Name: robust differentiation of human ipscs into a pure population of adipocytes to study adipocyte-associated disorders
Uploaded: 2022-02-09T14:45:00
Description: Watch this Scientific Journal Video about Robust Differentiation of Human iPSCs into a Pure Population of Adipocytes to Study Adipocyte-Associated Disorders at JoVE.com

Bu çalışma Katar Ulusal Araştırma Fonu'ndan (QNRF) bir hibe ile finanse edilmiştir (Hibe No. NPRP10-1221-160041). Maryam Aghadi Katar Ulusal Araştırma Fonu'ndan (QNRF) GSRA bursu ile desteklendi.

Çalışma, ilgili kurumsal araştırma etik komitesi tarafından onaylanmış ve 1964 Helsinki Deklarasyonu'nda ve daha sonraki değişikliklerinde veya karşılaştırılabilir etik standartlarda belirtilen etik standartlara uygun olarak gerçekleştirilmiştir. Protokol, HMC (no. 16260/16) ve QBRI (no. 2016-003) Kurumsal İnceleme Kurulu (IRB) tarafından onaylanmıştır. Bu çalışma aynı zamanda H1 ve H9 gibi hESC'ler için de optimize edilmiştir. Sağlıklı bireylerden tam bilgilendirilmiş onam alınarak kan ...

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Bu protokol, MSC'leri yüksek verim ve verimlilikte sağlama kabiliyeti nedeniyle büyük önem taşımaktadır. MSC'lerin bu kitlesel ölçekli üretimi, iPSC'lerden türetilmiş EB'lerin 10 μM RA14,15 ile geçici inkübasyonu ile mümkün olmuştur. 10 μM RA ile geçici işlem, MSC verimini 11,2 ila 1542 kat artırdı14,15, bu protokol hem iPSC'lerde hem de hPSC'lerde uygulanabilir. Bu doz ve tedavi s...

