Bu çalışma NIH hibe R01AR054593 ve Xu R01AR054593-S1 tarafından desteklenmektedir.

1. Doku Kültürü Plakalarının Hazırlanması <ol><li> Coat Her iyi Poly-D-Lizin (PDL) kuyunun dibine karşılamak için yeterli. Plakalar kaputu kurumasını bekleyin. </li><li> Kuyu kuruduktan sonra, steril su ile durulayın ve aspire. Plakalar kaputu kurumasını bekleyin. </li><li> Ne zaman fibronektin ile kuru kat (6 ml içinde 1 mg &#39;lik bir konsantrasyonda 37 ° C de bir gece Biowhittaker su içinde çözülür ki). </li><li> ŞÇUK orta ekleyin [95 ml DMEM/F12, 1 ml Penn / Strep (P / S), 1 ml N2, 2 ml B27, 100 ul merkaptoetanol (2ME, 50 mM stok), bFGF (20 ng / ml orta), IGF -1 (20 ng / orta ml) ve EGF (20 ng / ml orta)] plakaları içinde kuru önce fibronektin, </li></ol> 2. İnsan Derisi şehirlerinde Saç Folikül Hücreleri Özü <ol><li> Tecrit NCSCs arasında işlemine başlamadan önce, bir (bkz. Tablo 1) ilgili medias ve reaktifler hazırlamak gerekiyor. </li><li> Facelift prosedürleri veya fe Taze yetişkin insan saçlı derital kafa derisi doku PBS penisilin-streptomisin içeren suyla yıkandı, toplanır. </li><li> Deri daha sonra 50 ml tüp içine aktarılır ve 4 ° C de bir gece dispase (10 mg / ml) DMEM içinde inkübe edilir 37, 2-4 saat boyunca inkübasyon ° C &#39;de etkilidir. Deri parçaları nüfuz enzim sağlamak için en az 1 cm en fazla genişlikte olmalıdır. </li><li> Steril bir Petri kabı içine deri aktarın; cilt yüzeyine yakın saç şaft tutup sıkıca ve düzgün çekerek cilt her saç çekin. Saç köklerinin anajen (Şekil 1A) veya telojen (Şekil 1B) ya morfolojisi göstermektedir. </li><li> Periyodik sallama ile oda sıcaklığında 15-20 dakika için% 0.05 tripsin-EDTA (Invitrogen) içinde izole folikül parçaları inkübe, reaksiyonu durdurmak için% 10 FBS ile 4 ml DMEM ilave edin. Epitel tripsinize ve değişik boyut ve şekil hücreleri içeren tek bir hücre süspansiyonu elde etmek için 40 um filtre ile filtre edilir. </li><li> Dönüş5 dakika boyunca 200 x g, 1 ml PBS% 2 serumu (FBS) ihtiva eden yeniden askıya, dikkatlice süpernatan atılır. </li><li> Alternatif olarak, alınmış saç köklerinin in situ olarak saç büyümesini küreler tripsin sindirim olmaksızın kültür içinde yerleştirilebilir. </li></ol> 3. Akış Sitometrik Hücre Ayırma kullanılması Saç Folikül NCSCs İzolasyonu <ol><li> Saç folikülü tek bir hücre süspansiyonu tripsin ile sindirim ile elde edilen ve CD271 (APC-konjuge) / HNK1 (FITC-konjuge) ya da CD271 / alpha4 karanlıkta buz üzerinde 40 dakika süreyle integrin (PE ile konjuge) karşı antikorlar ile işaretlenmiştir. </li><li> Oda sıcaklığında, 200 x g&#39;de 5 dakika boyunca santrifüjden sonra süpernatan aspire. </li><li> PBS,% 2 serum (FBS) içeren içinde süspanse hücreleri, ve sıralama önce, PI ölü hücreleri kapı eklenir. </li><li> Flow sitometri (FACS) tarafından hücre sıralama yapın. CD271 toplayın +, HNK1 + çift pozitif hücreleri veya CD271 +, alpha4 integrin + çift pozitif hücreler (Şekil 2). </li><li> Sonra sıralamaing, CD271 +, HNK1 + çift pozitif hücreleri