The authors thank members of the Renee Reijo Pera laboratory for help during development of this protocol and during preparation of this manuscript. This work is supported by California Institute for Regenerative Medicine (CIRM) shared laboratory (CL-00518).

Kültür Medya 1. Hazırlık <ol><li> Aşağıdaki birleştirerek fare embriyonik akciğer fibroblast (MEF) orta hazırlayın: 445 mi DMEM, 50 ml cenin sığır serumu (FBS) ve 5 ml 100x penisilin / ampisilin stok solüsyonu. En fazla 14 gün boyunca 4 ° C&#39;de filtreyle sterilize orta tutun. </li><li> Hazırlama serum ihtiva eden, aşağıdaki birleştirerek hPSC kültür ortamı: 385 ml DMEM / F12 100 mi nakavt serum değiştirme (KSR), 5 mi 100x esansiyel olmayan amino asit stok çözeltisi, 5 ml 100x penisilin / ampisilin stok çözeltisi, 5 ml 100x stok çözeltisi p-merkaptoetanol, ve 10 ng / ml bFGF. En fazla 10 gün boyunca 4 ° C&#39;de filtreyle sterilize orta tutun. </li><li> Aşağıdaki birleştirerek mTeSR1 serumsuz ortam hazırlayın: 400 mi mTeSR1 bazal ortam, 5x 100 mi ek, ve 5 ml 100x penisilin / ampisilin stok çözeltisi. En fazla 10 gün boyunca 4 ° C&#39;de orta tutun. </li><li> 10x kollajenaz IV stok çözeltisi hazırlayın: 0.5 gr kollajenaz IV gücünü tartmak ve50 mi DMEM / F12 ve filtre ile sterilize çözülür. Hacimde yapın ve ay boyunca -20 ° C&#39;de stok onları. </li><li> % 0.1 jelatin solüsyonu hazırlayın: güç (sığır derisi, tip B&#39;den) 0.5 gr jelatin tartılır ve deiyonize su ile çözülür. Hafta boyunca oda sıcaklığında bir otoklav sterilize edilmiş bir çözeltisinin tutun. </li><li> Aşağıdaki birleştirerek KSR farklılaştırma ortamının hazırlanması: 410 ml DMEM, 75 mi KSR, 5 ml stok çözeltisi merkaptoetanol 100 x esansiyel olmayan amino asit stok çözeltisi, 5 ml 100x penisilin / ampisilin stok çözeltisi, 5 ml 100x. En fazla 10 gün boyunca 4 ° C&#39;de filtreyle sterilize orta tutun. NOT: KSR farklılaşma verimliliğini etkileyebilecek sürü sürü, değişir. Bu farklılaşma için en iyisini bulmak için ksr birkaç toplu test etmek daha iyidir. </li><li> Aşağıdaki birleştirerek, N2 farklılaşma ortamı hazırlayın: 98 ml DMEM, 1 ml 100x N2 takviyesi ve 1 ml 100x penisilin / ampisilin stok solüsyonu. 4 ° C&#39;de filtreyle sterilize orta tutunEn fazla 10 gün ° C. </li><li> 10 mi 50x B27 takviyesi, 5 ml stok çözeltisi Glutamax 100x, 100x ve 5 ml penisilin / ampisilin stok çözeltisi, 480 mi, Neuro temelli vasat: Aşağıdaki birleştirerek B27 farklılaştırma ortamı hazırlayın. En fazla 10 gün boyunca 4 ° C&#39;de filtreyle sterilize orta tutun. </li></ol> MEF besleyici hücreler HESC ve hiPSCs 2. Kültür H9 HESC WiCell Research Institute elde edilmiş ve hiPSCs Yamanaka retrovirüs aracılı iletimi yoluyla Reijo Pera laboratuarda kurulmuş faktörleri Zeiss OCT3 / 4, Sox2, Klf4 ve c-myc 18. <ol><li> En az bir günlük hücre geçmeden önce, 6 oyuklu plakaların 1 oyuğuna 1 mi,% 0.1 jelatin ilave edilerek bir jelatin hazırlık plakası ve en az 30 dakika boyunca 37 ° C&#39;de inkübe edin. </li><li> Inkübasyon plakalardan aspire jelatin ve tohum sonra ışınlama 1,6 x 10 5 hücre yoğunluğunda plakaları üzerine MEF hücreleri inaktive/ Iyi MEF ortamda hücreleri saymak için Hemasitometre kullanarak. NOT: İsteğe bağlı olarak erken 3 gün gibi hücre geçiş önce MEF besleyiciler hazırlamak. </li><li> Geçiş hücreler MEF besleyiciler kaplama bir gün sonra: hPSCs aspire orta, iyi / 1 ml hücreleri 1 mg / ml kollajenaz IV ekleyin ve 1 saat boyunca 37 ° C&#39;de kuluçkalayın. </li><li> Bu kuluçka sırasında, bir kez PBS ile yıkayın, MEF besleyiciler, iyice 1,5 ml / sıcak (37 ° C), serum ihtiva eden kültür ortamı hPSC ekleyin. </li><li> Farklılaşmamış kolonilerinin en plakalarından ayırmak böylece farklı koloniler bağlı olarak kaldığı, 1 saat sonra, kültür plakasının bir kaç kez dokunun. Dikkatlice yüzen koloniler toplamak ve 15 ml tüp içine aktarabilirsiniz. </li><li> Yavaşça sıcak (37 ° C) DMEM / F12 ile bir kez, plakayı yıkayın ve aynı 15 ml&#39;lik bir tüp içine DMEM / F12 aktarın. </li><li> 3 dakika boyunca 179 x g&#39;de santrifüje tüpler. </li><li> Pelet rahatsız etmemek için dikkatli olmak tüpleri, orta aspire. Savaş Eklem hPSC kültür ortamı, küçük kümeler halinde onları kırmak ve onları karıştırın ve birkaç kez aşağı hücreleri pipetle. </li><li> 1 yeni MEF besleyiciler üzerine hücreleri aktarın: 3-1: 6 oranı. </li><li> Her 5-6 gün her gün ve geçit hücreleri orta değiştirin. </li></ol> Farklılaşma için Hücrelerin 3. hazırlanması <ol><li> Hücre işlemınden önce en az bir gün, bir Matrigel plakaları (6 çukurlu bir levha, tipik olarak 3 kuyu) maddesinin hazırlanması. 1:40, soğuk (4 ° C) DMEM / F12 ile Matrigel seyreltin ve 6 oyuklu plakalar, oyuk başına 1 mi Matrigel ekleyin. 4 ° C sıcaklıkta gece boyunca Matrıgel inkübe edin. NOT: Bir Parafilm ile Matrıgel plakaları mühür ve sürece bir sürede hafta boyunca 4 ° C&#39;de onları stok olabilir. </li><li> 4 gün geçit sonra hücrelerin (Adım 2) kullanın. Plakalardan tüm farklı koloniler çıkarın. Kültür plakası orta aspire, 5 dakika boyunca 37 ° C&#39;de hücreler 1 mi Accutase başına ekleyin ve inkübe edilir. </li><li> İnkübasyon sırasında, bir jel hazırlamak6-delikli plakalar 1 ml jelatin ile 1 ilave ederek plakaları atin. Bu tabak ve inkübatör bir gün önce hazırlanan Matrigel tabak yerleştirin. </li><li> Hücreler hale geldiğinde yan-yüzer 5 dakikalık inkübasyondan sonra ya da pipet hücreler yukarı ve aşağı birkaç kez tek bir hücre içine bunları yapmak. </li><li> 3 dakika boyunca 258 x g hızında tek bir 15 ml&#39;lik tüplere hücreleri ve santrifüj toplayın. </li><li> Yeniden süspanse hPSC kültür ortamı içeren serum, uygun miktarda hücreleri ve 2 uM nihai konsantrasyona kadar Thiazovivin ekleyin. </li><li> 1 de, jelatin kaplı plakalar üzerine hücreleri aktarın: 30 dakika boyunca 37 ° C &#39;de 1 oranında ve MEF hücreleri inkübe besleyiciler kaldırın. </li><li> 15 ml konik tüp hücreleri aktarın, hPSC ortamı içeren uygun serumu ekleyin ve hemositometre ile hücreleri saymak. </li><li> 3 dakika boyunca 258 xg&#39;de hücreleri santrifüj. </li><li> Santrifüj sırasında, 2 uM&#39;lik bir konsantrasyona yapmak için uygun bir ortam için mTeSR1 Thiazovivin ekleyin. </li><li> Santrifüj, a sonraorta spirate ve / ml orta 1,8 x 10 5 hücre vardır, böylece Thiazovivin ile uygun mTeSR1 orta ekleyin. </li><li> , Inkübatör Matrigel plakalarını çıkartın Matrigel aspire ve 6 oyuklu plakaların 1 oyuk üzerine karışık hücre 2 ml ilave ediniz. Not: Hücre yoğunluğu 3.6 x 10 4 hücre / yüzey alanının cm2 olmalıdır. </li><li> Geri inkübatör plakaları dönmek ve hücreler 24 saat takmak edelim. </li><li> 24 saat hücreleri kaplama sonra, eski orta aspire ve kuyu başına 2 ml Yeni mTeSR1 orta ekleyin ve farklılaşmasını başlamadan önce hücreleri bir 24 saat boyunca büyümesine izin. </li></ol> Hücre Farklılaşma <ol><li> Matrıgel plakalar üzerine hücreleri replating sonra farklılaşma 48 saat başlatın. </li><li> Levhasından kültür ortamı aspire, hücreler bir kez PBS ile yıkayın ve oyuk başına aşağıdaki farklılaştırma ortamının 2 ml ekleyin: 10 uM SB431542 ve 100 nM LDN-193 ile takviye KSR farklılaştırma ortamının189. Mark farklılaşma D0 olarak bu gün. </li><li> D20 D0 her gün orta değiştirin. NOT: Bir seferde 2 günden fazla kullanım için orta hazırlayabilirsiniz. </li><li> 10 uM SB431542, 100 nM LDN-193.189, 0.25 uM, SAG, 2 uM purmorphamine