Mezenkimal kök hücreler (MSC'ler), adipositler, osteositler ve kondrositler gibi mezodermal kökenli hücrelerin üretilmesi için etkili bir geçici kaynak görevi görür ve bu hücreler kendi genetik bozukluklarını modellemek için daha fazla kullanılabilir. Bununla birlikte, önceki yaklaşımlar, bu MSC'lerin yetişkin dokulardan elde edilmesine dayanıyordu 1, bu da onları donörlerden yüksek sayılarda elde etme zorluğunu ve onları suboptimal in vitro kültür koşullarında işlevsel olarak canlı tutmanın sınırlandırılmasını getirdi 1,2. Bu engeller, in vitro MSC'ler üretmek için bir protokole sahip olma konusunda büyük bir talep yaratmıştır. İnsan kaynaklı pluripotent kök hücreler (iPSC'ler), MSC özelliklerini sergileyen değerli bir MSC kaynağı olarak kullanılabilir 3,4,5. iPSC'lerden türetilen MSC'ler çeşitli hastalıklarda terapötik bir seçenek olarak kullanılabilir. Ayrıca, iPSC'lerden türetilen MSC'lerin adiposit üretme yeteneği, onları insan adipogenezi, obezite ve adiposit ile ilişkili bozuklukları incelemek için değerli bir in vitro insan modeli haline getirir.
Adipositlerin mevcut farklılaşma protokolleri iki gruba ayrılabilir, bunlardan biri kimyasal veya protein bazlı kokteyller kullanılarak adipositlerin farklılaştırılmasını içerir ve sonuçta %30-%60 oranında 6,7,8,9 verim sağlarken, diğeri %80-%90 verim vermek üzere adipositlerin gelişimini yöneten anahtar transkripsiyon faktörlerinin sağlam indüksiyonu için genetik manipülasyonu içerir. 11. Bununla birlikte, genetik manipülasyon, adiposit farklılaşmasının doğal sürecini özetlemez ve genellikle adipogenez sırasında gelen ince paradigmaları maskeler, bu da hastalık modelleme amaçları için etkisiz hale getirir12,13. Bu nedenle, Nil kırmızısı kullanarak lipit taşıyan adipositleri floresan olarak etiketleyerek kimyasal olarak türetilmiş olgun adipositleri olgunlaşmamış olanlardan ayırmanın bir yolunu sunuyoruz.
Burada, iPSC'lerden türetilmiş embriyoid cisimlerin (EB'ler) all-trans retinoik asit ile geçici inkübasyonunu içeren bir protokol sunuyoruz ve bu da adipositlerin14 üretilmesinde daha fazla kullanılabilecek çok sayıda hızla çoğalan MSC'ler üretiyor. Ayrıca, kimyasal olarak türetilmiş olgun adipositleri, lipofilik bir boya kullanarak lipit damlacıklarını floresan olarak etiketleyerek heterojen farklılaşma havuzundan ayırmanın bir yolunu sunuyoruz; Nil kırmızısı. Bu, adipositle ilişkili metabolik bozuklukları doğru bir şekilde modellemek için gelişmiş işlevselliğe sahip saf bir olgun adiposit popülasyonunun oluşturulmasına izin verecektir.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adiponectin</td>
			<td>Abcam</td>
			<td>ab22554</td>
			<td>Adipocyte maturation marker</td>
		</tr>
		<tr>
			<td>anti-CD105</td>
			<td>BD Pharmingen</td>
			<td>560839</td>
			<td>MSC differentiation marker</td>
		</tr>
		<tr>
			<td>anti-CD14</td>
			<td>BD Pharmingen</td>
			<td>561712</td>
			<td>MSC differentiation marker</td>
		</tr>
		<tr>
			<td>anti-CD19</td>
			<td>BD Pharmingen</td>
			<td>555415</td>
			<td>MSC differentiation marker</td>
		</tr>
		<tr>
			<td>anti-CD34</td>
			<td>BD Pharmingen</td>
			<td>555824</td>
			<td>MSC differentiation marker</td>
		</tr>
		<tr>
			<td>anti-CD44</td>
			<td>abcam</td>
			<td>ab93758</td>
			<td>MSC differentiation marker</td>
		</tr>
		<tr>
			<td>anti-CD45</td>
			<td>BD Pharmingen</td>
			<td> 
			560975</td>
			<td>MSC differentiation marker</td>
		</tr>
		<tr>
			<td>anti-CD73</td>
			<td>BD Pharmingen</td>
			<td>550256</td>
			<td>MSC differentiation marker</td>
		</tr>
		<tr>
			<td>anti-CD90</td>
			<td>BD Pharmingen</td>
			<td>555596</td>
			<td>MSC differentiation marker</td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>R&#38;D</td>
			<td>233-FP</td>
			<td>MSC culture media supplement</td>
		</tr>
		<tr>
			<td>C/EBPA</td>
			<td>Abcam</td>
			<td>ab40761</td>
			<td>Adipocyte maturation marker</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Torics</td>
			<td>1126</td>
			<td>Adipocyte differentiation media supplement</td>
		</tr>
		<tr>
			<td>FABP4</td>
			<td>Abcam</td>
			<td>ab93945</td>
			<td>Adipocyte maturation marker</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>ThermoFisher</td>
			<td>10082147</td>
			<td>MSC culture media supplement</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>ThermoFisher</td>
			<td>35050-061</td>
			<td>MSC culture media supplement</td>
		</tr>
		<tr>
			<td>IBMX</td>
			<td>Sigma Aldrich</td>
			<td>I5879</td>
			<td>Adipocyte differentiation media supplement</td>
		</tr>
		<tr>
			<td>Indomethacin</td>
			<td>Sigma Aldrich</td>
			<td>I7378</td>
			<td>Adipocyte differentiation media supplement</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma Aldrich</td>
			<td>91077C</td>
			<td>Adipocyte differentiation media supplement</td>
		</tr>
		<tr>
			<td>Knockout DMEM</td>
			<td>ThermoFisher</td>
			<td>12660012</td>
			<td>Basal media for preparing matrigel</td>
		</tr>
		<tr>
			<td>Low glucose DMEM</td>
			<td>ThermoFisher</td>
			<td>11885084</td>
			<td>MSC culturing media</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354230</td>
			<td>Coating matrix</td>
		</tr>
		<tr>
			<td>MEM-alpha</td>
			<td>ThermoFisher</td>
			<td>12561056</td>
			<td>Adipocyte differentiation media</td>
		</tr>
		<tr>
			<td>Nilered</td>
			<td>Sigma Aldrich</td>
			<td>19123</td>
			<td>Sorting marker for adipocyte</td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td>ThermoFisher</td>
			<td>15140122</td>
			<td>MSC/Adipocyte media supplement</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>ThermoFisher</td>
			<td>14190144</td>
			<td>wash buffer</td>
		</tr>
		<tr>
			<td>Pierce&#8482; 20X TBS Buffer</td>
			<td>Thermo Fisher</td>
			<td>28358</td>
			<td>wash buffer</td>
		</tr>
		<tr>
			<td>PPARG</td>
			<td>Cell Signaling Technology</td>
			<td>2443</td>
			<td>Adipocyte maturation marker</td>
		</tr>
		<tr>
			<td>ReLeSR</td>
			<td>Stem Cell Technologies</td>
			<td>5872</td>
			<td>Dissociation reagent</td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma Aldrich</td>
			<td>R2625</td>
			<td>MSC differentiation media supplement</td>
		</tr>
		<tr>
			<td>Rock inhibitor</td>
			<td>Tocris</td>
			<td>1254/10</td>
			<td>hPSC culture media supplement</td>
		</tr>
		<tr>
			<td>Roziglitazone</td>
			<td>Sigma Aldrich</td>
			<td>R2408</td>
			<td>Adipocyte differentiation media supplement</td>
		</tr>
		<tr>
			<td>StemFlex</td>
			<td>ThermoFisher</td>
			<td>A334901</td>
			<td>hPSC culture media</td>
		</tr>
		<tr>
			<td>Triton</td>
			<td>Thermo Fisher</td>
			<td>28314</td>
			<td>Permebealization reagent</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>ThermoFisher</td>
			<td>25200072</td>
			<td>Dissociation reagent</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma Aldrich</td>
			<td>P7942</td>
			<td>Wash buffer</td>
		</tr>
	</tbody>
</table>

