REVIEW ARTICLE SERIES: STEM CELLS

Hematopoietic stem cells from induced pluripotent stem cells –

considering the role of microRNA as a cell differentiation regulatorAline F. Ferreira1, George A. Calin2, Virgınia Picanço-Castro3, Simone Kashima3, Dimas T. Covas3,4 andFabiola A. de Castro1,*

ABSTRACTAlthough hematopoietic stem cell (HSC) therapy for hematologicaldiseases can lead to a good outcome from the clinical point of view, thelimited number of ideal donors, the comorbidity of patients and theincreasing number of elderly patients may limit the application of thistherapy. HSCs can be generated from induced pluripotent stem cells(iPSCs), which requires the understanding of the bone marrow andliver niches components and function in vivo. iPSCs have beenextensively applied in several studies involving disease models, drugscreening and cellular replacement therapies. However, the somaticreprogramming by transcription factors is a low-efficiency process.Moreover, the reprogrammingprocess is also regulated bymicroRNAs(miRNAs), which modulate the expression of the transcription factorsOCT-4 (also known as POU5F1), SOX-2, KLF-4 and MYC, leadingsomatic cells to a pluripotent state. In this Review, we present anoverview of the challenges of cell reprogramming protocols with regardto HSC generation from iPSCs, and highlight the potential role ofmiRNAs in cell reprogramming and in the differentiation of inducedpluripotent stem cells.

KEY WORDS: HSC, iPSC, miRNA, Cell reprogramming

INTRODUCTIONStem cells are undifferentiated cells that present an indeterminateexpansion potential to produce progeny through self-renewal ordifferentiation (Sakaki-Yumoto et al., 2013). Furthermore, stemcells have a low-turnover profile, in contrast to their differentiatedprogeny (Eckfeldt et al., 2005; Cheng et al., 2000). We focus hereon the role of miRNAs in cell reprogramming and inducedpluripotent stem cell (iPSC) differentiation. The first study todemonstrate the formation of iPSCs upon viral-mediatedtransduction of murine embryonic and adult fibroblasts withoctamer-binding transcription factor 4 (OCT-4; also known asPOU5F1), sex-determining region Y-box 2 (SOX-2), v-myc avianmyelocytomatosis viral oncogene homolog (MYC) and Kruppel-like factor 4 (KLF-4) was published in 2006 (Takahashi andYamanaka, 2006). Although iPSCs are similar in morphology andpluripotent potential to embryonic stem cells (ESCs), these types ofstem cells are not identical and their gene expression profile,

microRNA (miRNA) expression and epigenetic markers aredistinct, indicating that the stem cell differentiation process needsto be further investigated.

Stem cells have proven to be a powerful tool in studies aimed atunderstanding in vitro cell differentiation, and advances indeveloping cell reprogramming protocols have meant that thesecells can now be induced to differentiate into a number of differenttissues in vitro (Takayama et al., 2010; Teng et al., 2014; Menonet al., 2016). iPSC generation has now been achieved by severalmethods, including through integration of viruses and episomalplasmids (Meng et al., 2012; Slamecka et al., 2016). Key aspects ofiPSC production, such as the target tissue, reprogramming factors,method of cell delivery, culture conditions and the biological assaysto confirm the resulting cell pluripotency potential, are all time-consuming, arduous and expensive (Maherali and Hochedlinger,2008). In fact, the technology used to integrate viruses for thegeneration of iPSCs represents the main bottleneck for thetherapeutic application of iPSCs owing to the possibility of viralvectors being integrated into the genome, which can result intumorigenesis (Maherali and Hochedlinger, 2008). By contrast,episomal plasmids do not integrate in the genome and typicallydisappear from iPSCs after 10 to 14 passages (Chou et al., 2011;Meng et al., 2012).

Somatic cell reprogramming and iPSC differentiation have thepotential to be used in awide range of therapeutic applications in vitro,including disease modelling, drug screening and cellular replacementtherapies (Maherali and Hochedlinger, 2008; Giani et al., 2016;Tiyaboonchai et al., 2014); however, the culture conditions of stemcells and the particular protocols applied have a great influence onwhether the desirable final results are obtained (Fig. 1).

