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(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization I International Bureau (10) International Publication Number (43) International Publication Date WO 2016/103269 Al 30 June 2016 (30.06.2016) P O P C T (51) International Patent Classification: AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, C12N 5/0793 (2010.01) CI2N 5/079 (2010.01) BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (21) International Application Number: HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, PCT/IL2015/05 1253 KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, (22) International Filing Date: MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, 23 December 2015 (23. 12.2015) PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, (25) Filing Language: English TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (26) Publication Language: English (84) Designated States (unless otherwise indicated, for every (30) Priority Data: kind of regional protection available): ARIPO (BW, GH, 62/096,184 23 December 2014 (23. 12.2014) US GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, (71) Applicant: RAMOT AT TEL-AVIV UNIVERSITY TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, LTD. [IL/IL]; P.O. Box 39296, 6139201 Tel-Aviv (IL). DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, (72) Inventors: ELKABETZ, Yechiel; 16/3 Dror Street, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, 6813520 Tel-Aviv (IL). EDRI, Reuven; c/o Ramot at Tel- GW, KM, ML, MR, NE, SN, TD, TG). Aviv University Ltd., P.O. Box 39296, 6139201 (IL). YAFFE, Yakey; 6 Ruchama Street, 5235944 Ramat-Gan Published: (IL). MEISSNER, Alexander; 134 Larch Road, Cam — with international search report (Art. 21(3)) bridge, Massachusetts 02138 (US). ZILLER, Michel J.; 89 Museum Street, Cambridge, Massachusetts 02138 (US). — before the expiration of the time limit for amending the claims and to be republished in the event of receipt of (74) Agents: EHRLICH, Gal et al; G.E. EHRLICH (1995) amendments (Rule 48.2(h)) LTD., 11 Menachem Begin Road, 5268104 Ramat Gan (IL). — with sequence listing part of description (Rule 5.2(a)) (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, (54) Title: POPULATIONS OF NEURAL PROGENITOR CELLS AND METHODS OF PRODUCING AND USING SAME FIG. 1A L-RG DO D12 P14 21 D2 D80 D220 P P P2 P3 P4 P10 P25 Neural Induction o Differentiation Differentiation Differentiation Differentiation (57) Abstract: An isolated population of cells is provided the cells comprise at least 10 % HES5+ cells, wherein said HES5+ cells o are: (i) early radial glial (E -RG) cells; (ii) mid radial glial (M-RG) cells; (iii) late radial glial (L-RG) cells; or (iv) long term neural progenitor (LNP) cells. Also provided are additional populations of neural cells and use of the populations and methods of produ cing same. POPULATIONS OF NEURAL PROGENITOR CELLS AND METHODS OF PRODUCING AND USING SAME FIELD AND BACKGROUND OF THE INVENTION The present invention, in some embodiments thereof, relates to populations of neural progenitor cells and methods of producing and using same. Human pluripotent stem cell derived models that accurately recapitulate neural development in vitro and allow for the generation of specific neuronal subtypes are of major interest to the stem ce l and biomedical community. To date, the process of neural differentiation has not been elucidated and therefore any attempts to accurately mimic i or manipulate it have failed. The identification of neural stem cells (NSCs) in the developing and adult brain has transformed the way the scientists understand central nervous system (CNS) development and regeneration. However, long following their isolation from the CNS or the derivation of neural progenitors from pluripotent stem cells (PSCs), the ability to address the dynamic changes in self-renewal and potency of distinct NSC types in vitro has remained poor. The exceptionally pioneering studies done in the NSC field in vivo have led to the identification of fundamental NSC types populating the germinal zones - neuroepithelial (NE) cells, radial glial (RG) cells, and adult NSCs (aNSCs). These studies provided the basis for understanding of the dynamic nature and lineage relationship of these distinct NSC types in vivo, describing the unique timing mechanism of neuronal cel type generation. However, in depth in vitro dissection of the molecular characteristics of each stage, particularly within the RG compartment, has been stalled mainly by the heterogeneity of NSC cultures and the lack of stage specific markers. In fact, despite being highly heterogeneous, distinct RG cell types as well as aNSCs are known to share similar RG cell markers rather than distinctive ones. The reporter gene- and surface marker-based prospective isolation of acute mouse aNSCs serves as a great example for a more in depth analysis of aNSC characteristics. However, applying such a study to human CNS derived RG cells is limited due to obvious shortage in early human CNS tissue. Thus, in depth understanding on human NSC ontogeny and dynamics in culture is still elusive. The advent of PSCs has brought the ability to direct early neural progenitors towards a range of neuronal cell fates including midbrain dopaminergic neurons spinal motoneurons and telencephalic cortical neurons. One remarkable study by Knoblich and co-workers allows monitoring early to mid gestation cerebral morphogenesis and neurogenesis, making up an attractive approach to model development and disease of the human brain (Lancaster, M . A et al. Cerebral organoids model human brain development and microcephaly. Nature 501, 373-379, doi:10.1038/naturel2517, 2013). Another recently published comprehensive work delineates the temporal transcriptome analysis of cerebral cortex neuronal subtypes derived from PSCs [van de Leemput, J. et al. CORTECON: A Temporal Transcriptome Analysis of In Vitro Human Cerebral Cort e Development from Human Embryonic Stem Cells. Neuron 83, 51-68, doi: 10. 