Katsutsugu Umeda

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 ε-globin-expressing erythroid cells from individual clones decreased, whereas adult-type β-globin-expressing cells increased to ≈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.

Generation of skeletal muscle stem/progenitor cells from murine induced pluripotent stem cells

Induced pluripotent stem (iPS) cells, which are a type of pluripotent stem cell generated from reprogrammed somatic cells, are expected to have potential for patient‐oriented disease investigation, drug screening, toxicity tests, and transplantation therapies. Here, we demonstrated that murine iPS cells have the potential to develop in vitro into skeletal muscle stem/progenitor cells, which are almost equivalent to murine embryonic stem cells. Cells with strong in vitro myogenic potential effectively were enriched by fluorescence‐activated cell sorting using the anti‐satellite cell antibody SM/C‐2.6. Furthermore, on transplantation into mdx mice, SM/C‐2.6+ cells exerted sustained myogenic lineage differentiation in injured muscles, while providing long‐lived muscle stem cell support. Our data suggest that iPS cells have the potential to be used in clinical treatment of muscular dystrophies.—Mizuno, Y., Chang, H., Umeda, K., Niwa, A, Iwasa, T., Awaya, T., Fukada, S., Yamamoto, H., Yamanaka, S., Nakahata, T., Heike, T. Generation of skeletal muscle stem/progenitor cells from murine induced pluripotent stem cells. FASEB J. 24, 2245–2253 (2010). www.fasebj.org

Development of primitive and definitive hematopoiesis from nonhuman primate embryonic stem cells in vitro

Although information about the development of primitive and definitive hematopoiesis has been elucidated in murine embryos and embryonic stem (ES) cells, there have been few in vitro studies of these processes in primates. In this study, we investigated hematopoietic differentiation from cynomolgus monkey ES cells grown on OP9, a stromal cell line deficient in macrophage colony-stimulating factor. Primitive erythrocytes (EryP) and definitive erythrocytes (EryD) developed sequentially from ES cells in the culture system; this was confirmed by immunostaining and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of embryonic, fetal and adult globin genes. EryP were detected on day 8 without exogenous erythropoietin (EPO), whereas EryD appeared on day 16 and had an indispensable requirement for exogenous EPO. RT-PCR analysis of the cultures revealed a sequential expression of genes associated with primitive and definitive hematopoietic development that was equivalent to that seen during primate ontogeny in vivo. Vascular endothelial growth factor (VEGF) increased, in a dose-dependent manner, not only the number of floating hematopoietic cells, but also the number of adherent hematopoietic cell clusters containing CD34-positive immature progenitors. In colony assays, exogenous VEGF also had a dose-dependent stimulatory effect on the generation of primitive erythroid colonies. More efficient primitive and definitive erythropoiesis was induced by re-plating sorted CD34-positive cells. Thus, this system reproduces early hematopoietic development in vitro and can serve as a model for analyzing the mechanisms of hematopoietic development in primates.

Generation of transplantable, functional satellite-like cells from mouse embryonic stem cells

Satellite cells are myogenic stem cells responsible for the postnatal regeneration of skeletal muscle. Here we report the successful in vitro induction of Pax7‐positive satellite‐like cells from mouse embryonic stem (mES) cells. Embryoid bodies were generated from mES cells and cultured on Matrigel‐coated dishes with Dulbeccos modified Eagle medium containing fetal bovine serum and horse serum. Pax7‐positive satellite‐like cells were enriched by fluorescence‐activated cell sorting using a novel anti‐satellite cell antibody, SM/C‐2.6. SM/C‐2.6‐positive cells efficiently differentiate into skeletal muscle fibers both in vitro and in vivo. Furthermore, the cells demonstrate satellite cell characteristics such as extensive self‐renewal capacity in subsequent muscle injury model, long‐term engraft‐ment up to 24 wk, and the ability to be secondarily transplanted with remarkably high engraftment efficiency compared to myoblast transplantation. This is the first report of transplantable, functional satellite‐like cells derived from mES cells and will provide a foundation for new therapies for degenerative muscle disor‐ders.—Chang, H.,Yoshimoto, M., Umeda, K., Iwasa, T., Mizuno, Y., Fukada, S., Yamamoto, H., Motohashi, N., Yuko‐Miyagoe‐Suzuki, Takeda, S., Heike, T., Nakahata, T. Generation of transplantable, functional satellite‐like cells from mouse embryonic stem cells. FASEB J. 23, 1907–1919 (2009)

A novel serum-free monolayer culture for orderly hematopoietic differentiation of human pluripotent cells via mesodermal progenitors.

