1: Impostazione (non mostrato in video) PREPARAZIONE MATERIALI E SOLUZIONI (prima di prendere le uova fuori dell&#39;incubatore) <ul><li> Le uova devono essere incubate in un incubatore umidificato per 11 giorni a 37 ° C (100 ° F). </li><li> Etanolo Spray 70%, piastre pulite, precise pinze pulite, 4 ° C (40 ° F) freddo sterile DMEM su ghiaccio e 60 ml siringhe sterili </li><li> 50 mL Provette sterili Corning devono essere riempito a metà con 4 ° C a freddo DMEM per miscelare gli embrioni macerare con un rapporto di 1:1 (massa macerata: DMEM). Non appena le uova vengono portati fuori dal dissezione incubatore di embrioni di pollo deve essere avviato. </li><li> Sterilizzare gli strumenti dissezione in autoclave o il giorno della dissezione, immergere gli strumenti in etanolo al 70% per 20 minuti. </li></ul> Parte 2: Preparazione del embrioni di pollo (dimostrata su Video) <ol><li> Bagnare le uova incubate con uno spray etanolo al 70% per almeno 30 secondi e asciugare accuratamente con asciugamani puliti carta morbida, mentre non scuotere troppo le uova. Nota: Si consiglia di non sezionare più di 60 embrioni di pollo contemporaneamente. </li><li> Aprire le uova con attenzione da lanciasse contro un bordo tagliente ed estrarre l&#39;embrione con cautela, non distruggendo il sacco vitellino e l&#39;embrione stesso pulcino. </li><li> Ricerca per l&#39;inizio del cordone ombelicale in modo rapido ed aprire la confezione trasparente, senza distruggere il sacco vitellino o le navi poco. Poi sezionare il cordone ombelicale con precise pinze pulite. </li><li> Prendete l&#39;embrione fuori dal sacco vitellino. Nota: Gli embrioni devono essere decapitato in fretta (non mostrato nel video). </li><li> Raccogliere gli embrioni in minima quantità di terreno necessario a 4 ° C. </li></ol> Parte 3: Macerazione delle embrioni di pollo <ol><li> Prendete lo stantuffo di una siringa da 60 ml sterile. </li><li> Trasferimento di circa 10 embrioni in una siringa da 60 ml. </li><li> Mettere lo stantuffo con attenzione nella siringa per non rischiare di perdere materiale macerato. </li><li> Macerare premendo lo stantuffo. </li><li> Raccogliere la massa macerare nel provette preparate corning mezza piena di DMEM ad un rapporto di 1:1 (massa macerata: DMEM). Utilizzando 10 embrioni un volume di circa 25 ml viene prodotto. </li><li> Mettere il composto in uno shaker per 45 minuti a 4 ° C. </li></ol> Parte 4: centrifugazione del embrione di pollo - Sospensione DMEM <ol><li> Trasferire la embrione di pollo - sospensione DMEM nei tubi centrifugazione. </li><li> Aggiungi ialuronidasi sterile ad una concentrazione di 1 mg per 25 mg di embrione. </li><li> Tara i tubi centrifugazione con estrema precisione. Nota: Questa operazione deve essere effettuata con cautela, poiché centrifugazione viene eseguita molto alto G-Force e di danni materiali o centrifuga potrebbe verificarsi se si utilizza tubi squilibrato. Con una elevata precisione di pesatura secondo macchina per quanto riguarda le tre posizioni dopo il punto decimale per tutti i tubi dovrebbero essere ricevuti regolando la quantità mancante con DMEM. </li><li> Ridurre la temperatura della centrifuga a 4 ° C. Nota: Anche il rotore centrifugazione devono essere conservati in una camera di raffreddamento o frigorifero per una notte. </li><li> Centrifugare 6 ore a meno di 180,000 xgx h. Nota: per risultati superiori per quanto riguarda la separazione delle singole fasi uso 266,000 xgx h. Se possibile, utilizzare la regolazione del vuoto. </li></ol> Parte 5: Filtrazione dell&#39;estratto embrione di pollo Dopo la centrifugazione, tre fasi si possono notare: La tomaia fase squallido liquida con frammenti di grasso, la fase liquido limpido in mezzo e il pellet alla base del tubo di centrifugazione. Nota: Shaking o in movimento vivace della miscela deve essere centrifugata assolutamente evitata, in quanto ciò avrebbe distrutto il risultato della fase di centrifugazione, perdendo la fase liquido limpido centrale. Nota: Tutti i passaggi che seguono devono essere eseguite in condizioni sterili con un banco d&#39;aria a flusso laminare: <ol><li> Pipettare la fase chiara liquido in un sistema di filtri con pori di 0,45 micron. Poi accendere la pompa del vuoto collegata. Nota: non toccare il pellet in basso. Ciò porterebbe a una contaminazione di estratto e un blocco del sistema di filtraggio. </li><li> Trasferire la pre-estratto in un sistema di filtri con pori di 0,22 micron e accendere la pompa del vuoto collegata di nuovo. </li><li> L&#39;aliquota raggiunta estrarre embrione di polloin sterili da 15 ml tubi Corning. </li><li> Memorizzare rapidamente le aliquote a -80 ° C (-62 ° F). Nota: L&#39;durare fino a due anni CEE se conservati a -80 ° C. </li></ol>

