Induced pluripotent stem cells reprogramming: Epigenetics and applications in the regenerative medicine

Células-tronco de pluripotência induzida: papel da epigenética na reprogramação e sua aplicabilidade clínica

Kátia Maria Sampaio Gomes Ismael Cabral Costa Jeniffer Farias dos Santos Paulo Magno Martins Dourado Maria Fernanda Forni Julio Cesar Batista Ferreira About the authors

Summary

Induced pluripotent stem cells (iPSCs) are somatic cells reprogrammed into an embryonic-like pluripotent state by the expression of specific transcription factors. iPSC technology is expected to revolutionize regenerative medicine in the near future. Despite the fact that these cells have the capacity to self-renew, they present low efficiency of reprogramming. Recent studies have demonstrated that the previous somatic epigenetic signature is a limiting factor in iPSC performance. Indeed, the process of effective reprogramming involves a complete remodeling of the existing somatic epigenetic memory, followed by the establishment of a "new epigenetic signature" that complies with the new type of cell to be differentiated. Therefore, further investigations of epigenetic modifications associated with iPSC reprogramming are required in an attempt to improve their self-renew capacity and potency, as well as their application in regenerative medicine, with a new strategy to reduce the damage in degenerative diseases. Our review aimed to summarize the most recent findings on epigenetics and iPSC, focusing on DNA methylation, histone modifications and microRNAs, highlighting their potential in translating cell therapy into clinics.

Keywords:
induced pluripotent stem cells; regenerative medicine; cell reprogramming; epigenetics; histones; microRNAs

Resumo

As células-tronco de pluripotência induzida (CTPI) ou do inglês induced pluripotent stem cells (iPSCs) são células somáticas reprogramadas para o estado embrionário por meio da expressão de fatores ectópicos de transcrição específicos, tornando-as um alvo promissor para a medicina regenerativa. Apesar das CTPI compartilharem características embrionárias, como pluripotência e capacidade de autorrenovação, elas possuem uma baixa eficiência de reprogramação, sendo a memória epigenética uma das principais barreiras nesse processo. A epigenética é caracterizada por alterações reversíveis e herdáveis no genoma funcional que não alteram a sequência de nucleotídeos do DNA. Dentre as diferentes modificações epigenéticas, destacam-se metilação de DNA, alterações em histonas e microRNA. Atualmente, sabe-se que o processo de reprogramação efetivo das CTPI envolve um completo remodelamento da memória epigenética somática existente, seguido pelo estabelecimento de uma "assinatura epigenética" que esteja de acordo com o novo tipo de célula a ser diferenciada. Modificações epigenéticas personalizadas são capazes de melhorar o rendimento e a efetividade das CTPI geradas, abrindo uma nova perspectiva para a terapia celular. Nesta revisão reunimos as principais informações sobre os fatores epigenéticos que afetam a reprogramação das CTPI, bem como seus benefícios na aplicação da terapia celular.

Palavras-chave:
células-tronco de pluripotência induzida; medicina regenerativa; reprogramação celular; epigenética; histonas; microRNA

Introduction

Human embryonic stem cells have great potential for self-renewal and the ability to differentiate into all tissues of the body (except embryonic attachments),11 Biehl JK, Russell B. Introduction to stem cell therapy. J Cardiovasc Nurs. 2014; 24(2):98-103; quiz 104-5. forming an important source of material for regenerative medicine and cell therapy. However, the use of embryonic stem cells is limited by ethical and religious conflicts, as well as immunological incompatibility.22 Lo B, Parham L. Ethical issues in stem cell research. Endocr Rev. 2009; 30(3):204-13. In order to reduce the damage caused by degenerative diseases, different strategies are being used in an attempt to optimize the use of embryonic stem cells.

The first strategy used was that of somatic cell nuclear content (SCNT) for unfertilized and enucleated oocytes.33 Gurdon JB, Elsdale TR, Fischberg M. Sexually mature individuals of Xenopus laevis from the transplantation of single somatic nuclei. Nature. 1958; 182(4627):64-5. However, the yield of this technique is still very low and the cells obtained can present phenotypic and gene expression abnormalities.44 Tachibana M, Amato P, Sparman M, Gutierrez NM, Tippner-Hedges R, Ma H, et al. Human embryonic stem cells derived by somatic cell nuclear transfer. Cell. 2014; 153(6):1228-38. Another strategy is the fusion of somatic cells with embryonic cells, reprogramming their genome.55 Cowan C a, Atienza J, Melton DA, Eggan K. Nuclear reprogramming of somatic cells after fusion with human embryonic stem cells. Science. 2005; 309(5739):1369-73. However, although fusion-induced reprogramming is very efficient (about 95%), the resulting hybrid cells lack therapeutic potential due to their tetraploidy, as well as the presence of exogenous genes from the pluripotent cells used in the fusion.55 Cowan C a, Atienza J, Melton DA, Eggan K. Nuclear reprogramming of somatic cells after fusion with human embryonic stem cells. Science. 2005; 309(5739):1369-73. Therefore, a search for new strategies for the efficient use of cells with an embryonic profile is still needed.