During differentiation, it is expected that stem cells lose theexpression of the pluripotency-related genes OCT4 and NANOGand begin to express markers associated with differentiation, such asGATA4 and GATA6 (Miyamoto et al., 2015). An important issueregarding epigenetic markers in stem cells is that the currentmethods of iPSC cultivation allow the maintenance of theepigenetic profile over a long period (Philonenko et al., 2017).Such epigenetic control may be also mediated by miRNAs (Fig. 1).

miRNAs are endogenous small non-coding RNA (ncRNAs)consisting of 20 to 22 nucleotides that impair protein expression bybinding to mRNAs and interfering with their translation (Ambros,2004). In this way, miRNAs are involved in fundamental biologicalprocesses, including tissue development, cell differentiation,proliferation and apoptosis. Besides all this knowledge with regardto miRNA function, their role in stem cells differentiation is not thatwell understood (Kimet al., 2017).Here,we aim tohighlightmiRNAsas a relevant regulator of iPSC generation and reprogramming.

Protocols for cell differentiation in vitro aim to generate specificcell types from undifferentiated stem cells by using embryoid bodiesas an initial step (Brickman and Serup, 2017). Embryonic bodies are

1Department of Clinical Analysis, Toxicology and Food Science, School ofPharmaceutical Sciences, University of Sa o Paulo (USP), Ribeirao Preto, Sa o Paulo14040-903, Brazil. 2Department of Experimental Therapeutics, MD AndersonCancer Center, Houston, TX 77054, USA. 3Center of Cell Therapy, Regional BloodCenter of Ribeirao Preto, Ribeirao Preto, Sa o Paulo 14051-140, Brazil. 4Departmentof Internal Medicine, School of Medicine of Ribeirao Preto, University of Sa o Paulo(USP), Ribeirao Preto, Sa o Paulo 14049-900, Brazil.

*Author for correspondence (castrofabiola@hotmail.com)

A.F.F., 0000-0001-6874-184X; G.A.C., 0000-0002-7427-0578; S.K., 0000-0002-1487-0141; D.T.C., 0000-0002-7364-2595; F.A.d., 0000-0003-3347-5873

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a three-dimensional, multicellular aggregates consisting of the threegerm-layers – endoderm, mesoderm and ectoderm – and areobtained from spontaneous differentiation of iPSCs when they arecultured under low-oxygen conditions (Hawkins et al., 2013).Besides the ability to obtain embryonic bodies from iPSCs,

another advantage in using iPSCs for in vitro cell differentiation isthat technical manipulations, such as selection of clones or cellsorting, do not interfere with their ability to differentiate intohematopoietic tissue (Philonenko et al., 2017). Even if iPSCs requiregenetic manipulations (e.g. the introduction of reprogrammingfactors to induce cell pluripotency and self-renewal, such asOCT-4, SOX-2 and KLF-4) their properties as iPSCs aremaintained due to their high genetic and epigenetic stability(Philonenko et al., 2017).

Althoughnumerous studies have linked iPSCsor iPSC-differentiatedcells with cell-based therapy, there are, however, still challenges andmethodology limitations to be overcome. We consider the mainchallenges in generating iPSCs to be: choosing the ideal somatic cellsource for iPSC generation, the reprogramming method used, and thecontrol and the optimization of iPSC differentiation (Box 1).Here, we discuss the processes and protocols currently employed

to obtain hematopoietic stem cells (HSCs) in vitro from iPSCs, andalso highlight the potential roles of miRNAs in cell reprogrammingand differentiation.

iPSCs as a potential tool for HSC generation in vitroThe current therapies for hematological neoplasms arechemotherapy, immunotherapy, tyrosine kinase inhibitors andtransplantation of hematopoietic stem cells (HSCs) obtained frombone marrow, peripheral blood or umbilical cord (Jaramillo et al.,2017; Ye et al., 2017; Baron and Nagler, 2017).Patients who receive allogeneic transplants of bone marrow or

umbilical cord may exhibit a rejection of the bone marrowtransplanted cells due to graft-versus-host disease (GVHD),toxicity owing to the conditioning regimen used, disease relapseor infections (van Bekkum andMikkers, 2012). In addition, patientsreceiving HSCs by allogeneic bone marrow transplantation shouldbe under 55 years old and should have a compatible humanleukocyte antigen (HLA) donor, which restricts the applicability ofthe procedure.

