この作品は、NIHの助成金R01AR054593とXuへR01AR054593-S1でサポートされています。

1。組織培養プレートの作製<ol><li>コー​​トはそれぞれに良く、ポリ-D-リジン（PDL）は井戸の底をカバーするのに十分。プレートがボンネットに乾燥することができます。 </li><li>井戸が乾燥した後、滅菌水で洗い流して、吸引します。プレートがボンネットに乾燥することができます。 </li><li>時フィブロネクチンと乾燥、コート（6ミリリットルで1ミリグラムの濃度で37℃で一晩biowhittakerの水に溶解したもの）。 </li><li> NCSC媒体を加える[95ミリリットルDMEM/F12、1ミリリットルペン/ストレプトマイシン（P / S）、1mlのN2は、2ミリリットルB27、100μlのメルカプトエタノール（2ME、50mMのストック）は、bFGF（20 ng / mlの培地）、IGF -1（20 / NG媒体のml）およびEGF（20 ngの/培地1ml）]プレートで乾燥したフィブロネクチンの前に、 </li></ol> 2。人間の頭皮から毛包細胞を抽出<ol><li>隔離NCSCsの手順を開始する前に、1（ 表1参照） は 、それぞれのメディアと試薬を準備する必要があります。 </li><li>フェイスリフトの手順やFEから新鮮なヒト成人頭皮TAL頭皮組織をPBSでペニシリン - ストレプトマイシンを含むで洗浄し、収集されます。 </li><li>皮膚を50 mlチューブに移し、4℃で一晩ディスパーゼ（10 mg / ml）を含むDMEM中で培養される37℃で2〜4時間のインキュベーション℃のも効果的です。皮膚片は、酵素が浸透することを可能にするために1cmの最大幅でなければなりません。 </li><li>滅菌シャーレに皮膚を移し、皮膚表面の近くに毛幹をつかみ、しっかりとスムーズに引っ張ることによって皮膚からそれぞれの髪をやってのける。毛包が成長期（ 図1A）または休止（ 図1B）のいずれかの形態を示している。 </li><li>定期的に振とうしながら室温で15〜20分間0.05％トリプシン-EDTA（Invitrogen）中で孤立した卵胞の断片をインキュベートし、反応を停止し、10％FBSを含むDMEM 4ミリリットルを追加します。濾胞上皮をトリプシン処理し、様々なサイズと形状の細胞を含む単細胞懸濁液を得るために、フィルタは40μmで濾過する。 </li><li>スピン5分間、200×gで、1 mlのPBSで2％血清（FBS）を含むで再懸濁、慎重に上清を捨てる。 </li><li>代わりに、羽をむしられた毛包は、 その場で毛球を成長させるためにトリプシン消化することなく、文化に配置することができます。 </li></ol> 3。フローサイトメトリー細胞選別を使用した毛包NCSCsの分離<ol><li>毛包単細胞懸濁液をトリプシン消化により得られ、暗闇の中で氷上で40分間CD271（APC標識）/ HNK1（FITC-結合）またはCD271 / alpha4インテグリン（PE-結合）に対する抗体を用いて標識した。 </li><li>室温で200×gで5分間遠心し、上清を吸引除去する。 </li><li> 2パーセント血清（FBS）を含むPBSで細胞を再懸濁して、ソート前に、PIは、死んだ細胞アウトゲートに追加されます。 </li><li>フローサイトメトリー（FACS）による細胞選別を行います。 CD271 +、HNK1 +ダブルポジティブ細胞またはCD271 +、alpha4インテグリン+二重陽性細胞（ 図2）を収集します。 </li><li>ソート後り、CD271 +、HNK1 +ダブルポジティブ細胞またはCD271 +、alpha4インテグリン+二重陽性細胞がNCSC培地で超低アタッチメントプレート中で培養される</li></ol> 4。プライマリNCSCsの文化<ol><li> NCSC培地中で超低アタッチメントプレートに細胞をソート文化引き離され濾胞細胞またはFACSは、毎日培地を変更してください。 </li><li>顕微鏡下で毎日細胞培養を確認してください。 NCSCsは、ドナー（ 図3A）の年齢に応じて培養液中で2〜5週間で数日間と整形球後に浮動小凝集体を形成するために開始されます。伸長はバルジ領域と数週間でin situで整形球（ 図3B）全体の毛包がNCSC培地中で培養さで数日内に表示されます。 </li></ol> 5。 NCSCsの拡大<ol><li>一度PBSで細胞を洗浄します。 </li><li>あらかじめ温めておいた追加（〜37℃、クリティカル）で37 accutaseインキュベートし5〜10℃のC単一のセル（アタッチメント培養NCSCのため、37℃でインキュベートaccutase細胞℃で5分間、毛包幹細胞が切り離さかどうかを確認するため）に解離してください神経幹細胞の球体を作るために定期的に振とうしながら分。 </li><li>残っaccutaseソリューションを削除するDMEM/F12培地において室温で200×gで5分間細胞を2回洗浄し、遠心分離する。 </li><li> NCSC培地で細胞を再懸濁する。 </li><li>浮遊培養では、超低アタッチメントプレートに細胞を入れた。添付ファイルのカルチャでは、前処理された組織培養プレートに細胞を入れた。 </li></ol> 6。代表的な結果 NCSC培地はフィーダー細胞を必要とせずに、未分化状態でhNCSCsを維持するのに十分である。ケラチノサイトは増殖し、徐々に培地中に死亡したことはありません。特定の小円形細胞が増殖し、3から5日後に、懸濁液中の小さな凝集体を形成することになる。これらの浮遊凝集体が徐々にiを増加我々は毛球（ 図3A）と呼ばれるn個のサイズ、生成された三次元の球体状の構造、。コー​​ティングされたプレートで培養した場合、NCSCsは、表面に付着し、どんな球を形成しません。添付NCSCsは、懸濁液中よりも速く成長します。球形成または接続された幹細胞はNCSCsマーカーを発現している。全体の毛包が文化である場合、球はバルジ領域（ 図3B）に対応する領域に形成されている。 <img alt="図1" src="/files/ftp_upload/3194/3194fig1.jpg" /> 図1。成長期（）および休止期（B）が毛包を羽をむしられた。 <img alt="図2" src="/files/ftp_upload/3194/3194fig2.jpg" /> 図2。 NCSCsのFACS分析の代表的な画像左のパネル：。細胞は、抗CD271と抗alphe 4インテグリン抗体を用いて、ゲートされています。右のパネル：細胞は、抗CD271と抗HNK1抗体を用いてゲートさ。 <img alt="図3" src="/files/ftp_upload/3194/3194fig3.jpg" /> 図3。毛球の形態浮遊毛球、左パネルの（A）の形態：低消費電力、右のパネル：ハイパワー（B）はバルジ領域、左側のパネルで、in situで毛球の形態学：休止期の膨らみの毛球、右のパネル：成長期毛包のバルジ領域の毛球。

