Takeo Kato

Generation of Pluripotent Stem Cells from Neonatal Mouse Testis

Although germline cells can form multipotential embryonic stem (ES)/embryonic germ (EG) cells, these cells can be derived only from embryonic tissues, and such multipotent cells have not been available from neonatal gonads. Here we report the successful establishment of ES-like cells from neonatal mouse testis. These ES-like cells were phenotypically similar to ES/EG cells except in their genomic imprinting pattern. They differentiated into various types of somatic cells in vitro under conditions used to induce the differentiation of ES cells and produced teratomas after inoculation into mice. Furthermore, these ES-like cells formed germline chimeras when injected into blastocysts. Thus, the capacity to form multipotent cells persists in neonatal testis. The ability to derive multipotential stem cells from the neonatal testis has important implications for germ cell biology and opens the possibility of using these cells for biotechnology and medicine.

Prospective characterization of neural stem cells by flow cytometry analysis using a combination of surface markers.

Neural stem cells (NSCs) with self‐renewal and multilineage differentiation properties can potentially repair degenerating or damaged neural tissue. Here, we have enriched NSCs from neurospheres, which make up a heterogeneous population, by fluorescence‐activated cell sorting (FACS) with antibodies against syndecan‐1, Notch‐1, and integrin‐β1, which were chosen as candidates for hematopoietic cell—or somatic stem cell—markers. Antigen‐positive cells readily initiated neurosphere formation, but cells lacking these markers did so less readily. Doubly positive cells expressing both syndecan‐1 and Notch‐1 underwent neurosphere formation more efficiently than did singly positive cells. The progeny of sorted cells could differentiate into neurons and glial cells both in vitro and in vivo. These antibodies were also useful for isolating cells from the murine embryonic day 14.5 brain that efficiently formed neurospheres. In contrast, there was no distinct difference in neurosphere formation efficiency between Hoechst 33342‐stained side population cells and main population cells, although the former are known to have a stem cell phenotype in various tissues. These results indicate the usefulness of syndecan‐1, Notch‐1, and integrin‐β1 as NSC markers.

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.

Relation between muscle thickness, spasticity, and activity limitations in children and adolescents with cerebral palsy

The aim of this study was to investigate the relation between muscle thickness of the quadriceps femoris muscle, knee joint function (spasticity and range of motion), and activity and participation measures in children and adolescents with cerebral palsy (CP). Thirty‐eight children and adolescents with mild to severe CP (20 males, 18 females; mean age 12y 8mo [SD 3y 7mo], range 6‐18y) participated. The severity and type of CP of participants covered all five levels of the Gross Motor Function Classification System and three types: spastic (quadriplegia, hemiplegia, and diplegia), athetotic, and hypotonic. The thickness of the quadriceps femoris muscle (MTQ) was measured from B‐mode ultrasound images. Activity limitations were evaluated by the Gross Motor Function Measurement‐66 (GMFM‐66) and the Pediatric Evaluation of Disability Inventory (PEDI). Spasticity was assessed with the modified Ashworth scale (MAS). After adjustment for age and body mass index, the MTQ showed significant positive correlations with GMFM‐66 and PEDI scores; however, there was no significant correlation with MAS ratings. The degree of knee flexion contracture correlated positively with the MAS rating of the knee flexor muscles and negatively with the MTQ. These results established the clinical relevance of assessment of muscle thickness across a broad spectrum of individuals with CP.

Intracranial aneurysms in Ehlers-Danlos syndrome type IV in early childhood

Ehlers-Danlos syndrome type IV is of special interest to neurologists because of the risk of cerebrovascular complications. We describe a 5-year-old female with Ehlers-Danlos syndrome type IV, demonstrating multiple intracranial aneurysms and right middle cerebral artery stenosis. The diagnosis of Ehlers-Danlos syndrome type IV was confirmed by electron microscopic examination of a skin biopsy. To our knowledge, this is the youngest reported patient with intracranial aneurysms associated with the Ehlers-Danlos syndrome type IV. Ehlers-Danlos syndrome type IV should be considered in the differential diagnosis of cerebrovascular disorder and stroke in early childhood.