veya CD271 +, alpha4 integrin + çift pozitif hücreler ŞÇUK ortamda ultra-düşük eki plakalar kültüre </li></ol> 4. İlköğretim NCSCs Kültürü <ol><li> Kültür ilişkisiz foliküler hücreleri veya FACS ŞÇUK ortamda ultra-düşük eki plakaları hücreleri kriteri, her gün orta değiştirin. </li><li> Mikroskop altında her gün hücre kültüründe kontrol edin. NCSCs birkaç gün ve bağışçılar (Şekil 3A) yaşına bağlı olarak kültürde 2-5 hafta içinde iyi biçimli küreler sonra yüzen küçük agregatlar oluşturması için başlayacaktır. Outgrowth çıkıntı bölge ve birkaç hafta içinde in situ olarak iyi biçimlenmiş küreler (Şekil 3B) tüm saç köklerinin ŞÇUK ortamda kültüre de birkaç gün içinde görünecektir. </li></ol> 5. NCSCs genişletilmesi <ol><li> Bir kez PBS ile hücreleri yıkayın. </li><li> Önceden ısıtılmış ekle (37 ° C, kritik) 37 accutase ve inkübe ° C 5-10 içintek bir hücreye emin Nöral kök hücre küre disosiye yapmak için periyodik sallayarak dk (ek kültürlü ŞÇUK, 37 accutase ile inkübe hücreleri için emin kıl folikülü kök hücreleri müstakil olmak için 5 dakika ° C). </li><li> Kalan çözelti accutase kaldırmak için DMEM/F12 ortamı içinde oda sıcaklığında 200 x g&#39;de 5 dakika boyunca iki kez yıkanır ve hücrelerin santrifüj. </li><li> ŞÇUK besiyeri ile hücreleri yeniden askıya. </li><li> Kayan kültürü için, ultra-düşük eki plakaları hücrelerin koydu. Eki kültürü için, ön işlem doku kültürü kabına hücreleri koydu. </li></ol> 6. Temsilcisi Sonuçlar ŞÇUK kültür ortamı besleyici hücreleri için gerek kalmadan bir farklılaşmamış bir durumda hNCSCs korumak için yeterlidir. Keratinositlerin prolifere ve yavaş yavaş orta ölmüş olmaz. Bazı küçük yuvarlak hücreler çoğalmaya başlar ve 3 ila 5 gün sonra süspansiyon küçük agrega oluşturacaktır. Bu yüzen agregalar yavaş i arttıBiz saç küreler (Şekil 3A) olarak adlandırılan n boyutu, oluşturulan üç-boyutlu küre benzeri yapılar. Kaplanmış plakalar içinde kültüre ise, NCSCs yüzeye yapışır ve herhangi küreleri oluşturmayacaktır. Ekli NCSCs süspansiyon daha hızlı büyür. Küre-şekillendirme ya da bağlı kök hücreler NCSCs işaretleri ifade eder. Tüm saç köklerinin kültürleri olduğunda, küreler çıkıntı bölgesi (Şekil 3B) karşılık gelen bölge de oluşturulur. <img alt="Şekil 1" src="/files/ftp_upload/3194/3194fig1.jpg" /> Şekil 1. Anajen (A) ve telojen (B), saç folikülleri çekti. <img alt="Şekil 2," src="/files/ftp_upload/3194/3194fig2.jpg" /> Şekil 2. NCSCs FACS analizi Örnek görüntüleri Sol panel:. Hücreleri, anti-CD271 ve anti-alphe 4 integrin antikorları ile kapı vardır. Sağ panel: hücrelerinin, anti-CD271 ve anti-HNK1 antikorlar kullanılarak kapı vardır. <img alt="Şekil 3" src="/files/ftp_upload/3194/3194fig3.jpg" /> Şekil 3,. Saç küreleri Morfoloji dalgalı saç küreleri, sol panel (A) Morfoloji:. Düşük güç, sağ panelde:. Yüksek güç (B) şişkinliği bölgede, sol paneldeki in situ olarak saç kürenin Morfoloji: telojen çıkıntı bir saç küre, Sağ panel: bir anagen kıl folikülünün şişkinliği bölgede bir saç küre.