In cases of autologous HSC transplantation, patients are notaffected by severe GVHD nor by risk of rejection. However, thedisease relapse index is higher in comparison with allogeneic HSCtransplantation, as no GVHD and graft-versus-leukemia effect(GVL) will occur (i.e. immune response against neoplastic cells)and any remaining leukemic cells could then induce the diseaserelapse. Taken together, all these disadvantages of bone marrowHSC transplantation highlight the requirement of an alternativesource of HSCs, which aims to reduce the rate of HSC rejection,disease relapse and bone marrow failure syndromes (graft failure orpoor graft function), as well as increasing the possibility ofobtaining HSCs more easily (van Bekkum and Mikkers, 2012;Masouridi-Levrat et al., 2016). In this regard, iPSCs cells represent asuitable source that may be used to generate sufficient amounts ofHSCs in vitro with limited immunogenicity (Araki et al., 2013).

Despite the success in obtaining in vitro hematopoietic cells fromiPSCs, the strategy used is laborious and expensive (Yang et al.,2017). Furthermore, there is a the lack of a specific surface markerfor the human hemangioblast, a single mesodermal cell that givesrise to blood cells and endothelium, which hinders the derivation ofHSCs from iPSCs in practice (Lacaud and Kouskoff, 2017).

A particular advantage for the use of iPSC-derived HSCs is thatthey do not induce GVHD because they are autologous. However,these cells nevertheless exhibit some bias, such as their inability toself-renew in culture and a failure to engraft and survive long-termafter the transplant (Shepard and Talib, 2014). Another advantage ofiPSCs as a source ofHSCs is their ability to differentiate into primitivecells, such as erythrocytes, which express fetal-type hemoglobin, anddefinitive cells including lymphocytes (Seiler et al., 2011).

iPSCs might be also useful as disease models, as the maintenanceof human primary cells in culture over long periods is difficult, andthe use of animal models often involves interspecies variabilities(De Lázaro et al., 2014). Moreover, numerous reports havedemonstrated the therapeutic potential of iPSCs in diversehematological conditions, such as myelodysplastic syndrome(MDS), sickle cell anemia and hemophilia (Kotini et al., 2015;Hanna et al., 2007; Park et al., 2014). iPSCs derived from a varietyof monogenic diseases can accurately recapitulate diseasephenotypes in vitro when differentiated into disease-relevant celltypes (Hamazaki et al., 2017).

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Fig. 1. General diagramof a somatic cell reprogramming protocol.Somatic cells from patients (fibroblasts or peripheral blood cells), can be reprogrammed byusing the transcription factors OCT-4, SOX-2, KLF-4 and MYC, together with a cocktail of miRNAs, or by the addition of miRNAs alone. The resulting iPSCs thathave been generated in vitro through somatic cell reprogramming have the potential to be used in a wide range of therapeutic applications and as a research tool(e.g. in vitro disease model).

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Based on these properties, iPSCs should be considered as apotential and relevant source for HSCs in vitro. Below, we willdiscuss the current protocols used for hematopoietic differentiationof pluripotent stem cells, both ESCs and iPSCs. We will alsoemphasize the role of miRNAs as reprogramming anddifferentiation regulators.

Current means of obtaining HSCs in vitroThe derivation of blood cells from iPSCs has been reported byseveral research groups, including ours (reviewed by Zhang, 2013;Smith et al., 2013; Sweeney et al., 2016).The embryoid bodies formed from iPSCs, as well as the culture of

iPSCs in a differentiation medium supplemented with human bonemorphogenetic protein 4 (hBMP-4), human vascular endothelialgrowth factor (hVEGF) and human WNT3A Wnt family member3A (hWnt3a), have been used to differentiate iPSCs into thehematopoietic lineage in vitro (Sweeney et al., 2016; Smith et al.,2013). Dissociation of embryonic bodies and culture of their cellswith OP9 cells (a marrow stromal cell line) have been successfullyused to obtain myeloid precursors, macrophages, eosinophils andneutrophils (Sweeney et al., 2016). These authors also reported thatthe potential for colony formation in iPSCs is associated with the co-expression of the hematopoietic markers CD34 and CD45 (alsoknown as PTPRC) during iPSC differentiation; however, theseiPSCs were not able to promote long-term hematopoietic engraftingin mice (Sweeney et al., 2016). In order to generate hematopoieticcells from iPSCs, growth factors, such as prostaglandin-E2 (PGE2)and StemRegenin 1 (SR1), have been used to increase the iPSCdifferentiation efficiency and to give rise to a long-term HSCphenotype (Zhang, 2013).

