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

Detta arbete finansierades av ett bidrag från Qatar National Research Fund (QNRF) (Grant No. NPRP10-1221-160041). Maryam Aghadi stöddes av GSRA-stipendium från Qatar National Research Fund (QNRF).

Studien har godkänts av vederbörande institutionella forskningsetiska kommitté och utförts i enlighet med de etiska normer som fastställs i Helsingforsdeklarationen från 1964 och dess senare ändringar eller jämförbara etiska normer. Protokollet godkändes av HMC:s Institutional Review Board (IRB) (nr. 16260/16) och QBRI (nr. 2016-003). Detta arbete är också optimerat för hESC som H1 och H9. Blodprover erhölls från friska individer med fullt informerat samtycke. iPSC genereras från mononukleära celler i pe...

<ol>
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Detta protokoll är av största vikt på grund av dess förmåga att tillhandahålla MSC i hög avkastning och effektivitet. Denna storskaliga produktion av MSC möjliggjordes genom övergående inkubation av iPSCs-härledda EB med 10 μM RA14,15. Övergående behandling med 10 μM RA ökade MSC-utbytet med 11,2 till 1542 gånger14,15, med detta protokoll tillämpligt på både iPSC och hPSC. Vid denna do...

Mesenkymala stamceller (MSC) fungerar som en effektiv övergående resurs för att producera celler av mesodermalt ursprung som adipocyter, osteocyter och kondrocyter, som vidare kan användas för att modellera deras respektive genetiska störningar. Tidigare tillvägagångssätt förlitade sig dock på att uppnå dessa MSC från vuxna vävnader 1, vilket innebar utmaningen att erhålla dem i stort antal från givarna och begränsningen att hålla dem funktionellt livskraftiga under suboptimala in vitro-odlingsförhållanden 1,2. Dessa hinder har skapat en stor efterfrågan på att ha ett protokoll för att generera MSC in vitro. Humant inducerade pluripotenta stamceller (iPSC) kan användas som en värdefull källa till MSC och uppvisar MSC-egenskaper 3,4,5. iPSCs-härledda MSC kan användas som ett terapeutiskt alternativ vid flera sjukdomar. Dessutom gör förmågan hos iPSC-härledda MSC att generera adipocyter dem till en värdefull in vitro human modell för att studera human adipogenesis, fetma och adipocytassocierade störningar.
Nuvarande differentieringsprotokoll för adipocyter kan klassificeras i två grupper, där den ena involverar differentiering av adipocyter med användning av kemiska eller proteinbaserade cocktails som ger ett resulterande utbyte på 30%-60%6,7,8,9, medan den andra involverar genetisk manipulation för robust induktion av viktiga transkriptionsfaktorer som styr adipocytutveckling för att ge ett utbyte på 80%-90%10, 11. Genetisk manipulation rekapitulerar emellertid inte den naturliga processen för adipocytdifferentiering och maskerar ofta de subtila paradigmerna som anländer under adipogenesis, vilket gör den ineffektiv för sjukdomsmodelleringsändamål12,13. Därför presenterar vi ett sätt att sortera kemiskt härledda mogna adipocyter från omogna genom att fluorescerande märka lipidbärande adipocyter med Nile röd.
Här presenterar vi ett protokoll som involverar övergående inkubation av iPSCs-härledda embryoidkroppar (EB) med all-trans retinsyra för att producera ett stort antal snabbt prolifererande MSC, som kan användas vidare för att generera adipocyter14. Vi presenterar också ett sätt att sortera kemiskt härledda mogna adipocyter från den heterogena differentieringspoolen genom att fluorescerande märka sina lipiddroppar med hjälp av ett lipofilt färgämne; Nilen röd. Detta skulle möjliggöra generering av en ren population av mogna adipocyter med förbättrad funktionalitet för att exakt modellera adipocytassocierade metaboliska störningar.

<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

De senaste framstegen inom inducerad pluripotent stamcellsteknik (iPSC) har möjliggjort generering av olika celltyper, inklusive adipocyter. De nuvarande differentieringsmetoderna har emellertid låg effektivitet och producerar inte en homogen population av adipocyter. Här kringgår vi detta problem genom att använda en all-trans retinoic-baserad metod för att producera mesenkymala stamceller (MSC) i hög avkastning. Genom att reglera vägar som styr cellproliferation, överlevnad och vidhäftning möjliggör vår differentieringsstrategi effektiv generering av embryonala kroppar (EB) som differentieras till en ren population av multipotenta MSC. Det stora antalet MSC som genereras med denna metod ger en idealisk källa för att generera adipocyter. Provheterogenitet som härrör från adipocytdifferentiering är dock fortfarande en utmaning. Därför använde vi en Nile rödbaserad metod för att rena lipidbärande mogna adipocyter med hjälp av FACS. Denna sorteringsstrategi gjorde det möjligt för oss att etablera ett tillförlitligt sätt att modellera adipocytassocierade metaboliska störningar med hjälp av en pool av adipocyter med minskad provheterogenitet och förbättrad cellfunktionalitet.