In 2006 Takahashi and Yamanaka managed to induce pluripotency in mouse fibroblasts (MEF) from the expression of four embryonic transcription factors (Oct4, Sox2, Klf4, c-Myc), currently known as OSKM or "Yamanaka factors." These cells were called induced pluripotent stem cells (iPSCs).66 Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006; 126(4):663-76. The iPSC are similar to embryonic cells in terms of morphology, gene expression, differentiation status and epigenetic pattern, both in culture, as well as in vivo.77 Patel M, Yang S. Advances in reprogramming somatic cells to induced pluripotent stem cells. Stem Cell Rev. 2010; 6(3):367-80. This mechanism of reprogramming somatic cells into embryonic stem cells resulted in the Nobel Prize in medicine for Yamanaka in 2012, shared with John b. Gurdon.88 Johnson MH, Cohen J. Reprogramming rewarded: the 2012 Nobel Prize for Physiology or Medicine awarded to John Gurdon and Shinya Yamanaka. Reprod Biomed Online. 2012; 25(6):549-50. Since then, several surveys have been developed to explore this technology. In fact, several researchers were able to reprogram somatic cells (postmitotic) into iPSC using the aforementioned strategy.99 Lowry WE, Plath K. The many ways to make an iPS cell. Nat Biotechnol. 2008; 26(11):1246-8.

10 Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007; 318(5858):1917-20.
-1111 Takahashi K, Yamanaka S. Induced pluripotent stem cells in medicine and biology. Development. 2013; 140(12):2457-61.

The advantage of using this method is that it allows the derivation of pluripotent cells from the donor, reducing the risk of rejection by the immune system. In addition, this method provides a platform to study the molecular mechanisms of genetic and chronic diseases, minimizing the ethical, religious and political conflicts and opening up new perspectives for regenerative medicine. However, although cell identity can be modified by the ectopic expression of transcription factors, the efficiency of reprogramming remains low (0.1 to 3%) and its cost is high.1111 Takahashi K, Yamanaka S. Induced pluripotent stem cells in medicine and biology. Development. 2013; 140(12):2457-61.,1212 Apostolou E, Hochedlinger K. Chromatin dynamics during cellular reprogramming. Nature. 2013; 502(7472):462-71.

The low reprogramming efficiency of iPSC is associated with residual epigenetic memory of the tissue from which they were derived, which complicates the reprogramming process. Recent studies show that despite iPSC sharing common characteristics of pluripotency and self-renewal capacity, these cells still retain an epigenetic memory.1313 Chin MH, Mason MJ, Xie W, Volinia S, Singer M, Peterson C, et al. Induced pluripotent stem cells and embryonic stem cells are distinguished by gene expression signatures. Cell Stem Cell. 2009; 5(1):111-23.

14 Kim K, Doi A, Wen B, Ng K, Zhao R, Cahan P, et al. Epigenetic memory in induced pluripotent stem cells. Nature. 2010; 467(7313):285-90.
-1515 Polo JJM, Liu S, Figueroa MME, Kulalert W, Eminli S, Tan KY, et al. Cell type of origin influences the molecular and functional properties of mouse induced pluripotent stem cells. Nat Biotechnol. 2010; 28(8):848-55. In addition, there is evidence that the reprogramming process involves complete remodeling of the existing somatic epigenetic memory, followed by the establishment of a new "epigenetic signature" that conforms to the type of cell to be differentiated.1616 Nashun B, Hill PWS, Hajkova P. Reprogramming of cell fate: epigenetic memory and the erasure of memories past. EMBO J. 2015; 34(10):1296-308. Therefore, the epigenetic memory becomes a barrier in the process of cellular reprogramming. This fact highlights the need for new studies investigating the epigenetic changes associated with cellular reprogramming in an attempt to improve the efficiency and effectiveness of the iPSC created, as well as their clinical application. As such, our review aimed to gather information about the epigenetic factors (DNA methylation, changes to histones and microRNAs) associated with iPSC reprogramming efficiency. In addition, we have brought together the clinical studies using iPSC as cell therapy.

Epigenetics and iPSC

Waddington was the first researcher to use the term epigenetics in 1942 to explain how the genome interacts with the environment during the development process.1717 Waddington CH. The epigenotype. 1942. Int J Epidemiol. 2012; 41(1):10-3. Therefore, any reversible and inheritable change in the functional genome that does not alter the sequence of DNA nucleotides refers to epigenetics.1818 Haig D. The (dual) origin of epigenetics. Cold Spring Harb Symp Quant Biol. 2004; 69:67-70. Several pathologies are associated with epigenetic changes.1919 Kim SY, Morales CR, Gillette TG, Hill JA. Epigenetic regulation in heart failure. Curr Opin Cardiol. 2016; 31(3):255-65.

