Pluripotency and Regenerative medicine

What is Pluripotency?

Pluripotency can be defined as the ability at the single cell level to generate all somatic cell lineages as well as germ cells. In the preimplantation embryo, pluripotency is established in the epiblast of the late inner cell mass (ICM). These cells can be captured and maintained in culture as embryonic stem cells (ESCs). Both ICM cells and ESCs can contribute to chimeras and colonize the germline reintroduction to the embryo, providing functional proof of their naïve pluripotency. Conversely, neither postimplantation epiblast nor the primed pluripotent stem cells derived from this tissue have the capacity to contribute efficiently to chimeras following blastocyst integration. Pluripotency is then lost in the embryo upon somatic differentiation and can only be reinstated experimentally by reprogramming strategies.

Potencial applications

Pluripotent stem cells present many exciting opportunities for fundamental studies as well as for their potential applications in regenerative medicine. The main applications of pluripotent cells are the following:

In fundamental research, the uniqueness of the pluripotent state promises to provide new insights into the mechanisms that regulate cell-fate decisions. Many questions regarding the differentiation outcome of pluripotent cells and how those decisions might be taken remain unsolved. 

The ability to differentiate into diverse cell types also provides a potential to generate replacement cells in the quest to repair diseased tissues. This might be probably the most immediate industrial applicability. 

Pluripotent stem cells are also being used to develop disease models to explore how various human diseases originate from the very beginning as a result of specific mutations and epimutations. Such disease models may in turn allow the development of new drugs to cure or even to prevent diseases. This application is also remarkable, considering that this filed is now emerging and there is a significant interest focused on it. 

Potential applications of pluripotent stem cells

What is Pluripotency?

Pluripotency can be defined as the ability at the single cell level to generate all somatic cell lineages as well as germ cells. In the preimplantation embryo, pluripotency is established in the epiblast of the late inner cell mass (ICM). These cells can be captured and maintained in culture as embryonic stem cells (ESCs). Both ICM cells and ESCs can contribute to chimeras and colonize the germline reintroduction to the embryo, providing functional proof of their naïve pluripotency. Conversely, neither postimplantation epiblast nor the primed pluripotent stem cells derived from this tissue have the capacity to contribute efficiently to chimeras following blastocyst integration. Pluripotency is then lost in the embryo upon somatic differentiation and can only be reinstated experimentally by reprogramming strategies.

Potencial applications

Pluripotent stem cells present many exciting opportunities for fundamental studies as well as for their potential applications in regenerative medicine. The main applications of pluripotent cells are the following:

Potential applications of pluripotent stem cells

In fundamental research, the uniqueness of the pluripotent state promises to provide new insights into the mechanisms that regulate cell-fate decisions. Many questions regarding the differentiation outcome of pluripotent cells and how those decisions might be taken remain unsolved.

The ability to differentiate into diverse cell types also provides a potential to generate replacement cells in the quest to repair diseased tissues. This might be probably the most immediate industrial applicability. 

Pluripotent stem cells are also being used to develop disease models to explore how various human diseases originate from the very beginning as a result of specific mutations and epimutations. Such disease models may in turn allow the development of new drugs to cure or even to prevent diseases. This application is also remarkable, considering that this filed is now emerging and there is a significant interest focused on it.

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).

To date, 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.

Our method and its advantages


We have identified a microRNA -previously described as an oncosuppressor- that is preferentially expressed in the 2C-morula stages during preimplantation development in the mouse embryo. By using a variety of in vitro and in vivo approaches, we have reported that transient exposure of already-established induced pluripotent stem cells (iPSC) or ESCs to this miRNA promotes naive pluripotency improving the ability of these PSCs to differentiate into multiple cell lineages and to reach further maturation properties without interfering with their self-renewal capacity.

Transient expression of the miRNA in PSCs leads to greater developmental potential in chimeric or tetraploid complementation assays. In addition, human iPSCs exposed to this miRNA generate interspecies human-mouse conceptuses with higher efficiency than control cells.

Mechanistically, these effects are mediated through the miRNA-dependent control of de novo DNA methyltransferases Dnmt3a and Dnmt3b, thereby regulating the DNA methylation landscape of these miRNA-treated PSCs. These observations suggest that the developmental and differentiation potential of already established PSCs can be readily enhanced by transient exposure to a single microRNA.

The novelty and advantages of this technology are as follows:

Our method can be used in already-established pluripotent clones and it is therefore an additive procedure to all protocols discussed above.

The expression of miRNA sequences can be achieved easily using synthetic small RNA molecules.

The expression of the microRNA is transient (5 days) avoiding long-term secondary effects of the microRNA in other pathways..

Mechanistically, this miRNA erases transiently the epigenetic memory of pluripotent cells; a factor known to act as a barrier in the establishment of pluripotent cells.

Pluripotent cells exposed to this miRNA display enhanced function both in vitro and in vivo in the generation of differentiated and functional cell types.

Our method and its advantages

We have identified a microRNA -previously described as an oncosuppressor- that is preferentially expressed in the 2C-morula stages during preimplantation development in the mouse embryo. By using a variety of in vitro and in vivo approaches, we have reported that transient exposure of already-established induced pluripotent stem cells (iPSC) or ESCs to this miRNA promotes naive pluripotency improving the ability of these PSCs to differentiate into multiple cell lineages and to reach further maturation properties without interfering with their self-renewal capacity.

Transient expression of the miRNA in PSCs leads to greater developmental potential in chimeric or tetraploid complementation assays. In addition, human iPSCs exposed to this miRNA generate interspecies human-mouse conceptuses with higher efficiency than control cells.

Mechanistically, these effects are mediated through the miRNA-dependent control of de novo DNA methyltransferases Dnmt3a and Dnmt3b, thereby regulating the DNA methylation landscape of these miRNA-treated PSCs. These observations suggest that the developmental and differentiation potential of already established PSCs can be readily enhanced by transient exposure to a single microRNA.

The novelty and advantages of this technology are as follows:

Our method can be used in already-established pluripotent clones and it is therefore an additive procedure to all protocols discussed above.

The expression of miRNA sequences can be achieved easily using synthetic small RNA molecules.

The expression of the microRNA is transient (5 days) avoiding long-term secondary effects of the microRNA in other pathways..

Mechanistically, this miRNA erases transiently the epigenetic memory of pluripotent cells; a factor known to act as a barrier in the establishment of pluripotent cells.

Pluripotent cells exposed to this miRNA display enhanced function both in vitro and in vivo in the generation of differentiated and functional cell types.

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, diabetes, and amyotrophic lateral sclerosis (ALS). In theory, easily-accessible cell types (such as skin fibroblasts) could be biopsied from a patient and reprogrammed, effectively recapitulating 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 originate within the patient, immune rejection of the differentiated derivatives would be minimized. 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.

Possible uses of this technology include:

In the field of cardiac regeneration, iPSCs created from human and murine fibroblasts can give rise to functional cardiomyocytes that display hallmark cardiac action potentials. However, the maturation process into cardiomyocytes is impaired when iPSCs are used—cardiac development of iPSCs is delayed compared to that seen with cardiomyocytes derived from ESCs or fetal tissue. Furthermore, variation exists in the expression of genetic markers in the iPSC-derived cardiac cells as compared to that seen in ESC-derived cardiomyocytes. Therefore, iPSC-derived cardiomyocytes demonstrate normal commitment but impaired maturation, and it is unclear whether observed defects are due to technical (e.g., incomplete reprogramming of iPSCs) or biological barriers (e.g., functional impairment due to genetic factors).

The treatment of diabetes is one of the most urgent needs of our society. Insulin-producing pancreatic β cells (IPCs) are of special economic and social interest due to the need for insulin to treat diabetes mellitus. Transplantation of IPCs might be of special interest in patients of type 1-diabetes as these cells could not only help to control blood sugar but could actually cure the disease if properly integrated in the body. Unfortunately, current procedures to differentiate such cells are very inefficient. Although the progress is encouraging, existing differentiation protocols still fall short of producing mature β cells and improvement remains a major challenge in the field. Taking these data together, one can easily speculate that exposure of PSCs to this miRNA might enhance not only the generation of insulin-producing pancreatic β cells but also its functionality, thus improving significantly current procedures to generate either general or patient- specific IPCs for treating diabetes.

Antitumoral differentiation-based therapy: One of our more recent open projects in the lab is aimed to investigate the potential role of this microRNA on dropping the cancer stem cell population in tumors. It is now widely accepted that cancer stem cells (CSCs) constitute the only subset of cancer cells truly immortal and capable of supporting cancer progression. We have found that this microRNA not only acts as a potent driver from pluripotency to expanded differentiation potential, but importantly it also blocks reprogramming from somatic to stem cells. Given that experimental induction of pluripotency and tumorigenesis entail obvious similar pathways, here we speculate that the miRNA might drop the CSC population within the tumor: unlocking the cellular differentiation programs that are normally inactivated in cancer stem cells and at the same time, blocking the reprogramming from somatic to cancer stem cells. Our hypothesis, if confirmed, would shed a new light on the differentiation-based antitumoral therapy.

Further reading

Takahashi, K., and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-676.

Nichols, J., and Smith, A. (2009). Naive and primed pluripotent states. Cell stem cell 4, 487-492.

Li, M., and Izpisua Belmonte, J.C. (2016). Looking to the future following 10 years of induced pluripotent stem cell technologies. Nature protocols 11, 1579-1585.

Takahashi, K., and Yamanaka, S. (2016). A decade of transcription factor-mediated reprogramming to pluripotency. Nature reviews Molecular cell biology 17, 183-193.

Theunissen, T.W., Friedli, M., He, Y., Planet, E., O’Neil, R.C., Markoulaki, S., Pontis, J., Wang, H., Iouranova, A., Imbeault, M., et al. (2016). Molecular Criteria for Defining the Naive Human Pluripotent State. Cell stem cell 19, 502-515.

Kilpinen, H. et al. (2017) Common genetic variation drives molecular heterogeneity in human iPSCs. Nature 

Sophie Morgani, Jennifer Nichols & Anna-Katerina Hadjantonakis. (2017) The many faces of Pluripotency: in vitro adaptations of a continuum of in vivo states. BMC Developmental Biology

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