Small molecules and human cardiomyogenesis: Is there a bottleneck in current research?
Human pluripotent stem cell derived cardiomyocytes (hPSC-derived CMs) have a vast potential in drug discovery, disease modeling and regenerative medicine. In recent years various differentiation protocols for hPSC-derived CMs have been developed. Most of them utilize the modulation of human cardiomyogenesis via small-molecule compounds. However, setbacks to the large-scale application of hPSC-derived CMs still abound: insufficient insight into important signaling pathways for cardiac lineage-specific differentiation and identification of suitable small-molecule modulators; inconsistent results due to unstandardised culturing techniques; lack of effective maturation of hPSC-derived CMs in vitro. So is there a bottleneck in current research? This paper attempts to answer this question.
Citation: Trifonov D. Small molecules and human cardiomyogenesis: Is there a bottleneck in current research? Biodiscovery 2015; 15: 2; DOI: 10.7750/BioDiscovery.2015.15.2
Copyright: © 2015 Trifonov D. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, provided the original authors and source are credited.
Received: 14 March 2015; Accepted: 30 March 2015; Available online /Published: 31 March 2015
Keywords: human pluripotent stem cells;cardiomyogenesis; stem cell-derived cardiomyocytes; differentiation; small molecules.
Abbreviations: BMP – bone morphogenic protein;CM – cardiomyocyte; EGFP – enhanced greenfluorescent protein;hPSC – human pluripotent stem cell; hPSC-derived CMs – human pluripotent stem cell-derived cardiomyocytes;HCS – high-content screening; HTS – high-throughput screening; KDR– kinase insert domain receptor; mTOR –mammalian target of rapamycin;PDGFR-α–platelet-derived growth factor receptor; SAR – structure-activity relationship; TGF-β–tumor growth factor β.
Corresponding Author: Dimitar Trifonov, E-mail: email@example.com.
Conflict of Interests: No potential conflict of interest was disclosed.
Recent advances in the development of novel or improved differentiation protocols for hPSC-derived CMs [7, 17] have only been made thanks to the knowledge gained by years of ongoing research focused on cardiomyogenic small-molecule compounds [4, 5, 11, 13, 15, 21]. Nevertheless, there are some researchers who point out several bottlenecks in current research [4, 8, 9, 13, 15, 22].
Bottleneck 1: Compound screening, available testbeds and druggable targets
In recent years there is an ongoing debate about how small-molecule compounds should be screened for cardiomyogenic properties [4, 8, 11, 12]. Small molecule screening techniques, application of various stem cell types as testbeds for identification of cardiomyogenic potential and validation of druggable targets are among the most commonly discussed topics.
Compound screening. Over the years both synthetic and natural small-molecule compounds have been identified as inducers of cardyomyogenesis [4, 5, 22, 23]. A classical method to investigate the cardiomyogenic potential of discrete, well-characterized chemical entities in pluripotent stem cells is the high-throughput screening (HTS) [5, 23]. In brief, this methodology allows the analysis of large amounts of data collected in a parallel fashion. It is typically achieved with the aid of fluorescent, luminescent or color label/s that mark the molecule/s of interest. The signal is read out by a detector/s that may record and interpret many datapoints at once, e.g. a plate reader. In terms of cell-based HTS, labelled reporter proteins are commonly used. Usually, GFP is a staple label in stem cell research, and studies of differentiation into the cardiogenic lineage are no exception . A variety of cardiomyogenic small molecules like ascorbic acid and classes of compounds like cardiogenols andsulfonyl-hydrazones have been discovered using HTS [5, 23]. However, despite the speed and simplicity of assay development  HTS is not particularly suitable for the identification of potent cardiomyogenesis-inducing molecules, as procardiogenic effects of such compounds could vary depending on culturing conditions . Another caveat is the often inconsistent production of late-stage cardiac mesoderm progenitors needed for the development of reliable assays . Therefore, the discovery of small molecules with procardiogenic potential via high-content screening (HCS) has been recently advocated [4, 13]. This methodology is based on the selection of cells which have a signal intensity corresponding to the content of a specific substance or metabolite measured by automated microscope imaging of reporter proteins [4, 13]. Advanced approaches to HCS also include flow cytometric analysis (e.g. counting differentiated versus undifferentiated cells) and, potentially, cell sorting, allowing for the identification of cellular subset sub-populations . In comparison to HTS, HCS is a more complex type of assay. It is more time-consuming and may not allow the screening of many compounds simultaneously [4, 13]. On the other hand, imaging analysis inherent to HCS enables morphological analysis as well . However, it is still to be decided among biomedical researchers whether one assay type should be looked upon more favorably than the other.
