Name: deriving retinal pigment epithelium (rpe) from induced pluripotent stem (ips) cells by different sizes of embryoid bodies
Uploaded: 2015-02-04T14:00:00
Description: Watch this Scientific Journal Video about Deriving Retinal Pigment Epithelium RPE from Induced Pluripotent Stem iPS Cells by Different Sizes of Embryoid Bodies at JoVE.com

The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense. This research was performed while the authors Alberto Mu&#241;iz, Ramesh R Kaini, Whitney A Greene and Jae-Hyek Choi held a National Research Council Postdoctoral Research Associateship at the USAISR. This work was supported by U.S. Army Clinical Rehabilitative Medicine Research Program (CRMRP) and Military Operational Medicine Research Program (MOMRP).

1. Voorbereiding van Cultuur Reagentia en Cultuur Platen <ol><li> Bereid voedingsvrije stamcellen kweekmedium door toevoeging van 100 ml 5x serumvrij supplement 400 ml stamcellen basaal medium. Het medium is stabiel bij 4 ° C gedurende maximaal 2 weken bij -20 ° C gedurende 6 maanden. </li><li> Voeg 10 pM oplossing van-rho verbonden, ook indien opgerold-coil met proteïne kinase (Rock) remmer (Y-27362) in de handel verkrijgbare embryoid lichaam (EB) vorming medium. </li><li> Bereid differentiatiemedium door 0,1 mM ?...

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Om de volledige belofte van pluripotente stamcellen voor celtherapie realiseren, is het noodzakelijk om hun differentiatie reguleren op consistente en reproduceerbare wijze. Dit rapport beschrijft protocollen op maat bediend EBS behulp microwell plate-technologie te vormen, initiëren differentiatie in de richting van RPE en identificeren van eiwit en gen markers van RPE. Om de in vitro differentiatie synchroniseren, werden homogene groottes van EBS gevormd door bekende aantallen iPS cellen gecentrifugeerd micr...

Geïnduceerde pluripotente stamcellen (iPS) cellen zijn een soort van pluripotente stamcellen afgeleid door het herprogrammeren van volwassen cellen met extrinsieke factoren 1. Daarentegen embryonale stamcellen (SER), een ander soort van pluripotente stamcellen, worden gegenereerd uit de binnenste celmassa van de blastocyst 2-3. Ondanks hun verschillende oorsprong, iPS cellen en SER vergelijkbaar in hun onbeperkte capaciteit tot replicatie in vitro en in hun vermogen om te differentiëren in elk celtype 4-5. Deze kenmerken van iPS-cellen maken ze ideale kandidaten voor toepassingen in gepersonaliseerde regeneratieve geneeskunde. Recent onderzoek inspanningen zijn gericht op het ontwikkelen van robuuste differentiatie protocollen voor het produceren van gespecialiseerde volwassen cellen waaronder netvliespigmentepitheel (RPE) 6-11. Voor potentiële klinische toepassingen van iPS afgeleide cellen, een gerichte differentiatie die specifiek celtype is essentieel. Er zijn verschillende methodengepubliceerd voor gerichte differentiatie van zowel SER&#39;s en iPS-cellen in RPE dat varieert enorm in hun efficiency 6-7, 12-16. We weten nog steeds niet veel van de moleculaire gebeurtenissen die de cel / weefsel lot regeren tijdens de ontwikkeling of differentiatie. De laatste jaren zijn pogingen gedaan om de differentiatie protocol dat de embryologische ontwikkeling zoveel mogelijk kunnen nabootsen ontwikkelen. Tijdens de blastocyst fase vastgelegde populatie van stamcellen bij elkaar in een driedimensionale micro. Zo werden verschillende strategieën toegepast op de ESC / iPS cellen geassembleerd samen te groeien en ze in drie dimensies. Deze stamcellen aggregaten worden embryoid instanties (EBS) genoemd. Studies hebben aangetoond dat EB differentiatie van stamcellen nabootsen vroeg stadium van embryo-ontwikkeling en kan spontaan ontstaan ​​primitieve endoderm op het buitenoppervlak geven. Later, zoals EB ontwikkeling vordert, gedifferentieerde cel fenotypes van drie kiem lijnen weergegeven 17-18. Therefore, EBS gebaseerd differentiatie protocollen hebben veel aandacht voor in vitro differentiatie van ESC / iPS-cellen aangetrokken en zijn een goede kandidaat voor RPE generatie van pluripotente stamcellen 13. EVSA&#39;s kunnen worden gemaakt met behulp van verschillende methoden van ESC / iPS-cellen. Aanvankelijk, EBS werden gemaakt door het afschrapen van aanhangend koloniën en het handhaven ervan in niet-klevende schorsing cultuur. Echter, deze aanpak levert heterogene populatie van EBS die lage reproduceerbaarheid veroorzaakt. Opknoping druppel celcultuur en microwell gebaseerd EVSA vorming zijn andere populaire technieken voor EVSA formatie die homogeen EVSA van gedefinieerde maten die zijn zeer reproduceerbaar opleveren. Bovendien kan de microwell techniek groot aantal aggregaten opleveren met minder inspanning. Differentiatie van cellen binnen EBS wordt geregeld door een multiplex van morfogenische signalen uit de extracellulaire en intracellulaire micro-omgeving. In tegenstelling tot differentiatie in een maolayer formaat, EBS een platform voor complexe samenstel van cellen en intercellulaire signalering optreden 17. Interessant is het aantal pluripotente stamcellen gebruikt om individuele EBS maken werd waargenomen dat het lot van cellen beïnvloeden. Bijvoorbeeld, in een hematopoietische differentiatie studie van de menselijke SER, werd waargenomen dat 500-cel EB bevorderde differentiatie naar myeloïde lijn terwijl 1000-cel EB geduwd richting erythroid lineage 20. In een andere studie, kleinere EBS begunstigd endoderm differentiatie terwijl grotere EVSA gepromoveerd naar neuro-ectoderm differentiatie 11, 17. Deze eerdere studies suggereren sterk dat het aantal ESC / iPS-cellen gebruikt om individuele EBS maken van invloed op de EBS gebaseerde differentiatie naar elke celtypen. Echter, voor zover wij weten, zijn momenteel geen studies die de effecten van EBS grootte zijn toegelicht in de neiging om te differentiëren naar RPE. Het doel van deze studie is om de invloed van de kenmerkenEB grootte op geïnduceerde pluripotente stamcellen (iPS-cellen) - netvliespigmentepitheel (iPS-RPE) differentiatie en om de optimale mobiele nummer aan de EBS te maken voor gerichte differentiatie naar RPE lineage identificeren.

