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

Diese Arbeit wurde durch einen Zuschuss des Qatar National Research Fund (QNRF) finanziert (Fördernummer NPRP10-1221-160041). Maryam Aghadi wurde durch ein GSRA-Stipendium des Qatar National Research Fund (QNRF) unterstützt.

Die Studie wurde von der zuständigen institutionellen Forschungsethikkommission genehmigt und nach den ethischen Standards durchgeführt, wie sie in der Deklaration von Helsinki von 1964 und ihren späteren Änderungen oder vergleichbaren ethischen Standards festgelegt sind. Das Protokoll wurde vom Institutional Review Board (IRB) der HMC (Nr. 16260/16) und QBRI (Nr. 2016-003) genehmigt. Diese Arbeit ist auch für hES-Zellen wie H1 und H9 optimiert. Die Blutproben wurden von gesunden Personen mit vollständiger Einverst...

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Dieses Protokoll ist von größter Bedeutung, da es in der Lage ist, MSCs mit hoher Ausbeute und Effizienz zu liefern. Diese Massenproduktion von MSCs wurde durch die transiente Inkubation von iPSCs-abgeleiteten EBs mit 10 μM RA14,15 ermöglicht. Die transiente Behandlung mit 10 μM RA erhöhte die MSC-Ausbeute um das 11,2- bis 1542-fache14,15, wobei dieses Protokoll sowohl auf iPS-Zellen als auch auf hP...

Mesenchymale Stammzellen (MSCs) fungieren als effektive transitorische Ressource für die Produktion von Zellen mesodermalen Ursprungs wie Adipozyten, Osteozyten und Chondrozyten, die für die Modellierung ihrer jeweiligen genetischen Erkrankungen verwendet werden könnten. Bisherige Ansätze beruhten jedoch auf der Gewinnung dieser MSCs aus adulten Geweben 1, was die Herausforderung mit sich brachte, sie in großer Zahl von den Spendern zu erhalten, und die Einschränkung der funktionellen Lebensfähigkeit unter suboptimalen In-vitro-Kulturbedingungen mit sich brachte 1,2. Diese Hindernisse haben zu einem großen Bedarf an einem Protokoll für die Herstellung von MSCs in vitro geführt. Humane induzierte pluripotente Stammzellen (iPS-Zellen) können als wertvolle Quelle für MSCs verwendet werden, die MSC-Eigenschaften aufweisen 3,4,5. Aus iPS-Zellen gewonnene MSCs können als therapeutische Option bei verschiedenen Krankheiten eingesetzt werden. Auch die Fähigkeit von aus iPS-Zellen gewonnenen MSCs, Adipozyten zu erzeugen, macht sie zu einem wertvollen In-vitro-Humanmodell zur Untersuchung von menschlicher Adipogenese, Fettleibigkeit und Adipozyten-assoziierten Erkrankungen.
Die derzeitigen Differenzierungsprotokolle von Adipozyten können in zwei Gruppen eingeteilt werden, wobei die eine die Differenzierung der Adipozyten unter Verwendung chemischer oder proteinbasierter Cocktails beinhaltet, die eine resultierende Ausbeute von 30%-60%6,7,8,9 ergeben, während die andere genetische Manipulation zur robusten Induktion von Schlüsseltranskriptionsfaktoren, die die Entwicklung von Adipozyten steuern, beinhaltet, um eine Ausbeute von 80%-90% zu erzielen10. 11. Sonstiges Die genetische Manipulation rekapituliert jedoch nicht den natürlichen Prozess der Adipozytendifferenzierung und verschleiert oft die subtilen Paradigmen, die während der Adipogenese auftreten, was sie für die Modellierung von Krankheiten unwirksam macht12,13. Daher stellen wir eine Möglichkeit vor, chemisch gewonnene reife Adipozyten von unreifen zu sortieren, indem lipidtragende Adipozyten fluoreszenzartig mit Nilrot markiert werden.
In dieser Arbeit stellen wir ein Protokoll vor, das die transiente Inkubation von iPS-abgeleiteten Embryoidkörpern (EBs) mit all-trans-Retinsäure beinhaltet, um eine große Anzahl schnell proliferierender MSCs zu produzieren, die zur Erzeugung von Adipozyten verwendet werden könnten14. Wir stellen auch eine Möglichkeit vor, chemisch gewonnene reife Adipozyten aus dem heterogenen Differenzierungspool zu sortieren, indem ihre Lipidtröpfchen mit einem lipophilen Farbstoff fluoreszenzmarkiert werden. Nilrot. Dies würde es ermöglichen, eine reine Population reifer Adipozyten mit verbesserter Funktionalität zu erzeugen, um Adipozyten-assoziierte Stoffwechselstörungen genau zu modellieren.