Schematisk och morfologi av celler under mesenkymal differentiering: Differentiering av iPSC till MSC involverar olika utvecklingsstadier som spänner över EB-bildning, MSC-differentiering och MSC-expansion (figur 1). Under dessa utvecklingsstadier förvärvar celler olika morfologi på grund av de olika stimulerande kemikalier de utsätts för. Vid initiering av differentiering pläteras cellerna i suspension och förväntas vara runda, med definierade cellgränser, samtidigt som de är sm...

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

Protokollet möjliggör generering av en ren adipocytpopulation från inducerade pluripotenta stamceller (iPSC). Retinsyra används för att differentiera iPSC till mesenkymala stamceller (MSC) som används för att producera adipocyter. Därefter används en sorteringsmetod baserad på Nile röd färgning för att erhålla rena adipocyter.

Robust differentiering av humana iPSC till en ren population av adipocyter för att studera adipocytassocierade störningar

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

Tidigare tillvägagångssätt förlitade sig på mycket invasiva metoder för att erhålla mesenkymala stamceller in vitro, men dessa mesenkymala stamceller hade en begränsning att producera heterogen pool på 30% med effektivitet från 30 till 60% Här presenterar vi ett protokoll för att producera ett stort antal snabbt prolifererande mesenkymala stamceller som kan producera en ren population av mogna adipocyter genom sortering med Nile röd. När iPSC har nått 80% sammanflöde, tvätta cellerna med DPBS och tillsätt dissociationsmedium innehållande EDTA. Inkubera cellerna vid 37 grader Celsius i en minut.Efter en minut, aspirera dissociationsreagenset och placera cellerna vid 37 grader Celsius i ytterligare en minut. Ta sedan bort plattan från inkubatorn. Tillsätt en milliliter StemFlex-media per brunn och samla försiktigt cellerna i ett 15-milliliter koniskt rör.Centrifugera cellerna. Ta bort supernatanten och suspendera dem försiktigt i tre milliliter MSC-differentieringsmedium innehållande 10 mikromol av en stenhämmare. Blanda cellsuspensionen och fördela 500 mikroliter per brunn i en 24-brunns ultralåg fästplatta.Placera sedan plattan i en inkubator vid 37 grader Celsius. Efter 24 timmar, för att inducera de uppnådda embryonala kropparna, samla dem i ett 15-milliliter rör och låt dem slå sig ner. Efter 15 minuter, ta bort supernatanten och tillsätt MSC-differentieringsmedium kompletterat med 10 mikromolar retinsyra.Resuspendera de embryonala kropparna försiktigt och fördela 500 mikroliter per brunn i samma 24-brunns ultralåga fästplatta. Placera sedan plattan i inkubatorn. Efter 48 timmar, samla de embryonala kropparna i ett 15-milliliter rör och låt dem slå sig ner i 15 minuter.Ta sedan bort supernatanten och tillsätt MSC-differentieringsmedium kompletterat med 0,1 mikromolär retinsyra. Efter resuspendering av de embryonala kropparna, fördela 500 mikroliter per brunn i samma 24-brunnsplatta och placera plattan i inkubatorn. Fyrtioåtta timmar efter den sista retinsyrabehandlingen, samla de embryonala kropparna, ta bort supernatanten och tillsätt DMEM lågglukosmedium utan cytokiner.Resuspendera försiktigt och fördela 500 mikroliter av cellsuspensionen per brunn i samma 24-brunns ultralåga fästplatta, inkubera sedan plattan vid 37 grader Celsius. Efter 48 timmar, för att platta de iPSC-härledda embryonala kropparna, samla de embryonala kropparna i ett 15-milliliter rör, låt dem sedimentera, ta sedan bort supernatanten och resuspendera i två milliliter färskt MSC-differentieringsmedium. Därefter överför suspensionen till två brunnar i en matrisbelagd platta med sex brunnar i basalmembranet och inkubera cellerna vid 37 grader Celsius.Efter fem dagar, ersätt det använda mediet med ett nytt MSC-differentieringsmedium innehållande 2,5 nanogram per milliliter basisk fibroblasttillväxtfaktor och fortsätt MCS-differentieringen i upp till 11 och 15 dagar. När de pläterade embryonala kropparna har nått 80 till 90% sammanflöde, passera dem genom att tvätta med DPBS, tillsätta trypsin-EDTA och inkubera vid 37 grader Celsius i en minut. Ta bort trypsin-EDTA från plattan och inkubera igen vid 37 grader Celsius i en minut.Lägg sedan till en milliliter media per brunn. Efter tre minuter, samla cellerna med MSC-differentieringsmedia i ett 15-milliliter koniskt rör och centrifugera cellerna. Resuspendera sedan cellerna i ett MSC-differentieringsmedium innehållande grundläggande fibroblasttillväxtfaktor och platta cellerna på matrisbelagda plattor med basalmembran i förhållandet ett till tre.Efter att MSC har nått 90% sammanflöde, fortsätt odla dem i ytterligare 48 timmar så att de kan genomgå en period av tillväxtstopp. Ta sedan bort mediet och tvätta cellerna med DBPS. Efter tvätten, tillsätt fullständigt adipocytdifferentieringsmedium till plattan och inkubera cellerna vid 37 grader Celsius.Byt medium varannan dag i 14 