HK1226443B - Method for producing engineered heart muscle (ehm) - Google Patents

Description

Methods for generating engineered myocardium (EHM)

Technical Field

The present invention provides a novel method for producing engineered cardiac muscle (EHM) under chemically well-defined conditions that are fully compliant with GMP regulations. The resulting human cardiac muscle is viable and exhibits typical properties of cardiac muscle.

Background Art

Heart disease is the leading cause of death in industrialized countries, and its prevalence is expected to rise despite the availability of comprehensive medications and interventional therapies. Therefore, new drug and non-drug treatment modalities are inevitably needed. Tissue-engineered myocardium can be used to identify new drugs or new drug targets for treating heart disease (substance screening/target confirmation), and can be directly applied to cardiac repair (regeneration/repair medicine) (Eschenhagen and Zimmermann, Circ Res, 97:1220-1231 (2005); Zimmermann et al., Cardiovasc Res, 71:419-429 (2006)). The main prerequisite for functional engineered myocardium is the ability to generate force like native myocardium.

Several myocardial tissue engineering models have been established over the past decade. However, reliable force generation has only been demonstrated using hydrogel-cell capture (Eschenhagen et al., Faseb J 11:683-694 (1997); Kofidis et al., J Thorac Cardiovasc Surg 124:63-69 (2002); Morritt et al., Circulation 115:353-360 (2007); Zimmermann et al., Biotechnol Bioeng 68:106-114 (2000); Tulloch et al., Circ Res 109:47-59 (2011)) or cell layer technology (Shimizu et al., Circ Res 90:e40 (2002)). The inventors and other researchers have provided evidence that the capture of cardiomyocytes in collagen-hydrogels provides a three-dimensional growth environment that promotes the assembly of multicellular, anisotropic cardiomyocytes and supports the high degree of maturation of immature cardiomyocytes (Tiburc et al., Circ Res 109:1105-1114 (2011)). The resulting engineered myocardium (EHM) preparation (formerly described as engineered heart tissue: EHT) ultimately facilitates the formation of contractile myocardial constructs with postnatal myocardial properties (Radisic et al., Proc Natl Acad Sci USA 101:18129-18134 (2004); Tiburcy et al., Circ Res, 109:1105-1114 (2011); Zimmermann et al., Circ Res 90:223-230 (2002)). Proof-of-principle animal studies have shown that, following implantation onto diseased hearts, EHMs not only electrically integrate but also improve cardiac function (Zimmermann et al., Nat. Med 12:452-458 (2006)).

In principle, the inventors have demonstrated that tissue-engineered myocardium could be a new therapeutic modality for heart disease. However, all publicly reported cardiac tissue engineering methods to date have relied on the use of unrestricted animal components, primarily animal matrices (e.g., rat collagen, bovine fibrin, mouse tumor-derived extracellular matrix), and animal serum (Tulloch et al., Circ Res 109:47-59 (2011); Zimmermann et al., Circ Res 90:223-230 (2002); Zimmermann et al., Nat. Med 12:452-458 (2006); Schaaf et al., PloS One 6:e26397 (2011); Soong et al., Curr Prot Cell Biol 23.8.1-23.8.21 (2012); WO 01/55297, WO 2007/054286, and WO 2008/058917). In the rat EHM model, the present inventors have conducted initial studies replacing animal serum with serum-free medium. Although the present inventors were able to obtain comparable force generation in the resulting tissue, the present inventors were unable to remove the animal component during the initial seven-day tissue formation phase (Naito et al., Circulation, 114: 172-78 (2006); Zimmermann, Hamburg Eppendorf, Habilitation (2006); Schneiderbanger, University of Hamburg, Thesis (2006)).

Recently, several serum-free, cytokine-guided protocols for more efficient cardiac differentiation have been described (Burridge et al., Cell Stem Cell 10: 16-28 (2012)), resulting in cultures containing up to 98% cardiomyocytes (Lian et al., Proc Natl Academic Sci USA (2012)). Importantly, these serum-free differentiation protocols offer potential clinical applicability due to the use of limited substances free of animal products. Although scaling up human cardiac cells under GMP conditions appears to be a solvable problem, the manufacture of force-generating human myocardium remains a challenge. Supporting the organotypic construction and high maturation of ESC-derived cardiomyocytes under serum-free defined culture conditions remains a key issue.

Summary of the Invention

Here, the inventors report a protocol for engineering human myocardium (human engineered myocardium: hEHM) using fully defined components, including a hydrogel matrix, human cells, and serum-free culture media in full compliance with GMP regulations. The resulting human myocardium is capable of generating force and exhibits typical myocardial properties.

More specifically, a method for producing an engineered myocardium (EHM) is provided, the method comprising the following steps:

(i) providing a serum-free reconstitution mixture in one or more molds, the reconstitution mixture comprising (a) serum-free minimal essential medium; (b) a serum-free supplement to give a final concentration of 0.5-50 mg/ml albumin, 1-100 μg/ml transferrin, 0.1-10 μg/ml ethanolamine, 0.003-0.3 μg/ml sodium selenite, 0.4-40 μg/ml L-carnitine hydrochloride, 0.1-10 μg/ml hydrocortisone, 0.05-5 μl/ml fatty acid supplement, 0.0001-0.1 μg/ml triiodo-L-thyronine (T3), and 0.2-2 mg/ml collagen; and (c) a mixture of human cardiomyocytes and human non-myocytes, wherein 20% to 80% of the total cell mixture are cardiomyocytes; wherein the pH of the reconstitution mixture is 7.2 to 7.6;

ii) incubating the serum-free reconstitution mixture in the one or more molds, thereby allowing the serum-free reconstitution mixture to clot for at least 15 minutes;

iii) culturing the mixture obtained in step (ii) in the one or more molds in serum-free EHM medium until the mixture solidifies to at least 50% of its original thickness, wherein the EHM medium comprises (a) a basal medium containing 0.5-3 mmol/L Ca2 ⁺ ; (b) a serum-free supplement as defined in (i)(b); (c) 0.5-10 mmol/L L-glutamine; (d) 0.01-1.0 mmol/L ascorbic acid; (e) 1-100 ng/ml IGF-1; and (f) 1-10 ng/ml TGFβ1;

iv) culturing the mixture obtained in step (iii) in a serum-free EHM medium as defined in steps (iii)(a)-(f) under mechanical tension, thereby forming a force-generating EHM.

DETAILED DESCRIPTION

More specifically, the present invention provides a method for producing an engineered myocardium (EHM), the method comprising the following steps:

(i) providing a serum-free reconstitution mixture in one or more molds, the reconstitution mixture comprising (a) serum-free minimum essential medium; (b) serum-free supplements to obtain a final concentration of

0.5-50 mg/ml albumin (preferably 1-40 mg/ml, more preferably 2-30 mg/ml, still more preferably 3-20 mg/ml, most preferably 4-10 mg/ml, even most preferably 4.5-7.5 mg/ml, such as about 5 mg/ml),

1-100 μg/ml transferrin (preferably 2-90 μg/ml, more preferably 3-80 μg/ml, even more preferably 4-70 μg/ml, still more preferably 5-60 μg/ml, more preferably 6-50 μg/ml, more preferably 7-40 μg/ml, more preferably 8-30 μg/ml, more preferably 9-20 μg/ml, such as about 10 μg/ml),

0.1-10 μg/ml ethanolamine (preferably 0.2-9 μg/ml, more preferably 0.3-8 μg/ml, even more preferably 0.4-7 μg/ml, still more preferably 0.5-6 μg/ml, more preferably 0.6-5 μg/ml, more preferably 0.7-4 μg/ml, more preferably 0.8-3 μg/ml, more preferably 1-2.5 μg/ml, such as about 2 μg/ml),

0.003-0.3 μg/ml sodium selenite (preferably 0.005-0.2 μg/ml, more preferably 0.01-0.1 μg/ml, even more preferably 0.02-0.05 μg/ml, most preferably about 0.03 μg/ml, such as about 0.032 μg/ml),

0.4-40 μg/ml L-carnitine hydrochloride (preferably 0.5-30 μg/ml, more preferably 1-20 μg/ml, even more preferably 2-10 μg/ml, most preferably 3-5 μg/ml, even most preferably about 4 μg/ml),

0.1-10 μg/ml hydrocortisone (preferably 0.2-9 μg/ml, more preferably 0.3-8 μg/ml, even more preferably 0.4-7 μg/ml, still more preferably 0.5-6 μg/ml, more preferably 0.6-5 μg/ml, more preferably 0.7-4 μg/ml, more preferably 0.8-3 μg/ml, more preferably 0.9-2 μg/ml, such as about 1 μg/ml),