<ol>
	<li>Cotsarelis, G., Cheng, S. Z., Dong, G., Sun, T. T., Lavker, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Existence+of+slow-cycling+limbal+epithelial+basal+cells+that+can+be+preferentially+stimulated+to+proliferate:+implications+on+epithelial+stem+cells.">Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: implications on epithelial stem cells.</a> Cell. 57, 201-209 (1989).</li><li>Cotsarelis, G., Sun, T. T., Lavker, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-retaining+cells+reside+in+the+bulge+area+of+pilosebaceous+unit:+implications+for+follicular+stem+cells,+hair+cycle,+and+skin+carcinogenesis.">Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis.</a> Cell. 61, 1329-1337 (1990).</li><li>Tanimura, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair+follicle+stem+cells+provide+a+functional+niche+for+melanocyte+stem+cells.">Hair follicle stem cells provide a functional niche for melanocyte stem cells.</a> Cell Stem Cell. 8, 177-187 (2011).</li><li>Yu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+novel+population+of+multipotent+adult+stem+cells+from+human+hair+follicles.">Isolation of a novel population of multipotent adult stem cells from human hair follicles.</a> Am. J. Pathol. 168, 1879-1888 (2006).</li><li>Yu, H., Kumar, S. M., Kossenkov, A. V., Showe, L., Xu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+with+neural+crest+characteristics+derived+from+the+bulge+region+of+cultured+human+hair+follicles.">Stem cells with neural crest characteristics derived from the bulge region of cultured human hair follicles.</a> J. Invest. Dermatol. 130, 1227-1236 (2010).</li><li>Wong, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest-derived+cells+with+stem+cell+features+can+be+traced+back+to+multiple+lineages+in+the+adult+skin.">Neural crest-derived cells with stem cell features can be traced back to multiple lineages in the adult skin.</a> J. Cell Biol. 175, 1005-1015 (2006).</li><li>Sieber-Blum, M., Grim, M., Hu, Y. F., Szeder, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotent+neural+crest+stem+cells+in+the+adult+hair+follicle.">Pluripotent neural crest stem cells in the adult hair follicle.</a> Dev. Dyn. 231, 258-269 (2004).</li><li>Amoh, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implanted+hair+follicle+stem+cells+form+Schwann+cells+that+support+repair+of+severed+peripheral+nerves.">Implanted hair follicle stem cells form Schwann cells that support repair of severed peripheral nerves.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 17734-17738 (2005).</li><li>Koliatsos, V. E., Xu, L., Yan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+stem+cell+grafts+as+therapies+for+motor+neuron+disease.">Human stem cell grafts as therapies for motor neuron disease.</a> Expert Opin. Biol. Ther. 8, 137-141 (2008).</li><li>McKenzie, I. A., Biernaskie, J., Toma, J. G., Midha, R., Miller, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin-derived+precursors+generate+myelinating+Schwann+cells+for+the+injured+and+dysmyelinated+nervous+system.">Skin-derived precursors generate myelinating Schwann cells for the injured and dysmyelinated nervous system.</a> J. Neurosci. 26, 6651-6660 (2006).</li><li>LeDouarin, N. 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Biz ifşa etmek başka bir şey var.