Another approach that has been reported for the generation ofhematopoietic cells in vitro is the co-culture of ESCs with pre-adipocytic stromal cells, favoring conditions conducive forhematopoietic differentiation without the need for to generateembryonic bodies (reviewed by Seiler et al., 2011).

However, the differentiation of iPSCs in vitro might be impairedby the transgenes that are used to reprogram somatic cells, such asSOX-2, OCT-4 and KLF-4, as these have been shown to reduce theability of mesodermal-like cells to differentiate in hematopoieticprogenitors (Ebina and Rossi, 2015). In fact, SOX-2downregulation is important in hematopoietic development,because its expression appears to be inversely related to thehematogenic potency of a cell (Seiler et al., 2011).

In addition to the need of establishing a standardized protocol forthe generation of HSCs in vitro, there is also a great demand formature blood cells, such as erythrocytes and platelets, that have beengenerated from hematopoietic progenitor cells, obtained either fromthe bone marrow, peripheral blood or cord blood. Pluripotent HSCsand early multipotent progenitors (MPPs) all originate fromerythroblasts, which generate erythrocytes (Palis, 2008).

Mouse ESCs have been successfully used for in vitrodifferentiation into erythroid cells by using two strategies(Nakano, 1996; Carotta et al., 2004; Kitajima et al., 2003). Thefirst one utilizes disaggregated embryonic bodies that have beencultured with erythropoietin (EPO) and Kit ligand (KL-1) tostimulate the growth and differentiation of erythroid progenitors(Carotta et al., 2004). EPO stimulates cell growth by binding toEPO-R in burst-forming unit-erythrocytes (BFU-Es) and colony-forming unit-erythrocyte (CFU-Es) (Elliott et al., 2008; Metcalf,2008). KL-1, in synergy with other cytokines, stimulates growth of

Box 1. Potential challenges in iPSC generationChoice of the somatic cell sourceInduced pluripotent stem cells can be generated from different sources of somatic cells (fibroblasts, blood cells, keratinocytes, melanocytes, liver, gastric,epithelial, neural, stomach and pancreatic-β cells) by using ectopic expression of reprogramming factors (Aoi et al. 2008; Stadtfeld et al., 2008; Polo et al.,2010; Bar-Nur et al., 2011). Fibroblasts arewidely used in the generation of iPSCs, but the procedure for collecting them is considered invasive. Blood is aneasily accessible tissue and some groups have already demonstrated the successful generation of iPSCs from peripheral blood cells (Loh et al., 2010;Dowey et al., 2012). However, the cell type and its source influence the molecular and functional properties of iPSCs (Polo et al., 2010; Araki et al., 2013).Thus, although iPSCs acquire markers of pluripotent cells and differentiate into the three embryonic germ layers, they alsomaintain the transcriptional andepigenetic memory of their cell of origin (Kim et al., 2010; Polo et al., 2010; Bar-Nur et al., 2011), which can influence their phenotype and differentiationpotential. Thus, the desired iPSC characteristics should be considered when choosing the ideal somatic cell source and application.Reprogramming methodSomatic cell reprogramming changes the cell fate, thus allowing iPSCs to differentiate into tissues distinct from their tissue of origin, and can be achieved bythe ectopic expression of defined factors, such as OCT4, SOX-2, KLF4 and MYC (reviewed by Patel and Yang, 2010). Some studies suggest that thetranscription factors should be chosen depending on the future application (i.e. in regenerative medicine or as a tool to study disease) (Saha and Jaenisch,2009; reviewed by Patel and Yang, 2010). Here, we also highlight the potential of miRNAs to boost reprogramming efficiency, particularly in iPSCs fromperipheral blood cells, which may be used for HSC production. In order to improve iPSC reprogramming efficiency, it is mandatory to establish a robust andreproducible reprogramming process. Themost commonmethod uses integration of viral vectors into the cell genome.However, viral vectors are associatedwithmutagenesis andoncogenesis potential in iPSCs (Maherali andHochedlinger, 2008; Zhang et al., 2012;Nishimori et al., 2014).Nonintegrativemethodscan be also used, including use of episomal DNA (Schlaeger et al., 2015, Goh et al., 2013). Furthermore, the factor delivery technique and electroporationparameters are also crucial; these include electrical parameters, DNA amount and purity, cell density and temperature (Yildirim et al., 2016).Culture optimizationThesomatic cell culture conditions and reagents usedduring iPSCgenerationalso need to beoptimized to avoidanyadverse reactionswith recipient tissuesand to generate the ideal target cell. Feeder cells can be replaced by human extracellular matrix proteins, and culture media containing serum can bereplaced with a serum-free medium with recombinant molecules (Nakagawa et al., 2014, Warren and Wang et al., 2013). In addition, variation betweenbatches is another important issue to consider (Warren andWang, 2013; Yang et al., 2011; Liang et al., 2013; Kim et al., 2017). Other factors that currentlylimit the wider use of iPSC technology include the cost of developing cell lines and the time needed to fully characterize them.Differentiation controlThe controlled differentiation of iPSCs into any desired cell type is the greatest technical challenge in iPSC-based approaches. After iPSC generation, theirdifferentiation potential needs to be evaluated in both animalmodels and pharmaceutical screens (Teng et al., 2014; Choi et al., 2017) to ensure the resultingiPSC is functional and capable of differentiating into the target cell. The generation of blood cells from iPSCs has been particularly challenging, and thegeneration of HSCs with long-term self-renewal capability, as well as their differentiation into the different blood cell types capable of effective oxygentransport and hemostasis still remains a significant challenge (Rowe et al., 2016).

