Name: generation of integration-free human induced pluripotent stem cells using hair-derived keratinocytes
Uploaded: 2015-08-20T13:00:00.000+00:00
Duration: 8 min 36 s
Description: Watch this Scientific Journal Video about Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes at JoVE.com

The authors wish to thank Harene Ranjithakumaran and Stacey Jackson for technical support. This work was supported in part by grants from the National Health and Medical Research Council (R.C.B. Wong, A. P&#233;bay), the University of Melbourne (R.C.B. Wong), Retina Australia (R.C.B. Wong, S.S.C. Hung, A. P&#233;bay) and the Ophthalmic Research Institute of Australia (R.C.B. Wong, S.S.C. Hung, A. P&#233;bay); Australian Research Council Future Fellowship (A. P&#233;bay, FT140100047), Cranbourne Foundation Fellowship (R.C.B. Wong); intramural funding from the National Institutes for Health (R.C.B. Wong, S.S.C. Hung) and operational infrastructure support from the Victorian Government.

個人からの人間の毛髪サンプルの収集は、ホスト機関における人間研究倫理委員会の倫理的な承認を必要とし、機関のガイドラインに準拠して行う必要があります。 撥弦楽器髪からケラチノサイトの1の単離<ol><li>アイスO / Nで（ すなわち、マトリゲル）雪解け細胞外マトリックス（ECM）ソリューションを提供します。 </li><li>予備冷却したピペットチップを使用して、冷却されたDMEM / F12培地12 mlに200μlのECMソリューションを追加します。コー​​ト希ECM溶液1 mlの12ウェルプレートの各ウェルに。 37℃でコーティングしたプレートO / Nをインキュベートします。使用前に予め温めておいたDMEM / F12培地でコーティングされたプレートを洗浄します。 </li><li>髪の根元に近い指を置き、1迅速かつ滑らかな動きで髪を摘採して、個々の頭から一次ヒトの毛髪を収集します。各収集髪はそのまま毛包が含まれていることを確認し、毛アンの成長期を決定するためにチェック解剖顕微鏡（ 図1）を DER。 <ol><li>ケラチノサイトを抽出するために、各個体から5-10成長期毛を収集します。プロセスは、ケラチノサイトの正常な誘導を確保するために、できるだけ早く毛を摘み取ら。 注： 図1は、成長サイクルの異なる段階にある毛の形態を示しています。成長期毛をORSとして知られている髪の長さを、周囲の上皮の目に見える外側の層と、成長段階にあります。一方、休止期の毛は休止期にある毛を表し、可視ORSはありませんが、休止期の膨らみとして知られている毛の根元を覆う細胞の明確なボールを持っています。 </li></ol></li><li>室温で5分間洗浄するために抗生物質ミックスの10ミリリットルを含むペトリ皿に毛髪サンプルを置きます。 </li><li>毛幹の1センチメートル - 滅菌ハサミとピンセットを使用して、無傷の毛包と約0.5とヘア断片を残し、過剰な毛をカット。 </li><li>慎重TRAピンセットを用いてECMコーティングされたプレートの各ウェルに1ヘア断片nsfer。 </li><li>毛髪試料に - （200μL〜100）を1mlピペットを用いて、ノックアウト血清代替（KSR）培地の約3滴を追加します。 注：添付ファイルとそれに続くケラチノサイト成長を可能にするために、プレートの表面に密着して毛髪断片を保管してください。この段階で過度のメディアを追加すると、髪の断片を浮遊させ、成功した髪の添付ファイルの可能性を減少させることができます。 </li><li> 5％CO 2で37℃インキュベーターで毛髪サンプルをインキュベートし、毛包は、O / Nを添付することができます。湿った毛髪サンプルを維持するために - （200μL〜100）は、次の日に、静かにKSR媒体の他の3滴を追加します。 </li><li>毛包が正常に取り付けられていることを確認するために毎日の毛髪試料を観察します。一般的に毛包の付着は、1-3日以内に明らかです。細かい毛が高い持っているように太い毛は、通常、微細な毛に比べて取り付けが容易です添付ファイルのプロセスを妨害するフロートする傾向。 <ol><li>髪をコーティングしたプレートに取り付けられた後、ウェルにKSR培地500μlを添加します。毛髪サンプルがしっかりと取り付けられていない可能性がありますようにメディアを追加するときに細心の注意を払ってください。この点から、2日おきにメディアを変更します。 注：ケラチノサイト増殖物は3-7日（ 図2A-B）後の毛髪断片のORS領域から見えるはずです。ケラチノサイトは、次のセクションで説明するように細胞を継代14日前までに成長することを可能にします。 </li></ol></li></ol> 2.メンテナンスとケラチノサイトの継代<ol><li>プレコートコーティングマトリックスキットを使用して、6ウェルプレート。コー​​ティングのマトリックス溶液6.8μlを、続いて、キットの各ウェルに希釈媒体の680μlを添加します。プレート上にコーティングマトリックス溶液の均一な分布を確実にするためにプレートを振ります。 </li><li>コー​​ティングされたプレートを室温で30分間インキュベートすることができます。削除しますケラチノサイトのメッキコーティング前Matrixソリューション。 </li><li> 37℃で2〜5分間、ウェル当たり0.25％トリプシン500μlので単一細胞懸濁液にケラチノサイトを解離します。血清（ 例えば、ウシ胎児血清（FBS）培地）を含む培地500μlにトリプシンを不活性化し、滅菌15mlチューブに集めます。滅菌ピンセットを用いて毛の断片を削除します。 </li><li>血球計数器を用いて採取した細胞の数を決定します。 </li><li>遠心分離機200×gで3分間の細胞。 PBSを繰り返し、遠心分離で細胞を洗浄します。 PBSを吸引し、新鮮なケラチノサイト培地中で細胞ペレットを再懸濁します。 </li><li>ダウン4×10 4ケラチノサイトケラチノサイト培地の存在下でECMコーティングされたプレートの1ウェルにプレート。 5％CO 2（ 図2C）を用いて37℃のインキュベーター内でケラチノサイトをしてください。文化があるときに、メディア毎日通路の細胞を変更〜80％コンフルエント、WHIchがケラチノサイトの通過数と増殖率に応じて約2-7日かかる場合があります。由来ケラチノサイトは、標準的なスロー凍結凍結保存方法16を使用して十分な凍結ストックを生成するように拡張されるべきです。 注：記載の培養条件は、典型的な敷石状の形態（ 図2C）と角化細胞の均一な集団の拡大をサポートしています。時折、いくつかの分化した大規模かつフラットな形態のケラチノサイト、および複製能力を失っているが、文化の中で観察することができます。以前17に記載されているように、ケラチノサイトの特徴付けは、サイトケラチン14に対する抗体を用いて免疫染色することにより行うことができます。確立されたケラチノサイト細胞株はまた、市販のキットを使用して1~3継代後マイコプラズマについて試験されるべきです。 </li></ol>エピソームベクターを使用したケラチノサイトからhiPSCsの3世代<ol><li> 6ウェルプレートでプレコート0.1％のゼラチン（2ミリリットル/ウェル）。室温で少なくとも20分間コートにゼラチンを許可します。 </li><li>再プログラミング実験のために、遅くとも通路6よりもケラチノサイトを使用しないで解離は、37℃で2〜5分間、ウェル当たり0.25％トリプシン1mlで単一細胞懸濁液にケラチノサイト。血清（ 例えば、FBS培地）を含む培地1mlのトリプシンを不活性化し、15mlチューブに集めます。 </li><li>ケラチノサイト培地中で200×gで再懸濁した細胞で3分間遠心分離した細胞。板ゼラチン化プレートO / Nでウェル当たり1×10 5 -1.5×10 5ケラチノサイト。 </li><li>次の日（0日目）に、細胞が50〜60％コンフルエントであることを確認するためにプレートをチェックしてください。 注：ケラチノサイトが、彼らはトランスフェクション後によく成長しない可能性があり〜50％コンフルエント未満である場合。 </li><li> 0日目に、2μgの全プラスミドDNAに8μlのトランスフェクション試薬の比を用いて、ケラチノサイトのエピソームリプログラミングベクターのトランスフェクションを行います。<ol><li> 4プラスミド（pCXLE-EGFP、pCXLE-hOct3 / 4-shP53F、pCXLE-HSK、pCXLE-HUL）のそれぞれについて、0.5μgのを取り、低血清培地（ 例えば、OPTI-MEM）100μlに希釈します。 注：プラスミドpCXLE-EGFPは、ケラチノサイトにおけるトランスフェクション効率をモニターするために使用され、成功した再プログラミングのために必要とされません。 </li><li>希釈したDNAへのトランスフェクション試薬の8μlを添加します。チューブをフリックすることにより混合します。 DNA反応混合物は室温で15分間邪魔されずに休ませることができます。 </li><li>新鮮な培地とケラチノサイト培地を交換してください。 </li><li>ゆっくりケラチノサイト培養物にDNA反応ミックスを添加し、4時間、5％CO 2、37℃のインキュベーター中でインキュベートします。 注：それは彼らが低生存トランスフェクション後に示すように、ケラチノサイトに長期のトランスフェクションのO / Nを実行することは推奨されません。 </li><li> 4時間後、トランスフェクションした後、新鮮な培地でケラチノサイト培地を交換してください。 </li></ol></li><li> 1日目（1日後、トランスフェクション）、ケラチノサイト培地を変更し、蛍光顕微鏡下でのeGFP陽性細胞の割合を推定することにより、トランスフェクション効率を確認してください。典型的には&gt; 60〜70％のトランスフェクション効率は、このプロトコルを使用して、初代ケラチノサイトで観察されます。 </li><li>ステップ3.5で説明したように2日目に、トランスフェクションを繰り返します。二つの連続した​​トランスフェクションは、典型的には&gt; 90％のトランスフェクション効率を達成します。ケラチノサイトの培養は、通常、第二のトランスフェクションの終了までにコンフルエンスに達しました。 <ol><li>各再プログラミング反応のために、マウス胚性線維芽細胞（MEF）フィーダーの10cmの皿を準備します。以前に18を説明したように、有糸分裂不活性化MEFフィーダーを準備します。 </li><li>少なくとも20分間、0.1％ゼラチンを5 mlのプレコート10cmディッシュ。 FBS培地中で10cmディッシュあたりプレート4×10 6有糸分裂的に不活性化MEF。細胞は、5％CO 2で37℃のインキュベーターでO / Nを解決し、添付することができます。 </li></ol></li><li> 3日目に、HARステップ3.2および3.3に記載されているように、0.25％トリプシンでケラチノサイトを権利が確定します。 KSR培地中のケラチノサイトを再懸濁し、KSR培地で10センチメートルMEFフィーダー皿に6ウェルプレートの1ウェルからケラチノサイトの90％をプレート。 </li><li>以降4日目から、毎日KSR培地を変更します。 <ol><li>あるいは、18日目の後に、一日おきに培地を交換してください。 注： - 21ヒト胚性幹細胞（ヒトES細胞）の似ている定義された境界を有するhiPSCコロニーは14日目から出現する一方、非再プログラムケラチノサイトは、増殖しなくなります。 </li></ol></li><li>以降21日目からの手動切開によりhiPSCコロニーを分離します。密接に一緒にクラスタ化されたhiPSCコロニーを避けるだけ明らかに他のものから分離されたhiPSCコロニーを選択します。 注：hiPSCコロニーは早く取り出すことができますが、定義された境界を有するコロニーは、小さなコロニーサイズ指定された以前の時点では明らかではないかもしれません。再プログラミング効率はデフの間で異なる場合がありますerent患者の角化細胞、およびそのような継代数またはケラチノサイトの増殖速度などの他の要因によって影響されます。 <ol><li> hiPSCコロニーのマニュアル解剖について、解剖顕微鏡下でhiPSCコロニーを切断するために26G針を使用しています。長さが600ミクロン - 約300小片にhiPSCコロニーをカットします。 hiPSCsのクローン株を確立するために、新しいMEFフィーダープレート（2.5×10 4 MEF / cm 2）を1にhiPSCコロニーから作品を転送します。最初に、文化は12ウェルプレートまたは器官培養皿の1つのウェル内の各hiPSCクローン株は、その後、6ウェルプレートフォーマットに展開します。 </li><li> MEFフィーダー上KSR培地中hiPSCsの確立クローン系を維持します。 hiPSCコロニーと80％の密集度、直径〜1.5ミリメートル、製造者の指示に従って、標準的な酵素継代方法（のAccutase、ディスパーゼまたはコラゲナーゼIV）を使用して、通路hiPSCs - 文化たら70に到達します。 </li></ol></li><li>確立されたhiPSCラインを特徴づけますSは多能性と、in vitroおよび / ​​またはin vivo 13,19 で三胚葉の細胞の代表に分化する能力を確認することができます。また、日常的にテストは、それらが病原体を含まないであることを確認するために、市販のキットを用いて、マイコプラズマのためのhiPSCラインを確立しました。日常核型分析またはアレイベースのコピー数変動（CNV）解析によりhiPSCラインのゲノムの安定性を監視します。 </li></ol>

