Name: generation of integration-free human induced pluripotent stem cells using hair-derived keratinocytes
Uploaded: 2015-08-20T13:00:00
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.

व्यक्तियों से मानव बाल के नमूने का संग्रह मेजबान संस्थानों में मानव अनुसंधान नैतिकता समिति द्वारा नैतिक अनुमोदन की आवश्यकता है और संस्थागत दिशा निर्देशों के अनुपालन में किया जाना चाहिए। ?...

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रोगी-विशिष्ट hiPSCs की पीढ़ी के लिए इन विट्रो में रोगग्रस्त प्रकार की कोशिकाओं में रोगजनन के अध्ययन के लिए एक अनूठा तरीका प्रदान करता है, और यह भी रोग phenotypes के बचाव कर सकते हैं कि उपन्यास के अणुओं की पहचान ?...

मानव प्रेरित स्टेम सेल (hiPSCs) की खोज के रोगी-विशिष्ट स्टेम कोशिकाओं को 1-3 की पीढ़ी के लिए एक व्यावहारिक विधि उपलब्ध कराने, पुनर्योजी चिकित्सा के क्षेत्र में क्रांति ला दी है। hiPSCs सफलतापूर्वक fibroblasts के 4,5, hematopoietic कोशिकाओं 6,7, मूत्र 8 से गुर्दे की उपकला कोशिकाओं और 9,10 केरेटिनकोशिकाओं सहित विभिन्न दैहिक प्रकार की कोशिकाओं से उत्पन्न किया गया है। तिथि करने के लिए, त्वचा fibroblasts और hematopoietic कोशिकाओं रोगी-विशिष्ट IPSCs पैदा करने के लिए सबसे अधिक इस्तेमाल सेल के सूत्रों का प्रतिनिधित्व करते हैं। बेशक, यह त्वचा बायोप्सी और रक्त संग्रह नियमित चिकित्सा प्रक्रियाओं और कई देशों में स्थापित किया गया है रोगी के रक्त या त्वचा के नमूनों की बड़ी biobanks कर रहे हैं कि इस तथ्य के कारण है। रक्त कोशिकाओं और आक्रामक निकासी तरीकों की आवश्यकता होती है जो त्वचा तंतुप्रसू के विपरीत, केरेटिनकोशिकाओं hiPSC पीढ़ी के लिए एक आसानी से सुलभ सेल प्रकार का प्रतिनिधित्व करते हैं। Keratinocytतों त्वचा की बाहरी एपिडर्मल बाधा के रूप में और भी नाखून और बालों 11 में पाए जाते हैं कि केरातिन युक्त उपकला कोशिकाओं रहे हैं। विशेष रूप से, केरेटिनकोशिकाओं बालों के रोम, भीतर जड़ म्यान (आईआरएस) कोशिकाओं (12, चित्रा 1) के साथ मिलकर बाल शाफ्ट कवर किया है कि एक बाहरी सेलुलर परत के बाहरी जड़ म्यान (ओआरएस) पर पाया जा सकता है। बाल संग्रह चिकित्सा कर्मियों की सहायता की आवश्यकता नहीं है कि एक साधारण प्रक्रिया है के रूप में रोगियों को इकट्ठा करने और बहुत hiPSC पीढ़ी के लिए रोगी के नमूने के संग्रह की सुविधा होगी जो प्रयोगशालाओं, के लिए अपने स्वयं के बाल के नमूने भेजने के लिए, यह एक अवसर प्रदान करता है। एपिडर्मल केरेटिनकोशिकाओं भी hiPSC पीढ़ी 9,13 के लिए शुरू की कोशिकाओं के रूप में केरेटिनकोशिकाओं उपयोग कर के लाभ के लिए, उनका कहना है fibroblasts की तुलना में एक उच्च reprogramming दक्षता और तेजी से reprogramming के कैनेटीक्स है। इसके अलावा, hiPSCs भी बाल कूप के भीतर अन्य सेल आबादी का उपयोग कर उत्पन्न किया जा सकता है,बाल कूप 14,15 के आधार पर स्थित त्वचीय papilla कोशिकाओं सहित। बाल-व्युत्पन्न कोशिकाओं का उपयोग IPSC पीढ़ी की पिछली रिपोर्टों अक्सर रेट्रोवायरल या lentiviral आधारित reprogramming के तरीकों को 9,14,15 का उपयोग। हालांकि, इन वायरल तरीकों reprogramming के दौरान विदेशी transgenes की अवांछनीय जीनोमिक एकीकरण परिचय। इसकी तुलना में, episomal वैक्टर का उपयोग एकीकरण मुक्त IPSCs 4 उत्पन्न करने के लिए एक व्यावहारिक, गैर वायरल reprogramming विधि का प्रतिनिधित्व करता है। हम पहले से कुशलतापूर्वक episomal वैक्टर 13 का उपयोग कर hiPSCs में keratinocyte reprogram करने के लिए एक सरल, लागत प्रभावी और गैर-वायरल तरीका विकसित किया है। यहाँ हम साहसपूर्ण बाल, विस्तार और केरेटिनकोशिकाओं के रखरखाव और hiPSCs उत्पन्न करने के लिए बाद में reprogramming से केरेटिनकोशिकाओं की व्युत्पत्ति सहित keratinocyte व्युत्पन्न hiPSCs, की पीढ़ी के लिए एक विस्तृत प्रोटोकॉल प्रदान करते हैं।