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hematopoietic progenitors in vitro and increases blood cellproduction in vivo in animals (Broxmeyer et al., 1991). Theprotocol involving EPO and KL-1 requires 10 days of culture toobtain erythroid colonies (Carotta et al., 2004). Using only EPOwithout KL-1 results in primitive erythroid colonies, which arecharacterized by their nucleated morphology and expression ofembryonic globin (Carotta et al., 2004). By contrast, definitiveerythroid colonies are composed of cells that express adult globin,which is an important featurewhen considering the use of these cellsfor hemotherapy (Carotta et al., 2004).The second protocol uses OP9 cells, which are able to create an

environment that can induce hematopoietic differentiation when co-cultured with ESCs. After 5 days of co-culturing, the presence ofcolonies formed of hematopoietic tissue can be noticed (Kitajimaet al., 2003). In this setup, the addition of cytokines, such as EPO andKL-1, also increases the potential of the culture to produce erythroidcolonies (Kitajima et al., 2003). Indeed, after 14 days, definitiveerythroid colonies are obtained, which can be separated from theprimitive cells by simply removing the nonadherent cell populationrepresenting the primitive erythroid progenitors (Carotta et al., 2004).In addition to the above protocols for generating mice erythroid

cells in vitro, humanmature erythroid cells had already been obtainedthrough human ESC differentiation in vitro nearly a decade ago (Luet al., 2008). Although enucleation, the final step in the developmentofmature erythrocytes remains poorly understood, these authors wereable to generate oxygen-carrying erythrocytes on a large scale.Furthermore, the obtained erythroid cells showed the capacity toexpress the adult β-globin chain upon further maturation in vitro,indicating their potential functionality (Lu et al., 2008). Blood cellsthat have been generated in vitro could serve as a disease model and,importantly, also pave the way for the large-scale manufacture of redblood cells, which is a global challenge in order to provide a safesupply of transfusable erythrocytes (Timmins et al., 2011).Although all the methods mentioned above yield functional

hematopoietic cells, they also have limitations including thepresence of byproducts from murine feeder cells, the need forlong culturing periods for embryonic body formation, and thepresence of animal-derived culture products, such as bovine serum;all of these will need to be overcome for any future clinicalapplications (Kim et al., 2017).Although the literature shows that it is feasible to generate blood

cells in vitro, the underlying molecular mechanisms, such as howthe Wnt signaling pathway, iron homeostasis and hypoxia affect theexpression of transcriptional factors that contribute to cell fate, arestill unclear (Tsiftsoglou et al., 2009a,b; Undi et al., 2016).The current view with regard to hematopoietic differentiation in

vitro demonstrates that ESCs and iPSCs are useful sources for bloodcells production. However, it would be desirable if blood cellexpansion protocols were feeder free, as this would greatly simplifythe commercial scalability, as well as reduce the cost of blood cellproduction.Besides this knowledge and the considerable advances in

protocols that drive derivation of hematopoietic stem cells (HSCs)from iPSCs, the generation of robust transplantable HSCs andmature blood cells production from iPSCs remains elusive.Researchers should discovery strategies to overcome thechallenges and obstacles to produce a functional HSC in vitro. Itis also important to optimize methodologies to control or stabilizecell epigenetic states and understand the pathways that are linked tofunctional HSC generation and differentiation. Here, we point outthe role of miRNAs in somatic cell reprogramming and HSCdifferentiation.

