Pluripotency and Regenerative medicine
State of the art
In 2006, Yamanaka´s group at Kyoto University identified the conditions in which adult cells, called induced pluripotent stem cells (iPSCs), were reprogrammed to an embryonic stem cell-like state by introducing certain genes important for maintaining the essential properties of embryonic stem cells (ESCs). Although much additional research is needed, researchers are focused on the potential utility of iPSCs as a tool for drug development, modeling of disease, and transplantation medicine. Of interest, ethical issues associated with the production of ESCs do not apply to iPSCs, which offer a non-controversial strategy to generate patient-specific stem cell lines.
However, before reprogramming can be considered for use as a clinical tool, the efficiency of the process must improve substantially.
In order to increase the reprogramming efficiency, researchers have developed many variants of the original Yamanaka´s protocol, including those using additional RNAs, proteins, microRNAs or small molecule inhibitors of epigenetic modifiers. For instance, mir-302-367 are directly linked to the levels of the three transcription factors Oct4, Sox2 and Nanog (Card et al, 2008; Marson et al, 2008). It has been found that one particular miRNA, miR-302, which is expressed abundantly in ESCs, is able to transform human cancer cell lines to cells that resemble ESCs (Lin et al, 2008). Very recent data suggest that genetic ablation of miR-34 in PSCs results in improved potential to form embryonic and extraembryonic tissues in part by promoting Gata2 expression (Choi YJ et al, 2017) although the conditions for applying this information to favor the differentiation potential of PSCs remain to be established.
Also, many small molecules inhibitors have been found to improve reprogramming efficiency, by inhibiting specific enzymes or signaling pathways. This group includes inhibitors of mitogen-activated protein kinase (MAPK), glycogen synthase kinase 3 beta (GSK3b), transforming growth factor beta (TGF-b), chromatin modifying HDACs or DNMTs, and many more that can also enhance the reprogramming efficiency in combination with the Yamanaka´s factors (Burdon et al, 1999; Sato et al, 2004; Kunath et al, 2007; Ying et al, 2008; Mikkelsen eta l, 2008; Hanna et al, 2009; Huangfu et al, 2008).
One of the most commonly used protocol to stabilize full pluripotency includes the use of Mek1/2 and Gsk3 inhibitors in the presence of the cytokine Lif (2i/L conditions; Ying et al, 2008). Although these conditions improve the stabilization of naive pluripotency in vitro, recent evidences suggest that prolonged inhibition of Mek1/2 may limit the developmental potency of PSCs in vivo, in part by inducing irreversible demethylation of imprinting control regions (ICRs) (Choi J et al, 2017; Yagi et al, 2017). Finally, a recent alternative proposal suggests the use of a chemical cocktail of inhibitors that also enhances the developmental potential of PSCs (Yang et al, 2017); however, the applicability of this method is still limited due to the lack of mechanistic details.
In summary, many technical and basic science issues remain before the promise offered by iPSC technology can be realized fully. So far, reprogramming has demonstrated a proof-of-principle, yet the process is currently too inefficient for routine clinical application.
Potential therapeutic uses of our method
Pluripotent stem cells have the potential to become research and clinical tools to understand and model diseases, develop and screen candidate drugs, and deliver cell-replacement therapy to support regenerative medicine. Reprogramming technology offers the potential to treat many diseases, including neurodegenerative diseases, cardiovascular disease, and amyotrophic lateral sclerosis (ALS). Yet while iPSCs have great potential as sources of adult mature cells, much remains to be learned about the processes by which these cells differentiate.
We have recently reported the relevance of a microRNA, miR-203, whose expression greatly improves the differentiation potential of pluripotent cells (Salazar-Roa et al., EMBO J, 2020). miR-203 represses de novo DNA methyltransferases thereby resulting in overall hypomethylation of DNA. A transient exposure of pluripotent cells to this microRNA improves their ability to form cardiomyocytes, bone marrow or pancreatic cells, thus opening a wide spectrum of possibilities for this technology in regenerative medicine.
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