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. Beredning av reagens kultur och odlingsplattor <ol><li> Förbered feeder-fria stammen cellodlingsmedium genom att tillsätta 100 ml av 5x serumfritt tillägg till 400 ml av stamceller basmedium. Mediet är stabil vid 4 ° C i upp till två veckor och vid -20 ° C under 6 månader. </li><li> Lägg 10 iM lösning av rho-associerad, ringlad-spole innehållande proteinkinas (Rock) hämmare (Y-27362) för att kommersiellt tillgänglig embryoid kropp (EB) bildande mediet. </li><li> Förbered differentieringsmedium genom ...

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För att förverkliga utsikterna av pluripotenta stamceller för cellterapi, är det nödvändigt att reglera deras differentiering på ett konsekvent och reproducerbart sätt. Denna rapport beskriver protokoll för att bilda storleksstyrda EB använder mikrobrunnsplatta teknik startar differentiering mot RPE och identifiera protein och genmarkörer av RPE. För att synkronisera differentiering in vitro, var homogena storlekar av EB bildas av kända antal iPS-celler centrifuge i mikroplattor med tvångs aggrege...

Inducerade pluripotenta stamceller (iPS-celler) är en typ av pluripotenta stamceller härstammar genom omprogrammering vuxna celler med yttre faktorer 1. I motsats, embryonala stamceller (ESCS), en annan typ av pluripotenta stamceller, genereras från den inre cellmassan av blastocysten 2-3. Trots deras olika ursprung, iPS-celler och ekonomiska och sociala råd är jämförbara i sin obegränsade förmåga att föröka sig in vitro och in deras förmåga att differentiera till någon celltyp 4-5. Dessa egenskaper hos iPS-celler gör dem idealiska kandidater för applikationer i personlig regenerativ medicin. Senaste forskningsinsatser är inriktade på att utveckla robusta differentieringsprotokoll för att producera specialiserade vuxna celler inklusive retinal pigment epitel (RPE) 6-11. För potentiella kliniska tillämpningar av iPS härledda celler, är väsentligt en riktad differentiering för den specifika celltyp. Det finns olika metoderpublicerat för riktad differentiering av både ekonomiska och sociala råd och iPS-celler i RPE som varierar stort i deras effektivitet 6-7, 12-16. Vi vet fortfarande inte många av de molekylära händelser som styr cellen / vävnaden öde under utveckling eller differentiering. Under de senaste åren har ansträngningar gjorts för att utveckla differentiering protokoll som kan härma embryologiska utveckling så mycket som möjligt. Under blastocysten fasen obekräftat population av stamceller är tillsammans i en tredimensionell mikroomgivning. Så har olika strategier för att göra dem ESC / iPS-celler monterade ihop och odla dem i tre dimensioner. Dessa stamcells aggregat kallas embryoidkroppar (EBS). Studier har visat att EB differentiering av stamceller härma tidigt stadium av embryots utveckling och kan spontant ge upphov till primitiva endoderm på sin utsida. Senare, när EB utveckling fortskrider, differentierade cell fenotyper av alla tre bakterie härstamningar visas 17-18. Therefore, har EBS baserade differentiering protokoll fått mycket uppmärksamhet för in vitro differentiering av ESC / iPS-celler och är en bra kandidat för RPE generering från pluripotenta stamceller 13. EB kan göras med hjälp av flera metoder från ESC / iPS-celler. Inledningsvis var EB gjordes genom att skrapa bort fastsittande kolonier och behålla dem i icke-vidhäftande suspensionskultur. Dock ger detta tillvägagångssätt heterogen population av EBS som orsakar låg reproducerbarhet. Hängande droppe cellodling och mikro baserade EB bildning är andra populära tekniker för EB formation som ger homogena EB av definierade storlekar som är mycket reproducerbara. Vidare kan mikrotekniken ge stort antal aggregat med mindre ansträngning. Differentiering av celler inom EBS regleras av en multiplex av morfogena signaler från den extracellulära och intracellulära mikromiljö. I motsats till differentiering i ett monolayer format, EBS ger en plattform för komplex sammansättning av celler och intercellulära signalering ske 17. Intressant nog var antalet av pluripotenta stamceller som används för att göra individuella EB observeras att påverka ödet för cellerna. Till exempel, i en hematopoetisk differentiering studiet av mänskliga ESCs, observerades det att 500-celler EB främjade differentiering mot myeloid härkomst medan 1000-cell EB skjuts mot erytroid härstamning 20. I en annan studie, mindre EB gynnade endoderm differentiering medan större EB främjas mot neuro ektoderm differentiering 11, 17. Dessa tidigare studier tyder starkt på att antalet ESC / iPS-celler används för att göra individuella EB påverkar EBS baserade differentiering eventuella celltyper. Men till vår kunskap, det finns inga aktuella studier som har klar effekten av EBS storlek i dess benägenhet att skilja mot RPE. Målet med denna studie är att karakterisera påverkan avEB storlek på inducerade pluripotenta stammen (iPS-celler) - retinal pigment epitel (iPS-RPE) differentiering och att identifiera den optimala antalet celler att göra EBS för riktad differentiering mot RPE härstamning.

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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.

I detta experiment, iPS-celler odlades och differentierats till RPE härstamning från EBS. EB av kontrollerade storlekar bildades med användning av mikrobrunnplattor. Såsom framgår av fig 1 EB Bildningen var homogena i mikrobrunnplattorna. Dessa EB uppsamlades därefter och ströks ut på 6-brunnars plattor (fig 2). RPE kan identifieras genom sin klassiska sexkantiga morfologi, pigmentering och uttryck av RPE markörer. Efter 12 veckors odling hade 200-c...

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

Härrör näthinnans pigmentepitel (RPE) från inducerade pluripotenta stamceller (iPS) Celler med olika storlekar av Embryoid organ

Syftet med denna rapport är att beskriva de protokoll att härleda näthinnans pigmentepitel (RPE) från inducerade pluripotenta stammen (iPS-celler) med olika storlekar av embryoidkroppar.

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

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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

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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

Analys av retinsyra-inducerade Neural differentiering av Mouse embryonala stamceller i två och tre-dimensionella Embryoid organ

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

Differentierande kondrocyter från perifert blod härledda humana inducerade plöripotenta stamceller

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

Kondrogen pelletsbildning från Cord Blood-härledda inducerade Pluripotenta stamceller

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

Snabb, regisserad differentiering av Retinal Pigment epitelial celler från mänskliga embryonala eller inducerade pluripotenta stamceller

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

Engineering Transplantation-lämplig Retinal Pigment epitel vävnad härrör från mänskliga embryonala stamceller

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

Generation av mänskliga primordiala könsceller-liknande celler på ytan av Embryoid kroppar från primas-pluripotency inducerade pluripotenta stamceller

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

Generation av 3D hud Organoid från sladd blod-derived inducerade pluripotenta stamceller

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

I Vitro Generation av Somite derivat från mänskliga inducerade stamceller pluripotenta

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

Hemogena Endotelium differentiering från mänskliga pluripotenta stamceller i en feeder-och Xeno-fritt definierade villkor

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