0.05-5 μl/ml fatty acid supplement (preferably 0.1-4 μl/ml, more preferably 0.2-3 μl/ml, even more preferably 0.3-3 μl/ml, most preferably 0.4-2 μl/ml, even most preferably 0.45-1 μl/ml, such as about 0.5 μl/ml),

0.0001-0.1 μg/ml triiodo-L-thyronine (T3) (preferably 0.001-0.01 μg/ml, more preferably 0.002-0.0075 μg/ml, even more preferably 0.003-0.005 μg/ml, most preferably about 0.004 μg/ml), and

0.2-2 mg/ml collagen (preferably 0.3-1.9 mg/ml, more preferably 0.4-1.8 mg/ml, even more preferably 0.4-1.7 mg/ml, still more preferably 0.5-165 mg/ml, more preferably 0.6-1.5 mg/ml, more preferably 0.7-1.4 mg/ml, more preferably 0.8-1.3 mg/ml, more preferably 0.9-1.2 mg/ml, such as about 1 mg/ml);

and (c) a mixture of human cardiomyocytes and human non-myocytes, wherein 20% to 80% of the total cell mixture are cardiomyocytes;

wherein the pH of the reconstituted mixture is 7.0 to 7.8, preferably 7.1 to 7.7, more preferably 7.2 to 7.6, even more preferably 7.3 to 7.5, most preferably about 7.4;

(ii) culturing the serum-free reconstitution mixture in the one or more molds, thereby allowing the serum-free reconstitution mixture to clot for at least 15 minutes, preferably 0.25-3 hours, more preferably 0.5-1.5 hours;

(iii) culturing the mixture obtained in step (ii) in the one or more molds in serum-free EHM medium until the mixture solidifies to at least 50% (preferably at least 55%, more preferably at least 60%, even more preferably at least 70%, most preferably at least 75%) of its original thickness,

The EHM medium comprises

(a) a basal medium comprising 0.5-3 mmol/L (preferably 1-2.5 mmol/L, more preferably about 2 mmol/L) Ca2 ⁺ ;

(b) a serum-free supplement as defined in (i)(b);

(c) 0.5-10 mmol/L (preferably 1-7 mmol/L, more preferably 2-6 mmol/L, even more preferably 3-5 mmol/L, still more preferably about 2 mmol/L) L-glutamine;

(d) 0.01-1.0 mmol/L (preferably 0.05-1.0 mmol/L, more preferably 0.1-0.5 mmol/L, even more preferably 0.2-0.4 mmol/L, still more preferably about 0.3 mmol/L) ascorbic acid;

(e) 1-100 ng/ml (preferably 2-90 ng/ml, more preferably 3-80 ng/ml, even more preferably 4-70 ng/ml, still more preferably 5-60 ng/ml, more preferably 6-50 ng/ml, more preferably 7-40 ng/ml, more preferably 8-30 ng/ml, more preferably 9-20 ng/ml, such as about 10 ng/ml) IGF-1; and

(f) 1-10 ng/ml (preferably 1-9 ng/ml, more preferably 2-8 ng/ml, even more preferably 3-7 ng/ml, most preferably 4-6 ng/ml, even most preferably about 5 ng/ml) TGFβ1;

(iv) culturing the mixture obtained in step (iii) in a serum-free EHM medium as defined in steps (iii)(a)-(f) under mechanical tension, thereby forming a force-generating EHM.

The minimum essential medium in step (i) can be selected from Iscove's medium, αMEM, DMEM and RPMI. In a preferred embodiment, the basal medium is Iscove's medium or αMEM. In a more preferred embodiment, the basal medium is Iscove's medium. However, any suitable minimum medium can be used in this method. Suitable minimum essential medium formulations are provided herein or are publicly available, for example, from the ATCC catalog.

Preferably, the serum-free supplement of step (i) further comprises one or more components selected from vitamin A, D-galactose, linoleic acid, linolenic acid, progesterone, and putrescine. These components all contribute to cell survival. The appropriate concentration of each component is known to those skilled in the art or can be readily determined using conventional means.

For the serum-free supplement in component (b) of step (i), a commercially available supplement or insulin-reducing supplement can be used. Alternatively, a custom supplement as shown in Table 2 can also be used. In a preferred embodiment, the supplement or insulin-reducing supplement is used as component (b) of step (i) of the above method in an amount of 2-6% (v/v). More preferably, the supplement or insulin-reducing supplement is used as component (b) of step (i) of the above method in an amount of 4% (v/v).

In addition, the reconstitution mixture of step (i) comprises 0.3-0.5 mg collagen/1.5×10 ⁶ cardiomyocytes and non-myocyte mixture. More preferably, the reconstitution mixture of step (i) comprises about 0.4 mg collagen/1.5×10 ⁶ cardiomyocytes and non-myocyte mixture.

The collagen in component (c) of the reconstructed mixture of step (i) is preferably medical grade and is selected from type I collagen, type III collagen, type V collagen and mixtures thereof. In a more preferred embodiment, component (c) of the reconstructed mixture of step (i) contains at least 90% of the collagen being type I collagen. However, the collagen may further comprise one or more extracellular matrix components selected from elastin, laminin, entactin, nidogen, proteoglycans and fibronectin. Generally, the exact composition of the collagen depends on the source, i.e. where it comes from. The collagen is preferably of human origin, but bovine or marine sources such as algae or fish are also considered.

Developing functional (i.e., force-generating), limited, serum-free human tissue-engineered myocardium for potential regenerative cardiac therapy requires overcoming many difficulties. The most important is a reliable source of human cardiac cells. To date, human pluripotent stem cells have become the main source of human cardiac cells. Pluripotent stem cells can differentiate into every type of cell in the body. Therefore, human pluripotent stem cells provide a unique opportunity to obtain true human cardiac cells. Currently, the most commonly used pluripotent stem cells are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). The human ESC system was first established by Thomson and colleagues (Thomson et al., Science 282:1145-1147 (1998); incorporated herein by reference in its entirety). Recently, human ESC research has achieved a new technology for reprogramming somatic cells into ES cell-like cells. This technology was first established by Yamanaka and colleagues in 2006 (Takahashi and Yamanaka Cell 126:663-676 (2006); incorporated herein by reference in its entirety). The induced pluripotent cells (iPSCs) produced show very similar behavior to ESCs and, importantly, can also differentiate into any cell of the body. Cardiac differentiation of ESCs and iPSCs occurs in embryoid bodies (Schroeder et al., Biotechnol Bioeng 92:920-933 (2005); incorporated herein by reference in their entirety) cultures, as more or less random events, producing cell populations containing 5-20% true cardiomyocytes (Kehat et al., J Clin Invest 108:407-414 (2001); Mummery et al., Circulation 107:2733-2740 (2003); Xu et al., Cir Res 91:501-508 (2002); all incorporated herein by reference). In addition, it has been reported that parthenogenetic stem cells may also be suitable for EHM production (Didié et al., J Clin Invest. doi: 10.1172/JCI66584; incorporated herein by reference in its entirety). Therefore, in a preferred embodiment, the cardiomyocytes are human cardiomyocytes. Preferably, the cardiomyocytes are derived from embryonic stem cells, wherein the cells are not produced using a process involving modification of human germline genetic characteristics or involving the use of human embryos for industrial or commercial purposes. In alternative embodiments, the cardiomyocytes are derived from induced pluripotent cells, parthenogenetic stem cells, or adult stem cells as described above.

Recently, several serum-free, cytokine-guided protocols for more efficient cardiac differentiation have been described (Burridge et al., Cell Stem Cell 10:16-28 (2012); incorporated herein by reference in its entirety), resulting in cultures containing up to 98% cardiomyocytes (Lian et al., Proc Natl Academic Sci USA (2012); incorporated herein by reference in its entirety). Therefore, in another preferred embodiment, cardiomyocytes can be obtained by serum-free differentiation. Alternatively, cardiomyocytes can be derived from non-human primate stem cells, embryonic or neonatal cardiomyocytes.

It has been shown that it is advantageous to provide the cardiomyocytes as a mixture with cells selected from one or more types of non-muscle cells, such as fibroblasts, endothelial cells, smooth muscle cells and mesenchymal stem cells. Therefore, preferably, the cardiomyocyte mixture comprises 20-80% cardiomyocytes, more preferably 30-70% cardiomyocytes, even more preferably 40-60% cardiomyocytes, and most preferably about 50% cardiomyocytes, wherein the non-muscle cells are fibroblasts or endothelial cells. In fact, a particularly preferred embodiment is that the non-muscle cells are fibroblasts. Suitable non-muscle cells can be identified by the expression of surface markers such as CD90. Suitable cells can be identified, for example, using techniques such as immunostaining or fluorescence activated cell sorting (FACS). The EHM obtained from the above mixture generally generates stronger force.

