Methods for increasing stemness and differentiation potential of pluripotent cells

Abstract

A method for enhancing stemness and differentiation potential of pluripotent cells. The present invention is based on the finding that increasing microRNA-203 levels in induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs) improves the quality of cell fate potential and the ability of these cells to differentiate into multiple cell lineages and achieve further maturation characteristics without disrupting their self-renewal properties. This effect is mediated through miR-203-dependent regulation of the de novo DNA methyltransferases Dnmt3a and Dnmt3b, which in turn regulates the methylation landscape of pluripotent cells. This effect can be achieved by overexpressing microRNA-203 or by adding microRNA-203 or its analogs to cell culture media and can be observed using a variety of cellular and in vivo models. The resulting cells are naive pluripotent cells with improved differentiation capacity, which can be used to obtain more efficiently differentiated and mature cells suitable for regenerative medicine strategies. [Selection diagram] None

Description

[Technical Field]

[0001] The present invention relates to methods for improving the potential of induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs), and more particularly to methods for increasing the differentiation potential of said cells. [Background technology]

[0002] Pluripotent stem cells offer significant promise for regenerative medicine due to their capacity for self-renewal and differentiation into multiple cell lineages.

[0003] Pluripotency can be defined as the ability at the single-cell level to generate multiple somatic lineages as well as germ cells. In preimplantation embryos, pluripotency is established in the epiblast of the late inner cell mass (ICM), which contains cells that can develop into all tissues except the placenta. These cells can be captured and maintained in culture as embryonic stem cells (ESCs). Both ICM cells and ESCs contribute to chimeras and, when reintroduced into embryos, can colonize the germ lineage, providing functional evidence of their naive pluripotency. In contrast, neither the postimplantation epiblast nor primed pluripotent stem cells derived from this tissue possess the ability to efficiently contribute to chimeras following incorporation into blastocysts. It is therefore well established that two distinct pluripotent states can be observed in pluripotent cells from mice: a ground or naive state exemplified by mouse embryonic stem cells (mESCs) and a primed pluripotent state represented by mouse postimplantation epiblast stem cells (mEpiSCs). The most obvious differences between the two states are colony morphology (compact, dome-shaped in the case of naive cells, flattened in the case of primed cells), growth factor requirements for maintenance of the pluripotent state (primed cells depend on activin / FGF2, whereas naive cells depend on LIF), and contribution to chimeras and germline transmission (naive cells can contribute to chimeras, whereas primed cells never can). Naive pluripotency is then lost in the embryo upon somatic differentiation and can only be experimentally restored by reprogramming strategies.

[0004] Reprogramming is the process by which mature cells are converted into cells that are in an embryonic stem cell-like state. Complete reprogramming of cells is closely related to, or can be considered synonymous with, improved functionality and naive pluripotency.

[0005] The original protocol for reprogramming somatic cells into induced pluripotent cells (iPSCs) was established in 2006 by Yamanaka and coworkers (Takahashi & Yamanaka, 2006), and is therefore commonly known as the Yamanaka original protocol. It was disclosed in European Patent EP10970446. It allows differentiated somatic cells to be converted to a pluripotent state. The original method is based on introducing certain genes important for maintaining essential properties of embryonic stem cells (ESCs) into mature cells, thereby contacting somatic cells (in the original disclosure, mouse fibroblasts) with nuclear reprogramming factors, including gene products from each of the following families: the Oct family, the Klf family, and the Myc family, and preferably also the Sox family. One of the most commonly used embodiments of such a method involves the use of the products of the genes Oct4, Sox2, Klf4, and c-Myc (also known to those skilled in the art as the four Yamanaka factors). The abbreviation OSKM is often used for the combination of the four aforementioned factors.

[0006] The resulting cells are called induced pluripotent stem cells (iPSCs) because their pluripotency and proliferation capacity are judged to be similar to that of embryonic stem cells (ESCs).

[0007] iPSCs have attracted much attention due to their potential utility as a tool for drug development, disease modeling, and transplantation medicine. Interestingly, the ethical issues associated with the production of ESCs do not apply to iPSCs, which offer an uncontroversial strategy for generating patient-specific stem cell lines. However, the efficiency of the process must be substantially improved before reprogramming can be considered for use as a clinical tool.

[0008] Therefore, since the first disclosure of reprogramming methods, multiple efforts have focused on improving reprogramming. A crucial issue is increasing the efficiency of cell reprogramming, which is usually low, resulting in very low numbers of naive iPSCs obtained from the starting differentiated somatic cells. Simply adding transcription factors to a population of differentiated cells does not guarantee reprogramming. The low efficiency of in vitro reprogramming suggests that additional rare events are required to generate naive iPSCs, and reprogramming efficiency is sometimes further reduced in fibroblasts cultured for long periods of time.

[0009] Furthermore, the differentiation stage of the starting cells appears to directly affect reprogramming efficiency. Mouse hematopoietic stem and progenitor cells generate naive iPSCs up to 300 times more efficiently than their terminally differentiated B and T cell counterparts. Importantly, reported reprogramming frequencies are often based on criteria such as alkaline phosphatase activity and reporter gene activation. Both naive iPSCs and ESCs demonstrate key characteristics of pluripotent stem cells, including expressing stem cell markers and forming tumors containing cell types from all three primitive germ layers. However, the naive pluripotency of iPSCs is not entirely clear, as the efficiency of producing viable progeny is greatly reduced compared to ESCs. Thus, chimerism contribution and germline transmission serve as more accurate demonstrations of efficient reprogramming, although quantification of such quality is technically challenging and not frequently used (reviewed by Bilic and Izpisua Belmonte, 2012).

[0010] Increasing the safety of the process by reducing the transforming potential of reprogramming factors and the vectors used for their expression has also been a major concern. Strategies that avoid the use of viral transduction or genomic integration are under active development to replace the integrative viral vectors initially used to introduce the genes encoding reprogramming factors into cells. Furthermore, several efforts have been made to improve the pluripotency properties of iPSCs by increasing their differentiation potential into various cell lineages or by improving their maturation characteristics into specific functional cell types (Li & Izpisua Belmonte, 2016).

[0011] For all these reasons, the original protocol for reprogramming differentiated cells into iPSCs has been subject to many modifications, most of which consist of replacing one or more of the original Yamanaka factors with different compounds or using additional RNAs encoding additional proteins to the original factors. Thus, there are now many variations of the iPS protocol, including those that use microRNAs or small molecule inhibitors of epigenetic modifiers.

[0012] Many small molecule inhibitors have been found to improve reprogramming efficiency by inhibiting specific enzymes or signaling pathways. This group includes inhibitors of mitogen-activated protein kinases (MAPKs), glycogen synthase kinase 3β (GSK3b), transforming growth factor β (TGF-β), chromatin-modifying HDACs (histone deacetylases) or DNMTs (DNA methyltransferases), and many that can be combined with Yamanaka factors to enhance reprogramming efficiency.

[0013] Negatively regulating the expression of epigenetic factors and tumor suppressors using RNA interference (RNAi) also enhances reprogramming. Several studies have shown that using siRNA (small interfering RNA) against differentiated lineage markers, together with the DNA methyltransferase inhibitor (5-aza-cytidine; AZA), helps achieve complete reprogramming of partially reprogrammed cells (Mikkelsen et al., 2008). RNA interference against the DNA methyltransferase Dnmt1 also aids in the transition from a partially reprogrammed state to a pluripotent state; indeed, the epigenetic memory of pluripotent cells is a factor known to act as a barrier to the establishment of pluripotent cells (Mikkelsen et al., 2008). Several previous studies demonstrate that manipulating DNA methylation during the reprogramming process influences the success of reprogramming somatic cells to pluripotent cells. Using demethylases during reprogramming can enhance reprogramming to pluripotent cells, but the resulting pluripotent cells are not useful for applications because the genetic approaches used in those cases are irreversible and pluripotent cells need to remethylate DNA for differentiation, making the resulting reprogrammed cells useless for regenerative medicine (Papp and Plath, 2011).

[0014] RNAi-mediated knockdown of the tumor suppressors p53 and p21 also accelerates the reprogramming process by increasing the rate of cell division (Hanna et al., 2009).

[0015] However, beyond their ability to promote effective reprogramming, several safety issues of small molecules need to be carefully addressed. For example, AZA is known to induce DNA damage and cell death (Mikkelsen et al., 2008). Permanent modifications (genomic or epigenomic) should be avoided, and to date, there are no studies directing the optimization of the dosage or duration of chemical treatment to avoid toxicity. Therefore, in addition to the above-mentioned small molecule alternatives, different strategies to inhibit differentiated states in cell reprogramming are needed, which should preferably improve the complete reprogramming of somatic cells to naive iPSCs.

[0016] MicroRNAs (often abbreviated to miRNAs, or miRs when the specific identification of one of them is accompanied) are small (15–25 nucleotides, very often 20–21 nt) non-coding RNAs that can regulate the expression of protein-coding RNAs and thus perform multiple functions in cells (Ambros, 2004). Recent evidence suggests that miRNAs also link pluripotency by controlling the expression of stemness transcription factors, epithelial-mesenchymal transdifferentiation, cell cycle progression, or the epigenetic landscape of cells (Shenoy & Blelloch, 2014; Leonardo et al., 2012).

[0017] For example, miR-302-367 is directly linked to the levels of three transcription factors, Oct4, Sox2, and Nanog (Card et al., 2008; Marson et al., 2008). One particular miRNA (miR-302, which is highly expressed in ESCs) was found to be able to transform human cancer cell lines into cells resembling ESCs (Lin et al., 2008). Other clusters (such as miR-290-295 or miR-106-363) are also co-occupied by the promoters of Oct4, Sox2, and Nanog (Marson et al., 2008).

[0018] Some methods for producing pluripotent stem cells are based on combining the introduction of at least one mRNA into target cells with the introduction of at least one miRNA into target cells. For example, the US patent application published under US20150232810A1 discloses the possibility of producing pluripotent stem cells using miRNA or miRNA mimics in combination with mRNA, and this application defines miRNA mimics as synthetic miRNAs with enhanced stability due to modified nucleotides or structural modifications (e.g., bulges or loops), and also as small, chemically modified double-stranded RNAs that mimic endogenous miRNAs and enable miRNA functional analysis by upregulating miRNA activity. The method disclosed in US20150232810A1 specifically relates to improving reprogramming, as it is said to enable the generation of iPSCs from cell lines that are refractory to methods involving mRNA or miRNA alone.

[0019] Other research on miRNA and stemness has focused on molecular signatures that characterize stemness.For example, the US patent application published under US20130345289A1 discloses a method for identifying the presence of such clinically applicable adult stem cells in a biological sample from an adult subject, based on the identification of stem cell-specific miRNA signatures that are uniquely expressed in adult stem cells.The method involves determining the level of miR gene products in a biological sample, comparing it with the level of corresponding miR gene products in an anatomically accurate control sample, and determining that the subject has clinically applicable adult stem cells if there is a difference in the level of specific gene products between the samples.One of the microRNAs that can be used as a marker for this purpose is hsa-mir-203-precNo1, which is expressed in differentiated cells but not in stem cell populations. From a comparison of the accession number contained in US20130345289A1 for hsa-mir-203-precNo1 with the information available in the miRbase database (http: / / www.mirbase.org / ) on March 12, 2017, it can be concluded that it is hsa-miR203a-3p (SEQ ID NO: 1, miRbase accession MIMAT0000264), the major mature sequence generated by the human hsa-miR-203a gene (SEQ ID NO: 2, miRbase accession MI0000283). A second mature sequence is generated from hsa-miR-203a and may be hsa-miR-203a-5p* (SEQ ID NO: 53, miRbase accession MIMAT0031890).

[0020] Thus, as occurs with many other microRNAs, two mature microRNAs can arise from opposite arms of the same pre-miRNA (has-miR-203a), and they are designated with the suffixes -3p or -5p. However, mature microRNAs found from one arm of the hairpin are usually much more abundant than those found from the other arm; in such cases, an asterisk following the name indicates the mature species found at lower levels from the opposite arm of the hairpin. In the case of miR-203, the most abundant mature form is miR-203a-3p, while the less abundant form is designated miR-203a-5p*.

[0021] Human miR-203 is expressed from chromosome 19. Its mouse counterpart, mmu-miR-203 (SEQ ID NO: 3, miRbase accession MI0000246), is expressed from chromosome 14 in Mus musculus. The major mature sequence of mmu-miR-203, mmu-miR-203-3p (SEQ ID NO: 4, miRbase accession MIMAT0000236), appears to be identical to that of hsa-miR203a-3p. There is experimental evidence that the second mature sequence, mmu-miR-203-5p* (SEQ ID NO: 54, miRbase accession MIMAT0004547), is shorter than hsa-miR-203a-5p (22 instead of 25 nucleotides) and differs slightly from its human counterpart in the remaining sequence (G at position 11 is replaced by A in hsa-miR203a-5p*).

[0022] miR-203 is a microRNA with hundreds of potential targets (some of which act in opposite directions in their corresponding pathways), as can be found using tools such as TargetScan (http: / / www.targetscan.org) or MiRanda (downloadable version, August 2010, e.g., from http: / / www.microrna.org / microrna / getDownloads.do). It was first identified as a skin-specific microRNA, forming an expression gradient that defines the boundary between proliferating epidermal basal progenitor cells and terminally differentiating suprabasal cells, thus limiting stemness potential in the skin (Yi et al., 2008). It has also been found to be upregulated in psoriasis and differentially expressed in several types of cancer.

[0023] Contrary to microRNAs traditionally associated with cellular reprogramming and / or the acquisition of ESC characteristics, miR-302, and members of the miR-302-367, miR-290-295 or miR-106-363 clusters, such as miR-203, are considered stemness repressors (Yi et al., 2008; Volinia et al., 2014), but their expression during early development has been unknown until now.

[0024] Several reviews of microRNA regulation of stem cells (Huang et al., 2011) refer to miR-203 as a microRNA that cooperates with miR200c and miR-183 in regulating Sox2 and Klf4. Indeed, such statements are made in reference to a study (Wellner et al., 2009) that characterized miR-203 as a stemness inhibitor rather than a regulator of Sox2 or Klf4. Specifically, Wellner et al. reported that ZEB1 represses the expression of miR-203, which inhibits stemness, and furthermore, miR-200c, miR-203, and miR-183 cooperate to repress the expression of stem cell factors in cancer cells and mouse embryonic stem (ES) cells, as demonstrated for the Polycomb repressor Bmi1.

[0025] The role of miR-203 as an inhibitor of stemness of pluripotent cells has also been suggested in the context of epidermal differentiation (Nissan et al., 2011), more specifically in the context of in vitro differentiation of hESCs into keratinocytes, where induction of miR-203 during epidermal differentiation occurs from the earliest stages but becomes significant in keratinocyte differentiation 3 days after treatment with BMP4 (i.e., once the hESCs are already committed to epidermal differentiation). Accordingly, miR-203 may be considered a key factor in blocking stemness of pluripotent cells by inducing epidermal differentiation.

[0026] Similarly, selective regulation of survivin isoform expression by miR-203 has been reported to contribute to the mechanism of human embryonic stem cell pluripotency by specifically suppressing hESC pluripotency (Kapinas et al., 2015). Experiments in which hESCs (H9) were transfected with a miR-203 inhibitor showed increased nuclear survivin levels, while assays in which miR-203 overexpression was achieved by transfecting hESCs with miR-203 precursors resulted in decreased nuclear survivin levels. As can be seen in the cited paper, these results led Kapinas et al. to hypothesize that miR-203 may inhibit pluripotency by negatively regulating survivin expression.

[0027] In iPSCs, miR-203 has been reported to contribute to the process opposite to stemness and inhibit iPSC function (senescence), and such effects are precisely exerted via the miR-203-survivin-p21 pathway (Xu et al., 2010).

[0028] miR-203 has been implicated in cancer and has been implicated in tumor suppressor functions in multiple cancers (Bueno et al., 2008; Michel & Malumbres, 2013). In addition to the importance of epigenetic inactivation of miR-203 for the development of Philadelphia chromosome-associated leukemia, the possibility of injecting miR-203 as a therapy for this disease has been explored, as restoration of miR-203 levels blocks the production of the oncogenic protein BCR-ABL, resulting in tumor cell proliferation halting, even in tumors resistant to other therapeutic approaches. Primary tumors with metastasis showed widespread suppression of hsa-miR-203a, and an asymmetric hsa-miR-302 (high) / hsa-miR-203a (low) ratio was found to be associated with stem cell markers, metastasis, and shorter survival in invasive ductal carcinoma (Volinia et al., 2014).

[0029] miR-203 has also been determined to be one of the microRNAs involved in regulating components of the epigenetic apparatus. Studies on the potential relationship between the expression of miR-203, miR-26, and miR-29 family members and genes Dnmt3a, Dnmt3b, Mecp2, and Ezh2 during cell transformation have shown that the microRNAs and their validated or predicted targets are inversely expressed, indicating that these molecules are involved in epigenetic reprogramming. For example, it has been reported that miR-203 downregulates Dnmt3b in mouse melanocyte cells (Gasque Schoof et al., 2015).

[0030] Unfortunately, and despite all the knowledge gained about reprogramming and the markers and determinants of stemness and pluripotency, many questions regarding the differentiation outcomes of pluripotent cells and how they are determined remain unanswered. While researchers have begun to identify numerous molecular pathways involved in somatic cell reprogramming, identifying the full spectrum of events that enable this process will require further, more fundamental research. The details of the reprogramming process and its dynamics, particularly epigenetic reprogramming, remain incompletely understood.

[0031] Finally, most strategies have been designed to improve the efficiency of reprogramming, but few have been applied to improve the differentiation potential of already established pluripotent cells. Maintaining full differentiation potential along with the ability to self-renew is a key characteristic of stem cells during development and regeneration, yet the available ESCs or iPSCs obtained after the reprogramming process are often of a quality that facilitates subsequent differentiation. The low quality of iPSCs, and the differences in quality among iPSCs derived from the same reprogramming process, are particularly important for their use in human regenerative therapies. Many authors believe that iPSCs are a subset of pluripotent stem cells influenced by the somatic cell of origin and cell culture conditions, and that these subsets exhibit differences even among iPSCs obtained during the same reprogramming process. Many iPSCs exhibit reduced differentiation potential (which may be explained by incomplete genomic reprogramming), resulting in iPSCs closer to a primed state than a naive state. Honda and colleagues (Honda et al., 2013) reported that the limited differentiation potential of iPSCs could be improved by serial passaging and conversion to a more immature, naive-like state. This conversion required the expression of OCT3 / 4 from lentivirus, passaging of iPSCs into mouse embryonic fibroblasts, and culturing in medium containing CHIR99021 (a GSK inhibitor) and leukemia inhibitory factor (LEF). These cells showed improved differentiation potential into mature oligodendrocytes, suggesting that naive-like conversion of iPSCs confers greater differentiation potential.

[0032] The in vitro maintenance of pluripotent cells in a naive state (either ESCs or iPSCs) is challenging and has been the subject of numerous studies, most of which focus on the addition of different compounds to the culture medium with the aim of achieving the maintenance of the broadest possible pluripotent potential. Mouse ESCs can be maintained long-term in a naive state when cultured in the presence of serum plus leukemia inhibitory factor (LIF) (Niwa et al., 2009). Notably, LIF alone is in many cases unable to block differentiation of mESCs and is even ineffective in iPSCs. This limitation can be partially overcome by adding two small molecule kinase inhibitors, termed "2i," together with LIF. 2i components include a specific inhibitor of the extracellular signal-regulated kinase (ERK1 / 2) / mitogen-activated protein kinase (MAPK) signaling pathway (MEKi, PD0325901) and a specific inhibitor of glycogen synthase kinase 3β (GSK3βi, CHIR99021), which can protect pluripotent cells from differentiation-promoting stimuli and select against differentiating cells (Ying et al., 2008). International application WO2012087965 discloses a similar method for maintaining or increasing cell potential, in which pluripotent cells are cultured in a feeder-free environment in the presence of at least one small molecule selected from an inhibitor of TGF-β, GSK3, MEK, or ROCK, which method primarily achieves increased viability and increased potential, the increased potential being characterized by one or more of the following features: a) expression of at least one pluripotent stem cell marker selected from the group consisting of endogenous Oct4, Nanog, SSEA4, Sox2, Klf4, Tral81, and Lin28; b) pluripotent stem cell morphology; c) the ability to contribute to germline transmission; d) teratoma formation; e) the ability to differentiate or transdifferentiate from the starting lineage to a different lineage; and f) in vitro tri-lineage differentiation, which may be considered a marker of the naive state of pluripotent stem cells.