<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

Jüngste Fortschritte in der Technologie der induzierten pluripotenten Stammzellen (iPSC) haben die Erzeugung verschiedener Zelltypen, einschließlich Adipozyten, ermöglicht. Die derzeitigen Differenzierungsmethoden haben jedoch eine geringe Effizienz und erzeugen keine homogene Population von Adipozyten. Hier umgehen wir dieses Problem, indem wir eine all-trans-retinische Methode verwenden, um mesenchymale Stammzellen (MSCs) in hoher Ausbeute herzustellen. Durch die Regulierung von Signalwegen, die die Zellproliferation, das Überleben und die Adhäsion steuern, ermöglicht unsere Differenzierungsstrategie die effiziente Erzeugung von embryonalen Körperchen (EBs), die sich zu einer reinen Population multipotenter MSCs differenzieren. Die hohe Anzahl von MSCs, die durch diese Methode erzeugt werden, bietet eine ideale Quelle für die Bildung von Adipozyten. Die Heterogenität der Proben, die sich aus der Differenzierung von Adipozyten ergibt, bleibt jedoch eine Herausforderung. Daher haben wir eine Nilrot-basierte Methode zur Reinigung lipidhaltiger reifer Adipozyten mit FACS verwendet. Diese Sortierstrategie ermöglichte es uns, eine zuverlässige Methode zur Modellierung von Adipozyten-assoziierten Stoffwechselstörungen unter Verwendung eines Pools von Adipozyten mit reduzierter Probenheterogenität und verbesserter Zellfunktionalität zu etablieren.

Schematische Darstellung und Morphologie von Zellen während der mesenchymalen Differenzierung: Die Differenzierung von iPS-Zellen in MSCs umfasst verschiedene Entwicklungsstadien, die sich über EB-Bildung, MSC-Differenzierung und MSC-Expansion erstrecken (Abbildung 1). Während dieser Entwicklungsstadien erhalten die Zellen aufgrund der verschiedenen stimulierenden Chemikalien, denen sie ausgesetzt sind, eine unterschiedliche Morphologie. Nach Beginn der Differenzierung werden die Zellen i...

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

Das Protokoll ermöglicht die Erzeugung einer reinen Adipozytenpopulation aus induzierten pluripotenten Stammzellen (iPSCs). Retinsäure wird verwendet, um iPS-Zellen in mesenchymale Stammzellen (MSCs) zu differenzieren, die für die Produktion von Adipozyten verwendet werden. Anschließend wird ein Sortieransatz auf Basis der Nilrotfärbung verwendet, um reine Adipozyten zu erhalten.

Robuste Differenzierung von humanen iPS-Zellen in eine reine Population von Adipozyten zur Untersuchung von Adipozyten-assoziierten Erkrankungen

