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.

Description

BACKGROUND OF THE INVENTION[0001]Human pluripotent stem cells (hPSCs) are now widely used to provide a theoretically endless and also large supply of human cardiomyocytes (Kehat et al. J Clin Invest 108, 407-414 (2001); Takahashi et al. Cell 131, 861-872 (2007); Zhang et al., Circ Res 104, e30-41 (2009)). Human cardiomyocytes have been derived from human embryonic stem cells (hESCs) (Thomson et al. Science 282, 1145-1147 (1998)) and induced pluripotent stem cells (hIPSCs) (Takahashi et al., Cell 131, 861-872 (2007)) and have a demonstrated use for multiple purposes including developmental models (Lian et al. Stem Cells 2012 (2012)), drug efficacy and / or safety screening (Schaaf et al. PLoS ONE 6, 20 (2011)), hypertrophy modelling and regenerative applications. Additionally, with recent advances in hIPSC technology, cardiomyocytes exhibiting heritable genetic disease phenotypes can be generated in vitro (Carvajal-Vergara, X. et al. Nature 465, 808-812 (2010); Itzhaki et al., Nature 4...

AI Technical Summary

Benefits of technology

[0013]The present data suggests that the BHM protocol disclosed herein is a robust, serum-free and reproducible way to produce human myocardium for multiple applications.

[0067]Apart from the above disclosed method, the invention further relates to a BHM produced by said method. Despite the increased maturity observed in our BHM protocol, it should also be noted that the BHM is still a relatively immature tissue. Compared to adult heart tissue the BHM still has an inferior β-MHC / α-MHC ratio, and low but still retained expression of progenitor genes (e.g. ISL1). However, prolonged culture under appropriate culture conditions with biophysical stimulation may further increase maturity. There is already morphological evidence suggesting that this may also be the case in the BHM system.

[0070]The BHM may provide a good model system for investigating mechanisms driving maturation in a serum-free environment, and we have already demonstrated that with increased culture periods maturity may be increased (we showed increase isoprenaline sensitivity and improve tissue morphology). The capability of long term BHM culture without loss of function (at least 63 days) also suggests that long term pharmacological safety and efficacy experiments are possible. Hence, in a preferred embodiment, the BHM obtained by the method disclosed herein can be maintained for at least 63 days.

[0079](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.

Problems solved by technology

Cardiovasc Res 58, 423-434 (2003)) and making it difficult to extrapolate results to the in vivo situation.

Tissue engineering may therefore be considered as an inefficient process for two reasons, 1) disassociation of a tissue / differentiation culture destroys the extracellular environment thus destroying developmental information (eg. cell-cell interconnectivity, geometric cell positioning, cell-ECM connectivity), this necessitates very large increases in extracellular matrix (ECM) production in order to re-build the environment (Hudson et al.

Tissue Eng Part A 17, 2279-2289 (2011)), and 2) the disassociation process is variable between hPSC lines and can lead to considerable cell death.

However, the inventor's results demonstrate that changes in differentiation protocol may greatly affect the cardiomyocyte phenotype (e.g. it is shown that dorsomorphin may greatly affect the bioengineered heart muscle (BHM)).

This may lead to changes in tissue engineered myocardial properties which may mask the effects of different experimental conditions or genetic disease models, therefore care must be taken when using different protocols in different lines.

However, pure cardiomyocytes do not facilitate the formation of functional tissue engineered myocardium and both cardiomyocytes and stromal cells are required for the formation of functional tissue engineered myocardium (Naito et al.

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