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deriving retinal pigment epithelium (rpe) from induced pluripotent stem (ips) cells by different sizes of embryoid bodies

Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of techniques to reprogram somatic cells into induced pluripotent stem (iPS) cells has generated interest and excitement towards the possibility of customized personal regenerative medicine. However, the efficiency of stem cell differentiation towards a desired lineage remains low. The purpose of this study is to describe a protocol to derive retinal pigment epithelium (RPE) from iPS cells (iPS-RPE) by applying a tissue engineering approach to generate homogenous populations of embryoid bodies (EBs), a common intermediate during in vitro differentiation. The protocol applies the formation of specific size of EBs using microwell plate technology. The methods for identifying protein and gene markers of RPE by immunocytochemistry and reverse-transcription polymerase chain reaction (RT-PCR) are also explained. Finally, the efficiency of differentiation in different sizes of EBs monitored by fluorescence-activated cell sorting (FACS) analysis of RPE markers is described. These techniques will facilitate the differentiation of iPS cells into RPE for future applications.

In dit experiment, iPS cellen werden gekweekt en gedifferentieerd in de RPE geslacht van EBS. EBS van gecontroleerde grootte gevormd met microwell platen. Zoals gezien in figuur 1 EB formatie homogeen in de platen met microputjes. Deze EBS werden vervolgens verzameld en uitgeplaat op platen met 6 putjes (figuur 2). RPE kunnen worden geïdentificeerd door hun klassieke hexagonale morfologie, pigmentatie en expressie van RPE merkers. Na 12 weken van de cultuur...

Watch this Scientific Journal Video about Deriving Retinal Pigment Epithelium RPE from Induced Pluripotent Stem iPS Cells by Different Sizes of Embryoid Bodies at JoVE.com

Deriving Retinal Pigment Epithelium RPE from Induced Pluripotent Stem iPS Cells by Different Sizes of Embryoid Bodies

Afleiden van retinale pigment epitheel (RPE) van induced pluripotent stem (iPS) cellen door verschillende maten van embryoiden Bodies

Het doel van dit rapport is om de protocollen beschrijven aan de netvliespigmentepitheel (RPE) van geïnduceerde pluripotente stamcellen (iPS) cellen met behulp van verschillende maten van embryoid lichamen af ​​te leiden.

Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of ...

Research

JoVE Journal

Developmental Biology

Deriving Retinal Pigment Epithelium (RPE) from Induced Pluripotent Stem (iPS) Cells by Different Sizes of Embryoid Bodies

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

Efficiënte Afleiding van retinale pigment epitheel cellen uit stamcellen

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Analyse van retinoïnezuur geïnduceerde neurale differentiatie van muis embryonale stamcellen in twee en drie-dimensionale embryoiden organen

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

Differentiatie van chondrocyten van perifere bloed afgeleide humane geïnduceerde pluripotente stamcellen

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Chondrogenische Pelletvorming van Koord Bloed afgeleide geïnduceerde Pluripotente Stamcellen

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

Snelle, gerichte differentiatie van de epitheliale cellen van de retinale Pigment van menselijke embryonale of geïnduceerde pluripotente stamcellen

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Engineering Transplantation-suitable Retinal Pigment Epithelium Tissue Derived from Human Embryonic Stem Cells

Transplantatie-geschikt retinale Pigment epitheel Tissue Engineering afgeleid van menselijke embryonale stamcellen

Several pathological conditions of the eye affect the functionality and/or the survival of the retinal pigment epithelium (RPE). These include some forms ...

Generation of Human Primordial Germ Cell-like Cells at the Surface of Embryoid Bodies from Primed-pluripotency Induced Pluripotent Stem Cells

Generatie van menselijke primordiale geslachtscellen-achtige cellen aan de oppervlakte van Embryoid instanties van primer-pluripotent geïnduceerde pluripotente stamcellen

Primordial germ cells (PGCs) are common precursors of all germline cells. In mouse embryos, a founding population of ~40 PGCs are induced from pluripotent ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

Generatie van 3D huid Organoid van koord bloed afkomstige geïnduceerde pluripotente stamcellen

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In Vitro generatie van Somiet derivaten uit menselijk geïnduceerde pluripotente stamcellen-cellen

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

Hemogenic endoteel differentiatie van menselijke pluripotente stamcellen in een voeder-en xeno-vrije gedefinieerde voorwaarde

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous ...