Differentiation of human Embryonic Stem Cells (hESC) into neural progenitors as a tool to study both the pathways during early brain development and the neuroteratogenic effects of ethanol Thesis: Jelena Kostic The main objective of this work is to use human neuroprogenitors (hNPs) cells from hESC as a tool to study the cellular and molecular events involved in early human neural development under physiological conditions and to study the teratogenic effects of ethanol during the initial formation of the CNS. Specific objectives include: Objectives - Development of an in vitro protocol of derivation of human neural progenitors (hNPs) from hESCs, which could mirror early stages of human brain development - Characterize the in vitro culture by evaluating the gene and protein expression of human Neuroprogenitors (hNPs) in culture during their proliferation and differentiation into mature cells (neurons, astrocytes and oligodendrocytes). - Assess whether the endocannabinoid system, including endocannabinoid receptors (CB1, CB2) and the enzymes involved in their synthesis (NAPE-PLD) and degradation (FAAH) of endocannabinoids (EC), are expressed in human neural progenitors during their differentiation to mature nervous cells. - Assess the gene and protein expression of TLR4 and TLR2 receptors during neural differentiation from hESC (in vitro) and during brain ontogeny in mice. - Finally, the last objective will investigate the actions of ethanol on the proliferation and differentiation of hNPs. Specifically we will assess the effects of different physiological concentrations of ethanol on: 1) gene and protein expressions during the derivation of hESC to NPs: 2) the proliferation and cells survival of the hNPs, 3) the NPs differentiation processes to mature neural cells, by assessing gene and protein expressions and morphological alterations, 4) the expression of endocannabinoid system and TLR4 and TLR2 in hESC, hNPs and brain development in mice. Results In this work we demonstrated the generation of human neural progenitors (hNPs) and differentiated neurons, oligodendrocytes and astrocytes from human embryonic stem cells (hESC) which mimics early brain development in humans. Neuroepithelial progenitors display the morphological and functional characteristics of their embryonic counterparts and the proper timing of neurons and glial cells generation. Immunocytochemical and real time (RT)-polymerase chain reaction analyses reveal that cells appeared as clusters during neuroepithelial cell proliferation and that the genes associated with the neuroectodermal (Pax-6) and the endodermic (α-fetoprotein) lineages decreased in parallel to the upregulation of the genes of hNPs (nestin and Tuj1), followed by their differentiation into neurons (MAP-2+, GABA+), oligodendrocytes [galactocerebroside (GalC+)], and astrocytes (GFAP+). We further demonstrate, for the first time, that human NPs express the endocannabinoid receptors (CB1 and CB2) and the enzymes involved in endocannabinoids synthesis (NAPE-PLD) and degradation (FAAH). Using this in vitro culture, we demonstrate that ethanol exposure impairs NPs survival, affects the differentiation of NPs into neurons and astrocytes, disrupts the actin cytoskeleton, and affects the expression of different genes associated with neural differentiation. The results provide new insights into the effects of ethanol on human embryogenesis and neuroprogenitors and offer an opportunity to delineate potential therapeutic strategies to restore early ethanol-induced brain damage. We also show for the first time that TLR4 and TLR2 are expressed in both hESC and hNPs. In parallel, we demonstrated that TLR4 and TLR2 are expressed in mice brain development (prenatal and postnatal period). Conclusions 1- We establish an in vitro model for the generation of neural progenitors (NPs) from hESCs. 2- Neuroepithelial progenitors display the morphological and functional characteristics of their embryonic counterparts, and the proper timing of neurons and glia cells generation. 3- Immunocytochemical and RT-PCR analyses reveal that hESC appeared as clusters during neuroepithelial cell proliferation, and that the genes associated with the neuroectodermal and endodermal lineages decrease in parallel with the up-regulation of the genes of neural progenitors, followed by their differentiation into neurons oligodendrocytes and astrocytes. 4- We demonstrate for the first time that human NPs express the endocannabinoid receptors (CB1, CB2) and the enzymes involved in endocannabinoids synthesis and degradation, as well as the TLR4 and TLR2 receptors. 5- By using this in vitro model, we show that ethanol exposure: - Impairs the derivation of hESCs towards neural cells, alters the disappearance of the endodermal marker AFP during NPs proliferation and up-regulates the transcription factors Nanog, Sox2 and Pax-6 during their differentiation. - Impairs NPs survival, affects the differentiation of NPs into neurons and astrocytes, disrupts the actin cytoskeleton and affects the expression of different genes associated with neural differentiation. 6- The results provide new insights into the effects of ethanol on early human embryogenesis and offer an opportunity to delineate potential therapeutic strategies to restore early brain damage induced by ethanol. 7- Finally, the present study provides not only a new tool to elucidate the mechanisms underlying early human neural and brain development, but also offers the possibility to assess the neuroteratogenic actions of ethanol during early embryogenesis. References Aguado T, Monory K, Palazuelos J, Stella N, Cravatt B, Lutz B, Marsicano G, Kokaia Z, Guzmán M, Galve-Roperh I (2005). The endoccanabinoid system drives neural progenitor proliferation FASEB J. (12):1704-6. Aguado T, Palazuelos J, Monory K, Stella N, Cravatt B, Lutz B, Marsicano G, Kokaia Z, Guzmán M, Galve-Roperh I (2006). The endoccanabinoid system promotes astroglial differentiation by acting on neural progenitor cells . J Neurosci. 1;26(5):1551-61. Alfonso-Loeches S, Pascual-Lucas M, Blanco AM, Sanches-Vera I, Guerri C. (2010) Pivotal role of TLR4 receptors in alchohol-induced neuroinflammation and brain damage. The Journal of Neuroscience, 30(24): 8285-8295. Carpenter MK, Inokuma MS, Denham J, Mujtaba T, Chiu CP, Rao MS (2001). Enrichement of neurons and neural precursors from human embryonic stem cells. Exp Neurol 172:383-397. Elkabetz Y, Panagiotakos G, Al Shamy G, D. Socci N, Tabar V and Lorenz Studer (2008). Human ES cell-derived neural rossetes reveal a functionally distinct early neural stem cell stage. Genes&Dev. 22:152-165. Guerri C, Alissa Bazinet and Edward P. Riley (2002). Foetal Alcohol Spectrum Disorders and alterations in brain and behaviour.Alcohol and alcoholism Mar-Apr;44(2):108-14. Guerri C. (2002) Mechanisms involved in central nervous system dysfunction induced by prenatal exposure. Neurotoxicology Res 4:327-35. Itskovitz-Eldor J, Schuldiner M, Karsenti D, Eden A, Yanuka O, Amit M, Soreq H, and Benvenisty N. (2000). Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol. Med. Feb;6(2):88-95. Nat R, M Nilbratt, S Narkilahti, B Winblad, O Hovatta and A Nordberg. (2007). Neurogenic neuroepithelial and radial glia cells generated from six human embryonic stem cell lines in serum free suspension and adherent cultures. Glia 55:385-99. Okun E, Griffoen KJ, Lathia JD, Tang SC, Matson MP and Arumugam TV. (2008) Toll-like receptors in neurodegeneration. Brain Res. Rev. 59(2): 278-292. Okun E et al. (2010). TLR2 activation inhibits embryonic neural progenitor cell proliferation. J. Cell Biol. 9, 1081-1088. Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145-1147. Wilson PG and Stice SS (2006). Development and differentiation of neural rosettes derived from human embryonic stem cells. Stem Cell Rev. 2(1):67-77. Zhang SC, Wernig M, Duncan ID, Brustle O, Thomson JA (2001). In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat Biotechnol.19:1129–1133.