Methods and compositions for generating or maintaining pluripotent cells

Abstract

Methods and compositions are provided for generating or maintaining human iPS cells in culture. Methods include the use of a low osmolality medium to make human iPS cells, or use of a low osmolality medium to maintain human iPS cells. Methods for making targeted genetic modification to human iPS cells cultured in low osmolality medium are also included. Compositions include human iPS cells cultured and maintained using the low osmolality medium defined herein.

1 . A method for modifying a target genomic locus in a human induced pluripotent stem cell (hiPSC), comprising:
 (a) providing a population of naïve hiPSCs that display a morphology characterized by compact, dome-shaped colonies,   wherein the hiPSCs are cultured in a low osmolality medium comprising a base medium and supplements,   wherein the low osmolality medium comprises:
 (i) a leukemia inhibitory factor (LIF) polypeptide; 
 (ii) a glycogen synthase kinase 3 (GSK3) inhibitor; and 
 (iii) a MEK inhibitor, and 
   wherein the low osmolality medium has an osmolality of about 200 mOsm/kg to about 250 mOsm/kg;   (b) introducing into the population of hiPSCs a targeting vector comprising an insert nucleic acid flanked by 5′ and 3′ homology arms corresponding to 5′ and 3′ target sites at the target genomic locus; and
 (c) identifying a genetically modified hiPSC comprising in its genome the insert nucleic acid integrated at the target genomic locus. 
   
     
     
         2 .- 31 . (canceled) 
     
     
         32 . A method for modifying a target genomic locus in a human induced pluripotent stem cell (hiPSC), comprising:
 (a) providing a population of naïve hiPSCs that display a morphology characterized by compact, dome-shaped colonies,   wherein the hiPSCs are cultured in a low osmolality medium comprising a base medium and supplements,   wherein the low osmolality medium comprises:
 (i) a leukemia inhibitory factor (LIF) polypeptide; 
 (ii) a glycogen synthase kinase 3 (GSK3) inhibitor; and 
 (iii) a MEK inhibitor, and 
   wherein the low osmolality medium has an osmolality of about 200 mOsm/kg to about 250 mOsm/kg;   (b) introducing into the population of hiPSCs a nuclease agent that induces one or more nicks or double-strand breaks at a recognition site at the target genomic locus; and   (c) identifying a genetically modified hiPSC comprising in its genome a modification at the target genomic locus.   
     
     
         33 .- 58 . (canceled) 
     
     
         59 . The method of  claim 1 , wherein the targeting vector is a large targeting vector (LTVEC), wherein:
 (I) the LTVEC is at least 10 kb in size;   (II) the LTVEC is from about 50 kb to about 300 kb in size;   (III) the sum total of the 5′ and 3′ homology arms is at least 10 kb;   (IV) the sum total of the 5′ and 3′ homology arms is from about 10 kb to about 200 kb;   (V) the 5′ homology arm is from about 5 kb to about 100 kb and/or the 3′ homology arm is from about 5 kb to about 100 kb; or   (VI) the LTVEC comprises a nucleic acid insert ranging from about 5 kb to about 200 kb.   
     
     
         60 . The method of  claim 1 , wherein the targeted genetic modification comprises:
 (a) deletion of an endogenous human nucleic acid sequence;   (b) insertion of an exogenous nucleic acid sequence; or   (c) replacement of the endogenous human nucleic acid sequence with the exogenous nucleic acid sequence.   
     
     
         61 . The method of  claim 1 , wherein the targeted genetic modification comprises insertion of an exogenous nucleic acid sequence, wherein the exogenous nucleic acid sequence comprises one or more of the following:
 (a) a nucleic acid sequence that is homologous or orthologous to the endogenous human nucleic acid sequence;   (b) a chimeric nucleic acid sequence;   (c) a conditional allele flanked by site-specific recombinase target sequences; and   (d) a reporter gene operably linked to a promoter active in the hiPSC.   
     
     
         62 . The method of  claim 1 , wherein introducing step (b) further comprises introducing a nuclease agent that promotes homologous recombination between the targeting vector and the target genomic locus in the hiPSC. 
     
     
         63 . The method of  claim 62 , wherein the nuclease agent comprises:
 (a) a Zinc Finger Nuclease (ZFN);   (b) a Transcription Activator-Like Effector Nuclease (TALEN);   (c) a meganuclease; or   (d) a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated (Cas) protein and a guide RNA (gRNA) comprising a CRISPR RNA (crRNA) that recognizes a genomic target sequence and a trans-activating CRISPR RNA (tracrRNA).   
     
     
         64 . The method of  claim 63 , wherein the nuclease agent comprises the Cas protein and the guide RNA, wherein the Cas protein is Cas9. 
     
     
         65 . The method of  claim 1 , wherein prior to step (b), the hiPSCs are enzymatically dissociated into a single-cell suspension and subcultured. 
     