Preferably, the cardiomyocytes provided in step (i) have a cell concentration of at least 2.7-20×10 ⁶ cells/ml. In a more preferred embodiment, the cardiomyocytes provided in step (i) have a cell concentration of at least 2.9-10×10 ⁶ cells/ml, an even more preferred cell concentration of at least 3.1-5×10 ⁶ cells/ml, and in a most preferred embodiment, a cell concentration of at least 3.3-3.4×10 ⁶ cells/ml.

The mold mentioned in the method may have any suitable form that allows the EHM to be incorporated into a host in need thereof. However, in a preferred embodiment, the mold is in the form of a ring, a polygon, a disk or a bag.

Cell culture is performed using common procedures and equipment generally known in the art. Typically, culture conditions include a temperature in the range of 30-40°C, preferably 36-38°C, most preferably about 37°C, in a humidified cell culture incubator in the presence of 5-10% _CO2 .

For the serum-free supplement mentioned in component (b) of step (iii), a commercially available supplement or insulin-reducing supplement can be used. Alternatively, a custom supplement as shown in Table 2 below can be used. In a preferred embodiment, the supplement or insulin-reducing supplement is used as component (b) in step (iii) of the above method in an amount of 2-6% (v/v). More preferably, the supplement or insulin-reducing supplement is used as component (b) in step (i) of the above method in an amount of 4% (v/v).

In addition, the serum-free supplement of step (iii) further comprises one or more components selected from vitamin A, D-galactose, L-carnitine, linoleic acid, linolenic acid, progesterone, and putrescine. As previously mentioned, these components all contribute to cell survival. The appropriate concentrations of the individual components are known to those skilled in the art or can be readily determined using conventional means.

The basal medium included in the EHM culture medium described in step (iii) can be selected from Iscove's culture medium, αMEM, DMEM and RPMI. Because RPMI generally has a lower concentration of calcium, it may be necessary to supplement calcium to the RPMI basal medium accordingly. If necessary, non-essential amino acids can be supplemented to the basal medium. If αMEM is used as the basal medium, the EHM culture medium does not need to be supplemented with non-essential amino acids separately. Non-essential amino acids can be commercially available as a combination supplement. The supplement, for example, includes 750mg/L glycine, 890mg/L L-alanine, 1320mg/L L-asparagine, 1330mg/L L-aspartic acid, 1470mg/L L-glutamic acid, 1150mg/L L-proline and 1050mg/L L-serine.

In a preferred embodiment, the basal medium contained in the EHM medium in step (iii) is Iscove's medium or αMEM. In a more preferred embodiment, the basal medium contained in the EHM medium in step (iii) is Iscove's medium. However, any basal medium can be used in this method. Suitable minimum essential medium formulations are provided herein or are publicly available, for example, from the ATCC catalog.

The examples below demonstrate that it is more advantageous if the serum-free EHM medium also contains VEGF, FGF, or both VEGF and FGF.Addition of VEGF and/or FGF has been shown to result in EHMs that exhibit higher force.

Typically, VEGF is added at a concentration of about 5-20 ng/ml VEGF, preferably 6-18 ng/ml, more preferably 7-16 ng/ml, even more preferably 8-14 ng/ml, most preferably 9-12 ng/ml, and even most preferably at a concentration of about 10 ng/ml.

FGF is added at a concentration of about 5-20 ng/ml FGF, preferably 6-18 ng/ml, more preferably 7-16 ng/ml, even more preferably 8-14 ng/ml, most preferably 9-12 ng/ml, and even most preferably at a concentration of about 10 ng/ml.

In principle, any type of VEGF, FGF, IGF1, and TGFβ1 can be used, as long as these growth factors are able to transmit signals through their corresponding receptors on the surface of the cardiomyocytes of EHM. However, in a preferred embodiment, the VEGF is human VEGF. In another preferred embodiment, the FGF is human FGF. In another preferred embodiment, the IGF1 is human IGF1. In yet another embodiment, the TGFβ1 is human TGFβ1. In a most preferred embodiment, all VEGF, FGF, IGF1, and TGFβ1 are human.

Typically, the culturing in step (iii) is performed for at least 3 days, preferably for about 3 to about 7 days.

In principle, the further culturing in step (iv) can be carried out for any suitable period of time. However, typically, the further culturing in step (iv) is carried out for a period of at least 3-60 days, preferably 4-30 days, and more preferably 5-20 days. In a most preferred embodiment, the further culturing is carried out for 7 days, as this period of time is the best balance between the preferred short culturing time and a period of time sufficient to obtain productive EHM.

Typically, step (iv) of the above method is performed on a stretching device, as is known in the art. Preferably, the stretching device applies static, phased, or dynamic stretching to the EHM. More specifically, the mechanical stretching can be (i) static, (ii) dynamic, or (iii) flexible against an elastic load.

As will be further demonstrated below, the EHM produced by the above method generates a force greater than 0.01 mN after induction with 3 mM calcium, as measured using the method described in Zimmermann et al., Circ. Res. 90, 223-230 (2002), preferably a force greater than 0.1 mN, more preferably greater than 0.2 mN, and most preferably greater than 0.3 mN, as measured using the method described in Zimmermann et al., Circ. Res. 90, 223-230 (2002).

Another prior art protocol suitable as a basis for the improved methods disclosed herein is described by Soong et al., Curr Prot Cell Biol, 55:23.8.1-23.8.21 (2012), which is hereby incorporated by reference in its entirety, particularly to “Basic Protocol 2” and “Supporting Protocol 2”.

Table 1: Comparison of protocols for human EHM generation

Table 2: Customized supplements to replace B27

Prepare 25× with water that meets cell culture requirements

Table 3: Iscove’s basal medium without glutamine (Biochrom)

Table 4: RPMI basal medium (Invitrogen)

Table 5: αMEM basal medium formula (Invitrogen)

Table 6: Serum-free defined medium for EHM generation

Table 7: Alternative serum-free defined medium for EHM production

The present invention is further described by the following embodiments:

1. A method for producing an engineered myocardium (EHM), the method comprising the following steps:

(i) providing a serum-free reconstitution mixture in one or more molds, the reconstitution mixture comprising (a) serum-free minimal essential medium; (b) a serum-free supplement to give a final concentration of 0.5-50 mg/ml albumin, 1-100 μg/ml transferrin, 0.1-10 μg/ml ethanolamine, 0.003-0.3 μg/ml sodium selenite, 0.4-40 μg/ml L-carnitine hydrochloride, 0.1-10 μg/ml hydrocortisone, 0.05-5 μl/ml fatty acid supplement, 0.0001-0.1 μg/ml triiodo-L-thyronine (T3), and 0.2-2 mg/ml collagen; and (c) a mixture of human cardiomyocytes and human non-myocytes, wherein 20% to 80% of the total cell mixture are cardiomyocytes; wherein the pH of the reconstitution mixture is 7.2 to 7.6;

(ii) incubating the serum-free reconstitution mixture in the one or more molds, thereby allowing the serum-free reconstitution mixture to clot for at least 15 minutes;

(iii) culturing the mixture obtained in step (ii) in the one or more molds in serum-free EHM medium until the mixture solidifies to at least 50% of its original thickness, wherein the EHM medium comprises (a) a basal medium containing 0.5-3 mmol/L Ca2 ⁺ ; (b) a serum-free supplement as defined in (i)(b); (c) 0.5-10 mmol/L L-glutamine; (d) 0.01-1.0 mmol/L ascorbic acid; (e) 1-100 ng/ml IGF-1; and (f) 1-10 ng/ml TGFβ1;

(iv) culturing the mixture obtained in step (iii) in a serum-free EHM medium as defined in steps (iii)(a)-(f) under mechanical tension, thereby forming a force-generating EHM.

2. The method of embodiment 1, wherein the minimal essential culture medium in step (i) is selected from Iscove’s medium, αMEM, DMEM and RPMI.

3. The method of embodiment 2, wherein the basal culture medium is Iscove’s medium or αMEM.

4. The method of embodiment 2, wherein the basal medium is Iscove’s medium.

5. The method according to any one of embodiments 1 to 4, wherein the serum-free supplement of step (i) further comprises one or more components selected from vitamin A, D-galactose, linoleic acid, linolenic acid, progesterone and putrescine.