<ol>
	<li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126, 663-676 (2006).</li><li>Takahashi, K., 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=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131, 861-872 (2007).</li><li>Yu, J., 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=Induced+pluripotent+stem+cell+lines+derived+from+human+somatic+cells.">Induced pluripotent stem cell lines derived from human somatic cells.</a> Science. 318, 1917-1920 (2007).</li><li>Okita, K., 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=A+more+efficient+method+to+generate+integration-free+human+iPS+cells.">A more efficient method to generate integration-free human iPS cells.</a> Nat Methods. 8, 409-412 (2011).</li><li>Park, I. 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=Reprogramming+of+human+somatic+cells+to+pluripotency+with+defined+factors.">Reprogramming of human somatic cells to pluripotency with defined factors.</a> Nature. 451, U141-U141 (2008).</li><li>Okita, K., 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=An+efficient+nonviral+method+to+generate+integration-free+human-induced+pluripotent+stem+cells+from+cord+blood+and+peripheral+blood+cells.">An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells.</a> Stem Cells. 31, 458-466 (2013).</li><li>Dowey, S. N., Huang, X., Chou, B. K., Ye, Z., Cheng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+integration-free+human+induced+pluripotent+stem+cells+from+postnatal+blood+mononuclear+cells+by+plasmid+vector+expression.">Generation of integration-free human induced pluripotent stem cells from postnatal blood mononuclear cells by plasmid vector expression.</a> Nat Protoc. 7, 2013-2021 (2012).</li><li>Zhou, T., 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=Generation+of+induced+pluripotent+stem+cells+from+urine.">Generation of induced pluripotent stem cells from urine.</a> J Am Soc Nephrol. 22, 1221-1228 (2011).</li><li>Aasen, T., 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=Efficient+and+rapid+generation+of+induced+pluripotent+stem+cells+from+human+keratinocytes.">Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes.</a> Nat Biotechnol. 26, 1276-1284 (2008).</li><li>Peters, A., Zambidis, E., Ye, K., Jin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+16.+Generation+of+nonviral+integration-free+induced+pluripotent+stem+cells+from+plucked+human+hair+follicles.">Chapter 16. Generation of nonviral integration-free induced pluripotent stem cells from plucked human hair follicles.</a> Human Embryonic and Induced Pluripotent Stem Cells: Lineage-Specific Differentiation Protocols.Springer Protocols Handbooks. , 203-227 (2012).</li><li>Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scratching+the+surface+of+skin+development.">Scratching the surface of skin development.</a> Nature. 445, 834-842 (2007).</li><li>Limat, A., Noser, F. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+cultivation+of+single+keratinocytes+from+the+outer+root+sheath+of+human+scalp+hair+follicles.">Serial cultivation of single keratinocytes from the outer root sheath of human scalp hair follicles.</a> J Invest Dermatol. 87, 485-488 (1986).</li><li>Piao, Y., Hung, S. S., Lim, S. Y., Wong, R. C., Ko, 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=Efficient+generation+of+integration-free+human+induced+pluripotent+stem+cells+from+keratinocytes+by+simple+transfection+of+episomal+vectors.">Efficient generation of integration-free human induced pluripotent stem cells from keratinocytes by simple transfection of episomal vectors.</a> Stem Cells Transl Med. 3, 787-791 (2014).</li><li>Higgins, C. A., 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=Reprogramming+of+human+hair+follicle+dermal+papilla+cells+into+induced+pluripotent+stem+cells.">Reprogramming of human hair follicle dermal papilla cells into induced pluripotent stem cells.</a> J Invest Dermatol. 132, 1725-1727 (2012).</li><li>Muchkaeva, I. A., 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=Generation+of+iPS+Cells+from+Human+Hair+Follice+Dermal+Papilla+Cells.">Generation of iPS Cells from Human Hair Follice Dermal Papilla Cells.