Hidetoshi Sakurai

Characterization of mesendoderm: a diverging point of the definitive endoderm and mesoderm in embryonic stem cell differentiation culture

Bipotent mesendoderm that can give rise to both endoderm and mesoderm is an established entity from C. elegans to zebrafish. Although previous studies in mouse embryo indicated the presence of bi-potent mesendoderm cells in the organizer region, characterization of mesendoderm and its differentiation processes are still unclear. As bi-potent mesendoderm is implicated as the major precursor of definitive endoderm, its identification is also essential for exploring the differentiation of definitive endoderm. In this study, we have established embryonic stem (ES) cell lines that carry GFP gene in the goosecoid (Gsc) gene locus and have investigated the differentiation course of mesendodermal cells using Gsc expression as a marker. Our results show that mesendoderm is represented as a Gsc-GFP+E-cadherin(ECD)+PDGFRα(αR)+ population and is selectively induced from ES cells under defined conditions containing either activin or nodal. Subsequently, it diverges to Gsc+ECD+αR- and Gsc+ECD-αR+ intermediates that eventually differentiate into definitive endoderm and mesodermal lineages, respectively. The presence of mesendodermal cells in nascent Gsc+ECD+αR+ population was also confirmed by single cell analysis. Finally, we show that the defined culture condition and surface markers developed in this study are applicable for obtaining pure mesendodermal cells and their immediate progenies from genetically unmanipulated ES cells.

Precise correction of the dystrophin gene in duchenne muscular dystrophy patient induced pluripotent stem cells by TALEN and CRISPR-Cas9.

Summary Duchenne muscular dystrophy (DMD) is a severe muscle-degenerative disease caused by a mutation in the dystrophin gene. Genetic correction of patient-derived induced pluripotent stem cells (iPSCs) by TALENs or CRISPR-Cas9 holds promise for DMD gene therapy; however, the safety of such nuclease treatment must be determined. Using a unique k-mer database, we systematically identified a unique target region that reduces off-target sites. To restore the dystrophin protein, we performed three correction methods (exon skipping, frameshifting, and exon knockin) in DMD-patient-derived iPSCs, and found that exon knockin was the most effective approach. We further investigated the genomic integrity by karyotyping, copy number variation array, and exome sequencing to identify clones with a minimal mutation load. Finally, we differentiated the corrected iPSCs toward skeletal muscle cells and successfully detected the expression of full-length dystrophin protein. These results provide an important framework for developing iPSC-based gene therapy for genetic disorders using programmable nucleases.

In vitro modeling of paraxial and lateral mesoderm differentiation reveals early reversibility

Endothelial cells (ECs) are thought to be derived mainly from the vascular endothelial growth factor receptor 2 (VEGFR‐2)+ lateral mesoderm during early embryogenesis. In this study, we specified several pathways for EC differentiation using a murine embryonic stem (ES) cell differentiation culture system that is a model for cellular processes during early embryogenesis. Based on the results of in vitro fate analysis, we show that, in the main pathway, committed ECs are differentiated through the VEGFR‐2+ platelet‐derived growth factor receptor α (PDGFR‐α)− single‐positive (VSP) population that is derived from the VEGFR‐2+PDGFR‐α+ double‐positive (DP) population. This major differentiation course was also confirmed using DNA microarray analysis. In addition to this main pathway, however, ECs also can be generated from the VEGFR‐2−PDGFR‐α+ single‐positive (PSP) population, which represents the paraxial mesodermal lineage and is also derived from the DP population. Our results strongly suggest that, even after differentiation from the common progenitor DP population into the VSP and PSP populations, these two populations continue spontaneous switching of their surface phenotype, which results in switching of their eventual fates. The rate of this interlineage conversion between VSP and PSP is unexpectedly high. Because of this potential to undergo fate switch, we conclude that ECs can be generated via multiple pathways in in vitro ES cell differentiation.

