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. Fremstilling af kultur Reagenser og dyrkningsplader <ol><li> Forbered fødefri stamcelle dyrkningsmediet ved tilsætning af 100 ml af 5x serumfrit supplement til 400 ml stamceller basal medium. Mediet er stabilt ved 4 ° C i op til 2 uger og ved -20 ° C i 6 måneder. </li><li> Tilsæt 10 uM opløsning af rho-associeret, coiled-coil indeholdende proteinkinase (Rock) inhibitor (Y-27362) til kommercielt tilgængeligt embryoid legeme (EB) dannelse medium. </li><li> Forbered differentiering medium ved tilsætning af 0,...

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For at realisere det fulde løfte om pluripotente stamceller til celleterapi, er det nødvendigt at regulere deres differentiering på en konsekvent og reproducerbar måde. Denne rapport beskriver protokoller til at danne størrelse-kontrollerede EB&#39;er der anvender mikrobrøndsplade teknologi, indlede differentiering mod RPE og identificere protein- og gen-markører for RPE. For at synkronisere den in vitro differentiering blev homogene størrelser EB&#39;er dannet af kendte antal iPS celler centrifugeret i...

Induceret pluripotente stamceller (IPS) celler er en type af pluripotente stamcelle afledt ved omprogrammering voksne celler med ydre faktorer 1. I modsætning embryonale stamceller (ESC), en anden type af pluripotente stamcelle, er genereret ud fra den indre cellemasse af blastocysten 2-3. Trods deres forskellige oprindelser IPS-celler og økonomiske og sociale råd er sammenlignelige i deres ubegrænset kapacitet til at replikere in vitro og i deres evne til at differentiere til en hvilken som helst celletype 4-5. Disse karakteristika iPS celler gør dem ideelle kandidater til applikationer i personlig regenerativ medicin. Nyere forskning er fokuseret på at udvikle robuste differentiering protokoller til at lave specialiserede voksne celler, herunder retinal pigment epitel (RPE) 6-11. For potentielle kliniske anvendelser af iPS afledte celler, en rettet differentiering for den specifikke celletype er afgørende. Der er forskellige metoderoffentliggjort rettet differentiering af både økonomiske og sociale råd iPS celler i RPE, der varierer meget i deres effektivitet 6-7, 12-16. Vi ved stadig ikke mange af de molekylære begivenheder, der styrer celler / væv skæbne under udvikling eller differentiering. I de senere år er der gjort en indsats for at udvikle den differentiering protokol, der kan efterligne den embryologiske udvikling så meget som muligt. I blastocyst-fase uncommitted population af stamceller er sammen i et tredimensionelt mikromiljø. Så blev forskellige strategier for at gøre ESC / iPS celler samles sammen og dyrke dem i tre dimensioner. Disse stamceller aggregater kaldes embryoide organer (EbS). Undersøgelser har vist, at EB differentiering af stamceller efterligne tidligt udviklingstrin embryo og kan spontant medføre primitive endoderm på dens ydre overflade. Senere, da EB udvikling skrider frem, differentierede cellefænotyper alle tre kim slægter vises 17-18. Therefore har EBS differentiering protokoller tiltrukket en masse opmærksomhed til in vitro differentiering af ESC / iPS-celler og er en god kandidat til RPE generation fra pluripotente stamceller 13. EB&#39;er kan gøres ved hjælp af flere metoder fra ESC / iPS-celler. Oprindeligt blev EB&#39;er fremstillet ved afskrabning adhærerende kolonier og fastholde dem i ikke-klæbende suspensionskultur. Men denne fremgangsmåde giver heterogen population af EB&#39;er, der forårsager lav reproducerbarhed. Hængende drop cellekultur og mikrobrond baseret EB&#39;er dannelse er andre populære teknikker til EB&#39;er dannelse, der giver homogene EB&#39;er af definerede størrelser, der er meget reproducerbare. Endvidere kan mikrobrønd teknik gav stort antal aggregater med mindre indsats. Differentiering af celler inden EB&#39;er reguleres af en multiplex af morfogene signaler fra det ekstracellulære og intracellulære mikromiljø. I modsætning til differentiering i en monolayer format, EB&#39;er skabe en platform for kompleks samling af celler og intercellulære signalering at forekomme 17. Interessant nok blev antallet af pluripotente stamceller, der anvendes til at foretage individuelle EB&#39;er observeret at påvirke skæbne af celler. For eksempel i en hæmatopoietisk differentiering undersøgelse af humane ESC, blev det observeret, at 500-celle EB fremmet differentiering i retning myeloid afstamning henviser 1.000 celle EB skubbes mod erythroid afstamning 20. I en anden undersøgelse mindre EB&#39;er stillede endoderm differentiering mens større EB&#39;er fremmes i retning neuro-ectoderm differentiering 11, 17. Disse tidligere undersøgelser tyder stærkt på, at antallet af ESC / iPS celler, der anvendes til at foretage individuelle EB&#39;er påvirker EBS differentiering eventuelle celletyper. Men så vidt vi ved, er der ingen aktuelle undersøgelser, der har belyst effekten af ​​EB&#39;er størrelse i sin tilbøjelighed til at differentiere i retning RPE. Målet med denne undersøgelse er at karakterisere indflydelseEB størrelse på induceret pluripotente stamceller (IPS) celler - retinal pigment epitel (IPS-RPE) differentiering og til at identificere den optimale celleantal at gøre EBS for rettet differentiering mod RPE afstamning.

