© 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs203018. doi:10.1242/jcs.203018 REVIEW ARTICLE SERIES: STEM CELLS Hematopoietic stem cells from induced pluripotent stem cells – considering the role of microRNA as a cell differentiation regulator Aline F. Ferreira1, George A. Calin2, Virgıniá Picanço-Castro3, Simone Kashima3, Dimas T. Covas3,4 and Fabiola A. de Castro1,* ABSTRACT microRNA (miRNA) expression and epigenetic markers are Although hematopoietic stem cell (HSC) therapy for hematological distinct, indicating that the stem cell differentiation process needs diseases can lead to a good outcome from the clinical point of view, the to be further investigated. limited number of ideal donors, the comorbidity of patients and the Stem cells have proven to be a powerful tool in studies aimed at in vitro increasing number of elderly patients may limit the application of this understanding cell differentiation, and advances in therapy. HSCs can be generated from induced pluripotent stem cells developing cell reprogramming protocols have meant that these (iPSCs), which requires the understanding of the bone marrow and cells can now be induced to differentiate into a number of different in vitro liver niches components and function in vivo. iPSCs have been tissues (Takayama et al., 2010; Teng et al., 2014; Menon extensively applied in several studies involving disease models, drug et al., 2016). iPSC generation has now been achieved by several screening and cellular replacement therapies. However, the somatic methods, including through integration of viruses and episomal reprogramming by transcription factors is a low-efficiency process. plasmids (Meng et al., 2012; Slamecka et al., 2016). Key aspects of Moreover, the reprogramming process is also regulated by microRNAs iPSC production, such as the target tissue, reprogramming factors, (miRNAs), which modulate the expression of the transcription factors method of cell delivery, culture conditions and the biological assays OCT-4 (also known as POU5F1), SOX-2, KLF-4 and MYC, leading to confirm the resulting cell pluripotency potential, are all time- somatic cells to a pluripotent state. In this Review, we present an consuming, arduous and expensive (Maherali and Hochedlinger, overview of the challenges of cell reprogramming protocols with regard 2008). In fact, the technology used to integrate viruses for the to HSC generation from iPSCs, and highlight the potential role of generation of iPSCs represents the main bottleneck for the miRNAs in cell reprogramming and in the differentiation of induced therapeutic application of iPSCs owing to the possibility of viral pluripotent stem cells. vectors being integrated into the genome, which can result in tumorigenesis (Maherali and Hochedlinger, 2008). By contrast, KEY WORDS: HSC, iPSC, miRNA, Cell reprogramming episomal plasmids do not integrate in the genome and typically disappear from iPSCs after 10 to 14 passages (Chou et al., 2011; INTRODUCTION Meng et al., 2012). Stem cells are undifferentiated cells that present an indeterminate Somatic cell reprogramming and iPSC differentiation have the expansion potential to produce progeny through self-renewal or potential to be used in a wide range of therapeutic applications in vitro, differentiation (Sakaki-Yumoto et al., 2013). Furthermore, stem including disease modelling, drug screening and cellular replacement cells have a low-turnover profile, in contrast to their differentiated therapies (Maherali and Hochedlinger, 2008; Giani et al., 2016; progeny (Eckfeldt et al., 2005; Cheng et al., 2000). We focus here Tiyaboonchai et al., 2014); however, the culture conditions of stem on the role of miRNAs in cell reprogramming and induced cells and the particular protocols applied have a great influence on pluripotent stem cell (iPSC) differentiation. The first study to whether the desirable final results are obtained (Fig. 1). demonstrate the formation of iPSCs upon viral-mediated During differentiation, it is expected that stem cells lose the transduction of murine embryonic and adult fibroblasts with expression of the pluripotency-related genes OCT4 and NANOG octamer-binding transcription factor 4 (OCT-4; also known as and begin to express markers associated with differentiation, such as POU5F1), sex-determining region Y-box 2 (SOX-2), v-myc avian GATA4 and GATA6 (Miyamoto et al., 2015). An important issue myelocytomatosis viral oncogene homolog (MYC) and Kruppel- regarding epigenetic markers in stem cells is that the current like factor 4 (KLF-4) was published in 2006 (Takahashi and methods of iPSC cultivation allow the maintenance of