Available testbeds. Nowadays biomedical researchers have access to various stem cell lines which can be used to produce stem-cell-derived cardiomyocytes for applications like drug discovery and regenerative medicine or to probe the signaling pathways behind cardiomyogenesis. However, the need to choose one characterized cell line from a pool of available stem cell types (e.g. murine or human; embryonic or induced pluripotent) can be rather daunting and results obtained in animal cell lines may not be translatable to human cell lines. For example, the cardiomyogenic effect of ascorbic acid was first discovered in stably transfected EGFP-mouse embryonic stem cells back in 2003 , but in mouse induced pluripotent stem cells the yield of cardiomyocytes using ascorbic acid has recently been reported to be inconsistent . Therefore, pre-existing data may indicate that ascorbic acid ought to promote cardiomyogenesis in mouse induced pluripotent stem cells, but that statement remains purely speculative, as the results apparently do not support this hypothesis. In this regard, multi-testbed screening of compounds can be a great time-saver as speculative assumptions will be quickly disproven by experimental data. Moreover, the use of both murine and human stem cell lines in the same experiment promises a faster generation of data about the stem cell biology of the two species. There have been reports that despite the apparent closeness of the two species, there are some essential differences on molecular level that show greatly in their capacity for maintenance of the undifferentiated state and targeting into differentiation into different cell types . The report of Kattmanet et al.  about the stage-specific optimization of Activin/Nodal and BMP signaling promoting cardiac differentiation of mouse and human pluripotent stem cell lines is an excellent example of multi-testbed-based research. Their results show that the formation of cardiac mesoderm in both mouse and human pluripotent stem cells can be monitored by co-expression of KDR and PDGFR-α. Additionally, it was found that efficient cardiac differentiation was dependent on optimal levels of Activin/Nodal and BMP signaling in all tested stem cell lines. In a nutshell, translational research like that of Kattmanet et al. has the potential to “revolutionize” our understanding not only of just cardiomyogenesis, but of stem cell biology as a whole.
Druggable targets. The discovery of new druggable targets influencing cardiac differentiation is often coupled with breakthroughs in small molecule HTS. However, the opposite may also be true . Interestingly, the biological effect of several small molecules involves the inhibition rather than activation of specific signaling pathways [22, 27]. In this regard one of the most studied and well-known molecular pathways is Wnt signaling [11, 21, 28, 29]. Up-to-date several cardiomyogenic Wnt inhibitors have been discovered and are currently being used in differentiation protocols [6, 16]. The success story of Wnt inhibitor mediated cardiac differentiation [7, 29] has tempted biomedical researchers to overlook other druggable targets. However, in recent years the interest toward previously poorly studied signaling pathways like MAPK has increased [30, 31]. Other druggable targets under investigation include: microRNAs [32, 33]; mTOR signaling pathway ; TGF-β signaling pathway ; Neuregulin/ErbB signaling pathway ; retinoid signaling . Additionally, the regulation of cardiac differentiation in stem cell via metabolic oxidation is also of key research interest [38, 39] as well as the role of mitochondria . Furthermore, the interplay between small-molecule compounds and signaling molecules has been found to have a profound effect on cell type specification [6, 41]. Therefore the structure-activity relationship (SAR) and pharmacological properties (e.g. selectivity, solubility etc.) of a tested compound are key to its’ effectiveness as an inducer of cardiac development in pluripotent stem cells. In this regard, molecular analogues of some previously tested small molecules have also been found to possess cardiomyogenic potential . Moreover, some intriguing compounds like angiotensin II  and ghrelin (28-amino-acid peptide)  have recently been identified as Cardiomyogenic. All in all, druggable targets and small molecules are only parts of the puzzle. Only by applying our knowledge of both medicinal chemistry and stem cell biology can we solve the puzzle: a reliable method to obtain hPSC-derived CMs on a large scale. Something that is hardly possible in the present!