robust differentiation of human ipscs into a pure population of adipocytes to study adipocyte-associated disorders

İndüklenmiş pluripotent kök hücre (iPSC) teknolojisindeki son gelişmeler, adipositler de dahil olmak üzere farklı hücre tiplerinin üretilmesine izin vermiştir. Bununla birlikte, mevcut farklılaşma yöntemleri düşük verimliliğe sahiptir ve homojen bir adiposit popülasyonu üretmemektedir. Burada, mezenkimal kök hücreleri (MSC'ler) yüksek verimde üretmek için all-trans retinoik bazlı bir yöntem kullanarak bu sorunu aşıyoruz. Hücre proliferasyonunu, hayatta kalmayı ve yapışmayı yöneten yolları düzenleyerek, farklılaşma stratejimiz, saf bir multipotent MSC popülasyonuna farklılaşan embriyonik cisimlerin (EB'ler) verimli bir şekilde üretilmesini sağlar. Bu yöntemle üretilen yüksek sayıda MSC, adiposit üretmek için ideal bir kaynak sağlar. Bununla birlikte, adiposit farklılaşmasından kaynaklanan örnek heterojenliği bir zorluk olmaya devam etmektedir. Bu nedenle, FACS kullanarak lipit taşıyan olgun adipositleri saflaştırmak için Nil kırmızısı bazlı bir yöntem kullandık. Bu sıralama stratejisi, adipositle ilişkili metabolik bozuklukları, azaltılmış numune heterojenliği ve gelişmiş hücre işlevselliği ile bir adiposit havuzu kullanarak modellemek için güvenilir bir yol oluşturmamızı sağladı.

Mezenkimal farklılaşma sırasında hücrelerin şematik ve morfolojisi: iPSC'lerin MSC'lere farklılaşması, EB oluşumu, MSC farklılaşması ve MSC genişlemesi boyunca uzanan çeşitli gelişim aşamalarını içerir (Şekil 1). Gelişimin bu aşamalarında, hücreler maruz kaldıkları farklı uyarıcı kimyasallar nedeniyle çeşitli morfolojiler kazanırlar. Farklılaşmayı başlattıktan sonra, hücreler süspansiyonla kaplanır ve çapı küçük ila orta büyüklükte iken, tan?...

