Name: feeder-free derivation of melanocytes from human pluripotent stem cells
Uploaded: 2016-03-03T15:00:00
Description: Watch this Scientific Journal Video about Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells at JoVE.com

Bu çalışma Joanna M. Nicolay Vakfı melanom araştırmacılar için bir burs ile ve Ruth L. Kirschstein Ulusal Araştırma Hizmet Ödülü F31 altında Ulusal Sağlık Enstitüleri tarafından desteklenmiştir. Bu çalışma ayrıca NYSTEM ve Tri-kurumsal kök hücre girişimi (Starr Vakfı) hibe yoluyla destek verdi.

NOT: Burada özetlenen melanosit protokolü ilk Mika ve arkadaşları tarafından gösterilmiştir. 1. Kültür Orta hazırlanması, Kaplamalı Züccaciye ve hPSCs Bakım <ol><li> orta Hazırlık Not: saklayın fazla 2 hafta boyunca karanlıkta 4 ° C&#39;de tüm orta. Sterilizasyon için tüm orta Filtresi. <ol><li> DMEM /% 10 FBS hazırlayın. 885 ml DMEM, 100 ml FBS, 10 mi Pen / Strep ve 5 ml L-glutamin karıştırın. Sterilizasyon Filtre. </li><li> hESC orta hazırlayın. 800 m...

<ol>
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hPSCs gelen melanosit başarılı farklılaşması için aşağıdaki önerileri dikkate alınmalıdır. İlk ve en önemlisi, her zaman steril kültür koşulları altında çalışmak için gereklidir. Buna ek olarak, pluripotent tam farklılaşmamış hPSCs ile başlamak için önemlidir; Başlangıç ​​nüfus farklılaşmış hücreleri içeriyorsa verimi kaçınılmaz kirletici melanosit yönelik edilemez ve hatta daha düzgün ayırt hücreleri bozabilir olarak düşecek. Hücreler...

Insan pluripotent kök hücreler (hPSCs) hastalığı modelleme, ilaç tarama ve hücre replasman tedavileri 1-6 için ölçeklenebilir bir şekilde normal farklılaşma taklit etmek bir platform sağlar. Özellikle ilgi çekici, hPSCs izole etmek zor veya hasta numunelerinin kıt nadir / geçici hücre tipleri çalışmak için yollar açmak. Ayrıca, uyarılmış pluripotent kök hücreler (iPSCs) benzersiz mekanizmaları 1,2,7-11 çözülmeye hasta belirli bir şekilde gelişmesini ve hastalık modelleme incelemek için araştırmacılar sağlar. HPSCs gelen melanosit farklılaşması için önce yayınlanan bir protokolün farklılaşmalar 6 haftaya kadar gerekmektedir ve L-Wnt3a hücreleri 12 kıvamlandırılmış ortamı ile kültür hücreleri içerir. Protokol ilk Mika ve arkadaşları tarafından sunulan. Ve tarif burada üç hafta içinde pigmentli hücreler üretir ve belirsizlik ve koşullandırılmış ortam ile ilişkili tutarsızlıkları ortadan kaldırır. melanosit arnöral, omurgalılar özgü hücrelerin göç nüfusu türetilen e. nöral krest gastrulasyon sırasında tanımlanan ve nöral ve nöral olmayan ektoderm arasında sınırındaki nöral plaka kenarında hücre popülasyonu temsil etmektedir. Nörülasyon sırasında, sinir dokusu nöral tüp 13,14 sonuçlanan dorsal orta hat yakınsama sinir kıvrımlar oluşturmak için bir nöral plaka gelişir. nöral krest hücreleri Notokordun karşısında, nöral tüpün çatı plakası ortaya ve farklılaşmış hücrelerin çeşitli bir nüfusa yol vermek için uzağa göç önce mezenkimal geçiş bir epitel uğrar. tepe hücreleri kaderi embriyo gövde ekseni boyunca tavan plakasının anatomik bölgeye kısmen tanımlanır. Nöral tepe hücresi türevleri, hem mezoderm karakteristik soy (düz kas hücreleri, osteoblastlar, adipositler, kondrositler) ve ektoderm hücrelerini içerir (melanosit Schwann Cıarşın, nöronlar) 14. Sinir tepe kök hücreler transkripsiyon faktörü SOX10 upregülasyon yapabileceğini ve p75 ve HNK1 antikorlar ile sıralama floresans ile aktive edilmiş hücre ile izole edilebilir. Melanosit bir melanoblast aşamadan geçmesi ve 6,21 MITF melanosit gelişim ana düzenleyicisidir ve bir transkripsiyon faktörü melanosit gelişiminin çok kontrol sorumludur KIT ve MITF (mikroftalmi ilişkili transkripsiyon faktörü) upregüle olmaya mahkum nöral krest hücreleri 22- 24. İnsan Melanoblastlardan onlar saç şişkinliği veya Epidermisin keratinositlerin çevrili olgun, pigmentli melanosit için öncüleri olarak hizmet vermeye (pigmentasyon birimleri oluşturan) ya ikamet epidermisin bazal tabakasındaki göç. pigmentli melanosit içine Melanoblastlardan farklılaşması ve olgunlaşması saç ampul kolonizasyon ve ifade melanin üretimi yolunun (TYRP1, TYR, OKA2&#39;den ve ile birlikte oluşurPMEL) 25,26. Hastaların insan melanosit ve Melanoblastlardan Ayırma, pahalı zor ve miktar sınırlayıcı olduğunu. Bu protokol hücre sıralama olmadan iyi tanımlanmış, hızlı, tekrarlanabilir, ölçeklenebilir ve ucuz bir yöntem melanosit veya melanosit öncüleri içine hPSCs (isteğe bağlı veya embriyonik) ayırt etmek araştırmacılar sağlar. protokol pigmentasyon bozukluğu olan hastaların iPSCs ayırt zaman hastalığa özgü kusurları tespit etmek, daha önce kullanıldı.