Bottleneck 2: Cardiomyocyte maturation in vitro
A common setback of most currently used differentiation protocols for hPSC-derived CMstoday is the insufficient cell culture maturation in vitro [11, 15-17]. In this regard Robertson et al.  propose that hPSC-derived CM cultures be defined as ‘early-phase’ or ‘late-phase’ based on specific a sets of biomarkers, e.g. membrane ion channels and sarcomeric organization. Early-phase hPSC-derived CM culture is defined by Robertson et al. as contractile cells possessing some proliferative capacity and electrophysiological properties similar to the properties of embryonic cells, whereas the late-phase culture is characterized by loss of proliferative capacity and electrophysiological properties similar to those of adult cells . The group of Robertson advocates the need for development of novel culturing methods. However, despite the recent advancement in chemically defined culture media  or improvements in differentiation protocols , the researchers still face many challenges, such as: mainly obtaining adult-like cardiomyocyte cells with morphology and electrophysiology typical of mature cardiomyocytes . There is also the problem of standardization. Without a robust universally accepted method for evaluation of the identity of cardiomyocyte-like cells the analysis of the pros and cons of different cardiomyogenesis protocols in terms of efficiency is difficult .
Presently we face more than one bottlenecks in development of potential applications of small molecule-mediated cardiomyogenesis in hPSCs for large scale production of human CMs for the purposes of drug discovery, safety pharmacology, disease modeling and regenerative medicine. Firstly, the spacial-temporal complexity of cardiomyogenesis in human stem cells significantly slows down the identification of differentiation pathways and differentiation modulating molecules, that may successfully be manipulated used in a large-scale production. In addition, the cost of technology transfer alone may prove to be prohibitive for many currently developed differentiation protocols. Secondly, without the methodology for reliable assessment of hPSC-derived CMs for adult-like morphological and electrophysiologyical properties, their potential use is questionable, especially in the field of regenerative and reparative medicine. It could be expected that those problems could be solved using an interdisciplinary approach to human cardiomyocyte differentiation complemented by tissue engineering.
- Trifonov D, Tummala H, Clements S, Zhelev N. Effect of roscovitine on cardiac hypertrophy in human stem cell derived cardiomyocytes. Curr. Opin. Biotechnol. 2013; 24(1): S114-S115.
- Chow MZ, Boheler KR, Li RA. Human pluripotent stem cell-derived cardiomyocytes for heart regeneration, drug discovery and disease modeling: from the genetic, epigenetic, and tissue modeling perspectives. Stem Cell Research 2013; 4: 97.
- Acimovic I, Vilotic A, Pesl M,Lacampagne A, Dvorak P, Rotrekl Vet al. Human Pluripotent Stem Cell-Derived Cardiomyocytes as Research and Therapeutic Tools. BioMed Research International 2014; Article ID 512831.
- Willems E, Bushway PJ, Mercola M. Natural and synthetic regulators of human embryonic stem cell cardiomyogenesis. Pediatr Cardiol. 2009; 30(5): 635–642.
- Hao J, Sawyer DB, Hatzopoulos AK, Hong CC. Recent progress on chemical biology of pluripotent stem cell self-renewal, reprogramming and cardiomyogenesis. Rec Pat Regen Med. 2011; 1(3): 263–274.
- Zhelev N., Tummala H., Trifonov D., D’ Ascanio I., Oluwaseun O.A., Fischer P.M. Recent advances in the development of cyclin-dependent kinase inhibitors as new therapeutics in oncology and cardiology. Curr Opin Biotech 2013; 24(1): S25
- Karakikes I, Senyei GD, Hansen J, Kong CW, Azeloglu EU, Stillitano F et al. Small molecule-mediated directed differentiation of human embryonic stem cells toward ventricular cardiomyocytes. Stem Cells Translational Medicine 2014; 3: 18-31.
- Young DA, DeQuach JA, Christman KL. Human cardiomyogenesis and the need for systems biology analysis. Wiley Interdiscip Rev Syst Biol Med. 2011; 3(6): 666–680.
- Redig JK, Adler E. Doing the dirty work: progress in the search for a reliable protocol for cardiomyogenesis. Stem Cell Research & Therapy 2011; 2: 35.
