Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

Abstract

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cellbased transplantation or by cardiac tissue engineering [1][2][3] . Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart [1][2][3] . The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery [1][2][3] . The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration 4,5 . Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity 6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic [9][10][11] . To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinicallysuitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules 12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

Efficient Generation of Human Embryonic Stem Cell-Derived Cardiac Progenitors Based on Tissue-Specific Enhanced Green Fluorescence Protein Expression

“…In order to produce CMs from pluripotent stem cells, differentiation has been reported to be efficiently directed toward the cardiac lineage by various subsets of morphogenes [7][8][9][10][11] or chemical agents. [12][13][14][15][16] By using these induction strategies alone, 60-90% of CM enrichment could be achieved in the final preparation. To achieve higher purity, CMs may be selected based on genetically introduced selectable markers 17 or the special properties of CMs; for example, the specific expression of cell surface proteins [18][19][20][21] or a high level of mitochondria.…”

Reviving Cell-Based Regenerative Medicine for Heart Reconstitution with Efficiency in Deriving Cardiac Elements from Pluripotent Human Embryonic Stem Cells

“…Therefore, the pharmacologic utility of human cardiac stem cells cannot be satisfied only by their chaperone activity, if any, to differentiate into nonfunctional smooth muscle cells or endothelial cells, or produce trophic or protective molecules to rescue endogenous dying cardiomyocytes that can simply be achieved by a compound drug of molecular entity. Although a vast sum of government and private funding has been spent on looking for adult alternates, such as reprogramming and trans-differentiation of fibroblasts or mature tissues, so far, only human cardiac stem/precursor/progenitor cells derived from embryooriginated hESCs have shown such cellular pharmacologic utility and capacity adequate for myocardium regeneration in pharmaceutical development of stem cell therapy for the damaged heart [1][2][3][4][5][6].…”

“…The intrinsic ability of a hESC to both unlimited self-renew and differentiation into cardiac elements makes it a practically inexhaustible source of replacement cells for the damaged heart. The hESC cardiac cell therapy derivatives are emerging as a new type of pharmacologic agent of cellular entity in cellbased regenerative medicine because they have direct pharmacologic utility and capacity for human myocardial tissue reconstitution and contractile function restoration that the conventional compound drug of molecular entity lacks [1][2][3][4][5][6]. The pharmacologic activity of human cardiac stem cells is measured by their extraordinary cellular ability to regenerate the functional and structural cardiac tissue element, thus, the contractile cardiomyocytes that has been damaged or lost.…”

“…Resolving minimal essential human requirements for sustaining embryonic pluripotence allows all poorly-characterized and unspecified biological components and substrates in the culture system, including those derived from animals, to be removed, substituted, and optimized with defined human alternatives for de novo derivation and long-term maintenance of GMP-quality xeno-free stable hESC lines and their human cell therapy derivatives [1,5]. Formulation of minimal essential defined conditions renders pluripotent hESCs be directly and uniformly converted into a specific neural or cardiac lineage by small signal molecule induction [1][2][3][4][5][6][7][8][9][10][11][12]. Such milestone advances and medical innovations in hESC research enable high efficient generation of a large supply of high purity clinical-grade hESC neuronal and heart muscle cell therapy products as powerful cellular medicines that offer adequate pharmacologic utility and capacity for CNS and heart regeneration in the clinical setting that no conventional drug of molecular entity can.…”

“…Recent advances and technology breakthroughs in hESC research have overcome some major obstacles in bringing hESC therapy derivatives towards clinical applications, including resolving minimal essential human requirements for de novo derivation of clinically-suitable stable hESC lines and direct conversion of such pluripotent hESCs into a large supply of clinical-grade functional human neuronal or cardiomyocyte cell therapy products to be translated to patients for CNS or heart repair [1,2,[4][5][6][7][8][9][10][11][12]. Without an understanding of the essential developmental components for sustaining hESC pluripotence and selfrenewal, hESC lines are at risk for becoming unhealthy and unstable after prolonged culturing under animal feeders, feeder-conditioned C a rd iovascu la r P h ar macol o g y : O pe n Acc es s media, or artificially-formulated chemically-defined conditions [1,5,13].…”