Generation of functional erythrocytes from human embryonic stem cell-derived definitive hematopoiesis.

A critical issue for clinical utilization of human ES cells (hESCs) is whether they can generate terminally mature progenies with normal function. We recently developed a method for efficient production of hematopoietic progenitors from hESCs by coculture with murine fetal liver-derived stromal cells. Large numbers of hESCs-derived erythroid progenitors generated by the coculture enabled us to analyze the development of erythropoiesis at a clone level and investigate their function. The results showed that the globin expression in the erythroid cells in individual clones changed in a time-dependent manner. In particular, embryonic epsilon-globin-expressing erythroid cells from individual clones decreased, whereas adult-type beta-globin-expressing cells increased to approximately 100% in all clones we examined, indicating that the cells undergo definitive hematopoiesis. Enucleated erythrocytes also appeared among the clonal progeny. A comparison analysis showed that hESC-derived erythroid cells took a similar differentiation pathway to human cord blood CD34(+) progenitor-derived cells when examined for the expression of glycophorin A, CD71 and CD81. Furthermore, these hESC-derived erythroid cells could function as oxygen carriers and had a sufficient glucose-6-phosphate dehydrogenase activity. The present study should provide an experimental model for exploring early development of human erythropoiesis and hemoglobin switching and may help in the discovery of drugs for hereditary diseases in erythrocyte development.

Novel method for efficient production of multipotential hematopoietic progenitors from human embryonic stem cells.

We propose a novel method for the efficient production of hematopoietic progenitors from human embryonic stem cells (hESC) via coculture with murine fetal liver-derived stromal cells, in which embryonic hematopoiesis dramatically expands at midgestation. We generated various hematopoietic progenitors in coculture, and this hematopoietic activity was concentrated in cobblestone-like cells derived from differentiated hESC. The cobblestone-like cells mostly expressed CD34 and retained an endothelial cell potential. They also contained hematopoietic colony-forming cells, especially erythroid and multilineage colony-forming cells at high frequency. The multipotential hematopoietic progenitors abundant among the cobblestone-like cells produced almost all types of mature blood cells, including adult-type alpha-globin-expressing erythrocytes and tryptase/chymase double-positive mast cells. These progenitors showed neither the immature properties of ESC nor the potential to differentiate into endoderm and ectoderm at a clonal level. The coculture system developed for hESC can provide a novel source of hematopoietic and blood cells for applications in cellular therapy and drug screening.

Screening of drugs to treat 8p11 myeloproliferative syndrome using patient-derived induced pluripotent stem cells with fusion gene CEP110-FGFR1.

Induced pluripotent stem (iPS) cells provide powerful tools for studying disease mechanisms and developing therapies for diseases. The 8p11 myeloproliferative syndrome (EMS) is an aggressive chronic myeloproliferative disorder (MPD) that is caused by constitutive activation of fibroblast growth factor receptor 1. EMS is rare and, consequently, effective treatment for this disease has not been established. Here, iPS cells were generated from an EMS patient (EMS-iPS cells) to assist the development of effective therapies for EMS. When iPS cells were co-cultured with murine embryonic stromal cells, EMS-iPS cells produced more hematopoietic progenitor and hematopoietic cells, and CD34+ cells derived from EMS-iPS cells exhibited 3.2-7.2-fold more macrophage and erythroid colony forming units (CFUs) than those derived from control iPS cells. These data indicate that EMS-iPS cells have an increased hematopoietic differentiation capacity, which is characteristic of MPDs. To determine whether a tyrosine kinase inhibitor (TKI) could suppress the increased number of CFUs formed by EMS-iPS-induced CD34+ cells, cells were treated with one of four TKIs (CHIR258, PKC 412, ponatinib, and imatinib). CHIR258, PKC 412, and ponatinib reduced the number of CFUs formed by EMS-iPS-induced CD34+ cells in a dose-dependent manner, whereas imatinib did not. Similar effects were observed on primary peripheral blood cells (more than 90% of which were blasts) isolated from the patient. This study provides evidence that the EMS-iPS cell line is a useful tool for the screening of drugs to treat EMS and to investigate the mechanism underlying this disease.

Impaired neutrophil maturation in truncated murine G-CSF receptor-transgenic mice.

Severe congenital neutropenia (SCN) is a hematopoietic disorder characterized by neutropenia in peripheral blood and maturation arrest of neutrophil precursors in bone marrow. Patients with SCN may evolve to have myelodysplastic syndrome or acute myelocytic leukemia. In approximately 20% of SCN cases, a truncation mutation is found in the cytoplasmic region of the granulocyte colony-stimulating factor receptor (G-CSFR). We then generated mice carrying murine wild-type G-CSFR and its mutants equivalent to truncations at amino acids 718 and 731 in human G-CSFR, those were reported to be related to leukemic transformation of SCN. Although numbers of peripheral white blood cells, red blood cells, and platelets did not differ among mutant and wild-type G-CSFR transgenic (Tg) mice, both of the mutant receptor Tg mice had one third of peripheral neutrophil cell counts compared with wild-type receptor Tg mice. The mutant receptor Tg mice also showed impaired resistance to the infection with Staphylococcus aureus. Moreover, bone marrow of these Tg mice had an increased percentage of immature myeloid cells, a feature of SCN. This maturation arrest was also observed in in vitro cultures of bone marrow cells of truncated G-CSFR Tg mice under G-CSF stimulation. In addition, clonal culture of bone marrow cells of the truncated G-CSFR Tg mice showed the hypersensitivity to G-CSF in myeloid progenitors. Our Tg mice may be useful in the analysis of the role of truncated G-CSFR in SCN pathobiology.