Disintegration of the NuRD Complex in Primary Human Muscle Stem Cells in Critical Illness Myopathy.

Critical illness myopathy (CIM) is an acquired, devastating, multifactorial muscle-wasting disease with incomplete recovery. The impact on hospital costs and permanent loss of quality of life is enormous. Incomplete recovery might imply that the function of muscle stem cells (MuSC) is impaired. We tested whether epigenetic alterations could be in part responsible. We characterized human muscle stem cells (MuSC) isolated from early CIM and analyzed epigenetic alterations (CIM n = 15, controls n = 21) by RNA-Seq, immunofluorescence, analysis of DNA repair, and ATAC-Seq. CIM-MuSC were transplanted into immunodeficient NOG mice to assess their regenerative potential. CIM-MuSC exhibited significant growth deficits, reduced ability to differentiate into myotubes, and impaired DNA repair. The chromatin structure was damaged, as characterized by alterations in mRNA of histone 1, depletion or dislocation of core proteins of nucleosome remodeling and deacetylase complex, and loosening of multiple nucleosome-spanning sites. Functionally, CIM-MuSC had a defect in building new muscle fibers. Further, MuSC obtained from the electrically stimulated muscle of CIM patients was very similar to control MuSC, indicating the impact of muscle contraction in the onset of CIM. CIM not only affects working skeletal muscle but has a lasting and severe epigenetic impact on MuSC.

LMNA Co-Regulated Gene Expression as a Suitable Readout after Precise Gene Correction.

LMNA-related muscular dystrophy is an autosomal-dominant progressive disorder caused by mutations in LMNA. LMNA missense mutations are becoming correctable with CRISPR/Cas9-derived tools. Evaluating the functional recovery of LMNA after gene editing bears challenges as there is no reported direct loss of function of lamin A/C proteins in patient-derived cells. The proteins encoded by LMNA are lamins A/C, important ubiquitous nuclear envelope proteins but absent in pluripotent stem cells. We induced lamin A/C expression in induced pluripotent stem cells (iPSCs) of two patients with LMNA-related muscular dystrophy, NM_170707.4 (LMNA): c.1366A > G, p.(Asn456Asp) and c.1494G > T, p.(Trp498Cys), using a short three-day, serum-induced differentiation protocol and analyzed expression profiles of co-regulated genes, examples being COL1A2 and S100A6. We then performed precise gene editing of LMNA c.1366A > G using the near-PAMless (PAM: protospacer-adjacent motif) cytosine base editor. We show that the mutation can be repaired to 100% efficiency in individual iPSC clones. The fast differentiation protocol provided a functional readout and demonstrated increased lamin A/C expression as well as normalized expression of co-regulated genes. Collectively, our findings demonstrate the power of CRISPR/Cas9-mediated gene correction and effective outcome measures in a disease with, so far, little perspective on therapies.

Generation of hiPSC-Derived Skeletal Muscle Cells: Exploiting the Potential of Skeletal Muscle-Derived hiPSCs.

Cell therapies for muscle wasting disorders are on the verge of becoming a realistic clinical perspective. Muscle precursor cells derived from human induced pluripotent stem cells (hiPSCs) represent the key to unrestricted cell numbers indispensable for the treatment of generalized muscle wasting such as cachexia or intensive care unit (ICU)-acquired weakness. We asked how the cell of origin influences efficacy and molecular properties of hiPSC-derived muscle progenitor cells. We generated hiPSCs from primary muscle stem cells and from peripheral blood mononuclear cells (PBMCs) of the same donors (n = 4) and compared their molecular profiles, myogenic differentiation potential, and ability to generate new muscle fibers in vivo. We show that reprogramming into hiPSCs from primary muscle stem cells was faster and 35 times more efficient than from blood cells. Global transcriptome comparison revealed significant differences, but differentiation into induced myogenic cells using a directed transgene-free approach could be achieved with muscle- and PBMC-derived hiPSCs, and both cell types generated new muscle fibers in vivo. Differences in myogenic differentiation efficiency were identified with hiPSCs generated from individual donors. The generation of muscle-stem-cell-derived hiPSCs is a fast and economic method to obtain unrestricted cell numbers for cell-based therapies in muscle wasting disorders, and in this aspect are superior to blood-derived hiPSCs.

The Lepidoptera of Cuatrociénegas Protected Area 1. A new species in the genus Callistege Hübner, [1823] (Erebidae, Erebinae, Euclidiini) from the Chihuahuan Desert, Coahuila, Mexico.