Anlatılan hücre izolasyonu ve kültürü yöntemleri tekrarlanabilir ve sağlamdır. Biz geniş bir yaş aralığında bireylerin onlarca NCSCs yarattı. Doğru doku hasat sonrası doku işlemek için iyi olmasına rağmen, biz kafa derisi dokularıyla güvenle hücre canlılığı üzerine minimal etkisi ile gecede ulaşım için buz üzerinde medya saklanabilir bulundu. Bu antibiyotik ile kafa derisi doku tedavisi ve potansiyel mikroorganizma kontaminasyonu önlemek için saç folikülü...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Invitrogen</td>
 <td>11965-092</td>
 </tr>
 <tr>
 <td>DMEM/F12</td>
 <td>Invitrogen</td>
 <td>11330-32</td>
 </tr>
 <tr>
 <td>Heat-inactivated FBS</td>
 <td>Hyclone</td>
 <td>SH30071.03</td>
 </tr>
 <tr>
 <td>B27 supplement</td>
 <td>Invitrogen</td>
 <td>17504044</td>
 </tr>
 <tr>
 <td>N2 supplement</td>
 <td>Invitrogen</td>
 <td>17502048</td>
 </tr>
 <tr>
 <td>bFGF</td>
 <td>Invitrogen</td>
 <td>PHG0026</td>
 </tr>
 <tr>
 <td>EGF</td>
 <td>R&amp;D system</td>
 <td>236-EG-01M</td>
 </tr>
 <tr>
 <td>IGF-I</td>
 <td>R&amp;D system</td>
 <td>291-G1-050</td>
 </tr>
 <tr>
 <td>0.05% Trypsin/EDTA</td>
 <td>Invitrogen</td>
 <td>25300-054</td>
 </tr>
 <tr>
 <td>Dispase</td>
 <td>Invitrogen</td>
 <td>17105041</td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin</td>
 <td>Invitrogen</td>
 <td>15070063</td>
 </tr>
 </tbody> 
</table>
Table 1.

isolation and culture of neural crest stem cells from human hair follicles

Saç köklerinin yaşam boyu büyümeye geçmesi ve saç döngüsü kök hücre çoğalması ve sükunet içeren iyi-kontrollü bir süreç olduğunu. Saç şişkinliği yetişkin kök hücreleri 1 için iyi karakterize niş. Dış kök kılıfı bu segment epitel kök hücreler 2, melanosit kök hücre 3 ve 4-7 kök hücreleri gibi nöral krest dahil kök hücreler, farklı türde bir dizi içerir. Saç köklerinin insan kök hücreleri farklı türleri için erişilebilir ve zengin bir kaynak oluşturmaktadır. Biz ve diğerleri 4,5 insan fetal ve yetişkin saç köklerinin gelen nöral krest kök hücreler (NCSCs) izole ettim. Bunlar insan kök hücreleri etiket tutucu hücreleri ve in vitro asimetrik hücre bölünmesi yoluyla kendini yenileme yeteneğine sahiptir. Onlar immatür nöral krest hücre belirteçleri değil farklılaşma işaretleri ifade eder. Bizim ekspresyon profili çalışma, fare derisi immatür nöral CRE ile benzer bir gen ekspresyon paterni paylaşmaktadır gösterdiST hücreleri. Onlar vitro klonal tek hücre kültürü sonrası, miyojenik melanositik ve nöronal hücre soylarının ortaya çıkmasına neden olabilir klonal multipotency sergilerler. Farklılaştırılmış hücreler sadece soy-spesifik belirteçler elde değil, aynı zamanda ex vivo koşullarda uygun işlevleri göstermek değildir. Buna ek olarak, bu NCSCs mezenkimal soy doğru farklılaşma potansiyeli gösteriyor. Farklılaştırılmış nöronal hücrelerin fare beyin inat ve nöronal farklılaşma işaretleri koruyabilirsiniz. Bu saç NCSCs sinir büyütme yardımcı olabilir türetilmiş folikülü ve onlar transecting omurilik yaralanması 8 takiben bu kök hücreler nakledildi farelerde motor fonksiyon geliştirmek olduğu gösterilmiştir. Ayrıca, periferik sinirlerin kök hücre greftleri 9 ve ezilmiş siyatik sinirleri bitişik cilt türetilmiş prekürsör hücrelerin implantasyonu ile tamir edilmiştir remiyelinizasyon 10 sonuçlandı. Bu nedenle, saç folikülü / cilt elde NCSCs zaten r için umut verici sonuçlar göstermiştirpreklinik modellerde egenerative tedavisi. Indüklenmiş pluripotent kök (iPS) hücreleri yeniden programlayarak Somatik Hücre rejeneratif tıp için muazzam potansiyel göstermiştir. Bununla birlikte, özellikle iPS hücreleri, onkogen / virüs entegrasyon ve pluripotent kök hücrelerin potansiyel tümör gelişimini ve uzun dönemli etkisi ile hala birçok sorunları yeterince ele alınmamıştır vardır. IPS hücreleri rejeneratif ilaç için kullanılmadan önce üstesinden gelinmesi gereken çok engel hala vardır. Yetişkin kök hücreleri güvenli olduğu bilinen ve bu gibi kemik iliği nakli gibi pek çok yıl için klinik olarak kullanılmaktadır ise. Birçok hasta zaten tedavi yararlanmıştır. Otolog yetişkin kök hücreler hâlâ nakli için tercih edilen hücrelerdir. Bu nedenle, insan deri / saç köklerinin kolayca erişilebilir ve genişletilebilir erişkin kök hücrelerin rejeneratif tıp için değerli bir kaynaktır.