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Antibiotic Mix:&#160;</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.&#160;</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:&#160;</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&#215; MEM non-essential amino acid</td>
			<td>Invitrogen</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;0.5&#215; Penicillin/Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;0.1 mM &#946;-mercaptoethanol</td>
			<td>Invitrogen</td>
			<td>21985</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;bFGF (10 ng/ml, added fresh)</td>
			<td>Millipore</td>
			<td>GF003</td>
			<td></td>
		</tr>
		<tr>
			<td>Keratinocyte medium:&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;EpiLife with 60&#160;&#181;M Calcium</td>
			<td>Invitrogen</td>
			<td>M-EPI-500-CA</td>
			<td></td>
		</tr>
		<tr>
			<td>1&#215; Human keratinocyte growth supplement (HKGS)</td>
			<td>Invitrogen</td>
			<td>S-001-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS) medium:&#160;</td>
			<td></td>
			<td></td>
			<td>FBS medium is filtered using Stericup (Millipore, #SCGPU05RE)&#160; before use.</td>
		</tr>
		<tr>
			<td>10% fetal bovine serum (FBS)</td>
			<td>Invitrogen</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM&#160;</td>
			<td>Invitrogen</td>
			<td>11995-073</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5&#215; 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&#160;</td>
			<td>354234</td>
			<td>Aliquot Matrigel stock and store in -80&#176;C following manufacturer&#8217;s instructions. Stock concentration of Matrigel varies slightly from batch to batch (~9mg/ml). We recommend to use 200&#181;l matrigel for coating a 12-well plate (~150&#181;g/well).&#160;</td>
		</tr>
		<tr>
			<td>Coating Matrix Kit&#160;</td>
			<td>Invitrogen</td>
			<td>R-011-K</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmids:&#160;</td>
			<td></td>
			<td></td>
			<td rowspan="5">Note that pCXLE-eGFP is only used for monitoring transfection efficiency and is not required for reprogramming.</td>
		</tr>
		<tr>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; pCXLE-eGFP</td>
			<td>Addgene</td>
			<td>27082</td>
		</tr>
		<tr>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; pCXLE-hOct3/4-shP53F</td>
			<td>Addgene</td>
			<td>27077</td>
		</tr>
		<tr>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; pCXLE-hSK</td>
			<td>Addgene</td>
			<td>27078</td>
		</tr>
		<tr>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; pCXLE-hUL</td>
			<td>Addgene</td>
			<td>27080</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.&#160;</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 10mg/ml</td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Invitrogen</td>
			<td>17104-019</td>
			<td>Use at 1mg/ml</td>
		</tr>
		<tr>
			<td>TRA-160 antibody</td>
			<td>Millipore</td>
			<td>MAB4360</td>
			<td>Use at 5&#181;g/ml</td>
		</tr>
		<tr>
			<td>OCT4 antibody</td>
			<td>Santa Cruz</td>
			<td>SC-5279</td>
			<td>Use at 5&#181;g/ml</td>
		</tr>
		<tr>
			<td>NANOG antibody</td>
			<td>R&#38;D Systems</td>
			<td>AF1997</td>
			<td>Use at 10&#181;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.

ऐनाजेन (विकास चरण), केटाजन (प्रतिगमन चरण) और टेलोजन (बाकी चरण) 20,21: बाल 3 अलग विकास चक्र के चरणों के माध्यम से चला जाता है। ऐनाजेन बाल कूप उपकला की कई परतें हैं; इन परतों ओआरएस, आईआरएस और बाल शाफ्ट (चित्र...

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.

बाल-व्युत्पन्न केरेटिनकोशिकाएं का प्रयोग एकता मुक्त मानव प्रेरित pluripotent स्टेम कोशिकाओं की पीढ़ी

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

Research

JoVE Journal

Developmental Biology

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

गैर समेकित करने Episomal प्लास्मिड का उपयोग करते हुए जमे हुए बफी कोट से प्रेरित pluripotent स्टेम कोशिकाओं की पीढ़ी

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

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

फीडर मुक्त परिस्थितियों में सेंडाइ वायरस का उपयोग मानव परिधीय टी कोशिकाओं से प्रेरित स्टेम कोशिकाओं की पीढ़ी

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

Generation of Induced-pluripotent Stem Cells Using Fibroblast-like Synoviocytes Isolated from Joints of Rheumatoid Arthritis Patients

प्रेरित pluripotent स्टेम तंतुकोशिका-तरह Synoviocytes का उपयोग कोशिकाओं की पीढ़ी रुमेटी गठिया के रोगियों के जोड़ों से पृथक

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated ...

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

मानव मेलेनोमा के ट्यूमर घुसपैठ लिम्फोसाइटों से प्रेरित pluripotent स्टेम कोशिकाओं की पीढ़ी

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

रक्त कोशिकाओं से प्रेरित pluripotent स्टेम सेल पीढ़ी सेंडाइ वायरस और centrifugation का उपयोग

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent ...

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

मानव परिधीय रक्त कोशिकाओं mononuclear से एकता मुक्त प्रेरित pluripotent स्टेम कोशिकाओं की पीढ़ी episomal वैक्टर का उपयोग

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

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

परिभाषित किया है और मानव स्टेम कोशिकाओं से Hepatocyte कोशिकाओं की तरह की स्केलेबल जनरेशन

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

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

लाइमिनिन 521 मैट्रिक्स का उपयोग करने वाले मानव प्रेरित प्लुरिपोटेंट स्टेम सेल का एकीकरण फ्री डेरीवेशन

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

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

मानव प्रेरित Pluripotent स्टेम कोशिकाओं से मानकीकृत और प्रतिलिपि Forebrain-प्रकार सेरेब्रल Organoids की पीढ़ी

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

मानव प्रेरित Pluripotent स्टेम सेल का उपयोग-व्युत्पंन Hepatocyte-दवा की खोज के लिए कोशिकाओं की तरह

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...