Watch this Scientific Journal Video about Robust Differentiation of Human iPSCs into a Pure Population of Adipocytes to Study Adipocyte-Associated Disorders at JoVE.com

Robust Differentiation of Human iPSCs into a Pure Population of Adipocytes to Study Adipocyte-Associated Disorders

Protokol, indüklenmiş pluripotent kök hücrelerden (iPSC'ler) saf bir adiposit popülasyonunun üretilmesine izin verir. Retinoik asit, iPSC'leri adiposit üretmek için kullanılan mezenkimal kök hücrelere (MSC'ler) ayırmak için kullanılır. Daha sonra, saf adipositler elde etmek için Nil kırmızısı boyamaya dayalı bir sıralama yaklaşımı kullanılır.

Adipositle İlişkili Bozuklukları İncelemek için İnsan iPSC'lerinin Saf Bir Adiposit Popülasyonuna Sağlam Farklılaşması

Recent advances in induced pluripotent stem cell (iPSC) technology have allowed the generation of different cell types, including adipocytes. However, the ...

Önceki yaklaşımlar, in vitro mezenkimal kök hücreler elde etmek için oldukça invaziv yöntemlere dayanıyordu, ancak bu mezenkimal kök hücrelerin% 30 ila% 60 arasında değişen verimlilikle% 30'luk heterojen havuz üretme sınırlaması vardıBurada, Nil kırmızısı kullanarak saf bir olgun adiposit popülasyonu üretebilen çok sayıda hızlı çoğalan mezenkimal kök hücre üretmek için bir protokol sunuyoruz. İPSC'ler% 80 birleşime ulaştığında, hücreleri DPBS ile yıkayın ve EDTA içeren ayrışma ortamı ekleyin. Hücreleri bir dakika boyunca 37 santigrat derecede inkübe edin.Bir dakika sonra, ayrışma reaktifini aspire edin ve hücreleri bir dakika daha 37 santigrat dereceye yerleştirin. Ardından, plakayı inkübatörden çıkarın. Kuyucuk başına bir mililitre StemFlex ortamı ekleyin ve hücreleri 15 mililitrelik bir konik tüpte dikkatlice toplayın.Hücreleri santrifüj edin. Süpernatanı çıkarın ve bir kaya inhibitörünün 10 mikromolünü içeren üç mililitre MSC farklılaşma ortamında yavaşça askıya alın. Hücre süspansiyonunu karıştırın ve 24 kuyucuklu ultra düşük bir bağlantı plakasında kuyucuk başına 500 mikrolitre dağıtın.Ardından plakayı 37 santigrat derecede bir inkübatöre yerleştirin. 24 saat sonra, elde edilen embriyonik cisimleri indüklemek için, onları 15 mililitrelik bir tüpte toplayın ve yerleşmelerine izin verin. 15 dakika sonra, süpernatantı çıkarın ve 10 mikromolar retinoik asit ile desteklenmiş MSC farklılaşma ortamı ekleyin.Embriyonik cisimleri nazikçe yeniden askıya alın ve aynı 24 kuyucuklu ultra düşük bağlantı plakasında kuyu başına 500 mikrolitre dağıtın. Sonra plakayı inkübatöre yerleştirin. 48 saat sonra, embriyonik cisimleri 15 mililitrelik bir tüpte toplayın ve 15 dakika boyunca yerleşmelerine izin verin.Daha sonra süpernatantı çıkarın ve 0.1 mikromolar retinoik asit ile desteklenmiş MSC farklılaşma ortamı ekleyin. Embriyonik cisimleri yeniden askıya aldıktan sonra, aynı 24 delikli plakaya kuyucuk başına 500 mikrolitre dağıtın ve plakayı inkübatöre yerleştirin. Son retinoik asit tedavisinden kırk sekiz saat sonra, embriyonik cisimleri toplayın, süpernatanı çıkarın ve sitokinsiz DMEM düşük glikozlu ortam ekleyin.Aynı 24 kuyucuklu ultra düşük bağlantı plakasında kuyu başına 500 mikrolitre hücre süspansiyonunu yavaşça askıya alın ve dağıtın, ardından plakayı 37 santigrat derecede inkübe edin. 48 saat sonra, iPSC türevi embriyonik cisimleri plakalamak için, embriyonik cisimleri 15 mililitrelik bir tüpte toplayın, yerleşmelerine izin verin, daha sonra süpernatanı çıkarın ve iki mililitre taze MSC farklılaşma ortamında yeniden askıya alın. Daha sonra, süspansiyonu bir bazal membran matris kaplı altı delikli plakanın iki kuyucuğuna aktarın ve hücreleri 37 santigrat derecede inkübe edin.Beş gün sonra, harcanan ortamı, mililitre bazik fibroblast büyüme faktörü başına 2.5 nanogram içeren taze bir MSC farklılaşma ortamı ile değiştirin ve MCS farklılaşmasına 11 ve 15 güne kadar devam edin. Kaplanmış embriyonik cisimler% 80 ila% 90 birleşime ulaştığında, DPBS ile yıkayarak, Tripsin-EDTA ekleyerek ve bir dakika boyunca 37 santigrat derecede inkübe ederek bunları geçirin. Tripsin-EDTA'yı plakadan çıkarın ve bir dakika boyunca 37 santigrat derecede tekrar inkübe edin.Ardından, kuyu başına bir