Elucidating the in vitro differentiation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells is important for understanding both normal and pathological hematopoietic development in vivo. For this purpose, a robust and simple hematopoietic differentiation system that can faithfully trace in vivo hematopoiesis is necessary. In this study, we established a novel serum-free monolayer culture that can trace the in vivo hematopoietic pathway from ES/iPS cells to functional definitive blood cells via mesodermal progenitors. Stepwise tuning of exogenous cytokine cocktails induced the hematopoietic mesodermal progenitors via primitive streak cells. These progenitors were then differentiated into various cell lineages depending on the hematopoietic cytokines present. Moreover, single cell deposition assay revealed that common bipotential hemoangiogenic progenitors were induced in our culture. Our system provides a new, robust, and simple method for investigating the mechanisms of mesodermal and hematopoietic differentiation.

Selective Development of Myogenic Mesenchymal Cells from Human Embryonic and Induced Pluripotent Stem Cells

Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are promising sources for the cell therapy of muscle diseases and can serve as powerful experimental tools for skeletal muscle research, provided an effective method to induce skeletal muscle cells is established. However, the current methods for myogenic differentiation from human ES cells are still inefficient for clinical use, while myogenic differentiation from human iPS cells remains to be accomplished. Here, we aimed to establish a practical differentiation method to induce skeletal myogenesis from both human ES and iPS cells. To accomplish this goal, we developed a novel stepwise culture method for the selective expansion of mesenchymal cells from cell aggregations called embryoid bodies. These mesenchymal cells, which were obtained by dissociation and re-cultivation of embryoid bodies, uniformly expressed CD56 and the mesenchymal markers CD73, CD105, CD166, and CD29, and finally differentiated into mature myotubes in vitro. Furthermore, these myogenic mesenchymal cells exhibited stable long-term engraftment in injured muscles of immunodeficient mice in vivo and were reactivated upon subsequent muscle damage, increasing in number to reconstruct damaged muscles. Our simple differentiation system facilitates further utilization of ES and iPS cells in both developmental and pathological muscle research and in serving as a practical donor source for cell therapy of muscle diseases.

Human chondrogenic paraxial mesoderm, directed specification and prospective isolation from pluripotent stem cells.

Directed specification and prospective isolation of chondrogenic paraxial mesoderm progeny from human pluripotent stem (PS) cells have not yet been achieved. Here we report the successful generation of KDR−PDGFRα+ progeny expressing paraxial mesoderm genes and the mesendoderm reporter MIXL1-GFP in a chemically defined medium containing the canonical WNT signaling activator, BMP-inhibitor, and the Nodal/Activin/TGFβ signaling controller. Isolated (GFP+)KDR−PDGFRα+ mesoderm cells were sensitive to sequential addition of the three chondrogenic factors PDGF, TGFβ and BMP. Under these conditions, the cells showed robust chondrogenic activity in micromass culture, and generated a hyaline-like translucent cartilage particle in serum-free medium. In contrast, both STRO1+ mesenchymal stem/stromal cells from adult human marrow and mesenchymal cells spontaneously arising from hPS cells showed a relatively weaker chondrogenic response in vitro, and formed more of the fibrotic cartilage particles. Thus, hPS cell-derived KDR−PDGFRα+ paraxial mesoderm-like cells have potential in engineered cartilage formation and cartilage repair.

Derivation of mesenchymal stromal cells from pluripotent stem cells through a neural crest lineage using small molecule compounds with defined media.

