Name: generation of multicellular human primary endometrial organoids
Uploaded: 2019-10-04T15:00:00
Description: Watch this Scientific Journal Video about Generation of Multicellular Human Primary Endometrial Organoids at JoVE.com

इस अध्ययन को एनआईईएचएस/एनआईएच/एनकैट्स UG3 अनुदान (ES029073) और नॉर्थवेस्टर्न फेनबर्ग स्कूल ऑफ मेडिसिन ब्रिज फंड (जेजेके) द्वारा वित्त पोषित किया गया था। हम पैराफिन embedding के लिए निश्चित organoids प्रसंस्करण के लिए उत्तर पश्चिमी पैथोलॉजी कोर सुविधा स्वीकार करना चाहते हैं. हम व्यावहारिक विचार विमर्श और सहयोग के लिए वुडरफ, Burdette, और Urbanek प्रयोगशालाओं सहित पूरे UG3 टीम को स्वीकार करना चाहते हैं.

एक संस्थागत समीक्षा बोर्ड द्वारा अनुमोदित प्रोटोकॉल के अनुसार, नॉर्थवेस्टर्न विश्वविद्यालय प्रेंटिस महिला अस्पताल में सौम्य गर्भाशय की स्थिति के लिए नियमित गर्भाशयोच्छेदन के दौर से गुजर रही प्री?...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+decidualization+of+the+human+endometrium+in+reproductive+health+and+failure.">Cyclic decidualization of the human endometrium in reproductive health and failure.</a> Endocrine Reviews. 35 (6), 851-905 (2014).</li><li>Li, Q., 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=The+antiproliferative+action+of+progesterone+in+uterine+epithelium+is+mediated+by+Hand2.">The antiproliferative action of progesterone in uterine epithelium is mediated by Hand2.</a> Science. 331 (6019), 912-916 (2011).</li><li>Wetendorf, M., DeMayo, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+progesterone+receptor+regulates+implantation,+decidualization,+and+glandular+development+via+a+complex+paracrine+signaling+network.">The progesterone receptor regulates implantation, decidualization, and glandular development via a complex paracrine signaling network.</a> Molecular and Cellular Endocrinology. 357 (1-2), 108-118 (2012).</li><li>Boretto, 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=Development+of+organoids+from+mouse+and+human+endometrium+showing+endometrial+epithelium+physiology+and+long-term+expandability.">Development of organoids from mouse and human endometrium showing endometrial epithelium physiology and long-term expandability.</a> Development. 144 (10), 1775-1786 (2017).</li><li>Turco, M. 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=Long-term,+hormone-responsive+organoid+cultures+of+human+endometrium+in+a+chemically+defined+medium.">Long-term, hormone-responsive organoid cultures of human endometrium in a chemically defined medium.</a> Nature Cell Biology. 19 (5), 568-577 (2017).</li><li>Kurita, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+progesterone+receptors+mediate+the+inhibitory+effects+of+progesterone+on+estrogen-induced+uterine+epithelial+cell+deoxyribonucleic+acid+synthesis.">Stromal progesterone receptors mediate the inhibitory effects of progesterone on estrogen-induced uterine epithelial cell deoxyribonucleic acid synthesis.</a> Endocrinology. 139 (11), 4708-4713 (1998).</li><li>Kim, J. J., Kurita, T., Bulun, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progesterone+action+in+endometrial+cancer,+endometriosis,+uterine+fibroids,+and+breast+cancer.">Progesterone action in endometrial cancer, endometriosis, uterine fibroids, and breast cancer.</a> Endocrine Reviews. 34 (1), 130-162 (2013).</li><li>Bui, H. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+serum+testosterone+during+the+menstrual+cycle+evaluated+by+daily+measurements+with+an+ID-LC-MS/MS+method+and+a+2nd+generation+automated+immunoassay.">Dynamics of serum testosterone during the menstrual cycle evaluated by daily measurements with an ID-LC-MS/MS method and a 2nd generation automated immunoassay.</a> Steroids. 78 (1), 96-101 (2013).</li><li>Wiwatpanit, T., Murphy, A. R., Lu, Z., Urbanek, M., Burdette, J. E., Woodruff, T. 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हमने बहिर्जात पाड़ सामग्री के उपयोग के बिना एंडोमेट्रियम के उपकला और स्ट्रोमल कोशिकाओं के शामिल मानव एंडोमेट्रियल organoids उत्पन्न किया है। हालांकि यह पहले से ही दिखाया गया है कि प्राथमिक एंडोमेट्रियल उ?...