A new species of Callistege Hübner, [1823] (Lepidoptera, Erebidae, Erebinae, Euclidiini) is described from Cuatrociénegas Protected Area and Biosphere Preserve in Coahuila, Mexico. Adult male and female moths are illustrated, including genitalia. Callistege clara Homziak & Metzler, sp. nov. is one of 27 new species of insects discovered during an inventory survey of arthropods of White Sands National Monument, USA, and Cuatrociénegas Protected Area (Mexico), funded by the U.S. National Park Service. The Cuatrociénegas Basin is known for high endemism of aquatic and wetland biota within the Chihuahuan Desert. Callistege clara Homziak & Metzler, sp. nov. was found in a wetland environment.

Base editing repairs an SGCA mutation in human primary muscle stem cells.

Skeletal muscle can regenerate from muscle stem cells and their myogenic precursor cell progeny, myoblasts. However, precise gene editing in human muscle stem cells for autologous cell replacement therapies of untreatable genetic muscle diseases has not yet been reported. Loss-of-function mutations in SGCA, encoding α-sarcoglycan, cause limb-girdle muscular dystrophy 2D/R3, an early-onset, severe, and rapidly progressive form of muscular dystrophy affecting both male and female patients. Patients suffer from muscle degeneration and atrophy affecting the limbs, respiratory muscles, and heart. We isolated human muscle stem cells from 2 donors, with the common SGCA c.157G>A mutation affecting the last coding nucleotide of exon 2. We found that c.157G>A is an exonic splicing mutation that induces skipping of 2 coregulated exons. Using adenine base editing, we corrected the mutation in the cells from both donors with > 90% efficiency, thereby rescuing the splicing defect and α-sarcoglycan expression. Base-edited patient cells regenerated muscle and contributed to the Pax7+ satellite cell compartment in vivo in mouse xenografts. Here, we provide the first evidence to our knowledge that autologous gene-repaired human muscle stem cells can be harnessed for cell replacement therapies of muscular dystrophies.

Generation of three age and gender matched pairs of human induced pluripotent stem cells derived from myoblasts (MDCi011-A, MDCi012-A, MDCi013-A) and from peripheral blood mononuclear cells (MDCi011-B, MDCi012-B, MDCi013-B) from the same donor.

We describe the generation and characterization of three pairs of human induced pluripotent stem cell (hiPSC) lines reprogrammed from myoblasts and from peripheral blood mononuclear cells (PBMCs) of the same donor. All donors were free of neuromuscular disorders, female and between 47 and 50 years of age. For reprogramming we used Sendai-virus delivery of the four Yamanaka factors. The pluripotent identity of the hiPSC lines was confirmed by the expression of pluripotency markers and their capacity to differentiate into all three germ layers. These hiPSCs constitute a tool to study tissue of origin specific differences in the identity of hiPSCs.

Generation of two human induced pluripotent stem cell lines derived from myoblasts (MDCi014-A) and from peripheral blood mononuclear cells (MDCi014-B) from the same donor.

We describe the generation and characterization of two human induced pluripotent stem cell (hiPSCs) lines reprogrammed from myoblasts and from peripheral blood mononuclear cells (PBMCs) from the same donor. The donor was free of neuromuscular disorders, male and 18 years of age. For reprogramming we used Sendai-virus delivery of the four Yamanaka factors. The pluripotent identity of the hiPSC lines was confirmed by the expression of pluripotency markers and their capacity to differentiate into all three germ layers. These hiPSCs constitute a tool to study tissue of origin specific differences in the identity of hiPSCs.

Human muscle-derived CLEC14A-positive cells regenerate muscle independent of PAX7.

Skeletal muscle stem cells, called satellite cells and defined by the transcription factor PAX7, are responsible for postnatal muscle growth, homeostasis and regeneration. Attempts to utilize the regenerative potential of muscle stem cells for therapeutic purposes so far failed. We previously established the existence of human PAX7-positive cell colonies with high regenerative potential. We now identified PAX7-negative human muscle-derived cell colonies also positive for the myogenic markers desmin and MYF5. These include cells from a patient with a homozygous PAX7 c.86-1G > A mutation (PAX7null). Single cell and bulk transcriptome analysis show high intra- and inter-donor heterogeneity and reveal the endothelial cell marker CLEC14A to be highly expressed in PAX7null cells. All PAX7-negative cell populations, including PAX7null, form myofibers after transplantation into mice, and regenerate muscle after reinjury. Transplanted PAX7neg cells repopulate the satellite cell niche where they re-express PAX7, or, strikingly, CLEC14A. In conclusion, transplanted human cells do not depend on PAX7 for muscle regeneration.

