Delayed Mesoderm and Erythroid Differentiation of Murine Embryonic Stem Cells in the Absence of the Transcriptional Regulator FUBP1

Hindawi Stem Cells International Volume 2017, Article ID 5762301, 12 pages https://doi.org/10.1155/2017/5762301 Research Article Delayed Mesoderm and Erythroid Differentiation of Murine Embryonic Stem Cells in the Absence of the Transcriptional Regulator FUBP1 Josephine Wesely,1 Marlene Steiner,1 Frank Schnütgen,2 Manuel Kaulich,3 Michael A. Rieger,2,4 and Martin Zörnig1,4 1 Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Paul-Ehrlich-Strasse 42-44, 60596 Frankfurt/Main, Germany 2 LOEWE Center for Cell and Gene Therapy Frankfurt and Department for Medicine, Hematology/Oncology, Goethe University Hospital Frankfurt/Main, 60590 Frankfurt/Main, Germany 3 Institute of Biochemistry II, Goethe University Frankfurt, 60590 Frankfurt/Main, Germany 4 German Cancer Consortium (DKTK), 69120 Heidelberg, Germany Correspondence should be addressed to Martin Zörnig; Received 21 December 2016; Revised 2 March 2017; Accepted 19 March 2017; Published 15 May 2017 Academic Editor: Zhaohui Ye Copyright © 2017 Josephine Wesely et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The transcriptional regulator far upstream binding protein 1 (FUBP1) is essential for fetal and adult hematopoietic stem cell (HSC) self-renewal, and the constitutive absence of FUBP1 activity during early development leads to embryonic lethality in homozygous mutant mice. To investigate the role of FUBP1 in murine embryonic stem cells (ESCs) and in particular during differentiation into hematopoietic lineages, we generated Fubp1 knockout (KO) ESC clones using CRISPR/Cas9 technology. Although FUBP1 is expressed in undifferentiated ESCs and during spontaneous differentiation following aggregation into embryoid bodies (EBs), absence of FUBP1 did not affect ESC maintenance. Interestingly, we observed a delayed differentiation of FUBP1-deficient ESCs into the mesoderm germ layer, as indicated by impaired expression of several mesoderm markers including Brachyury at an early time point of ESC differentiation upon aggregation to EBs. Coculture experiments with OP9 cells in the presence of erythropoietin revealed a diminished differentiation capacity of Fubp1 KO ESCs into the erythroid lineage. Our data showed that FUBP1 is important for the onset of mesoderm differentiation and maturation of hematopoietic progenitor cells into the erythroid lineage, a finding that is supported by the phenotype of FUBP1-deficient mice. 1. Introduction The far upstream element (FUSE) binding protein 1 (FUBP1) was identified as a transcriptional regulator that binds to the single-stranded AT-rich FUSE DNA sequence 1.5 kb upstream of the c-myc promoter [1]. We and others found FUBP1 to be upregulated in a number of tumor entities, such as hepatocellular carcinoma (HCC), prostate, and colorectal cancer [2–5]. Our studies demonstrated an essential role for FUBP1 in HCC tumorigenesis and established FUBP1 as a pro-proliferative and antiapoptotic oncoprotein [4]. In our recent work, we analyzed the physiological role of FUBP1 in two independent functional FUBP1 knockout mouse models. In both models, FUBP1 deficiency led to embryonic lethality around day E15.5 and a strong anemic phenotype [6]. The embryos displayed a reduced number of hematopoietic stem cells (HSCs) in the fetal liver, and in contrast to wildtype controls, the remaining FUBP1deficient HSCs were not able to repopulate the blood lineages in a competitive transplantation experiment. Our studies established FUBP1 as an important regulator of HSC self-renewal. In addition, we noticed that the ery- 2 throid lineage in the FUBP1 mutant E15.5 embryos showed a diminished proportion of mature cells, hinting towards an erythroid differentiation defect in the absence of FUBP1 [6]. The essential role of FUBP1 in HSC self-renewal raises the question about the potential role of the protein in other stem cells. Interestingly, the pathohistological analysis of Fubp1 knockout embryos showed abnormalities during the development of the placenta and of lymphoid tissue and an increased parenchymal cellularity in the brain [7]. Embryonic stem cells (ESCs) are pluripotent cells, that is, they possess an infinite self-renewal potential and can differentiate into cells of all three germ layers (ectoderm, endoderm, and mesoderm) and the germline, ultimately contributing to all lineages of the mature organism [8]. Since the 1980s, mouse ESCs can be isolated from the inner cell mass of blastocysts (most suitable at day E 3.5) and cultivated on feeder cells, which usually consist of replication-deficient fibroblasts. Addition of leukemia inhibitory factor (LIF) to the growth medium can substitute the feeder cells, and ESC lines cultured on gelatincoated plates in the presence of LIF still maintain their stemness [9, 10]. The recent progress in the ESC research field holds high promise for biomedicine and transplantation medicine as well as for the pharmaceutic developmental research [11, 12]. Discovering novel genes important for specific differentiation decisions led to huge efforts to employ ESCs for cellular therapies [13]. A number of protocols for the differentiation of ESCs into a variety of cell types were established in the last two decades of stem cell research [14–16] (for review of literature describing specifically the in vitro differentiation of ESCs towards the hematopoietic lineage see for example [17, 18]). However, the formation of EBs, which represents the early embryonic development, is a spontaneous germ layer differentiation induced by the absence of LIF and used in almost every differentiation protocol as a first step [19, 20]. The embryonic stem cells undergo a rapid differentiation process during the formation of EBs, and the stem cell markers such as Oct4 and Nanog are downregulated. In parallel, a rapid upregulation of markers for the three germ layers ectoderm, endoderm, and mesoderm occurs [21]. The aim of this study was to analyze the function of FUBP1 in murine embryonic stem cells during spontaneous differentiation upon aggregation to EBs in the absence of LIF. In addition, we wanted to employ the induction of erythropoiesis in ESCs as a suitable cell culture model to complement our in vivo studies on the role of FUBP1 during erythropoiesis in FUBP1-deficient mice [6]. We established Fubp1 knockout ESC clones with the help of the CRISPR/Cas9 technology [22] and analyzed the consequences of FUBP1 deficiency in ESCs and during EB formation using the stem cell markers Oct4 and Nanog [23–26] and a number of differentiation markers indicative for the mesoderm, ectoderm, and endoderm germ layer cells. Finally, we cocultured the ESCs with OP9 cells [27, 28] to study the direct effect of FUBP1 inactivity for erythroid differentiation. Stem Cells International 2. Materials and Methods 2.1. Cell Lines Used, Embryonic Stem Ce (...truncated)