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

Bisherige Ansätze stützten sich auf hochinvasive Methoden zur Gewinnung mesenchymaler Stammzellen in vitro, aber diese mesenchymalen Stammzellen hatten eine Begrenzung der Produktion eines heterogenen Pools von 30% mit einer Effizienz von 30 bis 60%. Hier stellen wir ein Protokoll zur Herstellung einer hohen Anzahl schnell proliferierender mesenchymaler Stammzellen vor, die durch Sortierung mit Nilrot eine reine Population reifer Adipozyten erzeugen können. Wenn die iPS-Zellen eine Konfluenz von 80 % erreicht haben, waschen Sie die Zellen mit DPBS und fügen Sie EDTA-haltiges Dissoziationsmedium hinzu. Inkubieren Sie die Zellen eine Minute lang bei 37 Grad Celsius.Aspirieren Sie nach einer Minute das Dissoziationsreagenz und platzieren Sie die Zellen für eine weitere Minute bei 37 Grad Celsius. Nehmen Sie als Nächstes die Platte aus dem Inkubator. Geben Sie einen Milliliter StemFlex-Medien pro Vertiefung hinzu und sammeln Sie die Zellen vorsichtig in einem konischen 15-Milliliter-Röhrchen.Zentrifugieren Sie die Zellen. Entfernen Sie den Überstand und resuspendieren Sie ihn vorsichtig in drei Millilitern MSC-Differenzierungsmedien, die 10 Mikromol eines Gesteinsinhibitors enthalten. Mischen Sie die Zellsuspension und verteilen Sie 500 Mikroliter pro Vertiefung in einer 24-Well-Ultra-Low-Attachment-Platte.Stellen Sie den Teller dann bei 37 Grad Celsius in einen Inkubator. Nach 24 Stunden, um die erhaltenen embryonalen Körper zu induzieren, sammeln Sie sie in einem 15-Milliliter-Röhrchen und lassen Sie sie sich beruhigen. Entfernen Sie nach 15 Minuten den Überstand und fügen Sie MSC-Differenzierungsmedium hinzu, das mit 10 Mikromolaren Retinsäure ergänzt ist.Resuspendieren Sie die Embryonalkörper vorsichtig und verteilen Sie 500 Mikroliter pro Well in derselben 24-Well-Platte mit extrem niedrigem Anhang. Legen Sie dann die Platte in den Inkubator. Nach 48 Stunden werden die Embryonalkörper in einem 15-Milliliter-Röhrchen gesammelt und 15 Minuten ruhen gelassen.Anschließend wird der Überstand entfernt und MSC-Differenzierungsmedium mit 0,1 Mikromolar Retinsäure zugegeben. Nach der Resuspendierung der Embryonalkörper verteilen Sie 500 Mikroliter pro Vertiefung in derselben 24-Well-Platte und legen Sie die Platte in den Inkubator. Achtundvierzig Stunden nach der letzten Retinsäurebehandlung sammeln Sie die Embryonalkörper, entfernen Sie den Überstand und fügen Sie DMEM-Medium mit niedrigem Glukosegehalt ohne Zytokine hinzu.500 Mikroliter der Zellsuspension pro Well werden vorsichtig resuspendiert und in derselben 24-Well-Platte mit extrem niedrigem Anhaftungsgrad verteilt, dann wird die Platte bei 37 Grad Celsius inkubiert. Nach 48 Stunden werden die Embryonalkörper in einem 15-Milliliter-Röhrchen gesammelt, um die aus iPSC gewonnenen Embryonalkörper zu plattieren, sie absetzen zu lassen, dann den Überstand zu entfernen und in zwei Millilitern frischem MSC-Differenzierungsmedium zu resuspendieren. Als nächstes wird die Suspension in zwei Vertiefungen einer mit einer Basalmembranmatrix beschichteten Sechs-Well-Platte überführt und die Zellen bei 37 Grad Celsius inkubiert.Ersetzen Sie nach fünf Tagen das verbrauchte Medium durch ein frisches MSC-Differenzierungsmedium, das 2,5 Nanogramm pro Milliliter basischen Fibroblasten-Wachstumsfaktors enthält, und setzen Sie die MCS-Differenzierung bis zu 11 und 15 Tage lang fort. Wenn die plattierten Embryonalkörper eine Konfluenz von 80 bis 90 % erreicht haben, passieren Sie sie, indem Sie sie mit DPBS waschen, Trypsin-EDTA hinzufügen und eine Minute lang bei 37 Grad Celsius inkubieren. Das Trypsin-EDTA von der Platte nehmen und erneut bei 37 Grad Celsius für eine Minute inkubieren.Als nächstes fügen Sie ein Milliliter-Medium pro Vertiefung hinzu. Nach drei Minuten werden die Zellen mit MSC-Differenzierungsmedien in einem konischen 15-Milliliter-Röhrchen entnommen und zentrifugiert. Anschließend werden die Zellen in einem MSC-Differenzierungsmedium mit basischem Fibroblasten-Wachstumsfaktor resuspendiert und die Zellen im Verhältnis eins zu drei auf mit Basalmembranmatrix beschichteten Platten plattiert.Nachdem die MSCs eine Konfluenz von 90 % erreicht haben, kultivieren Sie sie weitere 48 Stunden, damit sie eine Phase des Wachstumsstopps durchlaufen können. Entfernen Sie dann das Medium und waschen Sie die Zellen mit DBPS. Nach dem Waschen geben Sie das vollständige Adipozyten-Differenzierungsmedium auf die Platte und inkubieren die Zellen bei 37 Grad Celsius.Wechseln Sie das Medium 14 Tage lang jeden zweiten Tag. Differenzierung von iPSC-abgeleiteten MSCs in Adipozyten. Um die Adipozyten mit Nilrot zu sortieren, bereiten Sie Nilrot-Arbeitslösung in DMSO vor, wie im Textmanuskript beschrieben.Tauen Sie die Stammlösung vor der Verwendung auf und rekonstituieren Sie sie in DPBS, um eine Arbeitslösungskonzentration von 300 Nanomolar zu erreichen. Am oder nach dem 14. Tag der Adipozytendifferenzierung wird das Medium aus den Zellen verworfen und mit DPBS gewaschen. Fügen Sie dann Nilrote Arbeitslösung hinzu.Decken Sie die Platte ab und inkubieren Sie die Zellen bei 37 Grad Celsius. Ersetzen Sie nach 15 Minuten die Nilrotlösung durch Trypsin-EDTA und inkubieren Sie die Zellen vier Minuten lang bei 37 Grad Celsius. Sammeln Sie dann die Zellen mit DMEM mit 5%FBS in einem konischen 15-Milliliter-Röhrchen und zentrifugieren Sie die Zellen.Entfernen Sie den Überstand und resuspendieren Sie die Zellen in DPBS mit einer Dichte von 1 Million Zellen pro Milliliter. Isolieren Sie dann mit dem FACS-Sortierer die roten Nil-positiven Zellen mit dem FL-1-Kanal. Sammeln Sie die sortierten Zellen und kultivieren Sie sie in Adipozyten-Differenzierungsmedien oder fahren Sie mit der RNA-Extraktion fort, gefolgt von einer quantitativen Analyse der Adipozyten-Differenzierungsmarker.Nach Beginn der Differenzierung sind die in Suspension plattierten Zellen rund mit definierten Zellrändern und haben einen kleinen bis mittleren Durchmesser von kleiner bis mittlerer Größe. Die Lebensfähigkeit embryonaler Körper wird durch das schnelle Proliferationsverhalten beobachtet, das zu mehr MSCs führt. Dieses schnelle Proliferationsverhalten, zusammen mit ihrer eigentümlichen und länglichen Morphologie, bleibt auch nach dem Passieren der MSCs auf frischen, matrixbeschichteten Platten erhalten.Eine gute Differenzierung führt zu zuverlässigen MSCs mit einer Expressionseffizienz von mehr als 90% der mesenchymalen Oberflächenmarker CD73, CD44 und CD90. Darüber hinaus ergab die Untersuchung von Zellen auf das Fehlen von Oberflächenmarkern, die den hämatopoetischen Phänotyp CD14, CD34 und CD19 darstellen, eine Expressionseffizienz von weniger als 1%. Eine hohe Expression in der zytoplasmatischen Verteilung von FABP4, einem Marker für terminal differenzierte Adipozyten, deutet auf ihre Entwicklungsreife hin. Darüber hinaus deutet eine hohe Expression von Adiponektin, einem weiteren Marker der Adipozytenreife, darauf hin, dass die Adipozyten funktionsfähig genug sind, um als Reaktion auf die Glukosesignalisierung eine Lipidspeicherung und Adipogenese zu durchlaufen.Bei der Färbung bindet Nilrot ausschließlich an die lipidtragenden reifen Adipozyten, was es zu einem effektiven Werkzeug für die Sortierung reifer Adipozyten mittels fluoreszenzaktivierter Durchflusszytometrie macht. Die Nilrot-positiven Zellen zeigen eine signifikante Hochregulation der Reifungsmarker um mindestens das Doppelte im Vergleich zu unsortierten Zellen. Bei der Entnahme von Zellen während der embryonalen Körperbildung und der Dissoziation von mesenchymalen Zellen muss man sehr vorsichtig vorgehen, da dies die Überlebenseffizienz nach der Dissoziation von mesenchymalen Stammzellen stark beeinträchtigen würde.Reine Populationen von Adipozyten, die von mutationstragenden Patienten gewonnen wurden, können einer funktionellen und der gesamten Transkriptomsequenzierung unterzogen werden, um regulatorische und Signalwege zu bestimmen, die von einer bestimmten Mutation betroffen sind, ohne dass die Daten durch die Heterogenität der Proben beeinflusst werden. Diese Technik ermöglicht eine skalierbare Erzeugung von mesenchymalen Stammzellen in vitro, so dass sie nicht mehr von menschlichen Spendern entnommen werden müssen, um sie leicht in Adipozyten, Chondrozyten und Osteozyten zu differenzieren, um die Pathogenese von gewebebedingten Krankheiten zu verstehen.