6. The method of any one of embodiments 1-5, wherein the serum-free supplement in component (b) of step (i) is a supplement or an insulin-reducing supplement.

7. The method of embodiment 6, wherein the serum-free supplement in component (b) of step (i) is a 2-6% (v/v) supplement or a reduced insulin supplement.

8. The method of embodiment 6, wherein the serum-free supplement in component (b) of step (i) is a 4% (v/v) supplement or a reduced insulin supplement.

9. The method according to any one of embodiments 1 to 8, wherein the reconstitution mixture of step (i) comprises 0.3-0.5 mg collagen per 1.5×10 ⁶ mixture of cardiomyocytes and non-myocytes.

10. The method of embodiment 9, wherein the reconstitution mixture of step (i) comprises about 0.4 mg collagen per 1.5×10 ⁶ mixture of cardiomyocytes and non-myocytes.

11. The method of any one of embodiments 1-10, wherein in component (c) of the reconstitution mixture of step (i), the collagen is selected from type I collagen, type III collagen, type V collagen, and mixtures thereof.

12. The method of any one of embodiments 1-11, wherein in component (c) of the reconstitution mixture of step (i), at least 90% of the collagen is type I collagen.

13. The method of any one of embodiments 1-12, wherein the collagen in component (c) of the reconstitution mixture of step (i) is medical grade.

14. The method of any one of embodiments 1-13, wherein in component (c) of the reconstitution mixture of step (i), the collagen is of human, bovine or marine origin.

15. The method of any one of embodiments 1-14, wherein in component (c) of the reconstitution mixture of step (i), the collagen further comprises one or more extracellular matrix components selected from elastin, laminin, entactin, nidogen, proteoglycans and fibronectin.

16. The method of any one of embodiments 1-15, wherein the pH of the reconstituted mixture of step (i) is 7 to 7.8.

17. The method of embodiment 13, wherein the pH of the reconstituted mixture of step (i) is about 7.4.

18. The method of any one of embodiments 1-17, wherein the cardiomyocytes are human cardiomyocytes.

19. The method of any one of embodiments 1-18, wherein the cardiomyocytes are derived from embryonic stem cells, wherein the cells are not produced using a process involving the modification of human germline genetic characteristics or the use of human embryos for industrial or commercial purposes.

20. The method of any one of embodiments 1-19, wherein the cardiomyocytes are derived from induced pluripotent cells, parthenogenetic stem cells, or adult stem cells.

21. The method of any one of embodiments 1-20, wherein the cardiomyocytes are obtained by serum-free differentiation.

22. The method of any one of embodiments 1-21, wherein the cardiomyocytes are non-human primate stem cell-derived, embryonic or neonatal cardiomyocytes.

23. The method of any one of embodiments 1-22, wherein the cardiomyocytes are provided as a mixture with cells selected from one or more types of cells of non-muscle cells, such as fibroblasts, endothelial cells, smooth muscle cells, and mesenchymal stem cells.

24. The method of embodiment 23, wherein the cardiomyocyte mixture comprises 20-80% cardiomyocytes.

25. The method of embodiment 23, wherein the cardiomyocyte mixture comprises 30-70% cardiomyocytes.

26. The method of embodiment 23, wherein the cardiomyocyte mixture comprises 40-60% cardiomyocytes.

27. The method of embodiment 23, wherein the cardiomyocyte mixture comprises 50% cardiomyocytes.

28. The method of any one of embodiments 1-27, wherein the non-muscle cells are fibroblasts or endothelial cells.

29. The method of any one of embodiments 1-28, wherein the non-muscle cells are fibroblasts.

30. The method of any one of embodiments 1-29, wherein the non-muscle cells express CD90.

31. The method of any one of embodiments 1-30, wherein the cell concentration of the cardiomyocytes provided in step (i) is at least 2.7-20×10 ⁶ cells/ml.

32. The method of embodiment 31, wherein the cell concentration of the cardiomyocytes provided in step (i) is at least 2.9-10×10 ⁶ cells/ml.

33. The method of embodiment 31, wherein the cell concentration of the cardiomyocytes provided in step (i) is at least 3.1-5×10 ⁶ cells/ml.

34. The method of embodiment 31, wherein the cell concentration of the cardiomyocytes provided in step (i) is at least 3.3-3.4×10 ⁶ cells/ml.

35. The method according to any one of embodiments 1 to 34, wherein in step (ii), the mold is in the shape of a ring, a polygon, a disk or a bag.

36. The method according to any one of embodiments 1-35, wherein the culturing in step (ii) is carried out for 0.25-3 hours.

37. The method according to embodiment 36, wherein the culturing in step (ii) is carried out for 0.5-1.5 hours.

38. The method of any one of embodiments 1-37, wherein the culturing is performed at a temperature in the range of 30-40°C.

39. The method of embodiment 38, wherein the culturing is carried out at a temperature range of 36-38°C.

40. The method of embodiment 39, wherein the culturing is performed at about 37°C.

41. The method of any one of embodiments 1-40, wherein the culturing is performed in a humidified cell culture incubator in the presence of 5-10% _CO2 .

42. The method of any one of embodiments 1-41, wherein the serum-free supplement in component (b) of step (iii) is a supplement or an insulin-reducing supplement.

43. The method of embodiment 42, wherein the serum-free supplement in component (b) of step (iii) is a 2-6% (v/v) supplement or a reduced insulin supplement.

44. The method of embodiment 43, wherein the serum-free supplement in component (b) of step (iii) is a 4% (v/v) supplement or a reduced insulin supplement.

45. The method of any one of embodiments 1-44, wherein the serum-free supplement of step (iii) further comprises one or more components selected from vitamin A, D-galactose, L-carnitine, linoleic acid, linolenic acid, progesterone, and putrescine.

46. The method according to any one of embodiments 1 to 45, wherein the basal culture medium contained in the EHM culture medium in step (iii) is selected from Iscove’s medium, αMEM, DMEM and RPMI.

47. The method of embodiment 46, wherein the basal culture medium is Iscove’s medium or αMEM.

48. The method of embodiment 47, wherein the basal medium is Iscove’s medium.

49. The method of any one of embodiments 1-48, wherein the serum-free EHM medium comprises about 20 ng/ml IGF1.

50. The method of any one of embodiments 1-49, wherein the IGF1 is human IGF1.

51. The method of any one of embodiments 1-50, wherein the serum-free EHM medium comprises about 5 ng/ml TGFβ1.

52. The method of any one of embodiments 1-51, wherein the TGFβ1 is human TGFβ1.

53. The method of any one of embodiments 1-52, wherein the serum-free EHM medium further comprises VEGF, FGF, or both VEGF and FGF.

54. The method of any one of embodiments 1-53, wherein the VEGF is human VEGF.

55. The method of any one of embodiments 1-54, wherein the FGF is human FGF.

56. The method of embodiment 53, wherein the serum-free EHM medium comprises about 5-20 ng/ml VEGF.

57. The method of embodiment 56, wherein the serum-free EHM medium comprises about 10 ng/ml VEGF.

58. The method of embodiment 53, wherein the serum-free EHM medium comprises about 5-20 ng/ml FGF.

59. The method of embodiment 58, wherein the serum-free EHM medium comprises about 10 ng/ml FGF.

60. The method of any one of embodiments 1-59, wherein the serum-free EHM medium in step (iii) further comprises 750 mg/L glycine, 890 mg/L L-alanine, 1320 mg/L L-asparagine, 1330 mg/L aspartic acid, 1470 mg/L L-glutamic acid, 1150 mg/L L-proline, and 1050 mg/L L-serine.

61. The method of any one of embodiments 1-60, wherein the culturing in step (iii) is performed for at least 3 days.

62. The method of embodiment 61, wherein the culturing in step (iii) is performed for about 3 to about 7 days.

63. The method of any one of embodiments 1-62, wherein the culturing in step (iv) is performed for a period of at least 3-60 days.

64. The method of embodiment 63, wherein the further culturing is performed for 4-30 days.

65. The method of embodiment 63, wherein the further culturing is performed for 5-20 days.

66. The method of embodiment 63, wherein the further culturing is performed for 6-10 days.

67. The method of embodiment 63, wherein the further culturing is carried out for 7 days.

68. The method of any one of embodiments 1-67, wherein step (iv) is performed on a stretching device.

69. The method of embodiment 68, wherein the stretching device applies static, phasic, or dynamic stretching.

70. The method of any one of embodiments 1-69, wherein the EHM generates a force greater than 0.01 mN upon induction with 3 mM calcium as determined using the method described in Zimmermann et al., Circ. Res. 90, 223-230 (2002).