20 Abdolmaleky HM, Zhou J-R, Thiagalingam S. An update on the epigenetics of psychotic diseases and autism. Epigenomics. 2015; 7(3):427-49.

21 Faroogi AA, Tang JY, Li RN, Ismail M, Chang YT, Shu CW, et al. Epigenetic mechanisms in cancer: push and pull between kneaded erasers and fate writers. Int J Nanomedicine. 2015; 10:3183-91.
-2222 Coppedè F. The potential of epigenetic therapies in neurodegenerative diseases. Front Genet. 2014; 5:220.

The reprogramming efficiency of iPSC is also directly related to epigenetic changes such as DNA methylation and the epigenetic memory of the source cells.2323 Gładych M, Andrzejewska A, Oleksiewicz U, Estécio MRH. Epigenetic mechanisms of induced pluripotency. Contemp Oncol (Pozn). 2015; 19(1A):A30-8. The iPSC reprogramming process can be divided into three distinct phases, called pre-iPSC, intermediate and full reprogramming. The reprogramming process is extremely slow, with low efficiency (0.1 to 3%) and high cost,1111 Takahashi K, Yamanaka S. Induced pluripotent stem cells in medicine and biology. Development. 2013; 140(12):2457-61.,1212 Apostolou E, Hochedlinger K. Chromatin dynamics during cellular reprogramming. Nature. 2013; 502(7472):462-71. and depends on suitable levels of gene expression in each phase and specific epigenetic changes.2424 Djuric U, Ellis J. Epigenetics of induced pluripotency, the seven-headed dragon. Stem Cell Res Ther. 2010; 1(1):3. Djuric and Ellis compared the epigenetic changes that occur during the reprogramming process with a "seven headed dragon," where a series of changes is necessary for efficient reprogramming, namely: 1) Endogenous reactivation of genes related to cell pluripotency, Nanog and Oct4; 2) Chromatin changes, such as trimethylation in H3K27 and changes in H3K4; 3) Hypomethylation of heterochromatin; 4) Reactivation of the inactive X chromosome; 5) Maintenance of DNA methylation marks; 6) Silencing the retrovirus that induces pluripotency; and, finally, 7) Two- or three-dimensional chromatin changes and location of nuclear subdomains.2424 Djuric U, Ellis J. Epigenetics of induced pluripotency, the seven-headed dragon. Stem Cell Res Ther. 2010; 1(1):3. Therefore, the control of epigenetic factors during reprogramming may improve the induction of iPSC and their efficiency.2525 Liang G, Zhang Y. Embryonic stem cell and induced pluripotent stem cell: an epigenetic perspective. Cell Res. 2013; 23(1):49-69.

Epigenetic changes in iPSC reprogramming

DNA methylation in iPSC reprogramming

DNA methylation is an epigenetic mechanism involved in many important cellular processes such as cell proliferation and differentiation, transcriptional repression, genomic imprinting, organization of chromatin and inactivation of the X chromosome.2626 Hackett JA, Surani MA. DNA methylation dynamics during the mammalian life cycle. Philos Trans R Soc Lond B Biol Sci. 2013; 368(1609):20110328. Thus, changes in the DNA methylation profile are associated with the appearance of many degenerative diseases.

Studies show that DNA methylation is considered a crucial epigenetic barrier in the reprogramming of iPSC.2727 Nishino K, Toyoda M, Yamazaki-Inoue M, Fukawatase Y, Chikazawa E, Sakaguchi H, et al. DNA methylation dynamics in human induced pluripotent stem cells over time. PLoS Genet. 2011; 7(5):5-8.

28 Popp C, Dean W, Feng S, Cokus SJ, Andrews S, Pellegrini M, et al. Genomewide erasure of DNA methylation in mouse primordial germ cells is affected by AID deficiency. Nature. 2010; 463(7284):1101-5.

29 Doege CA, Inoue K, Yamashita T, Rhee DB, Travis S, Fujita R, et al. Early-stage epigenetic modification during somatic cell reprogramming by Parp1 and Tet2. Nature. 2012; 488(7413):652-5.