<ol>
	<li>Bronner-Fraser, M., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lineage+analysis+reveals+multipotency+of+some+avian+neural+crest+cells.">Cell lineage analysis reveals multipotency of some avian neural crest cells.</a> Nature. 335, 161-164 (1988).</li><li>Stemple, D. L., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+stem+cell+for+neurons+and+glia+from+the+mammalian+neural+crest.">Isolation of a stem cell for neurons and glia from the mammalian neural crest.</a> Cell. 71, 973-985 (1992).</li><li>Rao, M. S., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+and+controlled+in+vitro+differentiation+of+murine+multipotent+neural+crest+stem+cells.">Immortalization and controlled in vitro differentiation of murine multipotent neural crest stem cells.</a> J Neurobiol. 32, 722-746 (1997).</li><li>Paratore, C., Goerich, D. E., Suter, U., Wegner, M., Sommer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+and+glial+fate+acquisition+of+neural+crest+cells+are+regulated+by+an+interplay+between+the+transcription+factor+Sox10+and+extrinsic+combinatorial+signaling.">Survival and glial fate acquisition of neural crest cells are regulated by an interplay between the transcription factor Sox10 and extrinsic combinatorial signaling.</a> Cell. 128, 3949-3961 (2001).</li><li>Kim, J., Lo, L., Dormand, E., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SOX10+maintains+multipotency+and+inhibits+neuronal+differentiation+of+neural+crest+stem+cells.">SOX10 maintains multipotency and inhibits neuronal differentiation of neural crest stem cells.</a> Neuron. 38, 17-31 (2003).</li><li>Anderson, D. J., Groves, A., Lo, L., Ma, Q., Rao, M., Shah, N. M., Sommer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lineage+determination+and+the+control+of+neuronal+identity+in+the+neural+crest.">Cell lineage determination and the control of neuronal identity in the neural crest.</a> Review Cold Spring Harb Symp Quant Biol. 62, 493-504 (1997).</li><li>Dorsky, R. I., Moon, R. T., Raible, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+signals+and+cell+fate+specification+in+premigratory+neural+crest.">Environmental signals and cell fate specification in premigratory neural crest.</a> Review Bioessays. 22, 708-716 (2000).</li><li>Sieber-Blum, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+controlling+lineage+specification+in+the+neural+crest.">Factors controlling lineage specification in the neural crest.</a> Int Rev Cytol. 197, 1-33 (2000).</li><li>Dupin, E., Real, C., Ledouarin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neural+crest+stem+cells:+control+of+neural+crest+cell+fate+and+plasticity+by+endothelin-3.">The neural crest stem cells: control of neural crest cell fate and plasticity by endothelin-3.</a> Int Rev Cytol. 73, 533-545 (2001).</li><li>Maurer, J., Fuchs, S., J&#228;ger, R., Kurz, B., Sommer, L., Schorle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+controlled+differentiation+of+neural+crest+stem+cell+lines+using+conditional+transgenesis.">Establishment and controlled differentiation of neural crest stem cell lines using conditional transgenesis.</a> Differentiation. 75, 890-891 (2007).</li><li>Wu, Y. Y., Mujtaba, T., Rao, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+stem+and+precursor+cells+from+fetal+tissue.">Isolation of stem and precursor cells from fetal tissue.</a> Methods Mol Biol. 198, 29-40 (2002).</li></ol>

Nessun conflitto di interessi dichiarati.