miRNAs in cell reprogramming and differentiationAs cell reprogramming and stem cell differentiation may be affectedby miRNAs, we discuss here how this epigenetic regulator couldinterfere with these processes (Wang et al., 2015; Ong et al., 2016).

miRNAs have been reported to contribute to somatic cellreprogramming by upregulating the expression of the pluripotentreprogramming, factors OCT-4, SOX-2, KLF-4 and MYC, thuspromoting reprogramming (Vitaloni et al., 2014; Wang et al., 2015;Hu et al., 2013). In addition, miRNAs can also induce thepluripotent state of somatic cells in the absence of exogenousfactors, as has been reported for miR-302, which inhibits nuclearreceptor subfamily 2 group F member 2 (NR2F2) and promotespluripotency by upregulating OCT-4 (Vitaloni et al., 2014; Wanget al., 2015; Hu et al., 2013).

Furthermore, Miyoshi et al. have described the possibility ofreprogramming mature cells by inducing the ectopic expression ofmiR-200c, miR-302 and miR-369 in human adipose stromal cellsand human dermal fibroblasts (Miyoshi et al., 2011). ThesemiRNAs were able to promote iPSCs pluripotency and self-renewal by inducing the overexpression of OCT-3, stage-specificembryonic antigen 3 and 4 (SSEA-3 and -4), SOX-2 and NANOGtranscription factors, which resulted in the establishment of a stem-cell-like state (Miyoshi et al., 2011; reviewed by Yao, 2016).

Moreover, in mice, miR-93 andmiR-106 have been shown to alsogive rise to a pluripotent state of somatic cells by increasing themRNA levels ofOCT-4, SOX-2, KLF-4 andMYC (denoted OSKM)through targeting their regulator, the CDKN1A gene (encodingp21), which promotes the formation of iPSC colonies (Li et al.,2011). miR-302 appears to maintain the renewal capacity andpluripotency of human ESCs, as this miRNA was found to beassociated with the inhibition of premature zygotic celldifferentiation during embryonic development (Miyoshi et al.,2011). Finally, the miR-302/367 cluster is a direct target of OCT-4and SOX-2 in ESCs and iPSCs (Card et al., 2008) and has beenshown to be able to directly reprogram mouse and human somaticcells without the need for any pluripotent stem cell transcriptionfactors (Anokye-Danso et al., 2011).

It has been suggested that miRNA expression may beantagonized by pluripotent factors such as MYC (Chang et al.,2008). Indeed, Yang et al. transduced mouse embryonic fibroblasts(MEFs) with OCT4, SOX2 and KLF4 (denoted OSK) in thepresence or absence of MYC and observed that expression of miR-let-7a, miR-16, miR-21, miR-29a and miR-143 decreased duringreprogramming, confirming that MYC is involved in the regulationof miRNA expression in MEFs and is able to sustain MEFsreprogramming. Moreover, when the authors tested the efficiency ofthe process in the presence of a miR-21 inhibitor, they observed alarger number of iPSC colonies, confirming that miRNA inhibitionenhances cell reprogramming (Yang et al., 2011).

Similarly, downregulation of miR-29a improves reprogrammingin mouse fibroblasts, whereas its overexpression reduced it (Hysolliet al., 2016). Thus, this result suggests that depletion of miR-29aalters the DNA methylation profile in somatic cells and may affectthe expression of the genes responsible for cell reprogramming(Hysolli et al., 2016). These findings demonstrate the capacity ofmiRNAs to regulate DNA methylation and/or demethylation andhighlight the need for further studies in order to better characterizethe iPSC methylome.