Watch this Scientific Journal Video about Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles at JoVE.com

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

Bu makale, insan saç köklerinin gelen nöral krest kök hücrelerin izolasyonu ve kültür için sağlam bir protokol sunar.

İnsan Saç köklerinin arasından Sinir Crest Kök Hücre İzolasyonu ve Kültür

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

isolation-culture-neural-crest-stem-cells-from-human-hair

Research

JoVE Journal

Neuroscience

16.9K Görüntüleme.  School of Medicine, University of Pennsylvania. Bu makale, insan saç köklerinin gelen nöral krest kök hücrelerin izolasyonu ve kültür için sağlam bir protokol sunar.

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

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

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent progenitor cells to be an integral part of the neural crest1. Phenotypically, neural crest stem cells (NCSC) are defined by simultaneously expressing p75 (low-affine nerve growth factor receptor, LNGFR) and SOX10 during their migration from the neural crest2,3,4,5. These progenitor cells can differentiate into smooth muscle cells, chromaffin cells, neurons and glial cells, as well as melanocytes, cartilage and bone6,7,8,9. To cultivate NCSC in vitro, a special neural crest stem cell medium (NCSCM) is required10. The most complex part of the NCSCM is the preparation of chick embryo extract (CEE) representing an essential source of growth factors for the NCSC as well as for other types of neural explants. Other NCSCM ingredients beside CEE are commercially available. Producing CCE using laboratory standard equipment it is of high importance to know about the challenging details as the isolation, maceration, centrifugation, and filtration processes. In this protocol we describe accurate techniques to produce a maximized amount of pure and high quality CEE.

To cultivate neural crest stem cells (NCSC) in vitro, a special medium (NCSCM) is required. Essential part of NCSCM is chick embryo extract (CEE). We here describe accurate techniques to produce a maximized amount of pure and high quality CEE, including details as the isolation, maceration, centrifugation, and filtration processes.

Civciv Embriyo Özü murin Sinir Crest Kök Hücre Yetiştiriciliği Üretimi

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

Nörobilim

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

Atılan İnsan Fetal Kortikal Doku Sinir Kök Hücre Üretimi

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

Isolation and Culture of Human Fungiform Taste Papillae Cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells9. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C &beta;2, (PLC-&beta;2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-&beta;2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation.
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 201110.

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

İnsan Mantar şeklinde Lezzet papilla hücrelerinin izolasyonu ve Kültürü

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple ...

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

Sıçan Embriyonik Nöral Hücre İzolasyonu ve Kültür: Bir Hızlı Protokol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

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.

Embriyonik Mürin Nöral Tüp çıkan sinirsel Crest Hücre İzolasyonu ve Kültür

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

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

Bir Fare, İki Kültür: İzolasyon ve Yetişkin Nöral Kültür Bireysel Farelerde iki Nörojenik Bölgeler&#39;den Kök Hücre

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

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.

İnsan Pluripotent dan Sinir Crest Progenitör Hücreler besleyici ücretsiz Derivasyon Kök Hücre

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

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

Fare Subcallosal Zone İzolasyon ve Yetişkin Nöral Kök Hücre Kültürü

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

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.

Sıçan gözlerinin içine insan retina Pigment epitel hücrelerinin Trans-scleral Subretinal nakli için rafine bir protokol geliştirilmesi

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

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

İnsan yetişkin cilt fibroblastlar indüklenen Neurons yüksek verimli kültürünün basit üretimi

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...