ve 50 ng / ml ile takviye edilmiş FGF8b KSR farklılaştırma ortamı: D1 ve D2 aşağıdaki ortamı kullanın. </li><li> 10 uM SB431542, 100 nM LDN-193.189, 0.25 uM, SAG, 2 uM purmorphamine, 50 ng / ml arasında FGF8b ve 3 uM CHIR99021 ile takviye edilmiş bir ortam KSR farklılaşması: D3 ve D4 aşağıdaki ortamı kullanın. </li><li> 100 nM LDN-193.189, 0.25 uM, SAG, 2 uM purmorphamine, 50 ng / ml arasında FGF8b ve 3 uM CHIR99021 ile takviye edilmiş% 75 KSR farklılaştırma ortamı artı% 25 N2 farklılaştırma ortamının: D5 ve D6 aşağıdaki ortamı kullanın. </li><li> 100 nM LDN-193189 ve 3 uM CHIR99021 ile takviye edilmiş% 50 KSR farklılaştırma ortamı artı% 50 N2 farklılaştırma ortamının: D7 ve D8 aşağıdaki ortamı kullanın.</li><li> 100 nM LDN-193189 ve 3 mcM CHIR99021 ile desteklenmiş% 25 KSR farklılaşma ortamı artı 75% N2 farklılaşması orta: Aşağıdaki D9 için orta ve D10 kullanın. </li><li> 3 uM CHIR99021, 10 ng / ml, BDNF, 10 ng / ml, GDNF, 1 ng / ml TGF3, 0.2 mM askorbik asit ve 0.1 mM cAMP ile desteklenmiş B27 farklılaştırma ortamının D11 ve D12 için aşağıdaki ortamı kullanın. </li><li> 10 ng / ml, BDNF, 10 ng / ml, GDNF, 1 ng / ml TGF3, 0.2 mM askorbik asit ve 0.1 mM cAMP ile desteklenmiş B27 farklılaştırma ortamının farklılaştırma geri kalanı için aşağıdaki ortamı kullanın. </li><li> Farklılaşma yaklaşık D16, bazı Poli-L-ornitin / laminin / fibronektin hazırlık plakası. Poli-L-ornitin, soğuk (4 ° C) stok çözeltisi, 15 ug / ml PBS, gece boyunca 37 ° C&#39;de plakaları 6 oyuklu plakaların 1 oyuğuna 1 ml ilave edilir, ve inkübe seyreltin. </li><li> , Poli-L-ornitin çözüm aspire Plakalar steril su ile 3 kez yıkanır ve bundan sonra da hava plakaları kurutunuz. </li><li> Laminin stok sol sulandırmak1 ug / ml soğuk (4 ° C) ilave edilmiştir Katkı, 6 oyuklu plakaların bir oyuğuna 1 ml ilave edilir ve bir gece boyunca 37 ° C&#39;de inkübe edin. </li><li> , Laminin çözüm aspire Plakalar steril su ile 3 kez yıkanır ve bundan sonra da hava plakaları kurutunuz. </li><li> , 2 ug / ml soğuk (4 ° C) ilave edilmiştir fibronektin stok çözelti seyreltik 6 oyuklu plakaların 1 oyuğuna 1 ml ilave edilir ve bir gece boyunca 37 ° C&#39;de inkübe edin. </li><li> Parafilm ile sızdırmaz plakalar ve sürece iki hafta gibi kısa boyunca 4 ° C &#39;de koyun. </li><li> Farklılaşma 20 gün sonra, Poli-L-ornitin / laminin / fibronektin plakalara hücreleri Replate. Aspire farklılaştırma ortamı, 6 oyuklu plakaların 1 oyuğuna 1 ml sıcak (37 ° C) Accutase ekleyin ve 5 dakika boyunca 37 ° C&#39;de kuluçkalayın. </li><li> İnkübasyon sırasında, kuluçka makinesi içindeki Poli-L-ornitin / laminin / fibronektin plaka yerleştirin. </li><li> 5 dakikalık inkübasyondan sonra hücreler, ya da hale yukan ve aşağı doğru, birkaç kez pipet yavaşça hücreler yan-yüzertek hücre içine bunları yapmak. </li><li> 15 ml konik tüp içine tek hücreleri toplamak ve genişlemeye bağlı olarak değişecektir hücre sayımı için Neurobasal orta uygun miktarda ekleme, (farklılaşma 11 gün sonra, hücreler 15-20 kat genişletebilir). Hemasitometre kullanarak hücreleri saymak. </li><li> 3 dakika boyunca 258 xg&#39;de hücreleri santrifüj. B27 farklılaşma ortamı ile aspire süpernatan ve tekrar süspansiyon hücreleri hücre / ml konsantrasyonlarda ortamı 1 x 10 6 hücre olacak şekilde. </li><li> Plakalardan aspire fibronektin, PBS ile iki kez yıkanır ve 6 oyuklu plakaların 1 oyuğuna 2 mi hücreleri ekleyin. Not: şarjı için hücre yoğunluğu 2 x 10 5 hücre / yüzey alanı cm2 olmalıdır. </li><li> Birleşmeyen ölü hücreleri çıkarmak için 24 saat sonra orta değiştirin. </li><li> Belirli bir deney için istenilen zaman noktalarında kadar her gün orta değiştirin. </li></ol>