मानव एंडोमेट्रियम गर्भाशय गुहा को लाइनों और प्रत्यारोपण के दौरान भ्रूण के लिए पहला संपर्क के रूप में कार्य करता है। एंडोमेट्रियम में ज्योतिर्मय और ग्रंथियों की उपकला कोशिकाएं, सहायक स्ट्रॉमल फाइब्रोब्लास्ट, एंडोथेलियल कोशिकाएं और प्रतिरक्षा कोशिकाएं शामिल हैं। एक साथ, इन सेल प्रकार एंडोमेट्रियल ऊतक जो सेक्स स्टेरॉयड हार्मोन के लिए सबसे संवेदनशील ऊतकों में से एक है1बनाते हैं। प्रत्येक मासिक धर्म चक्र के दौरान होने वाले परिवर्तन हड़ताली हैं। एंडोमेट्रियम के उचित विकास और remodeling भ्रूण प्रत्यारोपण होने के लिए अनुमति देने के लिए आवश्यक है। एस्ट्रोजन और प्रोजेस्टेरोन के लिए एबरेंट प्रतिक्रिया एक रिफ्रैक्टरी एंडोमेट्रियम में परिणाम कर सकती है जो गर्भावस्था की सफल स्थापना की अनुमति नहीं देती है और एंडोमेट्रियल नियोप्लासिया सहित रोगों में भी परिणाम कर सकती है।
एंडोमेट्रियम में होने वाले हार्मोन प्रतिक्रियाओं और आवश्यक परिवर्तनों का अध्ययन करने के लिए, एंडोमेट्रियल ऊतकों से कोशिकाओं को सर्जरी या एंडोमेट्रियल बायोप्सी के दौरान रोगियों से उत्पादित सेल संस्कृति में प्रचारित किया गया है। एंडोमेट्रियल स्ट्रोमल कोशिकाओं को वरीयता से बढ़ने और आसानी से प्रचारित किया जा सकता है, और प्रोजेस्टेरोन द्वारा प्रेरित भेदभाव की प्रक्रिया को विट्रो में recapitulated किया जा सकता है। इसके परिणामस्वरूप, इस विभेदन प्रक्रिया के दौरान बहुत कुछ सीखा गया है, जिसे decidualization2,3 कहा जाताहै. एंडोमेट्रियम में अन्य प्रमुख सेल प्रकार, luminal और ग्रंथियों उपकला कोशिकाओं, तथापि, के रूप में अच्छी तरह से पारंपरिक monolayers विकसित नहीं करते, polarity खोने, जीर्ण हो जाते हैं, और सीमित proliferative क्षमता होने. नतीजतन, कम उनके जीव विज्ञान और मानव एंडोमेट्रियम में उनकी भूमिका के बारे में जाना जाता है. चूंकि कई नेओप्लासिया उपकला कोशिकाओं से उत्पन्न होते हैं, हाइपरप्लासिया या कैंसर कोशिकाओं में परिवर्तन से जुड़े तंत्र पूरी तरह से परिभाषित किए जाते हैं। इसके अलावा, अध्ययनों से यह साबित हो गया है कि हार्मोन की प्रतिक्रिया में एंडोमेट्रियम4,5के उपकला और स्ट्रोमल कोशिकाओं के बीच अंतरंग पैराक्रिन क्रियाएं शामिल हैं.
हाल ही में, एंडोमेट्रियल उपकला कोशिकाओं की एक तीन आयामी (3 डी) organoid संस्कृति दो स्वतंत्र समूहों6,7, जो एंडोमेट्रियल ऊतक से गठित organoids की पहली रिपोर्ट कर रहे हैं द्वारा स्थापित किया गया था। इन organoids एक प्रोटीन मैट्रिक्स के भीतर एम्बेडेड एंडोमेट्रियल उपकला कोशिकाओं के शामिल थे (सामग्री की तालिका) और एंडोमेट्रियल एंडोमेट्रियम के एक महत्वपूर्ण हार्मोनल उत्तरदायी डिब्बे शामिल नहीं था, stromal फाइब्रोब्लास्ट्स. मैट्रिक्स प्रोटीन बहुत से बहुत भिन्न हो सकते हैं और जरूरी ऊतक में नहीं होते हैं कि संकेतन रास्ते को गति प्रदान कर सकते हैं, यह एंडोमेट्रियम के घटकों के साथ मैट्रिक्स प्रोटीन की जगह आदर्श होगा. वर्तमान अध्ययन में, एक प्रोटोकॉल पाड़ मुक्त मानव एंडोमेट्रियल organoids उपकला और मानव एंडोमेट्रियम के stromal कोशिकाओं उत्पन्न करने के लिए प्रस्तुत किया है. स्ट्रोमल कोशिकाओं की उपस्थिति न केवल उपकला कोशिकाओं के लिए समर्थन प्रदान करती है बल्कि एंडोमेट्रियल हार्मोन प्रतिक्रिया4,8,9 के लिए महत्वपूर्ण होने के लिए स्थापित किए गए आवश्यक पैराक्रिन क्रियाएँ भी प्रदान करतीहै .
नई बहुकोशिकीय एंडोमेट्रियल ऑर्गनॉइड एंडोमेट्रियम की एक मॉडल प्रणाली प्रदान करती है जो उत्पन्न करने के लिए सरल है और इसमें उपकला और स्ट्रोमल दोनों कोशिकाओं को शामिल किया गया है। इन organoids लंबे समय तक हार्मोनल परिवर्तन और हार्मोनल असंतुलन या exogenous अपमान के कारण tumorigenesis जैसे रोग की प्रारंभिक घटनाओं का अध्ययन करने के लिए इस्तेमाल किया जा सकता है. इन organoids की जटिलता अंततः अन्य सेल प्रकार शामिल करने के लिए विस्तार किया जा सकता है, endothelial और संभवतः मायोमेट्रियल कोशिकाओं के साथ प्रतिरक्षा कोशिकाओं सहित वास्तव में मानव ऊतक शरीर क्रिया विज्ञान की नकल करने के लिए.

