Følger retinale pigmentepitel (RPE) fra induceret Pluripotente Stem (IPS) Celler ved forskellige størrelser af embryoide legemer
Summary
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.
Abstract
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.
Introduction
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'er kan gøres ved hjælp af flere metoder fra ESC / iPS-celler. Oprindeligt blev EB'er fremstillet ved afskrabning adhærerende kolonier og fastholde dem i ikke-klæbende suspensionskultur. Men denne fremgangsmåde giver heterogen population af EB'er, der forårsager lav reproducerbarhed. Hængende drop cellekultur og mikrobrond baseret EB'er dannelse er andre populære teknikker til EB'er dannelse, der giver homogene EB'er af definerede størrelser, der er meget reproducerbare. Endvidere kan mikrobrønd teknik gav stort antal aggregater med mindre indsats.
Differentiering af celler inden EB'er reguleres af en multiplex af morfogene signaler fra det ekstracellulære og intracellulære mikromiljø. I modsætning til differentiering i en monolayer format, EB'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'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'er stillede endoderm differentiering mens større EB'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'er påvirker EBS differentiering eventuelle celletyper. Men så vidt vi ved, er der ingen aktuelle undersøgelser, der har belyst effekten af EB'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.
Protocol
Representative Results
Discussion
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'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'er dannet af kendte antal iPS celler centrifugeret i…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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 Muñiz, 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).
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| mTeSR1 media + 5X supplement | Stem Cell Technologies | 5850 | |
| Y-27632 (Rock Inhibitor) | Stem Cell Technologies | 72304 | |
| DMEM/F12 | Life Technologies | 11330-032 | |
| 2-Mercaptoethanol | Sigma | M-7154 | |
| Non essential amino acids | Hyclone(Fisher) | SH30853.01 | |
| Knockout serum replacement | Life Technologies | 10828-028 | |
| Gentamicin | Life Technologies | 15750-060 | |
| L-Glutamine | Life Technologies | 25030-081 | |
| MEM media | Life Technologies | 10370-021 | |
| N1 supplement | Sigma | N-6530-5ML | |
| Taurine | Sigma | T-8691-25G | |
| Hydrocortisone | Sigma | H0888-1G | |
| Fetal bovine serum | Hyclone(Fisher) | SH3008803HI | |
| Triiodo-l-thyronine sodium salt | Sigma | T6397 | |
| Sodium hydroxide | Sigma | S5881 | |
| Dispase | Life Technologies | 17105-041 | |
| Matrigel | BD Biosciences | 354277 | |
| Phosphate buffered saline | Hyclone(Fisher) | 10010-023 | |
| Aggrewell 400 plate | Stem Cell Technologies | 27940 | |
| AggreWell medium | Stem Cell Technologies | 5893 | |
| Accutase | Stem Cell Technologies | 7920 | |
| BD Cytofix/Cytoperm Fixation/Permeabilization Kit | BD Biosciences | 554714 | |
| Mouse Anti-PAX6 antibody | Developmental Studies Hybridoma Bank | ||
| Rabbit Anti- RX antibody | Abcam | Ab23340 | |
| Mouse Anti-MITF antibody | Thermo Scientific | MS-772-P | |
| Rabbit Anti-ZO-1 antibody | Invitrogen | 40-2200 | |
| RNeasy plus mini kit | Qiagen | 74134 | |
| PCR master mix | promega | M7502 | |
| High capacity RNA to c DNA kit | Life Technologies | 4387406 | |
References
- Takahashi, K., et al. Induction of pluripotent stem cells from fibroblast cultures. Nat Protoc. 2 (12), 3081-3089 (2007).
- Reubinoff, B. E., et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol. 18 (4), 399-404 (2000).
- Thomson, J. A., et al. Embryonic stem cell lines derived from human blastocysts. Science. 282 (5391), 1145-1147 (1998).
- Takahashi, K., Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 126 (4), 663-676 (2006).
- Yu, J., et al. Induced pluripotent stem cell lines derived from human somatic cells. Science. 318 (5858), 1917-1920 (2007).
- Buchholz, D. E., et al. Derivation of functional retinal pigmented epithelium from induced pluripotent stem cells. Stem Cells. 27 (10), 2427-2434 (2009).
- Ukrohne, T. U., et al. Generation of retinal pigment epithelial cells from small molecules and OCT4 reprogrammed human induced pluripotent stem cells. Stem Cells Transl Med. 1 (2), 96-109 (2012).
- Subba, R. M., Sasikala, M., Nageshwar, R. D. Thinking outside the liver: induced pluripotent stem cells for hepatic applications. World J Gastroenterol. 19 (22), 3385-3396 (2013).
- Yoshida, Y., Yamanaka, S. iPS cells: a source of cardiac regeneration. J Mol Cell Cardiol. 50 (2), 327-332 (2011).
- Mak, S. K., et al. Small molecules greatly improve conversion of human-induced pluripotent stem cells to the neuronal lineage. Stem Cells Int. 2012, 140427 (2012).
- Bauwens, C. L., et al. Control of human embryonic stem cell colony and aggregate size heterogeneity influences differentiation trajectories. Stem Cells. 26 (9), 2300-2310 (2008).
- Cour la, M., Tezel, T. The Retinal Pigment Epithelium. Advances in Organ Biology. 10, 253-273 (2006).
- Vaajassari, V., et al. Toward the defined and xeno-free differentiation of functional human pluripotent stem cell-derived retinal pigment epithelial cells. Mol Vis. 17, 558-575 (2011).
- Carr, A. J., et al. Protective effects of human iPS-derived retinal pigment epithelium cell transplantation in the retinal dystrophic rat. PLoS One. 4 (12), e8152 (2009).
- Muniz, A., et al. Retinoid uptake, processing, and secretion in human iPS-RPE support the visual cycle. Invest Ophthalmol Vis Sci. 55 (1), 198-209 (2014).
- Meyer, J. S., et al. Modeling early retinal development with human embryonic and induced pluripotent stem cells. Proc Natl Acad Sci U S A. 106 (39), 16698-16703 (2009).
- Bratt-Leal, A. M., Carpenedo, R. L., McDevitt, T. C. Engineering the embryoid body microenvironment to direct embryonic stem cell differentiation. Biotechnol Prog. 25 (1), 43-51 (2009).
- Kurosawa, H. Methods for inducing embryoid body formation: in vitro differentiation system of embryonic stem cells. J Biosci Bioeng. 103 (5), 389-398 (2007).
- Itskovitz-Eldor, J., et al. Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol Med. 6 (2), 88-95 (2000).
- Ng, E. S., et al. Forced aggregation of defined numbers of human embryonic stem cells into embryoid bodies fosters robust, reproducible hematopoietic differentiation. Blood. 106 (5), 1601-1603 (2005).
- Maminishkis, A., et al. Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue. Invest Ophthalmol Vis Sci. 47 (8), 3612-3624 (2006).