<ol>
	<li>Freed, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Will+embryonic+stem+cells+be+a+useful+source+of+dopamine+neurons+for+transplant+into+patients+with+Parkinson's+disease.">Will embryonic stem cells be a useful source of dopamine neurons for transplant into patients with Parkinson's disease.</a> Proceedings of the National Academy of Sciences of the United States of America. 99, 1755-1757 (2002).</li><li>Perrier, A. L., 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=Derivation+of+midbrain+dopamine+neurons+from+human+embryonic+stem+cells.">Derivation of midbrain dopamine neurons from human embryonic stem cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 101, 12543-12548 (2004).</li><li>Park, C. 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=In+vitro+and+in+vivo+analyses+of+human+embryonic+stem+cell-derived+dopamine+neurons.">In vitro and in vivo analyses of human embryonic stem cell-derived dopamine neurons.</a> Journal of Neurochemistry. 92, 1265-1276 (2005).</li><li>Buytaert-Hoefen, K. A., Alvarez, E., Freed, C. R. <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+tyrosine+hydroxylase+positive+neurons+from+human+embryonic+stem+cells+after+coculture+with+cellular+substrates+and+exposure+to+GDNF.">Generation of tyrosine hydroxylase positive neurons from human embryonic stem cells after coculture with cellular substrates and exposure to GDNF.</a> Stem Cells. 22, 669-674 (2004).</li><li>Zeng, X., 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=Dopaminergic+differentiation+of+human+embryonic+stem+cells.">Dopaminergic differentiation of human embryonic stem cells.</a> Stem Cells. 22, 925-940 (2004).</li><li>Yan, 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=Directed+differentiation+of+dopaminergic+neuronal+subtypes+from+human+embryonic+stem+cells.">Directed differentiation of dopaminergic neuronal subtypes from human embryonic stem cells.</a> Stem Cells. 23, 781-790 (2005).</li><li>Schulz, T. C., 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=Differentiation+of+human+embryonic+stem+cells+to+dopaminergic+neurons+in+serum-free+suspension+culture.">Differentiation of human embryonic stem cells to dopaminergic neurons in serum-free suspension culture.</a> Stem Cells. 22, 1218-1238 (2004).</li><li>Park, 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=Generation+of+dopaminergic+neurons+in+vitro+from+human+embryonic+stem+cells+treated+with+neurotrophic+factors.">Generation of dopaminergic neurons in vitro from human embryonic stem cells treated with neurotrophic factors.</a> Neuroscience Letters. 359, 99-103 (2004).</li><li>Cho, M. 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=Highly+efficient+and+large-scale+generation+of+functional+dopamine+neurons+from+human+embryonic+stem+cells.">Highly efficient and large-scale generation of functional dopamine neurons from human embryonic stem cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 105, 3392-3397 (2008).</li><li>Cooper, O., 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=Differentiation+of+human+ES+and+Parkinson's+disease+iPS+cells+into+ventral+midbrain+dopaminergic+neurons+requires+a+high+activity+form+of+SHH,+FGF8a+and+specific+regionalization+by+retinoic+acid.">Differentiation of human ES and Parkinson's disease iPS cells into ventral midbrain dopaminergic neurons requires a high activity form of SHH, FGF8a and specific regionalization by retinoic acid.</a> Molecular and Cellular Neurosciences. 45, 258-266 (2010).</li><li>Chambers, S. M., 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=Highly+efficient+neural+conversion+of+human+ES+and+iPS+cells+by+dual+inhibition+of+SMAD+signaling.">Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling.</a> Nature Biotechnology. 27, 275-280 (2009).</li><li>Sanchez-Danes, 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=Efficient+generation+of+A9+midbrain+dopaminergic+neurons+by+lentiviral+delivery+of+LMX1A+in+human+embryonic+stem+cells+and+induced+pluripotent+stem+cells.">Efficient generation of A9 midbrain dopaminergic neurons by lentiviral delivery of LMX1A in human embryonic stem cells and induced pluripotent stem cells.</a> Human Gene Therapy. 23, 56-69 (2012).</li><li>Fasano, C. A., Chambers, S. M., Lee, G., Tomishima, M. J., Studer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+derivation+of+functional+floor+plate+tissue+from+human+embryonic+stem+cells.">Efficient derivation of functional floor plate tissue from human embryonic stem cells.</a> Cell Stem Cell. 6, 336-347 (2010).</li><li>Kriks, 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=Dopamine+neurons+derived+from+human+ES+cells+efficiently+engraft+in+animal+models+of+Parkinson's+disease.">Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease.</a> Nature. 480, 547-551 (2011).</li><li>Xi, 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=Specification+of+midbrain+dopamine+neurons+from+primate+pluripotent+stem+cells.">Specification of midbrain dopamine neurons from primate pluripotent stem cells.</a> Stem Cells. 30, 1655-1663 (2012).</li><li>Kirkeby, 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+regionally+specified+neural+progenitors+and+functional+neurons+from+human+embryonic+stem+cells+under+defined+conditions.">Generation of regionally specified neural progenitors and functional neurons from human embryonic stem cells under defined conditions.</a> Cell Reports. 1, 703-714 (2012).</li><li>Mendez, I., 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=Cell+type+analysis+of+functional+fetal+dopamine+cell+suspension+transplants+in+the+striatum+and+substantia+nigra+of+patients+with+Parkinson's+disease.">Cell type analysis of functional fetal dopamine cell suspension transplants in the striatum and substantia nigra of patients with Parkinson's disease.</a> Brain: a Journal of Neurology. 128, 1498-1510 (2005).</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>Nishimura, K., Takahashi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+application+of+stem+cell+technology+toward+the+treatment+of+Parkinson's+disease.">Therapeutic application of stem cell technology toward the treatment of Parkinson's disease.</a> Biological &amp; Pharmaceutical Bulletin. 36, 171-175 (2013).</li><li>Smidt, M. P., Burbach, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+make+a+mesodiencephalic+dopaminergic+neuron.">How to make a mesodiencephalic dopaminergic neuron.</a> Nature Reviews. Neuroscience. 8, 21-32 (2007).</li><li>Wang, G., 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=Noggin+and+bFGF+cooperate+to+maintain+the+pluripotency+of+human+embryonic+stem+cells+in+the+absence+of+feeder+layers.">Noggin and bFGF cooperate to maintain the pluripotency of human embryonic stem cells in the absence of feeder layers.</a> Biochemical and Biophysical Research Communications. 330, 934-942 (2005).</li><li>Chen, J. K., Taipale, J., Young, K. E., Maiti, T., Beachy, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule+modulation+of+Smoothened+activity.">Small molecule modulation of Smoothened activity.</a> Proceedings of the National Academy of Sciences of the United States of America. 99, 14071-14076 (2002).</li></ol>