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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Agarose HS, molecular biology grade</td>
			<td style="width:229px;">Denville Scientific</td>
			<td style="width:112px;">CA3510-6</td>
			<td style="width:320px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose molds</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td>Z764043</td>
			<td>(https://www.microtissues.com/)</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:320px;">Ammonium chloride (NH4Cl)</td>
			<td style="width:229px;">Amresco</td>
			<td style="width:112px;">0621</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">&#946;-Estradiol</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:112px;">E2257</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Collagenase, Type II, powder</td>
			<td style="width:229px;">Thermo Fisher Scientific</td>
			<td style="width:112px;">17101015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Dispase</td>
			<td style="width:229px;">Corning</td>
			<td>354235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">DNase I</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td>D4513</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">EDTA</td>
			<td style="width:229px;">Fisher Scientific</td>
			<td style="width:112px;">BP120-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Eosin Stain</td>
			<td style="width:229px;">VWR</td>
			<td style="width:112px;">95057-848</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Estrogen Receptor (SP1), rabbit monoclonal antibody</td>
			<td style="width:229px;">Thermo Fisher Scientific</td>
			<td>RM-9101-S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Fluoroshield with DAPI, histology mounting medium</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:112px;">F6057</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td style="width:229px;">Corning</td>
			<td style="width:112px;">21-022-CV</td>
			<td>1x without calcium, magnesium, and phenol red</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Hematoxylin Stain Solution</td>
			<td style="width:229px;">Thermo Fisher Scientific</td>
			<td style="width:112px;">3530-32</td>
			<td>Modified Harris formulation, mercury free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Heparin solution</td>
			<td style="width:229px;">STEMCELL Technologies</td>
			<td style="width:112px;">07980</td>
			<td>added to MammoCult media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Hydrocortisone stock solution</td>
			<td style="width:229px;">STEMCELL Technologies</td>
			<td style="width:112px;">07925</td>
			<td>added to MammoCult media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Organoid media - Mammocult</td>
			<td style="width:229px;">STEMCELL Technologies</td>
			<td style="width:112px;">05620</td>
			<td>supplemented with 2 &#181;L/mL heparin and 5 &#181;L/mL hydrocortisone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Paraformaldehyde, 16% solution</td>
			<td style="width:229px;">Electron Microscopy Sciences</td>
			<td style="width:112px;">15710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Penicillin-Streptomycin</td>
			<td style="width:229px;">Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Phosphate buffered saline, pH 7.4</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:112px;">P3813</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Progesterone Receptor, PgR 1294, unconjugated, culture supernatant</td>
			<td style="width:229px;">Agilent Technologies</td>
			<td>M356801-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">protein matrix - Matrigel</td>
			<td style="width:229px;">BD Biosciences</td>
			<td style="width:112px;">356231</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Purified mouse anti-E-cadherin antibody</td>
			<td style="width:229px;">BD Biociences</td>
			<td style="width:112px;">610181</td>
			<td>Clone 36</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Recombinant anti-vimentin antibody [EPR3776]</td>
			<td style="width:229px;">Abcam</td>
			<td style="width:112px;">ab92547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNA lysis and isolation kit</td>
			<td style="width:229px;">Zymo Research</td>
			<td>R2060</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:320px;">Sodium bicarbonate (NaHCO3)</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:112px;">S6014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Testosterone</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td>86500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Trichrome Stain</td>
			<td style="width:229px;">Abcam</td>
			<td style="width:112px;">ab150686</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Wax film - Parafilm</td>
			<td style="width:229px;">VWR</td>
			<td>52858-000</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of multicellular human primary endometrial organoids