dagar. Differentiera iPSC-härledda MSC till adipocyter. För att sortera adipocyterna med Nile röd, förbered Nile röd arbetslösning i DMSO som beskrivs i textmanuskriptet.Tina stamlösningen före användning och bereda den i DPBS för att uppnå en koncentration på 300 nanomolar arbetslösningar. På eller efter dag 14 av adipocytdifferentiering, kassera mediet från cellerna och tvätta med DPBS. Lägg sedan till Nile röd arbetslösning.Täck plattan och inkubera cellerna vid 37 grader Celsius. Efter 15 minuter, ersätt den Nilröda lösningen med trypsin-EDTA och inkubera cellerna vid 37 grader Celsius i fyra minuter. Samla sedan cellerna med DMEM innehållande 5% FBS i ett 15-milliliter koniskt rör och centrifugera cellerna.Ta bort supernatanten och suspendera cellerna i DPBS med en densitet av 1 miljon celler per milliliter. Använd sedan FACS-sorteraren, isolera de röda positiva cellerna i Nilen med FL-1-kanalen. Samla de sorterade cellerna och odla dem i adipocytdifferentieringsmedia eller fortsätt till RNA-extraktion följt av kvantitativ analys av adipocytdifferentieringsmarkörer.Vid initiering av differentiering är celler pläterade i suspension runda med definierade cellgränser och är små till medelstora i diameter. Livskraften hos embryonala kroppar observeras av det snabba spridningsbeteendet som ger upphov till fler MSC. Detta snabba spridningsbeteende, tillsammans med deras speciella och långsträckta morfologi, bibehålls även efter att MSC har passerat på färska matrisbelagda plattor.Bra differentiering ger tillförlitliga MSC med större än 90% uttryckseffektivitet för mesenkymala ytmarkörer CD73, CD44 och CD90. Dessutom visade bedömning av celler för frånvaro av ytmarkörer som visar den hematopoetiska fenotypen CD14, CD34 och CD19 mindre än 1% uttryckseffektivitet, Högt uttryck i cytoplasmatisk fördelning av FABP4, en markör för terminalt differentierade adipocyter, indikerar deras utvecklingsmognad. Dessutom indikerar högt uttryck av adiponectin, en annan markör för adipocytmognad, att adipocyterna är funktionella nog att genomgå lipidlagring och adipogenesis som svar på glukossignalering.Vid färgning binder Nilrött uteslutande till de lipidbärande mogna adipocyterna, vilket gör det till ett effektivt verktyg för sortering av mogna adipocyter med fluorescerande aktiverad flödescytometri. De röda positiva cellerna i Nilen visar en signifikant uppreglering av mognadsmarkörer med minst två gånger jämfört med osorterade celler. Du måste vara mycket försiktig när du samlar celler under embryonal kroppsbildning och mesenkymal celldissociation eftersom det i hög grad skulle påverka överlevnadseffektiviteten efter dissociation av mesenkymala stamceller.Ren population av adipocyter som uppnåtts från mutationsbärande patienter kan utsättas för funktionell och hela transkriptomsekvenseringen för att bestämma reglerings- och signalvägar som påverkas av en viss mutation utan att data påverkas av provets heterogenitet. Denna teknik möjliggör skalbar generering av mesenkymala stamceller in vitro, vilket tar bort behovet av att skörda dem från mänskliga givare för att enkelt skilja dem till adipocyter, kondrocyter och osteocyter för att förstå vävnadsrelaterad sjukdomspatogenes.

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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.

Generering av Human Fett stamceller genom dedifferentiering av mogna Adipocyter i Tak kulturer

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

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.

Differentiering av SH-SY5Y humana neuroblastom cellinje

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-differentiering av mänskliga pluripotenta stamceller till trophoblastic celler

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.

Generation av iPSC härledda mänskliga hjärnan Organoids till modell Tidig nervsystemets sjukdomar

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 för differentieringen av humant inducerade plöripotenta stamceller i blandade kulturer av neuroner och Glia för neurotoxicitetstestning

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 Differentiering av humana mesenkymala stamceller till funktionella hjärtmuskelcellen-liknande celler

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.

Dämpning av Pro-fibrotiska signalering potentierar faktor-medierad omprogrammering av mus embryonala fibroblaster till inducerad hjärtmuskelcellerna

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.

Effektiv differentiering av humana pluripotenta stamceller till leverceller

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.

Effektiv neurala differentiering med hjälp av Single-cell kultur av mänskliga embryonala stamceller

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.

Riktad differentiering av hemogena endotelceller från humana pluripotenta stamceller

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