Efficient and Reproducible Myogenic Differentiation from Human iPS Cells: Prospects for Modeling Miyoshi Myopathy In Vitro

The establishment of human induced pluripotent stem cells (hiPSCs) has enabled the production of in vitro, patient-specific cell models of human disease. In vitro recreation of disease pathology from patient-derived hiPSCs depends on efficient differentiation protocols producing relevant adult cell types. However, myogenic differentiation of hiPSCs has faced obstacles, namely, low efficiency and/or poor reproducibility. Here, we report the rapid, efficient, and reproducible differentiation of hiPSCs into mature myocytes. We demonstrated that inducible expression of myogenic differentiation1 (MYOD1) in immature hiPSCs for at least 5 days drives cells along the myogenic lineage, with efficiencies reaching 70–90%. Myogenic differentiation driven by MYOD1 occurred even in immature, almost completely undifferentiated hiPSCs, without mesodermal transition. Myocytes induced in this manner reach maturity within 2 weeks of differentiation as assessed by marker gene expression and functional properties, including in vitro and in vivo cell fusion and twitching in response to electrical stimulation. Miyoshi Myopathy (MM) is a congenital distal myopathy caused by defective muscle membrane repair due to mutations in DYSFERLIN. Using our induced differentiation technique, we successfully recreated the pathological condition of MM in vitro, demonstrating defective membrane repair in hiPSC-derived myotubes from an MM patient and phenotypic rescue by expression of full-length DYSFERLIN (DYSF). These findings not only facilitate the pathological investigation of MM, but could potentially be applied in modeling of other human muscular diseases by using patient-derived hiPSCs.

Neutrophil depletion delays wound repair in aged mice

One of the most important clinical problems in caring for elderly patients is treatment of pressure ulcers. One component of normal wound healing is the generation of an inflammatory reaction, which is characterized by the sequential infiltration of neutrophils, macrophages and lymphocytes. Neutrophils migrate early in the wound healing process. In aged C57BL/6 mice, wound healing is relatively inefficient. We examined the effects of neutrophil numbers on wound healing in both young and aged mice. We found that the depletion of neutrophils by anti-Gr-1 antibody dramatically delayed wound healing in aged mice. The depletion of neutrophils in young mice had less effect on the kinetics of wound healing. Intravenous G-CSF injection increased the migration of neutrophils to the wound site. While the rate of wound repair did not change significantly in young mice following G-CSF injection, it increased significantly in old mice.

Paraxial Mesodermal Progenitors Derived from Mouse Embryonic Stem Cells Contribute to Muscle Regeneration via Differentiation into Muscle Satellite Cells

Pluripotent embryonic stem (ES) cells hold great potential for cell‐based therapies. Although several recent studies have reported the potential of ES cell‐derived progenitors for skeletal muscle regeneration, how the cells contribute to reconstitution of the damaged myofibers has remained elusive. Here, we demonstrated the process of injured muscle regeneration by the engraftment of ES cell‐derived mesodermal progenitors. Mesodermal progenitor cells were induced by a conventional differentiation system and isolated by flow cytometer of platelet‐derived growth factor receptor‐α (PDGFR‐α), a marker of paraxial mesoderm, and vascular endothelial growth factor receptor‐2 (VEGFR‐2), a marker of lateral mesoderm. The PDGFR‐α+ population that represented the paraxial mesodermal character demonstrated significant engraftment when transplanted into the injured muscle of immunodeficient mouse. Moreover, the PDGFR‐α+ population could differentiate into the muscle satellite cells that were the stem cells of adult muscle and characterized by the expression of Pax7 and CD34. These ES cell‐derived satellite cells could form functional mature myofibers in vitro and generate myofibers fused with the damaged host myofibers in vivo. On the other hand, the PDGFR‐α−VEGFR‐2+ population that showed lateral mesodermal character exhibited restricted potential to differentiate into the satellite cells in injured muscle. Our results show the potential of ES cell‐derived paraxial mesodermal progenitor cells to generate functional muscle stem cells in vivo without inducing or suppressing gene manipulation. This knowledge could be used to form the foundation of the development of stem cell therapies to repair diseased and damaged muscles.

Differentiation of induced pluripotent stem cells to thymic epithelial cells by phenotype

Thymic epithelial cells (TECs) are present in both cortical and medullary thymic areas, and have crucial roles in functional T‐cell development. In this study, we studied the differentiation of induced pluripotent stem cells (iPSCs) to TEC. When iPSC were cultured for 4 days in collagen IV‐coated dishes in the presence of both activin A and lithium chloride (LiCl), the cells differentiated to definitive endoderm through mesendoderm. Further treatment with Fgf8 followed by Fgf7, Fgf10 and BMP4 differentiated iPSC to thymic epithelial progenitor cells (TEPCs) by phenotype. Gene expression of Hoxa3, Pax1 and Pax9 was observed and cell surface proteins EpCAM1 and MTS24 were detected at day 14 of iPSC differentiation. TEPCs differentiated to medullary TECs (mTECs) by phenotype following the addition of receptor activator nuclear factor B ligand with LiCl. Thus, we successfully induced efficient differentiation from mouse iPSC to TEPCs and mTEC by phenotype using chemically defined conditions.