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	<tbody>
		<tr>
			<td>Accutase</td>
			<td>Innovative Cell Technologies</td>
			<td>AT104</td>
			<td></td>
		</tr>
		<tr>
			<td>apo human transferrin</td>
			<td>Sigma</td>
			<td>T1147</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic Acid (L-AA)</td>
			<td>Sigma</td>
			<td>A4034</td>
			<td>100 mM</td>
		</tr>
		<tr>
			<td>B27 (B27 Supplement)</td>
			<td>Invitrogen</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Gibco-Life Technologies</td>
			<td>21985-023</td>
			<td>10 mg/ml</td>
		</tr>
		<tr>
			<td>BMP4</td>
			<td>R&#38;D Systems</td>
			<td>314-bp</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Tocris-R&#38;D Systems</td>
			<td>4423</td>
			<td>6 mM</td>
		</tr>
		<tr>
			<td>Cholera toxin</td>
			<td>Sigma</td>
			<td>C8052</td>
			<td>50 mg/ml</td>
		</tr>
		<tr>
			<td>cAMP (cyclicAMP)</td>
			<td>Sigma</td>
			<td>D0627</td>
			<td>100 mM</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma</td>
			<td>D2915-100MG</td>
			<td>50 &#956;M</td>
		</tr>
		<tr>
			<td>DMEM - Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Gibco-Life Technologies</td>
			<td>11985-092</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 - Dulbecco&#39;s Modified Eagle Medium: Nutrient Mixture F-12</td>
			<td>Gibco-Life Technologies</td>
			<td>1133--032</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 powder</td>
			<td>Invitrogen</td>
			<td>12500-096</td>
			<td></td>
		</tr>
		<tr>
			<td>EDN3 (Endothelin-3, human)</td>
			<td>American Peptide Company</td>
			<td>88-5-10B</td>
			<td>100 &#956;M</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>BD Biosciences</td>
			<td>356008</td>
			<td>200 &#956;g/ml</td>
		</tr>
		<tr>
			<td>gelatin (PBS without Mg/Ca)</td>
			<td>in house</td>
			<td></td>
			<td>0.1% in PBS</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Human insulin</td>
			<td>Sigma</td>
			<td>I2643</td>
			<td></td>
		</tr>
		<tr>
			<td>ITS+ Universal Culture Supplement Premix&#160;</td>
			<td>BD Biosciences</td>
			<td>354352</td>
			<td></td>
		</tr>
		<tr>
			<td>KSR (Knockout Serum Replacement)</td>
			<td>Gibco-Life Technologies</td>
			<td>10828-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout DMEM</td>
			<td>Gibco-Life Technologies</td>
			<td>10829-018</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco-Life Technologies</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>LDN193189</td>
			<td>Stemgent</td>
			<td>04-0074</td>
			<td>100 mM</td>
		</tr>
		<tr>
			<td>Low glucose DMEM</td>
			<td>Invitrogen</td>
			<td>11885-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel matrix</td>
			<td>BD Biosciences</td>
			<td>354234</td>
			<td>Dissolve 1:20 in DMEM/F12</td>
		</tr>
		<tr>
			<td>MCDB201 Medium</td>
			<td>Sigma</td>
			<td>M6770</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM minimum essential amino acids solution</td>
			<td>Gibco-Life Technologies</td>
			<td>11140-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse embryonic fibroblasts (7 million cells/vial)</td>
			<td>GlobalStem</td>
			<td>GSC-8105M</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Laminin-I</td>
			<td>R&#38;D Systems</td>
			<td>3400-010-01</td>
			<td>1 mg/ml</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Invitrogen</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin/Streptomycin</td>
			<td>Gibco-Life Technologies</td>
			<td>15140-122</td>
			<td>10,000 U/ml</td>
		</tr>
		<tr>
			<td>Poly-L Omithin hydrobromide</td>
			<td>Sigma</td>
			<td>P3655</td>
			<td>15 mg/ml&#160;</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P8783</td>
			<td>0.032g in 100ml 100% ethanol</td>
		</tr>
		<tr>
			<td>Putrescine dihydrochloride</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF2 (Recombinant human FGF basic)</td>
			<td>R&#38;D Systems</td>
			<td>233-FB-001MG/CF</td>
			<td>10 mg/ml</td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Tocris-R&#38;D Systems</td>
			<td>1814</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Selenite</td>
			<td>Sigma</td>
			<td>S5261</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>SCF (Stem Cell Factor, recombinant Human)&#160;</td>
			<td>Peprotech Inc.</td>
			<td>300-07</td>
			<td>50 &#956;g/ml</td>
		</tr>
		<tr>
			<td>TYRP1 (G-17) Antibody</td>
			<td>Santa Cruz</td>
			<td>10443</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>TYRP2 Antibody</td>
			<td>Abcam</td>
			<td>74073</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%)</td>
			<td>Gibco-Life Technologies</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632 dihydrochloride</td>
			<td>Tocris-R&#38;D Systems</td>
			<td>1254</td>
			<td>10 mM</td>
		</tr>
	</tbody>
</table>

