A 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 is applicable to multiple pluripotent stem cell lines. The present invention is also directed to the BHM produced by the method disclosed herein, as well as to uses of said BHM in pharmacologic and toxicity screenings, and its use in medicine.

1 . An in vitro bioengineered heart muscle (BHM), wherein the BHM is artificially moulded. 
     
     
         2 . The BHM of  claim 1 , wherein the BHM has a β-MHC/α-MHC ratio of more than 0.2 or more than 0.3, but less than the β-MHC/α-MHC ratio found in adult heart tissue. 
     
     
         3 . The BHM of  claim 1 , wherein the BHM has a low but still retained expression of progenitor genes as compared to adult heart tissue. 
     
     
         4 . The BHM of  claim 3 , wherein the BHM has a low but still retained expression of ISL-1 as compared to adult heart tissue. 
     
     
         5 . The BHM of  claim 1 , wherein the BHM does not exhibit blood perfusion. 
     
     
         6 . The BHM of  claim 1 , wherein the BHM is serum-free. 
     
     
         7 . The BHM of  claim 1 , wherein the BHM comprises cardiomyocytes and CD90 +  stromal cells. 
     
     
         8 . The BHM of  claim 7 , wherein the BHM is comprised of about 50% cardiomyocytes and the rest predominantly CD90 +  stromal cells. 
     
     
         9 . The BHM of  claim 1 , wherein the BHM is made of cells of primate origin. 
     
     
         10 . The BHM of  claim 1 , wherein the BHM is made of cells of human origin. 
     
     
         11 . The BHM of  claim 1 , wherein the BHM is moulded in a collagen matrix, wherein the collagen is selected from the group consisting of collagen type I, collagen type III, collagen type V, and a mixture thereof. 
     
     
         12 . The BHM of  claim 11 , wherein the BHM is moulded in a collagen matrix consisting of at least 90% collagen type I. 
     
     
         13 . The BHM of  claim 11 , wherein the collagen matrix further comprises one or more extracellular matrix components selected from the group consisting of elastin, laminin, entactin, nidogen, proteoglycan, and fibronectin. 
     
     
         14 . The BHM of  claim 11 , wherein the collagen is of human origin, bovine origin, porcine origin, or marine origin. 
     
     
         15 . The BHM of  claim 11 , wherein the collagen is of human origin. 
     
     
         16 . The BHM of  claim 11 , wherein the collagen is of algae origin or fish origin. 
     
     
         17 . The BHM of  claim 11 , wherein the collagen is recombinant collagen. 
     
     
         18 . The BHM of  claim 1 , wherein the BHM has a ring-shaped form. 
     
     
         19 . The BHM of  claim 1 , wherein the BHM is capable of being paced at multiple frequencies up to at least 3 Hz. 
     
     
         20 . The BHM of  claim 1 , wherein the BHM exhibits an increased twitch tension in response to increased resting length and resting tension. 
     
     
         21 . The BHM of  claim 1 , wherein the BHM exhibits a calcium EC 50  higher than 0.2 mM. 
     
     
         22 . The BHM of  claim 21 , wherein the BHM exhibits a calcium EC 50  in the range of 0.2-8 mM. 
     
     
         23 . The BHM of  claim 1 , wherein the BHM exhibits a twitch tension of more than 200 μN. 
     
     
         24 . The BHM of  claim 1 , wherein the BHM exhibits an inotropic response to 1 μM isoprenaline of more than 40 μN under paced conditions at 0.6 mM calcium. 
     
     
         25 . The BHM of  claim 24 , wherein the BHM exhibits an inotropic response to 1 μM isoprenaline of more than 45 μN under paced conditions at 0.6 mM calcium. 
     
     
         26 . The BHM of  claim 24 , wherein the BHM exhibits an inotropic response to 1 μM isoprenaline of more than 50 μN under paced conditions at 0.6 mM calcium. 
     
     
         27 . The BHM of  claim 1 , wherein the BHM can be maintained for at least 62 days. 
     
     
         28 . A method for screening drug toxicity, comprising the step of contacting a BHM according to  claim 1  with a drug to be screened. 
     
     
         29 . A method for testing of cardiac function modulation, comprising the step of contacting a BHM according to  claim 1  with a pharmacological candidate agent. 
     
     
         30 . A method for heart repair, comprising the step of incorporating the BHM according to  claim 1  into a heart of a patient in need thereof.