US 20160264934A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0264934 A1 GALLOURAKIS et al. (43) Pub. Date: Sep. 15, 2016 (54) METHODS FOR MODULATING AND Publication Classification ASSAYING MI6AIN STEM CELL POPULATIONS (51) Int. Cl. CI2N5/0735 (2006.01) (71) Applicants: THE GENERAL, HOSPITAL AOIN I/02 (2006.01) CORPORATION, Boston, MA (US); CI2O I/68 (2006.01) The Regents of the University of GOIN 33/573 (2006.01) California, Oakland, CA (US) CI2N 5/077 (2006.01) CI2N5/0793 (2006.01) (72) Inventors: Cosmas GIALLOURAKIS, Boston, (52) U.S. Cl. MA (US); Alan C. MULLEN, CPC ............ CI2N5/0606 (2013.01); CI2N5/0657 Brookline, MA (US); Yi XING, (2013.01); C12N5/0619 (2013.01); C12O Torrance, CA (US) I/6888 (2013.01); G0IN33/573 (2013.01); A0IN I/0226 (2013.01); C12N 2501/72 (73) Assignees: THE GENERAL, HOSPITAL (2013.01); C12N 2506/02 (2013.01); C12O CORPORATION, Boston, MA (US); 2600/158 (2013.01); C12Y 201/01062 The Regents of the University of (2013.01); C12Y 201/01 (2013.01) California, Oakland, CA (US) (57) ABSTRACT (21) Appl. No.: 15/067,780 The present invention generally relates to methods, assays and kits to maintain a human stem cell population in an (22) Filed: Mar 11, 2016 undifferentiated state by inhibiting the expression or function of METTL3 and/or METTL4, and mA fingerprint methods, assays, arrays and kits to assess the cell state of a human stem Related U.S. Application Data cell population by assessing mA levels (e.g. mA peak inten (60) Provisional application No. 62/131,490, filed on Mar. sities) of a set of target genes disclosed herein to determine if 11, 2015. the stem cell is in an undifferentiated or differentiated state. Patent Application Publication Sep. 15, 2016 Sheet 2 of 13 US 2016/0264934 A1 X- & FG. 2B FG, 2C ACN s ACN fic lett3 iype via FIG. 2E FG. 2F g 8 hasg g 08 3 -i-. is 3. s C - - 883-i- if 3 : s s 2 c - a. ;3. : Six a w kE E. : s as E a s 33is is s 8: isN see * : s i $2 s Patent Application Publication Sep. 15, 2016 Sheet 3 of 13 US 2016/0264934 A1 F.G. 3A 88 Narc 33 8 : 50 -. 8 3 3: 8. W 33 is: SS S. & E. c 8 i. s: 30 : g aw& " is a 20 3 4 s: is 8.3. is 10 a c 2 8 && Š.s: FG. 3B iQ sri A {E} 3. a. 80 3 c ; is $ 503 s s : 9 8 S. 4 Aay 8 3.: saE. i.; SSes 30 3.g iss is: 3 sx 20 asa as . t R s 838. F.G. 3D &:::::y: 3. g a 38 -------8 33.--------------------------- w838 ye ie:S F.G. 3E s: Patent Application Publication Sep. 15, 2016 Sheet 4 of 13 US 2016/0264934 A1 FG. 4A FG. AB & 838 & 3A RP : 8.-- 8 & ...-a-...--...------...-------------------...-------------------------. 1.s x $ 8. s s: s a. xy 3. : l a. š : - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ------- - - - - - - - - - - - - - - - -k . & 3: s ; ---8. - : s 8 s 3g2 33.3%. 38te:8:ty atog FG. AD FIG. 4C fat gets S. & its aba RNA sex. 8; Riccie.8 Boss a risihyatic: pya:32 ki3.3s pya:38 x8.3 FG. A.E x- F.G. 4F mA-8-Nanostring RA ests 8er tiavoiridot treat sett 8 Wild type 32. c B.S OS is x &lett:3 -i- Qs 3 8.8 85 3. E3 8 3.4 83.3 8s 8 V . 3.3 $3.3 c $ 3. 4 3 : 0.2 3. 8 t . 2. 5 3.3 B {} Narog Octa 8 Wild typs & Metti3 :- Patent Application Publication Sep. 15, 2016 Sheet 5 of 13 US 2016/0264934 A1 F.G. 5A 8: 8:8 F.G. SD ----------&-. F.G. SE 3. s 8 & s s 3. : s s F.G. SG F.G. SH FIG. 5 F.G. 5, Patent Application Publication Sep. 15, 2016 Sheet 6 of 13 US 2016/0264934 A1 & rSSC & ESS |----+--~~~~~~~~~ aga is: 88. s F.G. 6D F.G. 6E Rossa Sesii. iiia Speci 8. FIG. 6F is: 88s & .......S.S.C. A38:8 w kissets: 8kse: Patent Application Publication Sep. 15, 2016 Sheet 8 of 13 US 2016/0264934 A1 FIG. 8 Patent Application Publication Sep. 15, 2016 Sheet 9 of 13 US 2016/0264934 A1 Ajacg FIG.9B se k s :::::: 8 : SO FIG. 9F Excisgth its 8: 8. 88scing s & 8 hSassass -8-38.-2S 250 583. 750. 5' UTai cis 3' uri; Esince cast exor-exxiiikoi by FG. 9. RNA Polymerase E 3st Eartile w 2st quartile & st cartile s 3.3 -- fr&A frtified Patent Application Publication Sep. 15, 2016 Sheet 10 of 13 US 2016/0264934 A1 F.G. 10A fiss448 3 3.8 mala-Han SRNA taigeied region gr8A targeted fegion Adeiti3 / S. gasysexes s . Seri areas 38ere stated Eferentiate: six type & Aitk: -- i.18:34atti-i- is FIG. OF FG. LOG FIG. OH 5 exile.iii.333 as: -late:3:2 s g is 4.8 i. s. 3 aš& s ;: 3s is . s: 3 0:5 E3 Š s esses Asti ges 8 Patent Application Publication Sep. 15, 2016 Sheet 11 of 13 US 2016/0264934 A1 FIG. 11A KANS: i FG, 11B mRNA level in Metii3 -f- derived tumors compared to wildtype derived tumors. O 35 3. s ; : e a r 3. 