Tomonari Awaya

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

Aicardi-Goutières Syndrome Is Caused by IFIH1 Mutations

Aicardi-Goutières syndrome (AGS) is a rare, genetically determined early-onset progressive encephalopathy. To date, mutations in six genes have been identified as etiologic for AGS. Our Japanese nationwide AGS survey identified six AGS-affected individuals without a molecular diagnosis; we performed whole-exome sequencing on three of these individuals. After removal of the common polymorphisms found in SNP databases, we were able to identify IFIH1 heterozygous missense mutations in all three. In vitro functional analysis revealed that IFIH1 mutations increased type I interferon production, and the transcription of interferon-stimulated genes were elevated. IFIH1 encodes MDA5, and mutant MDA5 lacked ligand-specific responsiveness, similarly to the dominant Ifih1 mutation responsible for the SLE mouse model that results in type I interferon overproduction. This study suggests that the IFIH1 mutations are responsible for the AGS phenotype due to an excessive production of type I interferon.

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.

A Combination Therapy of Whole Lung Lavage and GM-CSF Inhalation in Pulmonary Alveolar Proteinosis

Systemic and inhalation therapy of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) is usually effective in controlling autoimmune pulmonary alveolar proteinosis (PAP), but some cases are refractory to GM‐CSF therapy and subjected to whole lung lavage (WLL). A 9‐year‐old girl developed severe respiratory failure due to autoimmune PAP was treated with inhalational 250 µg of GM‐CSF daily, however, it was ineffective. Unilateral WLL was performed three times and subsequent GM‐CSF inhalation therapy yielded marked physiological and radiological improvement and was continued for 1 year. Pediatr Pulmonol. 2008; 43:828–830.

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.

Heterozygous TREX1 p.Asp18Asn mutation can cause variable neurological symptoms in a family with Aicardi–Goutières syndrome/familial chilblain lupus

Marco U. Martı́nez-Martı́nez, Lourdes Baranda-Cándido, Roberto González-Amaro, Oscar Pérez-Ramı́rez and Carlos Abud-Mendoza Regional Unit of Rheumatology and Osteoporosis, Central Hospital ‘Dr. Ignacio Morones Prieto’ and Faculty of Medicine, Immunology, Faculty of Medicine and Hematology, Central Hospital ‘Dr Ignacio Morones Prieto’ and Faculty of Medicine, Universidad Autónoma de San Luis Potosı́, San Luis Potosı́, México. Accepted 10 May 2012 Correspondence to: Carlos Abud-Mendoza, Regional Unit of Rheumatology and Osteoporosis, Central Hospital ‘Dr Ignacio Morones Prieto’, Av. V. Carranza 2395, San Luis Potosı́, S.L.P., zc 78240, México. E-mail: c_abud@hotmail.com

Rare Noncoding Mutations Extend the Mutational Spectrum in the PGAP3 Subtype of Hyperphosphatasia with Mental Retardation Syndrome

HPMRS or Mabry syndrome is a heterogeneous glycosylphosphatidylinositol (GPI) anchor deficiency that is caused by an impairment of synthesis or maturation of the GPI‐anchor. The expressivity of the clinical features in HPMRS varies from severe syndromic forms with multiple organ malformations to mild nonsyndromic intellectual disability. In about half of the patients with the clinical diagnosis of HPMRS, pathogenic mutations can be identified in the coding region in one of the six genes, one among them is PGAP3. In this work, we describe a screening approach with sequence specific baits for transcripts of genes of the GPI pathway that allows the detection of functionally relevant mutations also including introns and the 5′ and 3′ UTR. By this means, we also identified pathogenic noncoding mutations, which increases the diagnostic yield for HPMRS on the basis of intellectual disability and elevated serum alkaline phosphatase. In eight affected individuals from different ethnicities, we found seven novel pathogenic mutations in PGAP3. Besides five missense mutations, we identified an intronic mutation, c.558‐10G>A, that causes an aberrant splice product and a mutation in the 3′UTR, c.*559C>T, that is associated with substantially lower mRNA levels. We show that our novel screening approach is a useful rapid detection tool for alterations in genes coding for key components of the GPI pathway.

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.

A nationwide survey of Aicardi–Goutières syndrome patients identifies a strong association between dominant TREX1 mutations and chilblain lesions: Japanese cohort study

OBJECTIVES Aicardi-Goutières syndrome (AGS) is a rare, genetically determined, early onset progressive encephalopathy associated with autoimmune manifestations. AGS is usually inherited in an autosomal recessive manner. The disease is rare, therefore the clinical manifestations and genotype-phenotype correlations, particularly with regard to autoimmune diseases, are still unclear. Here we performed a nationwide survey of AGS patients in Japan and analysed the genetic and clinical data. METHODS Patients were recruited via questionnaires sent to paediatric or adult neurologists in Japanese hospitals and institutions. Genetic analysis was performed and clinical data were collected. RESULTS Fourteen AGS patients were identified from 13 families; 10 harboured genetic mutations. Three patients harboured dominant-type TREX1 mutations. These included two de novo cases: one caused by a novel heterozygous p.His195Tyr mutation and the other by a novel somatic mosaicism resulting in a p.Asp200Asn mutation. Chilblain lesions were observed in all patients harbouring dominant-type TREX1 mutations. All three patients harbouring SAMHD1 mutations were diagnosed with autoimmune diseases, two with SLE and one with SS. The latter is the first reported case. CONCLUSION This study is the first to report a nationwide AGS survey, which identified more patients with sporadic AGS carrying de novo dominant-type TREX1 mutations than expected. There was a strong association between the dominant-type TREX1 mutations and chilblain lesions, and between SAMHD1 mutations and autoimmunity. These findings suggest that rheumatologists should pay attention to possible sporadic AGS cases presenting with neurological disorders and autoimmune manifestations.