Questo protocollo si basa su modificazioni delle procedure che sono state descritte in passato 10,11. Oltre alle pubblicazioni precedenti abbiamo qui mostrare le singole fasi diverse del processo più esteso. Questo sostiene lo sperimentatore con dettagli importanti garantendo procedimenti corretti e risultati affidabili. Inoltre, come il processo di dissezione è una parte essenziale di estrazione CEE descriviamo la procedura di presa degli embrioni fuori dal sacco vitellino in ogni dettaglio. Ci sono divers...

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<td>11 days incubated chicken eggs</td>
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<td>S&ouml;rries-Trockels Vermehrungszucht GmbH &amp; Co.KG</td>
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<td>Alcoholic disinfection spray</td>
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<td>Sch&uuml;lke &amp; Mayr GmbH</td>
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<td>DMEMGlutamax</td>
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<td>GIBCO Invitrogen</td>
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<td>Syringe sterile (60 ml)</td>
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<td>BD Biosciences</td>
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<td>Tubes sterile (50; 15 ml)</td>
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<td>Greiner Bio-One GmbH</td>
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<td>Pipet tips sterile</td>
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<td>Starlab GmbH</td>
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<td>Hyaluronidase typeIV-S</td>
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<td>Sigma-Aldrich</td>
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<td>Milipore stericups sterile (0.45; 0.22 &mu;l)</td>
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<td>Fisher Scientific AG</td>
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<td>Pipettes sterile (25; 10; 5 ml)</td>
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<td>Greiner Bio-One GmbH</td>
<td>&nbsp;</td>
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<td>Centrifuge L5-50</td>
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<td>Rotor type Ti-45</td>
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production of chick embryo extract for the cultivation of murine neural crest stem cells

La cresta neurale nasce dalla neuro-ectoderma durante l&#39;embriogenesi e persiste solo temporaneamente. Primi esperimenti già prova di cellule progenitrici pluripotenti di essere parte integrante della cresta neurale 1. Fenotipicamente, le cellule staminali neurali cresta (NCSC) sono definite dal simultaneamente esprimendo p75 (affine a basso fattore di crescita dei recettori nervosi, LNGFR) e SOX10 durante la loro migrazione dalla cresta neurale 2,3,4,5. Queste cellule staminali possono differenziarsi in cellule muscolari lisce, cellule cromaffini, neuroni e cellule gliali, così come melanociti, cartilagine e osso 6,7,8,9. Per coltivare NCSC in vitro, uno speciale medium cresta neurale delle cellule staminali (NCSCM) è richiesto 10. La parte più complessa del NCSCM è la preparazione di estratto pulcino dell&#39;embrione (CEE) che rappresenta una fonte essenziale di fattori di crescita per il NCSC così come per altri tipi di espianti neurale. Altri ingredienti NCSCM accanto CEE sono disponibili in commercio. Produrre CCE uso di apparecchiature di laboratorio standard è di grande importanza per conoscere i dettagli impegnativo come l&#39;isolamento, la macerazione, centrifugazione, filtrazione e processi. In questo protocollo si descrivono le tecniche accurate per produrre una quantità di massimizzare la qualità CEE pura e alta.

Watch this Scientific Journal Video about Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells at JoVE.com

Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

Per coltivare le cellule staminali neurali cresta (NCSC) in vitro, un mezzo speciale (NCSCM) è obbligatorio. Parte essenziale della NCSCM è embrione estratto di pulcino (CEE). Noi qui descrivere le tecniche accurate per produrre una quantità di massimizzare la qualità CEE pura e alta, compresi i dettagli come l&#39;isolamento, la macerazione, centrifugazione, filtrazione e processi.

Produzione di estratto di Chick dell&#39;embrione per la coltivazione delle cellule della cresta neurale Murine staminali

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent ...

production-chick-embryo-extract-for-cultivation-murine-neural-crest

Research

JoVE Journal

Neuroscience

12.6K Views.  University Children's Hospital Essen. Per coltivare le cellule staminali neurali cresta (NCSC) in vitro, un mezzo speciale (NCSCM) è obbligatorio. Parte essenziale della NCSCM è embrione estratto di pulcino (CEE). Noi qui descrivere le tecniche accurate per produrre una quantità di massimizzare la qualità CEE pura e alta, compresi i dettagli come l'isolamento, la macerazione, centrifugazione, filtrazione e processi.

