sign in sign up
<<
Home , Phenol Red

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 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 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 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 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 reprogram 0010. In various aspects, the invention(s) contemplated ming and in vitro culture-induced stress (Martins-Taylor and herein may include, but need not be limited to, any one or Xu (2012) Stem Cells 30:22-27; Lund et al. (2012) Nat. Rev. more of the following embodiments: Genet., 13: 732-744) in addition to possessing substandard 0011 Embodiment 1: A cell culture medium for the cul physiology (such as impaired cellular proliferation). The ture of stem cells with improved genetic stability, said culture issue of hiPSC genomic instability is one of the most impor medium including: a basal culture medium for stem cells, tant bottlenecks in advancing personalized iPSC-based where said culture medium is Supplemented with one or more regenerative therapies to the clinic (Martins-Taylor and Xu nucleoside triphosphates or one or more precursors thereof (2012) Stem Cells 30: 22-27; Lund et al. (2012) Nat. Rev. 0012 Embodiment 2: A nucleoside cocktail transmission Genet., 13: 732-744: Byrne (2013) Gene Therapy and Regu (NCT) medium for improving Somatic or stem cell genetic lation 7: 1230002), given the established link between stability said medium including: a cosmetic or pharmaceuti genomic instability and an increased risk of malignant trans cal vehicle; and one or more nucleoside triphosphates or formation. precursors thereof. 0004. The causes of genomic instability and impaired cel 0013 Embodiment 3: The cell culture medium of embodi lular physiology during hipSC reprograming and culture, and ment 1 or nucleoside cocktail transmission medium of indeed for other pluripotent stem cells (such as human embry embodiment 2, wherein said one or more nucleoside triphos onic stem cells) and Somatic stem cells (such as neural stem phates are independently selected from the group consisting cells, mesenchymal stem cells and dermal stem cells) are of deoxyadenosine triphosphate (dATP), deoxyguanosine unclear, and approaches to reduce and eventually eliminate triphosphate (dGTP), deoxycytidine triphosphate (NCTP), the occurrence of this highly deleterious phenomenon have deoxythymidine triphosphate (dTTP) deoxyuridine triphos yet to be developed. phate, triphosphate (ATP), triphosphate US 2015/O 105343 A1 Apr. 16, 2015

(GTP), triphosphate (CTP), 5-methyluridine triph 0023 Embodiment 13: The cell culture medium according osphate (m5UTP), and triphosphate (UTP). to any one of embodiments 1, and 3-12, or the NCT medium 0014 Embodiment 4: The cell culture medium of embodi according to any one of embodiments 2-12, wherein said ment 1 or nucleoside cocktail transmission medium of culture medium is supplemented with, or said NCT medium embodiment 2, wherein said one or more nucleoside triphos includes, deoxyguanosine triphosphate (dGTP), or a precur phates are independently selected from the group consisting sor thereof of deoxyadenosine triphosphate (dATP), deoxyguanosine 0024. Embodiment 14: The cell culture medium or NCT triphosphate (dGTP), deoxycytidine triphosphate (NCTP), medium according of embodiment 13, wherein said cell cul deoxythymidine triphosphate (dTTP) and deoxyuridine ture medium is supplemented with or said NCT medium triphosphate. includes deoxyguanosine triphosphate. 00.15 Embodiment 5: The cell culture medium of embodi 0025 Embodiment 15: The cell culture medium or NCT ment 1 or nucleoside cocktail transmission medium of medium according of embodiment 13, wherein said cell cul embodiment 2, wherein said one or more nucleoside triphos ture medium is supplemented with or said NCT medium phates are independently selected from the group consisting includes a precursor of deoxyguanosine triphosphate. of (ATP), (GTP), (CTP), 5-methyluridine triph 0026. Embodiment 16: The cell culture or NCT medium of embodiment 15, wherein said precursor of deoxygua osphate (m5UTP), and (UTP). nosine triphosphate selected from the group consisting of 0016 Embodiment 6: The cell culture medium according deoxyguanosine diphosphate, deoxyguanosine monophos to any one of embodiments 1 and 3-5 or nucleoside cocktail phate, deoxyguanosine, and transmission medium according to any one of embodiments 2-5, wherein said culture medium is Supplemented with, or 0027 Embodiment 17: The cell culture medium according said NCT medium includes, one or more precursors of to any one of embodiments 1, or 3-16, or the NCT medium nucleoside triphosphates independently selected from the according to any one of embodiments 2-16, wherein said group consisting of deoxyadenosine triphosphate (dATP), culture medium is supplemented with or said NCT medium deoxyguanosine triphosphate (dGTP), deoxycytidine triph includes deoxycytidine triphosphate (NCTP), or a precursor osphate (NCTP), deoxythymidine triphosphate (dTTP) deox thereof. yuridine triphosphate, adenosine triphosphate (ATP), gua 0028 Embodiment 18: The cell culture medium or NCT nosine triphosphate (GTP), cytidine triphosphate (CTP), medium of embodiment 17, wherein said cell culture medium 5-methyluridine triphosphate (mSUTP), and uridine triphos is supplemented with, or said NCT medium includes deoxy phate (UTP). cytidine triphosphate. 0017 Embodiment 7: The cell culture or NCT medium of 0029 Embodiment 19: The cell culture medium or NCT embodiment 6, wherein said one or more precursors of medium of embodiment 17, wherein said cell culture medium nucleoside triphosphates are independently selected from the is supplemented with, or said NCT medium includes a pre group consisting of deoxyadenosine triphosphate (dATP), cursor of deoxycytidine triphosphate. deoxyguanosine triphosphate (dGTP), deoxycytidine triph 0030 Embodiment 20: The cell culture medium or NCT osphate (NCTP), deoxythymidine triphosphate (dTTP) and medium of embodiment 19, wherein precursor of deoxycyti deoxyuridine triphosphate. dine is selected from the group consisting of deoxycytidine 00.18 Embodiment 8: The cell culture or NCT medium of diphosphate, deoxycytidine monophosphate, deoxycytidine, embodiment 6, wherein said one or more precursors of and . nucleoside triphosphates are independently selected from the group consisting of adenosine triphosphate (ATP), guanosine 0031 Embodiment 21: The cell culture medium according triphosphate (GTP), cytidine triphosphate (CTP), 5-methylu to any one of embodiments 1, and 3-20, or the NCT medium ridine triphosphate (mSUTP), and uridine triphosphate according to any one of embodiments 2-20, wherein said culture medium is supplemented with, or said NCT includes (UTP). deoxythymidine triphosphate (dTTP), or a precursor thereof. 0019 Embodiment 9: The cell culture medium according to any one of embodiments 1 and 3-8, or the NCT medium 0032 Embodiment 22: The cell culture medium or NCT according to any one of embodiments 3-8, wherein said cul medium of embodiment 20, wherein said culture medium is ture medium is supplemented with, or said NCT medium supplemented with, or said NCT medium includes deoxythy includes, deoxyadenosine triphosphate (dATP), or a precur midine triphosphate. sor thereof 0033 Embodiment 23: The cell culture medium or NCT 0020 Embodiment 10: The cell culture medium or NCT medium of embodiment 20, wherein said culture medium is medium of embodiment 9, said culture medium is Supple supplemented with, or said NCT medium includes a precur mented with, or said NCT medium includes, deoxyadenosine sor of deoxythymidine triphosphate. triphosphate. 0034 Embodiment 24: The cell culture medium or NCT 0021 Embodiment 11: The cell culture medium or NCT medium of embodiment 23, wherein said precursor of deox medium of embodiment 9, said culture medium is Supple ythymidine triphosphate is selected from the group consisting mented with, or said NCT medium includes, a precursor of of deoxythymidine diphosphate, deoxythymidine mono deoxyadenosine triphosphate. phosphate, deoxythymidine, and thymine. 0022. Embodiment 12: The cell culture medium or NCT 0035 Embodiment 25: The cell culture medium according medium of embodiment 11, wherein said precursor of deoxy to any one of embodiments 1, and 3-24, or the NCT medium adenosine triphosphate is selected from the group consisting according to any one of embodiments 2-24, wherein said of deoxyadenosine diphosphate, deoxyadenosine monophos culture or NCT medium is supplemented with deoxyuridine phate, deoxyadenosine, and . triphosphate, or a precursor thereof US 2015/O 105343 A1 Apr. 16, 2015

0036 Embodiment 26: The cell culture medium or NCT 0050 Embodiment 40: The cell culture medium or NCT medium of embodiment 25, wherein said culture medium is medium of embodiment 39, wherein said precursor of cyti supplemented with, or said NCT medium includes deoxyuri dine triphosphate is selected from the group consisting of dine triphosphate. , , cytidine, and 0037 Embodiment 27: The cell culture medium or NCT cytosine. medium of embodiment 25, wherein said culture medium is 0051 Embodiment 41: The cell culture medium according supplemented with, or said NCT medium includes a precur to any one of embodiments 1, and 3-40, or the NCT medium sor of deoxyuridine triphosphate. according to any one of embodiments 2-40, wherein said 0038 Embodiment 28: The cell culture medium or NCT culture or NCT medium is supplemented with 5-methyluri medium of embodiment 27, wherein said precursor of deox dine triphosphate (m,5UTP), or a precursor thereof. yuridine triphosphate is selected from the group consisting of 0052 Embodiment 42: The cell culture medium or NCT deoxyuridine diphosphate, deoxyuridine monophosphate, medium of embodiment 41, wherein said culture medium is deoxyuridine, and . supplemented with, or said NCT medium includes, 5-methy 0039 Embodiment 29: The cell culture medium according luridine triphosphate. to any one of embodiments 1, and 3-28, or the NCT medium 0053 Embodiment 43: The cell culture medium or NCT according to any one of embodiments 2-28, wherein said medium of embodiment 41, wherein said culture medium is culture or NCT medium is supplemented with adenosine supplemented with, or said NCT medium includes, a precur triphosphate (ATP) or a precursor thereof sor of 5-methyluridine triphosphate. 0040 Embodiment 30: The cell culture medium or NCT 0054 Embodiment 44: The cell culture medium or NCT medium of embodiment 29, wherein said culture medium is medium of embodiment 43, wherein said precursor of 5-me supplemented with, or said NCT medium includes, adenosine thyluridine triphosphate is selected from the group consisting triphosphate (ATP). of 5-methyluridine diphosphate, 5-methyluridine monophos 0041 Embodiment 31: The cell culture medium or NCT phate, 5-methyluridine, and thymine. medium of embodiment 29, wherein said culture medium is 0055 Embodiment 45: The cell culture medium according supplemented with, or said NCT medium includes, a precur to any one of embodiments 1, and 3-44, or the NCT medium sor of adenosine triphosphate. according to any one of embodiments 2-44, wherein said 0042 Embodiment 32: The cell culture medium or NCT culture medium is supplemented with, or said NCT medium medium of embodiment 31, wherein said precursor of includes uridine triphosphate (UTP), or a precursor thereof adenosine triphosphate (ATP) is selected from the group con 0056. Embodiment 46: The cell culture medium or NCT sisting of , , medium of embodiment 45, wherein said culture medium is adenosine, and adenine. supplemented with, or said NCT medium includes uridine 0043 Embodiment 33: The cell culture medium according triphosphate. to any one of embodiments 1, and 3-32, or the NCT medium 0057 Embodiment 47: The cell culture medium or NCT according to any one of embodiments 2-32, wherein said medium of embodiment 45, wherein said culture medium is culture medium is supplemented with, or said NCT medium supplemented with, or said NCT medium includes a precur includes guanosine triphosphate (GTP) or a precursor sor of uridine triphosphate. thereof. 