Indsamling af serum- og Feeder-fri mus embryonale stamceller-conditioned Medium for en celle-fri tilgang
Summary
Denne protokol giver en metode til indsamling af musen embryonale stamceller (Mesc) -konditioneret medium (Mesc-CM) afledt af serum (føtalt bovint serum, FBS) – og feeder (mus embryonale fibroblaster, MEF'er) -fri betingelser for en celle -fri tilgang. Det kan være anvendelig til behandling af aldring og aldring-associerede sygdomme.
Abstract
The capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate various cell types has opened new avenues in the field of regenerative medicine. However, despite their benefits, the tumorigenic potential of ESCs and iPSCs has long been a barrier for clinical applications. Interestingly, it has been shown that ESCs produce several soluble factors that can promote tissue regeneration and delay cellular aging, suggesting that ESCs and iPSCs can also be utilized as a cell-free intervention method. Therefore, the method for harvesting mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) with minimal contamination of serum components (fetal bovine serum, FBS) and feeder cells (mouse embryonic fibroblasts, MEFs) has been highly demanded. Here, the present study demonstrates an optimized method for the collection of mESC-CM under serum- and feeder-free conditions and for the characterization of mESC-CM using senescence-associated multiple readouts. This protocol will provide a method to collect pure mESC-specific secretory factors without serum and feeder contamination.
Introduction
Målet med denne protokol er at indsamle mus embryonale stamceller (Mesc) -konditioneret medium (Mesc-CM) fra serum- og feeder-fri dyrkningsbetingelser og at beskrive sine biologiske funktioner.
Generelt embryonale stamceller (EKSF) har et stort potentiale for regenerativ medicin og celleterapi på grund af deres pluripotens og evne til selv-fornyelse 1-3. Men den direkte transplantation af stamceller har adskillige begrænsninger, såsom immun afstødning og tumordannelse 4,5. Derfor kan en celle-fri tilgang giver en alternativ terapeutisk strategi for regenerativ medicin og aldrende interventioner 6,7.
Ældning ses som en cellulær modstykke til den aldrende væv og organer, kendetegnet ved en permanent tilstand af vækst anholdelse, ændret celle fysiologi og adfærd. Aldring er den vigtigste risikofaktor for udbredte sygdomme, herunder kræft, hjerte-karsygdomme, type 2-diabetes, og neurodegeneration 8. En af de åbenlyse karakteristika aldring er faldet i den regenerative potentiale af væv, som er forårsaget af stamceller aldring og udmattelse 9. Mange betydningsfulde undersøgelser har vist farmakologiske molekyler, såsom rapamycin 9, resveratrol 10, og metformin 11, og blodbårne systemiske faktorer, nemlig GDF11 12, der har evnen til konsekvent at forsinke ældning og forlænge levetiden.
I den foreliggende undersøgelse har Mesc-CM blevet høstet uden serum (føtalt bovint serum, FBS) og føder (muse embryonale fibroblaster, MEF'er) lag for at udelukke kontaminering af serum faktorer og sekretoriske faktorer fra MEF'er. Disse betingelser er tilladt for en serum- og feeder-fri CM, der dermed muliggjort præcis identifikation af Mesc-specifikke sekretoriske faktorer.
Denne foreslåede protokol er højeffektive, forholdsvis omkostningseffektive og letat operere. Denne teknik giver indblik i karakteriseringen af Mesc-afledte opløselige faktorer, som kan mediere en anti-senescens virkning, som kan anvendes til udvikling af et sikkert og potentielt fordelagtig cellefri terapeutisk tilgang mod indgreb for aging-associerede sygdomme og andre regenerativ behandlinger.
Protocol
Representative Results
Discussion
For en vellykket indsamling af serum- og feeder-fri Mesc-CM, bør tages hensyn til følgende forslag. Den mest kritiske faktor er at bruge tidlige passage mESCs for indsamling af Mesc-CM. Tidligere er det blevet vist, at tidlig passage Mesc-CM har bedre anti-aging effekter i forhold til sene passage mESCs. Passagen antal mESCs er blevet rapporteret til at påvirke deres udviklingsmæssige potentiale 16 og pluripotens 17.
Mens yderligere forskning er nødvendig for at an…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Denne forskning blev støttet af Basic Science Research Program (2013R1A1A2060930) og Medical Research Center Program (2015R1A5A2009124) gennem Grundforskningsfonden Korea (NRF), finansieret af Ministeriet for Videnskab, IKT, og fremtidig planlægning. Denne forskning understøttes også af en start-up driftstilskud fra Hospital for Sick Children (HK Sung). Vi vil gerne takke Laura Barwell og Sarah JS Kim for deres fremragende hjælp i at redigere dette manuskript og Dr. Andras Nagy for at give G4 Mesc linje.
