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. Utarbeidelse av kultur reagenser og kultur Plater <ol><li> Forbered mater fritt stamcellekulturmediet ved tilsetning av 100 ml 5 x serumfritt supplement til 400 ml stamcellebasalmedium. Mediet er stabile ved 4 ° C i opp til 2 uker, og ved -20 ° C i 6 måneder. </li><li> Tilsett 10 uM oppløsning av rho-assosiert, coiled-coil inneholdende protein kinase (Rock) inhibitor (Y-27362) med kommersielt tilgjengelig embryoid legeme (EB) dannelse medium. </li><li> Forbered differensieringsmedium ved tilsetning av 0,1 mM β...

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For å realisere den fulle løftet av pluripotente stamceller for celleterapi, er det nødvendig å regulere deres differensiering på en konsekvent og reproduserbar måte. Denne rapporten beskriver protokoller for å danne size-kontrollerte EBS bruker mikro plate-teknologi, starter differensiering mot RPE og identifisere protein og genmarkører av RPE. Å synkronisere in vitro differensiering, ble homogene størrelser av EBS dannet av kjente antall iPS-celler sentrifugert i mikroplater med tvungen aggregering....

Induserte pluripotent stilk (IPS) celler er en type av pluripotent stamcelle avledet ved reprogrammering voksne celler med ytre faktorer 1. I motsetning til dette, embryonale stamceller (ESCS), en annen type av pluripotent stamcelle, blir generert fra den indre cellemasse av blastocysten 2-3. Til tross for deres forskjellige opphav, IPS-celler og ESCs er sammenlignbare i deres ubegrenset kapasitet til å replikere in vitro og i sin evne til å differensiere til en hvilken som helst celletype 4-5. Disse egenskapene til iPS-celler gjør dem ideelle kandidater for applikasjoner i personlig regenerativ medisin. Nyere forskning innsats er fokusert på å utvikle robuste differensiering protokoller for å produsere spesialiserte voksne celler inkludert retinal pigment epitel (RPE) 6-11. For potensielle kliniske anvendelser av iPS avledet celler, er essensielt en rettet differensiering for den spesifikke celletype. Det finnes ulike fremgangsmåterpublisert for rettet differensiering av både ESCs og iPS-celler i RPE som varierer mye i deres effektivitet 6-7, 12-16. Vi vet fortsatt ikke mange av de molekylære hendelser som styrer celle / vev skjebne under utvikling eller differensiering. I de senere årene har det vært gjort for å utvikle differensiering protokoll som kan etterligne den embryologiske utviklingen så mye som mulig. Under blastocyst fase-iverk populasjon av stamceller er sammen i et tredimensjonalt mikromiljøet. Så ble forskjellige strategier anvendes for å gjøre de ESC / IPS-celler montert sammen og dyrke dem i tre dimensjoner. Disse stamcelle aggregater kalles embryoid organer (EBS). Studier har vist at EB differensiering av stamceller ligne tidlig stadium av embryo- utvikling og kan spontant gi opphav til primitive endoderm på sin ytre overflate. Senere, som EB utvikling utvikler seg, differensierte celle fenotyper av alle tre bakterie linjene vises 17-18. Therefore, har EBS basert differensiering protokoller tiltrakk seg mye oppmerksomhet for in vitro differensiering av ESC / iPS-celler og er en god kandidat for RPE generasjon fra pluripotente stamceller 13. EBS kan gjøres ved hjelp av flere metoder fra ESC / iPS-celler. Utgangspunktet, EBS ble gjort ved å skrape av heft kolonier og opprettholde dem i ikke-tilhenger suspensjon kultur. Imidlertid gir denne tilnærmingen heterogen befolkning på EBS som forårsaker lav reproduserbarhet. Hengende dråpe cellekultur og mikro basert EBS formasjon er andre populære teknikker for EBS formasjon som gir homogene EBS av definerte størrelser som er reproduserbar. Videre kan mikro teknikken gir stort antall aggregater med mindre anstrengelse. Differensiering av celler i EBS er regulert av en multipleks av morphogenic signaler fra den ekstracellulære og intracellulære mikromiljøet. I motsetning til differensiering i en Manolayer format, EBS gi en plattform for kompleks sammensetning av celler og intersignale å skje 17. Interessant nok var antallet pluripotente stamceller som brukes til å lage enkelt EBS observert å påvirke skjebnen til cellene. For eksempel, i et hematopoetisk differensiering undersøkelse av menneskelige ESCs, ble det observert at 500-celle EB fremmet differensiering mot myeloide cellelinjen, mens 1000-celle EB skjøvet mot erytroid herkomst 20. I en annen studie, mindre EBS favoriserte endoderm differensiering mens større EBS fremmet mot neuro-ektoderm differensiering 11, 17. Disse siste studiene tyder på at antall ESC / iPS-celler som brukes til å lage individuelle EBS påvirke EBS basert differensiering til eventuelle celletyper. Men til vår kunnskap, finnes det ingen aktuelle studier som har belyst virkningen av EBS størrelse i sin tilbøyelighet til å skille mot RPE. Målet med denne studien er å karakterisere påvirkning avEB størrelse på indusert pluripotent stilk (iPS-celler) - retinal pigment epitel (iPS-RPE) differensiering og å identifisere den optimale celle nummer for å gjøre EBS for rettet differensiering mot RPE avstamning.

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			<td>Abcam</td>
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			<td height="21" style="height:21px;width:239px;">Mouse&#160; Anti-MITF antibody</td>
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			<td>MS-772-P</td>
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			<td height="21" style="height:21px;width:239px;">Rabbit Anti-ZO-1 antibody</td>
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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 dette eksperimentet, IPS-celler ble dyrket og differensiert i RPE-linjen fra EBS. EBS av kontrollerte størrelser ble dannet ved hjelp av mikroplater. Som vist i figur 1 EB formasjonen var homogen i mikrobrønnplater. Disse EBS ble deretter oppsamlet og utsådd på 6-brønners plater (figur 2). RPE kan identifiseres ved deres klassiske heksagonal morfologi, pigmentering, og ekspresjon av RPE-markører. Etter 12 uker med kultur, hadde 200-celle EBS utviklet...

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

Stammer Netthinne Pigment Epitel (RPE) fra Induced pluripotent Stem (IPS) Cells av forskjellige størrelser av embryoid Bodies

Målet med denne rapporten er å beskrive protokoller å utlede retinal pigment epitel (RPE) fra indusert pluripotent stilk (iPS-celler) ved hjelp av forskjellige størrelser av embryoid organer.

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

Effektiv Utledning av retinal pigment epitelet Celler fra stamceller

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 av retinsyre-indusert Neural Differensiering av mus embryonale stamceller i to og tre-dimensjonale embryoidlegemer

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

Differensiering av kondrocytter fra perifert blod-avledede humane inducerte pluripotente 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 pelletsdannelse fra Cord Blood-avledede Induced Pluripotent 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

Rask, rettet differensiering av netthinnens Pigment epitelceller fra menneskelige embryonale eller indusert Pluripotent 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 transplantasjon-passer netthinnens Pigment epitel vev avledet fra menneskelige embryonale 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

Generasjon av menneskelige Primordial bakterie celle-lignende celler på overflaten av Embryoid organer fra primet pluripotency indusert Pluripotent 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

Generasjon 3D huden Organoid fra ledningen blod-avledet indusert Pluripotent 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 generasjon av Somite derivater fra menneske indusert stamceller Pluripotent

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 endotelet differensiering fra Human Pluripotent stamceller i en mater-og Xeno-fri definert tilstand

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