0058 Embodiment 48: The cell culture medium or NCT 0044. Embodiment 34: The cell culture medium or NCT medium of embodiment 47, wherein precursor of uridine medium of embodiment 33, wherein said culture medium is triphosphate is selected from the group consisting of uridine Supplemented with, or said NCT medium includes, , , uridine, and uracil. triphosphate (GTP). 0059 Embodiment 49: The nucleoside cocktail transmis 004.5 Embodiment 35: The cell culture medium or NCT sion (NCT) medium according to any one of embodiments medium of embodiment 33, wherein said culture medium is 2-48, wherein said NCT medium includes a vehicle formu supplemented with, or said NCT medium includes, a precur lated for administration via a route selected from the group sor of guanosine triphosphate. consisting of Subcutaneous administration, parenteral admin 0046 Embodiment 36: The cell culture medium or NCT istration, topical administration, oral administration, nasal or medium of embodiment 35, wherein said precursor of gua inhalation administration, local administration Such as by nosine triphosphate is selected from the group consisting of paint, aerosol, or transdermally. guanosine diphosphate, , gua 0060 Embodiment 50: The nucleoside cocktail transmis nosine, and guanine sion (NCT) medium according to any one of embodiments 0047 Embodiment 37: The cell culture medium according 2-48, wherein said NCT medium includes a vehicle formu to any one of embodiments 1, and 3-36, or the NCT medium lated for topical administration, Subdermal administration, or according to any one of embodiments 2-36, wherein said cell intradermal administration. culture medium is supplemented with, or said NCT medium 0061 Embodiment 51: The nucleoside cocktail transmis includes cytidine triphosphate (CTP), or a precursor thereof sion (NCT) medium of embodiment 50, wherein said medium 0048 Embodiment 38: The cell culture medium or NCT is formulated in a vehicle selected from the group consisting medium of embodiment 37, wherein said culture medium is of a mud, an herbal mixture, a fat, an emulsions, a lotion, a supplemented with, or said NCT medium includes, cytidine cream, a gel, a biological, a Solution, a spray, an ointment, a triphosphate. foams, a mousses, a liquid, a Suspensions, a dispersion, an 0049 Embodiment 39: The cell culture medium or NCT aerosol, a Soap, a shampoo, and a conditioner. medium of embodiment 37, wherein said culture medium is 0062 Embodiment 52: The nucleoside cocktail transmis supplemented with, or said NCT medium includes, a precur sion (NCT) medium of embodiment 50, wherein said medium sor of cytidine triphosphate. is formulated as a cream (e.g., a face cream). US 2015/O 105343 A1 Apr. 16, 2015

0063 Embodiment 53: The nucleoside cocktail transmis 0072 Embodiment 62: The cell culture medium according sion (NCT) medium according to any one of embodiments to any one of embodiments 1, 3-48, and 54-61, or the NCT 56-58, wherein said medium includes a formulation selected medium according to any one of embodiments 2-61, wherein from the group consisting of a wrinkle removing cream, a said cell culture medium, or said NCT medium is without dermal filler, a scar-reducing cream, and an acne treatment. animal or human derived serum albumin. 0064. Embodiment 54: The cell culture medium according 0073 Embodiment 63: The cell culture medium according to any one of embodiments 1, and 3-48 or the nucleoside to any one of embodiments 1, 3-48, and 54-62, wherein the cocktail transmission (NCT) medium according to any one of Supplemented medium is selected from the group consisting embodiments 2-53, wherein said nucleoside triphosphate or of DMEM (Dulbecco's Modified Eagle's Medium), MEM precursor thereof Supplementing said culture medium, or (Minimal Essential Medium), BME (Basal Medium Eagle), including said NCT medium, are present at a concentration RPMI 1640, DMEM/F-12 (Dulbecco's Modified Eagle's Sufficient to improve the short term and/or long term genetic Medium: Nutrient Mixture F-12), DMEM/F-10 (Dulbecco's stability of stem cells as compared to the same cells cultured Modified Eagle's Medium: Nutrient Mixture F-10), C-MEM in the same medium without Supplementation by a nucleoside (C.-Minimal essential Medium), G-MEM (Glasgow’s Mini triphosphate or precursor thereof mal Essential Medium), IMDM (Isocove’s Modified Dulbec 0065 Embodiment 55: The cell culture medium or the co's Medium), essential 8 (E8) medium, and KnockCut nucleoside cocktail transmission (NCT) medium of embodi DMEM. ment 54, wherein said stem cells are stem cells selected from 0074 Embodiment 64: The cell culture medium of the group consisting of somatic cells (e.g. fibroblasts) or stem embodiment 63, wherein the basal medium is DMEM/F12. cells (e.g., neural stem cells, mesenchymal stem cells, 0075 Embodiment 65: The nucleoside cocktail transmis hematopoietic stem cells, adipose stem cells, embryonic stem sion (NCT) medium of embodiment 2, wherein said NCT cells, cord stem cells, and induced pluripotent stem cells). medium comprises said vehicle and a cell culture medium 0066 Embodiment 56: The cell culture medium according according to any one of embodiments 1, 3-48, and 55-64. to any one of embodiments 1, 3-48, and 54-55, or the NCT 0076 Embodiment 66: A somatic or stem cell culture or medium according to any one of embodiments 2-55, wherein nucleoside cocktail transmission (NCT) culture, said cell cul each nucleoside triphosphate or precursor thereof Supple ture including cells in a cell culture medium according to any menting said culture medium, or including said NCT medium one of embodiments 1, 3-48, and 55-64, or including cells in is present at a concentration (e.g., either in transmission an NCT medium according to any one of embodiments 2-62, medium and/or resulting in Such a concentration on target and 65. cells in vivo, such as extrinsic delivery of nucleoside cocktail 0077. Embodiment 67: The cell culture or NCT culture of into human skin using a skin cream as the NCT medium) embodiment 66, wherein said somatic or stem cells are cells ranging from about 1 uM up to about 50 uM, or from about 1 selected from the group consisting of Somatic cells (e.g., uM to about 40 uM, or from about 1 uM up to about 35 uM, fibroblasts) or stem cells (e.g., neural stem cells, mesenchy or from about 1 uM up to about 30 uM. mal stem cells, hematopoietic stem cells, adipose stem cells, 0067 Embodiment 57: The cell culture medium according embryonic stem cells, cord stem cells, induced pluripotent to any one of embodiments 1, 3-48, and 54-55, or the NCT stem cells, and the like). medium according to any one of embodiments 2-55, wherein 0078 Embodiment 68: The cell culture or NCT culture of each nucleoside triphosphate or precursor thereof Supple embodiment 66, wherein said cells comprise stem cells. menting said culture medium, or included in said NCT 0079 Embodiment 69: The cell culture or NCT culture of medium, is at a concentration starting (stock) or final (extrin embodiment 68, wherein said stem cells are selected from the sic delivery) of about 50 uM or lower, or about 40 uM or group consisting of embryonic stem cells and adult stem cells, lower, or about 30 uM or lower or about 25 uM or lower, or including, but not limited to neural stem cells, hepatic stem about 20 uM or lower, or about 15uM or lower, or about 10 cells, hematopoietic stem cells, umbilical cord stem uM or lower, or about 5uM or lower. cells, epidermal stem cells, gastrointestinal stem cells, endot 0068 Embodiment 58: The cell culture medium, or NCT helial stem cells, muscle stem cells, mesenchymal stem cells, medium of embodiment 57, wherein each nucleoside triph and pancreatic stem cells. osphate or precursor thereof Supplementing said culture 0080 Embodiment 70: The cell culture or NCT culture of medium, or included in said NCT medium, is present at a embodiment 68, wherein said cells are induced pluripotent starting (stock) or final (extrinsic delivery) concentration stem cells (IPSCs) (e.g., such as autologous hIPSCs derived ranging from about 5 LIM to about 30 LIM. from a patient). 0069. Embodiment 59: The cell culture medium, or NCT 0081 Embodiment 71: The cell culture or NCT culture of medium of embodiment 57, wherein each nucleoside triph embodiment 70, wherein said IPSCs are reprogrammed from osphate or precursor thereof Supplementing said culture said cells selected from the group consisting offibroblasts, neural culture medium, or included in said NCT medium, is present stem cells, stomach cells, liver cells, keratinocytes, melano at a starting (stock) or final (extrinsic delivery) concentration cytes, amniotic cells, blood cells, 13-cells, and adipose cells. of about 30 uM. 0082 Embodiment 72: The cell culture or NCT culture of 0070 Embodiment 60: The cell culture medium, or NCT embodiments 70 or 71, wherein the IPSCs comprise cells medium of embodiment 57, wherein each nucleoside triph reprogramed two or more factors selected from the group osphate or precursor thereof Supplementing said culture said consisting of KLF4 (K), LIN28 (L), c-MYC (M), NANOG culture medium, or included said NCT medium, is present at (N); OCT4 (0), SOX2 (S), and valproic (VPA). a starting (stock) or final (extrinsic delivery) concentration of 0083. Embodiment 73: The cell culture or NCT culture of about 5uM. embodiment 72, wherein the ISPCs includes cells repro 0071 Embodiment 61: The cell culture medium according gramed using the four canonical Yamanaka factors KLF4 (K), to any one of embodiments 1, 3-48, and 54-60, or the NCT c-MYC (M),OCT4 (O), and SOX2 (S). medium according to any one of embodiments 2-60, wherein 0084 Embodiment 74: The cell culture or NCT culture of said cell culture medium, or said NCT medium is xenopatho embodiment 73, wherein the reprogramming factors further gen-free. comprise LIN28. US 2015/O 105343 A1 Apr. 16, 2015

0085 Embodiment 75: The cell culture or NCT culture the three germ layers observed in transgene-free hIPSC-de according to any one of embodiments 70-74, wherein said rived teratomas, (c) Karyotype analysis of the transgene-free IPSCs are reprogrammed using a vector selected from the iPSCs before and after CC-based stress. Red arrow highlights group consisting of an integrating Vector, a non-integrating extra chromosome 12. Scale bar equals 100 microns. vector, an excisable vector, and a DNA-free vector. 0100 FIG. 2: Significant reduction of dNTP pools follow I0086 Embodiment 76: A method of reducing genetic ing hIPSC reprogramming. *The size of the dNTP pool was instability of stem cells said method including culturing said standardized relative to the level observed in the original cells in a cell culture medium according to any one of embodi dermal fibroblasts. ments of embodiments a culture medium according to any 0101 FIG. 3A and 3B: Visualization (FIG.3A) and quan one of embodiments 1,3-48, and 55-64. tification (FIG. 3B) of YH2AX positive foci. Green staining 0087 Embodiment 77: A method of performing autolo highlights yH2AX foci, blue staining highlights DAPI gous stem cell transfer, said method including isolating stem stained nuclei. HDFs were reprogrammed into hiPSCs and cells from a subject or generating IPSCs from said subject and then cultured for four days with and without deoxyribo expanding and/or culturing said stem cells or IPSCs in a cell nucleoside (dN) Supplementation. culture medium according to any one of embodiments 1, 0102 FIG. 4: Single polymorphism (SNP)- 3-48, and 55-64, or in an NCT medium according to any one based loss of heterozygosity (LOH) analysis following “clini of embodiments 2-62, and 65. cal-grade conversion” (CC) based stress. 0088 Embodiment 78: A method of promoting regenera (0103 FIG. 5: Supplementing with 30 uM of dNs signifi tion and/or maintenance of tissues, said method including cantly increases hIPSC proliferation rate. First experiment administering to a subject, an NCT medium according to any treated cells with 0 uM, 30 uM, 100 uM, 200 uM dN over 5 one of embodiments 2-62, and 65. day period. 