71. The method of embodiment 70, wherein the EHM generates a force greater than 0.1 mN after induction with 3 mM calcium as measured using the method described in Zimmermann et al., Circ. Res. 90, 223-230 (2002).

72. The method of embodiment 70, wherein the EHM generates a force greater than 0.2 mN after induction with 3 mM calcium as measured using the method described in Zimmermann et al., Circ. Res. 90, 223-230 (2002).

73. The method of embodiment 70, wherein the EHM generates a force greater than 0.3 mN after induction with 3 mM calcium as measured using the method described in Zimmermann et al., Circ. Res. 90, 223-230 (2002).

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. hEHM formation. hEHM after 10 days of culture (3 days in a cast mold followed by 7 days on a custom spacer; culture time: 3+7 days); Scale bar: 1 mm.

Figure 2. Characterization of hEHM contractile function. (A) Chronotropic response after stimulation with compounds that increase cAMP (Iso: β-adrenergic stimulation; IBMX: phosphodiesterase inhibition, n = 6/group). (B) Inotropic response of hEHM to increasing concentrations of extracellular calcium, comparing hES2 (n = 12), hES3 (n = 12), and iPS I2 (n = 3). (C) Inotropic response of hEHM (hES2) to stimulation with isoproterenol (triangles) and carbachol (squares). Inset: Normalized contraction (twitch) under low calcium (lowest curve), stimulation with isoproterenol (top curve), and carbachol (middle curve). (D) Relaxation of hEHM under stimulation with isoproterenol (triangles) and carbachol (squares). Inset: normalized contraction *P<0.05 relative to baseline value (A: paired two-tailed Student's t-test; C, D: ANOVA followed by Dunnet's post hoc test).

Figure 3. Importance of non-myocytes for EHM formation. (A) Comparison of normalized force and percentage of cardiomyocytes (actinin+ cells) in EHM generated from the hES2 line (circles: monolayer cardiomyocyte differentiation protocol (Hudson et al., Stem Cell Dev 21:1513-1523 (2012)), squares: EB cardiomyocyte differentiation protocol (Yang et al., Nature 453:524-528 (2008))) and the iPS BJ line (triangles). (B) Contractile force of hEHM generated with 100% cardiomyocytes (CM 100%) and a mixture of 70% cardiomyocytes and 30% cardiac fibroblasts (CM/CF 70/30%), n = 1.

Figure 4. GMP-compliant hydrogel matrices. (A) Contractile force of hEHM prepared with rat collagen (0.4 mg/EHM, n = 12) or bovine collagen (0.4 mg/EHM, n = 14) at an extracellular calcium concentration of 2 mmol/L. (B) Contractile force of hEHM prepared with the “initial protocol” (rat collagen, n = 12) and the “matrix protocol” (bovine collagen, no, n = 9) at 2 mmol/L extracellular calcium. See Table 1. (C) Contractile force of hEHM prepared with bovine collagen alone (bov), bovine collagen plus Matrigel (10% v/v), bovine collagen plus fibronectin (5 μg/EHM), and bovine collagen plus fibronectin (5 μg/EHM) plus laminin (5 μg/EHM) at 2 mmol/L extracellular calcium, n = 4 per group.

Figure 5. Screening of serum-free growth supplements relative to Iscove's basal medium with 20% serum. (A) F.l.t.r.: Changes in cell death, percentage of cardiomyocytes (CM percentage), mean cardiomyocyte actin fluorescence (CM maturation), cardiomyocyte size based on side scatter area (CM size), and non-myocyte size based on side scatter area (NM size) in serum-free medium based on Iscove's, RPMI, and αMEM basal medium (see also Tables 2-4) compared to serum-supplemented EHM medium (see also Table 1).

(B) F.l.t.r.: Changes in cell death, percentage of cardiomyocytes (CM percentage), mean cardiomyocyte actin fluorescence (CM maturation), cardiomyocyte size based on side scatter area (CM size), and non-myocyte size based on side scatter area (NM size) in serum-free medium with 2% and 4% insulin-plus B27 (B27+) and insulin-minus B27 (B27-) compared to serum-containing EHM medium (see also Table 1).

Figure 6. Screening of peptide growth factors and fatty acid supplements. (A) Factors considered for serum-free, defined EHM medium. F.l.t.r.: Changes in cell death, percentage of cardiomyocytes (CM%), mean cardiomyocyte actin fluorescence (CM maturation), cardiomyocyte size based on side scatter area (CM size), and non-myocyte size based on side scatter area (NM size) with the indicated growth factors and fatty acid supplements compared to serum-free medium without factors. (b) Factors not considered for serum-free, defined EHM medium. F.l.t.r.: Changes in cell death, percentage of cardiomyocytes (CM%), mean cardiomyocyte actin fluorescence (CM maturation), cardiomyocyte size based on side scatter area (CM size), and non-myocyte size based on side scatter area (NM size) with the indicated growth factors and fatty acid supplements compared to serum-free medium without factors.

Figure 7. Serum-free, defined EHM generation from hESCs. (A) Contractility of hEHM (hES2) in the presence of increasing concentrations of extracellular calcium, comparing serum-containing medium (Serum), Iscove's-based serum-free medium (SF-IMDM, see also Table 5), aMEM-based serum-free medium (SF-aMEM, see also Table 6), and RPMI-based serum-free medium (SF-RPMI). For the respective _EC50 values of calcium, n=15/5/6/7 for Serum, SF-IMDM, SF-aMEM, and SF-RPMI, and nd is not determined. (B) Immunostaining of serum-free hEHM, demonstrating typical cardiac muscle properties (muscle bundles). Immunostaining was performed with an antibody against α-sarcomeric actinin, and nuclear staining was performed with DAPI. (C) Percent change in force after adrenergic stimulation with 1 μM isoproterenol in serum-containing EHM (Serum), serum-free EHM based on Iscove's (SF-IMDM), and serum-free EHM based on aMEM (SF-aMEM), n = 15/5/6, *p < 0.05. (D) Percent change in force after muscarinic stimulation with 10 μM carbachol in serum-containing EHM (Serum), serum-free EHM based on Iscove's (SF-IMDM), and serum-free EHM based on aMEM (SF-aMEM), n = 15/5/6. Scale bar: 20 μm

Figure 8. Effect of calcium on contractility of serum-free human engineered cardiac muscle. A) Contractility of hEHM (hES2) in the presence of increasing concentrations of extracellular calcium, comparing RPMI medium (~0.4 mM calcium, n=2) versus RPMI medium supplemented with 0.8 mM CaCl (final free calcium concentration ~1.2 mM calcium, n=2). B) Response of serum-free hEHM cultured in RPMI medium or RPMI + calcium medium to 1 μM isoproterenol.

Figure 9. Effect of TGFβ1 on hEHM force. (A) Contractile force of hEHM (iPS BJ) in the presence of increasing concentrations of extracellular calcium, comparing serum-free medium (SF) and serum-free medium containing 5 ng/ml TGFβ1, hEHM from culture days 0-3 (n=4/group, *p<0.05). (B) Contractile force of hEHM (hES2) from culture days 0-3 (SF+TGHβ1 Day 0-3) and 0-10 (SF+TGHβ1 Day 0-10, n=5/group) in serum-free medium containing TGFβ1.

Figure 10. Effects of FGF and VEGF on hEHM force. Contractile force of hEHM (hES2) in the presence of increasing concentrations of extracellular calcium, comparing serum-free medium (SF, n=3), serum-free medium containing 10 ng/ml FGF-2 (SF+FGF, n=3), and serum-free medium containing 10 ng/ml VEGF (SF+VEGF, n=3). *p<0.05, SF+FGF vs. SF.

Figure 11. Effect of B27 supplement concentration on hEHM force. Contractile force of hEHM (hES2) in the presence of increasing concentrations of extracellular calcium, comparing serum-supplemented medium (Serum), serum-free medium containing 2% B27 (SF-2%B27), and serum-free medium containing 4% B27 (SF-4%B27), n=17/9/11, *p<0.05 vs. 2% B27.

Figure 12. Effect of B27 composition on hEHM force. Contractile force of hEHM (hES2) in the presence of increasing concentrations of extracellular calcium, comparing serum-containing medium (n=9), serum-free medium containing complete B27 (n=5), serum-free medium containing reduced antioxidant B27 (n=5), and serum-free medium containing reduced insulin B27 (n=5).