30 Costa Y, Ding J, Theunissen TW, Faiola F, Hore TA, Shliaha PV, et al. NANOG-dependent function of TET1 and TET2 in establishment of pluripotency. Nature. 2013; 495(7441):370-4.
-3131 Gao Y, Chen J, Li K, Wu T, Huang B, Liu W, et al. Replacement of Oct4 by Tet1 during iPSC induction reveals an important role of DNA methylation and hydroxymethylation in reprogramming. Cell Stem Cell. 2013; 12(4):453-69. For the expression of genes essential for reprogramming, such as Oct3/4 and Nanog, demethylation of cytosines is necessary in the respective promoter regions.3232 Watanabe A, Yamada Y, Yamanaka S. Epigenetic regulation in pluripotent stem cells: a key to breaking the epigenetic barrier. Phil Trans R Soc. 2013; 368:(1609):20120292. Thus, inhibition of methylation by enzymes or interfering RNA may be an option to improve this process.3333 Mikkelsen TS, Hanna J, Zhang X, Ku M, Wernig M, Schorderet P, et al. Dissecting direct reprogramming through integrative genomic analysis. Nature. 2008; 454(7200):49-55. Mikkelsen et al. showed that the use of 5-azacytidine (AZA) inhibits the enzyme DNA methyltransferase 1, assisting in DNA demethylation, which may favor the reprogramming of iPSC.3333 Mikkelsen TS, Hanna J, Zhang X, Ku M, Wernig M, Schorderet P, et al. Dissecting direct reprogramming through integrative genomic analysis. Nature. 2008; 454(7200):49-55. Another study noted an improvement in reprogramming efficiency using DNA hydroxylase (Tet1), an enzyme that is able to oxidize 5-methylcytosine and, after subsequent replications, promotes DNA demethylation by reactivating Oct4 gene transcription, favoring the reprogramming process.3131 Gao Y, Chen J, Li K, Wu T, Huang B, Liu W, et al. Replacement of Oct4 by Tet1 during iPSC induction reveals an important role of DNA methylation and hydroxymethylation in reprogramming. Cell Stem Cell. 2013; 12(4):453-69. In addition to the use of AZA and the enzyme Tet1, other researchers have used ascorbic acid (vitamin C). Vitamin C acts as a cofactor, stimulating hypomethylation and consequently increasing the activity of histone H3K36 demethylase. This process results in an improvement of cellular reprogramming efficiency.3434 Wang T, Chen K, Zeng X, Yang J, Wu Y, Shi X, et al. The histone demethylases Jhdm1a/1b enhance somatic cell reprogramming in a vitamin-C-dependent manner. Cell Stem Cell. 2011; 9(6):575-87.

35 Esteban MA, Wang T, Qin B, Yang J, Qin D, Cai J, et al. Vitamin C enhances the generation of mouse and human induced pluripotent stem cells. Cell Stem Cell. 2010; 6(1):71-9.
-3636 Bagci H, Fisher AG. DNA demethylation in pluripotency and reprogramming: the role of Tet proteins and cell division. Cell Stem Cell. 2013; 13(3):265-9.

The aforementioned studies demonstrate the importance of DNA demethylation in cell reprogramming. Several studies have attempted to improve these and other epigenetic mechanisms in order to improve both the quality and efficiency of iPSC reprogramming. Another relevant topic for such improvement is the change in histones during the reprogramming process.

Changes to histones in iPSC reprogramming

Histones are basic proteins rich in lysine and may suffer several epigenetic changes. Most of these modifications happen in the N-terminal region of the histone, with the exception of ubiquitination, which occurs in the C-terminal region of H2A and H2B.3737 Sadakierska-Chudy A, Filip M. A comprehensive view of the epigenetic landscape. Part II: Histone post-translational modification, nucleosome level, and chromatin regulation by ncRNAs. Neurotox Res. 2014; 27(2):172-97. Epigenetic modifications to histones may either promote or inhibit gene transcription by changing the level of chromatin folding.3838 Eissenberg JC, Shilatifard A. Histone H3 lysine 4 (H3K4) methylation in development and differentiation. Dev Biol. 2010; 339(2):240-9.,3939 Becker JS, Nicetto D, Zaret KS. H3K9me3-dependent heterochromatin: barrier to cell fate changes. Trends Genet. 2016; 32(1):29-41.

Taking into consideration the epigenetic changes in histones in iPSC, H3 is the histone researched the most, as it is directly related to genes expressed during embryonic development, such as Oct3/4, Sox2 and Nanog. It has already been demonstrated that methylation of H3K27 is associated with the suppression of various genes, and that persistent trimethylation of the lysine 27 of histone 3 (H3K27me3) blocks reprogramming by repressing the chromatin region associated with the target genes of the stem cells. However, the methylation of H3K4 is associated with the activation of different embryonic genes.2424 Djuric U, Ellis J. Epigenetics of induced pluripotency, the seven-headed dragon. Stem Cell Res Ther. 2010; 1(1):3. In an attempt to improve the performance of iPSC and reach the ideal conditions for the induction of pluripotency, by reducing the "epigenetic memory" in somatic cells, different strategies that directly or indirectly affect the methylation/acetylation of H3 have been used.1212 Apostolou E, Hochedlinger K. Chromatin dynamics during cellular reprogramming. Nature. 2013; 502(7472):462-71. Several researchers have demonstrated that it is possible to perform the induction of pluripotency without the use of Yamanaka factors, using only chemical compounds/ molecules that interfere with the enzymes that control the chromatin structure.4040 Lin T, Wu S. Reprogramming with small molecules instead of exogenous transcription factors. Stem Cells Int. 2015; 2015:794632.

