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(12) Patent Application Publication (10) Pub. No.: US 2015/0105343 A1 Byrne Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2015/0105343 A1 Byrne Et Al US 2015O105343A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0105343 A1 Byrne et al. (43) Pub. Date: Apr. 16, 2015 (54) NUCLEOSDE SUPPLEMENTATION TO Publication Classification PROMOTE CELLULAR FUNCTION, GENETIC STABILITY AND REGENERATIVE (51) Int. Cl. APPLICATIONS CI2N5/074 (2006.01) A613 L/7064 (2006.01) Applicant: The Regents of the University of (52) U.S. Cl. (71) CPC .......... CI2N5/0696 (2013.01); A61K3I/7064 California, Oakland, CA (US) (2013.01); C12N 2500/42 (2013.01); C12N (72) Inventors: James A. Byrne, Santa Monica, CA 2500/98 (2013.01); C12N 2500/92 (2013.01) (US); Caius Radu, Los Angeles, CA (57) ABSTRACT (US); Patrick Lee, Los Angeles, CA In various embodiments, a cell culture medium, or a nucleo (US) side cocktail transmission (NCT) medium for the culture of stem cells with improved genetic stability, cellular function and regeneration ability is provided. Illustrative culture media (21) Appl. No.: 14/516,451 comprise a basal culture medium for stem cells, where the culture medium is Supplemented with one or more nucleoside triphosphates (e.g., dNTPs and/or NPs) or one or more pre (22) Filed: Oct. 16, 2014 cursors thereof. Illustrative NCT media comprise a delivery vehicle (e.g. skin creams or other vehicle) containing one or more nucleoside triphosphates (e.g., dNTPs and/or NPs) or Related U.S. Application Data one or more precursors thereof. The NCT medium provides, interalia, direct delivery of nucleoside cocktails into human (60) Provisional application No. 61/891,846, filed on Oct. tissues (such as skin) for regenerative, cosmetic and/or thera 16, 2013. peutic purposes. Patent Application Publication Apr. 16, 2015 Sheet 1 of 13 US 201S/O 105343 A1 ***** ** * ***** Patent Application Publication Apr. 16, 2015 Sheet 2 of 13 US 201S/O 105343 A1 dATP 35 dGTP dcTP :47% drop p40,05 32%. g S. circ (p.G.95) s room.ua wall. Patent Application Publication Apr. 16, 2015 Sheet 3 of 13 US 201S/O 105343 A1 Gamma histone 2AX staining Original tra tieria fircasts (HDRs) iPSCs minus (-) deoxyribonicleoside (di stippierretation hiFSCs plus (+) decxyriboriticieoside N suppie retation Fig. 3A Patent Application Publication Apr. 16, 2015 Sheet 4 of 13 US 201S/O 105343 A1 Human induced pluripotent stem cells (iPSCs) stressed for two & weeks with and without deoxynucleoside supplementation finiSix Arrows highlight chronosorai iotation of rowei SNP-based its Arrows highlight cit is both iES treated and iNSutreated Sf2. Y Arrows highlightioh only in ciNS intreated (5:12 Arrows highlight OH only in dMS treated (if 12 Fig. 4 Patent Application Publication Apr. 16, 2015 Sheet 5 of 13 US 201S/O 105343 A1 mRNA-iPSC growth rate with without i800. -- control 1600.nn - - 30 is . w 1400 h 75 it dATP pool dTTP pool dGTP pool dCTP pool increase: increase: increase: increase: 31%. 55%. 26%. 13% i. i 3 Patent Application Publication Apr. 16, 2015 Sheet 6 of 13 US 201S/O 105343 A1 O PSCS iPSCs yith PSCs with without di short term d\ long tern divi supplement supplement supplement Fig. 7 Patent Application Publication Apr. 16, 2015 Sheet 7 of 13 US 201S/O 105343 A1 Population doubligates of dermal fibroblasts Skir irobiasts 88................................................................. .2ik.ix88.i................................... xix. sasis 3. 8: : 3 & 888 8&ggier888. * : x888 as xxxixess 8. Patent Application Publication Apr. 16, 2015 Sheet 8 of 13 US 201S/O 105343 A1 Deoxyribonucedtide triphosphate pools (dNTP) in reprogrammed stem cells and parental dermal fibroblasts dCTP 3f. ciecease $fircease post-reprogramming wi pieties it is Site eii Site si to stippierrett with suppiesent dTTP it::::::ss. with stippieriento. Stee et Ce tax stippierrett (with stippiesaert * ifference betweetgaretai ittoriasts artistences with stippierient x 8.5 * * difference between step ceis withoutsippierrettanic stem ceis witi suppierrent p & (.5 Fig. 9 Patent Application Publication Apr. 16, 2015 Sheet 9 of 13 US 201S/O 105343 A1 Deoxyribonucedtide triphosphate pools (dNTP) in reprogrammed stem cells and parental dermal fibroblasts dATP xxxxxx 53% decrease 42% increase w post-repr is sis: Fire its Stett ei Ste. Cei v,v,' to stippiernet with stippietient GP Figgiast Site eii Step: eit to stippierrent with stippierrent * : iiifference between parental fibroixiasts artister tes with suppiement p. x. (.85 ** a difference between ster ceis without stippierientarid ster ceis witt stappientent p 8 (.5 Fig. 9, cont'd. Patent Application Publication Apr. 16, 2015 Sheet 10 of 13 US 201S/O 105343 A1 Fig. 10 Patent Application Publication Apr. 16, 2015 Sheet 11 of 13 US 201S/O 105343 A1 Percent damaged taclei for X-if its a ster ceis ** at p < 0.05 . : : i Step cei witt stem cell with stippierrett supplement Percent damaged nuclei for rixietierived in a ster teis m ** = p < 0.05 Ster ceii witt Site ceili with W. W. W. W. stippierrent stippierrett Fig. 10, cont'd. Patent Application Publication Apr. 16, 2015 Sheet 12 of 13 US 201S/O 105343 A1 ?