</a> Acta naturae. 6, 45-53 (2014).</li><li>Naaldijk, Y., Friedrich-Stockigt, A., Sethe, S., Stolzing, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+different+cooling+rates+for+fibroblast+and+keratinocyte+cryopreservation.">Comparison of different cooling rates for fibroblast and keratinocyte cryopreservation.</a> J Tissue Eng Regen. , (2013).</li><li>Sporl, F., 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=Real-time+monitoring+of+membrane+cholesterol+reveals+new+insights+into+epidermal+differentiation.">Real-time monitoring of membrane cholesterol reveals new insights into epidermal differentiation.</a> J Invest Dermatol. 130, 1268-1278 (2010).</li><li>Conner, D. A., et al., Ausubel, F. N., 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=Mouse+embryo+fibroblast+(MEF)+feeder+cell+preparation.">Mouse embryo fibroblast (MEF) feeder cell preparation.</a> Current protocols in molecular biology. 23, Unit 23 22 (2001).</li><li>Pebay, A., 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=Essential+roles+of+sphingosine-1-phosphate+and+platelet-derived+growth+factor+in+the+maintenance+of+human+embryonic+stem+cells.">Essential roles of sphingosine-1-phosphate and platelet-derived growth factor in the maintenance of human embryonic stem cells.</a> Stem Cells. 23, 1541-1548 (2005).</li><li>Myung, P., Ito, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+bulge+in+hair+regeneration.">Dissecting the bulge in hair regeneration.</a> J Clin Invest. 122, 448-454 (2012).</li><li>Alonso, L., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hair+cycle.">The hair cycle.</a> J Cell Sci. 119, 391-393 (2006).</li><li>Robinton, D. A., Daley, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+promise+of+induced+pluripotent+stem+cells+in+research+and+therapy.">The promise of induced pluripotent stem cells in research and therapy.</a> Nature. 481, 295-305 (2012).</li><li>Soares, F. A., Sheldon, M., Rao, M., Mummery, C., Vallier, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+coordination+of+large-scale+human+induced+pluripotent+stem+cell+initiatives:+Wellcome+Trust+and+ISSCR+workshops+white+paper.">International coordination of large-scale human induced pluripotent stem cell initiatives: Wellcome Trust and ISSCR workshops white paper.</a> Stem cell reports. 3, 931-939 (2014).</li><li>McKernan, R., Watt, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+is+the+point+of+large-scale+collections+of+human+induced+pluripotent+stem+cells.">What is the point of large-scale collections of human induced pluripotent stem cells.</a> Nat Biotechnol. 31, 875-877 (2013).</li><li>Utikal, J., 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=Immortalization+eliminates+a+roadblock+during+cellular+reprogramming+into+iPS+cells.">Immortalization eliminates a roadblock during cellular reprogramming into iPS cells.</a> Nature. 460, U1145-U1112 (2009).</li><li>Xu, 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=Proliferation+rate+of+somatic+cells+affects+reprogramming+efficiency.">Proliferation rate of somatic cells affects reprogramming efficiency.</a> J Biol Chem. 288, 9767-9778 (2013).</li><li>Liu, J., 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=Late+passage+human+fibroblasts+induced+to+pluripotency+are+capable+of+directed+neuronal+differentiation.">Late passage human fibroblasts induced to pluripotency are capable of directed neuronal differentiation.</a> Cell Transplant. 20, 193-203 (2011).</li><li>Huallachain, M., Karczewski, K. J., Weissman, S. M., Urban, A. E., Snyder, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+genetic+variation+in+somatic+human+tissues.">Extensive genetic variation in somatic human tissues.</a> Proc Natl Acad Sci U S A. 109, 18018-18023 (2012).</li><li>Abyzov, A., 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=Somatic+copy+number+mosaicism+in+human+skin+revealed+by+induced+pluripotent+stem+cells.">Somatic copy number mosaicism in human skin revealed by induced pluripotent stem cells.</a> Nature. 492, 438-442 (2012).</li></ol>