In order to evaluate the importance of Dicer, which is involved inmiRNA biogenesis, for cell reprogramming, Kim et al. investigatedthe reprogramming of Dicer-null MEFs, which do not have afunctional miRNA biogenesis pathway (Kim et al., 2012). Use of

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OCT-4, SOX-2, KLF-4, MYC and LIN-28 to reprogram Dicer-nullMEFs was unsuccessful, but reprogramming could be achievedwhen the human Dicer homolog was introduced in Dicer-null MEFsbefore their differentiation, raising the hypothesis that miRNAs areessential for iPSC generation (Kim et al., 2012; Judson et al., 2009;Pfaff et al., 2017).Altogether, the role of miR-125a, miR-125b, miR-155, miR-181,

miR-221, miR-222, miR-223 in HSC self-renewal and differentiationhas been extensively explored in in vitro experiments (Chen, 2004;Fazi et al., 2005; Felli et al., 2005; Shaham et al., 2012; Kozakowska,et al., 2014, Yao, 2016;Wojtowicz et al., 2016) (Fig. 2). For instance,the ectopic expression ofmiR-125a in humanmultipotent progenitors(MPPs) increased their self-renewal, and these cells were also able torepopulate transplanted mice with a robust long-term multi-lineageengraftment (Wojtowicz et al., 2016). Conversely, downregulationof miR-125a decreased HSC self-renewal and so impaired theproduction of blood cells (Wojtowicz et al., 2014, 2016) (Fig. 2).Furthermore, overexpression of miR-125b has been shown topromote the generation of blood cells such as megakaryocytes invitro and therefore constitutes a potential therapeutic target inblood disorders (Shaham et al., 2012). However, it is worth notingthat the expression of high levels of miR-125b alone in micecauses a very aggressive form of transplantable myeloid leukemia.miR-125b exerts this effect by upregulating the number ofcommon myeloid progenitors, while inhibiting the developmentof pre-B cells. In this context, miR-125b targets Lin28A, whosedownregulation can mimic the preleukemic state in mice(Chaudhuri et al., 2012).In addition, miR-155, miR-221 and miR-222 also regulate

megakaryocytic (Georgantas et al., 2007) and erythroiddifferentiation (Felli et al., 2005). miR-155 impaired bothprocesses when it was transduced into K562 cells, a model cellline for human hematopoiesis (Georgantas et al., 2007). Here, miR-155 was shown to interfere with the generation of colonies fromhematopoietic progenitor cells (CD34+) that have been transducedwith miR-155, thereby giving rise to only a few myeloid anderythroid colonies, demonstrating its role as a negative regulator ofnormal myelopoiesis and erythropoiesis (Georgantas et al., 2007).Furthermore, the ectopic expression of miR-221 and miR-222 in

CD34+ cells from cord blood results in the impaired proliferationand accelerated differentiation of erythroid cells, coupled withdown-modulation of Kit protein. miR-221 and miR-222 exert thiseffect bymodulating the expression of the Kit receptor, an importantprotein involved in HSC maintenance, erythropoiesis upregulationand erythroleukemic cell expansion (Felli et al., 2005; An et al.,2016) (Fig. 2).

The differentiation of lymphoid cells might also be regulated bymiR-181, which is highly expressed in thymus (Li et al., 2007;Henao-Mejia et al., 2013). Li et al. show that increasing miR-181aexpression in mature T cells augments their sensitivity to peptideantigens, whereas inhibitingmiR-181a expression in the immature Tcells reduces sensitivity and impairs both positive and negativeselection (Li et al., 2007). Another study has also reported therelevance of miR-181 in natural killer T cell (NKT cell) ontogenesisand lymphocyte T development (Henao-Mejia et al., 2013). Theauthors described a severe defect in lymphoid development and Tcell homeostasis in miR-181-deficient mice, which was linked toderegulation of the phosphoinositide 3-kinase pathway. Similarly,miR-223 has been identified as a hematopoietic-specific miRNAand has crucial functions in myeloid and lymphoid lineagedevelopment and their cell fate due to its location in the bonemarrow, which contains HSCs and hematopoietic cells at variousstages of maturation (Chen, 2004).

Thus, the abovementioned miRNAs might all be involved inregulating differentiation of myeloid and lymphoid hematopoietic cells.

The modulation of miRNAs could also be exploited for thetreatment of solid cancer and leukemias (Sun et al., 2017; Fan et al.,2017; Lu et al., 2016). Indeed, miR-223 modulates the differentiationof humanmyeloid progenitor cells during granulocytic differentiationof acute promyelocytic leukemia (APL) cells in response to theirtreatment with retinoic acid, as shown both in vitro and in APLpatients (Fazi et al., 2005). Myelopoiesis, the generation of myeloidleukocytes, which includes granulopoiesis, monocytopoiesis andmegakariocytopoiesis, is controlled by a unique combination oftranscription factors, such as nuclear factor 1 A-type (NFI-A),CCAAT-enhancer-binding proteins (C/EBPα), core-binding factor-β(CBF-β) and retinoic acid receptor-α (RAR-α), which cooperativelyregulate promoters and enhancers present on myeloid-specific-genes.