[0033] Other research groups have focused on modifying the culture medium of iPS cells to improve the efficiency of differentiation into specific lineages and / or select more suitable cells for the desired differentiation process. Most of them are based on the addition of compounds (such as those mentioned above). For example, WO2014201254A1 discloses a method involving microRNAs to derive cardiomyocytes from iPSCs or ESCs, in which microRNAs of the let-7 family are described as important microRNAs for in vitro cardiac maturation. EP249071A2 discloses methods and compositions for producing cardiomyocytes from pluripotent stem cells, in which pluripotent cells are differentiated into cardiomyocytes in the presence of a ROCK inhibitor. WO2014200030A1 discloses a method for obtaining hematopoietic stem cells and / or hematopoietic progenitor cells from pluripotent stem cells, which comprises culturing the pluripotent stem cells in the presence of IGF2 and selecting induced pluripotent stem cells with a high ability to differentiate into hematopoietic stem cells based on the expression level of one or more genes. EP2646543A1 discloses a method for generating corneal cells, in which human pluripotent stem cells are cultured in a corneal fibroblast-conditioned medium on a solid surface containing extracellular matrix components. KR2017011676A discloses a method for differentiating stem cells into hepatocytes using a culture composition containing a biocompatible solubilized scaffold extract derived from decellularized organ tissue. WO2017062374 discloses compositions and methods for generating oligodendrocyte precursors from pluripotent stem cells using a three-dimensional culture system comprising a biocompatible polymer and a combination of at least two factors that promote differentiation into oligodendrocytes selected from an agonist of the sonic hedgehog signaling pathway, an agonist of the wnt signaling pathway, retinoic acid, and an inhibitor of dual Smads.

[0034] Several groups have reported achieving improved pluripotent stem cell differentiation into multiple lineages by adding a single molecule to the culture medium. In 2013, Chetty et al. described improved competency for directed differentiation into multiple lineages in over 25 stem cell lines after culturing pluripotent stem cells in dimethyl sulfoxide (DMSO), where the proportion of cells in the early G1 phase of the cell cycle increased, resulting in improved competency for directed differentiation and even enhanced terminal differentiation into functional derivatives. Nevertheless, none of these strategies adequately achieved a naive state for the resulting pluripotent cells because the stem cell potential of the cells was not demonstrated (none of the features described above for naive cells were examined in these cases), differentiation improvements were not substantial, and previous studies generally lacked rigorous pluripotency assays (such as 4n complementation or chimeric contribution).

[0035] Thus, despite recent improvements in the in vitro culture of pluripotent cells through the addition of new small molecules (e.g., LIF, 2i, bFGF, TGFβ, JNKi, p38i, ROCKi, activin A) and / or sustained expression of transcription factors (e.g., KLF4, KLF2, OCT4, SOX2, NANOG), improving the differentiation potential of pluripotent cells remains an unsolved problem and an important goal to achieve, either during the reprogramming protocol or once pluripotent cells have been established. Before cultured pluripotent cells can be considered for use as clinical tools, the efficiency of generating differentiated cells must be substantially improved. Clinical application of pluripotent cells will require the safe and highly efficient generation of stem cells capable of differentiating into diverse cell types with the potential to generate replacement cells in the quest to repair diseased tissue. Improving the quality of pluripotent cells is also important to make them useful research tools for dissecting the mechanisms that regulate cell fate decisions or for developing disease models to investigate how various human diseases arise as a result of specific mutations and epimutations, and with that information to develop new drugs to cure or possibly prevent such diseases.

[0036] Although methods to improve differentiation potential based on the addition of DMSO represent a promising approach, alternative methods should preferably be developed.

[0037] Therefore, there is a need for methods to improve the quality of pluripotent cells (especially iPSCs) by increasing their stemness properties and / or their ability to differentiate into different cell types. Preferably, the methods should be easy to perform without requiring complex devices or very expensive products, and, if possible, feasible to practice with molecules that are as safe as possible.

[0038] The present invention provides a solution to this problem. Summary of the Invention

[0039] The present invention is based on the surprising finding disclosed in this application that transient expression or increased levels of a single microRNA (microRNA-203) can improve the function of either iPSCs or ESCs in multiple assays. The effect of transient exposure to miR-203 is twofold. First, increasing miR-203 levels in iPSCs induces a transcriptional profile closer to that of ES cells (a naive or naive-like state), including upregulation of stemness signatures. This increase in stemness characteristics is also observed when using the reported 2C-like stage (a hallmark of totipotent cells in two-cell embryos). Second, transient expression or increased levels of miR-203 in pluripotent cells leads to enhanced in vitro and in vivo differentiation into multiple lineages encompassing the three germ layers, as shown in embryoid bodies generated in vitro or in teratomas and embryo-like structures observed after injection of these cells into mice. Exposure to miR-203 improved the generation of rare differentiated tissues (such as pancreas, bone marrow, or trophoblast), and these structures formed even weeks after transient exposure to miR-203 in pluripotent cells. The specific differentiation into cardiomyocytes suggests that exposure to miR-203 supports not only differentiation but also maturation and functionality in the resulting cells.

[0040] Thus, in a first aspect, the present invention relates to a method for promoting stemness and / or differentiation potential of pluripotent cells, comprising exposing the cells to increased levels of microRNA-203 or an analog thereof. Preferably, the pluripotent cells are induced pluripotent stem cells (iPSCs) in culture, but they may also be embryonic stem cells (ESCs) in culture. Also preferably, the cells are transiently exposed (e.g., for 3 to 5 days) to increased levels of microRNA-203 or an analog thereof. The increased level of microRNA-203 to which the cells are exposed can be achieved by transducing or transforming the cells with an expression vector expressing microRNA-203 (and inducing such expression if inducible), or by adding microRNA-203 or an analog thereof to the culture medium of the iPSCs if they are in culture. Among possible analogs, small RNA molecules containing fragments with a high degree of homology to at least the sequence of the mature form of microRNA-203 (SEQ ID NO: 1), preferably those with chemical modifications, are preferred possible embodiments, and analogs also include double-stranded RNA mimics. The method of the present invention is compatible with obtaining iPSCs by any method, such as the original Yamanaka method (contacting somatic differentiated cells (preferably fibroblasts) with nuclear reprogramming factors including gene products from each of the following families: Oct family, Klf family, Myc family, and Sox family), or its modifications.

[0041] As described above, a preferred embodiment of the method of the present invention, compatible with all other embodiments, is that cells exposed to increased levels of microRNA-203 or its analogs are exposed to a mature form of miR-203 originating from at least the 3' arm or its analog. Such a mature form may be the mature form of miR-203 of any mammalian species. Since the pluripotent cells exposed to increased levels of miR-203 are preferably of human or mouse origin, exposure to at least hsa-miR-203a-3p (SEQ ID NO: 1) or mmu-miR-203-3p (SEQ ID NO: 4) or their analogs is preferred, which may be alone or in combination with other forms of miR-203 (including combinations of analogs of hsa-miR-203a-3p and / or mmu-miR-203-3p, mature forms of miR-203 originating from the 5' arm (such as hsa-miR-203a-5p* and / or mmu-miR-203-5p*), or their analogs). Even if the added molecule is a pre-miRNA of miR-203 or an analog thereof, cells may be exposed to the mature form due to processing of the pre-miRNA in the cell.

[0042] As previously commented, miR-203 is expressed in vivo as a pre-miRNA (represented by SEQ ID NO: 2 in the case of the human molecule and SEQ ID NO: 3 in the case of the mouse molecule), which gives rise to two different mature forms, one arising from the 5' arm of the pre-miRNA and the other from the 3' arm. The mature form arising from the 3' arm is the most abundant and, according to one of the assays in Example 1, is the form responsible, to a higher extent, for the effects of the present invention (at least those related to embryo-body formation). As can be seen in Figure 10, the sequence of miR-203 is highly conserved among different mammalian species (especially the seed region associated with Dnmt3a and Dnmt3b). Thus, for the purposes of the present invention, unless reference is made to a particular species or form of microRNA, the term "miR-203" or "microRNA-203" encompasses microRNA-203 expressed by any mammal and any of its possible forms, i.e., the pre-miRNA and / or the mature form resulting from either the 5' arm (represented by SEQ ID NO: 53 in the case of the human molecule (hsa-miR-203a-5p) and SEQ ID NO: 54 in the case of the mouse molecule) or the 3' arm (represented by SEQ ID NO: 1 and SEQ ID NO: 4, which are identical, for the human molecule (hsa-miR-203a-3p) or the mouse molecule (mmu-miR-203-3p)). Combinations of two or more of the aforementioned forms of miR-203, as well as, as in most examples of this application, very particularly combinations of the two mature forms resulting from the 5' arm and the 3' arm, are also encompassed by the term "miR-203" or "microRNA-203."The term "analogue of miR-203" or, where the premise warrants it, "analogue thereof" is intended to encompass a molecule or a mixture of molecules, each of which is an analog of any of the molecules encompassed by the term "microRNA-203" or "miR-203", preferably at least one of the molecules of the molecule or mixture is an analog or mimic (as defined below) of one mature form arising from the 3' arm of pre-miRNA-203, and most preferably at least one of the molecule or mixture of molecules defined by the term "analog" will be an analog of hsa-miR-203a-3p (SEQ ID NO: 1) or mmu-miR-203-3p (SEQ ID NO: 4).

[0043] Another possible embodiment of the present invention is pluripotent cells obtained after carrying out the methods of the present invention (especially after transient exposure to increased levels of miR-203), which are distinct from the reprogrammed iPSCs used as starting material. When such naive pluripotent cells are subjected to differentiation protocols (in vitro or in vivo), they exhibit expanded cell fate potential and generate cells characterized by Nanog, Oct, and Sox2 expression (well-established markers of pluripotency), while cells expressing nestin, Gata4, or Cd34 (well-established markers of differentiation) appear to coexist with them in the same culture population. It is also important to note that they exhibit signs of being naive-like cells, such as the characteristic cellular morphology of naive cells, the ability to contribute to chimeras, and germline transmission.

[0044] The naive pluripotent cells resulting from the method of the present invention exhibit improved differentiation and maturation outcomes, which can be particularly observed when differentiated specifically into cardiomyocytes. With this in mind, another aspect of the present invention is the use of the cells of the present invention to obtain differentiated cells (such as cardiomyocytes). Other possible embodiments are cells of the nervous system (e.g., neurons and glial cells), chondrocytes, or pancreatic beta cells.

[0045] Therefore, the method of the present invention can be considered as a strategy for obtaining naive pluripotent cells, as well as differentiated and / or mature cells, with improved stemness potential and increased differentiation potential. The differentiated or mature cells obtained by the method of the present invention are also included within the scope of the present invention. [Brief explanation of the drawings]

