The possibility of generating pluripotent cells from adult individual has obviously created enormous expectations, and has the potential to revolutionize the field of regenerative medicine. Once obtained, iPSCs are just the starting point for autologous cell therapies, and subsequent differentiation is essential. In other words, the final aim for cell-based therapies is to obtain enough committed progenitors or fully differentiated cells at will. In that sense, there are three possible sources from where to obtain these cells: (1) terminal differentiation from iPSCs (pluripotent state) to the required somatic cell types (ground state) [108
]; (2) indirect lineage conversion from a somatic cell to a dedifferentiated activated state (progenitor-like state) that allows the commitment into several final cell lines in response to environmental signals and/or transcription factors [22
]; and (3) somatic cell direct linage conversion or trans
-differentiation avoiding a progenitor-like state [23
] (Fig. ).
Fig. 4 Pathways to generate specific differentiated lineages from specific somatic cells. Fully differentiated target cells can be induced by three conceptually separate mechanisms: reprogramming (1) by ectopic expression of Yamanaka factors to induce a pluripotent (more ...)
Forced expression of reprogramming factors induces a global dedifferentiation phenotype, which involves the removal of epigenetic marks and the reestablishment of the pluripotency network [117
]. Most differentiation protocols are inefficient, and derivation to several cell types is often complex. Cardiomyocyte differentiation of human iPSCs was first achieved by Zhang et al. who used the spontaneous embryoid-body based differentiation method [109
]. Some of the generated myocytes have been demonstrated to display molecular, structural, and functional properties of early human cardiomyocytes, showing different electrophysiological properties with ventricular-like, atrial-like, and nodal-like potential features [108
]. Human-derived cardiomyocyte are also able to display functional syncytium with stable pacemaker activity and synchronize action potential propagation [118
]. RNA-based tools are the most efficient and safest methods for iPSC generation [40
] (Fig. ), and it is likely that these techniques could contribute greatly to the development of new differentiation procedures. Thus, it could be possible that SeV vectors or modRNA transient transfection encoding differentiation factors could be used to reset the epigenetic marks of a given cell type. Improvements in the differentiation efficiency would have a great impact on bringing iPSC technology closer to clinical application.
Direct lineage conversion, which does not involve an activation state, depends on whether defined factors are able to override epigenetic marks and drive in trans
the establishment of the new target cell's genomic identity. Mouse fibroblasts, for instance, have been directly converted into cardiomyocytes by forced expression of cardiac-specific transcription factors Gata4
, and Tbx5
], or together with Hand2 [26
]. Furthermore, in vivo trans
expression of these genes in mouse heart fibroblasts after myocardial infarction can induce transdifferentiation into functional cardiomyocytes and improve heart function [25
]. These examples point to SeV tools as ideal for future use during direct lineage-conversion for several reasons. First, to overcome the existing epigenetic marks, strong and continuous expression of the trans
-differentiating factors are crucial in order to induce direct lineage conversion. As discussed above, SeV constructs can drive ectopic overexpression of defined factors in any cell type that expresses tubulin. Second, specific epigenetic marks determine the accessible sites to which the trans
-differentiating factors are able to transcribe from. Then, for a given combination of factors, the success in establishing a new determined network typical of a target cell will depend on matching genome accessibility of the original cell type. Hence, finding the best cellular source in combination with specific factors to directly convert one cell type into another could be challenging, although in the cardiac setting, heart-resident fibroblasts appear as the best substrate. As a typical recombinant viral vector, SeV is able to deliver transgenes more efficiently than a nonviral system. Its exceptionally broad host range gives SeV system a significant advantage over other methods. Since direct lineage-conversion occurs in the absence of a pluripotent state and generates post-mitotic populations, it could also theoretically reduce the risk of uncontrolled post-transplantation cell proliferation.
Indirect lineage conversion requires an activation state that leads to the generation of cellular intermediates, in which epigenetic marks get re-written [21
]. In this case, activated cells acquire a precursor-like phenotype with multipotent differentiation capacity. This is important, as generation of progenitor cells with such capabilities will expand applications in regenerative medicine, specifically in cases where progenitor transplantation might be an advantage over fully differentiated cells. Recent reports have demonstrated that short temporal expression of pluripotency factors was enough to induce a partially de-differentiated state suitable for conversion into specific cell types by extracellular developmental signals [113
]. More striking was the fact that cells in a more pluripotent stage diminished their lineage conversion efficiency, like it has been shown for mouse cardiomyocyte differentiation [119
]. These data indicate that the process itself must be fine-tuned in order to achieve partial reprogramming and start a differentiation route on time to obtain the target cell of interest. Synthetic modRNA technology presents a number of characteristics that make it a potential powerful platform for this type of indirect linage conversion. These features include the fact that modRNA enables robust and dose-titrable translation of nearly any protein. Moreover, since modRNA combination of multiple transcripts can be transfected into cells at once, co-translation of several factors at desired stoichiometry is simply controlled by changing the dose of the relevant modRNAs [120
]. To our knowledge, no other reprogramming technology permits such control over reprogramming factor expression. Remarkably, the labile nature of modRNAs inside the cells, (its half-life of around 24 h was originally considered a serious handicap) has become a powerful characteristic, differentiating it from alternative reprogramming vectors. As a consequence, modRNA stands out as an ideal tool to temporally and quantitatively control the expression of any given combination of factors in order to redefine cellular fate.
It is also noteworthy that RNA-based reprogramming methodology could easily take advantage of synthetic biology for further technical development. As emphasized throughout this review, using DNA-free delivery techniques abolishes the risk of random genomic integration and opens up the opportunity to develop safe artificial tools for reprogramming and/or lineage conversion. A recent example has demonstrated that reprogramming could be enhanced using engineered variants of Oct4
fused to N-terminal MyoD
transactivation domain [121
]. It is known that ectopic expression of MyoD
is able to direct the fate of iPSCs towards a myogenic fate [122
], and is also able to induce trans
-differentiation. Hence, this synthetic transcription factor maintains the powerful transactivation activity of MyoD
, without losing the target specificity of Oct4
. By modRNA transfection of this engineered factor chromatin accessibility and recruitment of chromatin remodeling proteins to the Oct4
site can be increased, resulting in a radical acceleration of iPSCs derivation [105
Taken together, it seems possible that RNA-based technologies for reprogramming and encoding lineage specification factors could emerge as important tools for generating diverse cell types, either by terminal differentiation from iPSCs, or by direct or indirect lineage conversion, for experimental and future therapeutic applications.