the Yamanaka, 2006). Although iPSCs are similar in morphology and epigenetic profile over a long period (Philonenko et al., 2017). pluripotent potential to embryonic stem cells (ESCs), these types of Such epigenetic control may be also mediated by miRNAs (Fig. 1). stem cells are not identical and their gene expression profile, miRNAs are endogenous small non-coding RNA (ncRNAs) consisting of 20 to 22 nucleotides that impair protein expression by binding to mRNAs and interfering with their translation (Ambros, 1Department of Clinical Analysis, Toxicology and Food Science, School of 2004). In this way, miRNAs are involved in fundamental biological Pharmaceutical Sciences, University of São Paulo (USP), Ribeirão Preto, São Paulo 14040-903, Brazil. 2Department of Experimental Therapeutics, MD Anderson processes, including tissue development, cell differentiation, Cancer Center, Houston, TX 77054, USA. 3Center of Cell Therapy, Regional Blood proliferation and apoptosis. Besides all this knowledge with regard Center of Ribeirão Preto, Ribeirão Preto, São Paulo 14051-140, Brazil. 4Department to miRNA function, their role in stem cells differentiation is not that of Internal Medicine, School of Medicine of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, São Paulo 14049-900, Brazil. well understood (Kim et al., 2017). Here, we aim to highlight miRNAs as a relevant regulator of iPSC generation and reprogramming. *Author for correspondence ([email protected]) Protocols for cell differentiation in vitro aim to generate specific A.F.F., 0000-0001-6874-184X; G.A.C., 0000-0002-7427-0578; S.K., 0000-0002- cell types from undifferentiated stem cells by using embryoid bodies 1487-0141; D.T.C., 0000-0002-7364-2595; F.A.d., 0000-0003-3347-5873 as an initial step (Brickman and Serup, 2017). Embryonic bodies are Journal of Cell Science 1 REVIEW Journal of Cell Science (2018) 131, jcs203018. doi:10.1242/jcs.203018 Addition of OCT-4, SOX-2, KLF-4, MYC 5Ј 3Ј 3Ј 5Ј miRNA Fibroblasts regulation iPSC 5Ј 3Ј 3Ј 5Ј Blood cells 3Ј 5Ј 3Ј 5Ј 3Ј 5Ј Somatic cells 3Ј Ј 5 5Ј 3Ј PatientPa en Addition of 5Ј 3Ј miRNAs Fig. 1. General diagram of a somatic cell reprogramming protocol. Somatic cells from patients (fibroblasts or peripheral blood cells), can be reprogrammed by using 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 that have 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). a three-dimensional, multicellular aggregates consisting of the three In cases of autologous HSC transplantation, patients are not germ-layers – endoderm, mesoderm and ectoderm – and are affected by severe GVHD nor by risk of rejection. However, the obtained from spontaneous differentiation of iPSCs when they are disease relapse index is higher in comparison with allogeneic HSC cultured under low-oxygen conditions (Hawkins et al., 2013). transplantation, as no GVHD and graft-versus-leukemia effect Besides the ability to obtain embryonic bodies from iPSCs, (GVL) will occur (i.e. immune response against neoplastic cells) another advantage in using iPSCs for in vitro cell differentiation is and any remaining leukemic cells could then induce the disease that technical manipulations, such as selection of clones or cell relapse. Taken together, all these disadvantages of bone marrow sorting, do not interfere with their ability to differentiate into HSC transplantation highlight the requirement of an alternative hematopoietic tissue (Philonenko et al., 2017). Even if iPSCs require source of HSCs, which aims to reduce the rate of HSC rejection, genetic manipulations (e.g. the introduction of reprogramming disease relapse and bone marrow failure syndromes (graft failure or factors to induce cell pluripotency and self-renewal, such as poor graft function), as well as increasing the possibility of OCT-4, SOX-2 and KLF-4) their properties as iPSCs are obtaining HSCs more easily (van Bekkum and Mikkers, 2012; maintained due to their high genetic and epigenetic stability Masouridi-Levrat et al., 2016). In this regard, iPSCs cells represent a (Philonenko et al., 2017). suitable source that may be used to generate sufficient amounts of Although numerous studies have linked iPSCs or iPSC-differentiated HSCs in vitro with limited immunogenicity (Araki et al., 2013). cells with cell-based therapy, there are, however, still challenges and Despite the success in obtaining in vitro hematopoietic cells from methodology limitations to be overcome. We consider the main iPSCs, the strategy used is laborious and expensive (Yang et al., challenges in generating iPSCs to be: choosing the ideal somatic cell 2017). Furthermore, there is a the lack