Neural crest cells (NCCs) are an embryonic migratory cell population with the ability to differentiate into a wide variety of cell types that contribute to the craniofacial skeleton, cornea, peripheral nervous system, and skin pigmentation. This ability suggests the promising role of NCCs as a source for cell-based therapy. Although several methods have been used to induce human NCCs (hNCCs) from human pluripotent stem cells (hPSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), further modifications are required to improve the robustness, efficacy, and simplicity of these methods. Chemically defined medium (CDM) was used as the basal medium in the induction and maintenance steps. By optimizing the culture conditions, the combination of the GSK3β inhibitor and TGFβ inhibitor with a minimum growth factor (insulin) very efficiently induced hNCCs (70–80%) from hPSCs. The induced hNCCs expressed cranial NCC-related genes and stably proliferated in CDM supplemented with EGF and FGF2 up to at least 10 passages without changes being observed in the major gene expression profiles. Differentiation properties were confirmed for peripheral neurons, glia, melanocytes, and corneal endothelial cells. In addition, cells with differentiation characteristics similar to multipotent mesenchymal stromal cells (MSCs) were induced from hNCCs using CDM specific for human MSCs. Our simple and robust induction protocol using small molecule compounds with defined media enabled the generation of hNCCs as an intermediate material producing terminally differentiated cells for cell-based innovative medicine.

Human cord blood CD34+ cells develop into hepatocytes in the livers of NOD/SCID/γcnull mice through cell fusion

Several studies have shown that hepatocytes can be generated from hematopoietic stem cells, but this event is believed to be rare and to require hepatic damage. To investigate this phenomenon in human cells, we used a NOD/SCID/γcnull (NOG) mouse model that can achieve a tremendously high level of chimerism when transplanted with human hematopoietic cells. Even without hepatotoxic treatment other than irradiation, human albumin and α‐1‐antitrypsin‐positive cells were invariably detected in the livers of NOG mice after i.v. transplantation of human cord blood CD34+ cells. Human albumin was detected in the murine sera, indicating functional maturation of the human hepatocytes. Flow cytometric analysis of recipient liver cells in single‐cell suspension demonstrated that human albumin‐positive cells were also positive for both murine and human MHC and were negative for human CD45. PCR analysis of recipient livers revealed the expression of a wide variety of human hepatocyte‐ or cholangiocyte‐specific mRNAs. These results show that human CD34+ cells fuse with hepatocytes of NOG mice without liver injury, lose their hematopoietic phenotype, and begin hepatocyte‐specific gene transcription. These phenomena were not observed when CD34− cells were transplanted. Thus, our model revealed a previously unidentified pathway of human hematopoietic stem/progenitor cell differentiation.—Fujino, H., Hiramatsu, H., Tsuchiya, A., Niwa, A., Noma, H., Shiota, M., Umeda, K., Yoshimoto, M., Ito, M., Heike, T., Nakahata, T. Human cord blood CD34+ cells develop into hepatocytes in the livers of NOD/SCID/γcnull mice through cell fusion. FASEB J. 21, 3499–3510 (2007)

Orderly hematopoietic development of induced pluripotent stem cells via Flk-1+ hemoangiogenic progenitors†

Induced pluripotent stem (iPS) cells, reprogrammed somatic cells with embryonic stem (ES) cell‐like characteristics, are generated by the introduction of combinations of specific transcription factors. Little is known about the differentiation of iPS cells in vitro. Here we demonstrate that murine iPS cells produce various hematopoietic cell lineages when incubated on a layer of OP9 stromal cells. During this differentiation, iPS cells went through an intermediate stage consisting of progenitor cells that were positive for the early mesodermal marker Flk‐1 and for the sequential expression of other genes that are associated with hematopoietic and endothelial development. Flk‐1+ cells differentiated into primitive and definitive hematopoietic cells, as well as into endothelial cells. Furthermore, Flk‐1+ populations contained common bilineage progenitors that could generate both hematopoietic and endothelial lineages from single cells. Our results demonstrate that iPS cell‐derived cells, like ES cells, can follow a similar hematopoietic route to that seen in normal embryogenesis. This finding highlights the potential use of iPS cells in clinical areas such as regenerative medicine, disease investigation, and drug screening. J. Cell. Physiol. 221: 367–377, 2009.