     
         66 . The method of  claim 65 , wherein the enzymatic dissociation:
 (a) is performed using trypsin;   (b) is performed in the absence of a ROCK inhibitor; or   (c) a combination thereof, and   wherein the subcultured hiPSCs:   (a) continue to express one or more pluripotency markers;   (b) maintain a naïve state and display a morphology characterized by compact dome-shaped colonies; or   (c) a combination thereof, and   wherein the subcultured hiPSCs maintain a normal karyotype.   
     
     
         67 . The method of  claim 1 , wherein the hiPSCs have a normal karyotype, and wherein the hiPSCs:
 (a) express one or more pluripotency markers, wherein the pluripotency markers comprise NANOG, alkaline phosphatase, or a combination thereof;   (b) can differentiate into cells of any one of the endoderm, ectoderm, or mesoderm germ layers;   (c) have a doubling time of between about 16 hours and about 24 hours; or   (d) any combination of (a) to (c).   
     
     
         68 . The method of  claim 1 , wherein the hiPSCs are derived from non-pluripotent cells transformed to express a pluripotent state, wherein the transformed cells express reprogramming genes comprising Oct4, Sox2, Klf4, Myc, or any combination thereof. 
     
     
         69 . The method of  claim 1 , wherein the base medium has an osmolality of about 180 mOsm/kg to about 250 mOsm/kg. 
     
     
         70 . The method of  claim 69 , wherein the base medium comprises sodium chloride at about 3 mg/mL, sodium bicarbonate at about 2.2 mg/mL, and glucose at about 4.5 mg/mL, and has an osmolality of about 200 mOsm/kg. 
     
     
         71 . The method of  claim 1 , wherein the low osmolality medium has an osmolality of about 220 mOsm/kg to about 240 mOsm/kg. 
     
     
         72 . The method of  claim 71 , wherein the low osmolality medium has an osmolality of about 233 mOsm/kg. 
     
     
         73 . The method of  claim 1 , wherein:
 (a) the supplements comprise:
 (i) F-12 medium; 
 (ii) N2 supplement; 
 (iii) B-27 supplement; 
 (iv) L-glutamine; 
 (v) 2-mercaptoethanol; or 
 (vi) any combination of (i) to (v); 
   (b) the LIF polypeptide is a human LIF (hLIF) polypeptide;   (c) the GSK3 inhibitor comprises CHIR99021;   (d) the MEK inhibitor comprises PD0325901;   (e) the hiPSCs are cultured on newborn human foreskin fibroblast (NuFF) feeder cells; or   (f) any combination of (a) to (e).   
     
     
         74 . The method of  claim 1 , wherein the low osmolality medium comprises inhibitors consisting essentially of the glycogen synthase kinase 3 (GSK3) inhibitor and the MEK inhibitor. 
     
     
         75 . The method of  claim 1 , wherein the low osmolality medium comprises base medium at about 24.75% (v/v), F-12 medium at about 24.75% (v/v), N2 supplement at about 0.5% (v/v), B-27 supplement at about 1% (v/v), L-glutamine at about 2 mM, 2-mercaptoethanol at about 0.1 mM, hLIF at about 100 units/mL, CHIR99021 at about 3 and PD0325901 at about 0.5 optionally wherein the hiPSCs are cultured on MATRIGEL, newborn human foreskin fibroblast (NuFF) feeder cells, or GELTREX. 
     
     
         76 . The method of  claim 1 , wherein the low osmolality medium does not comprise one or more of the following: bFGF supplement; TGF-β1 supplement; JNK inhibitor; p38 inhibitor; ROCK inhibitor; and PKC inhibitor. 
     
     
         77 . The method of  claim 76 , wherein the low osmolality medium does not comprise bFGF supplement. 
     
     
         78 . The method of  claim 32 , wherein the nuclease agent comprises:
 (a) a Zinc Finger Nuclease (ZFN);   (b) a Transcription Activator-Like Effector Nuclease (TALEN);   (c) a meganuclease; or   (d) a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated (Cas) protein and a guide RNA (gRNA) comprising a CRISPR RNA (crRNA) that recognizes a genomic target sequence and a trans-activating CRISPR RNA (tracrRNA).   
     
     
         79 . The method of  claim 78 , wherein the nuclease agent comprises the Cas protein and the guide RNA, wherein the Cas protein is Cas9. 
     
     
         80 . The method of  claim 32 , wherein prior to step (b), the hiPSCs are enzymatically dissociated into a single-cell suspension and subcultured. 
     
     
         81 . The method of  claim 80 , wherein the enzymatic dissociation:
 (a) is performed using trypsin;   (b) is performed in the absence of a ROCK inhibitor; or   (c) a combination thereof, and   wherein the subcultured hiPSCs:   (a) continue to express one or more pluripotency markers;   (b) maintain a naïve state and display a morphology characterized by compact dome-shaped colonies; or   (c) a combination thereof, and   wherein the subcultured hiPSCs maintain a normal karyotype.   
     
