ABSTRACT
Induced pluripotent stem cells (iPSCs) were discovered in 2006 by Shinya Yakama and his student Kazutoshi Takahashi in Japan. It was found out that, with the expression of only four genes, Oct3/4, Sox2, c-Myc and Klf4, somatic stem cells could be reprogrammed into the pluripotent-like state. This process was first made through the insertion of retroviral vectors, but studies have shown many risks that may occur. Still considered a recent discovery, it has already revolutionized medical research and is considered to be the future “mouse models” of science. Their exploration has allowed the study of cells with similar states of pluripotency as found in embryonic stem cells (ESCs), breaking the ethical barrier that interfered with the study of ESCs, as well as their use in therapies, disease modeling and drug development.
INTRODUCTION
In mammal’s development there are three types of stem cells: totipotent, pluripotent and somatic stem cells. The first one is only found in the zygote, which will eventually develop and form the blastocyst. Pluripotent stem cells form part of the blastocyst, more specifically, the embryoblast, in which its cells are called embryonic stem cells (ESCs) and are considered pluripotent because they have various possible fates. Somatic ones are adult cells, bi or unipotent since they only have one or two viable outcomes and are more specialized than the other types of cells.
When stem cells were first discovered in 1961 by the biophysicist James Till and the cellular biologist Ernest McCulloch, the scientific community became quickly enthusiastic about its coming possibilities: with the ability to self-renew and replace damaged tissue in the body, these new-known cells came to be the core of the upcoming field of medical treatment, the regenerative medicine.
The ESCs got special attention because of all its available possibilities. These pluripotent cells, as they form the embryo, can turn into any other type of cell that exists in the human body, except for the totipotent type. Speaking on disease treatments, these cells could be used to treat several types of diseases, such as diabetes and Parkinson’s disease [4], since with them any type of tissue needed could be formed and used to replace a damaged one.
For a long time, however, this new discovery about the use of ESCs as a regenerative treatment encountered a challenge to its progress, mainly for ethical reasons.
Human embryonic stem cell research is ethically and politically controversial because since it involves the destruction of human embryos that have the potential to become human beings, it is the topic of several debates addressing morals and beliefs. Especially in religious faith, it’s believed that human life begins at conception and therefore, the embryo is a person and its rights must be respected. Then, to take a blastocyst and withdraw the inner cell mass to obtain an embryonic stem cell line is equal to muder [1].
The idea of inducing a pluripotent stem cell similar to the ESCs was executed by Shinya Yakama and his student Kazutoshi Takahashi at Kyoto University, Japan, in 2006. Their study “Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors” is considered one of the most important findings of the past century and made Dr. Yakama win the Nobel Prize in Physiology or Medicine of 2012. By taking somatic stem cells and activating specific genes [3], induced pluripotent stem cells (iPSCs) were originated, making these theories of regenerative treatments mentioned before a current reality. E.g in cardiac therapies, the induced pluripotent stem cell line promotes cardiac tissue regeneration and when this one is applied to the organ, it leads to improvements in cardiac function [2].
METHODS
First, a literature search was conducted on the discovery of pluripotent stem cells and research development through the upcoming years. Then, specific studies about IPSCs and their application in several therapies, as well as attestment in its relevance, were probed. For all these, NCBI databases and peer-reviewed publications were explored.
RESULTS AND DISCUSSION
Somatic stem cells can be reprogrammed to an embryonic-like state by the expression of four genes in the cell’s DNA and under ESCs cell culture conditions [3]: Oct 3/4 (regulatory gene responsible for the maintenance of the pluripotency and self-renewal properties [14]), Sox2 (transcription factor involved in the regulation of pluripotency state and determination of cell fate [15]), c-Myc ( regulator of cell proliferation [12]) and Klf4 (that regulates cell growth, proliferation, and differentiation [13]), unexpectedly showing that cell identity is indeed more malleable than predicted before [5]. The iPSCs acquired present the same morphology, marker genes and growth properties of ESCs [3].
During its first decade, the introduction of these genes was made through retroviral transduction [3], when retroviral vectors infect a cell and, with the support of the reverse transcriptase enzyme, polymerizes DNA molecules from RNA molecules, incorporating, then, the produced DNA in the host cell\’s genome [16] and effectively integrating the exogenous genes into it. However, the use of retrovirus may have risks that limit their use in patients: despite being silenced during the reprogramming, it’s not guaranteed that the transgenes will remain silenced forever and their reactivation may occur in between the cell differentiation from iPSC to a differentiated and specialized cell. Also, exogenous reprogramming factors could be expressed and that could trigger the expression of oncogenes, possibly changing cell’s properties and stimulating cancer growth [17].
Due to all this risks, this process and the method used in it have been strategically analyzed and evolved during the past years with the rising of new technologies: adenoviral vectors were used to transduce mouse fibroblasts and hepatocytes and generate mouse iPSCs [18]; Sendai virus was able to reprogram human skin fibroblasts into iPS cells without genome integration [19]; The use of two plasmids (one carrying the complementary DNAs of Oct3/4, Sox2 and Klf4; and the other carrying the c-Myc complementary DNA) was able to generate induced pluripotent stem cells without indication of plasmid integration [20]; And human iPSCs were achieved through the use of synthetic mRNA for the expression of the four genes (Oct3/4, Klf4, Sox2 and c-Myc) [21].
The reprogramming process, despite the method, is associated with the silencing of somatic genes in the state of heterochromatin and the expression of the pluripotency regulators in the euchromatin state [6].
Hard efforts, including several studies and analyzes, are being made to improve the reprogramming method, its efficiency and the occurrence of fewer or none genetic alterations.
USES OF iPSCs
On therapies, iPSC cells present a relevant and valuable advantage in clinical medicine, avoiding a problem faced in many treatments: because the somatic cells used in the process are taken from the patient\’s own body, when the induced cells are applied into the patient, there isn’t any immune rejection since the DNA between patient and the used cell is compatible [8].
Induced Pluripotent Stem Cells have been used to study monogenic and polygenic diseases because they can recreate the genetic background of patients [8], offering an unprecedented chance to study pathological human tissue formation of diseases such as Parkinson’s disease, Down syndrome (trisomy 21) and Becker muscular dystrophy (BMD) [10] and the modeling of pulmonary fibrosis [11]
In terms of disease modeling and drug discovery, induced pluripotent stem cells overcame the limitation of animal models, which cannot completely mirror human pathophysiology due to interspecies differences [7]. Most iPSC disease modeling are in 2D monolayers, but with the arise of new technologies, 3D models, E.g lung, myocardial and intestinal tissues, have been engineered [8] to constitute organoids and organs-on-chips [7]. On drug development, iPSCs cultures are candidates for drug and toxicity testing, as well as clinical trials to discover biomarkers of response [7]. Its use in the field has presented exciting results, such as the production of compounds for patients with neurological disorders [9].
- Neurological Applications
Human iPSCs present a positive potential for its application in neurological diseases. When talking about this field, iPSCs have been mainly used in disease modeling, drug testing and cell replacement therapy [22]. The biggest challenge for scientists is to obtain great percentages of mature neural cells of needed specific subtypes. It’s possible to achieve various neuronal types of cells from iPSCs, but new technologies are needed to achieve a high level of purity in the populations generated, that typically present both mature and immature cells [23]. To solve this problem, the sorting needed could be attained by the expression of specific genes or proteins [24]. iPSCs can be used to achieve dopaminergic neurons through differentiation and then handled to create a human disease model for Parkinson’s Disease (PD) to be utilized for drug screening and further studying before any clinical application is applied in patients. PD is considered one of the neurodegenerative diseases that will probably acquire great benefit from this use of iPSCs since it affects the substantia nigra in its early stage [25].
- Diabetes Mellitus Applications
As a global emerging disease, the research for a diabetes cure continues. With the scientific breakthrough of iPSCs, a new possibility arised: to induce pancreatic progenitor cells and create brand new functional β cells. Yet, this type of cell has a lack of phenotype which makes its use and induction difficult. Cellular reprogramming through iPSCs can be a possibility to create insulin-producing cells that are similar, morphologically and genetically speaking, to β cells [26]. An insulin-producing cell culture was actually generated from skin fibroblast-derived induced pluripotent stem cells, raising the chances that in the future, specific cells generated from iPSCs could provide a potential treatment for diabetes [27].
Other iPSCs-derived specific cells have been used in more fields of medicine, such as cardiac applications [28] and leukemia treatments [29], but these utilities in specific have not been explored in this article.
CONCLUSION
iPS cells seem a great solution to the evolution of the medical research field that stagnated due to the ethical problems involving the use of embryonic stem cells. The collected data suggests that it’s expected that these cells become what mouse models have been for science in the past years. Despite the risks regarding the use of retroviral vectors, iPS cells models have already been used to investigate several types of diseases. The development of the technique used is needed to decrease the possibility of occurrence of any risk and to turn their therapeutic application safe for patients. Also, since it is a new technology, long term data must be collected and analyzed to prove their efficacy, even after long periods of time, when applied in the human body to provide a safe treatment opportunity.
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