The Lepidoptera of White Sands National Monument, Otero County, New Mexico, USA 9. A new species of Givira Walker (Cossidae, Hypoptinae) dedicated to Delinda Mix, including a list of species of Cossidae recorded from the Monument.

The U.S. National Park Service initiated a 10-year study of the Lepidoptera at White Sands National Monument, Otero County, New Mexico in late 2006. Givira delindaesp. n., discovered in 2007 during the first year of study, is described here. The male and female adult moths and genitalia are illustrated. The name is dedicated to Delinda Mix, mother of Steve Mix. The species of Cossidae recorded from the Monument during the study are listed.

The Lepidoptera of White Sands National Monument, Otero County, New Mexico, USA 10. A remarkable new white species of Chionodes Hübner (Gelechiidae).

The U.S. National Park Service initiated a 10-year study, in late 2006, of the Lepidoptera at White Sands National Monument, Otero County, New Mexico. Chionodes bustosorum sp. n., described here, was discovered in 2010, during the third year of the study. The male imago and male genitalia are illustrated, and its DNA barcode is compared to that of seven other species of Chionodes from western North America.

A review of the genus Ogdoconta Butler (Lepidoptera, Noctuidae, Condicinae, Condicini) from North America north of Mexico with descriptions of three new species.

The species of the genus Ogdoconta Butler, 1891 (Lepidoptera, Noctuidae, Condicinae, Condicini) from North America north of Mexico are reviewed, and a description of the genus is given. Ogdoconta satana Metzler, Knudson & Poole, sp. n., is described from New Mexico and Texas, Ogdoconta rufipenna Metzler, Knudson & Poole, sp. n., is described from Arizona, and Ogdoconta fergusoni Metzler & Lafontaine, sp. n., is described from Florida, Mississippi, and Louisiana. A key to the species of Ogdoconta of North America north of Mexico is provided. Adult moths and male and female genitalia of Ogdoconta satana, Ogdoconta rufipenna, Ogdoconta fergusoni, Ogdoconta cinereola (Guenée, 1852), Ogdoconta moreno Barnes, 1907, Ogdoconta sexta Barnes & McDunnough, 1913, Ogdoconta altura Barnes, 1904, and Ogdoconta tacna (Barnes, 1904) are illustrated.

A new species of Elasmia Möschler from New Mexico and Texas, and a new subspecies of Elasmia mandela (Druce) from Texas and Oklahoma (Lepidoptera, Notodontidae, Nystaleinae).

Hippia packardii (Morrison) and Hippia insularis (Grote) are moved to the genus Elasmia Möschler as comb. n.Elasmia cave Metzler,sp. n. is described from New Mexico and Texas, and Elasmia mandela santaana Metzler & Knudson,ssp. n. is described from Texas and Oklahoma. A key to the species of Elasmia of southwestern U.S. is provided. Adult male and female moths of Elasmia from southwestern U.S. and their genitalia are illustrated.

The Lepidoptera of White Sands National Monument, Otero County, New Mexico, USA 2. Rediscovery and description of Sparkia immacula (Grote, 1883) (Noctuidae, Noctuinae, Hadenini).

In 2006 the U.S. National Park Service initiated a long term study of the Lepidoptera at White Sands National Monument, Otero County, New Mexico. Sparkia immacula (Grote, 1883), previously known only from historical specimens collected in Arizona and New Mexico, was discovered in the Monument in 2007 during the second year of the study. The adult moths and male and female genitalia are illustrated for the first time.

The Lepidoptera of White Sands National Monument, Otero County, New Mexico, USA 3. A new species of Aleptina Dyar, 1902 (Lepidoptera, Noctuidae, Amphipyrinae, Psaphidini).

In 2006 the US National Park Service initiated a long-term study of the Lepidoptera at White Sands National Monument, Otero County, New Mexico. Aleptina arenariasp. n., described here, was discovered in 2008, the second year of the study. The adult moths and male and female genitalia are illustrated.

The Lepidoptera of White Sands National Monument, Otero County, New Mexico, USA 4. A new species of Schinia Hübner, 1818 (Lepidoptera, Noctuidae, Heliothinae).

In 2006 the U.S. National Park Service initiated a long term study of the Lepidoptera at White Sands National Monument, Otero County, New Mexico. Schinia pogueisp. n., described here, was discovered in 2007, the second year of the study. The male and female adult moths and genitalia are illustrated.