     
         82 . The method of  claim 32 , wherein the hiPSCs have a normal karyotype, and wherein the hiPSCs:
 (a) express one or more pluripotency markers, wherein the pluripotency markers comprise NANOG, alkaline phosphatase, or a combination thereof;   (b) can differentiate into cells of any one of the endoderm, ectoderm, or mesoderm germ layers;   (c) have a doubling time of between about 16 hours and about 24 hours; or   (d) any combination of (a) to (c).   
     
     
         83 . The method of  claim 32 , wherein the hiPSCs are derived from non-pluripotent cells transformed to express a pluripotent state, wherein the transformed cells express reprogramming genes comprising Oct4, Sox2, Klf4, Myc, or any combination thereof. 
     
     
         84 . The method of  claim 32 , wherein the base medium has an osmolality of about 180 mOsm/kg to about 250 mOsm/kg. 
     
     
         85 . The method of  claim 84 , wherein the base medium comprises sodium chloride at about 3 mg/mL, sodium bicarbonate at about 2.2 mg/mL, and glucose at about 4.5 mg/mL, and has an osmolality of about 200 mOsm/kg. 
     
     
         86 . The method of  claim 32 , wherein the low osmolality medium has an osmolality of about 220 mOsm/kg to about 240 mOsm/kg. 
     
     
         87 . The method of  claim 86 , wherein the low osmolality medium has an osmolality of about 233 mOsm/kg. 
     
     
         88 . The method of  claim 32 , wherein:
 (a) the supplements comprise:
 (i) F-12 medium; 
 (ii) N2 supplement; 
 (iii) B-27 supplement; 
 (iv) L-glutamine; 
 (v) 2-mercaptoethanol; or 
 (vi) any combination of (i) to (v); 
   (b) the LIF polypeptide is a human LIF (hLIF) polypeptide;   (c) the GSK3 inhibitor comprises CHIR99021;   (d) the MEK inhibitor comprises PD0325901;   (e) the hiPSCs are cultured on newborn human foreskin fibroblast (NuFF) feeder cells; or   (f) any combination of (a) to (e).   
     
     
         89 . The method of  claim 32 , wherein the low osmolality medium comprises inhibitors consisting essentially of the glycogen synthase kinase 3 (GSK3) inhibitor and the MEK inhibitor. 
     
     
         90 . The method of  claim 32 , wherein the low osmolality medium comprises base medium at about 24.75% (v/v), F-12 medium at about 24.75% (v/v), N2 supplement at about 0.5% (v/v), B-27 supplement at about 1% (v/v), L-glutamine at about 2 mM, 2-mercaptoethanol at about 0.1 mM, hLIF at about 100 units/mL, CHIR99021 at about 3 and PD0325901 at about 0.5 optionally wherein the hiPSCs are cultured on MATRIGEL, newborn human foreskin fibroblast (NuFF) feeder cells, or GELTREX. 
     
     
         91 . The method of  claim 32 , wherein the low osmolality medium does not comprise one or more of the following: bFGF supplement; TGF-β1 supplement; JNK inhibitor; p38 inhibitor; ROCK inhibitor; and PKC inhibitor. 
     
     
         92 . The method of  claim 91 , wherein the low osmolality medium does not comprise bFGF supplement. 
     
     
         93 . A method for making a population of naïve human induced pluripotent stem cells (hiPSCs) that display a morphology characterized by compact, dome-shaped colonies, comprising culturing in vitro a population of non-pluripotent cells, transformed to express a pluripotent state, in a low osmolality medium comprising a base medium and supplements, wherein the low osmolality medium comprises:
 (a) a leukemia inhibitory factor (LIF) polypeptide; 
 (b) a glycogen synthase kinase 3 (GSK3) inhibitor; and 
 (c) a MEK inhibitor; 
 wherein the low osmolality medium has an osmolality of about 200 mOsm/kg to about 250 mOsm/kg. 
 
     
     
         94 . The method of  claim 93 , wherein the transformed cells are first cultured in a high osmolality medium prior to culturing in the low osmolality medium, wherein the high osmolality medium comprises bFGF and has an osmolality of at least about 290 mOsm/kg, and wherein:
 (a) the transformed cells are first cultured in the high osmolality medium until they express characteristics of a naïve state;   (b) the transformed cells are first cultured in the high osmolality medium for a period of about two months;   (c) the transformed cells are first cultured in the high osmolality medium until they display a morphology characterized by three-dimensional cell clumps; or   (d) a combination thereof.   
     
     
         95 . A method for maintaining in an in vitro culture a population of naïve human induced pluripotent stem cells (hiPSCs) that display a morphology characterized by compact, dome-shaped colonies, comprising culturing the population of hiPSCs in a low osmolality medium comprising a base medium and supplements, wherein the low osmolality medium comprises:
 (a) a leukemia inhibitory factor (LIF) polypeptide; 
 (b) a glycogen synthase kinase 3 (GSK3) inhibitor; and 
 (c) a MEK inhibitor; 
 wherein the low osmolality medium has an osmolality of about 200 mOsm/kg to about 250 mOsm/kg.