mililitre ortam ekleyin. Üç dakika sonra, MSC farklılaşma ortamını kullanarak hücreleri 15 mililitrelik bir konik tüpte toplayın ve hücreleri santrifüj edin. Daha sonra hücreleri temel fibroblast büyüme faktörü içeren bir MSC farklılaşma ortamında yeniden askıya alın ve hücreleri bazal membran matris kaplı plakalar üzerinde bir ila üç oranında plakalayın.MSC'ler% 90'a ulaştıktan sonra, bir büyüme durması dönemine girmelerine izin vermek için onları 48 saat daha kültürlemeye devam edin. Ardından ortamı çıkarın ve hücreleri DBPS ile yıkayın. Yıkamadan sonra, plakaya tam adiposit farklılaşma ortamı ekleyin ve hücreleri 37 santigrat derecede inkübe edin.Ortamı 14 gün boyunca her gün değiştirin. iPSC türevi MSC'leri adipositlere ayırın. Adipositleri Nil kırmızısı kullanarak sıralamak için, metin makalesinde açıklandığı gibi DMSO'da Nil kırmızısı çalışma çözeltisi hazırlayın.Kullanmadan önce, stok çözeltisini çözün ve 300-nanomolar çalışma çözeltisi konsantrasyonuna ulaşmak için DPBS'de yeniden oluşturun. Adiposit farklılaşmasının 14. gününde veya sonrasında, ortamı hücrelerden atın ve DPBS kullanarak yıkayın. Ardından Nil kırmızısı çalışma çözümünü ekleyin.Plakayı örtün ve hücreleri 37 santigrat derecede inkübe edin. 15 dakika sonra, Nil kırmızısı çözeltisini Tripsin-EDTA ile değiştirin ve hücreleri dört dakika boyunca 37 santigrat derecede inkübe edin. Daha sonra 15 mililitrelik bir konik tüpte% 5 FBS içeren DMEM kullanarak hücreleri toplayın ve hücreleri santrifüj edin.Süpernatantı çıkarın ve DPBS'deki hücreleri mililitre başına 1 milyon hücre yoğunluğunda yeniden askıya alın. Daha sonra FACS sıralayıcısını kullanarak, FL-1 kanalını kullanarak Nil kırmızı-pozitif hücrelerini izole edin. Sıralanmış hücreleri toplayın ve bunları adiposit farklılaşma ortamında yeniden kültüre alın veya RNA ekstraksiyonuna devam edin, ardından adiposit farklılaşma belirteçlerinin kantitatif analizini yapın.Farklılaşmayı başlattıktan sonra, süspansiyonla kaplanmış hücreler, tanımlanmış hücre kenarlıkları ile yuvarlaktır ve çapı küçük ila orta büyüklüktedir. Embriyonik cisimlerin yaşayabilirliği, daha fazla MSC'ye yol açan hızlı proliferasyon davranışı ile gözlenir. Bu hızlı proliferasyon davranışı, kendine özgü ve uzamış morfolojileri ile birlikte, MSC'leri taze matris kaplı plakalara geçirdikten sonra bile korunur.İyi farklılaşma, mezenkimal yüzey belirteçleri CD73, CD44 ve CD90'ın% 90'ından fazla ekspresyon verimliliğine sahip güvenilir MSC'ler üretir. Ek olarak, hematopoetik fenotip CD14, CD34 ve CD19'u gösteren yüzey belirteçlerinin yokluğu açısından hücrelerin değerlendirilmesi,% 1'den az ekspresyon etkinliği gösterdi, Terminal olarak farklılaşmış adipositler için bir belirteç olan FABP4'ün sitoplazmik dağılımında yüksek ekspresyon, gelişimsel olgunluklarını gösterir. Ek olarak, adiposit olgunluğunun bir başka belirteci olan adiponektinin yüksek ekspresyonu, adipositlerin glikoz sinyalizasyonuna yanıt olarak lipit depolanması ve adipogeneze maruz kalacak kadar işlevsel olduğunu gösterir.Boyama üzerine, Nil kırmızısı sadece lipit taşıyan olgun adipositlere bağlanır, bu da onu floresan ile aktive edilmiş akış sitometrisi kullanarak olgun adipositleri sıralamak için etkili bir araç haline getirir. Nil kırmızı-pozitif hücreleri, olgunlaşma belirteçlerinin sıralanmamış hücrelere kıyasla en az iki kat daha fazla düzenlenmesini gösterir. Embriyonik vücut oluşumu ve mezenkimal hücre ayrışması sırasında hücreleri toplarken çok nazik olmalısınız, çünkü bu mezenkimal kök hücrelerin ayrışmasından sonra hayatta kalma verimliliğini büyük ölçüde etkileyecektir.Mutasyon taşıyan hastalardan elde edilen saf adiposit popülasyonu, veriler örnek heterojenliğinden etkilenmeden belirli mutasyondan etkilenen düzenleyici ve sinyal yollarını belirlemek için fonksiyonel ve tüm transkriptom dizilemesine tabi tutulabilir. Bu teknik, mezenkimal kök hücrelerin in vitro olarak ölçeklenebilir bir şekilde üretilmesine izin verecek ve doku ile ilişkili hastalık patogenezini anlamak için onları adipositlere, kondrositlere ve osteositlere kolayca ayırt etmek için insan donörlerinden toplama ihtiyacını ortadan kaldıracaktır.

