Scholarly article on topic 'Cardiomyocyte generation from somatic sources — current status and future directions'

Cardiomyocyte generation from somatic sources — current status and future directions Academic research paper on "Basic medicine"

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Current Opinion in Biotechnology
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Abstract of research paper on Basic medicine, author of scientific article — Michael G Monaghan, Monika Holeiter, Shannon L Layland, Katja Schenke-Layland

Transdifferentiation of one cell type to another has garnered significant research efforts in recent years. As cardiomyocyte loss following myocardial infarction becomes debilitating for cardiac patients, the option of an autologous source of cardiomyocytes not derived from multi/pluripotent stem cell sources is an attractive option. Such direct programming has been clearly realized with the use of transcription factors, microRNAs and more recently small molecule delivery to enhance epigenetic modifications, all albeit with low efficiencies in vitro. In this review, we aim to present a brief overview of the current in vitro and in vivo transdifferentiation strategies in the generation of cardiomyocytes from somatic sources. The interdisciplinary fields of tissue, cell, material and regenerative engineering offer many opportunities to synergistically achieve directly programmed cardiac tissue in vitro and enhance transdifferentiation in vivo. This review aims to present a concise outlook on this topic with these fields in mind.

Academic research paper on topic "Cardiomyocyte generation from somatic sources — current status and future directions"

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Current Opinion in


Cardiomyocyte generation from somatic sources — current status and future directions

Michael G Monaghan1, Monika Holeiter1, Shannon L Layland1'2 and Katja Schenke-Layland1,2,3


Transdifferentiation of one cell type to another has garnered significant research efforts in recent years. As cardiomyocyte loss following myocardial infarction becomes debilitating for cardiac patients, the option of an autologous source of cardiomyocytes not derived from multi/pluripotent stem cell sources is an attractive option. Such direct programming has been clearly realized with the use of transcription factors, microRNAs and more recently small molecule delivery to enhance epigenetic modifications, all albeit with low efficiencies in vitro. In this review, we aim to present a brief overview of the current in vitro and in vivo transdifferentiation strategies in the generation of cardiomyocytes from somatic sources. The interdisciplinary fields of tissue, cell, material and regenerative engineering offer many opportunities to synergistically achieve directly programmed cardiac tissue in vitro and enhance transdifferentiation in vivo. This review aims to present a concise outlook on this topic with these fields in mind.


1 Department of Women's Health, Research Institute for Women's Health, Eberhard Karls University, Tübingen, Germany

2 Department of Cell and Tissue Engineering, Fraunhofer Institute for Interfacial Engineering and Biotechnology (IGB), Stuttgart, Germany

3 Department of Medicine/Cardiology, Cardiovascular Research Laboratories, University of California, Los Angeles, CA, USA

Corresponding author: Schenke-Layland, Katja (


Cardiovascular disease is one of the world's leading causes of mortality. Myocardial infarction (MI) is the death of heart tissue due to ischaemia, typically caused by the blockage of blood flow to an area in the heart. Resident cardiomyocytes have a very limited capacity to

proliferate in the adult heart, resulting in the lack of heart regeneration post-MI [1]. To date, the most efficient therapy for heart failure is whole organ transplantation, which is limited by donor hearts availability, compromised by immunosuppressant therapy and an invasive procedure not suitable for all patients.

Cell therapies have been ofinterest to researchers due to their variety of cell sources, the ability to scale-up in vitro and their potential to improve the regeneration oftissue. This has evolved from research with autologous stem cell sources — bone marrow-derived stem cells, adipose tissue-derived stem and progenitor cells, all of which have reached clinical trials [2]. Recent approaches to induce a pluripotent state in various adult somatic cells, termed induced-pluripotent stem cells (iPSCs), has resulted in exciting work towards clinical therapy and disease modelling [3**]; however, returning cells to a pluripotent state raises concerns of teratoma formation and possible unwarranted differentiation [4]. Prompted by the advent of iPSCs [3**], the concept of a direct transition from one determined cell type into another (transdifferentiation) by overexpressing transcription factors, microRNAs (miRs) and/or delivering small molecules has emerged [5**,6**,7**]. Almost 30 years ago, myogenic features in fibroblasts were being driven by introducing the expression of the muscle-specific transcription factor MyoD [8]. This direct conversion was achieved by epigenetic suppression of the fibroblast phenotype and progressive activation of the target cell via cDNA transfections. Transdifferentiation has since been reported for cell types such as pancreatic beta cells [9], neurons [10], hepatocyte-like cells [11], and haematopoietic progenitor cells [12]. Inducing functional car-diomyocytes (iCMs) directly from fibroblasts was first reported with murine cells in 2010 [5**]. Since then, substantial efforts have been applied to increase transdifferentiation efficiencies [13**]. Gradually, the incorporation of additional stimuli such as dynamic cultures, mechanical, topographical and extracellular matrix (ECM) cues, along with other lessons learned from stem cell and iPSC differentiation is slowly impacting the direct reprogramming protocols with increased efficiencies. In this review, we aim to discuss the important developments in the transdifferentiation of fibroblasts to iCMs in vitro and in vivo with the goal of highlighting developments in the field of tissue engineering and biomaterials design that could realize exciting accomplishments in this field.

Current Opinion in Biotechnology 2016, 40:49-55

This review comes from a themed issue on Tissue, cell and pathway engineering

Edited by April Kloxin and Kyongbum Lee

0958-1669/© 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://

Driving transdifferentiation using cardiac transcription factors

Typically, cardiac fibroblasts maintain the structural and paracrine sustenance of adjacent cardiomyocytes. However, activation of these fibroblasts occurs after MI and subsequently they migrate to the site of injury and synthesize fibrotic ECM as a compensatory structure for the compromised myocardium [1]. The abundance of cardiac fibroblasts in the injured heart intuitively highlights them as a target for reprogramming, whereby they could offer as a source for cardiac regeneration. Cardiac fibroblasts and cardiomyocytes, in theory, should share many epigenetic features as they both derive from a common progenitor cell population [14]. The significance of the originating cell type and its natural environment has been reported in myogenic [15] and pancreatic beta cell reprogramming [9]. In both cases, somatic cells originating from different germ layers to that of the envisaged cell type failed to yield successful transdifferentiation.

The most documented and the first factors to derive iCMs are the transcription factors Gata4, Mef2c and Tbx5 (GMT). Since their initial reporting [5**], many reprogramming cocktails have been tested, most of them virally delivered and based on the original combination of GMT but with additional factors (Mesp1, Hand1, Hand2, Nkx2.5, myocardin (Myocd), Smarcd3 or SRF) to improve reprogramming efficiencies [16**,17—19] (see Table 1 for an overview). G, M, and T are the prevailing regulators at the peak of the cardiac gene regulatory networks and their expression during normal development follows a delicate pattern [20**]. It is reported that GMT alone is inefficient to produce functional iCMs but results in a partially reprogrammed phenotype expressing transcripts such as cardiac TroponinT but not alpha myosin heavy chain (a-MHC) [21*]. Combining Myocd with Tbx5 and Mef2c to treat neonatal cardiac fibroblasts has resulted in a 2.5% yield of a-MHC-expressing cells 14 days post-transduction (GMT alone achieved 2.2%); however, complete transdifferentiation in the form of beating cells after four weeks was not obtained [22].

It is also reported that a fine balance of the GMT factors is required to accomplish more efficient transdifferentiation [20**,23]. Essentially, a high Mef2c protein level and lower expression level of Gata4 and Tbx5 transpired to be key in yielding iCMs in fibroblasts transduced by a polycistronic vector [20**]. Stoichiometry of the factors has also been found to have an effect through non-viral mRNA delivery [23]. Such a sensitive equilibrium may be one reason why GMT has yielded poor efficiency in other researchers' investigations. Repression of Snai1 has been implicated as an enhancer of GMT transdifferentiation as Snai1 is capable of inducing mesenchymal behaviour and fibrogen-esis during development and disease. Knocking down Snai1-expression with siRNA during GMT transduction