मानव एंडोमेट्रियम शरीर में सबसे हार्मोनल उत्तरदायी ऊतकों में से एक है और गर्भावस्था की स्थापना के लिए आवश्यक है। यह ऊतक भी रोगग्रस्त हो सकता है और रुग्णता और यहां तक कि मौत का कारण बन सकता है। मानव एंडोमेट्रियल जीव विज्ञान का अध्ययन करने के लिए मॉडल सिस्टम एकल प्रकार के इन विट्रो संस्कृति प्रणालियों तक सीमित किया गया है. इसके अलावा, उपकला कोशिकाओं, एंडोमेट्रियम के प्रमुख सेल प्रकार ों में से एक, अच्छी तरह से प्रचार या संस्कृति में उनके शारीरिक लक्षण बनाए रखने नहीं है, और इस प्रकार एंडोमेट्रियल जीव विज्ञान की हमारी समझ सीमित रहता है। हम उत्पन्न किया है, पहली बार के लिए, एंडोमेट्रियल organoids कि दोनों उपकला और मानव एंडोमेट्रियम के stromal कोशिकाओं से मिलकर बनता है. इन organoids किसी भी exogenous पाड़ सामग्री की आवश्यकता नहीं है और विशेष रूप से इतना है कि उपकला कोशिकाओं गोलाभ की तरह संरचना को शामिल करने और केंद्र है कि उत्पादन और स्रावित कोलेजन में stromal कोशिकाओं के साथ polarized हो व्यवस्थित. एस्ट्रोजन, प्रोजेस्टेरोन और एण्ड्रोजन रिसेप्टर्स उपकला और स्ट्रॉमल कोशिकाओं और एस्ट्रोजन और टेस्टोस्टेरोन के शारीरिक स्तर के साथ उपचार में व्यक्त कर रहे हैं organoids के संगठन को बढ़ावा देने. इस नए मॉडल प्रणाली के तरीके है कि पहले संभव नहीं थे में सामान्य एंडोमेट्रियल जीव विज्ञान और रोग का अध्ययन करने के लिए इस्तेमाल किया जा सकता है.

प्रोटोकॉल का एक योजनाबद्ध चित्र 1में दर्शाया गया है। पैथोलॉजिस्ट द्वारा जांच किए जाने के बाद गर्भाशय ऊतक सर्जरी से प्राप्त किया गया था। एंडोमेट्रियल अस्तर स्क्रैपिंग द्वारा मायोमेट्रिय?...