- Mummery CL, Zhang J, Ng ES, Elliott DA, Elefanty AG, Kamp TJ. Differentiation of human embryonic stem cells and induced pluripotent stem cell to cardiomyocytes: a methods overview. Circ Res. 2012; 111: 344-358.
- Chakarov S, Petkova R, Pankov R. Stem cell biology. In: Stem cells. 2nd Edition. Prof. Marin Drinov Publishing House of the Bulgarian Academy of Sciences, 2014, 39-137.
- Arabadjiev A, Petkova R, Chakarov S, Pankov R, Zhelev N. We heart cultured hearts. A comparative review of methodologies for targeted differentiation and maintenance of cardiomyocytes derived from pluripotent and multipotent stem cells. Biodiscovery 2014; 14: 2.
- Willems E, Lanier M, Forte E, Lo F, Cashman J, Mercola M. A chemical biology approach to myocardial regeneration. J.Cardiovasc.Transl. Res. 2011; 4(3): 340–350.
- Forough R. Scarcello C, Perkins M. Cardiac Biomarkers: a Focus on Cardiac Regeneration. J TehUniv Heart Ctr 2011; 6(4): 179-186.
- Robertson C, Tran DD, George SC. Concise review: Maturation phases of human pluripotent stem cell-derived cardiomyocytes. Stem Cells 2013; 31: 829–837.
- Sepac A, Si-Tayeb K, Sedlic F, Barrett S, Canfield S, Duncan SA et al. Comparison of cardiomyogenic potential among fuman ESC and iPSC lines. Cell Transplant. 2012; 21(11): 2523–2530.
- Burridge PW,Matsa E, Shukla P, Lin ZC, Churko JM, Ebert AD et al. Chemically defined generation of human cardiomyocytes. Nature Methods 2014; 11: 855–860.
- Chakarov S, Petkova R, Russev GCh. Individual capacity for detoxification of genotoxic compounds and repair of DNA damage. Commonly used methods for assessment of capacity for DNA repair. Biodiscovery 2014; 11: 2.
- Chakarov S, Petkova R, Russev GCh, Zhelev N. DNA repair and carcinogenesis. Biodiscovery 2014; 12: 1.
- Arabadjiev B, Petkova R, Chakarov S, Momchilova A, Pankov R. (2010) Do we need more human embryonic stem cell lines? Biotechnol. Biotechnol. Eq. 2010; 24(3): 1921-1927.
- Blauwkamp TA, Nigam S, Ardehali R, Weissman IL, Nusse R. Endogenous Wnt signalling in human embryonic stem cells generates an equilibrium of distinct lineage-specified progenitors.Nat. Commun. 2012; 3: 1070.
- Atkinson SP, Lako M, Armstrong L. Potential for pharmacological manipulation of human embryonic stem cells. Br J Pharmacol. 2013; 169(2): 269–289.
- Hao J, Zhou L, Hong CC. Chemical Biology of Pluripotent Stem Cells: Focus on Cardiomyogenesis. Atwood C, editor. Embryonic Stem Cells – Recent Advances in Pluripotent Stem Cell-Based RegenerativeMedicine. Rijeka: InTech Europe, 2011; p. 51-64.
- Skalova S., Svadlakova T., Shaikh Qureshi WM., Dev K. Mokry J. Induced pluripotent stem cells and their use in cardiac and neural regenerative medicine. Int J Mol Sci. 2015; 16(2): 4043-67
- Petkova R., Arabadjiev A., Chakarov S., Pankov R.. Current state of the opportunities for derivation of germ-like cells from pluripotent stem cells – are you a man, or a mouse? Biotechnol.Biotechnol Eq. 2014; 28(2): 184-191.
- Kattman SJ, Witty AD, Gagliardi M, Dubois NC, Niapour M, Hotta A et al. Stage-specific optimization of Activin/Nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Cell Stem Cell 2011; 8: 228-240.
- Tabar V, Studer L. Pluripotent stem cells in regenerative medicine: challenges and recent progress. Nat. Rev. Genet. 2014; 15: 82-92.
- Gessert S, Kühl M. The multiple phases and faces of Wnt signaling during cardiac differentiation and development. Circ Res. 2010; 107: 186-199.