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Fig. 2. Role of miRNAs in HSC self-renewal and differentiation.The expression of miR-125a increases the self-renewal andpluripotency capability of hematopoietic stem cells (HSCs) (brightblue). The targeting of LIN-28 by miR-125b overexpression inducesuncontrolled proliferation of myeloid progenitor cells (MPCs), leadingto myeloid leukemia (green). miR-155 targets the ETS1 and MEIStranscription factor genes that are responsible for megakaryocyte(MK) proliferation and differentiation (dark purple). Thus, enforcedexpression of miR-155 impairs MK development. In addition, miR-155 may also target additional genes, including CEBPB (encodingC/EBPβ), MEIS, CREB1, JUN, SPI1, AGTR1, AGTR2 and FOS,which regulate the differentiation of HSCs into myeloid progenitorcells (MPCs) and lymphoid progenitor cells (LPCs), and therebystimulates the formation of MPCs and erythroid colonies (EC)(orange). miR-181 and miR-223 regulate HCS differentiation intoMPCs and LPCs (dark blue), whereas miR-221 and miR-222 inhibiterythropoiesis by modulating KIT protein modulation (pink).

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Fazi et al. showed that granulocytic differentiation is controlled by aregulatory circuitry involving miR-223 and the NFI-A and C/EBPαtranscription factors (Fazi et al., 2005). NFI-A maintains miR-223 atlow levels, whereas, following retinoic acid (RA)-induceddifferentiation, it is replaced by C/EBPα, which in turn upregulatesmiR-223 expression (Fazi et al., 2005). In acute promyelocyticleukemia (APL) cells, overexpression of miR-223 induced bytreatment with RA allows leukemic promyelocytes and leukemicprogenitors to differentiate. The resulting mature leukemic myeloidcells expressing high levels of miR-223 are more sensitive to celldeath, and thus chemotherapy, and, consequently, patients exhibitdisease remission in response to retinoic acid (Fazi et al., 2005).In summary, these observations have clearly demonstrated the role

of miRNAs in hematopoiesis, cell reprogramming and differentiationregulation. In addition, these observations have pointed out howmiRNAs can be used as a tool or strategy to improve the success ofthe methods used for iPSCs generation in vitro.

Conclusions and perspectivesThe generation of iPSCs from somatic cells of a patient or from ahealthy donor is highly relevant for cell-based therapy approaches andfor understanding the molecular and cellular mechanisms involved indisease pathogenesis. It is also worthwhile pointing out that iPSCsmay also be used for development and testing of new therapeuticagents in vitro. Here, we have discussed the challenges of someprotocols used for iPSC generation in vitro and highlighted the role ofmiRNAs in iPSC cell reprogramming and hematopoiesis regulation.The major challenge in this field is to obtain mature functional

peripheral blood cells or other tissue-specific mature cells based oniPSC differentiation in vitro.In the future, the protocols for iPSC generation must be further

optimized to allow iPSCs to be used as an unlimited source indisease therapy, for instance, as a source of HSCs. To that end, newstrategies must be developed to improve the current protocols forcell reprogramming and differentiation, including the use of ectopicmiRNAs as epigenetic regulator.Clearly, the role of miRNAs in disease pathogenesis and, more

generally, in the regulation of cell differentiation and proliferationneeds to be explored in greater depth. Considering their relevance,we anticipate that in the near future, miRNAs will be used asbiomarkers for several diseases (e.g. autoimmune diseases,leukemia, myeloproliferative neoplasm and solid tumors), and astargets for therapy.

AcknowledgementsWe also thank you, Sandra Navarro for the figure designs, Fernanda TeresinhaUdinal and Andy Alastair Cumming for carefully reading the manuscript.

Competing interestsThe authors declare no competing or financial interests.

FundingWe are grateful to Fundaça o de Amparo a Pesquisa do Estado de Sa o Paulo(FAPESP) (2013/08135-2 and 2015/21237-4), Coordenaça o de Aperfeiçoamentode Pessoal de Nıvel Superior (1535985) and Conselho Nacional deDesenvolvimento Cientıfico e Tecnologico.

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