Embryonic stem cells (ESCs) can differentiate into all cell types of the body and, therefore, hold tremendous promise for cell-based regenerative medicine therapies. One significant challenge that should be addressed before using ESCs in the clinic is to improve methods of efficiently and effectively directing the differentiation of this heterogeneous cell population. The work presented here examines the potential of harnessing naturally derived extracellular vesicles to deliver genetic material from mature cells to undifferentiated ESCs for the purpose of manipulating stem cell fate. Vesicles were isolated from preosteoblast cells and were found to be ∼170 nm in diameter and to express the CD40 surface marker. Multiple interactions were visualized between vesicles and ESCs using confocal microscopy, and no significant difference in cell viability was noted. Incubation with vesicles caused significant changes in ESC gene expression, including persistence of pluripotent gene levels as well as increased neurectoderm differentiation. Genetic cargo of the vesicles as well as the cells from which they were derived were examined using a small microRNA (miRNA) gene array. Interestingly, ∼20% of the examined miRNAs were increased more than twofold in the vesicles compared with preosteoblast cells. Together, these results suggest that extracellular vesicles may be utilized as a novel method of directing stem cell differentiation. Future work examining methods for controlled delivery of vesicles may improve the clinical potential of these physiological liposomes for therapeutic applications.
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References
1.
EvansMJ and KaufmanMH. (1981). Establishment in culture of pluripotential cells from mouse embryos. Nature, 292:154–156.
2.
MartinGR. (1981). Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A, 78:7634–7638.
3.
WobusAM, HolzhausenH, JakelP and SchoneichJ. (1984). Characterization of a pluripotent stem cell line derived from a mouse embryo. Exp Cell Res, 152:212–219.
4.
Ben-DavidU, KopperO and BenvenistyN. (2012). Expanding the boundaries of embryonic stem cells. Cell Stem Cell, 10:666–677.
5.
MurryCE and KellerG. (2008). Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell, 132:661–680.
6.
CohenDE and MeltonD. (2011). Turning straw into gold: directing cell fate for regenerative medicine. Nat Rev Genet, 12:243–252.
KleinD, SchmandtT, Muth-KohneE, Perez-BouzaA, SegschneiderM, GieselmannV and BrustleO. (2006). Embryonic stem cell-based reduction of central nervous system sulfatide storage in an animal model of metachromatic leukodystrophy. Gene Ther, 13:1686–1695.
9.
GreberB, LehrachH and AdjayeJ. (2007). Silencing of core transcription factors in human EC cells highlights the importance of autocrine FGF signaling for self-renewal. BMC Dev Biol, 7:46
10.
LiuYP, DambaevaSV, DovzhenkoOV, GarthwaiteMA and GolosTG. (2005). Stable plasmid-based siRNA silencing of gene expression in human embryonic stem cells. Stem Cells Dev, 14:487–492.
11.
ZouGM and YoderMC. (2005). Application of RNA interference to study stem cell function: current status and future perspectives. Biol Cell, 97:211–219.
12.
GrivennikovIA. (2008). Embryonic stem cells and the problem of directed differentiation. Biochemistry (Mosc), 73:1438–1452.
13.
GiudiceA and TrounsonA. (2008). Genetic modification of human embryonic stem cells for derivation of target cells. Cell Stem Cell, 2:422–433.
14.
Al-NedawiK, MeehanB, MicallefJ, LhotakV, MayL, GuhaA and RakJ. (2008). Intercellular transfer of the oncogenic receptor EGFRvIII by microvesicles derived from tumour cells. Nat Cell Biol, 10:619–624.
15.
SkogJ, WurdingerT, van RijnS, MeijerDH, GaincheL, Sena-EstevesM, CurryWTJr., CarterBS, KrichevskyAM and BreakefieldXO. (2008). Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat Cell Biol, 10:1470–1476.
16.
LaiRC, ChenTS and LimSK. (2011). Mesenchymal stem cell exosome: a novel stem cell-based therapy for cardiovascular disease. Regen Med, 6:481–492.
17.
RatajczakJ, MiekusK, KuciaM, ZhangJ, RecaR, DvorakP and RatajczakMZ. (2006). Embryonic stem cell-derived microvesicles reprogram hematopoietic progenitors: evidence for horizontal transfer of mRNA and protein delivery. Leukemia, 20:847–856.
18.