The authors declare that they have no competing financial interests.

(HESC ve hiPSCs her ikisi de dahil olmak üzere), insan kök hücreleri en oluşturmak için in vitro olarak ayırt edilebilir, hepsi değilse de, daha önceki çalışmalarda 19&#39;da gösterilmiştir DA nöronlarının da dahil olmak üzere vücut, hücre türleri. Burada, diğer laboratuarlar 14,15 embriyonik dopaminerjik nöron gelişiminde 20 den bilgi ve yayınlanmış protokollere dayalı, biz hESC ve hiPSCs DA nöronların nesil için kültür koşullarını optimize. B...

Dopaminerjik (DA) nöronlar midbrain, hipotalamus, retina ve bulbuslarında dahil olmak üzere birçok beyin bölgelerinde bulunabilir. Ön beyinde striatum projelendirme ve ekstrapiramidal motor sistemini oluşturarak substantia nigra pars compacta (SNpc) kontrol davranış ve hareket A9 DA nöronlar. A9 DA nöronların dejenerasyonu ikinci en yaygın insan nörodejeneratif bir hastalıktır ve şu anda çaresiz Parkinson hastalığı (PH) yol açar. Hücre replasman tedavisi PH 1 tedavisi için en umut verici stratejilerinden biridir, bu nedenle, insan embriyonik kök hücreleri (hESC) ve hem de dahil olmak üzere, insan pluripotent kök hücreler (hPSCs) den A9 DA nöronlar kaynaklanan büyük bir ilgi olmuştur Son insan uyarılmış pluripotent kök hücreler (hiPSCs). Pek çok araştırma, çeşitli yöntemler kullanarak hPSCs adlı A9 DA nöronları türetmek için çalıştı. Bir sinir yoluyla erken raporlar tüm oluşturulan DA nöronlarırozet ata sahne. Ile birlikte bir ya da 2-4 hafta, L. ve arkadaşları Studer&#39;den ve başarılı bir şekilde sinir rozet üretmek için HESC uyarılan üç grup için dış büyüme faktörleri olmaksızın MS5 2 veya PA6 3-5 stromal hücreleri ile ortak-kültür. Daha sonra mekanik diseksiyon ya da daha fazla farklılaşması için enzimatik sindirim yoluyla bu rozetler zenginleştirilmiş. Diğer raporlarda, araştırmacılar kültür farklılaşmasını 6-9 yüzen embriyoit vücuda sinir rozetler (EB) oluşturulur. Daha sonra, araştırmacılar, ekstra hücresel matriks üzerinde HESC ve hiPSCs kaplama bir tek tabakalı göre ayırt etme yöntemini 10,11, in vivo oluşturulan embriyonik gelişme DA nöron taklit DA nöronlara hPSCs farklılaşmasını indüklemede kültür içinde değişik büyüme faktörleri ilave . Bütün bu çalışmalar, DA nöronlarının bazı özelliklere sahip tirosin hidroksilaz (TH) -expressing hücreleri elde edilmiş olmakla birlikte, bütün farklılaşma süreci, zaman ve iş gücü tüketengenellikle verimsiz ve daha da önemlisi, bu nöronların A9 kimlik LMX1a ektopik ifade 12 ile biri dışında en çalışmalarda gösterilmiştir değildi. Son zamanlarda, yeni bir taban plakası (FP) tabanlı protokol daha sonra DA nöron potansiyeli ile FP öncüleri ilk farklılaşma erken dönemde sonik kirpi ve doğal Wnt sinyal yollarının aktivasyonu tarafından oluşturulan edildiği 13-16, geliştirilen ve oldu Bu FP hücreler daha fazla DA nöronlarının için belirlenmiştir. Bu protokol daha verimli olmasına rağmen, hala bazı sorunlar var; Örneğin, tüm süreç farklılaşma uzun (en az 35 gün) sürer ve bağımlı besleyici hücre 15, ya da EB veya A9 kimliği 14 görülmemiştir 16 bağlıdır. Burada, in vivo embriyonik DA nöron gelişimi ve diğer araştırmacıların yayınlanan sonuçlarına bilgiye dayalı, biz eff için kültür koşullarını optimizeHESC ve hiPSCs hem DA nöronların icient üretimi. Biz ilk olarak küçük molekül CHIR99021 ve küçük moleküller SAG ve purmorphamine ile sinyalizasyon sonik kirpi ile kanonik Wnt sinyal aktivasyonu ile FP öncül hücreleri üretilir. Bu FP hücreler FOXA2, LMX1a, Corin, OTX2 ve Nestin ifade eder. Biz sonra vb BDNF, GDNF&#39;nin içeren büyüme faktörleri ile DA nöronlarının bu FP hücreleri belirtildi. Bunlar kalbindin 17 için ise negatif GIRK2 pozitif olarak üretilen DA nöronları hücre A9 tiptedir. Bu protokol, yüksek verimli ve yeniden üretilebilir bir besleyici hücre veya EB bağımsız vardır. Bu protokolü kullanarak, bir, ya da in vitro PH modelleme veya PH için potansiyel terapötik ajanların test için PD hastalarının hiPSCs gelen HESC veya hücre nakli çalışma için normal insanların hiPSCs daha az 4 hafta DA nöronları elde edebilirsiniz.