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			<td height="21" style="height:21px;">mTeSR1 media + 5X supplement</td>
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			<td>5850</td>
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			<td height="20" style="height:20px;">Y-27632 (Rock Inhibitor)</td>
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			<td>72304</td>
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			<td height="21" style="height:21px;">DMEM/F12</td>
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			<td>11330-032</td>
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			<td height="21" style="height:21px;width:239px;">Non essential amino acids</td>
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			<td height="21" style="height:21px;width:239px;">Knockout serum replacement</td>
			<td style="width:183px;">Life Technologies</td>
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			<td style="width:183px;">Life Technologies</td>
			<td style="width:140px;">15750-060</td>
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			<td height="21" style="height:21px;width:239px;">L-Glutamine</td>
			<td style="width:183px;">Life Technologies</td>
			<td style="width:140px;">25030-081</td>
			<td></td>
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			<td height="21" style="height:21px;width:239px;">MEM media</td>
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			<td style="width:183px;">Sigma</td>
			<td style="width:140px;">T-8691-25G</td>
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			<td style="width:140px;">H0888-1G</td>
			<td></td>
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			<td height="21" style="height:21px;width:239px;">Fetal bovine serum</td>
			<td style="width:183px;">Hyclone(Fisher)</td>
			<td>SH3008803HI</td>
			<td></td>
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			<td height="21" style="height:21px;">Triiodo-l-thyronine sodium&#160; salt</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:140px;">T6397</td>
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			<td style="width:183px;">Sigma</td>
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			<td></td>
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			<td height="21" style="height:21px;width:239px;">Dispase</td>
			<td style="width:183px;">Life Technologies</td>
			<td>17105-041</td>
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			<td height="21" style="height:21px;width:239px;">Phosphate buffered saline</td>
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			<td>10010-023</td>
			<td></td>
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			<td height="21" style="height:21px;width:239px;">Aggrewell 400 plate</td>
			<td style="width:183px;">Stem Cell Technologies</td>
			<td>27940</td>
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			<td>Stem Cell Technologies</td>
			<td>5893</td>
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			<td height="21" style="height:21px;width:239px;">Accutase</td>
			<td>Stem Cell Technologies</td>
			<td>7920</td>
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			<td height="42" style="height:42px;width:239px;">BD Cytofix/Cytoperm Fixation/Permeabilization Kit</td>
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			<td>554714</td>
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			<td height="21" style="height:21px;width:239px;">Mouse Anti-PAX6 antibody</td>
			<td colspan="2">Developmental Studies Hybridoma Bank</td>
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			<td height="21" style="height:21px;width:239px;">Rabbit Anti- RX antibody</td>
			<td>Abcam</td>
			<td>Ab23340</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Mouse&#160; Anti-MITF antibody</td>
			<td>Thermo Scientific</td>
			<td>MS-772-P</td>
			<td></td>
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			<td height="21" style="height:21px;width:239px;">Rabbit Anti-ZO-1 antibody</td>
			<td>Invitrogen</td>
			<td>40-2200</td>
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			<td>Qiagen</td>
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			<td height="21" style="height:21px;width:239px;">High capacity RNA to c DNA kit</td>
			<td style="width:183px;">Life Technologies</td>
			<td>4387406</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 eksperiment iPS-celler blev dyrket og differentieret til RPE afstamning fra EB&#39;er. EB&#39;er af kontrollerede størrelser blev dannet under anvendelse mikrobrøndsplader. Som det ses i figur 1 EB formation var homogen i mikrobrøndsplader. Disse EB&#39;er blev derefter opsamlet og udpladet på plader med 6 brønde (figur 2). RPE kan identificeres ved deres klassiske sekskantet morfologi, pigmentering, og udtryk for RPE markører. Efter 12 ugers d...

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

Følger retinale pigmentepitel (RPE) fra induceret Pluripotente Stem (IPS) Celler ved forskellige størrelser af embryoide legemer

Formålet med denne rapport er at beskrive de protokoller til at udlede det retinale pigment epitel (RPE) fra inducerede pluripotente stamceller (IPS) celler ved hjælp af forskellige størrelser af embryoide 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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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

Effektiv Udledning af retinale pigmentepitel 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 af retinoidsyre-induceret Neural Differentiation af muse embryonale stamceller i to og tre-dimensionelle embryoide legemer

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

Differentierende chondrocytter fra perifert blodafledte humane inducerede 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 ledningsblod-afledte inducerede pluripotente 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

Hurtig, instrueret differentiering af retinale Pigment epitel celler fra menneskelige embryonale eller induceret pluripotente 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-egnet retinale Pigment epitelvæv stammer 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

Generation af menneskelige primordiale kønsceller-lignende celler på overfladen af Embryoid organer fra primet pluripotency induceret pluripotente 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 af 3D hud Organoid fra ledningen blod-afledte induceret pluripotente 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 af Somite derivater fra menneskelige induceret celler pluripotente stamceller

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

Hemogen endothelium differentiering fra humane pluripotente stamceller i en feeder-og Xeno-fri defineret tilstand

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