Figure 13. Replacement of B27 with custom supplements in hEHM derived from hES and iPS cells. (A) Contractile force of hEHM (hES2) in the presence of increasing concentrations of extracellular calcium, comparing serum-containing medium (n=3), serum-free medium with insulin-minus B27 (B27-insulin, n=5), and serum-free medium with custom supplements (CMS; n=3). (B) Contractile force of hEHM derived from iPS cells (BJ) in the presence of increasing concentrations of extracellular calcium, comparing serum-containing medium (n=8), serum-free medium with insulin-minus B27 (n=8), and serum-free medium with custom supplements (CMS; n=4).

Figure 14. Maturation of serum-free hEHMs under prolonged culture. (A) Contractile force of hEHMs (hiPS-G1) cultured in serum-free conditions (n = 4-6/condition) at 2 weeks (lower curve), 4 weeks (middle curve), and 8 weeks (upper curve). (B) Brightfield images of individual cardiomyocytes from 8-week-old serum-free hEHMs. Scale bar: 20 μM.

The following examples are used to further illustrate, but not to limit, the present invention. These examples include multiple technical features, and it should be understood that the present invention also relates to the combination of technical features appearing in this illustrative section.

Example

Material

All materials used in this article are commercially available. For example, DMEM, RPMI, αMEM (Catalog No. 32561-029), streptomycin, penicillin, and B27 can be obtained from Invitrogen; medical-grade bovine collagen can be purchased from Devros Medical; fatty acid supplements can be ordered from Sigma (Catalog No. F7050); and various growth factors can be purchased from Peprotech (FGF2, AF-1ββ-18B; IGF-1, AF-100-11; TGFβ1, 100-21).

method

Human ESC and iPS-lines and culture

In this study (Robert-Koch Institute Grant W.-H.Z.: Grant #12; Reference Number: 1710-79-1-4-16), the inventors utilized H9.2 (Technion, Haifa, Israel), hES3 (Embryonic Stem Cell International, Singapore), and transgenic hES3-ENVY (Costa, M. et al., Nat Methods 2:259-260 (2005)), as well as hES2 lines (McEwen Centre for Regenrative Medicine, Toronto, Canada; Yang et al., Nature 453:2:5-528: (2008)). Differentiated EBs were transported to Hamburg/Göttingen at room temperature and delivered within 72-96 hours. iPS lines were from Toronto (iPS BJ) and Göttingen (iPS I2, Streckfuss-Bomeke et al., Eur Heart J (2012) doi: 10.1093/eurheartj/ehs203 and iPS Sendai).

EBs were digested with collagenase B (1 mg/ml; H9.2), collagenase I (2 mg/ml) and/or trypsin/EDTA (0.25%/1 mmol/l; hES3, hES3-ENVY, hES2, iPS NJ, iPS I2) as described elsewhere (Kehat et al., J Clin Invest 108:407-414 (2001); Mummery et al., Circulation 107:2733-2740 (2003); Xu et al., Circ Res 91:501-508 (2002); Yang et al., Nature 453:524-528 (2008); Passier et al., Stem Cells 23:772-780 (2005); each incorporated herein by reference). Cardiomyocytes were counted in representative aliquots of enzymatically dispersed cells after staining for tropomyosin and sarcomeric actinin.

Construction of basic human engineered myocardium (hEHM)

The present inventors employed a modified EHM-engineering protocol (Zimmermann et al., Circ Res 90:223-230 (2002), incorporated herein by reference) to construct hEHMs. Briefly, EHMs were prepared by pipetting a mixture containing freshly dispersed ESC derivatives (1×10 ⁴ -15×10 ⁶ cells in Iscove's medium containing 20% fetal bovine serum, 1% non-essential amino acids, 2 mmol/l glutamine, 100 μmol/l β-mercaptoethanol, 100 U/ml penicillin and 100 mg/ml streptomycin), pH-neutralized rat tail-derived type I collagen (0.4 mg/EHM), Matrigel ^™ (10% v/v; Becton Dickenson or tebu), and concentrated serum-containing medium (2×DMEM, 20% horse serum, 4% chicken embryo extract, 200 U/ml penicillin and 200 mg/ml streptomycin) into a ring mold (inner/outer diameter: 2/4 mm; height: 5 mm) (Table 1) (reconstitution volume: 450 μl). hEHMs rapidly solidified within the casting mold and were transferred to a static stretch apparatus (110% overhang) on day 3 of culture (Zimmermann et al., Nat Med 12:452-458 (2006), incorporated herein by reference). The culture medium was changed every other day. hEHM cultures were maintained under tension for 7 days.

Another prior art protocol suitable as a basis for the improved methods disclosed herein is described by Soong et al., Curr Prot Cell Biol. 23.8.1-23.8.21 (2012), which is incorporated herein by reference in its entirety, with particular reference to "Basic Protocol 2" and "Supporting Protocol 2."

Removal and replacement of xenogeneic matrix components

To meet pre-GMP conditions for hEHM, a protocol with reduced xenobiotic components was established (matrix protocol, Table 1). Cells were reconstituted in a mixture of pH-neutralized bovine collagen (Devros Medical, 0.4 mg/EHM) and concentrated serum-containing medium (2×DMEM, 40% fetal bovine serum, 200 U/ml penicillin, and 200 mg/ml streptomycin) and cultured in Iscove's medium containing 20% fetal bovine serum, 1% non-essential amino acids, 2 mmol/l glutamine, 0.3 mmol/l ascorbic acid, 100 μmol/l β-mercaptoethanol, 100 U/ml penicillin, and 100 μg/mL streptomycin.

Removal and replacement of xenogeneic culture medium components

To generate fully defined serum-free EHM, cells were reconstituted in a mixture of pH-neutralized bovine collagen (Devros Medical, 0.4 mg/EHM), concentrated serum-free medium (2×DMEM, 8% B27, 200 U/ml penicillin and 200 mg/ml streptomycin) and cultured in Iscove-medium (serum-free protocol, Table 1) containing 4% complete B27, 1% non-essential amino acids, 2 mmol/l glutamine, 0.3 mmol/l ascorbic acid, 20 ng/ml IGF-1, 10 ng/ml FGF2, 10 ng/ml VEGF, 5 ng/ml TGFb1 (culture days 0-3 only) and 100 U/ml penicillin, and 100 μg/ml streptomycin. B27 supplements contain vitamins (biotin, DL-alpha tocopherol, DL-alpha-tocopherol acetate, vitamin A), proteins, and enzymes (BSA, free fatty acid fraction V, catalase, human recombinant insulin, human transferrin, superoxide dismutase), as well as other cell-supporting components (corticosterone, D-galactose, ethanolamine, glutathione (reduced form), L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, and T3 (triiodo-I-thyronine). Complete B27 (Invitrogen, A1486701) was compared with antioxidant-free B27 (Invitrogen, #10889038) and insulin-free B27 (Invitrogen, #0050129SA), as indicated. The B27 supplement was replaced by a customized supplement consisting of albumin, transferrin, ethanolamine, sodium selenite, L-carnitine hydrochloride, hydrocortisone, fatty acid supplement, and triiodo-L-thyronine ( Table 2 ).

Contractile function analysis

As previously described, the present inventors analyzed contractile force and contractile kinetics (contraction time: time from 50% to maximal contraction; relaxation time: time from maximal contraction to 50% relaxation) under isometric conditions (Zimmermann et al., Circ Res 90: 223-230 (2002); incorporated herein by reference). Contraction frequency (unstimulated spontaneous contraction) was assessed by light microscopy immediately after the EHM was removed from the incubator.

Flow cytometry

EBs cultured under different culture conditions were prepared as described above to prepare single cell suspensions. Cells were fixed in 70% ice-cold ethanol under constant stirring. Cells were stained with sarcomere actin (Sigma) to mark cardiomyocytes and DAPI to analyze nuclear DNA content and exclude cell doublets. Cells were run on an LSRII flow cytometer (BD). At least 10,000 viable cells were analyzed. The following parameters were then analyzed, (1) cell death (percentage of apoptotic cells in the sub-G1 fraction), (2) percentage of cardiomyocytes and non-myocytes (actinin positive and negative cells, respectively), (3) cardiomyocyte maturation (mean actinin fluorescence), (4) cardiomyocyte size and (5) non-myocyte size (based on side scatter area, SSC-A).

Morphological analysis

As previously described (Zimmermann et al., Circ Res 90:223-230 (2002), incorporated herein by reference), hEHM were fixed in neutral 4% buffered formaldehyde/1% methanol, pH 7.4 for confocal laser scanning microscopy (CLSM; Zeiss 510 Meta LSM system or Zeiss 710 LSM). For CLSM, the present inventors prepared vibrating sections (100 μm; Leica VT1000 S) and immunofluorescently labeled them with antibodies against α-sarcomeric actinin (Sigma clone EA-53, 1:800; with appropriate secondary antibodies). Cell nuclei were stained with DAPI (4′, 6-diamidino-2-phenylindole; 1 μg/ml).