<table border="0" cellpadding="0" cellspacing="0" width="778">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Name of Material/ Equipment</td>
			<td style="width:184px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">DMEM</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">10569-010</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">FBS</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">26140</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">penicillin/ampicillin</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">DMEM/F12</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">10565-018</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">KSR</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">10828-028</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Non-essential amino acid</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">11140-050</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">b-mercaptoethanol</td>
			<td style="width:184px;">Millipore</td>
			<td style="width:119px;">ES-007-E</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">bFGF</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">233-FB-025</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:308px;">mTesR1</td>
			<td style="width:184px;">STEMCELL Technologies</td>
			<td style="width:119px;">5850</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Collagenase IV</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">17104-019</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Gelatin</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">G9391</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">N2 supplement</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">17502-048</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">B27 supplement</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">17504-044</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Neurobasal</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">21103-049</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Glutamax</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">35050-061</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">PBS</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">10010-023</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Growth factor reduced matrigel</td>
			<td style="width:184px;">BD Biosciences</td>
			<td style="width:119px;">354230</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:308px;">Accutase</td>
			<td style="width:184px;">MP Biomedicals</td>
			<td style="width:119px;">1000449</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:308px;">Thiazovivin</td>
			<td style="width:184px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-361380</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">SB431542</td>
			<td style="width:184px;">Tocris Bioscience</td>
			<td style="width:119px;">1614</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">LDN-193189</td>
			<td style="width:184px;">Stemgent</td>
			<td style="width:119px;">04-0074</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">SAG</td>
			<td style="width:184px;">EMD Millipore</td>
			<td style="width:119px;">566660-1MG</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:308px;">Purmorphamine</td>
			<td style="width:184px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-202785</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">FGF8b</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">423-F8-025</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">CHIR99021</td>
			<td style="width:184px;">Cellagen Technology</td>
			<td style="width:119px;">C2447-2s</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">BDNF</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">248-BD-025</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">GDNF</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">212-GD-010</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">TGF-beta3</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">243-B3-002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Ascorbic acid</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">A4034</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">cAMP</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">D0627</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:308px;">Mouse anti human NESTIN antibody</td>
			<td style="width:184px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-23927</td>
			<td style="width:167px;">1/1000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Rabbit anti human OTX2 antibody</td>
			<td style="width:184px;">Millipore</td>
			<td style="width:119px;">AB9566</td>
			<td style="width:167px;">1/2000 dilutiion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Goat anti human FOXA2 antibody</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">AF2400</td>
			<td style="width:167px;">1/200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">rabbit anti human LMX1a antibody</td>
			<td style="width:184px;">Millipore</td>
			<td style="width:119px;">AB10533</td>