Video: Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

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.

L&#39;isolamento e la coltura di cellule staminali neurali Crest di follicoli dei capelli umani

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

Ricerca

Neuroscienze

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

Isolamento e la coltura delle cellule della cresta neurale da Embryonic Murine tubo neurale

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

Derivazione libero-Feeder della cresta neurale progenitrici Cellule pluripotenti da cellule staminali umane

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Enumeration of Neural Stem Cells Using Clonal Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres. Finally, the NSC frequency is calculated based on neurosphere formation and multipotency capabilities. Utilities of this protocol include the evaluation of candidate NSC markers, purification of NSCs, and the ability to distinguish NSCs from NPs. This method takes 13 days to perform, which is much shorter than current methods to enumerate NSC frequency.

Neural stem cells (NSCs) refer to cells&#160;which can self-renew and differentiate into the three neural lineages. Here, we describe a protocol to determine NSC frequency in a given cell population using neurosphere formation and differentiation under clonal conditions.

Conteggio di cellule staminali neurali L&#39;utilizzo clonali Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

Sviluppo di un protocollo di raffinata per Trans-sclerale Subretinal trapianto delle cellule epiteliali del pigmento retinico umano negli occhi di topo

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

Il rilevamento di 5-Hydroxytethylcytosine in cellule staminali neurali e cervelli di topi

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the lack of proper animal models that fully mimic human diseases and the poor availability of post-mortem human brain tissue. Adult human nervous tissue culture offers the possibility to study different aspects of neurological disorders. Molecular, cellular, and biochemical mechanisms could be easily addressed in this system, as well as testing and validating drugs or different treatments, such as cell-based therapies. This method combines long-term organotypic cultures of the adult human cortex, obtained from epileptic patients undergoing resective surgery, and ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors. This method will allow the study of cell survival, neuronal differentiation, the formation of synaptic inputs and outputs, and the electrophysiological properties of human-derived cells after transplantation into intact adult human cortical tissue. This approach is an important step prior to the development of a 3D human disease modeling platform that will bring basic research closer to the clinical translation of stem cell-based therapies for patients with different neurological disorders and allow the development of new tools for reconstructing damaged neural circuits.

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

This protocol describes long-term organotypic cultures of adult human cortex combined with ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors, which present a novel methodology to further test stem cell-based therapies for human neurodegenerative disorders.

Colture organotipiche della corteccia umana adulta come modello ex vivo per il trapianto e la validazione di cellule staminali umane

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the ...

Derivazione di Neuroni Enterici da Cellule Staminali Pluripotenti Umane

Differentiation of Enteric Nervous System Lineages from Human Pluripotent Stem Cells

The human enteric nervous system, ENS, is a large network of glial and neuronal cell types with remarkable neurotransmitter diversity. The ENS controls bowel motility, enzyme secretion, and nutrient absorption and interacts with the immune system and the gut microbiome. Consequently, developmental and acquired defects of the ENS are responsible for many human diseases and may contribute to symptoms of Parkinson's disease. Limitations in animal model systems and access to primary tissue pose significant experimental challenges in studies of the human ENS. Here, a detailed protocol is presented for effective in vitro derivation of the ENS lineages from human pluripotent stem cells, hPSC, using defined culture conditions. Our protocol begins with directed differentiation of hPSCs to enteric neural crest cells within 15 days and yields diverse subtypes of functional enteric neurons within 30 days. This platform provides a scalable resource for developmental studies, disease modeling, drug discovery, and regenerative applications.

Autore in primo piano: Coltura robusta di neuroni enterici umani da cellule staminali pluripotenti umane

Deriving enteric nervous system (ENS) lineages from human pluripotent stem cells (hPSC) provides a scalable source of cells to study ENS development and disease, and to use in regenerative medicine. Here, a detailed in vitro protocol to derive enteric neurons from hPSCs using chemically defined culture conditions is presented.

Differenziazione delle linee del sistema nervoso enterico dalle cellule staminali pluripotenti umane

The human enteric nervous system, ENS, is a large network of glial and neuronal cell types with remarkable neurotransmitter diversity. The ENS controls ...