The authors have no conflict of interest.

患者特有のhiPSCsの生成は、in vitroでの病気の細胞型において病因を研究するためのユニークなアプローチを提供し、また、疾患の表現型を救出することができる新規分子を同定するための薬物スクリーニングのためのプラットフォームを提供します。 hiPSCsを使用して、この疾患のモデリングアプローチは、QT延長症候群、ハンチントン病、パーキンソン病および筋萎縮性側索硬化症...

ヒト誘導多能性幹細胞（hiPSCs）の発見は、患者特異的幹細胞1-3の生成のための実行可能な方法を提供し、再生医療の分野に革命をもたらしました。 hiPSCsは正常線維芽細胞4,5を含む種々の体細胞タイプ、造血細胞6,7、尿8およびケラチノサイト9,10からの腎上皮細胞から生成されています。これまで、皮膚線維芽細胞および造血細胞は、患者固有のiPS細胞を生成するための最も一般的に使用される細胞の供給源を表します。おそらく、これは、皮膚生検及び採血ルーチン医療処置および多くの国で確立された患者の血液または皮膚サンプルの大規模なバイオバンクであるという事実によるものです。 侵襲的な抽出方法を必要とする血液細胞および皮膚線維芽細胞とは対照的に、ケラチノサイトはhiPSCの生成のために容易にアクセスできる細胞型を表します。 KeratinocytESは、皮膚の外側表皮バリアを形成し、また、爪及び髪11に見出されるケラチン豊富な上皮細胞です。特に、ケラチノサイトは毛包、内毛根鞘（IRS）細胞（12、 図1）と一緒に毛幹をカバーし、外部の細胞膜の外毛根鞘（ORS）に記載されています。ヘアコレクションは医療関係者の支援を必要としない簡単な手順であるように、患者が収集し、大幅hiPSCの世代のための患者サンプルの収集を容易にするであろう実験室に自分の毛髪サンプルを送信するための機会を提供しています。表皮角化細胞はまた、hiPSCの生成9,13のための出発細胞としてのケラチノサイトを使用する利点に加えて、線維芽細胞と比較してより高い初期化の効率と高速再プログラミング速度を持っています。さらに、hiPSCsはまた毛包内の他の細胞集団を使用して生成することができ、毛包14,15の基部に位置毛乳頭細胞を含みます。 毛由来の細胞を用いてiPS細胞の発生以前の報告は、多くの場合、レトロウイルスまたはレンチウイルスベースの再プログラミング法9,14,15を利用します。しかし、これらのウイルスの方法は、再プログラミング中に外国の導入遺伝子の望ましくないゲノム組込みをご紹介します。対照的に、エピソームベクターの使用は、統合フリー性IPSC 4を生成するために実行可能な、非ウイルス性の再プログラミング方法を示しています。我々は以前に効率的にエピソームベクター13を使用してhiPSCsにケラチノサイトを再プログラムするための簡単な、費用対効果および非ウイルス法を開発しました。ここでは、hiPSCsを生成するためのケラチノサイトおよびその後の再プログラミングの撥髪、拡張やメンテナンスからケラチノサイトの導出を含むケラチノサイト由来hiPSCsの生成のための詳細なプロトコルを提供します。