of MEFs significantly increased the reprogramming efficiency compared to GMT alone [24]. In contrast, over-expressing Snai1 during transdifferentiation inhibited cardiac gene expression and spontaneous beating. Other researchers have noted a fivefold improvement of iCM induction has been achieved via inhibition of TGF-b using SB431542 with transfection of GMT + Hand2 + Nkx2.5 [25]. TGF-b acts as an activator of Snai1. Therefore both studies establish that the repression of Snai 1 is important to stop the maintenance of the fibroblast phenotype. Additionally, a more recent study found that although GMT and Hand2 transdifferentiated fibroblasts into beating cells expressing cardiac markers (5%), genes associated with fibrosis were also upregulated in the first week of culture [26**]. On the basis of the hypothesis that fibrotic signalling was hindering transdifferentiation, small molecules to silence TGF-b and Rho associated kinase signalling yielded an efficiency of 60% functional cardiomyocytes from mouse embryonic fibroblasts [26**].

microRNA mediated transdifferentiation

The role of miRs and the disruption of their endogenous levels and cell-specific functions following MI are well reported [27]. The regulatory role of miRs in the suppression of mRNA translation plays an important role in cell fate decisions, which can have a knock-on/off effect on the presence of transcription factors and other stimulatory factors. Jayawardena et al. were the first who identified a cocktail of miRs (miR-1, -133, -208, -499) that seemed to preferably transdifferentiate fibroblasts into iCMs [6**]. Within this study; cardiac protein expression, rhythmic calcium oscillations and beating clusters were observed in about 1-2% of the cell population [6**]. Notably, the introductory method of the miRs in this study (non-viral delivery of mature miR mimics) necessitated a single transient transfection.

Muraoka et al. investigated the effect of miR-1, -133,-208, and -499 on mouse embryonic fibroblasts (MEFs) isolated from a-MHC promoter-driven eGFP transgenic mice in generating iCMs [24]. This study was not successful in generating iCMs using this defined cocktail of miRs. However, combining GMT viral delivery with just miR-133 (non-viral mature miR mimic) resulted in significantly enhanced transdifferentiation efficiencies in murine and human fibroblasts [24]. When investigating the cardiomyocyte subtype they observed mostly iCMs of an atrial phenotype. Interestingly, the study detected beating events in GMT+ miR-133 transduced MEFs as early as day 10 post-induction; whereas cells treated with GMT alone did not exhibit beating cells until four weeks post-induction.

Another approach in converting fibroblasts to iCMs is the combination of transiently overexpressing factors generally recognized for iPSC generation, with culture conditions and factors specific to cardiac differentiation, but

obviating a pluripotent state. Efe et al. retrovirally transduced MEFs with Oct4, Sox2, Klf4 (OSK) and cultured under defined conditions (LIF-free cardiomyogenic media) using small molecules and growth factors [28*] and induced spontaneously contracting patches of cardiac cells. This study found that small molecule inhibition of JAK-STAT (Janus kinase-signal transducer and activator of transcription) during the initial nine day period and supplementation of BMP4 from day nine gave more beating cells. However, regardless of the culture time, the expression of late stage markers (Mlc-2a) suggested that the generated iCMs were of an atrial subtype. As early as 11 days after transduction, spontaneous contractions were observed and many colonies were beating by day 15. The authors speculated that pluripotency reprogramming factors (especially Oct4) initially remove the cell's identity but epigenetic mechanisms, and soluble factors in a staged protocol of differentiation media are then capable of inducing the desired cell type. More recently, this group demonstrated combining Oct4 [29] with a small molecule cocktail consisting of SB431542 (ALK4/5/7 inhibitor), CHIR99021 (GSK3 inhibitor), par-nate (LSD1/KDM1 inhibitor), and forskolin (adenylyl cyclase activator) collectively known as SCPF [29], was sufficient to wipe the fibroblast epigenetic memory, thus enabling improved cell transdifferentiation with cardio-myogenic signals (small molecules and growth factors). In this case, BMP4 was added from day 6 after transduction to induce a cardiomyocyte phenotype. The group observed contracting clusters from day 20 and generated 99 ± 17 beating foci on day 30 after 1 x 104 MEFs were initially plated. Most of the derived cells indicated a ventricular subtype with hardly any displaying atrial or nodal features.