Recently, Rais et al. showed that the inhibition of Mbd3 - a subunit of the NuRD complex responsible for the deacetylation of histones, remodeling of nucleosomes and gene expression inhibition - is able to reactivate the genes expressed during embryonic development and improve the efficiency of iPSC reprogramming by almost 90%, both in human as well as mouse cells.4141 Rais Y, Zviran A, Geula S, Gafni O, Chomsky E, Viukov S, et al. Deterministic direct reprogramming of somatic cells to pluripotency. Nature. 2013; 502(7469):65-70.

Many other strategies have been tested in order to improve the iPSC reprogramming process, such as the use of small molecules like Forskolin (FSK),4242 Hou P, Li Y, Zhang X, Liu C, Guan J, Li H, et al. Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds. Science. 2013; 341(6146):651-4. BIX-01294,4343 Shi Y, Desponts C, Do JT, Hahm HS, Schöler HR, Ding S. Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule compounds. Cell Stem Cell. 2008; 3(5):568-74. valproic acid (VPA - HDAC histone deacetylase inhibitor)4444 Huangfu D, Maehr R, Guo W, Eijkelenboom A, Snitow M, Chen AE, et al. Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds. Nat Biotechnol. 2008; 26(7):795-7. and vitamin C.4545 Chen J, Liu H, Liu J, Qi J, Wei B, Yang J, et al. H3K9 methylation is a barrier during somatic cell reprogramming into iPSCs. Nat Genet. 2013; 45(1):34-42. Therefore, the induction of pluripotency of iPSC can only occur with the use of small molecules.4646 Liang G, Taranova O, Xia K, Zhang Y. Butyrate promotes induced pluripotent stem cell generation. J Biol Chem. 2010; 285(33):25516-21.

47 Onder TT, Kara N, Cherry A, Sinha AU, Zhu N, Bernt KM, et al. Chromatin-modifying enzymes as modulators of reprogramming. Nature. 2012; 483(7391):598-602.
-4848 Liang G, He J, Zhang Y. Kdm2b promotes induced pluripotent stem cell generation by facilitating gene activation early in reprogramming. Nat Cell Biol. 2012; 14(5):457-66. The authors advocate the use of such because they are not immunogenic, with greater yield and easy production. Thus, the use of chromatin modulators can increase efficiency in the iPSC reprogramming process.4949 Zare M, Soleimani M, Akbarzadeh A, Bakhshandeh B, Aghaee-Bakhtiari SH, Zarghami N. A novel protocol to differentiate induced pluripotent stem cells by neuronal microRNAs to provide a suitable cellular model. Chem Biol Drug Des. 2015; 86(2):232-8. To do so, it is necessary to use a small molecule that is able to demethylate the DNA in the promoter region and change specific regions in histones.

MicroRNAs in iPSC reprogramming

MicroRNA or miRNA are important tools for regulating gene expression in post-transcriptional iPSC by promoting pluripotency to modulate the stability of messenger RNA (mRNA) and suppress the signs of differentiation during the self-renewal of embryonic stem cells.

MiRNAs also modulate the signaling cascades that are necessary for maintaining the pluripotent state.5050 Li MA, He L. microRNAs as novel regulators of stem cell pluripotency and somatic cell reprogramming. Bioessays. 2012; 34(8):670-80. Wang et al. noted that the loss of function in the enzyme Dicer and DGCR8, proteins essential for the biogenesis of microRNAs in the embryonic stem cells of mice, present two different phenotypes: 1) reduction in proliferation due to cell cycle arrest in G1; and 2) resistance to differentiation, which reveals a close relationship between microRNAs, differentiation and the pluripotency of cells.5151 Wang Y, Medvid R, Melton C, Jaenisch R, Blelloch R. DGCR8 is essential for microRNA biogenesis and silencing of embryonic stem cell self-renewal. Nat Genet. 2007; 39(3):380-5.,5252 Wang Y, Baskerville S, Shenoy A, Babiarz JE, Baehner L, Blelloch R. Embryonic stem cell-specific microRNAs regulate the G1-S transition and promote rapid proliferation. Nat Genet. 2008; 40(12):1478-83.

MicroRNAs are small non-coding RNA molecules. They have 18-25 nucleotides (nt), and are derived from a larger precursor. The processing of microRNAs occurs as follows:

  1. After DNA transcription by RNA polymerase II or III the primary microRNA is formed (pri-miRNA). This may be presented in the shape of a fork. The pri-miRNA first undergoes processing by the enzyme ribonuclease (RNase) nuclear III-like DROSHA. The specificity of the cleavage in this step is guided by the DGCR8 protein, which acts as a "molecular ruler," positioning the DROSHA at a distance of 11 nucleotides from the base of the pri-miRNA loop. After cleavage, a premiRNA (precursor miRNA) is released, formed by about 60-70 nt.5353 He L, Hannon GJ. MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet. 2004; 5(7):522-31.