<table><tbody><tr><td>Antibiotic Mix:&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>250 ng/ml Antimycotic amphotericin B</td><td>Sigma</td><td>A2942-20ml</td><td>Antibiotic mix is made up in PBS.&amp;nbsp;</td></tr><tr><td>1X Penicillin/Streptomycin</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>PBS (-)</td><td>Invitrogen</td><td>14190-144</td><td></td></tr><tr><td>Knockout Serum Replacement (KSR) medium:&amp;nbsp;</td><td></td><td></td><td>KSR medium is filtered using Stericup (Millipore, #SCGPU05RE) before use. bFGF is added fresh to the media before use.</td></tr><tr><td>20% knockout serum replacement (KSR)</td><td>Invitrogen</td><td>10828-028</td><td></td></tr><tr><td>DMEM/F12 with glutamax</td><td>Invitrogen</td><td>10565-042</td><td></td></tr><tr><td>1&amp;times; MEM non-essential amino acid</td><td>Invitrogen</td><td>11140-050</td><td></td></tr><tr><td>&amp;nbsp;0.5&amp;times; Penicillin/Streptomycin</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>&amp;nbsp;0.1 mM &amp;beta;-mercaptoethanol</td><td>Invitrogen</td><td>21985</td><td></td></tr><tr><td>&amp;nbsp;bFGF (10 ng/ml, added fresh)</td><td>Millipore</td><td>GF003</td><td></td></tr><tr><td>Keratinocyte medium:&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;EpiLife with 60&amp;nbsp;&amp;micro;M Calcium</td><td>Invitrogen</td><td>M-EPI-500-CA</td><td></td></tr><tr><td>1&amp;times; Human keratinocyte growth supplement (HKGS)</td><td>Invitrogen</td><td>S-001-5</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS) medium:&amp;nbsp;</td><td></td><td></td><td>FBS medium is filtered using Stericup (Millipore, #SCGPU05RE) before use.</td></tr><tr><td>10% fetal bovine serum (FBS)</td><td>Invitrogen</td><td>26140079</td><td></td></tr><tr><td>DMEM&amp;nbsp;</td><td>Invitrogen</td><td>11995-073</td><td></td></tr><tr><td>0.5x Penicillin/Streptomycin</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>2 mM L-glutamine</td><td>Invitrogen</td><td>25030</td><td></td></tr><tr><td>0.25% trypsin-EDTA</td><td>Invitrogen</td><td>25200-056</td><td></td></tr><tr><td>Extracellular Matrix (ECM):</td><td></td><td></td><td></td></tr><tr><td>Matrigel</td><td>Corning&amp;nbsp;</td><td>354234</td><td>Aliquot Matrigel stock and store in -80&amp;deg;C following manufacturer&amp;rsquo;s instructions. Stock concentration of Matrigel varies slightly from batch to batch (~9mg/ml). We recommend to use 200&amp;micro;l matrigel for coating a 12-well plate (~150&amp;micro;g/well).&amp;nbsp;</td></tr><tr><td>Coating Matrix Kit&amp;nbsp;</td><td>Invitrogen</td><td>R-011-K</td><td></td></tr><tr><td>Plasmids:&amp;nbsp;</td><td></td><td></td><td>Note that pCXLE-eGFP is only used for monitoring transfection efficiency and is not required for reprogramming.</td></tr><tr><td>-&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; pCXLE-eGFP</td><td>Addgene</td><td>27082</td><td></td></tr><tr><td>-&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; pCXLE-hOct3/4-shP53F</td><td>Addgene</td><td>27077</td><td></td></tr><tr><td>-&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; pCXLE-hSK</td><td>Addgene</td><td>27078</td><td></td></tr><tr><td>-&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; pCXLE-hUL</td><td>Addgene</td><td>27080</td><td></td></tr><tr><td>Transfection reagent Fugene HD</td><td>Promega</td><td>E231B</td><td></td></tr><tr><td>Gelatin (from porcine skin)</td><td>Sigma</td><td>G1890</td><td>Make up 0.1% gelatin in distilled water. Autoclave before use.&amp;nbsp;</td></tr><tr><td>Reduced Serum medium: OPTI-MEM</td><td>Invitrogen</td><td>31985062</td><td></td></tr><tr><td>Accutase</td><td>Sigma</td><td>A6964-100ml</td><td></td></tr><tr><td>Mouse embryonic fibroblast (MEF) feeder</td><td></td><td></td><td>MEF can be inactivated by mitomycin C treatment or irradiation as described previously 16.</td></tr><tr><td>26G needle</td><td>Terumo</td><td>NN2613R</td><td></td></tr><tr><td>6-well plate (tissue culture treated)</td><td>BD Biosciences</td><td>353046</td><td></td></tr><tr><td>12-well plate (tissue culture treated)</td><td>BD Biosciences</td><td>353043</td><td></td></tr><tr><td>10 cm dish (tissue culture treated)</td><td>BD Biosciences</td><td>353003</td><td></td></tr><tr><td>Dispase</td><td>Invitrogen</td><td>17105-041</td><td>Use at 10 mg/ml</td></tr><tr><td>Collagenase IV</td><td>Invitrogen</td><td>17104-019</td><td>Use at 1 mg/ml</td></tr><tr><td>TRA-160 antibody</td><td>Millipore</td><td>MAB4360</td><td>Use at 5 &amp;micro;g/ml</td></tr><tr><td>OCT4 antibody</td><td>Santa Cruz</td><td>SC-5279</td><td>Use at 5 &amp;micro;g/ml</td></tr><tr><td>NANOG antibody</td><td>R&amp;amp;D Systems</td><td>AF1997</td><td>Use at 10 &amp;micro;g/ml</td></tr><tr><td>MycoAlert Detection kit</td><td>Lonza</td><td>LT07-418</td><td></td></tr></tbody></table>

generation of integration-free human induced pluripotent stem cells using hair-derived keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

成長期（成長期）、退行期（退行期）および休止期（休止期）20,21：髪は成長サイクルの3つの異なる段階を通過します。成長期毛包上皮の複数の層を含んでいます。これらの層は、ORS、IRSと毛幹（ 図1）を含みます。成長期の毛は最終的ORSと毛幹分化の終了のアポトーシスによってマークされて退行期への移行受けます。最後に、アポトーシスが停止し、休止期の毛包が?...

