Method to direct differentiation of pluripotent stem cells into functional heart muscle

Abstract

The present invention is directed to a method for producing bioengineered heart muscle (BHM) from pluripotent stem cells, generally comprising the steps of inducing mesoderm differentiation, cardiac differentiation, and cardiac maturation by directed tissue formation. The method is a robust, serum-free and reproducible way to produce BHM for multiple applications, and closed herein, as well as to uses of said BHM in pharmacologic and toxicity screenings, and its use in medicine.

1 .- 15 . (canceled) 
     
     
         16 . A bioengineered heart muscle (BHM) produced by a method comprising the steps of
 (i) cultivating pluripotent stem cells in a basal medium comprising an effective amount of (a) BMP4, Activin A, FGF2, a GSK3-inhibitor, and (b) a serum-free supplement resulting in a final concentration of 0.5-50 mg/ml albumin, 1-100 μg/ml transferrin, 0.1-10 μg/ml ethanol amine, 0.003-0.3 μg/ml sodium selenite, 0.4-40 μg/ml L-Carnitine HCl, 0.1-10 μg/ml Hydrocortisone, 0.05-5 μl/ml Fatty acid supplement, and 0.0001-0.1 μg/ml triodo-L-thyronine (T3), thereby inducing mesoderm differentiation of said pluripotent stem cells;   (ii) cultivating the cells obtained in step (i) in a basal medium comprising an effective amount of an inhibitor of the Wnt-signaling pathway and a serum-free supplement as in (i), thereby inducing cardiac differentiation of the cells; and   (iii) cultivating the cells obtained in step (ii) in a basal medium comprising an effective amount of a serum-free supplement as in (i), under mechanical stimulation, thereby promoting cardiac maturation.   
     
     
         17 . The BHM of  claim 16 , wherein the BHM
 a) is capable of being paced at multiple frequencies up to at least 3 Hz; or   b) exhibits an increased twitch tension in response to increased resting length and resting tension; or   c) exhibits a calcium EC 50  higher than 0.2 mM; or   d) exhibits a twitch tension of more than 200 μN; or   e) exhibits an inotropic response to 1 μM isoprenaline of more than 40 μN under paced conditions at 0.6 mM calcium; or   f) comprises cardiomyocytes and CD90 +  stromal cells; or   g) has a combination of at least two of (a) to (f).   
     
     
         18 . The BHM of  claim 16 , wherein the pluripotent stem cells are selected from embryonic stem cells, induced pluripotent stem cells, and parthenogenetic stem cells. 
     
     
         19 . The BHM of  claim 16 , wherein the pluripotent stem cells are pluripotent stem cells of primate origin. 
     
     
         20 . The BHM of  claim 16 , wherein the pluripotent stem cells are human pluripotent stem cells. 
     
     
         21 . The BHM of  claim 16 , wherein the basal medium in step (i) comprises
 1-20 ng/ml BMP4;   0.1-10 ng/ml FGF2; and   1-20 ng/ml Activin A.   
     
     
         22 . The BHM of  claim 21 , wherein the basal medium in step (i) comprises about 5 ng/ml BMP4; or about 5 ng/ml FGF2; or about 9 ng/ml Activin A; or a combination thereof. 
     
     
         23 . The BHM of  claim 16 , wherein the GSK3-inhibitor in the basal medium of step (i) is selected from the group consisting of CHIR99021, CHIR98014, SB216763, TWS119, Tideglusib, SB415286, and LY2090314. 
     
     
         24 . The BHM of  claim 23 , wherein the basal medium in step (i) comprises 0.1-10 μM CHIR99021. 
     
     
         25 . The BHM of  claim 16 , wherein the inhibitor of the Wnt-signaling pathway in the basal medium of step (ii) is selected from the group consisting of IWP4, IWP2, IWR-1, IWP1, IWP3, IWR-2, IWR-3, IWR-4, IWR-5, XAV939, DKK1, quercetin, ICG-001, pyrvinium, CCT031374, iCRT-3, 5, 14, CPG049090, NC043. 
     
     
         26 . The BHM of  claim 25 , wherein the basal medium of step (ii) comprises 0.1-10 μM IWP4. 
     
     
         27 . The BHM of  claim 16 , wherein the basal medium of step (iii)
 a) further comprises 0.1-10 ng/ml TGFβ1; or   b) does not comprise an effective amount of FGF2; or   c) comprises 0.5-3 mM Ca 2+ ; or   d) comprises a combination of any one of a), b), and c).   
     
     
         28 . The BHM of  claim 27 , wherein the basal medium of step (iii) comprises about 1 ng/ml TGFβ1; or about 1.2 mM Ca 2+ ; or a combination thereof. 
     
     
         29 . The BHM of  claim 16 , wherein the mechanical stimulation in step (iii) is dynamic mechanical stimulation or static stretch. 
     
     
         30 . The BHM of  claim 29 , wherein the mechanical stimulation in step (iii) is dynamic mechanical stimulation. 
     
     
         31 . The BHM of  claim 16 , wherein the basal medium of any one of step (i), (ii) or (iii) comprises 10-1000 μM of ascorbic acid or a salt or a derivative thereof. 
     
     
         32 . The BHM of  claim 16 , wherein the basal medium used in any one of step (i), (ii) or (iii) is DMEM/F12, Iscove's medium, αMEM, DMEM, and RPMI. 
     
     
         33 . The BHM of  claim 32 , wherein the basal medium used in any one of step (i), (ii) or (iii) is RPMI supplemented with pyruvate. 
     
     
         34 . The BHM of  claim 16 , wherein the method comprises prior to step (i) a seeding step, wherein said pluripotent stem cells are seeded in a ratio of (2.5-6×10 6  cells/1 mg collagen)/1 ml medium in a suitable mould. 
     
     
         35 . The BHM of  claim 34 , wherein the seeding step is carried out 18-30 h prior to step (i). 
     
     
         36 . The BHM of  claim 34 , wherein the medium used in the seeding step further comprises a ROCK-inhibitor. 
     
     
         37 . The BHM of  claim 36 , wherein the ROCK-inhibitor is selected from Y27632, H-1152P, Thiazovivin, Fasudil, Hydroxyfasudil, GSK429286A, and RKI-1447. 
     
     
         38 . The BHM of  claim 36 , wherein the medium used in the seeding step comprises 1-50 μM Y27632. 
     
     
         39 . The BHM of  claim 16 , wherein said pluripotent stem cells are not produced using a process which involves modifying the germ line genetic identity of human beings or which involves use of a human embryo for industrial or commercial purposes.