			<td style="width:167px;">1/1000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Rabbit anti human TH antibody</td>
			<td style="width:184px;">Pel Freez</td>
			<td style="width:119px;">P40101</td>
			<td style="width:167px;">1/500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Chicken anti human TH antibody</td>
			<td style="width:184px;">Millipore</td>
			<td style="width:119px;">AB9702</td>
			<td style="width:167px;">1/500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Mouse anti human TUJ1 antibody</td>
			<td style="width:184px;">Covance</td>
			<td style="width:119px;">MMS-435P</td>
			<td style="width:167px;">1/2000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Rabbit anti human GIRK2 antibody</td>
			<td style="width:184px;">Abcam</td>
			<td style="width:119px;">ab30738</td>
			<td style="width:167px;">1/300 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Rabbit anti human Calbindin antibody</td>
			<td style="width:184px;">Abcam</td>
			<td style="width:119px;">ab25085</td>
			<td style="width:167px;">1/400 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Centrifuge</td>
			<td style="width:184px;">Eppendorf</td>
			<td style="width:119px;">5804</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

directed dopaminergic neuron differentiation from human pluripotent stem cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

Farklılaşma protokolünün bir genel görünüşü Şekil 1&#39;de gösterilmiştir. Burada yer alan farklılaşma protokolünün verimi başlangıç ​​hücrelerin durumuna dayanır. Bu nedenle, ilk ayrıştırma için tek bir hücre içine hPSCs ayrıştırma önce tüm farklı koloniler kaldırır, ve ikinci olarak, bir bütün halinde, MEF besleyici hücreler, jelatin kaplı plakalar üzerinde hücrelerin inkübe edilmesi ile, çoğu tüketir olduğundan emin olmak için önemlidir 30 dakika ve...

Watch this Scientific Journal Video about Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells at JoVE.com

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

İnsan Pluripotent Kök Hücreleri dopaminerjik Nöron Farklılaşma Yönetmen

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

directed-dopaminergic-neuron-differentiation-from-human-pluripotent

Research

JoVE Journal

Neuroscience

16.4K Görüntüleme.  Stanford University School of Medicine. We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson’s disease.

Video: Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

Küçük Molekül İndüksiyon Pluripotent İnsan Embriyonik Kök Hücreleri İnsan Nöronal atalarıdır ve Nöronlar Verimli türetilmesi

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Nörobilim

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

İnsan Pluripotent Kök Hücreler Telencephalic glutamaterjik Nöron Şartnamesi

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as &#8220;chopping&#8221; that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.&#160; Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

İnsan Nöral Agregalarının bir cGMP uygulanabilir Açılım Yöntemi kök hücrelerin pluripotent kök hücreleri veya Fetal Beyin dokusundan türetilen

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

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 ...

Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model system to explore disease mechanisms and carry out high content screening (HCS) to interrogate compound libraries or identify gene mutation phenotypes. However, with human cells the transition from NPC to functional, mature neuron requires several weeks. Synapses typically start to form after 3 weeks of differentiation in monolayer culture, and several neuron-specific proteins, for example the later expressing pan-neuronal marker NeuN, or the layer 5/6 cerebral cortical neuron marker CTIP2, begin to express around 4-5 weeks post-differentiation. This lengthy differentiation time can be incompatible with optimal culture conditions used for small volume, multi-well HCS platforms. Among the many challenges are the need for well-adhered, uniformly distributed cells with minimal cell clustering, and culture procedures that foster long-term viability and functional synapse maturation. One approach is to differentiate neurons in a large volume format, then replate them at a later time point in HCS-compatible multi-wells. Some main challenges when using this replating approach concern reproducibility and cell viability, due to the stressful disruption of the dendritic and axonal network. Here we demonstrate a detailed and reliable procedure for enzymatically resuspending human induced pluripotent stem cell (hiPSC)-derived neurons after their differentiation for 4-8 weeks in a large-volume format, transferring them to 384-well microtiter plates, and culturing them for a further 1-3 weeks with excellent cell survival. This replating of human neurons not only allows the study of synapse assembly and maturation within two weeks from replating, but also enables studies of neurite regeneration and growth cone characteristics. We provide examples of scalable assays for neuritogenesis and synaptogenesis using a 384-well platform.