Statistical analysis

Data are presented as mean ± standard error of the mean. Statistical differences were determined using paired and unpaired two-tailed Student's t-tests or ANOVA followed by Dunnet's post hoc test. P values < 0.05 were considered statistically significant.

result

Generation of human engineered myocardium (hEHM)

EBs were generated in Haifa (H9.2;), Singapore (hES3 and hES3-ENVY; Costa et al., Nat Methods 2:259-260 (2005)), and Toronto (hHES2; iPS) and shipped to Hamburg/Göttingen in sealed containers filled with culture medium at room temperature by courier. Arrival was ensured within 72-96 hours. Upon arrival, EBs were transferred to fresh culture medium and allowed to recover for 24-48 hours. During this time, EBs resumed spontaneous contractile activity. EBs were enzymatically dissociated, and the resulting single-cell suspensions were used for hEHM generation or cell histology. An initial series of experiments explored the required cell number per hEHM (1×10 ⁴ -15×10 ⁶ cells) and the utility of different ESC lines (H9.2, hES2, hES3, hES3-ENVY) and iPS lines (I2, BJ, Sendai) for hEHM construction (n=67). Within 48 hours of hEHM casting, spontaneous beating of areas of varying sizes was observed in all cultures. However, force-producing hEHMs were formed using only 1.25-15×10 ⁶ cells/EHM ( FIG. 1 ). Cell density can be easily adjusted based on the size of the EHM.

Organotypic function of hEHM

hEHM contracted stably and rhythmically in culture for at least 3 weeks (0.8±0.05 Hz at 37°C; n=14). The present inventors performed detailed functional characterization over 10 days. Incubation with isoproterenol increased the spontaneous beating frequency to 1.2±0.1 Hz (n=6; P<0.01, Figure 2A). Addition of 3-isobutyl-1-methylxanthine (IBMX; 100 μmol/l) to inhibit phosphodiesterase further increased the spontaneous beating rate to 1.6±0.1 Hz (n=6; P<0.001, Figure 2A). At 2.4 mmol/l calcium, hEHM produced a maximal mean contractile force of 130±13 μN ( _EC50 : 0.8±0.04 mmol/l, n=17; Figure 2B). At sub- _EC50 extracellular calcium (0.4 mmol/l), β-adrenergic stimulation with 1 μmol/l isoproterenol increased contractility by 47±12% (n=12; Figure 2C) and shortened relaxation to 113±2.3 ms (n=12; Figure 2D).

Importance of non-muscle cells in EHM formation

All ESC- and IPS-derived lines used in this experiment appeared suitable for hEHM generation. To test which cardiomyocyte content was optimal for force-generating tissue, the inventors plotted the force generated versus the percentage of cardiomyocytes. Interestingly, a bell-shaped distribution was obtained, with the greatest force generated at a cardiomyocyte percentage of 40-80% ( FIG3A ).

The above findings suggest that non-myocytes play an important role in proper tissue formation. To investigate this, the present inventors conducted experiments with human cardiomyocytes purified by the surface marker CD172a (SIRPα) (Dubois et al., Nat Biotechnol 29:1011-1018 (2011)). EHM generated from purified cardiomyocytes did not form force-generating tissue (Figure 3B). However, supplementation with 30% human cardiac fibroblasts produced a more rigid tissue with good force generation. These results emphasize that non-myocytes are crucial for myocardial tissue formation and that these cells also need to be supported under serum-free conditions.

hEHM production using GMP-compliant matrices

Based on the operating protocol that the inventors have established in the neonatal rat heart cell model, the inventors initially constructed hEHM (Zimmermann et al., Biotechnol Bioeng 68: 106-114 (2000)). This operating protocol includes several non-human components (including rat collagen, Matrigel, horse serum, fetal bovine serum and chicken embryo extract) that do not meet the requirements of "therapeutic application" in vivo. To address this issue, a series of experiments were first conducted to directly test whether the non-human components of the hEHM matrix could be reduced. Rat collagen was replaced with medical grade (GMP) bovine collagen without loss of performance (Figure 4A). Similarly, horse serum and chicken embryo extract could be eliminated without negatively affecting the formation and function of hEHM ("Matrix Operating Protocol", Figure 4B, Table 1). Supplementing bovine collagen with other extracellular matrix proteins such as fibronectin and laminin, one of the main components, did not produce additional benefits (Figure 4C).

Definition of serum-free culture medium supporting hEHM formation

To further define human EHM culture and make it GMP-compliant, the present inventors sought to replace all non-restricted serum components with chemically defined supplements. The present inventors proposed a simplified screening algorithm based on three-dimensional human embryoid body (EB) culture to screen these supplements. ESCs used for this screening were cultured under serum-free conditions (Yang et al., Nature 453:524-528 (2008); Kattman et al., Cell Stem Cell 8:228-240 (2011)). The reference for screening was our serum-containing EHM medium (Table 1): (1) Iscove's, (2) 2 mmol/L glutamine, (3) 20% FBS, (4) 1% non-essential amino acids, (5) 0.3 mmol/L ascorbic acid, (6) 100 μmol/l β-mercaptoethanol, and (7) 100 U/ml penicillin/100 mg/ml streptomycin. As a readout of the favorable or unfavorable effects of basal medium and supplements, a flow cytometry-based protocol was established (Tiburcy et al., Circ Res 109:1105-1114 (2011); incorporated herein by reference) to measure (1) cell death (based on sub-G1 DNA content), (2) cardiomyocyte content (based on pro-actin expression), (3) cardiomyocyte maturation (based on pro-actin mean fluorescence per cardiomyocyte), (4) cardiomyocyte size, and (5) non-myocyte size (based on side scatter area).

The inventors first screened three basal medium formulations with and without B27 supplementation (Iscove's, RPMI, αMEM: Tables 3-5). B27 has been used by several research groups for the differentiation of human ESCs and iPSCs (Burridge et al., Cell Stem Cell 10: 16-28 (2012)). The screening showed that B27 was essential for the formation of embryoid bodies (EBs), regardless of the basal medium tested. Iscove's and RPMI showed comparable results, while aMEM appeared to cause slightly higher cell death. On the other hand, αMEM was superior for cardiac pro-actin expression (Figure 5A).

Based on the initial finding that EHM cultured in RPMI basal medium performed less than ideally ( FIG. 7 ), the inventors continued screening using Iscove’s or aMEM as basal medium. The inventors next carefully examined the efficacy of B27 in its standard formulation with and without insulin. Compared to B27 without insulin and a lower B27 (2%) supplementation concentration, 4% B27 supplementation with insulin (final concentration 10 μg/ml) showed the lowest cell death and potentially greater support for non-myocytes (reduced percentage of cardiomyocytes, suggesting a shift towards more non-myocytes) ( FIG. 5B ). The inventors then screened the efficacy of six peptide growth factors and fatty acid supplements at concentrations above the EC50. PDGF-BB, CT-1, and neuregulin-1 caused extensive cell death, while IGF-1 appeared to protect against cell death ( FIG. 6A , B ). TGFβ1, VEGF, and fatty acid supplementation enhanced pro-actin expression in individual cardiomyocytes. FGF-2 supported non-myocyte growth and increased cardiomyocyte size ( FIG. 6A ). TGFβ2 had no beneficial effect (Figure 6B). All compounds tested or their combinations are involved in cardiac development and EHM generation (Naito et al., Ciirculation 114:172-78 (2006); Shimojo et al., Am J Physiol Heart CircPhysiol 293:H474-481 (2007); Vantleret et al., J Mol Cell Cardiol 48:1316-1323 (2010); Odiete et al., Circ Res 111:1376-1385 (2012); Wollert and Chien J Mol Med (Berl) 75:492-501 (1997); Price et al., Anat Rec A Discov Mol Cell Evol Biol 272:424-433 (2003); Molin et al., Dev Dyn 227:431-444 (2003); Corda et al., Circ Res 81:679-687 (1997); Lopaschuk and Jaswal J Cardiovasc Pharmacol 56:130-140; herein incorporated by reference in their entireties).