Watch this Scientific Journal Video about Generation of Multicellular Human Primary Endometrial Organoids at JoVE.com

Generation of Multicellular Human Primary Endometrial Organoids

मानव प्राथमिक एंडोमेट्रियल organoids कि उपकला और stromal कोशिकाओं से मिलकर बनता है और देशी एंडोमेट्रियल ऊतक की विशेषताओं को बनाए रखने उत्पन्न करने के लिए एक प्रोटोकॉल प्रस्तुत किया है. यह प्रोटोकॉल गर्भाशय ऊतक अधिग्रहण से एंडोमेट्रियल organoids के हिस्टोलॉजिक प्रसंस्करण के तरीकों का वर्णन करता है।

बहुकोशिकीय मानव प्राथमिक एंडोमेट्रियल ऑर्गेनॉइड्स का उत्पादन

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can ...

generation-of-multicellular-human-primary-endometrial-organoids

Research

JoVE Journal

Developmental Biology

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that limits the translation of basic brain research into clinical application. While induced pluripotent stem cells (iPSCs) have replaced the ethically questionable human embryonic stem cells, iPSC-based neuronal differentiation studies remain descriptive at the cellular level but fail to adequately provide the details that could be derived from a complex, 3D human brain tissue. 
This gap is now filled through the application of iPSC-derived, 3D brain organoids, "Brains in a dish," that model many features of complex human brain development. Here, a method for generating iPSC-derived, 3D brain organoids is described. The organoids can help with modeling autosomal recessive primary microcephaly (MCPH), a rare human neurodevelopmental disorder. A widely accepted explanation for the brain malformation in MCPH is a depletion of the neural stem cell pool during the early stages of human brain development, a developmental defect that is difficult to recreate or prove in vitro. 
To study MCPH, we generated iPSCs from patient-derived fibroblasts carrying a mutation in the centrosomal protein CPAP. By analyzing the ventricular zone of microcephaly 3D brain organoids, we showed the premature differentiation of neural progenitors. These 3D brain organoids are a powerful in vitro system that will be instrumental in modeling congenital brain disorders induced by neurotoxic chemicals, neurotrophic viral infections, or inherited genetic mutations.

Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described.

IPSC व्युत्पन्न मानव मस्तिष्क Organoids की पीढ़ी जल्दी neurodevelopmental विकार मॉडल

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that ...

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

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

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. ...

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

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

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

प्रौढ़ त्वचा से प्राथमिक मानव Keratinocytes की पीढ़ी और संवर्धन

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

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for embryo implantation into the uterine lining of the maternal womb. Improper decidualization has been established as a root cause for implantation failure and subsequent early embryo miscarriage. Therefore, understanding the molecular mechanisms underlying decidualization is advantageous to improving the rate of successful births. In vivo based studies of artificial decidualization are often limiting due to ethical dilemmas associated with human research, as well as translational complications within animal models. As a result, in vitro assays through primary cell culture are often utilized to explore the modulation of decidualization via hormones. This study provides a detailed protocol for the isolation of HESC and subsequent artificial decidualization via the supplementation of hormones to the culturing medium. Further, this study provides a well-designed method to knockdown any gene of interest by utilizing lipid-based siRNA transfections. This protocol permits the optimization of culture purity as well as product yield, thereby maximizing the ability to utilize this model as a reliable method to understand the molecular mechanisms underlying decidualization, and the subsequent quantification of secreted agents by decidualized endometrial stromal cells.

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

This study presents a validated and optimized procedure for the isolation and culture of human endometrial stromal cells to conduct in vitro decidualization assay. Further, this study provides a detailed method to efficiently knockdown a specific gene using siRNAs in human endometrial stromal cells.

आइसोलेशन इन विट्रो Decidualization के लिए मानव एंडोमेट्रियल Stromal कोशिकाओं का

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for ...

Generation of Organoids from Mouse Extrahepatic Bile Ducts

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have no effective therapeutic options. Tools to study EHBD are very limited. Our purpose was to develop an organ-specific, versatile, adult stem cell-derived, preclinical cholangiocyte model that can be easily generated from wild type and genetically engineered mice. Thus, we report on the novel technique of developing an EHBD organoid (EHBDO) culture system from adult mouse EHBDs. The model is cost-efficient, able to be readily analyzed, and has multiple downstream applications. Specifically, we describe the methodology of mouse EHBD isolation and single cell dissociation, organoid culture initiation, propagation, and long-term maintenance and storage. This manuscript also describes EHBDO processing for immunohistochemistry, fluorescent microscopy, and mRNA abundance quantitation by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). This protocol has significant advantages in addition to producing EHBD-specific organoids. The use of a conditioned medium from L-WRN cells significantly reduces the cost of this model. The use of mouse EHBDs provides almost unlimited tissue for culture generation, unlike human tissue. Generated mouse EHBDOs contain a pure population of epithelial cells with markers of endodermal progenitor and differentiated biliary cells. Cultured organoids maintain homogenous morphology through multiple passages and can be recovered after a long-term storage period in liquid nitrogen. The model allows for the study of biliary progenitor cell proliferation, can be manipulated pharmacologically, and may be generated from genetically engineered mice. Future studies are needed to optimize culture conditions in order to increase plating efficiency, evaluate functional cell maturity, and direct cell differentiation. Development of co-culture models and a more biologically neutral extracellular matrix are also desirable.