1016/j.neuron.2014.05.013 (2014)]. These two latter advancements have significantly helped to demonstrate the capability of iESC differentiation strategies to recapitulate major hallmarks of in vivo neural development and serve as a valuable resource for modeling development and disease of the human brain. Further to these important findings, however, there is a need to better understand how different types of progenitors emerge and exert their full potential while progressing through distinct competences during development. Addressing such an aim requires employing differentiation culture strategies tha allow distinguishing primary progenitor cells holding extensive proliferation capacity and broad differentiation potential from the bulk of accompanying progenitors that lack these abilities. The present inventors have previously isolated an early progenitor cell type from PSCs that exhibits considerable self-renewal capacity (termed rosette-neural stem cells (R-NSCs)), and showed their developmental potential and distinct molecular signature [Elkabetz, Y. et al. Human ES cell-derived neural rosettes reveal a functionally distinct early neural stem cell stage. Genes & Development 22, 152-165 (2008)]. However, also the R-NSC stage exhibits high heterogeneity with respect to NSC potential and corresponds to a transient stage in vitro. Currently there is no list of genes at high confidence that are known for specific types of neural progenitors emerging in culture, stressing the need to unravel generalized networks and pathways involved in the extensively changing dynamics of early neuroepithelial (NE) cells. Taken together, despite many years of NSC research, the heterogeneity and rapid transition through distinct neural stem and progenitor cell types still impedes the understanding of origin, lineage transitions, and the key factors hat maintain or alter the epigenetic stability of early NE cells. SUMMARY OF THE INVENTION According to an aspect of some embodiments of the present invention there is provided a method of isolating neural progenitor cells, the method comprising: (a) culturing p impotent stem cells having been transformed to express a Notch-activated reporter under culture conditions suitable for differentiation of the pluripotent stem ce s into neural progenitor cells; and (b) successively isolating progenitor cells of interest based on activation of the Notch-activated reporter. According to an aspect of some embodiments of the present invention there is provided an isolated population of cells comprising at least 10 % HES5+ cells, wherein the HES5+ cells are: (i) early radial glial (E-RG) cells; (ii) mid radial glial (M-RG) cells; (iii) late radial glial (L-RG) cells; or (iv) long term neural progenitor (LNP) cells. According to an aspect of some embodiments of the present invention there is provided an isolated population of cells comprising at least 10 % HES5- cells, wherein the HESS- cells are: (i) non-CNS cells comprising neural crest cells, placodal cells, non- neuroepithelial cells; and CNS cells which exhibit an NEUROD4+/NGN 1+/NGN2+/TB 2+ DCX+ expression signature and which form neurons of layers 1 and 6 of the brain cortex; (ii) neural progenitor cells which belong to the CNS, having a lower proliferative capacity as compared to the HES5+ ERG cells, which form layers 1, 5 and 6 of the brain cortex: (iii) intermediate progenitor cells (INPs) which belong to the CNS, and which are capable of differentiating into the neurons forming layers 4, and 2 of the brain cortex; (iv) HES5- neurons and astrocytes, wherein the neurons form layers 2, 4 and (v) neurons, oligodendrocyte and astrocytes, wherein the neurons comprise neurons reaching the olfactory bulb.
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  • AURKA Mrna Expression Is an Independent Predictor of Poor Prognosis in Patients with Non-Small Cell Lung Cancer

    AURKA Mrna Expression Is an Independent Predictor of Poor Prognosis in Patients with Non-Small Cell Lung Cancer

    ONCOLOGY LETTERS 13: 4463-4468, 2017 AURKA mRNA expression is an independent predictor of poor prognosis in patients with non-small cell lung cancer AHMED S.K. AL‑KHAFAJI1,2, MICHAEL W. MARCUS1, MICHAEL P.A. DAVIES1, JANET M. RISK1, RICHARD J. SHAW1, JOHN K. FIELD1 and TRIANTAFILLOS LILOGLOU1 1Department of Molecular and Clinical Cancer Medicine, Institute of Translational Medicine, University of Liverpool, Liverpool L7 8TX, UK; 2Department of Biology, College of Science, University of Baghdad, Al‑Jadriya, Baghdad 10070, Iraq Received September 12, 2016; Accepted January 17, 2017 DOI: 10.3892/ol.2017.6012 Abstract. Deregulation of mitotic spindle genes has been Introduction reported to contribute to the development and progression of malignant tumours. The aim of the present study was to Lung cancer is the most common cause of cancer-associated explore the association between the expression profiles of mortality in the UK for both males and females (1), and Aurora kinases (AURKA, AURKB and AURKC), cytoskel- >1/5 patients with cancer succumb to this malignancy world- eton-associated protein 5 (CKAP5), discs large-associated wide (2). Non-small cell lung carcinoma (NSCLC) accounts protein 5 (DLGAP5), kinesin-like protein 11 (KIF11), micro- for 80-85% of all cases of lung cancer, and develops through tubule nucleation factor (TPX2), monopolar spindle 1 kinase the accumulation of molecular alterations, which may serve as (TTK), and β-tubulins (TUBB) and (TUBB3) genes and clini- prognostic biomarkers for NSCLC outcome (3). copathological characteristics in human non-small cell lung Mitotic spindle formation and the spindle checkpoint are carcinoma (NSCLC). Reverse transcription-quantitative poly- critical for the maintenance of cell division and chromosome merase chain reaction‑based RNA gene expression profiles of segregation (4).