  2. The pre-miRNA is actively transported to the cytoplasm by exportin-5 (Exp5), when this protein is linked to its Ran-dependent GTP cofactor. In the cytoplasm, it undergoes another cleavage process, where it loses the loop and is reduced to a miRNA duplex approximately 18-25pb in length. This last stage of processing is conducted by DICER, an RNAse-III type enzyme, aided by the TRBP protein.5353 He L, Hannon GJ. MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet. 2004; 5(7):522-31.

  3. Finally, the RNA-binding protein TRBP recruits a multimeric complex denominated RISC (RNA-induced silence complex), which includes the protein Argonaute 2 (in mammals) as the main component. Only one of the microRNA duplex strands remains in the RISC complex (guide strand), with the other being degraded (passenger strand). The RISC complex is able to identify and bind to target messengers RNAs in region 3'UTR through complementarity of bases in the "SEED" region of the miRNA (nucleotides 2-8 from the 5' end) in order to inhibit its translation or promote its adenylation and degradation.5353 He L, Hannon GJ. MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet. 2004; 5(7):522-31.

MicroRNAs that promote the reprogramming of iPSC

  • miR 290-295 (cluster): These constitute more than 70% of the entire population of microRNAs in the embryonic stem cells of mice. miR 291-3 p, miR-294 and miR-295 are part of this cluster and indirectly promote the transition of genes associated with entry into the G1-S phase, blocking Cdkna (p21), a suppressor of the Cyclin E/Cdk2 complex, and regulator of the cell cycle. After its transfection into MEFs there is an increase of 0.01-0.05% to 0.1-0.3% in cell reprogramming efficiency.5454 Judson RL, Babiarz JE, Venere M, Blelloch R. Embryonic stem cell-specific microRNAs promote induced pluripotency. Nat Biotechnol. 2009; 27(5):459-61.

  • miR 302-367 and miR 371-373 (cluster): These miRNAs suppress the expression of MBD2 (methyl-CPG-binding domain protein 2), which works like a demethylase in cells, resulting in increased expression of Nanog and conversion of completely reprogrammed iPSC. They are also able to reduce expression of the inhibitors in the G1-S phase, as well as increasing the kinetics of the mesenchymal-epithelial transition (MET) required for reprogramming.5555 Subramanyam D, Lamouille S, Judson RL, Liu JY, Bucay N, Derynck R, et al. Multiple targets of miR-302 and miR-372 promote reprogramming of human fibroblasts to induced pluripotent stem cells. Nat Biotechnol. 2011; 29(5):443-8.,5656 Lin SL, Chang DC, Lin CH, Ying SY, Leu D, Wu DTS. Regulation of somatic cell reprogramming through inducible mir-302 expression. Nucleic Acids Res. 2011; 39(3):1054-65. Only the use of miR-302a/b/c/d and miR367 is able to reprogram adult cells, and with greater efficiency, when compared to the Yamanaka method.5757 Anokye-Danso F, Trivedi CM, Juhr D, Gupta M, Cui Z, Tian Y, et al. Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency. Cell Stem Cell. 2011; 8(4):376-88. Data demonstrate that miR302 in conjunction with Yamanaka factors inhibits NR2F2 (nuclear receptor subfamily 2, group F, member 2) and improves reprogramming efficiency through indirect positive regulation of Oct4.5858 Hu S, Wilson KD, Ghosh Z, Han L, Wang Y, Lan F, et al. MicroRNA-302 increases reprogramming efficiency via repression of NR2F2. Stem Cells. 2013; 31(2):259-68.

  • miR-200b and -200c, miR-205: These promote MET via signaling of transforming growth factor β (TGF-β) and, in conjunction with the Yamanaka factors, exclude the need for signaling of bone morphogenetic protein (BMP) during the initial reprogramming phase.5959 Samavarchi-Tehrani P, Golipour A, David L, Sung HK, Beyer TA, Datti A, et al. Functional genomics reveals a BMP-driven mesenchymal-to-epithelial transition in the initiation of somatic cell reprogramming. Cell Stem Cell. 2010; 7(1):64-77.

  • miR-93, miR-106: They suppress the expression of TGF-β and p21, leading to MET and increased proliferation.6060 Li Z, Yang C, Nakashima K, Rana TM. Small RNA-mediated regulation of iPS cell generation. EMBO J. 2011; 30(5):823-34.