Materials
| DMEM | Invitrogen | #11960-044 | |
| FBS | Invitrogen | #30044333 | 20%, ES cell quality |
| Penicillin and streptomycin | Invitrogen | #15140 | 50units/ml penicillin and 50mg/ml strepto |
| -mycin. | |||
| L-glutamine | Invitrogen | #25030 | 2mM |
| Nonessential amino acids (NEAA) | Invitrogen | #11140 | 100uM |
| β-mercaptoethanol | Sigma | #M3148 | 100uM |
| Leukemia inhibitory factor | Millipore | #ESG1107 | 100units/ml |
| OPTI-MEM | Invitrogen | #22600 | |
| X-gal | Sigma | #B4252 | 1mg/ml |
| Paraformaldehyde (PFA) | Sigma | P6148 | 3.70% |
| Dimethylformamide (DMF) | Sigma | #D4551 | |
| Potassium ferricyanide | Aldrich | #455946 | 5mM |
| potassium ferrocyanide | Aldrich | #455989 | 5mM |
| NaCl | Sigma | #S7653 | 150mM |
| MgCl2 | Sigma | #M2393 | 2mM |
| Mytomycin C | Sigma | #M4287 | 10ug/ml |
| Propidium iodide | Sigma | #P4170 | 50ug/ml |
| TRIzol | Ambion | #15596018 | |
| M-MLV reverse transcript-tase | Promega | #M170B | |
| Power SYBR Green PCR master mix | Applied Biosystems | #4367659 | |
| HDFs, NHDF-Ad-Der-Fibroblast | LONZA | #CC-2511 | |
| Bottle top filter, | Corning | #430513 | 0.2μm |
References
- Thomson, J. A., et al. Embryonic stem cell lines derived from human blastocysts. Science. 282, 1145-1147 (1998).
- Lavasani, M., et al. Muscle-derived stem/progenitor cell dysfunction limits healthspan and lifespan in a murine progeria model. Nat Commun. 3, 608 (2012).
- Woo, D. H., et al. Direct and indirect contribution of human embryonic stem cell-derived hepatocyte-like cells to liver repair in mice. Gastroenterology. 142, 602-611 (2012).
- Lee, A. S., Tang, C., Rao, M. S., Weissman, I. L., Wu, J. C. Tumorigenicity as a clinical hurdle for pluripotent stem cell therapies. Nat Med. 19, 998-1004 (2013).
- Moon, S. H., et al. A system for treating ischemic disease using human embryonic stem cell-derived endothelial cells without direct incorporation. Biomaterials. 32, 6445-6455 (2011).
- Tongers, J., Roncalli, J. G., Losordo, D. W. Therapeutic angiogenesis for critical limb ischemia: microvascular therapies coming of age. Circulation. 118, 9-16 (2008).
- Lazarous, D. F., et al. Basic fibroblast growth factor in patients with intermittent claudication: results of a phase I trial. J Am Coll Cardiol. 36, 1239-1244 (2000).
- Adams, P. D. Healing and hurting: molecular mechanisms, functions, and pathologies of cellular senescence. Mol Cell. 36, 2-14 (2009).
- Harrison, D. E., et al. Rapamycin fed late in life extends lifespan in genetically heterogeneous mice. Nature. 460, 392-395 (2009).
- Baur, J. A., Ungvari, Z., Minor, R. K., Le Couteur, D. G., de Cabo, R. Are sirtuins viable targets for improving healthspan and lifespan. Nat Rev Drug Discov. 11, 443-461 (2012).
- Martin-Montalvo, A., et al. Metformin improves healthspan and lifespan in mice. Nat Commun. 4, 2192 (2013).
- Loffredo, F. S., et al. Growth differentiation factor 11 is a circulating factor that reverses age-related cardiac hypertrophy. Cell. 153, 828-839 (2013).
- Jozefczuk, J., Drews, K., Adjaye, J. Preparation of mouse embryonic fibroblast cells suitable for culturing human embryonic and induced pluripotent stem cells. J Vis Exp. , (2012).
- Debacq-Chainiaux, F., Erusalimsky, J. D., Campisi, J., Toussaint, O. Protocols to detect senescence-associated beta-galactosidase (SA-betagal) activity, a biomarker of senescent cells in culture and in vivo. Nat Protoc. 4, 1798-1806 (2009).
- Bae, Y. U., Choi, J. H., Nagy, A., Sung, H. K., Kim, J. R. Antisenescence effect of mouse embryonic stem cell conditioned medium through a PDGF/FGF pathway. FASEB J. 30, 1276-1286 (2016).
- Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W., Roder, J. C. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc Natl Acad Sci U S A. 90, 8424-8428 (1993).
- Li, X. Y., et al. Passage number affects the pluripotency of mouse embryonic stem cells as judged by tetraploid embryo aggregation. Cell Tissue Res. 327, 607-614 (2007).
- Mbeunkui, F., Fodstad, O., Pannell, L. K. Secretory protein enrichment and analysis: an optimized approach applied on cancer cell lines using 2D LC-MS/MS. J Proteome Res. 5, 899-906 (2006).
- Makridakis, M., Vlahou, A. Secretome proteomics for discovery of cancer biomarkers. J Proteomics. 73, 2291-2305 (2010).
- Kim, K. S., et al. Regulation of replicative senescence by insulin-like growth factor-binding protein 3 in human umbilical vein endothelial cells. Aging Cell. 6, 535-545 (2007).
- Kim, K. S., et al. Induction of cellular senescence by insulin-like growth factor binding protein-5 through a p53-dependent mechanism. Mol Biol Cell. 18, 4543-4552 (2007).
- Eiselleova, L., et al. Comparative study of mouse and human feeder cells for human embryonic stem cells. Int J Dev Biol. 52, 353-363 (2008).