This protocol describes the production of a mouse extrahepatic bile duct 3-dimensional organoid system. These biliary organoids can be maintained in culture to study cholangiocyte biology. Biliary organoids express markers of both progenitor and biliary cells and are composed of polarized epithelial cells.

माउस बहिर्यकृत पित्त नलिकाओं से ऑर्गेनॉइड का उत्पादन

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have ...

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

अंतर ट्राइप्सिनाइजेशन द्वारा मोज़ेक मैमेरी ऑर्गेनॉइड की पीढ़ी

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells

The sense of taste is mediated by taste buds on the tongue, which are composed of rapidly renewing taste receptor cells (TRCs). This continual turnover is powered by local progenitor cells and renders taste function prone to disruption by a multitude of medical treatments, which in turn severely impacts the quality of life. Thus, studying this process in the context of drug treatment is vital to understanding if and how taste progenitor function and TRC production are affected. Given the ethical concerns and limited availability of human taste tissue, mouse models, which have a taste system similar to humans, are commonly used. Compared to in vivo methods, which are time-consuming, expensive, and not amenable to high throughput studies, murine lingual organoids can enable experiments to be run rapidly with many replicates and fewer mice. Here, previously published protocols have been adapted and a standardized method for generating taste organoids from taste progenitor cells isolated from the circumvallate papilla (CVP) of adult mice is presented. Taste progenitor cells in the CVP express LGR5 and can be isolated via EGFP fluorescence-activated cell sorting (FACS) from mice carrying an Lgr5EGFP-IRES-CreERT2 allele. Sorted cells are plated onto a matrix gel-based 3D culture system and cultured for 12 days. Organoids expand for the first 6 days of the culture period via proliferation and then enter a differentiation phase, during which they generate all three taste cell types along with non-taste epithelial cells. Organoids can be harvested upon maturation at day 12 or at any time during the growth process for RNA expression and immunohistochemical analysis. Standardizing culture methods for production of lingual organoids from adult stem cells will improve reproducibility and advance lingual organoids as a powerful drug screening tool in the fight to help patients experiencing taste dysfunction.

The protocol presents a method for culturing and processing lingual organoids derived from taste stem cells isolated from the posterior taste papilla of adult mice.

वयस्क माउस स्वाद स्टेम सेल से प्राप्त भाषाई ऑर्गेनॉइड की पीढ़ी और संस्कृति

The sense of taste is mediated by taste buds on the tongue, which are composed of rapidly renewing taste receptor cells (TRCs). This continual turnover is ...

Generation of a Mouse Artificial Decidualization Model with Ovariectomy for Endometrial Decidualization Research

Endometrial decidualization is a unique differentiation process of the endometrium, closely related to menstruation and pregnancy. Impairment of decidualization leads to various endometrial disorders, such as infertility, recurrent miscarriage, and preterm birth. The development and use of the endometrial decidualization model in reproductive studies have been a highlight for reproductive researchers for a long time. The mouse has been extensively used in studying reproduction and decidualization. There are three well-established mouse models regarding decidualization, namely natural pregnancy decidualization (NPD), artificial decidualization (AD), and in vitro decidualization (IVD). Among them, AD is considered a reliable model for mouse decidualization, which is easy to implement and close to NPD. This paper focuses on a modified method of the generation and application process of the mouse artificial decidualization model with ovariectomy to avoid ovarian effects, which can obtain highly reproducible results with small within group variances. This method provides a good and reliable animal model for the study of endometrial decidualization.

Here, we describe the method of generating an artificial decidualization model using the ovariectomized mouse, a classic endometrial decidualization experiment in the research field of endometrial decidualization.