- Lian X, Hsiao C, Wilson G, Zhu K, Hazeltine LB, Azarin SM et al.Robust cardiomyocyte differentiation from humanpluripotent stem cells via temporal modulation ofcanonical Wnt signaling. PNAS 2012; E1848- E1857.
- Gaur M, Ritner C, Sievers R, Pedersen A, Prasad M, Bernstein HS et al. Timed inhibition of p38MAPK directs accelerated differentiation ofhuman embryonic stem cells into cardiomyocytes. Cytotherapy 2010; 12(6): 807–817.
- Yeghiazarians Y, Gaur M, Zhang Y, Sievers R, Ritner C, Prasad M et al. Myocardial improvement with human embryonic stem cellderivedcardiomyocytes enriched by p38MAPK inhibition. Cytotherapy 2012; 14(2): 223–231.
- Coppola A, Romito A, Borel C, Gehrig C, Gabnebin M, Falconnet E et al. Cardiomyogenesis is controlled by themiR-99a/let-7c cluster andepigenetic modifications. Stem Cell Research 2014; 12: 323-337.
- van Mil A, Doevendans PA, Sluijter JP. Letter by van Mil et al Regarding, ”Dynamic microRNA expression programs during cardiac differentiation of human embryonic stem cells: Role for miR-499”. Circ Cardiovasc Genet. 2011; 4: e3.
- Padmasekar M, Sharifpanah F, Finkensieper A, Wartenberg M, Sauer H. Stimulation of cardiomyogenesis of embryonic stem cells by nitric oxide downstream of AMP-activated protein kinase and mTOR signaling pathways. Stem Cells and development 2011; 20(12): 2163-2175.
- Willems E, Cabral-Teixeira J, Shade D, Cai W, Reeves P, Bushway PJ et al. Small molecule-mediated TGF-β Type II receptor degradation promotes cardiomyogenesis in embryonic stem cells. Cell Stem Cell 2012; 11: 242-252.
- Zhu WZ, Xie Y, Moyes KW, Gold JD, Askari B, Laflamme MA. Neuregulin/ErbB signaling regulates cardiac subtype specification in differentiating human embryonic stem cells. Circ Res. 2010; 107: 776-786.
- ZhangQ, JiangJ, HanP, YianQ, ZhangJ, ZhangXetal. Direct differentiation of atrial and ventricular myocytesfrom human embryonic stem cells by alternating retinoidsignals. Cell Research 2011; 21: 579-587.
- Yanes O, Clark J, Wong DM, Patti GJ, Sanchez-Ruiz A, Benton HP et al. Metabolic oxidation regulates embryonic stem celldifferentiation. Nat Chem Biol. 2010; 6(6): 411–417.
- JiAR, KuSY, ChoMS, KimYY, KimYJ, OSKetal. Reactive oxygen species enhance differentiation of humanembryonic stem cells into mesendodermal lineage. Experimental and Molecular Medicine 2010; 42(3): 175-186.
- Kasahara A, Sipolat S, Chen Y, Dorn II GW, Scoranno L. Mitochondrial Fusion Directs Cardiomyocyte Differentiation via Calcineurin and Notch Signaling. Science 2013; 342 (6159): 734-737.
- Kim MS, Horst A, Blinka S, Stamm K, Mahnke D, Schuman J et al. Activin-A and Bmp4 levels modulate cell type specification during CHIR-induced cardiomyogenesis. PLoS ONE 2015; 10(2): e0118670.
- Low JL, Jürjens G, Seayad J, Seow J, Ting S, Laco F et al. Tri-substituted imidazole analogues of SB203580 as inducers of cardiomyogenesis of human embryonic stem cells. Bioorg. Med. Chem. Lett. 2013; 23(11): 3300-3303.
- Wu L, Jia Z, Yan L, Wang W, Wang J, Zhang Y et al. Angiotensin II promotes cardiac differentiation of embryonic stem cells via angiotensin type 1 receptor. Differentiation 2013; 86(1-2): 23-29.
- Yang J, Liu GQ, Wei R, Hou WF, Gao MJ, Zhu M et al. Ghrelin promotes differentiation of human embryonicstem cells into cardiomyocytes. Acta Pharmacologica Sinica 2011; 32: 1239–1245.