Watch this Scientific Journal Video about Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes at JoVE.com

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

髪の由来ケラチノサイトを使用した統合フリーヒト誘導多能性幹細胞の生成

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

generation-integration-free-human-induced-pluripotent-stem-cells

Research

JoVE Journal

Developmental Biology

11.5K ビュー.  University of Melbourne. This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors. 

ビデオ: Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

非統合ソームプラスミドを用いて凍結軟膜から誘導多能性幹細胞の生成

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

発生生物学

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

エピソームベクトルを用いたヒト末梢血単核細胞からの統合フリー人工多能性幹細胞の生成

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

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.

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 ...

神経科学

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

ラミニン521マトリックスを用いたヒト誘導多能性幹細胞の統合された誘導

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

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

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

世代、大人の肌からプライマリ ヒト角化培養

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

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

臍帯血由来から 3 D 肌 Organoid の生成誘導多能性幹細胞

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

成人ヒト線維芽細胞の導出と拡張神経前駆細胞へのそれらの直接変換

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS cell cultures to feeder-free while still maintaining your use of Knockout SR.

The following protocol provides instruction for adapting human induced Pluripotent Stem (iPS) Cells to feeder-free culture using complete KnockOut Serum Replacement Feeder-Free medium (KSR-FF). Once adapted, instructions for continual maintenance are also provided.

完全ノックアウト血清代替フィーダーを含まない培地を用いてヒトiPS細胞のフィーダーフリー適応、文化と継

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

生物学

Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS and hES cell cultures to feeder-free while still maintaining your use of Knockout SR.

This protocol describes the detailed procedure for cryopreserving human iPS cells in KnockOut SR cryopreservation medium and recovering these cells in complete KnockOut SR Feeder Free (KSR-FF) medium or feeder-based KnockOut SR medium.

完全ノックアウト血清代替のフィーダーを含まない培地を用いてヒトiPS細胞のCryopreservingおよびリカバリ

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

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

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

腹部皮膚からの成人ヒト皮膚線維芽細胞の分離と非積分法を用いた人工多能性幹細胞の生成

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

髪の由来ケラチノサイトを使用した統合フリーヒト誘導多能性幹細胞の生成

要約

さらに動画を探す

この動画の章

非統合ソームプラスミドを用いて凍結軟膜から誘導多能性幹細胞の生成

エピソームベクトルを用いたヒト末梢血単核細胞からの統合フリー人工多能性幹細胞の生成

人間の毛包から神経堤幹細胞の単離および培養

ラミニン521マトリックスを用いたヒト誘導多能性幹細胞の統合された誘導

世代、大人の肌からプライマリヒト角化培養

臍帯血由来から 3 D 肌 Organoid の生成誘導多能性幹細胞

成人ヒト線維芽細胞の導出と拡張神経前駆細胞へのそれらの直接変換

完全ノックアウト血清代替フィーダーを含まない培地を用いてヒトiPS細胞のフィーダーフリー適応、文化と継

完全ノックアウト血清代替のフィーダーを含まない培地を用いてヒトiPS細胞のCryopreservingおよびリカバリ

腹部皮膚からの成人ヒト皮膚線維芽細胞の分離と非積分法を用いた人工多能性幹細胞の生成

髪の由来ケラチノサイトを使用した統合フリーヒト誘導多能性幹細胞の生成

要約

さらに動画を探す

この動画の章

非統合ソームプラスミドを用いて凍結軟膜から誘導多能性幹細胞の生成

エピソームベクトルを用いたヒト末梢血単核細胞からの統合フリー人工多能性幹細胞の生成

人間の毛包から神経堤幹細胞の単離および培養

ラミニン521マトリックスを用いたヒト誘導多能性幹細胞の統合された誘導

世代、大人の肌からプライマリ ヒト角化培養

臍帯血由来から 3 D 肌 Organoid の生成誘導多能性幹細胞

成人ヒト線維芽細胞の導出と拡張神経前駆細胞へのそれらの直接変換

完全ノックアウト血清代替フィーダーを含まない培地を用いてヒトiPS細胞のフィーダーフリー適応、文化と継

完全ノックアウト血清代替のフィーダーを含まない培地を用いてヒトiPS細胞のCryopreservingおよびリカバリ

腹部皮膚からの成人ヒト皮膚線維芽細胞の分離と非積分法を用いた人工多能性幹細胞の生成

世代、大人の肌からプライマリヒト角化培養