PCDH19 mutation in Japanese females with epilepsy

PURPOSE To determine the significance of PCDH19 mutations in Japanese females with epilepsy and to delineate their phenotypes. METHODS PCDH19 sequencing analysis was performed in 116 females with various epilepsies, including 97 with Dravet syndrome (83.6%). They were referred for SCN1A analysis, and 52 carried SCN1A mutations. RESULTS Seven heterozygous mutations in exon 1 were identified in 7 patients (6.0%): 2 frameshift, 2 nonsense, and 3 missense mutations. One patient was a monozygotic twin, and her sister with mild phenotype carried the same mutation. The main clinical features among these 8 patients included early seizure onset (≤25 months of age), seizure clusters (7/8), fever-associated seizures (7/8), single seizure type (6/8), and late deterioration of intellect (5/8). Seizure durations were generally up to a few minutes, and only one patient developed status epilepticus once. The main seizure types were generalized tonic-clonic (4/8), tonic (3/8) and focal seizures, with (2/8) or without secondary generalization (3/8). Myoclonic, atonic and absence seizures were extremely rare. Two patients had Dravet syndrome (25%), and this proportion was significantly smaller than that in the total subjects (p<0.01). CONCLUSION PCDH19 mutation is a relatively frequent cause of epilepsy in Japanese females. Dravet syndrome was rare in our cohort.

Loss of Borealin/DasraB leads to defective cell proliferation, p53 accumulation and early embryonic lethality

Borealin/DasraB is a member of the chromosomal passenger protein complex (CPC) required for proper segregation of chromosomes during mitosis. In Drosophila melanogaster, inactivation of Borealin/DasraB results in polyploidy, delayed mitosis and abnormal tissue development, indicating its critical role for cell proliferation. However, the in vivo role of mammalian Borealin/DasraB remains unclear. Here, we analyzed the expression of Borealin/DasraB and found that borealin is widely expressed in embryonic tissues and later restricted to adult tissues which relies on rapid cell proliferation. To determine the role of borealin during mouse development, we generated borealin-null mice through targeted disruption. While heterozygous mice developed normally, disruption of both borealin alleles resulted in early embryonic lethality by 5.5 dpc (days postcoitus) due to mitotic defects and apoptosis in blastocyst cells that showed microtubule disorganization and no CPC enrichment. At 5.5 dpc, borealin-null embryos exhibited excessive apoptosis and elevated expression of p53. However, loss of p53 did not abrogate or delay embryonic lethality, revealing that Borealin/DasraB inactivation triggered impaired mitosis and apoptosis though p53-independent mechanisms. Our data show that Borealin/DasraB is essential for cell proliferation during early embryonic development, and its early embryonic lethality cannot be rescued by the loss of p53.

Subclinical central pontine myelinolysis following liver transplantation

Central pontine myelinolysis (CPM) is a demyelinating disorder of unknown origin that almost exclusively affects the central portion of the basis pontis, and is one of the fatal neurological complications after liver transplantation. We describe two children with CPM detected incidentally after liver transplantation. To our knowledge, this is the first report of CPM diagnosed antemortem in children who had undergone liver transplantation. In our patients, there were no clinical manifestations associated with CPM. We conclude that, including silent cases such as our patients, CPM may be more prevalent than previously appreciated following liver transplantation.

Ttyh1, a Ca2+-binding protein localized to the endoplasmic reticulum, is required for early embryonic development

Using comprehensive genetic studies on neuronal stem/progenitors cells through genome‐wide screening with oligonucleotide arrays, we identified an endoplasmic reticulum (ER) ‐resident protein, Tweety homologue 1 (ttyh1). Ttyh1 encodes a glycosylated protein composed of five predicted transmembrane segments and a C‐terminus that is enriched in negatively charged residues capable of Ca2+ binding. Ttyh1‐containing membranes changed to segmented tubuloreticular structures during mitosis, suggesting that the ER‐containing Ttyh1 could be responsible for Ca2+ sequestration and Ca2+ concentration regulation during mitosis. Ttyh1 inactivation in mice resulted in early embryonic lethality before organization of the nervous system, revealing that ttyh1 is essential in murine embryonic development. Our findings indicate that Ttyh1 plays an indispensable role during mitosis in early embryogenesis, possibly by maintaining Ca2+ homeostasis in the ER. Developmental Dynamics 239:2233–2245, 2010.

Successful cord blood transplantation using a reduced-intensity conditioning regimen for advanced childhood-onset cerebral adrenoleukodystrophy.

Awaya T, Kato T, Niwa A, Hiramatsu H, Umeda K, Watanabe K‐i, Shibata M, Yamanaka Y, Maruya E, Saji H, Nakahata T, Adachi S. Successful cord blood transplantation using a reduced‐intensity conditioning regimen for advanced childhood‐onset cerebral adrenoleukodystrophy.
Pediatr Transplantation 2011: 15: E116–E120.