Chemically achieved transdifferentiation

Suppression of the starting cell epigenetic signature is paramount to overcoming one major molecular roadblock for successful transdifferentiation; namely the shutdown of the fibroblast program, before an adoption of the desired cell fate becomes possible. Cells not only undergo tran-scriptional changes but also exhibit epigenetic changes in DNA methylation and histone modifications [30,31], and it are primarily these changes that convert the epigenetic pattern ofsomatic cells to an embryonic stem cell-like state. Several small molecules that block and inhibit enzymes involved in epigenetic modifications, including histone methylation or demethylation, can increase the efficiency of transdifferentiation and can sometimes functionally replace ectopic expression of certain transcription factors. Routinely, G9a-mediated H3K9 methylation is necessary for heterochromatinization and silencing of key pluripo-tency genes, such as Oct4 and Rex1 during early embryogenesis [32]. DNA methyltransferase inhibitor, 5-azacytidine, or histone deacetylase inhibitors (suberoyla-nilide hydroxamic acid, trichostatin A and valproic acid) improved reprogramming efficiency after transduction of

the four iPSC transcription factors in MEFs [33]. A cell's epigenetic memory can be essentially erased by treating established iPSCs with 5-azacytidine and trichostatin A [33]. The use of small molecule compounds in cell transdifferentiation, which could be better accepted for clinical translation, has recently been highlighted with the complete generation of iPSCs and neural progenitor cells via small molecules [34-36]. More recently, transdifferentiat-ing MEFs into cardiomyocytes (sometimes beating) using chemically defined cocktails has been achieved with a transition via a cardiac progenitor cell stage but not that of a pluripotent stage [7**]. Yet still, the induction efficiencies of iCMs using this, and other methods in vitro remain disappointingly low.

In vivo efforts

Interestingly, in vivo approaches of direct cardiac reprogramming applied after experimental MI in mice obtain higher efficiencies than in vitro approaches. Considering that the fibrotic scar is primarily composed of ECM-producing fibroblasts, this is indeed promising. Qian et al. [37**] and Song et al. [16**] have both used genetic lineage tracing to ascertain that in mouse infarcted hearts, transdifferentiation of non-myocytes into functional iCMs occurred. Both studies document improved functional recovery and reduced fibrotic scar tissue. Since then, other improvements to in vivo GMT transdifferentiation have been made with respect to the delivery vector [38,39] and preconditioning the myocardium with angiogenic factors [40,41]. miR-based transdifferentiation in vivo has also been reported by Jayawardena et al. whereby their initial study determined that 1% of the iCMs were of a fibroblast origin [6**] and a more recent study of the therapeutic effect of this treatment found progressive improvement in cardiac function. These conversion rates in vivo (1-35%) are encouraging; however, to generate disease-in-a-dish models and in vitro iCM yields suitable for transplantation, increased in vitro efficiencies are required to achieve large-scale cultures.

The influenoe of ECM signalling

Many strategies have potential regarding transdifferentiation to generate iCMs in vitro from somatic sources and the direct reprogramming of resident cells in vivo (Figure 1). The ECM serves as an important component of all tissues, and its composition and mechanical properties play significant roles in the self-renewal or differentiation of cells. ECM composition and signalling in stem cell niches promotes the self-renewal of stem or progenitor cells and this knowledge has been utilized early on in embryonic stem cell culture for ESC maintenance in vitro using MEFs secreting ECM [42], ECM-based substrates such as Matrigel® [43], specific ECM proteins such as laminins, collagen type I, or vitronectin [44-46]. ECM proteins have also been utilized to guide stem cell differentiation to somatic cell types, including cardiomyo-cytes [47-50]. For instance, collagen type IV has been

Figure 1

Multimodal Delivery ECM Cue Enhancement

ECM/Synthetic Biomaterial

Multimodal Delivery ECM Cue Enhancement

Induced Cardiomyoctes

Mechanical/Dynamic Stimulation Mimicking In Vivo Conditiions

Upscaling Production Biophysical Cues

2D/3D In Vitro Test System


In Vitro Transdifferentitation

Biomaterials/ Tissue Engineering

In Vivo Transdifferentiation

Current Opinion in Biotechnology

Direct reprogramming of fibroblasts to a functional cardiomyocyte with or without a progenitor cell intermediate has been widely reported with increasing efficiencies using transcription factors, miRs and small molecules. The evolution of these protocols will benefit greatly by the use of cardio-stimulatory environments with biomaterials, extracellular matrices and dynamic cultures based on lessons learned in vivo, which could yield significant efficiencies suitable for implantation. Additionally, using delivery vehicles of transdifferentiation factors that are based on biomaterial and extracellular matrices, which are favourable towards cardiomyogenesis could further improve direct reprogramming in vivo.

shown to increase the differentiation of mouse embryonic stem cells into cardiac progenitor cells (CPCs) while fibronectin can enhance CPC differentiation to cardio-myocytes [51]. Additionally, to reprogram somatic cells at least partially to multipotent cells, the use of embryonic stem cell extracts [52] or animal oocyte extracts [53,54] has been described. Zhang et al. induced multipotency in fibroblasts by extracellular delivery of the ECM component fibromodulin [55]. Interestingly, the multipotent cells differentiated into derivatives of all three germ layers including cardiomyocytes, skeletal myocytes, neurons, pancreatic lineage cells, osteoblasts, and adipocytes in vitro while omitting the risk of teratoma formation in vivo [55]. More delivery approaches become available when considering ECM-enhanced iCM generation as the ECM can also serve as a depot for growth factors, transcription factors and nucleic acid vectors (viruses and plasmid constructs) for gene therapy [56]. However, one

such factor alone is not enough and therefore payloads that can achieve sustained and programmed release of many molecules at the same, or at staged time intervals are paramount to the correct transdifferentiation of cells in vivo [57,58].

Incorporation of biomechanical cues

Research focused on the interplay between physical and developmental cues has demonstrated that mechanical forces generated by cells or tissues are crucial for the control of embryological development, morphogenesis and tissue patterning [59]. The importance of the mechanical properties of a cell's or tissue's microenvironment has been recognized by many in the field of tissue engineering [51,60]. This has resulted in the design of elaborate systems to mimic a native environment with defined mechanical cues of surface rigidity, stretch and strain. Some of these cues exist already in vivo, which

could be a strong justification as to why transdifferentiation is more successful in vivo. Ruan et al. have recently shown that cyclic mechanical stress in 3D in vitro cultures of ESC and hiPSC-derived CPCs favoured cardiac differentiation and promoted cardiomyocyte structural and functional maturation [61*]. It is difficult to recapitulate small molecule interventions in vivo, which suggests other epigenetic occurrences present in the myocardium. Again, such influences in vivo could be ECM signalling. Recently it was shown that topography plays an instrumental role in the epigenetic state of the cell whereby the study ofMorez et al. cultured adult heart-derived progenitor cells on microgrooves (10 mm wide, 3 mm deep) to enhance histone acetylation and cardiomyocyte differentiation [62*]. The growing range of functional biomaterials that can release drugs, proteins, growth factors and ECM components, or that display an improved mechanical functionality, is currently the focus of tissue engineering and regenerative medicine [63*]. A temporally and spatially controlled release of bioactive molecules from such functional biomaterials can be achieved through the combination of different mechanisms, like diffusion-based release, biomaterial-degradation, or cell-triggered release [64].


The generation of functional cardiac tissue in vitro by transdifferentiating somatic cell sources can only be truly realized and up-scaled by combining lessons learned from cardiomyocyte derivation from iPSCs or stem cells, whereby ECM biophysical cues and dynamic cultures have yielded more mature iCMs with higher efficiencies. This would enable the generation ofpatient-specific drug testing systems and personalized engineering of cardiac tissue in vitro.


The authors would like to thank Daniel Alejandro Carvajal Berrio (University Hospital Tubingen) for creating Figure 1. This work was financially supported by the European Union (AMCARE, 604531, FP7-NMP-2013-SME-7 to KS-L.; Marie Curie IEF, 331430 to MGM); Fraunhofer-Gesellschaft Internal programs (FFE to SLL), the Ministry of Science, Research and the Arts of Baden-Württemberg (33-729.55-3/214), and the Deutsche Forschungsgemeinschaft (SCHE 701/10-1) (all to KS-L).

Appendix A. Supplementary data

Supplementary material related to this article can be found, in the online version, at 1016/j.copbio.2016.02.014.

References and recommended reading

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