robust-differentiation-human-ipscs-into-pure-population-adipocytes-to

Research

JoVE Journal

Developmental Biology

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

Tavan Kültürlerde Olgun adipositlerinde ve dediferansiyonunu ile İnsan adipoz Kök Hücre Üretimi

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

Gelişim Biyolojisi

Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and the neurosciences. Traditional in vitro experimental models used in neurobiology include primary neuronal cultures from rats and mice, neuroblastoma cell lines including rat B35 and mouse Neuro-2A cells, rat PC12 cells, and short-term slice cultures. While many researchers rely on these models, they lack a human component and observed experimental effects could be exclusive to the respective species and may not occur identically in humans. Additionally, although these cells are neurons, they may have unstable karyotypes, making their use problematic for studies of gene expression and reproducible studies of cell signaling. It is therefore important to develop more consistent models of human neurological disease. 
The following procedure describes an easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y. This method integrates several previously described methods1-4 and is based on sequential removal of serum from media. The timeline includes gradual serum-starvation, with introduction of extracellular matrix proteins and neurotrophic factors. This allows neurons to differentiate, while epithelial cells are selected against, resulting in a homogeneous neuronal culture. Representative results demonstrate the successful differentiation of SH-SY5Y neuroblastoma cells from an initial epithelial-like cell phenotype into a more expansive and branched neuronal phenotype. This protocol offers a reliable way to generate homogeneous populations of neuronal cultures that can be used for subsequent biochemical and molecular analyses, which provides researchers with a more accurate translational model of human infection and disease.

