Establishment of Mouse Embryonic Stem Cell-Derived Erythroid Progenitor Cell Lines Able to Produce Functional Red Blood Cells Takashi Hiroyama, Kenichi Miharada, Kazuhiro Sudo, Inaho Danjo, Naoko Aoki, Yukio Nakamura* Cell Engineering Division, RIKEN BioResource Center, Tsukuba, Ibaraki, Japan Background. The supply of transfusable red blood cells (RBCs) is not sufficient in many countries. If erythroid cell lines able to produce transfusable RBCs in vitro were established, they would be valuable resources. However, such cell lines have not been established. To evaluate the feasibility of establishing useful erythroid cell lines, we attempted to establish such cell lines from mouse embryonic stem (ES) cells. Methodology/Principal Findings. We developed a robust method to obtain differentiated cell lines following the induction of hematopoietic differentiation of mouse ES cells and established five independent hematopoietic cell lines using the method. Three of these lines exhibited characteristics of erythroid cells. Although their precise characteristics varied, each of these lines could differentiate in vitro into more mature erythroid cells, including enucleated RBCs. Following transplantation of these erythroid cells into mice suffering from acute anemia, the cells proliferated transiently, subsequently differentiated into functional RBCs, and significantly ameliorated the acute anemia. In addition, we did not observe formation of any tumors following transplantation of these cells. Conclusion/Significance. To the best of our knowledge, this is the first report to show the feasibility of establishing erythroid cell lines able to produce mature RBCs. Considering the number of human ES cell lines that have been established so far, the intensive testing of a number of these lines for erythroid potential may allow the establishment of human erythroid cell lines similar to the mouse erythroid cell lines described here. In addition, our results strongly suggest the possibility of establishing useful cell lines committed to specific lineages other than hematopoietic progenitors from human ES cells. Citation: Hiroyama T, Miharada K, Sudo K, Danjo I, Aoki N, et al (2008) Establishment of Mouse Embryonic Stem Cell-Derived Erythroid Progenitor Cell Lines Able to Produce Functional Red Blood Cells. PLoS ONE 3(2): e1544. doi:10.1371/journal.pone.0001544 INTRODUCTION RBC transfusion was the first established transplantation procedure in clinical history, and is a common and indispensable clinical pro- cedure. However, the supply of transfusable RBCs is insufficient in many countries. Thus, there is interest in the development of in vitro procedures for the generation of functional RBCs from hematopoietic stem and/or progenitor cells present in bone marrow or umbilical cord blood [1–3]. Human ES cells possess the potential to produce various differentiated cells able to function in vivo and thus represent another promising resource to produce functional RBCs. Hematopoietic cells including cells of the erythroid lineage have been generated from mouse [4–7], non-human primate [8–10], and human ES cells [11–16]. We have recently established a method to culture hematopoietic cells derived from non-human primate ES cells long term in vitro [17]. The efficiency of generation of erythroid progenitors and/or RBCs varies based on the methods and ES cell lines used. Even with optimal experimental procedures and the most appropriate ES cell line, however, the generation of abundant RBCs directly from primate ES cells is a time-consuming process [17]. If human erythroid progenitor cell lines were established that could produce transfusable and functional RBCs efficiently, they would represent a much more useful resource to produce RBCs than ES cell lines. Several mouse and human erythroid cell lines have been established. However, to the best of our knowledge, there is no cell line that can efficiently differentiate into enucleated RBCs. It is generally difficult to establish hematopoietic cell lines from adult hematopoietic stem or progenitor cells, since these somatic cells are quite sensitive to DNA damage and are unable to maintain the length of telomere repeats on serial passage [18]. By contrast, ES cells are quite resistant to DNA damage and maintain telomere length on serial passage [18]. Therefore, we speculated that these characteristics of ES cells may be advantageous for the establishment of cell lines, since differentiated cells derived from ES cells may retain such characteristics. In addition, mouse cells tend to immortalize more readily than human cells, as has been shown to be the case following the induction of pluripotent stem cell lines from somatic cells [19–22]. Hence, we attempted to evaluate the feasibility of establishing hematopoietic cell lines, erythroid cell lines in particular, from mouse ES cells. RESULTS AND DISCUSSION Establishment of erythroid progenitor cell lines from mouse ES cells To induce differentiation of hematopoietic cells from mouse ES cells, we cultured the latter cells using OP9 cells as feeder cells [5,6,23] in the presence of specific factors (Table 1). OP9 cells were used not only for induction of hematopoietic differentiation but also for establishment of cell lines in the early phase of long term culture of the induced hematopoietic cells (Table 1). In most Academic Editor: Simon Williams, Texas Tech University Health Sciences Center, United States of America Received November 14, 2007; Accepted January 3, 2008; Published February 6, 2008 Copyright: 2008 Hiroyama et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology in Japan. Competing Interests: The authors have declared that no competing interests exist. * To whom correspondence should be addressed. E-mail: yukionak@brc.riken.jp PLoS ONE | www.plosone.org 1 February 2008 | Issue 2 | e1544