एंडोमेट्रियल निर्णय अनुसंधान के लिए ओवरेक्टोमी के साथ एक माउस कृत्रिम निर्णायक मॉडल की पीढ़ी

Endometrial decidualization is a unique differentiation process of the endometrium, closely related to menstruation and pregnancy. Impairment of ...

Generation of Retinal Organoids from Healthy and Retinal Disease-Specific Human-Induced Pluripotent Stem Cells

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative therapies. This protocol describes a simpler, robust, and stepwise method of generating retinal organoids in a hybrid culture system consisting of adherent monolayer cultures during the first 4 weeks of retinal differentiation till the emergence of distinct, self-organized eye field primordial clusters (EFPs). Further, the doughnut-shaped, circular, and translucent neuro-retinal islands within each EFP are manually picked and cultured under suspension using non-adherent culture dishes in a retinal differentiation medium for 1-2 weeks to generate multilayered 3D optic cups (OC-1M). These immature retinal organoids contain PAX6+ and ChX10+ proliferating, multipotent retinal precursors. The precursor cells are linearly self-assembled within the organoids and appear as distinct radial striations. At 4 weeks after suspension culture, the retinal progenitors undergo post-mitotic arrest and lineage differentiation to form mature retinal organoids (OC-2M). The photoreceptor lineage committed precursors develop within the outermost layers of retinal organoids. These CRX+ and RCVRN+ photoreceptor cells morphologically mature to display inner segment-like extensions. This method can be adopted for generating retinal organoids using human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). All steps and procedures are clearly explained and demonstrated to ensure replicability and for wider applications in basic science and translational research.

This protocol describes an efficient method of differentiating hiPSCs into eye field clusters and generating neuro-retinal organoids using simplified culture conditions involving both adherent and suspension culture systems. Other ocular cell types, such as the RPE and corneal epithelium, can also be isolated from mature eye fields in retinal cultures.

स्वस्थ और रेटिना रोग-विशिष्ट मानव-प्रेरित प्लुरिपोटेंट स्टेम सेल से रेटिना ऑर्गेनोइड्स का उत्पादन

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative ...

प्राइमेट सेरेब्रल ऑर्गेनोइड का आनुवंशिक संशोधन

Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification

The cerebral cortex is the outermost brain structure and is responsible for the processing of sensory input and motor output; it is seen as the seat of higher-order cognitive abilities in mammals, in particular, primates. Studying gene functions in primate brains is challenging due to technical and ethical reasons, but the establishment of the brain organoid technology has enabled the study of brain development in traditional primate models (e.g., rhesus macaque and common marmoset), as well as in previously experimentally inaccessible primate species (e.g., great apes), in an ethically justifiable and less technically demanding system. Moreover, human brain organoids allow the advanced investigation of neurodevelopmental and neurological disorders.
As brain organoids recapitulate many processes of brain development, they also represent a powerful tool to identify differences in, and to functionally compare, the genetic determinants underlying the brain development of various species in an evolutionary context. A great advantage of using organoids is the possibility to introduce genetic modifications, which permits the testing of gene functions. However, the introduction of such modifications is laborious and expensive. This paper describes a fast and cost-efficient approach to genetically modify cell populations within the ventricle-like structures of primate cerebral organoids, a subtype of brain organoids. This method combines a modified protocol for the reliable generation of cerebral organoids from human-, chimpanzee-, rhesus macaque-, and common marmoset-derived induced pluripotent stem cells (iPSCs) with a microinjection and electroporation approach. This provides an effective tool for the study of neurodevelopmental and evolutionary processes that can also be applied for disease modeling.

लेखक स्पॉटलाइट: जेनेटिक संशोधन के लिए प्राइमेट सेरेब्रल ऑर्गेनोइड के लक्षित माइक्रोइंजेक्शन और इलेक्ट्रोपोरेशन  

The electroporation of primate cerebral organoids provides a precise and efficient approach to introduce transient genetic modification(s) into different progenitor types and neurons in a model system close to primate (patho)physiological neocortex development. This allows the study of neurodevelopmental and evolutionary processes and can also be applied for disease modeling.

आनुवंशिक संशोधन के लिए प्राइमेट सेरेब्रल ऑर्गेनोइड्स का लक्षित माइक्रोइंजेक्शन और इलेक्ट्रोपोरेशन

The cerebral cortex is the outermost brain structure and is responsible for the processing of sensory input and motor output; it is seen as the seat of ...