YuanA, FarberEL, RapoportAL, TejadaD, DeniskinR, AkhmedovNB and FarberDB. (2009). Transfer of microRNAs by embryonic stem cell microvesicles. PLoS One, 4:e4722
19.
Chavez-MunozC, MorseJ, KilaniR and GhaharyA. (2008). Primary human keratinocytes externalize stratifin protein via exosomes. J Cell Biochem, 104:2165–2173.
20.
FaureJ, LachenalG, CourtM, HirrlingerJ, Chatellard-CausseC, BlotB, GrangeJ, SchoehnG, GoldbergY, et al. (2006). Exosomes are released by cultured cortical neurones. Mol Cell Neurosci, 31:642–648.
21.
EL AndaloussiS, MagerI, BreakefieldXO and WoodMJ. (2013). Extracellular vesicles: biology and emerging therapeutic opportunities. Nat Rev Drug Discov, 12:347–357.
22.
ValadiH, EkstromK, BossiosA, SjostrandM, LeeJJ and LotvallJO. (2007). Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol, 9:654–659.
23.
AntonyakMA, LiB, BoroughsLK, JohnsonJL, DrusoJE, BryantKL, HolowkaDA and CerioneRA. (2011). Cancer cell-derived microvesicles induce transformation by transferring tissue transglutaminase and fibronectin to recipient cells. Proc Natl Acad Sci U S A, 108:4852–4857.
24.
Muralidharan-ChariV, ClancyJW, SedgwickA and D'Souza-SchoreyC. (2010). Microvesicles: mediators of extracellular communication during cancer progression. J Cell Sci, 123:1603–1611.
25.
KubikovaI, KonecnaH, SedoO, ZdrahalZ, RehulkaP, HribkovaH, RehulkovaH, HamplA, ChmelikJ and DvorakP. (2009). Proteomic profiling of human embryonic stem cell-derived microvesicles reveals a risk of transfer of proteins of bovine and mouse origin. Cytotherapy, 11:330–340, 1 p following 340.
26.
BrunoS, GrangeC, CollinoF, DeregibusMC, CantaluppiV, BianconeL, TettaC and CamussiG. (2012). Microvesicles derived from mesenchymal stem cells enhance survival in a lethal model of acute kidney injury. PLoS One, 7:e33115
27.
ChenTS, LaiRC, LeeMM, ChooAB, LeeCN and LimSK. (2010). Mesenchymal stem cell secretes microparticles enriched in pre-microRNAs. Nucleic Acids Res, 38:215–224.
28.
CollinoF, DeregibusMC, BrunoS, SterponeL, AghemoG, ViltonoL, TettaC and CamussiG. (2010). Microvesicles derived from adult human bone marrow and tissue specific mesenchymal stem cells shuttle selected pattern of miRNAs. PLoS One, 5:e11803
29.
KimHS, ChoiDY, YunSJ, ChoiSM, KangJW, JungJW, HwangD, KimKP and KimDW. (2012). Proteomic analysis of microvesicles derived from human mesenchymal stem cells. J Proteome Res, 11:839–849.
30.
LiT, YanY, WangB, QianH, ZhangX, ShenL, WangM, ZhouY, ZhuW, LiW and XuW. (2013). Exosomes derived from human umbilical cord mesenchymal stem cells alleviate liver fibrosis. Stem Cells Dev, 22:845–854.
31.
ZhangB, YinY, LaiRC, TanSS, ChooAB and LimSK. (2014). Mesenchymal stem cells secrete immunologically active exosomes. Stem Cells Dev [Epub ahead of print]; DOI:10.1089/scd.2013.0479.
32.
MontecalvoA, LarreginaAT, ShufeskyWJ, StolzDB, SullivanML, KarlssonJM, BatyCJ, GibsonGA, ErdosG, et al. (2012). Mechanism of transfer of functional microRNAs between mouse dendritic cells via exosomes. Blood, 119:756–766.
33.
TheryC, OstrowskiM and SeguraE. (2009). Membrane vesicles as conveyors of immune responses. Nat Rev Immunol, 9:581–593.
34.
AliottaJM, PereiraM, JohnsonKW, de PazN, DoonerMS, PuenteN, AyalaC, BrilliantK, BerzD, et al. (2010). Microvesicle entry into marrow cells mediates tissue-specific changes in mRNA by direct delivery of mRNA and induction of transcription. Exp Hematol, 38:233–245.