  • miR-135b: This is highly expressed during the reprogramming process, regulating the expression of TGF-β, IGFBP5 (insulin-like growth factor binding protein 5) and Wisp1 (inducible-signaling pathway protein 1), which are involved in the expression of extracellular matrix genes.6161 Li Z, Dang J, Chang K, Rana TM. MicroRNA-mediated regulation of extracellular matrix formation modulates somatic cell reprogramming. RNA. 2014; 20(12):1900-15.

MicroRNAs that are barriers to iPSC reprogramming

  • miR Let-7 (cluster): This inhibits the Pou5f1/Oct4, Sox2, Nanog and Tcf3 targets - pluripotency factors - stabilizing a differentiated state. In addition, this miRNA inhibits the translation of CDK4, repressing the transition of the G1-S-phase. MiR Let-7 forms a negative feedback circuit, providing a molecular mechanism that facilitates the decision between self-renewal and differentiation of cells.5050 Li MA, He L. microRNAs as novel regulators of stem cell pluripotency and somatic cell reprogramming. Bioessays. 2012; 34(8):670-80.,6262 Zhong X, Li N, Liang S, Huang Q, Coukos G, Zhang L. Identification of microRNAs regulating reprogramming factor LIN28 in embryonic stem cells and cancer cells. J Biol Chem. 2010; 285(53):41961-71.

  • miR-34a, miR-34b/c: miR-34a represses the expression of Nanog, Sox2 and c-Myc. Taken in conjunction, miR-34a and miR-34b/c target p53 (tumor suppressor gene), holding an essential role in the containment of somatic reprogramming.6363 Choi YJ, Lin C, Ho JJ, He X, Okada N, Bu P, et al. miR-34 miRNAs provide a barrier for somatic cell reprogramming. Nat Cell Biol. 2011; 13(11):1353-60.

These studies show that miRNAs can be important tools in the mediation of iPSC reprogramming without the need for the ectopic expression of pluripotency induction factors, including OSKM factors. The tables below present a summary of different approaches and their effects on iPSC reprogramming. Table 1 is related to changes in DNA methylation, Table 2 is related to modifications in histones and Table 3 is related to the use of miRNAs. In addition, Figure 1 presents these changes in summarized form.

TABLE 1
Epigenetic DNA changes.
TABLE 2
Epigenetic changes in histones.
TABLE 3
Epigenetic changes using microRNAs.

FIGURE 1
Epigenetic factors and iPSC reprogramming efficiency. The ectopic expression of Yamanaka factors, Oct4, Sox2, Klf4 and c-Myc (OSKM) are able to lead to DNA demethylation and reprogramming of somatic cells. A. The use of Tet1 (DNA hydroxylase) and of 5-azacytidine (inhibitor of the enzyme DNA methyltransferase) improves the reprogramming efficiency of iPSC cells. B. The use of valproic acid (VPA), BIX-01294 and vitamin C favors reprogramming through inhibition of histone deacetylase (HDAC), histone methyltransferase (G9a) and activation of the demethylases Kdm3/Kdm4, respectively. C. miRNAs 302-367, 290-295, 135b are able to increase reprogramming efficiency by promoting the progression of the cell cycle. miR Let-7 inhibits the cell cycle, and miR-34 inhibits the translation of p53, decreasing reprogramming efficiency.

Clinical application of iPSCs

The main discussion about the use of iPSC in regenerative medicine is related to their ability to transform into cancer cells. Incomplete reprogramming of iPSC may be associated with the emergence of various mutations.

In addition to the impact on iPSC reprogramming, OSKM factors are associated with the development of tumors. Oct4 is highly expressed in the cervical cancer cell line. Deletion of Sox2 is associated with regression of melanomas. The Klf4 and c-Myc factors regulate genes involved in cell growth and proliferation.6464 Curry EL, Moad M, Robson CN, Heer R. Using induced pluripotent stem cells as a tool for modelling carcinogenesis. World J Stem Cells. 2015; 7(2):461-9. However, over the last 10 years following the discovery of iPSC, this technology has undergone several changes, such as the use of episomal plasmids that are not integrated into the genome, diminishing the carcinogenic potential of these cells.6565 Takahashi K, Yamanaka S. A decade of transcription factor-mediated reprogramming to pluripotency. Nat Rev Mol Cell Biol. 2016; 17(3):183-93. Currently, several methods are used to develop iPSC lines, including the use of plasmids, transposons, adenovirus, Sendai virus, miRNA and chemical compounds, minimizing mutagenic factors. Given these advances, iPSC have been used in pre-clinical tests and clinical trials.

The first transplant of iPSC in humans occurred in Japan in the second half of 2014, in a 70-year-old patient with age-related macular degeneration (AMD). This pathology affects around 700,000 people in Japan, and is the most common form of blindness in people aged over 60, causing progressive loss of the retinal pigment epithelial monolayer. The transplant lasted around two hours and, according to the researchers, the patient did not suffer adverse effects, and there were an improvement in the morphology of the macula and neovascularization.6666 First iPS cell transplant patient makes progress one year on. The Japan Times [internet]. 2015 [cited 23 May 2016]. Available from: http://www.japantimes.co.jp/news/2015/10/02/national/science-health/first-ips-cell-transplant-patient-makes-progress-one-year#.WJxP59JVikq.
http://www.japantimes.co.jp/news/2015/10...