Bidirectional induction toward paraxial mesodermal derivatives from mouse ES cells in chemically defined medium.

Embryonic stem cells (ESCs) are a renewable cell source of tissue for regenerative therapies. The addition of bone morphogenetic protein 4 (BMP4) to serum-free ESC cultures can induce primitive streak-like mesodermal cells. In differentiated mouse ESCs, platelet-derived growth factor receptor-alpha (PDGFR-alpha) and E-cadherin (ECD) are useful markers to distinguish between paraxial mesodermal progenitor cells and undifferentiated and endodermal cells, respectively. Here, we demonstrate methods for BMP4-mediated induction of paraxial mesodermal progenitors using PDGFR-alpha and ECD as markers for purification and characterization. Serum-free monolayers of ESCs cultured with BMP4 could efficiently promote paraxial mesodermal differentiation akin to embryonic mesodermal development. BMP4 treatment alone induced paraxial mesodermal progenitors that could differentiate into osteochondrogenic cells in vitro and in vivo. Furthermore, early removal of BMP4 followed by lithium chloride (LiCl) promoted the differentiation to myogenic progenitor cells. These myogenic progenitors were able to differentiate further in vitro into mature skeletal muscle cells. Thus, we successfully induced the efficient bidirectional differentiation of mouse ESCs toward osteochondrogenic and myogenic cell types using chemically defined conditions.

Metalloprotease-Dependent Onset of Blood Circulation in Zebrafish

The primitive blood circulation requires intravascular plasma flow. However, it remains unclear whether the onset of earliest blood circulation is dependent solely on establishment of a functional circulatory organ or whether it also requires active processes inherent in blood cells. In this study, we present novel mechanisms for the onset of blood circulation by monitoring fluorescently labeled blood precursors and blood vessels in zebrafish. The earliest blood circulation occurs synchronously. This synchrony is achieved by the retention of erythroid precursors on the lumen of the vasculature after their invasion from the subaortic region, and then by simultaneous release of these precursors into the flow. Morphological and biochemical analyses suggest that the onset of blood circulation accompanies disruption of blood cell-to-vessel adhesion and requires metalloprotease-dependent processes. ADAM8, a member of the a disintegrin and metalloprotease (ADAM) family, mediates the onset of blood circulation. In ADAM8-depleted embryos, erythroid cells fail to detach from the vascular lumen and stagnate. Expression of a protease-defective ADAM8 in erythroid cells causes dominant-negative effects on blood circulation, suggesting cell-autonomous roles of ADAM8. Based on these findings, we propose that the first erythroid cells require both flow-dependent passive and proteolysis-dependent active processes to enter the circulation.

Early pathogenesis of Duchenne muscular dystrophy modelled in patient-derived human induced pluripotent stem cells.

Duchenne muscular dystrophy (DMD) is a progressive and fatal muscle degenerating disease caused by a dystrophin deficiency. Effective suppression of the primary pathology observed in DMD is critical for treatment. Patient-derived human induced pluripotent stem cells (hiPSCs) are a promising tool for drug discovery. Here, we report an in vitro evaluation system for a DMD therapy using hiPSCs that recapitulate the primary pathology and can be used for DMD drug screening. Skeletal myotubes generated from hiPSCs are intact, which allows them to be used to model the initial pathology of DMD in vitro. Induced control and DMD myotubes were morphologically and physiologically comparable. However, electric stimulation of these myotubes for in vitro contraction caused pronounced calcium ion (Ca2+) influx only in DMD myocytes. Restoration of dystrophin by the exon-skipping technique suppressed this Ca2+ overflow and reduced the secretion of creatine kinase (CK) in DMD myotubes. These results suggest that the early pathogenesis of DMD can be effectively modelled in skeletal myotubes induced from patient-derived iPSCs, thereby enabling the development and evaluation of novel drugs.