This protocol describes a detailed procedure for resuspending and culturing human stem cell derived neurons that were previously differentiated from neural progenitors in vitro for multiple weeks. The procedure facilitates imaging-based assays of neurites, synapses, and late-expressing neuronal markers in a format compatible with light microscopy and high-content screening.

Neuritogenez ve Sinaps Matürasyonu yüksek içerikli Tarama için İnsan Pluripotent Kök Hücre Kaynaklı Nöronların Post-differentiation Rekaplamag

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

Fare Embriyonik Kök Hücre in vitro nöronal farklılaşma

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 &#937; x cm2. BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.

This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

İnsan İndüklenen Pluripotent Kök Hücrelerden Beyin Mikrovasküler Endotel Hücrelerinin Derivasyonu, Genişlemesi, Kriyoprezervasyonu ve Karakterizasyonu

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it contributes to disease development and progression, particularly in the gray matter of the brain, remains largely unknown. The dysfunction of oligodendrocyte lineage cells is hallmarked by deficiencies in myelination and impaired self-renewal of oligodendrocyte precursor cells (OPCs). These two defects are caused at least in part by the disruption of interactions between neuron and oligodendrocytes along the buildup of pathology. OPCs give rise to myelinating oligodendrocytes during CNS development. In the mature brain cortex, OPCs are the major proliferative cells (comprising ~5% of total brain cells) and control new myelin formation in a neural activity-dependent manner. Such neuron-to-oligodendrocyte communications are significantly understudied, especially in the context of neurodegenerative conditions such as AD, due to the lack of appropriate tools. In recent years, our group and others have made significant progress to improve currently available protocols to generate functional neurons and oligodendrocytes individually from human pluripotent stem cells. In this manuscript, we describe our optimized procedures, including the establishment of a co-culture system to model the neuron-oligodendrocyte connections. Our illustrative results suggest an unexpected contribution from OPCs/oligodendrocytes to the brain amyloidosis and synapse integrity and highlight the utility of this methodology for AD research. This reductionist approach is a powerful tool to dissect the specific hetero-cellular interactions out of the inherent complexity inside the brain. The protocols we describe here are expected to facilitate future studies on oligodendroglial defects in the pathogenesis of neurodegeneration.

The neuron-glial interactions in neurodegeneration are not well understood due to inadequate tools and methods. Here, we describe optimized protocols to obtain induced neurons, oligodendrocyte precursor cells, and oligodendrocytes from human pluripotent stem cells and provide examples of the values of these methods in understanding cell-type-specific contributions in Alzheimer&#8217;s disease.

Nöron-Oligodendrosit Etkileşimlerini Modellemek için Pluripotent Kök Hücrelerden İnsan Nöronlarının ve Oligodendrositlerin Üretilmesi

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it ...

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple neurological diseases. Studying human microglia in health and disease represents a challenge due to the extremely limited supply of human cells. Induced pluripotent stem cells (iPSCs) derived from human individuals can be used to circumvent this barrier. Here, it is demonstrated how to differentiate human iPSCs into microglia-like cells (iMGs) for in vitro experimentation. These iMGs exhibit the expected and physiological properties of microglia, including microglia-like morphology, expression of proper markers, and active phagocytosis. Additionally, documentation for isolating and labeling synaptosome substrates derived from human iPSC-derived lower motor neurons (i3LMNs) is provided. A live-cell, longitudinal imaging assay is used to monitor engulfment of human synaptosomes labeled with a pH-sensitive dye, allowing for investigations of iMG's phagocytic capacity. The protocols described herein are broadly applicable to different fields that are investigating human microglia biology and the contribution of microglia to disease.

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

This protocol describes the differentiation process of human induced pluripotent stem cells (iPSCs) into microglia-like cells for in vitro experimentation. We also include a detailed procedure for generating human synaptosomes from iPSC-derived lower motor neurons that can be used as a substrate for in vitro phagocytosis assays using live-cell imaging systems.

İnsan Mikroglia Benzeri Hücreler: İnsan Sinaptozomları Kullanılarak İndüklenmiş Pluripotent Kök Hücrelerden ve In Vitro Canlı Hücre Fagositoz Analizinden Farklılaşma

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple ...

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

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

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

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

İnsan Kök Hücre Nakli ve Validasyonu için Ex vivo Model Olarak Yetişkin İnsan Korteksinin Organotipik Kültürleri

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