This clinical trial was carried out by the group of Professor Takahashi, co-author of the manuscripts that won Professor Yamanaka the Nobel Prize in Medicine in 2012. However, in March 2015, clinical testing intended to treat six patients was suspended due to regulatory changes in regenerative medicine in Japan.6767 Garber K. RIKEN suspends first clinical trial involving induced pluripotent stem cells. Nat Biotechnol. 2015; 33(9):890-1.

Other clinical trials are currently underway to test the effectiveness of cellular therapy with iPSC in the treatment of AMD, Parkinson's disease, spinal cord injury, diabetes and myocardial infarction.6868 Trounson A, DeWitt ND. Pluripotent stem cells progressing to the clinic. Nat Rev Mol Cell Biol. 2016; 17(3):194-200. Preliminary results have not yet been presented.

Maintenance of the epigenetic factors of the iPSC after reprogramming has also been used to understand the molecular pathways involved in the development of diseases, development of new drugs and personalized medicine.6969 Avior Y, Sagi I, Benvenisty N. Pluripotent stem cells in disease modelling and drug discovery. Nat Rev Mol Cell Biol. 2016; 17(3):170-82. The first study conducted of this kind used a model of neuropathic disease. The authors reprogrammed fibroblasts from patients with Riley-Day syndrome and monitored in vitro splicing of the IKBKAP (mutation associated with the disorder). Furthermore, the researchers also evaluated candidate drugs for reversal of the splicing. The study of iPSC gains relevance in this case, due to the inability of accessing the tissues affected by Riley-Day syndrome.7070 Lee G, Papapetrou EP, Kim H, Chambers SM, Tomishima MJ, Fasano CA, et al. Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs. Nature. 2009; 461(7262):402-6. Other studies have been developed along this line,6969 Avior Y, Sagi I, Benvenisty N. Pluripotent stem cells in disease modelling and drug discovery. Nat Rev Mol Cell Biol. 2016; 17(3):170-82. and are promising in the context of drug development. Figure 2 summarizes the use of iPSC.

FIGURE 2
Somatic cells are reprogrammed into induced pluripotent stem cells (iPSCs). These cells are differentiated in the tissue of interest and transplanted in an attempt to reduce the damage caused by degenerative diseases. In addition, iPSCs are also being used in pre-clinical and clinical tests.

Conclusion

Despite the improvement recently seen in iPSC reprogramming by up to 100 times, when compared to Yamanaka factors,4444 Huangfu D, Maehr R, Guo W, Eijkelenboom A, Snitow M, Chen AE, et al. Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds. Nat Biotechnol. 2008; 26(7):795-7. the yield remains relatively low and high costs. Another problem is the long period for full iPSC reprogramming and the high cells proliferation rates associated with a greater chance of developing cancer.7171 Wasik AM, Grabarek J, Pantovic A, Cieslar-Pobuda A, Asgari HR, Bundgaard-Nielsen C, et al. Reprogramming and carcinogenesis - parallels and distinctions. Int Rev Cell Mol Biol. 2014; 308:167-203. Takahashi and Yamanaka suggest considering using iPSC with allogeneic transplantation in regenerative medicine, which would improve the effectiveness of the treatment of certain diseases such as spinal cord injury, which requires quick treatment without waiting for the time taken for reprogramming.1111 Takahashi K, Yamanaka S. Induced pluripotent stem cells in medicine and biology. Development. 2013; 140(12):2457-61. Furthermore, there is a need for better understanding of how the reprogramming interventions influence the epigenetic memory of the reprogrammed cells. Despite advances in iPSC reprogramming, certain questions have emerged: 1) Is it possible to completely erase the somatic epigenetic memory by associating the different treatments mentioned?; 2) Could "forced" reprogramming cause long-term damage, such as the development of cancer or other diseases?; 3) Is it possible to replace embryonic stem cells with iPSC in regenerative medicine? In spite of the extraordinary progress achieved recently in the use of iPSC, the deepening of ongoing studies and realization of new studies are necessary in order to elucidate the mechanisms of the aforementioned changes, optimizing their application in regenerative medicine.

  • Study conducted at Universidade de São Paulo (USP), São Paulo, SP, Brazil

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Publication Dates

  • Publication in this collection
    Feb 2017

History

  • Received
    23 May 2016
  • Accepted
    30 May 2016
Associação Médica Brasileira R. São Carlos do Pinhal, 324, 01333-903 São Paulo SP - Brazil, Tel: +55 11 3178-6800, Fax: +55 11 3178-6816 - São Paulo - SP - Brazil
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