[0046] [Figure 1] Mouse alleles generated for the assay. a) Schematic depiction of different alleles generated at the mouse mu-miR203 locus for this application. b) Representative photomicrograph showing dysgenetic tail skin in miR-203(- / -) mice. Hematoxylin-eosin staining; scale bar, 50 μm. c) Schematic depiction of the inducible miR-203 knock-in model generated in this study. In the miR-203 KI [ColA1(miR-203 / miR-203); Rosa26(rtTA / rtTA)] model, the reverse tetracycline transactivator is expressed from the Rosa26 locus, while miR-203 is driven by a tetracycline operator downstream of the ColA1 locus. d) MiR-203 RNA expression as determined by quantitative PCR in miR-203 WT, KO, and KI (after induction with doxycycline; Dox) MEFs (left panel), or miR-203 KI ESCs and iPSCs (right panel) treated with or without doxycycline. RNA expression is normalized by control miRNA (miR-142). ***P<0.001 (Student's t-test). [Figure 2]The effect of transient induction of miR-203 on the pluripotency and differentiation potential of iPSCs and ESCs. a) Protocol for reprogramming miR-203-mutant MEFs into pluripotent iPSCs and subsequent differentiation into embryoid bodies. Wild-type (WT), knockout (KO), and knockin (KI) MEFs were transduced with viruses expressing Oct4, Sox2, Klf4, and c-Myc (OSKM). The resulting iPSCs were then treated with 1 μg / ml doxycycline (Dox) for 5 days. tKI indicates transient miR-203 expression in knockin cells for the indicated 5 days. Dox was removed for 15–30 days before the initiation of the embryoid body generation protocol. Samples for RNA sequencing were collected 30 days after Dox withdrawal. b) Principal component analysis of RNA sequencing data from WT iPSCs (n = 3 clones), tKI iPSCs (n = 4), and WT ESCs (n = 3). c) Unbiased clustering of genome-wide RNA sequencing data (left) and a heatmap plot showing the comparative expression of 450 genes associated with pluripotency (stemness signature; 2 clones per sample). The tKI iPSC profile is similar to that observed in ES cells, as demonstrated by the hierarchical tree. d) Representative images of embryoid bodies (EBs) derived from WT, KO, and tKI iPSCs after 10 or 15 days of differentiation. Scale bar, 500 μm. The histogram in the middle shows the number of EBs generated from the three genotypes at day 10 of differentiation. Data are means ± standard deviations (n ​​= 9 independent experiments). The increase in EB size (compared to day 0 of differentiation) is shown in the right panel. Data are presented as the mean ± standard deviation (n = 3 independent experiments). e, Representative immunofluorescence detection of Cd34 (mesoderm; green), Gata4 (endoderm; red), and Pax6 (ectoderm; blue) in EBs derived from WT, KO, and tKI iPSCs. Scale bar, 20 μm. f, Immunohistochemical detection of Cd34 (mesoderm), Gata4 (endoderm), and nestin (ectoderm) in tKI EBs. Scale bars are 500 μm for the upper images and 100 μm (Cd34, Gata4) or 50 μm (nestin) for the lower images.g, Representative images of EBs derived from KI iPSCs and ESCs treated with vehicle (KI) or Dox (tKI) at different time points during differentiation. Scale bar, 500 μm. h, Quantification of the percentage of EBs displaying long internal cavities and beating EBs over the indicated time course. Data are presented as mean ± standard error (n = 3 independent experiments). i, Representative immunofluorescence analysis of ESCs stably expressing the 2C::tdTomato reporter and transiently transduced with GFP or miR-203-GFP viruses. Scale bar, 10 μm. The plot on the right shows the percentage of tdTomato-positive cells among all GFP-positive cells. Data are presented as mean ± standard error (n = 3 independent experiments). In d, h, and i, *P < 0.05; **P < 0.01; ***P < 0.001 (Student's t test). [Figure 3]Improved embryoid body generation after transient gene induction of miR-203 in iPSCs. a) Experimental design for generating embryoid bodies (EBs) from miR-203 KI iPSCs. Cells were treated or not with doxycycline (Dox) for 5 days, then Dox was removed for the following 2 weeks before the initiation of the embryoid body formation assay. b) Representative images of uninduced (-Dox) or transiently induced (+Dox) EBs derived from five different KI clones at different time points during the differentiation process. Scale bar, 500 μm. c) Quantification of EBs with long internal cavities, beating EBs, and EB size during the differentiation process. Data are means ± standard error (n = 5 independent experiments). **P < 0.01 (Student's t test). d) Top categories in gene ontology analysis of significantly upregulated genes in induced vs. uninduced KI iPSCs (four independent clones were analyzed). e, Heatmap showing the expression profiles of genes included in GO:0048513 (organ development) in uninduced embryoid bodies (EBs) as well as uninduced (KI) or transiently induced (tKI) iPSCs. The profiles observed for tKI and KI EBs are more similar than when compared to control KI iPS. f, Heatmap showing the expression profiles of genes included in the stemness signature in the indicated samples. Only tKI iPS express high levels of genes included in this signature. [Figure 4]Improved embryoid body formation after transient exogenous expression of miR-203. a) Schematic depiction of the experimental design used to generate embryoid bodies (EBs) from wild-type iPSCs or ESCs that were either retrovirally transduced or transfected with miRNA mimics for transient expression of miR-203. b) Expression of miR-203 RNA in wild-type iPSCs transduced with pMCSV or pMCSV-miR-203 or transfected with control mimics or miR-203 mimics. RNA expression is normalized by control miRNA (miR-142). ***P<0.001 (Student's t-test). c) Representative images of EBs derived from either wild-type iPSCs (left) or ESCs (right) transduced with empty pMCSV vector, pMCSV-miR-203, or transfected with control mimics or miR-203 mimics at different time points during the differentiation process. Scale bar, 500 μm. d, Quantification of EBs with long cavities and beating EBs during the differentiation process. Data are mean ± standard error (n = 3 independent experiments). **P < 0.01 (in both iPS cells and ES cells; Student's t-test). e, Representative photomicrographs of EBs generated using the conventional protocol. Note the complexity in structure and the formation of long cavities shown in the lower panel. Scale bar, 200 μm (upper panel) and 500 μm (lower panel) or 50 μm (inset). [Figure 5] Improved embryoid body formation after transient exogenous expression of miR-203a-3p versus miR-203a-5p. Representative images of EBs derived from wild-type ESCs transfected with control mimic, miR-203a-3p, or miR-203a-5p mimic at different time points during the differentiation process. Scale bar, 500 μm. [Figure 6]Transient exposure of iPSCs to miR-203 results in complex teratomas in vivo. a) Representative images of teratomas generated 20–25 days after subcutaneous injection of WT, KO, and tKI (GFP-expressing) iPSCs. Scale bar, 5 mm. The bottom image shows an example of GFP-expressing tKI iPSC-derived embryonic structures. Scale bar, 1 mm. The plot on the right shows tumor volume (mm3) measured at the end of the experiment. Data are presented as mean ± standard error (n = 8 tumors per genotype). b) Prevalence of specific highly differentiated tissues in teratomas. The number of tumors included in the analysis is indicated in the panel. c) Representative examples of highly differentiated teratomas generated from tKI iPSCs after intraperitoneal injection in nude mice. Most of these complex teratomas were detected near the uterus or as ovarian cysts in the host mice. Panels show higher magnifications (H&E staining) of multiple differentiated tissues and cells observed in teratomas. Scale bar, 1 mm (center image) or 50 μm (inset). d, Immunohistochemical detection of pluripotency markers (Nanog, Oct4, Sox2) and markers of differentiation into three germ layers (Nestin, Gata4, CD34) in teratomas derived from WT, KO, and tKI iPSCs. Scale bar, 2000 μm, and 100 μm for higher magnifications. In a and b, **P<0.01; ***P<0.001 (Student's t-test). [Figure 7]Differentiation characteristics in teratomas generated from miR-203 tKI iPSCs. a) Histopathological examples (H&E staining) of specific tissues found in tKI iPSC-derived teratomas. Scale bar, 100 μm. Also shown is a magnification of trophoblast stained with placental lactogen-1 (PL-1) (scale bar, 50 μm). b) Gene ontology analysis of significantly altered genes in miR-203 tKI-derived teratomas compared to wild-type teratomas. c) Immunohistochemical analysis of teratomas derived from wild-type, KO, or tKI iPSCs. Antibodies against the proliferation marker Ki67 or the terminal differentiation marker for smooth or skeletal muscle (actin) were used. Scale bar, 2000 μm, and 100 μm for higher magnifications. d, Immunodetection of CD31 (bone marrow), CD73 (pancreas), type I collagenase (cartilage), and insulin (producing cells) in teratomas generated from tKI iPSCs. Scale bar, 20 μm. [Figure 8] Transient in vitro exposure to miR-203 results in embryonic-like structures in host mice similar to those induced by in vivo-generated iPSCs. a) Representative example of E-Ls generated after intraperitoneal injection of tKI GFP-expressing iPSCs. H&E (hematoxylin-eosin). The following antigens were detected by immunohistochemistry: GFP, Sox2 (ectoderm), Cd34 (mesoderm), Gata4 (endoderm), AFP, and CK8 (visceral endoderm of the yolk sac), and Ter119 (nucleated erythroid cells). Scale bar, 500 μm, and 100 μm for higher magnification. b) Representative example of a viable whole-iPSC mouse (black) generated from tKI iPSCs in an embryonic tetraploid complementation assay. [Figure 9]DNA methyltransferases 3a and 3b are targets of miR-203, which is involved in the control of pluripotency and differentiation. a) Venn diagram showing common genes downregulated in tKI versus WT iPSCs and upregulated in K versus WT iPSCs, predicted as targets of miR-203 (a list of 35 common transcripts, including Dnmt3a and Dnmt3b, is shown in Table 6). b, c) Relative luciferase units (RLU; normalized to Renilla luciferase and relative to DNA content) in 293T cells transfected with DNA constructs carrying the wild-type 3' UTR from the indicated transcripts (b) or mutant versions of the 3' UTR of Dnmt3a and Dnmt3b downstream of a luciferase reporter (c). Cells were cotransfected with Renilla luciferase as a transfection control and plasmids expressing GFP or miR-203-GFP. Data are presented as mean ± standard deviation (n = 3 independent experiments). d. Representative images of embryoid bodies (EBs) derived from tKI iPSCs transiently cotransduced with Dnmt3a and Dnmt3b cDNAs or empty vector and treated with vehicle or Dox as indicated. Scale bar, 500 μm. Histograms show quantification of EBs with long cavities, beating EBs, and EB size at different time points. e. Immunodetection of GFP or tdTomato in ESCs stably expressing the 2C::tdTomato reporter and transiently transduced with either pMCSV-GFP, pMCSV-miR-203-GFP, or pMCSV-miR-203-GFP + Dnmt3a / b cDNA. Scale bar, 16 μm. The plot shows the percentage of Tomato-positive cells among all GFP-positive cells. Data are presented as mean ± standard error (n = 3 independent experiments). f, Venn diagram showing common genes downregulated in tKI iPSCs, predicted as targets of miR-203, and also involved in epigenetic regulation of gene transcription (GO:0040029).g, Principal component analysis from RNA sequencing data encompassing profiles from wild-type iPSCs, tKI iPSCs, and wild-type iPSCs transfected with either control siRNA (siC) or specific siRNAs against Dnmt3a (siDnmt3a), Dnmt3b (siDnmt3b), or both (siDnmt3a / b). h, Representative images of embryoid bodies (EBs) derived from wild-type iPSCs in which expression of Dnmt3a and Dnmt3b was transiently silenced by siRNA. Scale bar, 500 μm. Histograms show quantification of EB size and the percentage of EBs with large cavities or beating at different time points during the differentiation process. i, Detection of tdTomato and DAPI in ESCs stably expressing the 2C::tdTomato reporter and transiently transfected with either a control mimic, a miR-203 mimic, a miR-203 mimic plus Dnmt3a and Dnmt3b cDNAs, or siRNA against both Dnmt3a and Dnmt3b transcripts. Scale bar, 50 μm. j, The histogram on the left shows the percentage of Tomato-positive cells in the assay in panel i) 5 days after transfection. The histogram on the right shows the percentage of Tomato-positive colonies, where Tomato is expressed only at the periphery of the clone (black) or in the majority of cells comprising the colony (gray). Data are means ± standard error (n = 3 independent experiments; 486 colonies for control mimic, 504 colonies for miR-203 mimic, 515 colonies for miR-203 mimic + Dnmt3a / 3b cDNA, and 449 colonies for Dnmt3a / 3b siRNA). k, Transcript expression of miR-203, Dnmt3a, and Dnmt3b in wild-type iPSCs transiently transfected as indicated in (i). RNA expression was measured 24 hours after the transfection protocol and normalized by control miRNA (miR-142) or GAPDH mRNA, respectively. In b–e, h, j, k, *P < 0.05; **P < 0.01; ***P < 0.001 (Student's t-test). [Figure 10] Alignment between miR-203 and the 3'-UTR sequences of Dnmt3a / b. a, Alignment of the 3'UTRs of Dnmt3a and Dnmt3b in several representative species (Hsa: Homo sapiens; Mmu: Mus musculus; Rno: Rattus norvegicus; Ocu: Oryctolagus cuniculus; Ptr: Pan troglodytes (chimpanzee); Mml: Macaca mulatta; Oga: Otolemur garnetti; Tbe: Tupaia belangeri; Eeu: Erinaceus europaeus; Cfa: Canis familiaris (dog); Eca: Equus caballus; Bta: Bos taurus (cow); Ete: Echinops telfairi; Fca: Felis catus (house cat)). The seed regions of miR-203 target sites contained in these 3'-UTRs are highlighted in bold font and aligned with the corresponding miR-203 seed sequences. b, Schematic depiction of luciferase reporters carrying the complete 3'-UTR of wild-type Dnmt3a (left) or Dnmt3b (right) or the corresponding mutated versions downstream of the luciferase gene. Mutated residues are underlined. [Figure 11]Transient expression of miR-203 induces genome-wide hypomethylation in iPSCs. a) Experimental design for genome-wide DNA methylation analysis of WT and tKI iPSCs and their derived embryoid bodies (EBs). Cells (two independent tKI clones and two WT technical replicates) were transiently treated with Dox for 5 days, followed by Dox withdrawal for an additional 20 days before the initiation of the EB formation protocol. Samples for DNA and RNA analysis were collected at the indicated time points: before Dox (t = 0), 5 days after Dox withdrawal (t = 10), 20 days after Dox withdrawal (t = 25), or 7 days after the initiation of the EB generation protocol (t = 32). b) Genome-wide DNA methylation data showing the number and size of DNA methylation valleys (DMVs) and partially methylated domains (PMDs). c) DNA methylation distribution of the indicated samples, smoothed across 100-kb blocks. d, Principal component analysis showing the distribution of differential methylation profiles in the indicated samples (wt and tKI samples are grouped separately for clarity). e, Number of differentially methylated single CpG sites (DMPs) and variably methylated regions (DMRs) in the indicated comparisons. f, Experimental protocol followed to test DNA methylation rescue by miR-203-resistant Dnmt3a / b cDNA. Specific methylation variably regions (DMRs) at the Sirt6 and Elf5 loci were analyzed by PCR amplification and sequencing of bisulfite-modified DNA. Quantification of methylated versus unmethylated CpGs is shown in the histogram. [Figure 12]Genome-wide methylation of iPSCs or embryoid bodies after transient exposure to miR-203. a, Expression levels of transcripts for miR-203 and DNA methyltransferases Dnmt1, Dnmt3a, Dnmt3b, Dnmt3a2, and Dnmt3l, or markers of pluripotency (Dazl) and differentiation (Gata6), as determined by quantitative PCR. KI iPSCs treated with or without doxycycline (Dox) and co-transduced with Dnmt3a / b cDNA or empty vector were used, as shown in the schematic depiction of the experimental design. The first shading (light gray, pink in the original) starting from the left indicates the time course, in which cells were treated with or without Dox and transduced with or without Dnmt3a / b cDNA. The second shading (dark gray, orange in the original) from the left indicates the differentiation process into embryoid bodies. Data are expressed as the average of three technical replicates per experiment (n = 2 independent experiments). b, Representative genomic region (telomere of chromosome 10) showing methylation patterns in tKI iPSCs and embryoid bodies. Time refers to Figure 11a. DMV, DNA methylation valley; PMD, partially methylated domain. [Figure 13]Genome-wide methylation and gene expression changes in iPSCs or embryoid bodies. a) Venn diagram showing common up-regulated (data from RNA sequencing studies) and hypomethylated (data from genome-wide methylation studies) genes in tKI iPSCs 20 days after Dox withdrawal. A total of 235 genes were DNA hypomethylated and up-regulated under these conditions. Gene ontology analysis of this list is presented in the right panel. b) Methylation data in the Elf5 genomic region in KI iPSCs before induction (t=0, upper line), 10 or 25 days after transient miR-203 induction (lower signal), and in embryoid bodies (EBs, t=32), according to the experimental design shown in Figure 11a. Two Elf5 transcripts are shown. c, Experimental design used for validation of methylation data at the indicated Elf5 methylation variable regions (DMRs; boxes in b and the first part of the arrow representing Elf5 in c). DNA was isolated as indicated and sequenced after bisulfite modification. Eight to ten independent clones per condition were sequenced. Histograms show the percentage of DNA methylation at Elf5 DMRs in different conditions. [Figure 14]Transient exposure of progenitor cells to miR-203 mimics promotes their late differentiation into mature cardiomyocytes. a) Representative immunofluorescence images showing EdU (green in the original) and nuclear (DAPI, blue in the original) staining of primary cardiomyocytes extracted at postnatal day 1 and transiently transfected with control or miR-203 mimics 24 h after extraction. Photographs were taken 3 days after transfection. Scale bar, 60 μm. Histograms show the percentage of EdU-positive cells at different days after transfection. Data are means ± standard deviations (n ​​= 2 independent experiments, each with 6 replicates). b) RNA expression of miR-203 (24 h after transfection) and Ccnb1, Myh6, and Myh7 transcripts (5 days after transfection) as determined by quantitative PCR. The Myh6 / Myh7 ratio is calculated as an index of cardiomyocyte maturation. Data are means ± standard deviations (n ​​= 3 independent experiments). c, Experimental protocol followed for differentiation of cardiomyocytes from iPSCs in the absence or presence of miR-203 mimics and Dnmt3a / b cDNA. d, Representative immunofluorescence images showing cardiac troponin T (cTnT, green in the original) and nuclear (DAPI, blue in the original) staining of in vitro-generated cardiomyocytes derived from WT iPSCs transiently transfected with either control mimics, miR-203 mimics, or miR-203 mimics plus Dnmt3a / b cDNA. Photographs were taken on day 15 of differentiation. The bottom panel shows a magnified detail of cTnT staining in each condition. Scale bar, 68 μm (inset, 25 μm). The cTnT-positive areas in these cardiomyocytes are shown in the histograms on the right. Data are presented as means ± standard deviations (n ​​= 2 independent experiments, each with 6 replicates). e, miRNA or mRNA levels determined by quantitative PCR of the indicated transcripts at different time points during cardiomyocyte differentiation in the indicated samples. Data are presented as mean ± standard deviation (n = 2 independent experiments, each with 6 replicates). *P < 0.05; **P < 0.01; ***P < 0.001 in a, b, d, and e (Student's t-test). [Figure 15] Improved cardiomyocyte differentiation and maturation after transient expression of miR-203. a, Representative immunofluorescence images showing phosphorylated histone 3 (Ser-10; pH3) (green in the original), cardiac troponin T (cTnT, red in the original), and Hoescht for nuclei staining (blue in the original) (as indicated on the left side of the image) in primary cardiomyocytes extracted at postnatal day 1 and transiently transfected with control or miR-203 mimics 24 h after extraction. Images were taken 3 days after transfection. Scale bar, 64 μm. White arrows point out cardiomyocytes positive for pH3. The histogram in the middle shows the proliferation rate, measured as the percentage of pH3-positive cells relative to the total number of cTnT-positive cells 3 days after transfection. Data are means ± standard deviations (n ​​= 2 independent experiments). The plot on the right shows the total number of cells 3 days after transfection. Data are presented as mean ± standard deviation (n = 2 independent experiments). b, mRNA levels as determined by quantitative PCR of the indicated transcripts at different time points before and during cardiomyocyte differentiation. iPSCs were transfected with either control mimic or miR-203 mimic, maintained in culture for 15 days, and then differentiated in vitro. c, Left panel: mRNA levels of the indicated transcripts at different time points during cardiomyocyte differentiation. Right panel: The beating frequency (measured as the number of beats per 5 seconds) of these cardiomyocytes at day 15 of differentiation is also shown (n = 8 different clones). In b-c, data are presented as mean ± standard deviation (n = 2 independent experiments, each with 6 replicates). In a-c, *P < 0.05, **P < 0.01 (Student's t-test). [Figure 16]Transient exposure to miR-203 promotes differentiation into mature cardiomyocytes and improves cardiac regeneration. a) Representative image of a postnatal day 8 heart (7 days after cryoinjury). The cryoinjury area is highlighted. b) Representative images of cardiac sections from vehicle- and Dox-treated mice stained with Sirius Red (scale bar, 500 μm). Magnification details show the fibrotic area in white light (left) and polarized light (right). Scale bar in inset, 100 μm. c) Representative "black and white" images of cardiac sections stained with Sirius Red from a representative vehicle- or Dox-treated mouse, showing the fibrotic area in white 7 days after cryoinjury. Scale bar, 250 μm. Histograms show quantification of the percentage of fibrotic area relative to the total cardiac area for n = 15 mice per condition 7 days after cryoinjury. **P < 0.01 (Student's t-test). d, Immunohistochemical detection of Cd34 (mesodermal progenitor cells) and Sirius red staining of cardiac sections from vehicle- and Dox-treated mice. Representative images from three different mice are shown. Scale bar, 100 μm. Fibrotic areas are highlighted. e, Quantification of the percentage of survivors per litter 1 day after cryoinjury. Five litters per condition were tested. P = 0.053 (Student's t-test). [Figure 17] miR-203 is induced at the 2C stage during embryonic development. miR-203 expression in five different stages of normal early development as determined by qPCR: oocyte, 2-cell embryo, morula, compact morula, and blastocyst. RNA was extracted from 30 different embryos and pooled in two independent groups for analysis by qPCR. RNA expression was normalized by a control miRNA (miR-16). Data represent six different qPCR measurements. P=0.05 comparing 2C / morula vs. compact morula / blastocyst (Student's t-test). [Figure 18]Transient exposure to miR-203 induces 2C-like markers. a) Representative immunofluorescence analysis of ESC colonies stably expressing the 2C::tdTomato reporter and transiently transduced with GFP or miR-203-GFP viruses. Scale bar, 10 μm. The plot on the right shows the percentage of tdTomato-positive colonies among all GFP-positive colonies 24 h after transduction. Data are presented as mean ± standard error (n = 3 independent experiments). ***P < 0.001 (Student's t test). b) RNA expression of the indicated transcripts (known to harbor proximal upstream MERVL elements (Tcstv3, Zfp352, and Cml2) or intronic MERVL elements (Abcb5 and Chit1)) as determined by RNA sequencing. Data represent three independent wild-type (blue) or tKI (red) iPSC clones. *P<0.05; **P<0.01 (Student's t-test). c, Enrichment plot of the 2C signature (Biase et al., 2014) of 282 genes 10 and 25 days after Dox withdrawal in tKI iPSCs. d, Expression of the indicated transcripts encompassed in the 2C signature as determined by RNA sequencing. Data are mean ± standard error (n=3 independent experiments). *P<0.05; **P<0.01 (Student's t-test). e, Representative images of human pluripotent stem cells (hiPSCs) expressing the long terminal repeat (LTR7) of the HERVH endogenous retrovirus tagged with GFP. Cells were transfected with either control (left) or miR-203 mimic (right). White fields and GFP expression for the same colony are shown. Scale bar, 10 μm. f, The plot on the left shows the percentage of HERVH-GFP-positive colonies in the assay described in panel (E) 5 days after miRNA transfection. The plot on the right shows the percentage of HERVH-GFP-positive colonies, where GFP is expressed only at the periphery of the clone (black) or in the majority of cells comprising the colony (gray).Data are means ± standard error (n = 3 independent experiments; 508 colonies were counted for control mimics and 575 colonies for miR-203 mimics, as indicated). ***P<0.001 (Student's t-test). g, Representative images of EBs derived from HERVH hiPSCs transiently transfected with either control (left) or miR-203 mimics (right), as indicated in (E, F), at different time points during the differentiation process. Scale bar, 500 μm. h, Quantification of EB size from panel g) and the percentage of EBs presenting large internal cavities during the indicated differentiation time course. Data are means ± standard error (n = 3 independent experiments). ***P<0.001 (Student's t-test). [Figure 19] miR-203 induces naive pluripotency in cells cultured in 2i / LIF medium. a) Significantly deregulated pathways from the Gene Ontology database in tKI iPSCs (doxycycline-induced iPSCs) versus uninduced iPSCs (both cultured in 2i / L condition). b) Representative images of EBs derived from uninduced or doxycycline-induced iPSCs (tKI iPSCs) cultured in 2i / L condition for 10 passages at different time points during the differentiation process. Photomicrographs are representative of three different experiments. Scale bar, 500 μm. c) Quantification of EB size and the percentage of EBs displaying large internal cavities or beating from the same cells used in panels a) and b). Data are means ± standard error (n = 3 independent experiments). **P < 0.01 (Student's t-test). [Figure 20]miR-203 induces mild hypomethylation at imprinted genes. Heatmaps depicting methylation levels at the most variably methylated regions (DMRs; n = 100; left panel) or imprinted control regions (ICRs; n = 103 different imprinted loci; right panel) in the indicated samples. Grayscale is applicable to both heatmaps. Left panel: In tKI iPSCs, t = 0 has a methylation level close to 1.00, while at t = 10 it gradually decreases, and at t = 25 the methylation level approaches 0.00. In control KI iPSCs, methylation levels are between 0.75 and 1.00 at all indicated timepoints. Right panel: Slight hypomethylation is observed at the ICR at t = 25 in tKI iPSCs, not comparable to that detected at the DMRs shown in the left panel. DETAILED DESCRIPTION OF THE INVENTION

[0047] (Detailed Description of the Invention) The present invention, as described above, provides methods for improving the efficiency of pluripotent cells (particularly iPSCs) and therefore generating more suitable naive pluripotent cells for therapeutic approaches, most particularly for differentiation and maturation into differentiated cells useful for regenerative purposes.

[0048] The present invention is based on the results of the assays disclosed in the Examples of this application, which demonstrate that simple transient exposure to miR-203 sequences enhances the stemness potential of pluripotent cells and supports differentiation.

[0049] In the following examples, we demonstrate how transient overexpression of a single microRNA (miR-203) or its analogs enhances differentiation of pluripotent cells (both embryonic stem cells and induced pluripotent cells) while maintaining their stemness potential. miR-203 not only significantly improves stem cell marker expression, teratoma formation containing cell types from the three primitive germ layers, and differentiation efficiency into any lineage. More importantly, transient expression of miR-203 in iPSCs supports the stemness potential of cells, particularly those that produce viable offspring (as ESCs do). When iPSCs or ESCs are exposed to transient induction of the microRNA, both chimerism (particularly interestingly in tetraploid complementation assays) and germline transmission are dramatically increased. Thus, it can be said that pluripotent cells resulting from application of the methods of the present invention to iPSCs (i.e., by transiently subjecting iPSCs to increased levels of miR-203) enable the appropriate characteristics of pluripotent cells in a naive state, such that the resulting cells can be determined to be naive pluripotent cells. These results are consistent with additional studies performed by the present inventors and disclosed in the present application, in which miR-203 is identified as a microRNA preferentially expressed in mouse embryos at the 2C to morula stage during preimplantation development.

[0050] It is noteworthy that the effects mentioned (increasing the differentiation capacity of pluripotent cells and enhancing stemness potential in particular) are achieved by increasing the levels of miR-203 to which the cells are exposed (either by transient expression from a vector previously introduced into the cells or by adding miR-203 to the cell culture medium), since miR-203 has been considered by some authors as a stemness repressor (see Yi et al., 2008; Volinia et al., 2014), which limits stemness potential in the skin, and the asymmetric hsa-miR-302 (high) / has-miR-203a (low) has been found to be associated with stemness. In the same vein, even if a person skilled in the art had knowledge of the review by Huang et al. (Huang et al., 2011) and had read that miR-203 cooperatively regulates Sox2 and Klf4, without verifying what was actually stated in the source cited for that statement (Wellner et al., 2009, which states that miR-203 cooperates with miR-200c and miR-183 to repress stem factors, as commented above), that person would have expected that increased miR-203 would lead to poor stemness because miR-203 represses its targets. It could be said that all the information in the prior art leads one to predict that miR-203 expression, regardless of the cellular environment or differentiation state, would repress, not promote, stemness potential.