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.

Generation of Human Fettgewebestammzellen durch Dedifferenzierung der Reife Adipozyten in Decken Kulturen

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

Forschung

Entwicklungsbiologie

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.

Differenzierung der SH-SY5Y humanen Neuroblastom-Zelllinie

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.

In vitro Differenzierung von humanen pluripotenten Zellen in Trophoblastische Stammzellen

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.

Die Erzeugung von iPS-derived Human Brain Organoide auf Modell Frühe Störungen der neurologischen Entwicklung

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.

Protokoll zur Differenzierung von human induzierten pluripotenten Stammzellen in Mischkulturen von Neuronen und Glia für Neurotoxizitätstests

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.

In-vitro- Differenzierung humaner mesenchymaler Stammzellen in funktionellen Cardiomyocyte-ähnlichen Zellen

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.

Unterdrückung der Pro-fibrotische Signalisierung potenziert Faktor-vermittelten Reprogrammierung von Maus embryonalen Fibroblasten in induzierte Kardiomyozyten

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.

Effiziente Differenzierung menschlicher pluripotenter Stammzellen in Leberzellen

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.

Effiziente neuronale Differenzierung mit einzelzellischer Kultur menschlicher embryonaler Stammzellen

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.

Gerichtete Differenzierung hämogener Endothelzellen aus humanen pluripotenten Stammzellen

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