feeder-free derivation of melanocytes from human pluripotent stem cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

Bu protokol, bir in vitro besleyici içermeyen, düşük maliyetli, ve tekrarlanabilir bir şekilde hPSCs tamamen pigmentli olgun melanosit türetilmesi için bir yöntem temin etmektedir. HPSC türetilmiş melanosit için daha önce oluşturulan Fang ve diğerleri. Protokolünün aksine, ana hatları çizilen protokol, koşullu ortam gerektirir ve zaman gereksinimi azaltır etmez. Fang ve diğerleri. Protokol WNT3A üreten fare hücre hattı kıvamlan...

Watch this Scientific Journal Video about Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells at JoVE.com

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Insan pluripotent kök hücreleri gelen melanosit besleyici içermeyen türetilmesi

Bu çalışma, bir nöral ile insan pluripotent kök hücreleri pigmentli olgun melanosit üretilmesi ve besleyici içermeyen, 25 günlük protokolünü kullanarak ara madde aşama melanoblast için bir in vitro farklılaşma protokolü tarif eder.

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Research

JoVE Journal

Developmental Biology

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Embriyonik Kök Hücreleri Kardiyak Progenitör Hücrelerinin türetilmesi

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

Kök Hücreleri Retina pigment epiteli hücrelerinin Verimli türetilmesi

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

Küçük Molekül-modüle Farklılaşma ve sonraki Glikoz şiddetli açlık kullanma İnsan uyarılmış pluripotent kök hücrelerin yüksek oranda saf kardiyomiyositlerde türetilmesi

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes ...

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

İnsan uyarılmış pluripotent kök hücrelerin türetilmiş Piramidal Nöronlar gelen Dendritik Spine Üç boyutlu Niceleme

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Besleyici içermeyen şartlar Sendai virüs kullanılarak insan periferik T hücrelerinden uyarılmış pluripotent kök hücrelerin üretilmesi

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Tanımlı ve hepatosit benzeri Hücrelerinin Ölçeklenebilir Nesil insan pluripotent kök hücreleri gelen

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

Periferal Kan Türevi İnsan Kaynaklı Pluripotent Kök Hücrelerden Kondrositlerin Farklılaşması

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

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

Laminin 521 Matrisini Kullanarak İnsan Tarafından Oluşturulan Pluripotent Kök Hücrelerin Entegrasyon Serbest Türevi

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

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

Insan pluripotent kök hücrelerinin karaciğer hücrelerine verimli farklılaşma

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

Somite türevleri indüklenen insan nesil Vitro Pluripotent kök hücreler

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...