200 uM was observed to be toxic (data not 0089 Embodiment 79: The method of embodiment 78, shown). Second experiment: treated cells with OuM, 30 uM, wherein said NCT medium includes a vehicle formulated for 50 uM, 75uM, 100 uM dN over 5 day period administration via a route selected from the group consisting 0104 FIG. 6 illustrates a significant reduction of dNTP of Subcutaneous administration, parenteral administration, pools following hIPSC reprogramming and dNTP rescue topical administration, oral administration, nasal or inhala with deoxyribonucleoside (dN) culture media supplementa tion administration, local administration Such as by paint, tion. The size of the dNTP pool was standardized relative to aerosol, or transdermally. the level observed in the original skin cells. 0090 Embodiment 80: The method of embodiment 78, 0105 FIG. 7 shows visualization and quantification of wherein, wherein said NCT medium includes a vehicle for YH2AX positive foci (indicative of double strand breaks). mulated for topical administration, subdermal administra 01.06 FIG.8 shows that use of deoxyribonucleoside cock tion, or intradermal administration. tail (DC) can stimulate both skin fibroblasts and human 0091 Embodiment 81: The method of embodiment 80, induced pluripotent stem cells to divide more quickly. Cell wherein said NCT medium is formulated in a vehicle selected counts of fibroblasts and stem cells grown with and without from the group consisting of a mud, an herbal mixture, a fat, Supplement were taken at 0 and 48 hours post-plating. For the an emulsions, a lotion, a cream, a gel, a biological, a solution, AD1 stem cells, one Subclone that had been reprogrammed a spray, an ointment, a foams, a mousses, a liquid, a suspen with culture Supplement was taken off Supplement tempo sions, a dispersion, an aerosol, a soap, a shampoo, and a rarily to conduct doubling rate experiments. The equation conditioner. used to calculate population doubling time (PDT) 48 your 0092. Embodiment 82: The method of embodiment 80, post plating was 48/(Log (Ts)-Log (T)). To calculate dou wherein said NCT medium is formulated as a cream (e.g., a bling rate per 24 hours: 24/PDT. face cream). 0107 FIG. 9 shows that a nucleoside cocktail (NC) can 0093 Embodiment 83: The method of embodiment 80, significantly rescue dNTP pools in hIPSCs. wherein said NCT medium includes a formulation selected 0.108 FIG. 10 shows visualization and quantification of from the group consisting of a wrinkle removing cream, a YH2AX positive foci (indicative of double strand breaks) dermal filler, a scar-reducing cream, and an acne treatment. reducing in the presence of a nucleoside coctail. The inci 0094. Embodiment 84: The method according to any one dence of DNA damage (via YH2AX expression) was of embodiments 78-83, wherein said NCT is applied to the decreased with culture supplement. DNA damage levels were skin of a human. measured via YH2AX expression (green) which is indicative 0095 Embodiment 85: The method of embodiment 84, of double-stranded breaks. Blind counts were conducted to wherein said NCT is applied to reduce scarring. determine percent damaged nuclei. Supplement causes a 0096 Embodiment 86: The method of embodiment 84, decrease in damage in two separate stem cell lines. wherein said NCT is applied to reduce wrinkles 0109 FIG. 11 shows differentiation of pluripotent stem 0097 Embodiment 87: The method according to any one cells into a therapeutic target cell type (in this case hepato of embodiments 78-83, wherein said NCT is applied intrad cyte-like cells, hereby called hepatocytes). ermally or Subdermally to increase tissue volume. 0110 FIG. 12 shows alpha-fetoprotein expressing hepa tocytes can be derived within two weeks when DC supple BRIEF DESCRIPTION OF THE DRAWINGS ment used, but not without DC. 0098. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent DETAILED DESCRIPTION application publication with color drawing(s) will be pro 0111. In various embodiments, the methods an decompo vided by the Office upon request and payment of the neces sitions described herein pertain to the discovery that deoxyri sary fee. bonucleoside (dN) and/or nucleoside (ribonucleoside) 0099 FIG. 1: Characterization of human induced pluripo Supplementation can increase genetic Stability in stem cells tent stem cells (hIPSCs). (a) Morphology and immunocy including induced pluripotent stem cells (IPSCs) in culture. tochemical detection of stem cell markers (insert is DAPI); 0.112. As demonstrated herein, it was determined that (b) Histological characterization following H&E staining of deoxyribonucleotide triphosphate (dNTP) pools in rapidly US 2015/O 105343 A1 Apr. 16, 2015 dividing hIPSCs are significantly smaller in size than the the culture medium with one or more deoxyribonucleotide dNTP pools of cells (e.g., dermal fibroblasts, from which the substrates or precursors thereof will increase dNTP pools and hIPSCs are derived (see, e.g., FIGS. 2 and 6). It was also will alleviate replication stress, DNA damage and genomic determined that hIPSCs demonstrate significantly more instability in stem cells (including iPSCs) in culture. double strand breaks (DSBs) than the fibroblasts of origin I0120 Similarly, it is believed that administration of a (see, e.g., FIGS. 3A and 3B). nucleoside cocktail transmission medium (NCT), e.g., a phar 0113 Data presented herein indicate that: 1) hIPSCs can maceutical or cosmetic formulation comprising one or more utilize exogenous NTP precursors added to the hIPSC culture deoxynucleosides or precursors thereofas described herein or DCT medium in the form of a mixture of deoxyribonucleo will alleviate replication stress, DNA damage and genomic sides (dNs) and 2) that culture medium supplementation with instability in stem cells and Somatic cells (e.g., fibroblasts) in deoxyribonucleoside (dN) and/or nucleosides or precursors V1VO. thereof can both ameliorate the hIPSC dNTP pool deficiency 0.121. It was discovered that hIPSCs cultured under and significantly reduce the genomic instability experienced nucleoside-free conditions demonstrate dNTP deficiency by these cells (see, e.g., FIG. 4). (FIGS. 2 and 6), and high levels of genomic instability espe 0114. It was also discovered that exogenous NTP precur cially when placed under “clinical conversion” (CC)-based sors can be incorporated into delivery vehicles (e.g., pharma stress (FIG.1). However, when standard hiPSC culture media ceutical delivery vehicles, cosmetic delivery vehicles, cos was supplemented with different concentrations of dNs, it metics, etc.) to form a nucleoside cocktail transmission was found that 30 uM of each dN could increase hIPSC (NCT) medium that can significantly stimulate proliferation proliferation rate and genetic stability over 4-5 days (see, of stimulate cellular proliferation of both human skin cells Example 1). It was also demonstrated that lower doses of dNs and human induced pluripotent stem cells (see, e.g., FIG. 8). (e.g., 5uM) will ensure genetic stability of hIPSCs (or other As used here, the terms nucleoside cocktail transmission stem cells) over longer periods of time while also still signifi (NCT) or nucleoside cocktail delivery (NCD) medium refers cantly reducing the incidence of genomic damage as assayed to a formulation comprising a vehicle Supplemented with via gammaH2AX staining of double strand breaks. nucleosides or precursors thereofor deoxynucleosides or pre 0.122 The data presented herein show that a simple and cursors thereof formulated for administration in vivo to a cost-effective culture media Supplementation approach that Subject (e.g., a human, a non-human mammal, etc.). Deoxy can be easily implemented by members of the stem cell biol nucleoside cocktail transmission (DCT) medium or deoxy ogy and regenerative medicine field, can increase the clinical nucleoside cocktail delivery (DCD) medium refers to to a potential of personalized hiPSC-based cellular therapeutics. formulation comprising a vehicle Supplemented with deoxy I0123. The data presented herein show that a simple and nucleosides or precursors thereof formulated for administra cost-effective nucleoside supplementation approach that can tion in Vivo to a Subject (e.g., a human, a non-human mammal, be easily implemented by members of the cosmetic, regen etc.). erative and therapeutic fields to directly stimulate regenera 0115. It is believed that a nucleoside cocktail can also tion of human tissues (such as skin) Specifically, the direct stimulate cellular proliferation of other cells invitro as well as topical application of a nucleoside cocktail (and/or other in stimulating cellular proliferation and/or tissue rejuvenation/ Vivo delivery mechanisms) will induce regeneration through regeneration in vivo (such as following direct topical appli the stimulation of proliferation of skin fibroblasts (FIG. 8), cation of an NCT in the form of for example, a skin cream). mesenchymal stem cells and/or other cells in the skin. 0116. Data presented herein indicate that nucleoside 0.124. It is believed that nucleoside (e.g., dN) supple Supplementation can significantly rescue dNTP pools in mented cells have improved genetic stability and pose sig human induced pluripotent stem cells (see, e.g., FIG.9) and it nificantly less risk of neoplasm formation following person is believed that nucleoside Supplementation can also augment alized stem cell-based (e.g., hIPSC-based) therapeutics, than dNTP pool size in a wide range of other human and non stem cells derived, cultured and differentiated using the exist human cell types. ing art (nucleoside-free (e.g., dN-free) culture media). 0117 Data presented herein indicate that nucleoside 0.125. Accordingly, in various embodiments, culture Supplementation can significantly reduce the incidence of media is provided that enhances the short term or long term genetic damage in human induced pluripotent stem cells, e.g., genetic stability of stem cells (e.g., embryonic stem cells, as measured by gammaH2AX immunocytochemistry (see, adult stem cells, induced pluripotent stem cells, etc.) cultured e.g., FIG. 10) and it is believed that nucleoside supplementa in that medium. The culture media comprises essentially any culture medium utilized for the culture of stem cells (or other tion can also reduce the incidence of genetic damage in a wide cells) where that medium is medium is supplemented with range of other human and non-human cell types. one or more deoxynucleoside or nucleoside (triphosphates) 0118 Data presented herein indicate that nucleoside and/or precursors thereof Supplementation can significantly augment the differentia 0.126 Similarly, the nucleoside cocktail transmission tion of hIPSCs into hepatocyte-like cells (see, e.g., FIGS. 11 (NCT) medium comprises a pharmaceutical or cosmetic for and 12) and it is believed that nucleoside DC supplementation mulation (e.g., as described herein) containing one or more can also augment differentiation into a wide range of other deoxynucleoside or nucleoside (triphosphates) and/or precur human and non-human cell types. sors thereof 0119 Mammalian cells synthesize dNTPs via two path ways: the de novo pathway (DNP), which uses glucose and Culture Media. amino , and the nucleoside Salvage pathway (NSP), which uses preformed dNs from the extracellular environ I0127. In various embodiments the culture media comprise ment. Without being bound to a particular theory, it is essentially any culture medium utilized for the culture of stem believed that: 1) insufficient production of dNTP pools via the cells, where that medium is Supplemented with one or more DNP is an important cause of replication stress, DNA damage deoxynucleoside or nucleoside (triphosphates) and/or precur and genomic instability in rapidly dividing hIPSCs (or other sors thereof. In certain embodiments the culture medium is stem cells in culture) and 2) enabling the utilization by the supplemented with two different deoxynucleoside and/or hIPSC cells (or other stem cells) of the NSP by supplementing nucleoside (triphosphates) and/or precursors thereof. In cer US 2015/O 105343 A1 Apr. 16, 2015