It is critical in neurobiology and neurovirology to have a reliable, replicable in vitro system that serves as a translational model for what occurs in vivo in human neurons. This protocol describes how to culture and differentiate SH-SY5Y human neuroblastoma cells into viable neurons for use in in vitro applications.

SH-SY5Y İnsan Nöroblastom Hücre Hattı Farklılaşma

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

Insan pluripotent İn Vitro Farklılaşma Trofoblastik Hücreleri içine Kök Hücreler

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

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 türetilmiş İnsan Beyin Organoids oluşturulması Erken Nörogelişimsel Bozuklukları Model

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

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

Nörotoksisite Testi İçin İnsan Kaynaklı Pluripotent Kök Hücrelerin Nöronlar ve Glia Karışık Kültürlerine Ayrıştırılması Protokolü

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

Vitro İnsan Mezenkimal Kök hücre farklılaşma fonksiyonel Cardiomyocyte benzeri hücre içine

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that mouse embryonic, dermal, and cardiac fibroblasts can be reprogrammed into functional induced-cardiomyocyte-like cells (iCMs) through overexpression of cardiogenic transcription factors including GATA4, Hand2, Mef2c, and Tbx5 both in vitro and in vivo. However, these previous studies have shown relatively low efficiency. In order to restore heart function following injury, mechanisms governing cardiac reprogramming must be elucidated to increase efficiency and maturation of iCMs.
We previously demonstrated that inhibition of pro-fibrotic signaling dramatically increases reprogramming efficiency. Here, we detail methods to achieve a reprogramming efficiency of up to 60%. Furthermore, we describe several methods including flow cytometry, immunofluorescent imaging, and calcium imaging to quantify reprogramming efficiency and maturation of reprogrammed fibroblasts. Using the protocol detailed here, mechanistic studies can be undertaken to determine positive and negative regulators of cardiac reprogramming. These studies may identify signaling pathways that can be targeted to promote reprogramming efficiency and maturation, which could lead to novel cell therapies to treat human heart disease.

Here we present a robust method to reprogram primary embryonic fibroblasts into functional cardiomyocytes through overexpression of GATA4, Hand2, Mef2c, Tbx5, miR-1, and miR-133 (GHMT2m) alongside inhibition of TGF-&#946; signaling. Our protocol generates beating cardiomyocytes as early as 7 days post-transduction with up to 60% efficiency.

Pro-fibrotik sinyal bastırma faktörü-aracılı fare embriyonik fibroblastlar indüklenen Cardiomyocytes yeniden programlama Potentiates

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like 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 ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

İnsan Embriyonik Kök Hücrelerinin Tek Hücre Kültürünü Kullanarak Etkin Nöral Farklılaşma

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial cells, which line blood vessel lumens, from human pluripotent stem cells is crucial for a multitude of tissue engineering and regeneration applications. In vivo, primordial endothelial cells are derived from the mesodermal lineage and are specified toward specific subtypes, including arterial, venous, capillary, hemogenic, and lymphatic. Hemogenic endothelial cells are of particular interest because, during development, they give rise to hematopoietic stem and progenitor cells, which then generate all blood lineages throughout life. Thus, creating a system to generate hemogenic endothelial cells in vitro would provide an opportunity to study endothelial-to-hematopoietic transition, and may lead to ex vivo production of human blood products and reduced reliance on human donors. While several protocols exist for the derivation of progenitor and primordial endothelial cells, generation of well-characterized hemogenic endothelial cells from human stem cells has not been described. Here, a method for the derivation of hemogenic endothelial cells from human embryonic stem cells in approximately 1 week is presented: a differentiation protocol with primitive streak cells formed in response to GSK3&#946; inhibitor (CHIR99021), then mesoderm lineage induction mediated by bFGF, followed by primordial endothelial cell development promoted by BMP4 and VEGF-A, and finally hemogenic endothelial cell specification induced by retinoic acid. This protocol yields a well-defined population of hemogenic endothelial cells that can be used to further understand their molecular regulation and endothelial-to-hematopoietic transition, which has the potential to be applied to downstream therapeutic applications.

Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week.

Hemojenik Endotel Hücrelerinin İnsan Pluripotent Kök Hücrelerinden Yönlendirilmiş Farklılaşması

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial ...