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).
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.
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.
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