tain embodiments the culture medium is Supplemented with TABLE 1-continued three different deoxynucleoside and/or nucleoside (triphos phates) and/or precursors thereof. In certain embodiments the Composition of DMEMF 12 basal medium. culture medium is supplemented with four different deoxy Molecular Concentration nucleoside and/or nucleoside (triphosphates) and/or precur Components Weight (mg/L) mM sors thereof. In certain embodiments the culture medium is supplemented with even more different deoxynucleoside and/ L-Tyrosine disodium salt 261 55.79 0.213755 or nucleoside (triphosphates) and/or precursors thereof dihydrate 0128. As indicated above, in various embodiments, the L-Valine 117 52.85 O4S1709 basal culture medium can be any culture medium capable of Vitamins: expanding, maintaining, or differentiating stem cells includ Biotin 244 O OOOOO14 ing IPSCs. In certain embodiments the basal medium may be Choline chloride 140 8.98 O.O64143 manually prepared according to conventional methods. In D- pantothenate 477 2.24 O.OO4696 Folic Acid 441 2.65 O.OO6009 certain embodiments the basal medium may be a commer Niacinamide 122 2.02 O.O16557 cially available medium or a mixture thereof. For example, Pyridoxine hydrochloride 2O6 2.01 O.OO9772 the basal medium may be selected from the group consisting Riboflavin 376 O.22 O.OOOS82 of DMEM (Dulbecco's Modified Eagle's Medium; GIBCO), Thiamine hydrochloride 337 2.17 O.OO6439 MEM (Minimal Essential Medium; GIBCO), BME (Basal Vitamin B12 1,355 O.68 O.OOOSO2 Medium Eagle; GIBCO), RPMI 1640 (GIBCO), DMEM/F- i- 18O 12.6 O.O7 12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture norganic salts: F-12: GIBCO), DMEM/F-10 (Dulbecco's Modified Eagle's Calcium Chloride (CaCl2) 111 116.6 1.OSO45 Medium: Nutrient Mixture F-10; GIBCO), C-MEM (O-Mini (anhyd.) mal essential Medium; GIBCO), G-MEM (Glasgow’s Mini Cupric (CuSO4-5H2O) 250 O OOOOOOS mal Essential Medium; GIBCO), IMDM (Isocove’s Modi Ferric Nitrate (Fe(NO3) 404 O.OS OOOO124 3"9H2O) fied Dulbecco's Medium; GIBCO), and KnockCut DMEM Ferric sulfate (FeSO4-7H2O) 278 O.42 O.OO15 (GIBCO), essential 8 (E8) medium, and the like. Chloride 95 28.64 O3O1474 0129. In various embodiments, the basal medium may (anhydrous) contain one or more Supplements, which includes, but not Magnesium Sulfate (MgSO4) 120 48.84 O.407 limited to, KnockCut Serum Replacement (GIBCO), Knock (anhyd.) Potassium Chloride (KCI) 75 311.8 4.157333 Out SR XenoFree (GIBCO), KnockCut SR XenoFree Sodium Bicarbonate (NaHCO3) 84 2,438 29.02381 Growth Factor Cocktail (GIBCO), N2 supplement (GIBCO), Sodium Chloride (NaCl) 58 6,995.5 120.612O7 B27 supplement (GIBCO), and so on. In certain embodi Sodium Phosphate dibasic 142 71.02 OSOO141 ments the basal medium is a Xenopathogen-free medium (i.e., (Na2HPO4) anhydrous xeno-free medium), in order to avoid any safety problem by Sodium Phosphate monobasic 138 62.5 O4S2899 materials derived from animal source. Typically such Xeno (NaH2PO4-H2O) free medium does not include Xenopathgen(s). Such as bovine sulfate (ZnSO4-7H2O) 288 O.43 O.OO15 serum albumin, and recombinant proteins purified from ani Other components: mal cells. D-Glucose (Dextrose) 18O 3,151 17.505.556 0130. As indicated above, such media can routinely be Na 159 2.39 O.O15031 prepared in the laboratory, or can be commercially supplied. Linoleic Acid 28O O.04 O.OOO15 Lipoic Acid 2O6 O.1 O.OOOS1 By way of non-limiting illustration, the composition of Red 376.4 8.1 O.O2152 DMEM/F 12 is shown in Table 1. Putrescine 2HCI 161 O.08 O.OOOSO3 Sodium Pyruvate 110 55 O.S TABLE 1. Thymidine 242 O.36 O.OO1508 Composition of DMEMF 12 basal medium. References: 1. Dulbecco, R. and Freeman, G., (1959) Plaque formation by the polyomavirus. Virology 8: 396. Molecular Concentration 2. Ham, R. G., (1965) Clonal growth of mammalian cells in a chemically defined synthetic Components Weight (mg/L) mM medium, Proc. Natl. Acad. Sci., 53: 288, 3. Morton, H. J. (1970) A survey of commercially available tissue culture media. In vitro 6(2): 89. Amino acids: 4. Smith, J. D., Freeman, G., Vogt, M., et al., (1960) The nucleic acid of polyoma virus. Virology 12:185. Glycine 75 18.75 O.25 L-Alanine 89 4.45 O.OS L-Arginine hydrochloride 211 147.5 O.699052 I0131 The foregoing basal culture media are intended to be L-Asparagine-H2O 150 7.5 O.OS illustrative and non-limiting. One of skill will readily recog L-Aspartic acid 133 6.65 O.OS nize that the supplements described herein can be used with L-Cysteine hydrochloride-H2O 176 17.56 0.099773 virtually any culture media or incorporated into a pharmaceu L-Cystine 2HCl 313 31.29 O.O99968 tical or cosmetic to form any of a number of NCT media. L-Glutamic Acid 147 7.35 O.OS L-Glutamine 146 365 2.5 L-Histidine hydrochloride-H2O 210 31.48 O.149905 Nucleoside Cocktail Transmission (Delivery) Medium. L-Isoleucine 131 S447 0.4158O2 L-Leucine 131 59.05 O.4SO763 (0132. In certain embodiments the nucleoside cocktail L-Lysine hydrochloride 183 912S O.498.634 transmission (NCT) medium comprises a delivery vehicle L-Methionine 149 17.24 0.115705 L-Phenylalanine 16S 35.48 O.21SO3 (e.g., a cosmetic formulation) containing one or more deoxy L-Proline 115 17.25 O.15 nucleoside or nucleoside (triphosphates) and/or precursors L-Serine 105 26.25 O.25 thereof. The NCT medium is formulated to deliver the L-Threonine 119 53.45 O44916 nucleoside(s) or nucleoside precursor(s) to a desired location L-Tryptophan 204 9.02 O.044216 in or on a mammal and to provide appropriate local concen tration of the nucleoside or nucleoside precursor(s). US 2015/O 105343 A1 Apr. 16, 2015

0133. A “delivery vehicle' refers to, for example, a dilu including hydroxyethyl cellulose, hydroxymethyl cellulose, ent, adjuvant, excipient, auxiliary agent or carrier with which carboxymethyl cellulose, hydroxypropylmethyl cellulose one or more of the nucleosides and/or nucleoside precursors and mixtures thereof. described herein is administered. 0.141. In certain embodiments the vehicle comprises a 0134. The NCTs can be formulated for subcutaneous, lotion. Lotions can contain finely powdered substances that parenteral, topical, oral, nasal (or otherwise inhaled), or for are insoluble in the dispersion medium through the use of mulated for local administration, Such as by aerosol or trans Suspending agents and dispersing agents. Alternatively, dermally, e.g., to stimulate cellular proliferation of stem cells lotions can have as the dispersed phase liquid Substances that or other cells in vitro as well as to stimulate cellular prolif are immiscible with the vehicle and are usually dispersed by eration and/or tissue rejuvenation/regeneration in Vivo, and/ means of emulsifying agents or other Suitable stabilizers. In or to improve cellular maintenance of introduced exogenous one embodiment, the lotion is in the form of an emulsion cells (e.g., stem cells), endogenous stem cells, somatic cells, having a viscosity of between 100 and 1000 centistokes. The and the like in a variety of contexts. The compositions can be fluidity of lotions permits rapid and uniform application over administered in a variety of dosage/concentration forms a wide Surface area. Lotions are typically intended to dry on depending upon the method of administration. Suitable unit the skin leaving a thin coat of their medicinal components on dosage forms, include, but are not limited to powders, tablets, the skin's Surface. pills, capsules, lozenges, Suppositories, patches, nasal sprays, 0142. In certain embodiments, the vehicle comprises a injectables, implantable Sustained-release formulations, lipid topical cream. Creams may contain emulsifying agents and/ complexes, etc. or other stabilizing agents. In one embodiment, the formula 0135. In certain embodiments, the NCTs are formulated tion is in the form of a cream having a viscosity of greater than for topical administration, Subdermal administration, or intra 1000 centistokes, typically in the range of 20,000-50,000 dermal administration. In certain formulation for administra centistokes. Creams are often time preferred over ointments tion to wound sites, including for example, acute wound sites, as they are generally easier to spread and easier to remove. Surgical Sounds, burn sites, e.g., to promote healing and The basic difference between a cream and a lotion is the reduce Scarring is contemplated. Viscosity, which is dependent on the amount/use of various 0136. In certain embodiments the NCTs are formulated oils and the percentage of water used to prepare the formula with one or more pharmaceutical agents. Illustrative pharma tions. Creams are typically thicker than lotions, may have ceutical agents include, but are not limited to, agents used for various uses and often one uses more varied oils/butters, the treatment of wound sites, burn sites, acne, and other depending upon the desired effect upon the skin. In a cream topical disfigurements (e.g., various kinds of dermal scar formulation, the water-base percentage is about 60-75% and ring). Illustrative agents include, but are not limited to for the oil-base is about 20-30% of the total, with the other example, topical tamoxifen for dermal scarring, benzyol per percentages being the emulsifier agent, preservatives and oxide and antibiotics for acne, and the like. additives for a total of 100%. 0137 Illustrative NCT vehicles, include cosmetic muds, 0143. In various embodiments the vehicle comprises an herbal mixtures, fats (e.g., animal fats), emulsions, lotions, ointment. Examples of Suitable ointment bases include creams, gels, biologicals, gels, Solutions, sprays, ointments, hydrocarbon bases (e.g., petrolatum, white petrolatum, yel foams, mousses, liquids, Suspensions, dispersions, aerosols, low ointment, and mineral oil); absorption bases (hydrophilic Soaps, shampoos, conditioners, cleansers, and general cos petrolatum, anhydrous lanolin, lanolin, and cold cream): metic products. In certain embodiments, vehicles comprising water-removable bases (e.g., hydrophilic ointment), and agents for the reduction of wrinkles, and/or skin discolora water-soluble bases (e.g., polyethylene glycol ointments). tion, and/or dermal fillers are contemplated. Pastes typically differ from ointments in that they contain a 0138 Suitable vehicles to which the nucleoside(s) and/or larger percentage of solids. Pastes are typically more absorp nucleoside precursor(s) can be added include, but are not tive and less greasy that ointments prepared with the same limited to, mud, herbal mixtures, animal fats, emulsions, components. lotions, creams, gels, biologicals, gels, Solutions, sprays, 0144. In certain embodiments the vehicle comprises a gel. ointments, foams, mousses, liquids, Suspensions, disper Some emulsions may be gels or otherwise include a gelcom sions, aerosols, soaps, shampoos, conditioners, cleansers, ponent. Some gels, however, are not emulsions because they and general cosmetic products. do not contain a homogenized blend of immiscible compo 0139. In certain embodiments, suitable vehicles include nents. Suitable gelling agents include, but are not limited to, any carrier or vehicle commonly used as a base for creams, modified celluloses, such as hydroxypropyl cellulose and lotions, sprays, foams, gels, emulsions, lotions or paints for hydroxyethyl cellulose; Carbopol homopolymers and topical administration. Examples include emulsifying agents, copolymers; and combinations thereof. Suitable solvents in inert carriers including hydrocarbon bases, emulsifying the liquid vehicle include, but are not limited to, diglycol bases, non-toxic solvents or water-soluble bases. Particularly monoethyl ether, alklene glycols, such as propylene glycol, suitable examples include pluronics, HPMC, CMC and other dimethyl isosorbide; alcohols, such as isopropyl and cellulose-based ingredients, lanolin, hard paraffin, liquid par . The solvents are typically selected for their ability to affin, soft yellow paraffin or soft white paraffin, white bees dissolve the drug. ther additives, which improve the skin feel wax, yellow beeswax, cetostearyl alcohol, cetyl alcohol, and/or emolliency of the formulation, may also be incorpo dimethicones, emulsifying waxes, isopropyl myristate, rated. Examples of such additives include, but are not limited, microcrystalline wax, oleyl alcohol and Stearyl alcohol. Also isopropyl myristate, ethyl acetate, C12-C15 alkylbenzoates, contemplated are nonionic polyoxyethylene-polyoxypropy mineral oil, squalane, cyclomethicone, capric/caprylic trig lene copolymers, also referred to as poloxamers. One illus lycerides, and combinations thereof. trative poloxamer is poloxamer 407, also known as 0145. In certain embodiments the vehicle comprises a 0140 Pluronic F-127 (BASF). Additional carriers, foam. Foams consist of an emulsion in combination with a include, but are not limited to, alginates, polyvinyl alcohol, gaseous propellant. The gaseous propellant consists prima hydrogels, including hydrogels that contain a cellulose rily of hydro fluoroalkanes (HFAs). Suitable propellants derivative and/or polyacrylic acid; cellulose-based carrier, include HFAs such as 1,1,1,2-tetrafluoroethane (HFA 134a) US 2015/O 105343 A1 Apr. 16, 2015 and 1, 1, 1,2,3,3,3-heptafluoropropane (HFA 227), but mix mal and vegetable oils, and various polar compounds. Suit tures and admixtures of these and other HFAs that are cur able emulsifiers include acacia, anionic emulsifying wax, rently approved or may become approved for medical use are calcium Stearate, carbomers, cetostearyl alcohol, cetyl alco suitable. The propellants preferably are not hydrocarbon pro hol, cholesterol, diethanolamine, ethylene glycol palmito pellant gases which can produce flammable or explosive Stearate, glycerin monostearate, glyceryl monooleate, vapors during spraying. Furthermore, the compositions pref hydroxpropyl cellulose, hypromellose, lanolin, hydrous, erably contain no volatile alcohols, which can produce flam lanolin alcohols, lecithin, medium-chain triglycerides, meth mable or explosive vapors during use. ylcellulose, mineral oil and lanolin alcohols, monobasic 0146 It will be recognized that, in various embodiments Sodium phosphate, monoethanolamine, nonionic emulsify the vehicles can contain additional ingredients, e.g., to ing wax, oleic acid, poloxamer, poloxamers, polyoxyethyl enhance shelf-life, reduce biological contamination, improve enealkyl ethers, polyoxyethylene castor oil derivatives, poly wetting, maintain optimum pH, Viscosity, maintain or oxyethylene Sorbitan fatty acid esters, polyoxyethylene improve color, smell, texture, and the like. Illustrative, addi Stearates, propylene glycol alginate, self-emulsifying glyc tional ingredients include, but are not limited to excipients, eryl monostearate, Sodium citrate dehydrate, sodium lauryl diluents, emollients, Surfactants, emulsifier, buffers, preser Sulfate, Sorbitan esters, Stearic acid, Sunflower oil, tragacanth, Vatives, penetration enhancer, scents, colorants, and the like. triethanolamine, Xanthan gum and combinations thereof. In 0147 Appropriate excipients are selected based on the one embodiment, the emulsifier is glycerol Stearate. type of formulation. Standard excipients include gelatin, 0152 “Buffers are used to control pH of a composition. casein, lecithin, gum acacia, cholesterol, tragacanth, Stearic Preferably, the buffers buffer the composition from a pH of acid, benzalkonium chloride, calcium Stearate, glyceryl about 4 to a pH of about 7.5, more preferably from a pH of monostearate, cetostearyl alcohol, cetomacrogol emulsifying about 4 to a pH of about 7, and most preferably from a pH of wax, Sorbitan esters, polyoxyethylene alkyl ethers, polyoxy about 5 to a pH of about 7. In a preferred embodiment, the ethylene castor oil derivatives, polyoxyethylene sorbitan fatty buffer is triethanolamine. acid esters, polyethylene glycols, polyoxyethylene Stearates, (O153 “Preservatives' can be used to prevent the growth of colloidol silicon dioxide, phosphates, sodium dodecyl Sul fungi and microorganisms. Suitable antifungal and antimi fate, carboxymethylcellulose calcium, carboxymethylcellu crobial agents include, but are not limited to, benzoic acid, lose sodium, methylcellulose, hydroxyethylcellulose, butylparaben, ethyl paraben, methyl paraben, propylparaben, hydroxypropylcellulose, hydroxypropylmethycellulose Sodium benzoate, sodium propionate, benzalkonium chlo phthalate, noncrystalline cellulose, magnesium aluminum ride, benzethonium chloride, benzyl alcohol, cetylpyridinium silicate, triethanolamine, polyvinyl alcohol, polyvinylpyr chloride, chlorobutanol, phenol, phenylethyl alcohol, and rolidone, Sugars, and starches. thimerosal. 0148 “Diluents’ may be included in the formulations to 0154 “Penetration enhancers' are frequently used to pro dissolve, disperse or otherwise incorporate the carrier. mote transdermal delivery of drugs across the skin, in par Examples of diluents include, but are not limited to, water, ticular across the stratum corneum. A penetration enhancer buffered aqueous Solutions, organic hydrophilic diluents, may be added to enable the active agents to cross the barrier Such as monovalent alcohols, and low molecular weight gly of the stratum corneum. Some penetration enhancers cause cols and polyols (e.g. propylene glycol, polypropylene gly dermal irritation, dermal toxicity and dermal allergies. How col, glycerol, butylene glycol). ever, the more commonly used ones include urea, (carbonyl 0149 “Emollients’ are an externally applied agent that diamide), imidurea, N, N-diethylformamide, N-methy 1-2 softens or soothes skin and are generally known in the art and -pyrrolidine, 1-dodecal-azacyclopheptane-2-one, calcium listed in compendia, such as the “Handbook of Pharmaceuti thioglycate, 2-pyyrolidine, N,N-diethyl-m-toluamide, oleic cal Excipients, 4th Ed., Pharmaceutical Press, 2003. These acid and its ester derivatives, such as methyl, ethyl, propyl. include, without limitation, almond oil, castor oil, ceratonia isopropyl, butyl, vinyl and glycerylmonooleate, Sorbitan extract, cetostearoyl alcohol, cetyl alcohol, cetyl esters wax, esters, such as Sorbitan monolaurate and Sorbitan cholesterol, cottonseed oil, cyclomethicone, ethylene glycol monooleate, other fatty acid esters such as isopropyl laurate, palmitostearate, glycerin, glycerin monostearate, glyceryl isopropyl myristate, isopropyl palmitate, diisopropyl adipate, monooleate, isopropyl myristate, isopropyl palmitate, lano propylene glycol monolaurate, propylene glycol lin, lecithin, light mineral oil, medium-chain triglycerides, monooleatea and non-ionic detergents such as BRIJ(R) 76 mineral oil and lanolin alcohols, petrolatum, petrolatum and (stearyl poly(10 oxyethylene ether), BRIJ(R) 78 (stearyl poly lanolin alcohols, soybean oil, starch, Stearyl alcohol, Sun (20)oxyethylene ether), BRIJR 96 (oleyl poly(10)oxyethyl flower oil, xylitol and combinations thereof. In one embodi ene ether), and BRIJR 721 (stearyl poly (21) oxyethylene ment, the emollients are ethylhexylstearate and ethylhexyl ether) (ICI Americas Inc. Corp.). palmitate. 0.155. It will be recognized that the composition of the 0150 “Surfactants’ are surface-active agents that lower delivery vehicle will vary with the modality of administra Surface tension and thereby increase the emulsifying, foam tion, site of application, and the like. Methods of preparing ing, dispersing, spreading and wetting properties of a product. pharmaceutical and cosmetic vehicles are well known to Suitable non-ionic Surfactants include emulsifying wax, those of skill in the art (see, e.g., Remington's Pharmaceuti glyceryl monooleate, polyoxyethylene alkyl ethers, polyoxy cal Sciences, 18th Ed. Mack Printing Company, 1990; Barel, ethylene castor oil derivatives, polysorbate, Sorbitan esters, Handbook of Cosmetic Science and Technology, 3rd Ed., benzyl alcohol, benzyl benzoate, cyclodextrins, glycerin CRC Press; and Rosen, Delivery System Handbook for Per monostearate, poloxamer, povidone and combinations sonal Care and Cosmetic Products. Technology, Applications thereof. In one embodiment, the non-ionic Surfactant is and Formulations, Elsevier Science (2005)). Stearyl alcohol. 0156 The foregoing formulations and administration 0151. “Emulsifiers' are surface active substances which methods are intended to be illustrative and not limiting. It will promote the Suspension of one liquid in another and promote be appreciated that, using the teaching provided herein, other the formation of a stable mixture, or emulsion, of oil and Suitable formulations and modes of administration can be water. Common emulsifiers are: metallic Soaps, certain ani readily devised. US 2015/O 105343 A1 Apr. 16, 2015

Nucleoside Supplementation. selected from the group consisting of guanosine diphosphate, guanosine monophosphate, and guanosine. O157. In various embodiments, the culture medium is supplemented with, or the NCT medium comprises one or 0166 For cytidine triphosphate (CTP): cytidine diphos more deoxynucleoside or nucleoside (triphosphates) and/or phate, cytidine monophosphate, cytidine, cytosine, and the precursors thereof. Illustrative deoxyriboneucleoside triph like, or any combination thereof. In some embodiments the osphates include, but need not be limited to deoxyadenosine precursors include one or more precursors selected from the triphosphate (dATP), deoxyguanosine triphosphate (dGTP), group consisting of cytidine diphosphate, cytidine mono deoxycytidine triphosphate (dCTP), deoxythymidine triph phosphate, and cytidine. osphate (dTTP), and deoxyuridine triphosphate (dUTP). (0167 For 5-methyluridine triphosphate (m.5UTP): 5-me Illustrative precursors of the dNTPs include, but are not lim thyluridine diphosphate, 5-methyluridine monophosphate, ited to: 5-methyluridine, thymine, and the like, or any combination 0158 For deoxyadenosine triphosphate (dATP): deoxyad thereof. In some embodiments the precursors include one or enosine diphosphate, deoxyadenosine monophosphate, more precursors selected from the group consisting of 5-me deoxyadenosine, adenine, and the like. In some embodiments thyluridine diphosphate, 5-methyluridine monophosphate, the precursors include one or more precursors selected from and 5-methyluridine. the group consisting of deoxyadenosine diphosphate, deoxy 0168 For uridine triphosphate (UTP): uridine diphos adenosine monophosphate, and deoxyadenosine, or any com phate, uridine monophosphate, uridine, uracil, and the like, or bination thereof any combination thereof. In some embodiments the precur 0159 For deoxyguanosine triphosphate (dGTP): deox sors include one or more precursors selected from the group yguanosine diphosphate, deoxyguanosine monophosphate, consisting of , uridine monophosphate, deoxyguanosine, guanine, and the like. In some embodiments and uridine. the precursors include one or more precursors selected from 0169. In certain embodiments the precursor does not the group consisting of deoxyguanosine diphosphate, deox include the base alone. yguanosine monophosphate, and deoxyguanosine, or any (0170. In certain embodiments the culture medium is combination thereof supplemented with, or the NCT medium comprises two dif 0160 For deoxycytidine triphosphate (dCTP): deoxycyti ferent deoxynucleoside or nucleoside (triphosphates) and/or dine diphosphate, deoxycytidine monophosphate, deoxycy precursors thereof, or with three different deoxynucleoside or tidine, cytosine, and the like. In some embodiments the pre nucleoside (triphosphates) and/or precursors thereof, or with cursors include one or more precursors selected from the four (or more) different deoxynucleoside or nucleoside group consisting of deoxycytidine diphosphate, deoxycyti (triphosphates) and/or precursors. In certain embodiments, dine monophosphate, and deoxycytidine, or any combination the culture medium is supplemented with, or the DCT thereof medium comprises, at least, a and a pyramidine (or 0161 For deoxythymidine triphosphate (dTTP): deox precursors thereof). In certain embodiments the culture ythymidine diphosphate, deoxythymidine monophosphate, medium is supplemented with, or the DCT medium com deoxythymidine, thymine, and the like. In some embodi prises, at least, two (or precursors thereof). In certain ments the precursors include one or more precursors selected embodiments the culture medium is Supplemented with, or from the group consisting of deoxythymidine diphosphate, the NCT medium comprises, at least, two pyramidines (or deoxythymidine monophosphate, and deoxythymidine, or precursors thereof). In certain embodiments the culture any combination thereof medium is supplemented with, or the NCT medium com 0162 For deoxyuridine triphosphate deoxyuridine prises, at least, two purines (or precursors thereof) and at least diphosphate, deoxyuridine monophosphate, deoxyuridine, a pyramidine (or precursor thereof). In certain embodiments uracil, and the like. In some embodiments the precursors the culture medium is supplemented with, or the NCT include one or more precursors selected from the group con medium comprises, at least, two pyramidines (or precursors sisting of deoxyuridine diphosphate, deoxyuridine mono thereof) and a purine (or precursor thereof). In certain phosphate, and deoxyuridine, or any combination thereof embodiments the culture medium is Supplemented with, or 0163. In various embodiments, the basal culture medium the NCT medium comprises, at least, two purines (or precur is additionally or alternatively supplemented with, or the sors thereof)and two pyramidines (or precursors thereof). NCT comprises one or more nucleoside triphosphates and/or 0171 By way of illustration, in some embodiments the precursors thereof. Illustrative nucleoside triphosphates Supplement (e.g., incorporated the culture medium or com include, but need not be limited to adenosine triphosphate prising the NCT medium) comprises or consists of one or (ATP), guanosine triphosphate (GTP), cytidine triphosphate more of the following: (CTP), 5-methyluridine triphosphate (m,5UTP), uridine (0172 1) dATP (or a precursor thereof): triphosphate (UTP), and the like. In certain embodiments (0173 2) dGTP (or a precursor thereof): when ATP is present, at least one other nucleoside and/or (0174 3) dCTP (or a precursor thereof): deoxynucleoside triphosphate or precursor thereof is also (0175 4) dTTP (or a precursor thereof): present as a supplement. Illustrative precursors of the NTPs (0176 5) dUTP (or a precursor thereof): include, but are not limited to: 0164. For adenosine triphosphate (ATP): adenosine (0177 6) dATP (or a precursor thereof) and dGTP (or a diphosphate, adenosine monophosphate, adenosine, adenine, precursor thereof): and the like, or any combination thereof. In some embodi (0178 7) dATP (or a precursor thereof) and dCTP (or a ments the precursors include one or more precursors selected precursor thereof): from the group consisting of adenosine diphosphate, adenos (0179 8) dATP (or a precursor thereof) and dTTP (or a ine monophosphate, and adenosine. precursor thereof): 0.165 For guanosine triphosphate (GTP): guanosine 0180 9) dATP (or a precursor thereof) and dUTP (or a diphosphate, guanosine monophosphate, guanosine, gua precursor thereof): nine, and the like, or any combination thereof. In some 0181. 10) dGTP (or a precursor thereof) and dCTP (or a embodiments the precursors include one or more precursors precursor thereof): US 2015/O 105343 A1 Apr. 16, 2015

0182 11) dGTP (or a precursor thereof) and dTTP (or a uM, or about 4 LM, or about 5uM, or about 6 uM, or about 7 precursor thereof): uM, or about 8 LM, or about 9 uM, or about 10 uM, or about 0183) 12) dGTP (or a precursor thereof) and dUTP (or a 11 M, or about 12 uM, or about 13 uM, or about 14 uM, or precursor thereof): about 15 M, or about 16 uM, or about 17 uM, or about 18 0184 13) dCTP (or a precursor thereof) and dTTP (or a uM, or about 19 uM, or about 20 uM, or about 21 uM, or about precursor thereof): 22 uM, or about 23 uM, or about 24 uM, or about 25uM, or 0185. 14) dCTP (or a precursor thereof) and dUTP (or a about 26 M, or about 27 uM, or about 28 uM, or about 29 precursor thereof): uM, or about 30 uM, or about 31 uM, or about 32 uM, or about 0186 15) dTTP (or a precursor thereof) and dUTP (or a 33 uM, or about 34 uM, or about 35uM, or about 36 uM, or precursor thereof): about 37 uM, or about 38 LM, or about 39 uM, or about 40 0187. 16) dATP (or a precursor thereof), dGTP (or a pre uM, or about 41 uM, or about 42 uM, or about 43 uM, or about cursor thereof), and dCTP (or a precursor thereof); 44 uM, or about 45 uM, or about 46 uM, or about 47 uM, or 0188 17) dATP (or a precursor thereof), dGTP (or a pre about 48 uM, or about 49 uM, or about 50 uM. cursor thereof), and dTTP (or a precursor thereof); 0202. In certain embodiments the NTPs and/or dNTPs or (0189 18) dATP (or a precursor thereof), dCTP (or a pre precursor(s) thereof Supplementing the culture medium or cursor thereof), and dTTP (or a precursor thereof); comprising the NCT medium are each present at a concen (0190. 19) dGTP (or a precursor thereof), dCTP (or a pre tration ranging from about L1 LM, 1 M, or about 2 LM, or cursor thereof), and dTTP (or a precursor thereof); about 3 uM, or about 4 LM, or about 5uMup to about 50 uM, (0191) 20) dATP (or a precursor thereof), dGTP (or a pre or up to about 40 uM, or up to about 30 uM, or up to about 25 cursor thereof), and dCTP (or a precursor thereof); uM, or up to about 20 uM, or up to about 15uM. (0192. 21) dATP (or a precursor thereof), dGTP (or a pre 0203. In certain embodiments the total NTPs and/or cursor thereof), and dUTP (or a precursor thereof): dNTPs or precursor(s) thereof supplementing the culture (0193 22) dATP (or a precursor thereof), dCTP (or a pre medium or comprising the NCT medium is present at a con cursor thereof), and dUTP (or a precursor thereof): centration ranging from about 1 LM, or about 2 LM, or about (0194 23) dGTP (or a precursor thereof), dCTP (or a pre 3 uM, or about 4 LLM, or about 5uM, or about 6 uM, or about cursor thereof), and dUTP (or a precursor thereof): 7 uM, or about 8 LM, or about 9 uM, or about 10M, or about (0195 24) dATP (or a precursor thereof), dGTP (or a pre 11 M, or about 12 uM, or about 13 uM, or about 14 uM, or cursor thereof), dCTP (or a precursor thereof), and dTTP (or about 15 M, or about 16 uM, or about 17 uM, or about 18 a precursor thereof); uM, or about 19 uM, or about 20 Mup to about 200 uM, or (0196. 25) dATP (or a precursor thereof), dGTP (or a pre up to about 180 uM, or up to about 150 uM, or up to about 145 cursor thereof), dCTP (or a precursor thereof), and dUTP (or uM, or up to about 140 uM, or up to about 135 uM, or up to a precursor thereof); about 130 uM, or up to about 125uM, or up to about 120 uM, 0197) It will be recognized that in any of the foregoing or up to about 115uM, or up to about 110 uM, or up to about Supplements, the deoxynucleoside can be a nucleoside or a 105 uM, or up to about 100 uM. In certain embodiments, a precursor thereof. These supplements are intended to be illus stem cell culture is also provided herein, where the stem cell trative and non-limiting. culture comprises stem cells in a culture medium Supple (0198 Typically, the dNTPs and/or NTPs are present in the mented with one or more dNTPs and/or NTPs as described culture or NCT medium in an amount sufficient to improve herein. In certain embodiments the stem cells can be in vivo the short term and/or long term genetic stability of stem cells and exposed to an NCT as described herein. In various as compared to the same cells cultured in the same culture embodiments, the stem cells may be embryonic stem cells or medium or exposed to the NCT medium without supplemen adult stem cells including, but not limited to neural stem cells, tation by a nucleoside triphosphate or precursor thereof and/ hepatic stem cells, hematopoietic stem cells, umbilical cord or to improve proliferation rate, and/or to improve viability. blood stem cells, epidermal stem cells, gastrointestinal stem (0199. In certain embodiments the NTPs and/or dNTPs or cells, endothelial stem cells, muscle stem cells, mesenchymal precursor(s) thereof supplementing the culture or DCT stem cells, pancreatic stem cells, and the like. In certain medium are each, independently, present at a concentration embodiments the stem cells include induced pluripotent stem ranging from about luM up to about 50 uM, or from about 1 cells, especially human IPSCs. The stem cells (including uM to about 40 uM, or from about 1 uM up to about 35 uM, IPSCs) can be non-human animal stem cells or human stem or from about 1 uM up to about 30 uM. In certain embodi cells. ments the NTPs and/or dNTPs or precursor(s) thereof supple 0204. In certain embodiments the stem cells are induced menting the culture or DCT medium are each, independently, pluripotent stem cells, especially human IPSCs. Illustrative present at a concentration of about 50 uM or lower, or about stem cells include, but are not limited to, stem cells are 40 uM or lower, or about 35 uM or lower, or about 30 uM or selected from the group consisting of embryonic stem cells lower or about 25uM or lower, or about 20 uM or lower, or and adult stem cells, including, but not limited to neural stem about 15uM or lower, or about 10 uM or lower, or about 5uM cells, hepatic stem cells, hematopoietic stem cells, umbilical or lower (with the understanding that at least 0.1 uMofat least cord blood stem cells, epidermal stem cells, gastrointestinal one nucleoside triphosphate or precursor thereof is present stem cells, endothelial stem cells, muscle stem cells, mesen such that “lower” is not meant to be construed as an absence chymal stem cells, and pancreatic stem cells. of every nucleoside triphosphate or precursor thereof). 0205. In certain embodiments, where the stem cells are 0200. In certain embodiments the NTPs and/or dNTPs or IPSCs the IPSCs are reprogrammed from cells selected from precursor(s) thereof Supplementing the culture medium or the group consisting offibroblasts, neural stem cells, stomach comprising the NCT medium are each independently present cells, liver cells, keratinocytes, melanocytes, amniotic cells, at a concentration ranging from about 5uM to about 30 uM. blood cells, B-cells, and adipose cells. In certain embodi 0201 In certain embodiments the NTPs and/or dNTPs or ments the IPSCs comprise cells reprogramed two or more precursor(s) thereof Supplementing the culture medium or factors selected from the group consisting of KLF4 (K), comprising the NCT medium are each independently present LIN28 (L), c-MYC (M), NANOG (N); OCT4 (O), SOX2 (S), at a concentration of about 1 uM, or about 2 uM, or about 3 and valproic acid (VPA). In certain embodiments the ISPCs US 2015/O 105343 A1 Apr. 16, 2015

comprises cells reprogramed using the four canonical Molecular Genetics 17: R37-41). However, our findings Yamanaka factors KLF4 (K), c-MYC (M).OCT4 (O), and (FIG. 1, Preliminary Data), and those of others (Martins SOX2 (S). Taylor and Xu (2012) Stem Cells 30:22-27; Lundet al. (2012) 0206 Methods of reprogramming cells to produce IPSCs Nat. Rev. Genet., 13: 732-744), indicate that essentially all well knownto those of skill in the art. In certain embodiments iPSC clones derived using current approaches acquire reprogramming can be accomplished using for example inte genomic instability during reprogramming and in vitro cul grating vectors (e.g., lent viral vectors, inducible lentiviral ture-induced stress. The issue of hiPSC genomic instability is vectors, and the like), excisable vectors (e.g., transposon vec one of the most important bottlenecks in advancing person tors, loxP-flanked lentiviral vectors, and the like), non-inte alized iPSC-based regenerative therapies to the clinic (Mar grating vectors (e.g., adenoviral vectors, plasmid vectors, tins-Taylor and Xu (2012) Stem Cells 30: 22-27; Lund et al. etc.), DNA free vectors (e.g., Sendai virus, protein vectors, (2012) Nat. Rev. Genet., 13: 732-744: Byrne (2013) Gene modified mRNA vectors, microRNA vectors, and the like). It Therapy and Regulation 7: 1230002), given the established is noted that a number of reprogramming kits are commer link between genomic instability and an increased risk of cially available (e.g., (EpiSTM Episomal iPSC Reprogram malignant transformation. ming Kit and the CytoTune(R)-iPS Sendai Reprogramming 0215. The causes of genomic instability during hIPSC Kit from Life Technologies, and the like). reprograming and culture remain unknown, and approaches 0207. In various embodiments, methods of reducing the to reduce and eventually eliminate the occurrence of this genetic instability of induced stem cells (including pluripo highly deleterious phenomenon have yet to be developed. In tent stem cells) are provided where the method comprises ongoing experiments, we find that deoxyribonucleotide triph culturing the cells in a cell culture medium Supplemented osphate (dNTP) pools in rapidly dividing hiPSCs are signifi with NTPs and/or dNTPs and/or precursor(s) thereof as cantly smaller in size than the dNTP pools of dermal fibro described herein or contacting the cells, e.g., in Vivo, with a blasts, from which the hIPSCs are derived (FIG. 2). We also NCT medium comprising one or more NTPs and/or dNTPs find that hIPSCs demonstrate significantly more double and/or precursor(s) thereofas described herein. strand breaks (DSBs) than the fibroblasts of origin (FIGS. 3A 0208 Invarious embodiments a stem cell (e.g., an induced and 3B). Our observations of decreased dNTP pools and pluripotent stem cell) present in a cell culture medium Supple increased DNA damage in hIPSCs may provide new mecha mented with, or contacted with a NCT medium comprising, nistic insight into the etiology of genomic instability of hP NTPs and/or dNTPs and/or precursor(s) thereofas described SCs, and present a method for preventing this phenomenon. herein is provided. Thus, our data indicate that: 1) hIPSCs can utilize exogenous 0209. Also provided are methods of providing autologous dNTP precursors added to the hIPSC culture medium in the stem cell transfer. The methods typically involve providing form of a mixture of deoxyribonucleosides (dNs) and 2) that stem cells isolated from a subject or IPSCs generated from a culture medium supplementation with dNs can both amelio Subject and expanding and/or culturing said stem cells or rate the hIPSC dNTP pool deficiency and significantly reduce IPSCs in a cell culture medium or an NCT medium supple the genomic instability experienced by these cells (FIG. 4). mented with NTPs and/or dNTPs and/or precursor(s) thereof as described herein. 0216 Hypotheses and Rationale. 0210. In each of the embodiments described herein, the 0217 Mammalian cells synthesize dNTPs via two path subject matter (i.e., cell culture, DCT medium, cells, meth ways: the de novo pathway (DNP), which uses glucose and ods, etc.) may be free of any NTPs and/or dNTPs and/or amino acids, and the nucleoside Salvage pathway (NSP), precursor(s) thereof that have been radiolabeled. Thus, in which uses preformed dNs from the extracellular environ some embodiments, the NTPs and/or dNTPs and/or precursor ment. Without being bound by a particular theory, we hypoth (s) thereof used in such embodiments have not or are not esize that: 1) insufficient production of dNTP pools via the linked or incorporating any radiolabel (such as, for example, DNP is an important cause of replication stress, DNA damage H, Cr, or 'P, and the like). Thus, the embodiments can be and genomic instability in rapidly dividing hiPSCs and 2) free of, for example, H-dTTP or H-TTP or any precursor enabling the utilization by the hIPSC cells of the NSP by thereof Supplementing the culture medium with a defined mixture of 0211. The embodiments described herein are intended to dN substrates of the NSP will increase dNTP pools and will be illustrative and non-limiting. Using the teachings provided alleviate replication stress, DNA damage and genomic insta herein, numerous variations of the compositions and methods bility. described herein will be available to one of skill in the art. 0218. Without being bound by a particular theory, it was hypothesized that: 1) insufficient production of dNTP pools EXAMPLES from glucose and amino acids via the be novo pathway 0212. The following examples are offered to illustrate, but (DNP), is an important cause of replication stress, DNA dam not to limit the claimed invention. age and genomic instability in hiPSCs and 2) enabling the hIPSCs to also use a second dNTP biosynthetic pathway, the Example 1 nucleoside salvage pathway (NSP) in addition to the DNP will alleviate replication stress, DNA damage, and genomic Nucleoside Supplementation. In hIPSC Culture Or In instability. In an illustrative embodiment, enabling the use of Nucleoside Cocktail the NSP was accomplished by supplementing the hiPSC cul ture medium with a mixture of deoxyribonucleoside (dNs) 0213 Transmission (NCT) Medium To Promote Genetic Substrates of the NSP. Stability, Enhance Cellular Function and Enhance Regenera tion 0219. In summary, a simple, effective, generally appli cable, and cost-efficient solution to the problem of genomic Significance instability is provided that is useful for hIPSCs produced using the current methodology. Specifically, it is shown that 0214 Human induced pluripotent stem cells (hIPSCs) the occurrence of genomic instability in stem cells and hiP have significant therapeutic potential (Byrne (2008) Human SCs can be prevented by enabling these cells to synthesize US 2015/O 105343 A1 Apr. 16, 2015 dNTPs via the NSP. This can be achieved by supplementing Genes involved in dNTP Biosynthesis are Expressed at Simi the culture media with an optimized mixture of dN substrates lar Levels in Rapidly Dividing hiPSCs and in HDFs with a of the NSP Slower Rate of Cell Division 0223) An Affymetrix global transcriptional analysis on Derivation and Characterization of Human Induced HDFs and derived early passage hIPSCs (performed as pre Pluripotent StemCells viously described (Awe etal. (2013) Stem Cell Res. & Theap., 4:15)) showed significant upregulation of key pluripotency 0220 We have reprogrammed adult human dermal fibro specific markers; the expression of key fibroblast-specific blasts (HDFs) into human induced pluripotent stem cells markers was decreased following hiPSC reprogramming (hIPSCs) using either a LoxP-flanked polycistronic repro (Table 2). We also observed upregulation of genes previously gramming vector (which was then removed from the genome linked to the induction of genomic instability (CCNE1, E2F2, using Cre recombinase (Sommer et al. (2010) Stem Cells 28: E2F35). However, we did not observe any significant differ 64-74)), or by using a synthetic mRNA based approach (War ences in the expression of key dNTP biosynthetic genes ren et al. (2010) Cell Stem Cell, 7: 618-630). All reprogram (Table 2). As hIPSCs have a faster average population dou ming and Subsequent culture were performed in standard bling time (PDT) than HDFs (18 hrs vs. 48 hrs), these data hIPSC culture medium, which, according to the current stan support our hypothesis that the small dNTP pools in hIPSCs dard practice, was not Supplemented with any nucleosides may result from 1) a faster rate of cell proliferation which (www.stemgent.com/products/show/69. NUTRISTEMTM increases the usage of dNTPs for DNA replication, and 2) an XF/FF Culture Medium). Both transgene-free hIPSC lines inability to upregulate the expression of genes involved in demonstrated Standard characterization parameters, as previ dNTP biosynthesis. ously described (Byrne et al. (2009) PloS One 4: e7118). These parameters included embryonic stem cell (ESC)—like TABLE 2 morphology, significant expression of the key pluripotency markers (NANOG, OCT4, SSEA3, SSEA4, Tra-1-60 and Change in gene expression following hiPSC reprogramming. Tra-1-81), which were not detected in the original skin fibro blasts (FIG. 1, panel A), and differentiation into representa Gene tives of all three germ layers following teratoma formation in l8le Gene ID Type Fold change p-Value severe combined immunodeficiency (SCID) mice (FIG. 1, POUSF1 HS.450254 Pluripotency specific 40 fold increase 0.01 panel B). We then converted our transgene-free hIPSC lines NANOG Hs.661360 Pluripotency specific 100 fold increase 0.01 SOX2 Hs.518438 Pluripotency specific 30 fold increase 0.004 from research-grade conditions (containing animal-derived COL1A1 Hs. 172928 Fibroblast specific 20 fold decrease 0.03 epitopes: Matrigel substrate and Knockout Serum Replace COL3A1 HS.443625 Fibroblast specific 170 fold decrease 0.001 ment, KSR, containing culture medium) into putative clini COL6A3 Hs.233240 Fibroblast specific 40 fold decrease 0.02 cal-grade animal epitope-free"xeno-free' defined conditions CCNE1 Hs.244723 Genomic instability 7 fold increase 0.03 (CellStart substrate and an optimized xeno-free media, 50% E2F2 Hs.194333 Genomic instability 8 fold increase 0.02 E2F3 Hs.269408 Genomic instability 3 fold increase 0.01 mTeSRI, Stemcell Technologies, and 50% NutriStem, Stem dCK HS.709 Nucleotide metabolism No difference NA gent), as previously described (Karumbayaram et al. (2012) TK1 Hs.515122 Nucleotide metabolism No difference NA Stem Cells Trans. Med. 1: 36-43). Both hIPSC lines were UPP1 Hs.488240 Nucleotide metabolism No difference NA karyotyped, as we have previously described (Byrne et al. RM1 Hs.445705 Nucleotide metabolism No difference NA (2009) PloS One 4: e7118) both before and after “clinical RRM2 Hs.226390 Nucleotide metabolism No difference NA grade conversion” (CC). 0221 Following CC-induced stress and expansion both Elevated levels of DNA damage in hIPSCs vs. HDFs: dN lines went from being karyotypically normal (46XY) to pos supplementation reduces DNA damage in hiPSCs sessing an extra chromosome 12 (FIG. 1, panel C). The 0224. We utilized gamma-histone 2A.X (yH2A.X) stain observed genomic instability correlates closely with several ing, as previously described (Bester et al. (2011) Cell, 145: published studies showing that hIPSCs are genetically 435-446), in order to estimate the number of double strand unstable, particularly when placed under conditions of extrin breaks (DSBs) in HDFs and early passage hIPSCs. Signifi sic stress (Nagaria et al. (2013) Biochimica et Biophysica cantly more yH2AX positive foci were detected in hIPSCs Acta, 1830: 2345-2353), and preferentially duplicate chro than in the HDFs of origin. We also observed significantly mosome 12 (Draper et al. (2004) Nat. Biotechnol. 22:53-54). lessyH2AX positive foci following 4 days of dN supplemen It has previously been suggested that all chromosomes in tation (FIG. 3A, compare middle and lower panels). This hIPSCs have the same initial incidence of chromosomal finding provides additional Support for the hypothesis that translocation and duplication, but that duplication of chromo hIPSCs demonstrate higher levels of genomic instability than Some 12, when it occurs, results in a preferential expansion dermal fibroblasts and genomic instability could be prevented throughout the hiPSC population following extended in vitro with dN supplementation of the cell culture medium. We also culture through a selection process (Id.). It should be noted observed significantheterogeneity in the number of yH2AX that we consider CC-based stress to be a useful model for positive foci within the hiPSC population, supporting, our longer term culture (>6-12 month) induced stress, which also hypothesis that the incidence of genomic instability is non tends to result inhIPSCs and hESCs accumulating karyotypic uniform across the hiPSC population, and that a subpopula abnormalities, especially chromosome 12, as we observed tion of hiPSCs are more likely to incur additional genomic following CC-based stress over a 2 month period. DNA damage, as assayed by yH2AX staining, and thus rep Smaller dNTP Pools in hIPSCs vs. HDFs of Origin resent the cells most Susceptible to developing karyotypic 0222 We quantified deoxyribonucleotide triphosphate abnormalities. (dNTP) pools in the original HDFs and the early passage dNSupplementation Promotes hiPSC Genomic Stability Fol (none-CC-stressed) hIPSCs as described previously (Austin lowing CC-Based Stress et al. (2012) J. Exp. Med., 209: 2215-2228). We observed a 0225. We cultured our hIPSCs in conditions with and significant drop (between 32-52%) in levels of all four dNTPs without dNs in order to investigate whether dN supplemen after hIPSC reprogramming (FIG. 2). tation could prevent genomic instability observed during CC US 2015/O 105343 A1 Apr. 16, 2015

based stress. It should be noted that, while hPSCs have been mental towards safely unlocking the future promise of per demonstrated to accumulate genomic damage during repro sonalized pluripotent stem cell-based therapeutics for gramming and extended culture (Ben-David et al. (2010) Cell patients. Cycle (Georgetown, Tex.) 9: 4603-4604), we observed the most extreme genomic damage (karyotypic abnormalities), Example 2 in two independent hIPSC lines, when we stressed the cells by performing CC. This observation provides a rapid and con Obtaining Stable Stem dN Concentration sistent experimental assay to induce severe genomic damage over a relatively short period of time (2 months). Human 0230 We have obtained multiple different STAB iPSCs were first cultured on Matrigel, in a combination of LESTEMTM concentrations, one for short term culture (30 mTeSRI (Stemcell Technologies) and NutriStem (Stemgent). uM of each dN), one for long term culture (5uM of each dN) They were then divided into two groups grown in the presence as well as a suitable 30:30:5:5 cocktail that has proven robust or absence of exogenously supplied dNs in the cell culture in enhancing genomic stability, increasing cellular function medium (30 M of each dN). After one week of supplemen and improving differentiation potential across multiple cell tation, we began the conversion process to Xeno-free condi types (see, e.g. Table 3). tions, which entailed switching to CellStart as a basement membrane and NutriStem medium only. After two weeks of TABLE 3 culture, genomic DNA (gDNA) was extracted for single nucleotide polymorphism (SNP)-based loss-of-heterozygos dN concentrations. ity (LOH) analysis, which was previously shown to be an Culture Dose of 3-5 day cell 3+ weeks cell accurate measure of DSB-based genomic instability (Bester Supplement each dN culture culture et al. (2011) Cell, 145:435-446). To determine levels of LOH between the two conditions (+/-dNs), analysis was per Short term 30 M Good Not good formed using Affymetrix’s SNP 6.0 array as previously Long term 5 M Good Good described (Id.). Supplementation with dNs reduced the inci dence of genomic instability, as assayed by LOH, to 20% of 0231. The long term STABLESTEMTM culture media that observed in unsupplemented medium (FIG. 4). Together supplement allows human iPS cells to proliferate for at least with the significant decrease in yH2AX positive foci follow 3 weeks, while dramatically reducing the amount of genomic ing 4 days of dN supplementation (FIGS. 3A and 3B), the damage the cells are exposed to (FIG. 7); as measured by reduction in LOH provides evidence in support of our hypoth YH2A.X-positive double strand breaks. The causes of esis that dN supplementation reduces replication stress and genomic instability during hPSC reprograming and culture the associated genomic instability in hPSC reprogramming have been unclear. However, we recently discovered that and culture. deoxyribonucleotide triphosphate (dNTP) pools in rapidly dividing hIPSCs are significantly smaller in size than the 0226. It is important to note that previous research has dNTP pools of the skin cells from which the hIPSCs are correlated the incidence of DSB with LOH in human cancer derived. In addition, we discovered that Supplementing the cells (Id.), highlighting the value of SNP-based LOH mea stem cell culture media with deoxyribonucleosides (dNs) can Surements as a quantifiable method for assaying genomic significantly rescue the dNTP pools (FIG. 6). instability. In our study of CC-stressed hiPSCs with and 0232. It is understood that the examples and embodiments without dN supplementation, we observed a five-fold described herein are for illustrative purposes only and that increase in genomic stability, as assayed by the reductions in various modifications or changes in light thereofwill be Sug LOH, following CC-induced stress. This can be observed in gested to persons skilled in the art and are to be included FIG. 4, where 12 LOH chromosomal loci were identified within the spirit and purview of this application and scope of following CC-based stress. Five times as many LOH loci, the appended claims. All publications, patents, and patent which are indicative of genomic damage, were observed in applications cited herein are hereby incorporated by refer the untreated hiPSCs (identified by green arrows), than were ence in their entirety for all purposes. observed in the dN-treated hIPSCs (identified by the red 1. A cell culture medium for the culture of stem cells with arrow). This provides strong evidence in Support of our improved genetic stability, said culture medium comprising: hypothesis that dN Supplementation can ameliorate CC-in a basal culture medium for stem cells, where said culture duced genomic instability inhIPSCs. However, it is important medium is supplemented with one or more nucleoside to note that half of the LOH loci (as identified by the blue triphosphates or one or more precursors thereof. arrows) were still unstable in both dN treated and untreated 2. A nucleoside cocktail transmission (NCT) medium for samples. improving Somatic or stem cell genetic stability said medium 0227. While efficacy has been demonstrated, it is believed comprising: the dN supplementation procedure can be further optimized. a cosmetic or pharmaceutical delivery vehicle; and Data generated thus far support the hypothesis that dNTP one or more nucleoside triphosphates or precursors pool deficiency plays a critical role in the genomic instability thereof. we and others have observed in hIPSCs following general 3. The cell culture medium of claim 1, wherein said one or culture and induced stress. more nucleoside triphosphates are independently selected from the group consisting of deoxyadenosine triphosphate 0228 Moreover, our preliminary data also Suggest a (dATP), deoxyguanosine triphosphate (dGTP), deoxycyti simple and generally applicable culture medium deoxyribo dine triphosphate (NCTP), deoxythymidine triphosphate nucleoside Supplementation procedure to enhance the (dTTP) deoxyuridine triphosphate, adenosine triphosphate genomic stability of clinical grade hiPSCs and their deriva (ATP), guanosine triphosphate (GTP), cytidine triphosphate tives. (CTP), 5-methyluridine triphosphate (mSUTP), and uridine 0229. In summary, we propose that establishing such an triphosphate (UTP). optimized media system capable of maintaining genetically 4. The nucleoside cocktail transmission medium of claim stable stem cells and hPSC and derivatives will be instru 2, wherein said one or more nucleoside triphosphates are US 2015/O 105343 A1 Apr. 16, 2015

independently selected from the group consisting of deoxy 54. The cell culture medium of claim 1, wherein said adenosine triphosphate (dATP), deoxyguanosine triphos nucleoside triphosphate or precursor thereof Supplementing phate (dGTP), deoxycytidine triphosphate (NCTP), deox said culture medium, is presentata concentration Sufficient to ythymidine triphosphate (dTTP) deoxyuridine triphosphate, improve the short term and/or long term genetic stability of adenosine triphosphate (ATP), guanosine triphosphate stem cells as compared to the same cells cultured in the same (GTP), cytidine triphosphate (CTP), 5-methyluridine triph medium without Supplementation by a nucleoside triphos osphate (m5UTP), and uridine triphosphate (UTP). phate or precursor thereof. 5. (canceled) 55-60. (canceled) 6. The cell culture medium claim 1, wherein said culture 61. The cell culture medium of claim 1, wherein said cell medium is Supplemented with one or more precursors of culture medium is Xenopathogen-free. nucleoside triphosphates independently selected from the group consisting of deoxyadenosine triphosphate (dATP) 62. The cell culture medium of claim 1, wherein said cell precursor, deoxyguanosine triphosphate (dGTP) precursor, culture medium, is without animal or human derived serum deoxycytidine triphosphate (NCTP) precursor, deoxythymi albumin. dine triphosphate (dTTP) precursor, deoxyuridine triphos 63. The cell culture medium of claim 1, wherein the supple phate (dUTP) precursor, adenosine triphosphate (ATP) pre mented medium is selected from the group consisting of cursor, guanosine triphosphate (GTP) precursor, cytidine DMEM (Dulbecco's Modified Eagle's Medium), MEM triphosphate (CTP) precursor, 5-methyluridine triphosphate (Minimal Essential Medium), BME (Basal Medium Eagle), (m5UTP) precursor, and uridine triphosphate (UTP) precur RPMI 1640, DMEM/F-12 (Dulbecco's Modified Eagle's SO. Medium: Nutrient Mixture F-12), DMEM/F-10 (Dulbecco's 7. The NCT medium of claim 2, wherein said NCT medium Modified Eagle's Medium: Nutrient Mixture F-10), C-MEM comprises one or more precursors of nucleoside triphos (C.-Minimal essential Medium), G-MEM (Glasgow’s Mini phates independently selected from the group consisting of mal Essential Medium), IMDM (Isocove’s Modified Dulbec deoxyadenosine triphosphate (dATP) precursor, deoxygua co's Medium), essential 8 (E8) medium, and KnockCut nosine triphosphate (dGTP) precursor, deoxycytidine triph DMEM. osphate (NCTP) precursor, deoxythymidine triphosphate 64-65. (canceled) (dTTP) deoxyuridine triphosphate (dUTP) precursor, 66. A somatic or stem cell culture or nucleoside cocktail adenosine triphosphate (ATP) precursor, guanosine triphos transmission (NCT) culture, comprising: phate (GTP) precursor, cytidine triphosphate (CTP) precur cells in a basal culture medium for stem cells, where said sor, 5-methyluridine triphosphate (m,5UTP) precursor, and culture medium is Supplemented with one or more uridine triphosphate (UTP) precursor. nucleoside triphosphates or one or more precursors 8. The cell culture medium of claim 6, wherein said one or thereof and/or more precursors of nucleoside triphosphates comprise a cells in a medium comprising a cosmetic orpharmaceutical nucleoside diphosphate or a nucleoside monophosphate. delivery vehicle; and one or more nucleoside triphos 9. The NCT medium of claim 7, wherein said one or more phates or precursors thereof. precursors of nucleoside triphosphates comprise a nucleoside diphosphate or a nucleoside monophosphate. 67. The cell culture or NCT culture of claim 66, wherein 10-48. (canceled) said cells are cells selected from the group consisting of 49. The nucleoside cocktail transmission (NCT) medium Somatic cells or stem cells. of claim 2, wherein said NCT medium comprises a vehicle 68-75. (canceled) formulated for administration via a route selected from the 76. A method of reducing genetic instability of stem cells group consisting of Subcutaneous administration, parenteral said method comprising culturing said cells in a cell culture administration, topical administration, oral administration, medium comprising a basal culture medium for stem cells, nasal or inhalation administration, local administration Such Supplemented with one or more nucleoside triphosphates or as by paint, aerosol, or transdermally. one or more precursors thereof. 50. (canceled) 77. A method of performing autologous stem cell transfer, 51. The nucleoside cocktail transmission (NCT) medium said method comprising isolating stem cells from a subject or of claim 49, wherein said medium is formulated in a vehicle generating IPSCs from said subject and expanding and/or selected from the group consisting of a mud, an herbal mix culturing said stem cells or IPSCs in a cell culture medium ture, a fat, an emulsions, a lotion, a cream, a gel, a biological, comprising a basal culture medium for stem cells, Supple a solution, a spray, an ointment, a foams, a mousses, a liquid, mented with one or more nucleoside triphosphates or one or a Suspensions, a dispersion, an aerosol, a Soap, a shampoo, more precursors thereof. and a conditioner. 78. A method of promoting regeneration and/or mainte 52. The NCT medium of claim 2, wherein said nucleoside nance of tissues, said method comprising administering to a triphosphate or precursor thereof is presentata concentration Subject, an NCT medium comprising a cosmetic or pharma Sufficient to improve the short term and/or long term genetic ceutical delivery vehicle and one or more nucleoside triphos stability of stem cells as compared to the same cells in the phates or precursors thereof. same medium without Supplementation by a nucleoside triph osphate or precursor thereof. 79-87. (canceled) 53. (canceled)