Establishment of mouse embryonic stem cell-derived erythroid progenitor cell lines able to produce functional red blood cells

Abstract

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.

Figure 1. Characteristics of erythroid cell lines derived from mouse ES cells, MEDEP.

(A) Morphology of two erythroid cell lines, MEDEP-E14 and MEDEP-BRC5. Wright-Giemsa staining. (B) Cytokine dependent proliferation. Cells (1×10⁵ cells/ml) were cultured in various conditions for three days. The added factor(s) is shown at the bottom. None, no specific factor. SCF, stem cell factor. EPO, erythropoietin. Broken line, the number of cells at the start of culture. Values are mean±S.D. Results shown are representative of several independent experiments performed at different time points after establishment of the cell lines. (C) RT-PCR analyses. Oct-3/4 and Nanog, transcription factors specific for ES cells. GATA-1 and EKLF (Erythroid Krüppel-like factor), transcription factors specific for erythroid cells. EPOR, erythropoietin receptor. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. NC, negative control without cDNA. Day 0, E14TG2a cells before differentiation. Day 4, 7, 10, 14 and 21, the cells following induction of differentiation into hematopoietic cells from E14TG2a by the method described in Table 1 (Method A). The cycle numbers performed in each PCR are shown at the right. Results shown are representative of two independent experiments.

Figure 2. In vitro differentiation of MEDEP.

The in vitro differentiation of MEDEP-E14 was performed by culture for two days after deprivation of erythropoietin (EPO). The in vitro differentiation of MEDEP-BRC5 was performed by culture for three days after deprivation of stem cell factor (SCF) and addition of EPO. (A) Flow cytometric analyses. Control, results with isotype controls. Before and After, the cells before and after in vitro differentiation. CD71, transferrin receptor. c-Kit, receptor for SCF. TER119, a cell surface antigen specific for mature erythroid cells. (B) Cell pellets before and after in vitro differentiation. The method for in vitro differentiation of MEDEP-BRC4 is described in Figure S4. (C) Morphology of the cells after in vitro differentiation. Arrows indicate enucleated red blood cells. (A–C) Results shown are representative of three independent experiments.

Figure 3. In vivo proliferation and differentiation of MEDEP.

A transformant of MEDEP-E14 expressing Venus as a marker was established, MEDEP-E14-Venus. (A) The in vitro differentiation of MEDEP-E14-Venus was performed by culture for two days after deprivation of erythropoietin. Control, results with isotype controls. Before and After, the cells before and after in vitro differentiation. (B) In vivo differentiation of MEDEP-E14-Venus cells. Acute anemia was induced in an immuno-deficient mouse (NOD-SCID) and the next day MEDEP-E14-Venus cells (2×10⁷ cells/mouse) were transplanted into the anemic mouse. Three days after cell transplantation, bone marrow and spleen cells were subjected to flow cytometric analyses. Control mouse, NOD-SCID mouse without cell transplantation. The vast majority of Venus-positive cells in the spleen show differentiation into CD71⁺TER119⁺ mature erythroid cells. (A, B) CD71 and TER119, see legend of Figure 2A. Results shown are representative of three independent experiments. (C) In vivo proliferation of MEDEP-E14-Venus cells. Cell transplantation was performed as in (B). We determined the proportion (%) of Venus-positive cells and calculated the absolute number of Venus-positive cells in the spleen. Day 1 and Day 3, one day and three days following cell transplantation, respectively. Values are mean±S.D. (N = 3).

Figure 4. Amelioration of anemia by transplantation of MEDEP.

(A) MEDEP-E14 cells (2×10⁷ cells/mouse) were transplanted into an immuno-deficient mouse (NOD-SCID) 24 hours after the induction of hemolysis by phenylhydrazine (60 mg/kg body weight) injection. Day 5 and Day 26, five and twenty-six days after cell transplantation. RBC, red blood cell. White bars (n = 10) and black bars (n = 14), the data obtained from the mice transplanted with control cells and MEDEP-E14 cells, respectively. Values are mean±S.D. * p<0.01 (by the Student's t-test) (B) Increased survival of mice transplanted with MEDEP cells following induction of severe acute anemia. MEDEP-E14 cells (2×10⁷ cells/mouse) were transplanted into an NOD-SCID mouse 24 hours following the first induction of hemolysis by phenylhydrazine (60 mg/kg body weight) injection. Five days following the cell transplantation, the second induction of hemolysis by phenylhydrazine (80 mg/kg body weight) injection was performed. Statistical analysis was performed using the chi-square test. (A, B) Control cell, mast cell line derived from mouse ES cells (MEDMC-NT2) (Figures S1 and S2). MEDMC-NT2 cells (2×10⁷ cells/mouse) were transplanted similarly as a control experiment.