<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. 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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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我々は、開示することは何もない。

記載の細胞単離および培養方法は再現性があり、堅牢です。私たちは、幅広い年齢層を横断して、個人の数十からNCSCsを生成している。それが右の組織の収穫後に組織を処理するのが最善の方法ですが、私たちは、頭皮の組織が安全に、細胞生存率への影響を最小限に抑えて一晩輸送のための氷の上でメディアに格納することができることがわかった。 抗生物質で、頭皮...

<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

毛包は、生涯成長を受け、毛周期は、幹細胞の増殖と休止を伴うよく制御されたプロセスである。髪の膨らみは、成体幹細胞1のためのよく特徴付けニッチです。外毛根鞘のこのセグメントは、上皮幹細胞2、色素幹細胞3と4-7幹細胞のような神経堤幹細胞を含む、さまざまな種類の数が含まれています。毛包は、ヒト幹細胞の種類ごとにアクセス可能で、豊富な情報源を表しています。我々や他のは、人間の胎児と成人の毛包からの神経堤幹細胞（NCSCs）4,5単離した。これらのヒト幹細胞は、ラベル保細胞であり、in vitroにおける非対称細胞分裂を介して自己複製することが可能である。彼らは未熟な神経堤細胞のマーカーではなく、分化マーカーを発現している。当社の発現プロファイリングのための研究では、彼らがマウス皮膚未熟な神経CREと同様の遺伝子発現パターンを共有していることを示したstの細胞。彼らは 、in vitroクローン単一細胞培養での後、筋原性メラニン細胞、および神経細胞系譜を生じさせることができるクローンの多能性を示す。分化した細胞は、系統特異的マーカーを取得するだけでなく、ex vivoでの条件で適切な機能を発揮しないだけ。さらに、これらのNCSCsは間葉系統に向かって分化能を示しています。分化した神経細胞は、マウスの脳に固執し、ニューロンの分化マーカーを保持することができます。それは髪がNCSCsが神経再生を助けることができる派生卵胞、そして、彼らはtransecting脊髄損傷8以下、これらの幹細胞を移植したマウスの運動機能を改善することが示されている。さらに、末梢神経は、幹細胞移植片9と、押しつぶさ坐骨神経に隣接する皮膚由来前駆細胞の移植で修復されました再ミエリン10をもたらした。そのため、毛包/皮膚由来NCSCsはすでにRのための有望な結果が示されている前臨床モデルでegenerative療法。 人工多能性幹（iPS）細胞に再プログラミング体細胞は、再生医療のための巨大な可能性を示している。しかし、癌遺伝子/ウイルス統合および多能性幹細胞の潜在的な発癌性の長期的な効果が十分に対処されていない特に、iPS細胞に関する多くの問題は、まだあります。 iPS細胞は再生医療に使用することができます前に、克服すべき多くのハードルが残っています。成体幹細胞は、安全であることが知られており、それらはそのような骨髄移植のように、長年にわたり臨床的に使用されなければならないということです。多くの患者は、すでに治療の恩恵を受けている。自家成体幹細胞はまだ移植に適した細胞である。したがって、ヒトの皮膚/毛包において容易にアクセス可能で、拡張可能な成体幹細胞は、再生医療のための貴重な情報源です。

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

この記事では、人間の毛包からの神経堤幹細胞の単離および培養のための堅牢なプロトコルを提案する。

人間の毛包から神経堤幹細胞の単離および培養

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

16.8K ビュー.  School of Medicine, University of Pennsylvania. この記事では、人間の毛包からの神経堤幹細胞の単離および培養のための堅牢なプロトコルを提案する。

ビデオ: 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.

マウス神経堤幹細胞の培養のためのニワトリ胚抽出物の製造

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

神経科学

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.

廃棄されたヒト胎児脳組織から神経幹細胞の生成

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

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.

ラット胚神経細胞の分離と文化：クイックプロトコル

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.

胎児マウス神経管から神経堤細胞の単離および培養

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.

マウスを一回、二つの文化：個々のマウスの二つの神経原性のゾーンから成体神経幹細胞の単離および培養

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.

ヒト多能性幹細胞からの神経堤前駆細胞のフィーダーフリー導出

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.

マウス梁下ゾーンから成体神経幹細胞の単離および培養

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.

強膜網膜移植ひと網膜色素上皮細胞のラットの目の洗練されたプロトコルの開発

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

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

マウス萌芽期の神経幹細胞の分離と培養

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

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.

ヒト包皮からの初代シュワン細胞、ケラチノサイト、および線維芽細胞の単離、培養、および特性評価

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