4. 3. Patent Application Publication Sep. 15, 2016 Sheet 12 of 13 FG, 2A LNCE:08:MA s LINQRoš 50 Set isfai exis asses FG, 12E it:8 scietscore Patent Application Publication Sep. 15, 2016 Sheet 13 of 13 US 2016/0264934 A1 F.G. 13A F.G. 13D ***&&$33.33% F.G. 13B F.G. 13E FG, 13C 28 23.3 82.3.g US 2016/0264934 A1 Sep. 15, 2016 METHODS FOR MODULATING AND functional characterization of the YTH domain family of ASSAYING MI6AN STEM CELL “reader proteins which specifically bind mA sites and POPULATIONS recruit the linked transcripts to RNA decay bodies (Kang et al., 2014; Wang et al., 2014a). CROSS REFERENCE TO RELATED 0006 Whereas the DNA methylome undergoes dramatic APPLICATIONS reprogramming during early embryonic life, the developmen tal origins and functions of mA in mammals are incom 0001. This application claims benefit under 35 U.S.C. pletely understood. Furthermore, the degree of evolutionary S119(e) of U.S. Provisional Application No. 62/131,490 filed conservation of mA sites is not known in ESCs. Therefore, on Mar. 11, 2015, the contents of each of which are incorpo there is a need in the art for effective and efficient methods for rated herein by reference in their entireties. assessing mA mRNA methylome in stem cells and human GOVERNMENT SUPPORT stem cells, for example, to characterize and validate cells, including human pluripotent stem cells, and for determining 0002 This invention was made, in part, with government the quality and cell State of a human stem cell populations, support under NIH Grant Number DK090122 awarded by e.g., prior to its use, e.g., in therapeutic administration, dis National Institutes of Health. The Government of the U.S. has ease modeling, drug development and screening and toxicity certain rights in the invention. assays etc. FIELD OF THE INVENTION SUMMARY OF THE INVENTION 0003. The present invention relates to arrays and methods 0007. The present invention is directed to, in part, meth for characterizing stem cell populations assessing transcrip ods, compositions and kits to maintain a stem cell population, tionwide distribution of mA methylation to characterize and Such as a human stem cell population, in an undifferentiated permit selection of stem cell lines for further use, and to state, comprising contacting the stem cell population with an modulation of METTL3, e.g., inhibition to maintain stem inhibitor of METTL3 or METTL4. In some embodiments, cells in an undifferentiated state or activation of METTL3 to the methods, compositions and kits as disclosed herein relate promote differentiation along endoderm lineages. to methods to prevent a stem cell population differentiating along an endoderm lineage. Other aspects of the technology BACKGROUND OF THE INVENTION described herein relates to methods, compositions and kits to 0004 Reversible chemical modifications on messenger promote a stem cell population to differentiate along an endo RNAS have emerged as prevalent phenomena that may open derm lineage. Moreover, another aspect of the technology a new field of “RNA epigenetics', akin to the diverse roles described herein relates to methods, assays, arrays and kits that DNA modifications play in epigenetics (reviewed by Fu for performing mA analysis of RNA from stem cell popula and He, 2012; Sibbritt et al., 2013). N6-methyl-adenosine tions to characterize the cell state of the cell population, (mA) is the most prevalent modification of mRNAs in which can be used, for example, as a quality control for the Somatic cells, and dysregulation of this modification has stem cell population. In some embodiments, the stem cell already been linked to obesity, cancer, and other human dis population is a human stem cell population, e.g., a hESC cell eases (Sibbritt et al., 2013). mA has been observed in a wide population or other human stem cell line. range of organisms, and the known methylation complex is 10008 N6-methyl-adenosine (mA) is the most abundant conserved across eukaryotes (Bokar et al., 1997, Bujnicki, covalent modification on messenger RNAS in Somatic cells 2002 #375). In budding yeast, the mA methylation program and is linked to human diseases, but its functions in mamma is activated by starvation and required for sporulation (Agar lian development are poorly understood.