‘61-I Patent Application Publication Apr. 16, 2015 Sheet 13 of 13 US 201S/O 105343 A1 Alpha-fetoprotein positive hepatocytes 2 weeks if titure & 8 3-. ------------------------------xx--------------, iS-tieriyet iSO-cerific Hepatocytes Hepatocytes without supplement (with supplement x 3 3 Fig. 12 US 2015/O 105343 A1 Apr. 16, 2015 NUCLEOSDE SUPPLEMENTATION TO SUMMARY PROMOTE CELLULAR FUNCTION, GENETIC STABILITY AND REGENERATIVE 0005. It was discovered that the genomic integrity, stabil ity and functionality, of stem cells and somatic cells can be APPLICATIONS augmented by exogenous Supplementation of nucleosides CROSS-REFERENCE TO RELATED (e.g., deoxyribonucleosides). Such a nucleoside Supplement APPLICATIONS (refrred to as a nucloside cocktail (NC) or, in certain embod imetns a deoxyribonucleosideCocktail (DC) can be used to 0001. This application claims benefit of and priority to augment cell culture media or can be combined with a deliv U.S. Ser. No. 61/891,846, filed on Oct. 16, 2013, which is ery vehicle to produce a nucleoside cocktail transmission incorporated herein by reference in its entirety for all pur (NCT) medium, or in certain embodiments a deoxyriob poses. nucleoside transmission cocktail (DCT). Such Supplementa tion, in addition to improving genomic stability can enhance STATEMENT OF GOVERNMENTAL SUPPORT cellular function (for example, by speeding up cell prolifera tion, promoting differentiation into target therapeutic cell Not Applicable types (such as hepatocytes), and/or promoting human tissue regeneration (Such as stimulating division of human skin cells BACKGROUND in vivo for regenerative purposes). 0002 Although induced pluripotent stem cells (iPSCs) 0006 Moreover it was observed that exposure of human share great similarities with embryonic stem cells (ESCs), the skin fibroblasts to a nucleoside cocktail can stimulate prolif somatic cell reprogramming methods used for iPSC deriva eration at a significantly augmented rate, and this phenom tion have caused both scientific interest and concerns about enon, when combined with nucleoside transmission media the new cell type. There are important biosafety concerns Such as a skin cream, can prove very useful in directly regen about the genomic integrity and stability of iPSCs, as well as erating/rejuvenating human skin or other tissues. ESCs, during their derivation and prolonged culture, in addi 0007. The nucleoside cocktail not only improves genomic tion to significant concerns regarding Substandard/subphysi stability, it also significantly augments proliferation rate of a ological cellular functions (such as Suboptimal proliferation wide variety of somatic and stem cells in addition to augment and differentiation) in Suboptimally supplemented environ ing differentiation of pluripotent stem cells into target cell mental conditions. The usefulness of human induced pluri types (such as hepatocytes for treatment of liver disease). potent stem cells (or other stem or somatic cells) in research, 0008 Thus, some embodiments provide a basal culture cosmetic and therapeutic/regenerative applications highly medium for Somatic and stem cells, where the culture relies on their genomic integrity, stability and functionality. medium is Supplemented with one or more nucleoside triph Certain genomic or epigenomic abnormalities may not only osphates (e.g., dNTPs and/or NTPs) or one or more precur compromise the differentiation potential but also cause tum sors thereof. Other embodiments combine the one or more origenesis in the recipients of iPSC-based therapies. nucleoside triphosphates (e.g., dNTPs and/or NTPs) or one or Genomic abnormalities have been observed as karyotypic more precursors thereof with topical and/or delivery mecha aberrations such as changes in chromosomal number and nisms in vivo use inclusive of, but not limited to: 1) skin structures, copy number variations (CNVs) such as Subkaryo creams, 2) topical cosmetics and/or 3) extrinsic derivery typic or Subchromosomal amplifications and deletions, loss mechanisms (EDMs). Such as injectables, ingestion, inhala of heterozygosity (LOH) due to acquired uniparental disomy, tion or other. and random or site-specific integration of alien DNA into the 0009. Accordingly, in various aspects, the invention(s) host genome. contemplated herein may include, but need not be limited to, 0003 Studies indicate that various somatic and stem cells and essentially all IPSC clones derived using current any one or more of the following embodiments: approaches acquire genomic instability during
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