35.
GattiS, BrunoS, DeregibusMC, SordiA, CantaluppiV, TettaC and CamussiG. (2011). Microvesicles derived from human adult mesenchymal stem cells protect against ischaemia-reperfusion-induced acute and chronic kidney injury. Nephrol Dial Transplant, 26:1474–1483.
36.
SlotJW and GeuzeHJ. (1985). A new method of preparing gold probes for multiple-labeling cytochemistry. Eur J Cell Biol, 38:87–93.
37.
SlotJW and GeuzeHJ. (1981). Sizing of protein A-colloidal gold probes for immunoelectron microscopy. J Cell Biol, 90:533–536.
38.
HoubaviyHB, MurrayMF and SharpPA. (2003). Embryonic stem cell-specific MicroRNAs. Dev Cell, 5:351–358.
39.
SunX, FuX, HanW, ZhaoY and LiuH. (2010). Can controlled cellular reprogramming be achieved using microRNAs?. Ageing Res Rev, 9:475–483.
40.
MallannaSK and RizzinoA. (2010). Emerging roles of microRNAs in the control of embryonic stem cells and the generation of induced pluripotent stem cells. Dev Biol, 344:16–25.
41.
HuoJS and ZambidisET. (2013). Pivots of pluripotency: the roles of non-coding RNA in regulating embryonic and induced pluripotent stem cells. Biochim Biophys Acta, 1830:2385–2394.
42.
MeltonC and BlellochR. (2010). MicroRNA regulation of embryonic stem cell self-renewal and differentiation. Adv Exp Med Biol, 695:105–117.
43.
IveyKN and SrivastavaD. (2010). MicroRNAs as regulators of differentiation and cell fate decisions. Cell Stem Cell, 7:36–41.
44.
CalabreseJM, SeilaAC, YeoGW and SharpPA. (2007). RNA sequence analysis defines Dicer's role in mouse embryonic stem cells. Proc Natl Acad Sci U S A, 104:18097–18102.
45.
ChenX, LiangH, ZhangJ, ZenK and ZhangCY. (2012). Secreted microRNAs: a new form of intercellular communication. Trends Cell Biol, 22:125–132.
MeltonC, JudsonRL and BlellochR. (2010). Opposing microRNA families regulate self-renewal in mouse embryonic stem cells. Nature, 463:621–626.
48.
WestonMD, PierceML, Jensen-SmithHC, FritzschB, Rocha-SanchezS, BeiselKW and SoukupGA. (2011). MicroRNA-183 family expression in hair cell development and requirement of microRNAs for hair cell maintenance and survival. Dev Dyn, 240:808–819.
49.
RasmussenKD, SimminiS, Abreu-GoodgerC, BartonicekN, Di GiacomoM, Bilbao-CortesD, HorosR, Von LindernM, EnrightAJ and O'CarrollD. (2010). The miR-144/451 locus is required for erythroid homeostasis. J Exp Med, 207:1351–1358.
50.
AbdellatifM. (2010). The role of microRNA-133 in cardiac hypertrophy uncovered. Circ Res, 106:16–18.
DeregibusMC, CantaluppiV, CalogeroR, Lo IaconoM, TettaC, BianconeL, BrunoS, BussolatiB and CamussiG. (2007). Endothelial progenitor cell derived microvesicles activate an angiogenic program in endothelial cells by a horizontal transfer of mRNA. Blood, 110:2440–2448.
53.
ZhangHC, LiuXB, HuangS, BiXY, WangHX, XieLX, WangYQ, CaoXF, LvJ, et al. (2012). Microvesicles derived from human umbilical cord mesenchymal stem cells stimulated by hypoxia promote angiogenesis both in vitro and in vivo. Stem Cells Dev, 21:3289–3297.
54.
LiuL, ShaoX, GaoW, ZhangZ, LiuP, WangR, HuangP, YinY and ShuY. (2012). MicroRNA-133b inhibits the growth of non-small-cell lung cancer by targeting the epidermal growth factor receptor. FEBS J, 279:3800–3812.
55.
TaoJ, WuD, XuB, QianW, LiP, LuQ, YinC and ZhangW. (2012). microRNA-133 inhibits cell proliferation, migration and invasion in prostate cancer cells by targeting the epidermal growth factor receptor. Oncol Rep, 27:1967–1975.
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