The development of EHM is characterized by two stages. The first stage is the "condensation stage", in which isolated cells "settle" in the matrix, reconstruct themselves and the matrix, and may also be accompanied by a large amount of cell death. This stage is greatly influenced by non-muscle cells. The second stage is tissue maturation under mechanical load. This stage is characterized by hypertrophic growth and myocyte maturation, positioning, increasing force development, and matrix stabilization (Tiburcy et al., Circ Res 109: 1105-1114 (2011)).

The inventors reasoned that different culture medium conditions might be required depending on the stage. To prevent cell death during the condensation phase, the inventors selected a combination of culture medium components that were neutral or even reduced cell death in the initial screening. Namely, Iscove's basal medium, 4% B27, and IGF-1. Simultaneously, factors that increase the number and/or size of non-myocytes (IGF-1, TGF-β1, FGF-2 in the first stage) were supported by matrix remodeling and condensation of non-myocytes. VEGF was added to support the maturation of cardiomyocytes (Table 5). The ability of this culture medium to support the formation of force-generating EHMs was then tested. Serum-free EHMs beat continuously at a spontaneous beat frequency of 113±12 bpm, n=7. Serum-containing EHMs beat significantly faster (199±8 bpm, n=8). The inventors found similar maximum force generation and calcium sensitivity compared to the serum-containing control group (Figure 7A). Both Iscove's and αMEM basal media supported the formation of EHMs, and the contractile force results of the two were comparable. RPMI did not support force-generating tissue (Figure 7A, Tables 5 and 6). Morphologically, serum-free EHM contained well-developed muscle bundles with anisotropically arranged cardiomyocytes (Figure 7B). Importantly, serum-free EHM produced responses to adrenergic and muscarinic stimulation as expected for myocardium. In fact, the response of αMEM EHM was significantly superior to that of serum-containing controls (Figures 7C and D).

The inventors speculated that the poor performance of RPMI medium was due to the subphysiological free calcium concentration of RPMI medium (0.424 mmol/L, Table 4). To verify whether this speculation was correct, the inventors conducted an additional experiment comparing RPMI medium with RPMI medium supplemented with 0.8 mM CaCl (final free calcium concentration of 1.242 mmol/L). Although EHMs in the RPMI condition showed little contraction, calcium supplementation resulted in measurable maximum force and better responsiveness to isoproterenol (Figure 8). In fact, the maximum force generated in the supplemented RPMI condition was comparable to that of IMDM or αMEM medium (Figure 7A), indicating that a range of ~1–2 mmol/L calcium is necessary for proper tissue function.

To validate the initial screening results, the inventors also tested the effects of key factors on the formation of functional EHMs. Addition of TGFβ1 from day 0 to day 3 was essential, but prolonged TGFβ1 treatment did not provide additional benefits (Figures 9A, B). Both FGF and VEGF increased force generation, confirming that both contribute significantly to tissue formation (Figure 10).

Increasing the concentration of B27 additive to 4% was superior to 2% B27 (Figure 11). To test which components of B27 are critical for EHM function, the inventors first conducted experiments on different commercially available B27 formulations. The inventors compared complete B27 with insulin-reduced B27 and antioxidant-reduced B27. Antioxidant-reduced B27 performed comparable to complete B27, suggesting that antioxidants are not essential. Surprisingly, insulin-reduced B27 performed better than complete B27, suggesting that insulin supplementation is also not necessary (Figure 12).

Based on these results, the inventors developed a custom serum supplement to replace B27 (Table 7). When the inventors tested this custom serum supplement against serum-containing medium and serum-free medium containing insulin-minus B27, the inventors found that maximum force generation was comparable, suggesting that B27 can be omitted from serum-free EHM culture and all replaced by custom serum supplements (Figure 13A). These findings were confirmed by hEHM from iPSC, which demonstrated the effectiveness of generating limited engineered myocardium from different pluripotent stem cell sources. (Figure 13B).

To explore whether serum-free hEHM supports long-term culture and maturation of cardiomyocytes, the inventors tested the force production of serum-free hEHM from hIPS-G1 at culture weeks 2, 4, and 8. The inventors observed a robust increase in force production ( FIG14A ) and a rod-like morphology with regular striations of individual hEHM-derived cardiomyocytes, indicating good, advanced maturation under serum-free conditions.

in conclusion

This study demonstrates for the first time that differentiated, force-producing human cardiac muscle can be generated in vitro under fully defined, serum-free conditions. The protocol is applicable to cardiac muscle derived from both embryonic stem (ESC) and induced pluripotent stem (iPS) cells.

This is a major breakthrough that not only enables future in vitro studies to investigate, for example, maturation and hypertrophy without the confounding effects of serum factors, but also enables the potential for in vivo applications and therapeutic approaches under GMP requirements.

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Claims (14)

1. A method for producing an engineered myocardium (EHM), the method comprising the steps of: (i) providing a serum-free reconstitution mixture in one or more molds, the reconstitution mixture comprising (a) serum-free minimal essential medium; (b) a serum-free supplement to give a final concentration of 0.5-50 mg/ml albumin, 1-100 μg/ml transferrin, 0.1-10 μg/ml ethanolamine, 0.003-0.3 μg/ml sodium selenite, 0.4-40 μg/ml L-carnitine hydrochloride, 0.1-10 μg/ml hydrocortisone, 0.05-5 μl/ml fatty acid supplement, 0.0001-0.1 μg/ml triiodo-L-thyronine (T3), and 0.2-2 mg/ml collagen; and (c) a mixture of human cardiomyocytes and human non-myocytes, wherein 20% to 80% of the total cell mixture are human cardiomyocytes; wherein the cardiomyocytes were not produced using a process that involves modification of human germline genetic characteristics or that involves the use of human embryos for industrial or commercial purposes; wherein the pH of the reconstituted mixture is 7.2 to 7.6; (ii) incubating the serum-free reconstitution mixture in the one or more molds, thereby allowing the serum-free reconstitution mixture to clot for at least 15 minutes; (iii) culturing the mixture obtained in step (ii) in the one or more molds in serum-free EHM medium until the mixture solidifies to at least 50% of its original thickness, wherein the EHM medium comprises (a) a basal medium containing 0.5-3 mmol/L Ca2 ⁺ ; (b) a serum-free supplement as defined in step (i)(b); (c) 0.5-10 mmol/L L-glutamine; (d) 0.01-1.0 mmol/L ascorbic acid; (e) 1-100 ng/ml IGF-1; and (f) 1-10 ng/ml TGFβ1; (iv) culturing the mixture obtained in step (iii) in a serum-free EHM medium as defined in steps (iii)(a)-(f) under mechanical tension, thereby forming a force-generating EHM. 2. The method according to claim 1, wherein the minimum essential medium in step (i) is selected from the group consisting of Iscove's medium, αMEM, DMEM and RPMI. 3. The method according to claim 1, wherein the serum-free supplement of step (i) and/or step (iii) further comprises one or more components selected from vitamin A, D-galactose, linoleic acid, linolenic acid, progesterone and putrescine. 4. The method of claim 1, wherein the serum-free supplement in component (b) of step (i) and/or component (b) of step (iii) is a supplement or an insulin-reducing supplement. 5. The method according to claim 1, wherein the reconstitution mixture of step (i) comprises 0.3-0.5 mg collagen per ^1.5 x 106 mixture of cardiomyocytes and non-myocytes. 6. The method of claim 1, wherein in component (c) of the reconstitution mixture of step (i), the collagen is medical grade and is selected from type I collagen, type III collagen, type V collagen, and mixtures thereof. 7. The method of claim 1, wherein the pH of the reconstituted mixture of step (i) is 7.4. The method of claim 1 , wherein the cardiomyocytes are human cardiomyocytes. 9. The method of claim 1, wherein the human non-muscle cells are fibroblasts, endothelial cells, smooth muscle cells or mesenchymal stem cells. 10. The method according to claim 1, wherein the culturing in step (ii) is performed for 0.25-3 hours. 11. The method of claim 1, wherein the serum-free EHM medium comprises (i) 20 ng/ml human IGF1; (ii) 5 ng/ml human TGFβ1; and/or (iii) 5-20 ng/ml human VEGF and/or 5-20 ng/ml human FGF. 12. The method of claim 1 , wherein the serum-free EHM medium in step (iii) further comprises 750 mg/L glycine, 890 mg/L L-alanine, 1320 mg/L L-asparagine, 1330 mg/L aspartic acid, 1470 mg/L L-glutamate, 1150 mg/L L-proline, and 1050 mg/L L-serine. 13. The method according to claim 1, wherein the culturing in step (iii) is performed for at least 3 days. 14. The method of claim 1, wherein the culturing in step (iv) is performed for a period of at least 3-60 days; Wherein step (iv) is carried out on a stretching device.