[0051] Therefore, it was not predictable that increasing miR-203 levels in ESCs or iPSCs that can be determined to be truly pluripotent and have not yet begun the process of commitment to a specific differentiated cell type or tissue (such as keratinocytes) would lead to improved stemness. In this regard, it is important to note that the assays disclosed in the present application and the results obtained are different from and do not interfere with those described by Nissan et al. (Nissan et al., 2011). This is because, as discussed above, Nissan et al. reported that miR-203 becomes significant in keratinocyte differentiation once hESCs have already been committed to epidermal differentiation by additional treatment (particularly treatment with BMP4), a condition under which miR-203 becomes a key factor involved in early keratinocyte commitment, which can be considered to be already known since 2008 (see Yi et al., 2008). This is different from what is disclosed in the present application, where transient induction of miR-203 is shown to be valid for the pursuit of a pluripotent naive state resulting from the improvement of recent differentiation commitment. These two concepts, as well as their significance in regenerative medicine, are entirely different. In other words, transient expression of miR-203 acts as a stimulator of the stemness potential of pluripotent cells, regardless of their subsequent differentiation commitment. This is demonstrated, for example, in the present application's assays involving embryoid bodies and teratomas, where improved differentiation potential into the three germ layers of pluripotent cells briefly exposed to miR-203 can be observed compared to their control counterparts.

[0052] Furthermore, it was difficult to assume that the results of the assays disclosed in this application were based on knowledge of the conclusions reached after studying the effect of miR-203 on nuclear survivin levels in hESCs (Kapinas et al., 2015), which led to the proposal of miR-203 as an inhibitor of pluripotency that acts by negatively regulating survivin expression.

[0053] Importantly, de novo DNA methyltransferases likely play a key role in the effects observed after transient overexpression of miR-203. The significant improvement in the efficiency of iPS cells in chimera generation and tetraploid complementation assays (which allows these cells to form complex teratomas and embryo-like structures in vivo) appears to be mechanistically mediated by direct miR-203-dependent repression of the de novo DNA methyltransferases Dnmt3a and Dnmt3b, leading to the erasure of global DNA methylation in pluripotent cells.

[0054] DNA methylation dynamics have been widely described to regulate both pluripotency and differentiation processes. Global DNA hypomethylation and maintenance of genomic imprints are known to be essential for ensuring chimeric contribution and germline transmission as a hallmark of pluripotency. While the naive pluripotent state is characterized by global DNA hypomethylation, the differentiated state correlates with higher levels of methylation and upregulation of the de novo methyltransferases Dnmt3a and Dnmt3b, as well as their counteracting protein Dnmt3l.

[0055] Interestingly, these de novo Dnmt downregulations, accompanied by upregulation of pluripotency-associated genes, are observed in cells of the early preimplantation epiblast. These characteristics are recapitulated in the naive state and can be sustained in vitro under certain conditions. The in vitro derivation of embryonic stem cells with intact genomic imprints is challenging, even in the context of global DNA hypomethylation. Imprint instability is routinely assessed in several ESC lines and cultures, where the DNA methylation profile is generally altered, leading to low-to-moderate efficiencies in chimerism, germline transmission, and differentiation. However, in our system, we observed global DNA hypomethylation in pluripotent cells transiently overexpressing miR-203, even though genomic imprints remained unchanged. We have detailed how miR-203 targets the de novo methyltransferases Dnmt3a and Dmt3b, reducing global DNA methylation and thus supporting naive pluripotency. Dnmt1 remains unaffected in these conditions, likely avoiding DNA methylation loss on genomic imprints. This is another proof-of-concept for the naive state of iPSCs and ESCs transiently overexpressing miR-203.

[0056] The lack of these DNA methyltransferases in ESCs is known to induce progressive chromatin hypomethylation with passage (Liao et al., 2015). Expression of miR-203-resistant Dnmt3a and Dnmt3b cDNAs rescues the miR-203-induced phenotype (Figures 9-15). Notably, while the severe and irreversible hypomethylation observed in Dnmt3a / b knockout cells blocks differentiation (Jackson et al., 2004; Okano et al., 1999), the miR-203-induced hypomethylated state is reversible, and differentiation is highly efficient and occurs accompanied by strong DNA methylation (Figure 7).

[0057] Thus, the method of the present invention has several important differences from previously known methods directed to improving the quality of cultured pluripotent cells, which can be summarized as follows. i) This method can be used on already established pluripotent clones (iPSCs and ESCs) and is therefore an additional step to the methods discussed above and can be combined with them. This is an important difference with respect to most variations of Yamanaka's original protocol, which attempt to increase the efficiency or safety of the method by replacing some of the original Yamanaka factors with other compounds or by adding additional compounds to carry out the reprogramming process. In this case, an additional compound (miR-203) is added to already established iPSCs or ESCs. ii) Pluripotent cells exposed to miR-203 exhibit enhanced function in generating differentiated and functional cells both in vitro and in vivo. iii) The effect of exposing pluripotent cells to increased levels of miR-203 can also be readily achieved through the use of synthetic small RNA molecules that are analogs (e.g., specific mimetics) of commercially available miR-203. iv) Mechanistically, miR-203 has been observed to exert its effect by erasing the epigenetic memory of pluripotent cells, a factor known to act as a barrier to the establishment of pluripotent cells. This is another important difference from methods using demethylases, for example, during the pluripotent cell reprogramming process, because the demethylation is either irreversible or induces cytotoxicity, rendering the resulting cells useless for regenerative medicine. This method, in which established iPSCs or ESCs are transiently exposed to increased levels of miR-203 (or its analogs), induces transient genome-wide hypomethylation, which improves their pluripotency. Because such hypomethylation is transient and reversible, it also allows for increased differentiation potential. Thus, this method generates naive pluripotent cells, which may be useful for obtaining differentiated cells applicable in regenerative medicine. v) Exposure of already obtained pluripotent cells to increased levels of miR-203 (or its analogs) (e.g., by transient expression, preferably) represents a safer alternative to some known methods, such as the use of DNA methylation inhibitors (e.g., AZA) during reprogramming, because AZA is known to induce cell death. In this sense, it should also be pointed out that miR-203, unlike other factors, is a well-established tumor suppressor, and its use also makes it possible to avoid problems associated with the use of oncogenic factors during reprogramming (Tapia et al., 2016).

[0058] As shown in the examples below, when compared to their control counterparts, the pluripotent cells obtained after carrying out the methods of the present invention exhibit improved stemness potential, which can be observed due to the following features, which are characteristic of pluripotent cells in the naive state: (i) In vitro differentiation into embryoid bodies (EBs) is significantly increased compared to control iPSCs. EBs generated from iPSCs that have undergone the steps of the method of the present invention (especially tKI iPSC-derived EBs) proliferate faster, differentiate better, beat more efficiently, and display a complete organization of the three germ layers. Their cellular differentiation and functionality are also evidenced by the formation of long cavities, which are rarely found in control iPSC-derived EBs. (ii) When iPSCs (especially tKI iPSCs) undergoing the steps of the method of the present invention are injected into mice, their in vivo differentiation into teratomas is also dramatically improved compared to their respective controls. Although the level of differentiation of the three germ layers is notoriously low, several atypical tissues (such as pancreas, bone marrow, cartilage, or even extraembryonic tissue (placenta)) are readily found in tKI iPSC-derived teratomas. When these tKI iPSCs are injected intraperitoneally into mice, they are able to generate complex embryo-like structures (never found with wild-type iPSC injections). These structures are characterized by the expression of multiple markers of embryonic development, the three germ layers, and extraembryonic tissues. (iii) Expression of embryonic 2-cell stage markers (e.g., certain retrotransposons) is higher in tKI iPSCs compared to their WT counterparts. Furthermore, transcriptome profiles of these iPSCs demonstrate how tKI iPSCs activate many signaling networks involved in development, morphogenesis, stemness maintenance, chromatin organization, and cell fate commitment, demonstrating their naïve state. Interestingly, principal component analysis, hierarchical clustering, and heatmap analysis of RNA-seq samples demonstrated significant proximity between naïve tKI iPSCs and ES cells compared to WT iPSCs. The tetraploid complementation assay is the most stringent assay for testing the pluripotent potential of iPSCs. Here, tetraploid blastocysts are produced via the fusion of 2-cell stage embryos and are developmentally defective by forming only extraembryonic tissues in vivo. As expected, fully pluripotent ES cells compensate for the developmental defects of tetraploid embryos, and full-term organisms can be produced from ESCs along with extraembryonic tissues derived from such tetraploid embryos. While the efficiency of generating live-born offspring is approximately 20% for ESCs in such assays, iPSCs typically fail this rigorous test and do not support "all-iPSC" mice. However, iPSCs transiently expressing miR-203 successfully generate live-born offspring by 4n complementation to an extent similar to that achieved using wild-type ESCs. Furthermore, ESCs exposed to transient miR-203 induction exhibit significantly higher competency in this test compared to wild-type ESCs. This data clearly asserts that miR-203 improves the efficiency of iPSC technology. (iv) Chimera contribution is arguably the best proof of concept to demonstrate the value of pluripotency (iPS or ES cells). Cell aggregation and, even more importantly, tetraploid complementation assay experiments demonstrate how tKI iPSCs contribute to significantly more chimera generation and germline transmission than their respective control iPSCs.

[0059] Additional data reinforcing the finding that exposure of ESC-iPSCs to increased levels of miR-203 promotes naive pluripotency and that the methods of the present invention are advantageous over other previously described methods aimed at sustaining pluripotency in vitro are as follows: (i) miR-203 expression peaks at the 2-cell stage and morula, consistent with data showing that this microRNA supports the expression of 2-cell stage-typical transcripts. This observation supports the fact that miR-203 is relevant for acquiring naive pluripotency in vivo and therefore can be used to advantage to boost naive pluripotency in vitro. (ii) Expression of miR-203 in PSCs induces the expression of genes typically expressed at the two-cell stage of the embryo. In addition to specific two-cell reporters, the expression of transcripts contained in the "two-cell transcriptome" was analyzed in detail. A notable finding is the fact that miR-203 induces 281 of the top 282 genes contained in the two-cell signature. Again, these observations reinforce the idea that miR-203 promotes naive pluripotency. (iii) miR-203 also significantly improves developmental potential in 2i / LIF conditions (the previous standard for maintaining stem cell potential). In addition, analysis of imprinting control regions indicates that miR-203 has little effect in these regions compared to 2i / LIF conditions, thus explaining the significant improvement of miR-203-treated cells in multiple in vivo assays. These results support the preferred use of miR-203 over previous methods aimed at sustaining in vitro pluripotency. (iv) The effects of miR-203 have also been tested in human cells, showing similar effects on the expression of two cell markers and differentiation potential, thus extending the observations in mouse pluripotent cells to human pluripotent cells. (v) The relevance of Dnmt3a / b as a significant target is demonstrated by rescue assays as well as by knockdown of Dnmt3a / b in WT PSCs, thereby mimicking miR-203. These data reinforce the identification of de novo DNA methyltransferase as a target of miR-203 responsible for the described phenotype.

[0060] In addition, pluripotent cells exposed to miR-203 exhibited enhanced differentiation and maturation potential, as demonstrated by the effects found below. (i) The number and level of differentiation of tissues is increased in tKI cells (pluripotent cells that have undergone the steps of the method of the present invention), as observed in embryoid body differentiation assays, the formation of complex teratomas, and embryo-like structures (including differentiated tissues not normally observed, such as bone marrow, placental tissue, or the exocrine pancreas). In addition, teratomas derived from tKI cells contain cells positive for the hormone insulin, whereas control teratomas do not, suggesting the presence of differentiated pancreatic β cells, further illustrating the increased ability of tKI cells to differentiate into multiple cell lineages. (ii) Differentiation and maturation of iPSCs into cardiomyocytes is improved, as detected by the expression of maturation markers in addition to functional assays. (iii) The differentiation of neonatal cardiomyocytes as well as the level of maturation they reach after exposure to miR-203 is improved. (iv) Tissue regeneration assays (assays typically not included in reports of pluripotent cell improvement given the complexity of properly performing complex in vivo regeneration assays with in vitro improved cells) showed that miR-203 expression significantly enhanced cardiac regeneration and overall survival after cardiac injury; the results enhanced the differentiation potential of miR-203-treated pluripotent cells and their functionality in vivo, suggesting the relevance of this microRNA in regenerative medicine.

[0061] It is noteworthy that the method of the present invention differs from the method and means for deriving cardiomyocytes from iPSCs or ESCs disclosed in International Patent Application WO2014201254A1 (in which microRNAs of the let-7 family are the only microRNAs mentioned in said international patent application as important for in vitro cardiac maturation starting from pluripotent cells, and no other microRNAs are mentioned). The present invention then provides an alternative to previously suggested methods for cardiomyocyte differentiation, starting from pluripotent cells that have previously undergone a transient increase in the level of miR-203 (either by transient expression in the cells or by transient exposure of the cells to miR-203 or its analogs by addition to their culture medium), which pluripotent cells subsequently undergo cardiomyocyte differentiation, achieving improved efficacy since the resulting cardiomyocytes are mature and functional.

[0062] Considering their potential usefulness for future clinical applications, it can be determined that an important embodiment of the method of the present invention is the promotion of the differentiation potential of cells, characterized by improved differentiation efficiency, for example, into cardiomyocytes, and also into other differentiated cells, particularly those of clinical interest (cells of the nervous system (e.g., neurons and glial cells), chondrocytes, pancreatic beta cells, etc.).

[0063] miR-203 improves the potential of established ES cells and iPS cells to contribute to multiple cell lineages. Therefore, the methods of the present invention can also be defined as methods for improving the stemness properties / potential of established pluripotent cells (as they operate on embryonic stem cells and induced pluripotent stem cells) and / or promoting the differentiation and / or maturation potential of pluripotent cells. Thus, pluripotent cells that have undergone the steps of the methods of the present invention exhibit a) a higher percentage, faster proliferation, better differentiation, more efficient beating, and better architecture of the three germ layers when differentiated into embryoid bodies compared to control cells; b) improved in vivo differentiation into teratomas; and c) higher expression of two-cell stage markers. Here, with regard to enhanced differentiation and / or maturation potential, pluripotent cells that have undergone the steps of the methods of the present invention exhibit: a) an increased number of tissues and higher levels of differentiation compared to controls when differentiated into embryoid bodies; b) the formation of complex teratomas and embryo-like structures containing differentiated tissues not normally observed in control conditions; c) improved differentiation and maturation potential (including faster increased expression of maturation markers and faster acquisition of their functionality) when differentiated specifically into cardiomyocytes.

[0064] As commented above, the effects of exposure to increased levels of miR-203 or its analogs (by overexpression or addition to the culture medium) can be observed in both induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs). For the purposes of the present invention, both iPSCs and ESCs can be cells derived from any mammal. Human ESCs can be optionally excluded. If the ESCs are human ESCs, it is preferable that the human ESCs used in the methods of the present invention are obtained by a method that does not involve the destruction of embryos (such as the method described in Chung et al., 2008). However, due to the moral and legal issues related to the use of human ESCs and for handling reasons, it is preferred that the methods of the present invention be performed with iPSCs. For ease of generation and handling, it is particularly preferred that iPSCs are in culture when the methods of the present invention are applied thereto. It is also preferred that the generation of said iPSCs occurs in vitro from cultured differentiated cells (such as fibroblasts), although the use of in vivo-generated iPSCs (Abad et al., 2013) can be considered compatible with and within the scope of the methods of the present invention. To obtain iPSCs generated in vitro (in culture), any of the known reprogramming procedures can be used, including the original Yamanaka method (contacting somatically differentiated cells with nuclear reprogramming factors comprising gene products from each of the following families: Oct family, Klf family, Myc family, and Sox family) and any of its variations.

[0065] The present examples include several assays in which increased levels of microRNA-203 were obtained thanks to genetically induced overexpression in iPSC cells, resulting from the fact that such cells were reprogrammed in vitro from differentiated cells (fibroblasts) extracted from a mouse model (containing a doxycycline-inducible cassette for miR-203 expression). In that case, the increased levels then result directly from intracellular overexpression of miR-203, and the increase occurs transiently due to the fact that doxycycline is only added for 3-5 days. Other assays (such as some of the assays described in Example 4) were performed with wild-type iPSCs in culture, and exposure of iPSCs to increased levels / amounts of miR-203 was achieved by adding miR-203 (particularly chemically modified double-stranded miR-203) to the culture medium. The results obtained with this mimic of native miR-203 indicate that adding miR-203 to the culture medium is a possible embodiment of the method of the present application and also solves the same technical problem. Because miR-203, or its analogs or mimics, must enter cells to exert their effects, the amount of endogenous miR-203 (naturally present in cells) plus the amount of miR-203 resulting from the penetration of added compounds from the culture medium analogously results in the cells being exposed to increased levels of miR-203 (or analog compounds with the same type of activity). For that reason, a necessary step in carrying out the method of the present invention was defined as exposing cells to increased levels of miR-203. Even if the starting level is determined to be zero (0) (as in the culture medium or in some of the models used in the following examples), it can be determined to increase from 0 to a certain amount.

[0066] Such an increase in miR-203 levels can be achieved by using expression vectors known in the state of the art in which miR-203 can be expressed, for example, coding sequences for immature precursor forms (e.g., hsa-miR203 represented by SEQ ID NO: 2 in the case of humans or mmu-miR-203 represented by SEQ ID NO: 3 in the case of Mus musculus), which will give rise to the corresponding mature forms in the cells (hsa-miR-203a-3p represented by SEQ ID NO: 1 for humans or mmu-miR-203-3p represented by SEQ ID NO: 4 for Mus musculus). It can be observed that the mature forms in humans and mice are identical, so the use of certain precursor and / or mature forms that differ from the species of origin of the pluripotent cells, particularly in humans, is compatible with and falls within the scope of the method of the present invention, both when the microRNA is added directly to the culture medium or when it is produced from a vector or as an expression product from a vector.

[0067] A similarly possible embodiment is to start with a mutated differentiated cell in whose genome an expression cassette is inserted, from which a microRNA or its precursor is expressed, as in some examples of the present invention.

[0068] Because transient expression (e.g., 3-5 days) is preferred, the coding sequence giving rise to the microRNA or its precursor is preferably under the control of an inducible promoter (such as the tetracycline-inducible promoter (e.g., inducible by doxycycline) used in the examples of this application), so that the duration of expression is easily controllable and can be terminated by removing the inducible compound. The expression vector can also be a plasmid or a non-integrating virus-derived vector (e.g., derived from a virus such as an adenovirus or adeno-associated virus), which by itself facilitates transient expression without additional control and may be considered another possible embodiment.

[0069] The products of expression can be miRNA and mRNA, which presents a portion corresponding to the molecule's coding sequence for a protein or tag that can be used, for example, as a marker.

[0070] Another possible embodiment (and one of the preferred ones) is to add miR-203 or its analog to the culture medium of already generated pluripotent cells and, if transient exposure is desired, remove it from the culture medium upon completion of the desired exposure time (e.g., 3-5 days, as previously commented). Transient transfection of such a microRNA mimic allows for transient exposure (approximately 3-5 days, as previously commented). In this case, both mature and precursor miR-203 molecules can be added. As discussed above, in the case of miR-203, the most abundant mature form is that arising from the 3' arm of the pre-miRNA (hsa-miR-203a-3p in humans and mmu-miR-203-3p in mice), which is the form responsible, at least to a high degree, for the effects described in this application. It is therefore the form preferentially used in the present method, and it is a possible embodiment of the method in which, as in some assays of the present application, pluripotent cells are subjected to a mixture of both mature forms (those arising from the 3' arm and those arising from the 5' arm), thereby faithfully mimicking the endogenous situation in the cell.

[0071] As demonstrated in some examples of the present invention, synthetic compounds can be used in place of naturally occurring molecules of miR-203. The use of "analogs" is particularly contemplated. RNA analogs are molecules that resemble naturally occurring microRNAs but contain at least modifications that make them distinct and distinct. Included within the definition of an RNA analog are RNA molecules in which at least one nucleotide is replaced by another. Given that the complementarity between microRNAs and the fragments of the 3' UTR of mRNAs with which they base pair is rarely perfect (100%), changes in the microRNA sequence can be tolerated as long as base pairs are still present, so that the RNA analog can be at least 50-60% similar to naturally occurring microRNAs, provided that its function is still achieved. For that purpose, one must consider that there is always a region of 6-8 nucleotides (known as the seed region) where the complementarity within the microRNA and mRNA 3'UTR is perfect (100%) or almost perfect (see Figure 10 for the seed region for the 3'UTR of Dnmt3a and Dnmt3b), and consequently it is recommended not to modify the nucleotides in the seed region.

[0072] More commonly, but compatible with the preceding modifications, are chemical modifications in nucleotides (units of microRNA) that result in nucleotide analogs. Such modifications are typically performed to increase stability and / or resistance to nucleases, facilitate entry into cells, increase the strength of interaction between microRNA and mRNA, increase the desired activity of microRNA, and / or bias the processing of microRNA through specific cellular pathways (such as the uptake of small interfering RNA or one strand of microRNA and use it as a template for recognition of complementary mRNA, which then activates RNase and cleaves the RNA, RISC (RNA-induced silencing complex)). Such modifications are typically present in the sugar moiety and / or phosphate linkage and include the addition of one or more non-nucleotide moieties. Some common modifications include phosphorothioate bonds, which are commonly used instead of phosphate bonds; modifications at the 2' position of the sugar moiety (such as 2'-O-methyl or 2'-O-methoxyethyl); modifications in which the ribose presents a link connecting the 2' oxygen and the 4' carbon, thus blocking the ribose in the 3'-endo conformation (LNA: locked nucleic acid) or 2'-O,4'-C ethylene-bridged nucleic acid (ENA); replacement of the sugar backbone with an amide-containing backbone (such as the aminoethylglycine backbone in peptide nucleic acid (PNA)); the use of PMOs (nucleic acids in which the ribose moiety is replaced by a morpholine group); and other modifications well known to those skilled in the art, which can be found, for example, in the reviews by Kole et al. (2012). Because nitrogenous bases are less commonly modified elements in nucleotide analogs, comparisons of their homology or identity with fragments or entire sequences of natural oligonucleotides or polynucleotides are more appropriately performed with respect to the sequence of nitrogenous bases.Modifications at at least one of the chain ends (such as the addition of one or more unit moieties of compounds such as cholesterol, cholestanol, stigmasterol, cholanic acid, and ergosterol, and optionally further linker moieties that attach additional conjugate moieties to the chain) are also particularly common to facilitate entry of the microRNA analog into cells.

[0073] RNA mimics, particularly microRNA mimics (miRNA mimics), are also encompassed within the microRNA analogs used in this application. RNA mimics (as commented above in relation to US20130345289A1) are synthetic miRNAs with enhanced stability due to modified nucleotides or structural modifications (e.g., bulges or loops), and are also double-stranded RNAs chemically modified with small molecules that mimic endogenous miRNAs and enable miRNA functional analysis by upregulating miRNA activity. MicroRNA mimics added to the culture medium are taken up by cells and act directly as double-stranded molecules (cell expression is not required) that faithfully mimic the respective microRNA (in this case, miR-203). As in some examples of the present invention (e.g., Example 4), a method in which miR-203 mimics (single or a mixture thereof) are added to the culture medium of iPSCs is a preferred embodiment of the present invention, particularly when the mimics are characterized by: a. An RNA modified molecule in which at least one of the nucleotides is replaced by a chemically modified nucleotide, wherein the chemical modification is i. replacement of one or more phosphate linkages with phosphorothioate linkages; ii. one or more modifications at the 2' position of the sugar moiety selected from a 2'-O-methyl modification or a 2'-O-methoxyethyl modification; and / or iii. One or more modifications in the ribose moiety selected from the group consisting of those that create a link connecting the 2' oxygen and the 4' carbon, thus blocking the ribose in the 3'-endo conformation (LNA: locked nucleic acid) or 2'-O,4'-C ethylene-bridged nucleic acid (ENA); replacement of the sugar backbone with an amide-containing backbone (such as the aminoethylglycine backbone in peptide nucleic acid (PNA)); and the use of PMO (nucleic acid in which the ribose moiety is replaced by a morpholine group), and combinations thereof. selected from the group the molecule; b. A double-stranded molecule having a double-helical region of 16 to 31 nucleotides in length, which contains a fragment that is at least 50% identical in sequence to the nitrogenous base sequence of an RNA molecule represented by SEQ ID NO: 1 (hsa-miR-203a-3p) or SEQ ID NO: 4 (mmu-miR-203-3p); c. optionally, at least one end of at least one of the chains comprises a conjugate moiety comprising one or more units of cholesterol, cholestanol, stigmasterol, cholanic acid, and ergosterol, and optionally further comprises a linker moiety further attaching the conjugate moiety to the chain; d. Optionally, further presenting one or more mismatches in the two strands.

[0074] Examples of specific microRNA mimetics, which are specific examples of the mimetics described above, can be found, for example, in US Patent Applications US2009 / 0209626 and US2011 / 0263675 (both of which are published by Dharmacon, Inc.). Highly preferred are microRNA mimetics such as Dharmacon's miRIDIAN series of miR-203 mimics (http: / / dharmacon.gelifesciences.com / rnai-and-custom-rna-synthesis / microrna / miridian-microrna-mimics / ), which commercialize miR-203 mimics for Homo sapiens, Mus musculus, and other species (such as Rattus norvegicus) and are used in the assays of the present application. miRIDIAN mimics are chemically facilitated by an ON-TARGET modification pattern to preferentially program RISC with the active microRNA strand. This modification encompasses RNA in which the first and second nucleotides of each sense region have a 2'-O-methyl moiety, and the antisense strand is phosphorylated at its 5' end, whereby this on-target modification is also referred to as the dedicated modification created by On-Target™ (Dharmacon, Inc.). In any event, on-target modification can be used to help reduce off-target effects by blocking the sense (passenger) strand from being incorporated into the RISC process. In any case, other examples of microRNA mimics can be found, for example, at Sigma Aldrich, which also commercializes hsa-miR-203a mimics (HMI0357).

[0075] The naive pluripotent cells resulting from the practice of the methods of the invention starting from iPSCs differ from the starting iPSCs, as seen in Table 3. The resulting naive pluripotent cells, when subjected to well-established differentiation protocols, continue to express pluripotency markers (such as Nanog, Oct, and Sox2), but they coexist with cells expressing differentiation markers (such as Nestin, Gata4, and CD34). Thus, resulting cell populations (particularly those obtained by the methods of the invention) characterized by the expression of both pluripotency and differentiation markers are also a goal of the invention.

[0076] The assays disclosed in the Examples below demonstrate that transient regulation of miR-203 expression in iPSCs and also in ESCs improves the ability of these cells to differentiate into multiple cell lineages and achieve further mature characteristics without interfering with their self-renewal properties. Thus, the method of the present invention and the pluripotent stem cells obtained therefrom open up new possibilities for the clinical application of pluripotent stem cells and resolve some of the remaining challenges for seriously considering and planning their use for clinical purposes.

[0077] Pluripotent stem cells have the potential to become research and clinical tools for understanding model diseases, developing and screening drug candidates, and providing cell replacement therapies to support regenerative medicine. Reprogramming technology offers the potential to treat many diseases, including neurodegenerative diseases, cardiovascular diseases, diabetes, and amyotrophic lateral sclerosis (ALS). Theoretically, readily accessible cell types (such as skin fibroblasts) could be biopsied from patients, reprogrammed, and effectively reproduce the patient's disease in a culture dish. Such cells could then serve as the basis for autologous cell replacement therapy. Because the source cells are endogenous to the patient, immune rejection of differentiated derivatives would be minimized. Furthermore, while iPSCs have great potential as a source of adult, mature cells, much remains to be learned about the differentiation process of these cells. The methods of the present invention and the pluripotent cells obtained therefrom can be considered new and improved research tools that can be used to understand the differentiation process of pluripotent cells and to make useful improvements to facilitate and make more feasible the clinical application of these differentiated cells, particularly for regenerative medicine. Thus, methods of obtaining differentiated cells from iPSCs, in addition to the use of pluripotent cells of the invention to obtain differentiated cells, including steps such as those characterizing the methods of the invention (exposing cells to increased levels of miR-203), methods of obtaining differentiated cells using pluripotent cells of the invention as starting material, are within the scope of the invention.

[0078] Possible uses of the present technology (the methods of the present invention and the cells obtained therefrom) include the following: In the field of cardiac regeneration, iPSCs generated from human and mouse fibroblasts can give rise to functional cardiomyocytes that exhibit hallmark cardiac action potentials. However, when iPSCs are used, the maturation process into cardiomyocytes is impaired. Cardiac development of iPSCs is delayed compared to that observed in cardiomyocytes derived from ESCs or embryonic tissue. Furthermore, variability exists in the expression of genetic markers in iPSC-derived cardiac cells compared to that observed in ESC-derived cardiomyocytes. Thus, while iPSC-derived cardiomyocytes demonstrate normal commitment but impaired maturation, it is unclear whether the observed defects are due to technical issues (e.g., incomplete reprogramming of iPSCs) or biological barriers (e.g., functional defects due to genetic factors). Neuroregeneration, including the modeling and treatment of neurodegenerative diseases (such as Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, or spinal muscular atrophy), is currently being studied. Patient-derived iPSCs can be reverted to affected neuronal subtypes via in vitro differentiation, or iPSCs can be regenerated using gene targeting to repair disease-causing mutations. Protocols for the application of iPSC-based regeneration in neurodegenerative disorders remain a long way off. Furthermore, greater progress has been made in generating distinct mature populations of neurons and glia for the purpose of screening drugs and replacement therapies. However, these cells may not faithfully reflect the cellular responses to compounds present in the body at physiological levels. Finally, researchers still face the challenges of low conversion efficiency and the associated labor. The average conversion efficiency of such methods is less than 1%, further limiting the widespread use of the technology. Remodeling in cartilage regeneration systems. Because articular cartilage does not recover through spontaneous healing, numerous attempts have been made to improve the quality of this tissue repair. Autologous cartilage transplantation has shifted the treatment paradigm for cartilage defects from repair to regeneration, as demonstrated in a randomized trial that proved the concept of tissue regeneration with a cell therapy approach. In this scenario, we speculate that miR-203 treatment of autologous chondrocytes prior to transplantation may facilitate remodeling of the damaged tissue and its complete regeneration.

[0079] Since transient control of miR-203 expression in iPSCs and also in ESCs improves the ability of these cells to differentiate into multiple cell lineages (especially into cardiomyocytes) and achieve further mature properties without interfering with their self-renewal properties, the method of the present invention represents an important improvement for the generation of differentiated cells applicable in the above-mentioned fields. Therefore, the use of pluripotent cells obtained by the method of the present invention to obtain differentiated cells applicable in the above-mentioned fields (cardiac regeneration, nerve regeneration, and cartilage regeneration) (i.e., cardiomyocytes, nerve or glial cells, chondrocytes, insulin-producing cells) is a preferred embodiment of the use of the pluripotent cells of the present invention.

[0080] It should be noted that, as shown in the examples of the present application relating to cardiomyocyte differentiation and maturation (especially Example 4), transient exposure to miR-203 sequences improves both functional differentiation and maturation. Thus, the possible therapeutic use of this microRNA in regenerative medicine (such as nerve regeneration, cartilage regeneration, replacement of insulin-secreting cells, and very particularly cardiac regeneration) is supported by the assays disclosed in the present application.

[0081] Taken together, it can be concluded that modulating the DNA methylation landscape of pluripotent cells by exposure to small molecule microRNA mimics can promote the performance of these cells in multiple functional assays, including differentiation and maturation into multiple cell lineages of interest in regenerative medicine.

[0082] The invention will be explained in more detail by means of the examples and figures described below. [Example]

[0083] Summary of Experimental Procedure We generated a mouse model in which miR-203 expression can be regulated by doxycycline (DOX) treatment. These knock-in animal models (ColA_miR203; Rosa26_rtTA) allow for over 100-fold overexpression of this microRNA whenever the animal or cells derived from the animal are exposed to doxycycline treatment. Our first approach was to extract mouse fetal fibroblasts from these mice and reprogram them in vitro using Yamanaka factors to generate miR-203 knock-in inducible pluripotent cells (miR-203 KI iPSCs). These iPSCs were then treated with DOX for 3–5 days and then exposed to DOX withdrawal for multiple passages. For simplicity, we termed these iPSCs "transient KI (tKI) iPSCs." Multiple readouts were then analyzed to demonstrate how the tKI iPSCs exhibit improved stemness potential when compared with their control counterparts (identical iPSC clones treated with vehicle): (i) In vitro differentiation into embryoid bodies (EBs) is significantly increased in tKIs compared with control iPSCs. tKI iPSC-derived EBs proliferate faster, differentiate better, beat at a higher percentage and efficiency, and display a complete organization of the three germ layers. Their cellular differentiation and functionality are also manifested by the formation of long cavities, which are rarely found in control iPSC-derived EBs. (ii) Compared with their respective controls, tKI iPSCs also dramatically improve in vivo differentiation into teratomas when injected into mice. Although the level of differentiation of the three germ layers is notoriously low, several atypical tissues, such as pancreas, bone marrow, cartilage, or even extraembryonic tissue (placenta), are readily found in tKI iPSC-derived teratomas. When these tKI iPSCs were injected intraperitoneally in mice, they were able to generate complex embryonic-like structures (never observed with WT iPSC injections) characterized by the expression of multiple markers of embryonic development, the three germ layers, and extraembryonic tissues.(iii) Expression of two-cell stage markers (e.g., certain retrotransposons) is higher in tKI iPSCs compared to their WT counterparts. Furthermore, transcriptome profiles of these iPSCs demonstrate how tKI iPSCs activate many signaling networks involved in development, morphogenesis, stemness maintenance, chromatin organization, and cell fate commitment, demonstrating their naïve state. Interestingly, principal component analysis, hierarchical clustering, and heatmap analysis of RNA-seq samples demonstrated significant proximity between tKI iPSCs and ES cells compared to WT iPSCs; (iv) chimera contribution is arguably the best proof of concept for demonstrating the value of iPS or ES cells. The provided experiments on cell aggregation and, more importantly, tetraploid complementation assays demonstrated how tKI iPSCs contribute significantly more to chimera generation and germline transmission than their respective control iPSCs.

[0084] Our second approach to further validate the data was to replicate these proof-of-concept findings in several other different models. Thus, we demonstrated that the same effect was observed when miR-203 was transiently overexpressed in multiple clones of wild-type iPSCs or wild-type ESCs. The quality of the pluripotent cells (either iPSCs or ESCs) transiently transfected with miR-203 was, in all cases, better than their control counterparts. We also used a silencing retrovirus (pMCSV) in pluripotent cells several days after transduction to replicate the overexpression, allowing for transient expression of microRNAs. Finally, and very importantly, the data were replicated using miR-203 transfection with microRNA mimics and conventional methods for RNA transfection, such as Lipofectamine RNAi Max (Invitrogen) or Dharmafect (Dharmacon).

[0085] The example assays described below were performed using the following materials and methodology.

[0086] Animal models and procedures. A miR-203 conditional knockout model was generated by flanking the mmu-mir203 locus with loxP sites using homologous recombination into ES cells (ET recombination; Genebridges, Heidelberg, Germany) (Fig. 1a). A neomycin resistance cassette was used for selection of recombinant clones. Recombination of the frt site was achieved using a CAG-Flpe transgene (Rodriguez et al., 2000), and recombination of the loxP site was achieved using an EIIa-Cre transgene (Schwenk et al., 1995), resulting in the miR-203(- / -) allele (Fig. 1a). Following a previously reported strategy (Beard et al., 2006), we generated an miR-203-inducible model by cloning the genomic mmu-mir203 sequence (chromosome 12: 112130880-112130955; miRBase database release 21.0) into the pBS31 vector for recombination into the ColA1 locus in ES cells. The resulting knock-in allele [ColA1(miR-203)] was combined with the Rosa26-M2rtTA allele [Rosa26(rtTA)] for doxycycline-dependent induction, as previously described (Beard et al., 2006) (see Figure 1c for details). These animals were maintained on a mixed genetic background of C57BL6 / J × 129 × CD1.

[0087] Animal experiments were performed in accordance with protocols approved by the CNIO-ISCIII Ethics Committee for Research and Animal Welfare (CEIyBA).

[0088] For subcutaneous teratomas, iPSCs were trypsinized, and 2–3 million cells were suspended in 100 μl of PBS supplemented with 0.1% glucose and injected subcutaneously into both flanks of athymic nude mice, Crl:NU(NCr)-Foxn1nu (provided by Charles River). Teratomas were monitored daily, measured with a caliper, and finally isolated when they reached a diameter of 1.5 cm. Animals were euthanized at that time, and teratomas were weighed and processed for RNA extraction or histopathological analysis.

[0089] For intraperitoneal administration, wild-type athymic mice were given 4–5 × 10 cells resuspended in 100 μl of PBS supplemented with 0.1% glucose. 5 Mice were injected with iPSCs. They were monitored daily from the day of injection. Typically, 30 or 40 days after injection, mice were euthanized, and visceral teratomas and embryonic structures were isolated and processed for either RNA extraction or histopathological analysis.

[0090] For chimera generation, iPSCs or ESCs (5–7 cells per embryo, 10 passages) were microinjected into C57BL / 6J-Tyrc-2J / J blastocysts and transferred into Crl:CD1(ICR) pseudopregnant females.

[0091] For tetraploid complementation studies, zygotes were cultured overnight until they reached the two-cell stage, or two-cell stage Hsd:ICR(CD-1) embryos were harvested from pregnant females at E1.5 and electrofused in 0.3 M mannitol using a BLS CF-150 / B cell fusion instrument equipped with a 250 μm electrode chamber. The electric pulse conditions were 30 V amplitude, 31 μs duration, and 1.5 AC voltage. After 1 h, one-cell (tetraploid) embryos were carefully identified and separated from unsuccessfully fused embryos and cultured in KSOM for an additional 1 or 2 days. The following day, four-cell stage embryos were selected and aggregated with ESCs or iPSCs. The aggregated embryos were then injected into pseudopregnant females 24 h later.

[0092] To study germline contribution, black ES-iPS mice (from tetraploid complementation assays) or chimeras (from microinjection of diploid embryonic ES-iPS) were crossed with albino B6 or C57BL / 6J-Tyrc-2J / J females, and the color of the offspring was tested.

[0093] Cell culture and gene expression. Primary mouse embryonic fibroblasts (MEFs) were obtained from E13.5 embryos and cultured in DMEM supplemented with 10% FBS and penicillin / streptomycin. Cultures were routinely tested for mycoplasma. Reprogramming was induced in these MEF cultures by Oct4-Sox2-Klf4-cMyc (OSKM) lentiviral transduction (Takahasi & Yamanaka, 2006). For lentiviral transduction, HEK293T cells were transfected with Tet-O-FUW-OSKM (Addgene #20321) and packaging vectors using Lipofectamine 2000 (Invitrogen). Viral supernatants were collected twice daily for two consecutive days, starting 24 hours after transfection, and used to infect MEFs pre-plated at a density of 250,000 cells per well in 6-well plates. Prior to infection, polybrene was added to the viral supernatant at a concentration of 2 μg / ml. The infected MEFs were then cultured in IPSC medium (KO-DMEM (Gibco), 50 mM 2-mercaptoethanol (Invitrogen), non-essential amino acid MEM NEAA (Invitrogen), penicillin and streptomycin (5000 μg / ml, Invitrogen), LIF (leukemia inhibitory factor, ESGRO, Millipore), and 20% Knockout serum replacement (KSR, Invitrogen)). The medium was changed every 24 hours, and plates were stained for alkaline phosphatase activity (AP detection kit, Sigma-Aldrich) to confirm reprogramming efficiency. Once colonies were picked, IPSCs were cultured in IPSC medium on mitomycin C (Roche)-inactivated feeder cells. G4 ESCs were cultured on mitomycin C-inactivated feeders in the presence of ESC medium (containing KO-DMEM, 2-mercaptoethanol, non-essential amino acids, Glutamax, penicillin and streptomycin, LIF, and 10% fetal bovine serum (Hyclone)).Where indicated, culture medium for pluripotent cells contained 2i factors (1 μM MEK inhibitor PD0325901, 3 μM Gsk3 inhibitor CHIR99021) and mouse LIF (as above) in N2B27 medium as previously described ( Ying et al., 2008 ).

[0094] To induce transient miR-203 overexpression in miR-203 KI cells, cultures of ColA1 (miR-203 / miR-203), Rosa26 (rtTA / rt / TA) iPSCs, or ESCs were treated with doxycycline (1 μg / ml; Invitrogen) for 5 days. Afterward, doxycycline withdrawal was standard for subsequent culture passages (15–30 days). Doxycycline treatment was also applied to wild-type ESCs or iPSCs to assess the effect of the treatment itself.

[0095] For overexpression experiments, full-length cDNAs of miR-203 and Dnmt3a and Dnmt3b were subcloned into the retroviral vector pMCSV-PIG (available via Addgene plasmid 21654: https: / / www.addgene.org / 21654 / ) (Abad et al., 2013) by restriction-directed subcloning using the pCMV-Sport6-mDnmt3a (MGC clone: ​​5662) and attB-mCh-mDnmt3b-Poly(A)-NeoR (Addgene plasmid 65553) plasmids as templates, respectively. Upon transduction with these retroviral vectors, both ESCs and iPSCs were sorted by FACS, and GFP-positive cells were selected for subsequent culture and analysis.

[0096] For retroviral transduction, HEK293T cells were transfected with pMCSV-PIG vectors (Abad et al., Nature 2013) expressing GFP reporters (GFP alone; miR-203-GFP; Dnmt3a-GFP; or Dnmt3b-GFP) and the packaging vector pCL-ECO using Lipofectamine 2000 (Invitrogen). Viral supernatants were collected twice daily for two consecutive days, starting 24 h after transfection, and used to infect either ESCs or iPSCs pre-plated on feeders in six-well plates. Prior to infection, polybrene was added to the viral supernatant at a concentration of 2 μg / ml.

[0097] For mimic transfection, we used the miRIDIAN microRNA human hsa-miR-203a-5p (C-302893-00) / hsa-miR-203a-3p (C-300562-03) mimics from Dharmacon, or a mimic transfection control with Dy547 (CP-004500-01). Thus, cells received a mixture of analogs / mimics of both mature forms of microRNA-203, closely mimicking the endogenous situation in cells. Transfection was performed using either Dharmafect transfection reagent (Dharmacon) or Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions. Transfection efficiency was assessed by Dy547 fluorescence 24 hours after transfection, and experiments were then performed as indicated in the figures.

[0098] For RNA interference assays, ON-TARGETplus SMARTpools for non-targeting control siRNA (D-001810-01, 02, 03, 04), Dnmt3a siRNA (J-065433-09, 10, 11, 12), and Dnmt3b siRNA (J-044164-05, 06, 07, 08) from Dharmacon were used. Transfection was performed using Darmafect transfection reagent (Dharmacon) according to the manufacturer's instructions. Transfection efficiency was assessed by PCR 24 hours after transfection using the primers indicated in Table 2.

[0099] Luciferase reporter assays were performed in HEK293T cells. Briefly, 200,000 cells per well were seeded onto a 6-well plate, and the cells were transfected the next day using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The 3'UTR regions from mouse genes Dnmt3a, Dnmt3b, Dnmt3l, and Dnmt1 were amplified using cDNA clones (pCMV-Sport6-mDnmt3a, cDNA clone MGC: 5662; pBluescript-mDnmt3l, RIKEN clone: ​​2410006021; pYX-Asc-mDnmt1, MGC clone: ​​62302) or mouse cDNA (in the case of Dnmt3b) using specific primers (Dnmt3a_EcoRI-Fw: 5'-GAATTCAGGGACATGGGGGCAAACTGAA-3' (SEQ ID NO: 55); Dnmt3a_NdeI-Rv: 5'-CATATGCTGAGGCAGTCATTTTAGATTCAT-3' (SEQ ID NO: 56); Dnmt3b_EcoRI-Fw: 5'-GAATTC TTTAGCTCACCTGTGTGGGG-3' (SEQ ID NO: 57); Dnmt3b_NdeI-Rv: 5'-CATATGCCAGAAAGGTAAACTCTGGGCA-3' (SEQ ID NO: 58); Dnmt3l_EcoRI-Fw: 5'-GAATTCGAAATGAATCACCATAAGATGAAAG-3' (SEQ ID NO: 59); Dnmt3l_NdeI-Rv: 5'-CATATGAACAATCCTATGATATATTTGAAAAA-3' (SEQ ID NO: 60); Dnmt1_EcoRI-Fw: 5'-GAATTCGTGCTCTCACCCAGAGCCCCA-3' (SEQ ID NO: 61); Dnmt1_NdeI-Rv: 5'-CATATGGCTTGACAGAAGCGCTTTATTTTG-3' (SEQ ID NO: 62)). The PCR product was verified by sequencing and ligated into the pGL3-Control vector (Promega) downstream of the luciferase reporter gene. Mutations in the miR-203 binding site (Figure 10) were generated by site-directed mutagenesis and subsequently verified by sequencing.Transfection was performed with either the pMCSV-GFP or pMCSV-miR-203-GFP vectors combined with pGL3-derived vectors and Renilla as a control. Luciferase measurements were achieved 48 hours after transfection using a luminescence microplate reader (Biotek). Finally, to detect endogenous expression of the 2C stage retrotransposon MuERV-L, ES cells expressing the 2C::tdTomato reporter were cultured in 2i medium on feeders as previously reported (Macfarlan et al., 2012). This retrotransposon is specifically induced in totipotent two-cell blastomeres. MERVL expression is rarely detectable in pluripotent stem cells in culture, but only in a very low proportion (less than 0.5% of the culture).

[0100] Human iPSCs expressing the long terminal repeat (LTR7) of the HERVH endogenous retrovirus fused to a GFP reporter (Wang et al., 2016) were cultured in mTeSRTM1 medium (Stem Cell Technologies) on a Matrigel base (Corning). Experiments with human cells were performed according to protocols approved by the ISCIII Ethics Committee for Research (CEI; number PI 61_2017).

[0101] Embryoid body generation. Briefly, when wild-type iPSCs or ESCs were used, they were either pre-transduced with a retrovirus expressing miR-203 (pMSCV-miR-203) or transfected with a miRNA mimic for transient expression of miR-203, as can be seen in the schematic representation in Figure 4a. iPSCs or ESCs were trypsinized and resuspended to a concentration of 200,000 cells / ml in the presence of complete growth medium lacking leukemia inhibitory factor (LIF). Small droplets (approximately 25 μl) of this suspension were collected and seeded onto the lids of 10 mm plates, generating hanging drops of approximately 5,000 cells per droplet. After 4 days, aggregates were already visible at the bottom of the droplets, which were removed and transferred to non-adherent plates containing DMEM and 10% fetal bovine serum. There, they were maintained in suspension for the indicated times, and beating, size, and cavity formation were assessed as described in the figure. Briefly, embryoid bodies were observed every 5 days and measured for size, beating, and cavity formation. 20–30 EBs per condition were analyzed, and the percentage of beating or cavity-forming EBs was calculated for all time points. EB size was measured using Image J software.

[0102] Immunofluorescence and immunohistochemistry. Cells previously seeded on coverslips were fixed in 4% paraformaldehyde for 15 minutes, permeabilized using 0.1% Triton X-100 in PBS for 15 minutes, and blocked in BSA for 1 hour at room temperature. Primary antibody incubation was performed overnight at 4°C in all cases, followed by secondary antibody incubation at room temperature for 1 hour. Nuclear staining using Hoescht or DAPI was included in the final PBS wash. Primary antibodies used in this study were against Cd34 (Abcam), Gata4 (Santa Cruz), Pax6 (Abcam), nestin (Millipore), cTnT (Abcam), and phosphorylated histone H3 (Millipore). Table 1 below provides detailed information about these and other antibodies used in the assays of this application. Cells were examined under a Leica SP5 microscope equipped with a white light laser and hybridization detection.

[0103] For immunohistochemistry, tissue samples were fixed in 10% formalin, paraffin-embedded, and cut into 3-μm sections, which were mounted and rehydrated in Super-Frost-Plus porta-objects. Serial sections were stained with hematoxylin and eosin (H&E), or immunohistochemistry was performed using an automated immunostaining platform (Ventana Discovery XT, Roche, or Autostainer Plus Link 48). Depending on the primary antibody, antigen retrieval was first performed with high or low pH buffer (CC1m, Roche, or low pH antigen retrieval buffer, Dako), endogenous peroxidase was blocked (3% hydrogen peroxide), and slides were then stained with antibodies against Nanog (Cell Signaling Biotechnology, 8822), cytokeratin 8 (CK8; CNIO Monoclonal Antibodies Core Unit, AM-TROMA I), GFP (Roche, 11814460001), Sox2 (Cell Signaling Technology, 3728), alpha-fetoprotein (AFP; R&D Systems, AF5369), Oct4 (Santa Cruz Biotechnology, sc-9081), KI-67 (Master Diagnostica, 0003110QD), nestin (Millipore MAB353), Cd31 (Abcam), Cd34 (ABCAM), and other antibodies. Primary antibodies against IL-11 (ab8158), Cd73 (Cell Signaling Technology), type I collagenase (Rockland), Gata4 (Santa Cruz Biotechnology, sc-1237), insulin (Dako A0564), smooth muscle actin (Thermo Scientific RB-9010-PO), skeletal actin (Dako, M0635), and Ter119 (LY-76; BD Bioscience, 550565) were used for incubation. Detailed information about these antibodies can also be found below in Table 1.The slides were then incubated with the corresponding horseradish peroxidase-conjugated secondary antibody (OmniRabbit Ventana, Roche). The immunohistochemical reaction was developed using 3,30-diaminobenzidine tetrahydrochloride (DAB) as the chromogen (Chromomap DAB, Ventana, Roche, or DAB solution, Dako). Nuclei were counterstained with Carazzi's hematoxylin. Finally, the slides were dehydrated, cleared, and mounted with permanent mounting medium for microscopic evaluation. For Sirius Red staining, the slides were incubated with Weigert's hematoxylin for 8 minutes and Picro / Sirius Red for 1 hour, followed by a 10-minute wash in water. Images were acquired using a slide scanner (AxioScan Z1, Zeiss). Sirius Red staining was measured using both brightfield and polarized light. Images were captured and quantified using Zen software (Zeiss). [Table 1]

[0104] Analysis of mRNA and microRNA levels. RNA was extracted from cell or tissue samples using Trizol (Invitrogen) or the miRVana miRNA isolation kit (Thermo Fisher) according to the manufacturer's recommendations. Reverse transcription to cDNA was performed using M-MLV reverse transcriptase (Promega) according to the manufacturer's protocol. Quantitative real-time PCR was performed using Syber Green Power PCR Master Mix (Applied Biosystems) in an ABI PRISM 7700 thermocycler (Applied Biosystems). The housekeeping gene Gapdh was used for normalization. The oligonucleotide primers used in the assays of this application are listed in Table 2 below. [Table 2-1] [Table 2-2]

[0105] For reverse transcription of microRNAs, we used the Taqman small RNA assay (ThermoFisher Scientific, 4366596), which contained specific commercial oligonucleotides for mmu-miR-203-5p and 3p (ThermoFisher Scientific, 002580 and 000507) and housekeeping RNA sno-202 or sno-142 (ThermoFisher Scientific). Conditions for miRNA amplification were as follows: 16°C for 30 min; 42°C for 30 min; and a final step at 85°C for 5 min. Quantitative real-time PCR was then performed using Taqman Universal PCR Master Mix (434437) according to the manufacturer's instructions in an ABI PRISM 7700 thermocycler (Applied Biosystems).

[0106] For RNA sequencing, total RNA was extracted using the miRVana miRNA Isolation Kit (ThermoFisher) according to the manufacturer's recommendations. 0.8–1 μg of total RNA was extracted from iPSCs, ESCs, or teratomas, with a RIN (Ratio of RNA Integrity Number) ranging from 7–10 (Agilent 2100 Bioanalyzer). The polyA+ fraction was purified, randomly fragmented, converted to double-stranded cDNA, and processed using Illumina's "TruSeq Stranded mRNA Sample Preparation Part # 15031047 Rev.D" kit. The adapter-ligated libraries were completed by PCR (8–11 cycles) with Illumina PE primers, and the resulting directional cDNA libraries were sequenced for 50 bases in single-read format (Illumina HiSeq2000) and analyzed by nextpresso (available at http: / / bioinfo.cnio.es / nextpresso / ) ( Grana et al., 2017 ). The quality of the reads was checked using FastQC (http: / / www.bioinformatics.babraham.ac.uk / projects / fastqc) and aligned to the mouse genome (GRCm38 / mm10) using Bowtie 1.0.0 (available for download from different internet resources, e.g., https: / / slackbuilds.org / repository / 14.2 / academic / bowtie / ) (Langmead et al., 2009) and Samtools 0.1.19 (https: / / sourceforge.net / projects / samtools / files / samtools / 0.1.19 / ) (Li et al., 2009)) with TopHat-2.0.10 (available at https: / / ccb.jhu.edu / software / tophat / ) (Trapnell et al., 2012), allowing for two mismatches and five multiple hits.Transcript assemblies, their abundance estimates, and differential expression were calculated using Cufflinks 2.2.1 (available at http: / / cole-trapnell-lab.github.io / cufflinks / releases / v2.2.1 / ) (Trapnell et al., 2012) using the mouse genome annotation dataset GRCm38 / mm10 from the UCSC Genome Browser (https: / / genome.ucsc.edu / ) (Rosenbloom et al., 2015). A false discovery rate (FDR) of 0.05 was used as the threshold for significance in differential expression. Heatmaps were subsequently constructed using GENE-E (http: / / www.broadinstitute.org / cancer / software / GENE-E / index.html). RNA sequencing data were deposited in the GEO repository (accession number GSE81571).

[0107] Bisulfite conversion, genome-wide DNA methylation, and DMR validation. DNA samples were prepared for whole-genome bisulfite sequencing using the TrueMethyl® Whole Genome Kit (CEGX®) according to the manufacturer's instructions. Briefly, 200 ng of genomic DNA was sheared to 800 bp using an M220 Focused-ultrasonicator™ (Covaris®). The fragmented DNA was then denatured and oxidized with a chemical oxidant to convert 5-hydroxymethylcytosine to 5-formylcytosine (5fC). The purpose of oxidation was to ensure pure information about the methylation of 5'-methylcytosine, rather than an indistinguishable pattern of a combination of 5'-methylcytosine and 5'-hydroxymethylcytosine. Following oxidation, the DNA underwent bisulfite conversion to deaminate cytosine and 5fC to uracil. The bisulfite-converted DNA was desulfonated and purified before proceeding to library preparation. In this "post-bisulfite conversion" library preparation method, fragmented single-stranded bisulfite-converted DNA was fitted with a sequencing adapter at the 3' end, followed by an extension step and finally adapter ligation at the 5' end of the molecule. Finally, the library was indexed and amplified. Ten cycles of PCR were performed, followed by bead-based purification. An additional purification and size selection step using Agencourt AMPure XP beads (Beckman Coulter, Cat: A63881) was performed to remove adapter dimers. The purified library was eluted in a final volume of 14 μl of purified water. The quality of the resulting library was checked using a DNA high-sensitivity chip on an Agilent Bioanalyzer. The library was quantified using Qubit and the KAPA Biosystems Library quantification kit (#KK4824) according to the manufacturer's instructions. A total of 12 pMol of multiplexed libraries was loaded onto an Illumina HiSeq2500 in a 125 bp PE rapid run.Adapter sequences were removed using cutadapt version 1.9.1 (Martin, 2011) in paired-end mode with the parameters "-m 15 -u 6 -U 6." Reads were then aligned to mm10 using bwa-meth (Pedersen et al., 2014) with default parameters. PCR duplicates were removed using Picard v1.91 (http: / / broadinstitute.github.io / picard). A count table of the number of methylated and unmethylated bases sequenced at each CpG site in the genome was constructed using bwa-meth and the "tabulation" module of BisSNP-0.82.2 (Liu et al., 2012) with default parameters. All libraries passed basic quality control checks with a minimum of 82.7% uniquely aligning read pairs. DMRs were called using the WGBS module of DMRcate ( Peters et al., 2015 ) with parameters of lambda = 1000 and C = 50, DMPs were called using DSS ( Feng et al., 2014 ), PMDs, LMRs and UMRs were called using MethylSeekR ( Burger et al., 2013 ), and PMDs were called using the R package “aaRon” ( https: / / github.com / astatham / aaRon ).

[0108] To verify DMRs, bisulfite sequencing of clones was performed at two specific loci: the promoter regions of Elf5 (chromosome 2: 103, 423, 778-103, 424, 180) and Sirt6 (chromosome 10: 81, 624, 595-81, 625, 547) (Genome Browser: Mouse mm10 Dec. 2011. Genome Reference Consortium GRCm38). 100 ng of DNA was bisulfite-treated using the EZ DNA-Methylation-Lighting™ Kit (Zymo Research) according to the manufacturer's instructions. The bisulfite-converted DNA was then analyzed by bisulfite PCR analysis. Triplicate PCR amplifications were performed using semi-nested bisulfite conversion-specific primers listed in the last part of Table 2 ("DNA methylation analysis (mouse gene)") according to PCR conditions previously described for Elf5 (Lee et al., 2011) or the following protocol for Sirt6: 95°C for 4 min; 5 cycles (95°C for 45 s, 54°C for 1.5 min, 72°C for 2 min); 25 cycles (95°C for 45 s, 54°C for 1.5 min; 72°C for 1.5 min) with a final extension at 72°C for 4 min. The methylation status of PCR amplicons was determined by Sanger clonal sequencing of pooled PCR products to ensure representative clonal analysis using between 8 and 10 clones per sample. Analysis of clonal methylation status was performed using a BiQ Analyzer (Bock et al., 2005).

[0109] Neonatal cardiomyocyte isolation and differentiation studies. Neonatal mouse and rat cardiomyocytes were prepared as previously described (Huang et al., 2015). Briefly, neonatal cardiomyocytes were isolated by enzymatic dissociation of 1-day-old neonatal mouse or rat hearts using the Neonatal Cardiomyocyte Isolation System (Cellutron Life Technology). Cardiomyocytes were pre-plated for 2 hours to remove fibroblasts. Cells were then plated on 1% gelatin-coated plates in medium containing 10% horse serum and 5% fetal bovine serum. 18 hours after plating, cells were switched to serum-free medium and then transfected with 50 nM miR-203a-3p and miR-203a-5p mimics or control mimics (all from Dharmacon) using Lipofectamine RNAiMAX transfection reagent according to the manufacturer's instructions. After 6 hours, the transfection reagent-containing medium was removed and replaced with medium containing 1% serum. Cells from different time points (1, 3, or 5 days after transfection, as indicated in Figure 13a) were collected for subsequent RNA extraction and EdU staining / immunofluorescence. For EdU staining, a Click-iT EdU staining kit (Invitrogen) was used according to the manufacturer's recommendations. Immunofluorescence for a cardiomyocyte marker (troponin T) was then performed, and finally, Hoescht was used for nuclear staining.

[0110] For in vitro differentiation of mouse iPSCs into cardiomyocytes, wild-type mouse iPSCs were transfected with either a control mimic or miR-203a-3p / -5p mimics before differentiation. In some experiments, pMCSV-Dnmt3a and 3b or an empty pMCSV vector were additionally transiently transfected 24 hours after mimic transfection. The iPSCs were then maintained in culture under standard conditions (in 2i / LIF medium) for 15 days before differentiation began. The cells were then plated in 6-well plates pre-coated with Matrigel. Once they reached confluence, the medium was changed to RPMI 1640 / B27 medium (Thermo Fisher Scientific, 61870127 for RPMI 1640 medium and A1895601 for B27) containing 5 μM CHIR99021 (Stem Cell Technologies, 72054). After two days, cells were washed and medium without CHIR99021 was added. On day 3 of differentiation, the medium was supplemented with 5 μM IWR1 (Stem Cell Technologies, 72564) and maintained under these conditions for another two days. On day 7 of differentiation, the medium was changed to RPMI 1640 / B27 + insulin. Then, from days 9 to 15, cells were cultured in RPMI 1640 / B27 with medium changes every two days. From days 16 to 18 of differentiation, cells were cultured in DMEM containing 4 mM lactate without glucose. From days 18 to 21, the medium was changed to RPMI / 1640 / B27 and changed daily. Finally, from days 22 to 28, the monolayer cells were dissociated into cell clusters and maintained in low-adherent condition for another week.

[0111] Cardiac cryoinjury in neonatal mice Cardiac cryoinjury experiments in neonatal mice were performed as previously described (Polizzotti et al., 2016). Briefly, on postnatal day 1, neonates were placed on a heated water blanket set to 37°C and covered with bedding from the mother's nest. Pups were then anesthetized by placing them in an ice-water bath for 3 minutes. Afterwards, pups were dried using sterile gauze pads and placed in the surgical field in a supine position with their arms, legs, and tail immobilized. A horizontal skin incision across the chest was made using microscissors, and a lateral thoracotomy was then carefully performed by making a small incision at the fourth or fifth intercostal space. The pericardium was then carefully removed, and the heart was exteriorized by gentle abdominal compression. The left ventricle was identified, and a pre-cooled cryoprobe was then applied to the ventricular surface for exactly 2 seconds. An 8-0 non-absorbable Prolene suture was used to close the chest wall, and a Webglue suture was used to close the skin. After closure, the surgical area was washed with wet gauze to remove any residual blood. The pups were quickly warmed for several minutes and then placed back onto the heating blanket with the other pups and covered with bedding from the mother's cage. Once all pups had fully recovered from surgery, they were replaced into their mother's cage. Seven days after freeze injury, the pups were euthanized by decapitation, and the hearts were collected and fixed in formaldehyde. The hearts were then routinely processed for paraffin embedding as described above.

[0112] statistics. Normal distribution and equal variance were confirmed before using Student's t-test (unpaired, two-tailed) to infer statistical significance. For contingency tables, Fisher's exact test was used. Statistical analysis was performed using Prism (GraphPad Software, La Jolla, CA).

[0113] Example 1. miR-203 promotes pluripotent cell function in vitro. To study miR-203 in vivo, we first generated a conditional knockout mouse model in which the gene encoding miR-203 could be ablated after expression of Cre recombinase (see Figure 1a). Although genetic ablation of miR-203 in mice did not result in major abnormalities, miR-203-null mice [miR-203(- / -);KO] exhibited mild developmental defects in the skin (Figure 1b).

[0114] We also generated an inducible knock-in model (KI:ColA_MiR203;Rosa26_rtTA) in which the miR-203 coding sequence was inserted downstream of the type I collagen gene [ColA1(miR-203 / miR-203)] under the control of a tetracycline-responsive sequence in the presence of the tetracycline reverse transactivator expressed from the Rosa26 locus [Rosa26(rtTA / rtTA)] (Fig. 1c). Thus, the KI model allows for over 100-fold overexpression of miR-203 whenever animals or cells derived from animals are exposed to doxycycline treatment.

[0115] The first approach was to extract mouse embryonic fibroblasts (MEFs) from those mice (KO and KI) and reprogram them in vitro using viral versions of Yamanaka factors to generate miR-203 knock-in inducible pluripotent cells (miR-203 KI iPSCs).

[0116] Treatment of mouse embryonic fibroblasts (MEFs) isolated from these mice with doxycycline led to a 1000-fold induction of miR-203 levels (FIG. 1d).

[0117] To directly study the effects of miR-203 expression in pluripotent cells, we generated iPSCs from MEFs isolated from these miR-203 KO and KI models. Wild-type, KO, and uninduced KI MEFs were transduced with lentiviral vectors expressing Oct4, Sox2, Klf4, and Myc to generate iPSCs. KI cultures were subsequently treated with doxycycline (DOX) for 3–5 days and then exposed to DOX withdrawal for multiple passages, thus obtaining transiently induced KI iPSCs (or tKI iPSCs for short) (see Figure 2a for a schematic depiction of the protocol used).

[0118] Although it was previously proposed that miR-203 blocks the stemness potential of skin progenitors ( Yi et al., 2008 ), RNA sequencing of these iPSC clones, in addition to wild-type embryonic stem cells (ESCs), unexpectedly revealed that tKI cells are transcriptionally closer to ESCs than WT iPSCs ( Figure 2 b), both at the genome-wide level and when determining the previously defined naive pluripotency signature ( Chung et al., 2012 ) ( Figure 2 c).

[0119] To study the differentiation potential of mutant iPSCs, wild-type and mutant (KO and tKI) iPSCs were tested in an in vitro embryoid body formation assay. In vitro differentiation into embryoid bodies (EBs) was significantly increased in tKI compared to control iPSCs. Furthermore, tKI iPSC-derived EBs proliferated faster, differentiated better, beat more efficiently, and displayed a more complete organization of the three germ layers. Thus, while the absence of miR-203 resulted in poor formation of embryoid bodies, transient induction of miR-203 for 5 days 2 weeks prior to the assay resulted in significantly greater numbers and volumes of embryoid bodies (Figure 2d). tKI embryoid bodies displayed a complex organization with high expression of primitive endoderm (Gata4), mesoderm (Cd34), and ectoderm (Pax6) or neuroectoderm (Nestin) markers (Figure 2e, f). Similar results were found when comparing identical ColA1 (miR-203 / miR-203); Rosa26 (rtTA / rtTA) iPSC clones that were untreated (KI) or transiently induced with doxycycline (tKI; Fig. 3a). Furthermore, in this case, transient induction of miR-203 resulted in more efficient formation of larger embryoid bodies with elongated cavities and enhanced beating frequency (Fig. 3b, c). Thirty days after doxycycline withdrawal, comparison of expression profiles in tKI clones versus uninduced KI clones suggested higher expression of genes related to tissue morphogenesis and embryonic development in clones in which miR-203 was transiently induced (Fig. 3d, e). Table 3 shows gene ontology analysis of genes upregulated in tKI iPSCs (5 days in the presence of doxycycline and 30 days after doxycycline withdrawal) versus KI iPSCs (untreated). [Table 3-1] [Table 3-2] [Table 3-3]

[0120] Interestingly, the upregulation of developmental and morphogenetic pathways was accompanied by enhanced expression of pluripotency genes after transient induction of miR-203 in these iPSCs (FIG. 3f).

[0121] We next tested whether miR-203 has a similar effect in ES cells. ES cells were generated from ColA1 (miR-203 / miR-203); Rosa26 (rtTA / rtTA) mice and were either left untreated (KI) or transiently treated with doxycycline for 5 days (tKI) 2 weeks before performing the embryoid body assay. As shown in Figure 2g,h, transient induction of miR-203 in ESCs resulted in larger embryonic bodies that beat more efficiently at earlier timepoints than untreated ESCs. Interestingly, transient expression of ectopic miR-203 using a CMV-driven retroviral vector or RNA mimic also promoted embryoid body formation in wild-type iPSCs or ESCs (Figure 4a–e), indicating that these effects were not unique to inducible genetic models. As depicted in Figure 4, both miR-203a-3p and -5p were used in these assays to faithfully mimic the endogenous situation in which both mature forms are expressed. However, the most abundant form (miR-203a-3p) is responsible for the effects observed in this study (Figure 5). Finally, transient expression of miR-203-GFP in wild-type ES cells significantly increased the number of 2C (two-cell embryo)-like cells (Figure 2i), as determined by expression of a mouse endogenous retrovirus (MuERV-L; 2C::tdTomato reporter) with a leucine tRNA primer (Macfarlan et al., 2012).

[0122] Taken together, these results suggest that transient induction of miR-203 sequences improves pluripotency in iPSCs and ESCs and supports the differentiation of these cells into multiple lineages in vitro.

[0123] Example 2. miR-203 improves pluripotent function of iPS cells in vivo. The potential of tKI iPSCs (miR-203 expression was induced in vitro for only 5 days) was then tested after subcutaneous injection in mice, and it was found that compared with the respective controls, tKI IP also dramatically improved in vivo differentiation into teratomas when injected into mice.

[0124] Although teratoma formation was reduced in miR-203 KO cells, tKI iPSCs formed significantly larger tumors in these assays (Fig. 6a). Interestingly, not only were these teratomas large, but they also contained tissues not typically found in control iPSC-derived teratomas, such as bone marrow, cartilage, or pancreas, as well as insulin-positive cells (representing pancreatic β cells) and trophoblasts (representing extraembryonic tissue, placenta), as confirmed by PL-1 expression (Fig. 6b, c and Fig. 7a, d).

[0125] Transcriptome studies of these teratomas suggested upregulation of genes involved in embryonic development and organ morphogenesis when derived from tKI iPSCs (Fig. 7b). Immunohistochemistry studies showed elevated expression of multiple differentiation markers representing ectoderm, mesoderm, and endoderm in tKI teratomas (Fig. 6d and Fig. 7c). Interestingly, the expression levels of in vivo pluripotency markers (e.g., Nanog, Oct4, or Sox2) were also elevated (Fig. 6d), suggesting that these structures contain a complex mixture of undifferentiated and differentiated cells.

[0126] It was recently reported that iPSCs generated in vivo from the OSKM transgene were capable of forming small embryo-like structures (containing tissues derived from three germ layers) when inoculated intraperitoneally (Abad et al., 2013). In a previous assay performed by our group, wild-type iPSCs generated in vivo were able to form embryo-like structures in 11% of injected mice (an efficiency similar to that previously reported (Abad et al., 2013)). However, in vitro-generated tKI iPSCs were much more efficient, exhibiting embryo-like structures (complex embryo-like structures characterized by the expression of multiple markers or embryonic development, including the three germ layers and even extraembryonic tissues) in 83% of injected mice, which are not found after WT iPSC injection (see Figure 8a, where it can be observed that the tKI embryo-like structures were positive for specific markers of the three germ layers, as in the case of in vivo-formed iPSCs). Table 4 below shows the frequency of nude mice with embryo-like structures (E-Ls) in the peritoneal cavity 20-30 days after intraperitoneal (ip) injection of 400,000-500,000 WT iPSCs, iKI iPSCs, or in vivo (iv) generated WT iPSCs. The number of independent clones tested per condition is indicated in the panel (each animal was inoculated with a different clone). [Table 4]

[0127] We next tested whether miR-203 also affects the potential of pluripotent cells in forming chimeras. Table 5 summarizes the results, showing the frequency of chimera contributions exhibited by KI iPSCs and ESCs transiently treated in vitro with vehicle (KI) or Dox (tKI) as indicated (two independent clones per condition were analyzed). From Table 5, it can be seen that tKI ES cells were 27.5% successful in forming 100% chimeras (as determined by coat color), compared with 11.8% success for control, uninduced KI ES cells. Furthermore, tKI iPSCs were 5.8% successful in generating 100% chimeras, while control, uninduced iPSCs were 1.5% successful in these assays. All chimeras generated in these experiments were germline-transmitted. [Table 5]

[0128] Finally, the performance of tKI iPSCs was tested in a tetraploid complementation assay with WT iPSCs, tKI iPSCs, or WT ESCs (n = 3 clones per condition). Tetraploid embryo complementation represents the most stringent test for pluripotency and developmental potential. iPSCs are highly inefficient in this assay, where any viable, live-born animals uniquely develop from diploid iPS (or ES) cells injected into tetraploid blastocysts. As shown in Table 6, while no viable pups were obtained with control iPSCs, tKI iPSCs were able to form complete chimeras (one of which is shown in Figure 8b) with a success rate of 2.8%, closer to the 11.9% achieved with control ESCs. [Table 6]

[0129] Example 3. miR-203 effects on pluripotency are Dnmt3a / b-dependent. To identify miR-203 targets that may be involved in controlling pluripotency, we searched for predicted miR-203 targets among transcripts that were upregulated in miR-203-null iPSCs (more than two-fold increase relative to wild-type iPSCs) and downregulated in the miR-203 tKI-inducible model (less than two-fold decrease relative to the wild-type situation).

[0130] 678 transcripts were found to be upregulated in miR-203-null iPSCs and downregulated in miR-203 tKI iPSCs, 35 of which were predicted to be miR-203 targets (Figure 9a) (in silico predictions were based on the miRanda, Target Scan, and MicroTar databases), as shown in Table 7a below.

[0131] When restricting the search to identify predicted miR-203 targets among transcripts downregulated in tKI iPSCs (FIG. 9f), selection from that list of genes involved in epigenetic regulation of expression (GO0040029) resulted in 18 GO0040029 transcripts that were downregulated in tKI iPSCs and predicted miR-203 targets according to the same microRNA target prediction algorithm used for Table 7a. The list of the 18 transcripts is shown below in Table 7b. [Table 7] [Table 8]

[0132] Among the transcripts deregulated in these two standard analyses, we identified the de novo DNA methyltransferase Dnmt3a (DNA methyltransferase 3α, EC number 2.1.1.37; Human Gene HGNC:2978, Ensembl:ENSG00000119772, NCBI Gene:1788, NCBI Accession Number:NM_022552 version NM_022552.4 07, October 2016; Mus Mus musculus gene MGI:1261827, Ensembl:ENSMUSG00000020661; NCBI gene:13435, NCBI accession number:NM_001271753 version NM_001271753.1, 15 February 2015) and Dnmt3b (DNA methyltransferase 3β, EC number 2.1.1.37; human gene HGNC:2979, Ensembl:ENSG00000088305 version ENSG00000088305.18; NCBI gene:1789, NCBI accession number:NM_006892 version NM_006892.3 03, November 2016; Mus musculus gene MGI:1261819, Ensembl We decided to focus on miR-203 (ENSMUSG00000027478 version ENSMUSG00000027478.15, NCBI Gene 13436, NCBI Accession Number: NM_001003961 version NM_001003961, February 15, 2015) (data based on the following databases: HUGO Gene Nomenclature Committee (http: / / www.genenames.org / ); MGI Mouse Genome Informatics (http: / / www.informatics.jax.org / ), updated March 13, 2017; and Ensembl (www.ensembl.org) release 87, December 2016). This decision was based on the relevance of chromatin modifications in pluripotency and the long-term effects of transient expression of miR-203, which is suggestive of epigenetic modifications.Additionally, these two transcripts were significantly downregulated after expression of miR-203 mimics in wild-type iPSCs (log2 fold change = -0.24 for Dnmt3a and -0.22 for Dnmt3b transcripts). Human miR-203 and DNMT3a / b transcripts have previously been shown to exhibit opposite expression profiles in cancer cells (Sandhu et al., 2012; Gasque Schoof et al., 2015), and DNMT3b was recently shown to be a direct miR-203 target in human colon cancer cells (To et al., 2016).

[0133] Both Dnmt3a and Dnmt3b transcripts contain conserved miR-203 sites in their 3'-UTRs (Fig. 10a) (Dnmt3a: SEQ ID NO: 49; Dnmt3b: SEQ ID NO: 51), and exogenous expression of miR-203 led to a decrease in the signal of luciferase constructs fused to these sequences, but not to the 3'-UTR sequences from the related genes Dnmt3l or Dnmt1 (Fig. 9b, c). When the putative miR-203 binding site was mutated and luciferase constructs were fused to the mutated 3'-UTR sequences of Dnmt3a (SEQ ID NO: 50) or Dnmt3b (SEQ ID NO: 52) (Fig. 10b), this regulation was abolished (Fig. 9c), indicating direct control of these transcripts by miR-203. Notably, transient doxycycline treatment concomitant with overexpression of miR-203-resistant Dnmt3a and Dnmt3b cDNAs blocked the expansion of 2C-like cells induced by retroviral expression of miR-203 in wild-type ESCs (Fig. 9e), in addition to the corresponding overgrowth of embryoid bodies (Fig. 9d).

[0134] To test whether downregulation of Dnmt3a / b could mimic the effects of miR-203, we knocked down these de novo DNA methyltransferases by RNA interference (siDnmt3a / b). RNA sequencing of these samples revealed that siDnmt3a / b iPSCs exhibited a transcriptome profile similar to that of tKI iPSCs (Figure 9g). In addition, downregulation of Dnmt3a / b induced increased EB proliferation, long cavity formation, and beating to a degree similar to that observed in tKI iPSC-derived EBs (Figure 9h), whereas individual knockdown of these transcripts exhibited only partial effects (quantified in Figure 9h).

[0135] Knockdown of Dnmt3a / b also increased the 2C-like population in wild-type ESCs, as measured by MERVL element expression (Figure 9i, j, k), but to a lesser extent than miR-203. It is currently unclear whether these differences result from variable efficiency in regulating gene expression of additional miR-203 targets. Furthermore, expression of miR-203-resistant Dnmt3a / b cDNA significantly blocked the expression of 2C-associated markers (Figure 9i, j), thereby suggesting that Dnmt3a / b de novo methyltransferases are important targets of miR-203 in inducing naive pluripotency.

[0136] Because Dnmt3a / b are de novo methyltransferases involved in DNA methylation, we next analyzed the genome-wide methylation profiles of wild-type and tKI iPSCs (before and after induction with doxycycline), as well as embryoid bodies derived from them (Fig. 11a). Wild-type and tKI iPSCs displayed similar levels of methylation before Dox, and wild-type cells were unaffected by this treatment. In contrast, transient induction of miR-203 for 5 days resulted in genome-wide hypomethylation in tKI iPSCs, which was surprisingly reduced even 20 days after Dox withdrawal (t = 25; Fig. 11b-e), a time point at which Dnmt3a / b transcription levels had already recovered after suppression in the presence of Dox (Fig. 12a). Notably, the number of DNA methylation valleys (DMVs; (Xie et al., 2013)) and partially methylated domains (PMDs; (Lister et al., 2009)) was progressively higher in tKI iPSCs over time (Figures 11b and 12b). For example, 131 PMDs were found at t = 0, whereas 548 and 6,555 PMDs were found at t = 10 and t = 25, respectively. DNA methylation comparison between groups showed that 128 of 131 total DMRs (97.7%; t = 10 vs. t = 0) and 12,542 of 12,549 DMRs (99.9%; t = 25 vs. t = 0) were hypomethylated in tKI iPSCs as a result of prior exposure to miR-203 (Figures 11e and 12b). Transcriptional analysis of these samples suggested that genes deregulated by the affected DMRs were significantly enriched in chromatin regulators or genes involved in DNA replication, cell division, or embryonic morphogenesis, among other pathways (Fig. 13a).

[0137] Validating these observations, bisulfite sequencing confirmed miR-203-dependent hypomethylation of the locus encoding E74-like ETS transcription factor 5 (Elf5), a protein involved in epithelial cell differentiation and trophoblast stem cell renewal and differentiation (Figure ​(Figure13b,c). 13b,c). Interestingly, embryoid bodies derived from tKI iPSCs exhibited higher genome-wide DNA methylation (Figure 11b,c), consistent with upregulation of Dnmt3a and Dnmt3b transcripts (Figure 12a). Also observed on a genome-wide scale, the Elf5 DMR was hypermethylated in embryoid bodies generated from tKI iPSCs compared to wild-type-derived structures (Figure 13b,c).

[0138] Previous data suggest that disrupting the expression or activity of DNA methyltransferases leads to global hypomethylation in the genome (Blattler et al., 2014; Liao et al., 2015; Mikkelsen et al., 2008). To further analyze whether methylation changes in tKI iPSCs are related to miR-203-mediated suppression of Dnmt3a / b, we investigated DMR methylation changes after overexpression of miR-203-resistant forms of these DNA methyltransferases in tKI iPSCs. As depicted in Figure 11f, exogenous Dnmt3a / b expression rescued the hypomethylation observed after miR-203 induction in both the Elf5 DMR and the DMR located adjacent to the histone deacetylase Sirt6 gene, suggesting that these de novo DNA methyltransferases are important targets of miR-203 in inducing genome-wide hypomethylation.

[0139] Example 4. Transient expression of miR-203 improves cardiomyocyte differentiation and maturation. Because transient expression of miR-203 improves pluripotent cell function in multiple assays (Figures 2–8), we decided to directly test the effects of expressing this miRNA during cardiomyocyte differentiation. We first tested the effects of miR-203 in primary cardiomyocytes isolated from neonatal (P1) rats, which undergo further expansion and differentiation when cultured in vitro. miR-203 mimics triggered a transient burst of cell proliferation, as measured by incorporation of the nucleotide analog EdU (Figure 14a) and mitotic markers such as cyclin B1 (Figure 14b). Importantly, this increase in proliferation occurred in cardiac troponin T (cTnT)-positive cells (Figure 15a) and resulted in cells with an increased ratio of Myh6 to Myh7 myosin heavy chain genes (a developmentally regulated switch that correlates with cardiomyocyte maturation and cardiac workload (Miyata et al., 2000)) (Figure 14b).

[0140] We also tested a cardiomyocyte differentiation protocol from wild-type iPSCs (Kattman et al., 2011). Mouse iPSCs were transiently transfected with miR-203 mimics or control mimics and differentiated into cardiomyocytes 15 days later using specific media and culture conditions (see Methods). This differentiation was accompanied by increased expression of cardiomyocyte differentiation transcripts (e.g., myosin heavy chain (Myh), atrial natriuretic peptide (Nppa), and cardiac troponin T (encoded by the Tnnt2 gene)) and markers for cardiac progenitor cells (e.g., insulin gene enhancer transcription factors Isl1 and Tbx5) in iPSCs previously treated with miR-203 mimics (Figure 15b). Importantly, transient exposure to miR-203 resulted in higher expression of not only differentiation markers but also maturation markers (e.g., potassium channel components encoded by the Kcnh2, Hcn1, and Kcna4 genes (Otsuji et al., 2010)), which were only minimally induced in cells treated with control mimic RNA (Fig. ​(Fig.15c). 15c). Consistent with these data, beating frequency in cardiomyocytes derived from miR-203-treated iPSCs was significantly higher, suggesting enhanced functionality (Fig. 15d). Expression of miR-203-resistant forms of Dnmt3a and Dnmt3b in parallel with miR-203 (Fig. 14c) prevented the upregulation of these differentiation and maturation markers (Fig. 14d, e), suggesting the relevance of the miR-203-Dnmt3a / b axis in the functional differentiation and maturation of cardiomyocytes from pluripotent cells.

[0141] The fact that miR-203 renders iPSCs more naive and increases their differentiation potential and plasticity led us to test the effect of miR-203 on myocardial regeneration after injury. Myocardial cryoinjury in neonatal mice allows for the testing of molecular interventions that stimulate regeneration in a model with characteristics similar to those observed in pediatric heart disease (Polizzotti et al., 2016). Cardiomyocyte death was induced by cryoinjury in ColA1(miR-203 / miR-203);Rosa26(rtTA / rtTA) mice on postnatal day 1 (Figure 16a), and neonates were treated with vehicle (control) or Dox (induction of miR-203 expression) for 7 days. After 1 week of recovery, control pups presented significantly larger areas of fibrosis in the heart, whereas wound healing was significantly improved in Dox-treated neonates (Figures 16b, c). Identification of CD34-positive cardiac progenitor cells by co-staining with Sirius Red revealed the accumulation of undifferentiated progenitor cells in the scar of control hearts. However, treatment with doxycycline led to a significant reduction in fibrosis and better recovery of normal tissue, accompanied by a reduction in the presence of these progenitor cells in the injury site (Fig. 16d). Interestingly, the percentage of surviving pups in the operated litters was higher in the Dox-treated group compared to controls (Fig. 16e), suggesting the therapeutic effect of inducing miR-203 during myocardial regeneration and the possible relevance of this microRNA in regenerative medicine.

[0142] Example 5. miR-203 induces a basal naive state in vitro. To enable a proper comparison of the characteristics of pluripotent cells obtained after transient exposure to increased levels of miR-203 with those of natural cells in the ground state, quantitative PCR analysis was performed on mouse embryos isolated at different stages. The results (Figure 17) showed that miR-203 expression was low in oocytes, significantly induced at the two-cell stage, and exhibited a gradual reduction in morula and blastocysts.

[0143] Expression of miR-203 at the 2-cell stage led us to analyze the expression of 2C markers in cultured tKI mouse PSCs. Transient expression of miR-203-GFP in wild-type ES cells significantly increased the number of 2C-like cells (Figure 18a), as determined by expression of a mouse endogenous retrovirus (MuERV-L; 2C::tdTomato reporter) with a leucine tRNA primer (Macfarlan et al., 2012). Consistent with these observations, exposure to miR-203 induced the expression of genes harboring proximal upstream or intronic MERVL elements (Figure 18b). In addition, miR-203 exposure induced the expression of a significant number of genes characteristic of totipotent 2C blastomeres (Biase et al., 2014) (Figures 18c, d). Dramatically, almost all transcripts contained in the 282-gene signature of 2C cells were induced by miR-203 10 days after Dox withdrawal, and their expression gradually decreased over time (Fig. 18c). Interestingly, transient exposure to miR-203 mimics also drove human pluripotent cells to a basal naive state, as measured by expression of HERVH, a family of human endogenous retroviruses (HERVs) involved in maintaining human naive pluripotency (Wang et al., 2016) (Fig. 18e, f). miR-203 mimics induced expression of the HERVH-GFP reporter in a significant number of colonies, in many cases not only at the periphery but also throughout most of the colony's cells. When the differentiation potential of these cultures was tested, human tKI iPSCs generated significantly larger EBs with larger internal cavities than control counterparts (Fig. 18g, h). As such, the results indicate that observations in mouse pluripotent cells can be extended to human pluripotent cells.

[0144] Example 6. miR-203 induces naive pluripotency in cells cultured in 2i / LIF medium. Since the combination of LIF with the MEK inhibitor PD0325901 and the GSK3 inhibitor CHIR99021 (2i / L conditions) has previously been shown to make iPSCs more similar to ESCs (Ying et al., 2008), and therefore it can be considered the previous standard for maintaining stemness potential, we decided to also test the effect of miR-203 under 2i / L conditions.

[0145] We observed that tKI iPSCs grown in 2i / L also exhibited enriched transcription of stemness factors and developmental pathways when compared with wild-type iPSCs grown in the same conditions (Fig. 19a). In addition, pretreatment of tKI iPSCs cultured in 2i / L conditions with Dox for 5 days, 2 weeks prior to EB assay, significantly increased EB size and the formation of large internal cavities, as well as enhanced beating (Fig. 19b, c), suggesting an additive effect of miR-203 over 2i / L conditions.

[0146] Example 7. miR-203 has little effect in the ICR region compared to the 2i / L condition. Given recent findings showing that widespread loss of methylation in PSC cultures can be deleterious when accompanied by large-scale erasure of genomic imprints ( Choi et al., 2017 ; Yagi et al., 2017 ), we decided to test methylation levels at 103 different imprinting control regions (ICRs) in tKI iPSCs.

[0147] tKI iPSCs displayed progressive demethylation of genes (darker signals, red in the original; Figure 20; left panel, at t = 10 and t = 25), whereas ICR demethylation was very limited at t = 10 and moderate at t = 25 in the same samples (right panel). Importantly, demethylation was fully restored upon differentiation in all cases (tKI iPSC-derived EBs; Figure 20), suggesting that demethylation of tKI iPSCs (both DMR and ICR) is manageable and reversible and does not impair iPSC quality or differentiation competence.

[0148] Thus, it can be observed that miR-203 has little effect in the ICR region compared to the 2i / L condition, thus explaining the significant improvement of miR-203-treated cells in multiple in vivo assays.

[0149] conclusion In conclusion, using a variety of cellular and in vivo models, we have disclosed and demonstrated that transiently controlling miR-203 expression in induced pluripotent stem (iPS) or embryonic stem (ES) cells improves the ability of these cells to differentiate into multiple cell lineages and achieve further mature characteristics without interfering with their self-renewal properties. This effect is mediated through miR-203-dependent regulation of the de novo DNA methyltransferases Dnmt3a and Dnmt3b, which in turn regulate the methylation landscape of pluripotent cells.

[0150] References JPEG0007735050000012.jpg227153 JPEG0007735050000013.jpg232153 JPEG0007735050000014.jpg232153 JPEG0007735050000015.jpg233153 JPEG0007735050000016.jpg150153 [Sequence List Free Text]

[0151] SEQ ID NO:1: <223> hsa-miR203a-3p: miRBase accession number MIMAT0000264 SEQ ID NO:2: <223> hsa-miR203a, miRBase accession number MI0000283 SEQ ID NO:3: <223> mmu-miR-203, miRBase accession number MI0000246 SEQ ID NO:4: <223> mmu-miR-203-3p: miRbase accession number MIMAT0000236 SEQ ID NO:5: <223> Forward primer for RT-PCR of mouse gene Dazl SEQ ID NO:6: <223> Reverse primer for RT-PCR of mouse gene Dazl SEQ ID NO:7: <223> Forward primer for RT-PCR of mouse gene Dnmt1 SEQ ID NO:8: <223> Reverse primer for RT-PCR of mouse gene Dnmt1 SEQ ID NO:9: <223> Forward primer for RT-PCR of mouse gene Dnmt3a SEQ ID NO:10: <223> Reverse primer for RT-PCR of mouse gene Dnmt3a SEQ ID NO:11: <223> Forward primer for RT-PCR of mouse gene Dnmt3a2 SEQ ID NO:12: <223> Reverse primer for RT-PCR of mouse gene Dnmt3a2 SEQ ID NO: 13: <223> Forward primer for RT-PCR of mouse gene Dnmt3b SEQ ID NO: 14: <223> Reverse primer for RT-PCR of mouse gene Dnmt3b SEQ ID NO: 15: <223> Forward primer for RT-PCR of mouse gene Dnmt3l SEQ ID NO: 16: <223> Reverse primer for RT-PCR of mouse gene Dnmt3l SEQ ID NO: 17: <223> Forward primer for RT-PCR of mouse gene Ecat1 SEQ ID NO: 18: <223> Reverse primer for RT-PCR of mouse gene Ecat1 SEQ ID NO: 19: <223> Forward primer for RT-PCR of mouse gene Eras SEQ ID NO:20: <223> Reverse primer for RT-PCR of mouse gene Eras SEQ ID NO:21: <223> Forward primer for RT-PCR of mouse gene Esg1 SEQ ID NO:22: <223> Reverse primer for RT-PCR of mouse gene Esg1 SEQ ID NO:23: <223> Forward primer for RT-PCR of mouse gene Fgf4 SEQ ID NO:24: <223> Reverse primer for RT-PCR of mouse gene Fgf4 SEQ ID NO:25: <223> Forward primer for RT-PCR of mouse gene Gapdh SEQ ID NO:26: <223> Reverse primer for RT-PCR of mouse gene Gapdh SEQ ID NO:27: <223> Forward primer for RT-PCR of mouse gene Gata6 SEQ ID NO:28: <223> Reverse primer for RT-PCR of mouse gene Gata6 SEQ ID NO:29: <223> Forward primer for RT-PCR of mouse gene Gdf3 SEQ ID NO:30: <223> Reverse primer for RT-PCR of mouse gene Gdf3 SEQ ID NO:31: <223> Forward primer for RT-PCR of mouse gene Hcn1 SEQ ID NO:32: <223> Reverse primer for RT-PCR of mouse gene Hcn1 SEQ ID NO:33: <223> Forward primer for RT-PCR of mouse gene Isl1 SEQ ID NO:34: <223> Reverse primer for RT-PCR of mouse gene Isl1 SEQ ID NO:35: <223> Forward primer for RT-PCR of mouse gene Kcna4 SEQ ID NO:36: <223> Reverse primer for RT-PCR of mouse gene Kcna4 SEQ ID NO:37: <223> Forward primer for RT-PCR of mouse gene Kcnh2 SEQ ID NO:38: <223> Reverse primer for RT-PCR of mouse gene Kcnh2 SEQ ID NO:39: <223> Forward primer for RT-PCR of mouse gene Myh SEQ ID NO:40: <223> Reverse primer for RT-PCR of mouse gene Myh SEQ ID NO:41: <223> Forward primer for RT-PCR of mouse gene Nanog SEQ ID NO:42: <223> Reverse primer for RT-PCR of mouse gene Nanog SEQ ID NO:43: <223> Forward primer for RT-PCR of mouse gene Nppa SEQ ID NO:44: <223> Reverse primer for RT-PCR of mouse gene Nppa SEQ ID NO:45: <223> Forward primer for RT-PCR of mouse gene Tbx5 SEQ ID NO:46: <223> Reverse primer for RT-PCR of mouse gene Tbx5 SEQ ID NO:47: <223> Forward primer for RT-PCR of mouse gene Tnnt2 SEQ ID NO:48: <223> Reverse primer for RT-PCR of mouse gene Tnnt2 SEQ ID NO:49: <223> A fragment of the 3'UTR of Dnmt3a <223> miR-203 seed region SEQ ID NO:50: <223> Mutated versions of the 3'-UTR of Mus musculus Dnmt3a <223> Substitution of A by C with respect to the native sequence of the 3'UTR of Dnmt3a mRNA <223> Substitution of U by A with respect to the native sequence of the 3'UTR of Dnmt3a mRNA <223> Substitution of C by A with respect to the native sequence of the 3'UTR of Dnmt3a mRNA SEQ ID NO:51: <223> 3'UTR of Dnmt3b <223> miR-203 seed region SEQ ID NO:52: <223> Mutated versions of the 3'UTR of Dnmtb <223> Substitute A with C with respect to the native sequence of the 3'UTR of Dnmt3b mRNA <223> Substitution of U by A with respect to the native sequence of the 3'UTR of Dnmt3b mRNA <223> Substitution of C by A with respect to the native sequence of the 3'UTR of Dnmt3b mRNA SEQ ID NO:53: <223> hsa-miR203a-5p: miRBase accession number MIMAT0031890 <223> Substitution of a by g in mmu-miR203-5p <223> Nucleotides absent in mmu-mi203-5p SEQ ID NO:54: <223> mmu-miR-203-5p, miRBase accession number MIMAT0004547 <223> Substitution of g by a in hsa-miR203a-5p SEQ ID NO:55: <223> Primer Dnmt3a EcoRI-Fw for PCR amplification of Dnmt3a SEQ ID NO:56: <223> Primers for PCR amplification of Dnmt3a: Dnmt3a EcoRI-Rv SEQ ID NO:57: <223> Primer Dnmt3b_EcoRI-Fw for PCR amplification of Dnmt3b SEQ ID NO:58: <223> Primers Dnmt3b_NdeI-Rv for PCR amplification of Dnmt3b SEQ ID NO:59: <223> Primer Dnmt31_EcoRI-Fw for PCR amplification of Dnmt31 SEQ ID NO:60: <223> Primers for PCR amplification of Dnmt31: Dnmt31_NdeI-Rv SEQ ID NO:61: <223> Primers Dnmt1_NdeI-Rv for PCR amplification of Dnmt1 SEQ ID NO:62: <223> Primers Dnmt1_NdeI-Rv for PCR amplification of Dnmt1 SEQ ID NO:63: <223> Forward primer for RT-PCR of mouse gene Ecat1 SEQ ID NO:64: <223> Reverse primer for RT-PCR of mouse gene Ecat1 SEQ ID NO:65: <223> Forward primer for RT-PCR of mouse gene Eras SEQ ID NO:66: <223> Reverse primer for RT-PCR of mouse gene Eras SEQ ID NO:67: <223> Forward primer for RT-PCR of mouse gene Esg1 SEQ ID NO:68: <223> Reverse primer for RT-PCR of mouse gene Esg1 SEQ ID NO:69: <223> Forward primer for RT-PCR of mouse gene Fgf4 SEQ ID NO:70: <223> Reverse primer for RT-PCR of mouse gene Fgf4 SEQ ID NO:71: <223> Forward primer for RT-PCR of mouse gene Gdf3 SEQ ID NO:72: <223> Reverse primer for RT-PCR of mouse gene Gdf3 SEQ ID NO:73: <223> Forward primer for RT-PCR of rat gene Ccnb1 SEQ ID NO:74: <223> Reverse primer for RT-PCR of rat gene Ccnb1 SEQ ID NO:75: <223> Forward primer for RT-PCR of rat gene Gapdh SEQ ID NO:76: <223> Reverse primer for RT-PCR of rat gene Gapdh SEQ ID NO:77: <223> Forward primer for RT-PCR of rat gene Myh6 SEQ ID NO:78: <223> Reverse primer for RT-PCR of rat gene Myh6 SEQ ID NO:79: <223> Forward primer for RT-PCR of rat gene Myh7 SEQ ID NO:80: <223> Reverse primer for RT-PCR of rat gene Myh7 SEQ ID NO:81: <223> Forward primer for DNA methylation analysis of the mouse gene Elf5(a) SEQ ID NO:82: <223> Reverse primer for DNA methylation analysis of the mouse gene Elf5(a) SEQ ID NO:83: <223> Forward primer for DNA methylation analysis of the mouse gene Elf5(b) SEQ ID NO:84: <223> Reverse primer for DNA methylation analysis of the mouse gene Elf5(b) SEQ ID NO:85: <223> Forward primer for DNA methylation analysis of the mouse gene Sirt6 SEQ ID NO:86: <223> Reverse primer for DNA methylation analysis of the mouse gene Sirt6

Claims

1. 1. A method for promoting stemness of pluripotent stem cells, comprising transiently expressing microRNA-203 or an analog thereof at an increased level in the cells for 3 to 5 days and maintaining the cells in culture in a medium for pluripotent cells for 15 to 30 days; The analog is an RNA modified molecule in which at least one of the nucleotides is replaced by a chemically modified nucleotide, wherein the chemical modification is i. replacement of one or more phosphate linkages with phosphorothioate linkages; ii. one or more modifications at the 2' position of the sugar moiety selected from a 2'-O-methyl modification or a 2'-O-methoxyethyl modification; and / or iii. One or more modifications in the ribose moiety selected from the group consisting of those that create a link connecting the 2' oxygen and the 4' carbon, thus blocking the ribose in the conformation 3'-endo (LNA: Locked Nucleic Acid) or 2'-O,4'-C Ethylene-Bridged Nucleic Acid (ENA); replacement of the sugar backbone with an amide-containing backbone; and the use of PMO (nucleic acids in which the ribose moiety is replaced by a morpholine group); The molecule is selected from the group consisting of:

2. 2. The method of claim 1, wherein the cells are induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs) in culture.

3. The method of claim 1 or 2, wherein microRNA-203 is transiently expressed in the cells.

4. The method of claim 1 or 2, wherein the cells are transduced with an exogenous gene encoding microRNA-203 or an analog thereof.

5. The method of any one of claims 1 to 4, wherein the cells are transduced with an expression vector that expresses microRNA-203.

6. The method according to any one of claims 1 to 5, wherein the promotion of the differentiation potential of the cells is characterized by an improvement in the efficiency of differentiation into cardiomyocytes.

7. 7. The method of any one of claims 1 to 6, wherein the pluripotent stem cells are iPSCs obtained by contacting somatic differentiated cells with nuclear reprogramming factors including Oct, Klf, Myc, and Sox genes.

8. 8. The method of any one of claims 1 to 7, wherein the pluripotent stem cells are iPSCs, and the cells are in a naive-like state after expressing microRNA-203 or an analog thereof.

9. A method for obtaining differentiated and / or mature cells, comprising carrying out the method according to any one of claims 1 to 8.

10. 10. The method of claim 9, wherein the differentiated cells are selected from the group consisting of cardiomyocytes, neural or glial cells, chondrocytes and pancreatic cells.

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