Isolation of Primary Human Skeletal Muscle Cells

Spinazzola, Joseph; Gussoni, Emanuela

doi:10.21769/bioprotoc.2591

Cited by 38 publications

(29 citation statements)

References 7 publications

(7 reference statements)

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“…All rights reserved. 4 hPSCs-derived myogenic progenitors, but the specific tetanic force reported in this study is approximately 28-fold lower than that generated by adult human muscle [8] . Therefore, methods that enhance functional muscle differentiation and force generation of 3D muscle constructs engineered from hPSCs-derived myogenic progenitors are highly desirable.…”

Section: Introductioncontrasting

confidence: 55%

“…In the past decade, limited availability of human myogenic cells has been one of the major hurdles to the progress of human muscle tissue engineering. Engineering 3D muscle requires a large amount of myogenic cells, but isolation of human myogenic cells from muscle biopsy is a destructive method with low yield, and the cells have limited expansion capacity [4] .…”

Section: Introductionmentioning

confidence: 99%

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Skeletal Muscle Constructs Engineered from Human Embryonic Stem Cell Derived Myogenic Progenitors Exhibit Enhanced Contractile Forces When Differentiated in a Medium Containing EGM‐2 Supplements

Zhang

Perlingeiro

2019

Adv. Biosys.

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Three-dimensional skeletal muscle constructs engineered from myogenic progenitors derived from human pluripotent stem cells (hPSCs) have a wide range of applications, but to date such constructs generate lower specific tetanic force than adult human muscles. Methods enhancing functional muscle differentiation and force generation of these constructs are highly desirable. The finding of this study is that addition of the supplements in the EGM-2 medium to the myogenic differentiation medium could substantially enhance contractile force generation. For constructs differentiated for four weeks, addition of the EGM-2 supplements in the first two weeks leads to 10-fold and 7-fold increases in twitch and tetanic forces, respectively. The specific tetanic force generated by these constructs is 33 mN mm-2 , which is significantly higher than previously reported. These constructs show wider myotubes and higher gene expression levels for all skeletal muscle-specific myosin heavy chain isoforms, suggesting that a more mature differentiation stage of the cells underlies the greater contractile force generation. The constructs exposed to these supplements for four weeks do not generate as high contractile forces, suggesting that prolonged treatment is not beneficial. These results suggest that temporal conditioning with the EGM-2 supplements assists functional development of hPSC-derived skeletal muscle constructs.

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Section: Introductioncontrasting

confidence: 55%

Section: Introductionmentioning

confidence: 99%

Skeletal Muscle Constructs Engineered from Human Embryonic Stem Cell Derived Myogenic Progenitors Exhibit Enhanced Contractile Forces When Differentiated in a Medium Containing EGM‐2 Supplements

Zhang

Perlingeiro

2019

Adv. Biosys.

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“…Briefly, the connective tissue was removed from the muscle, which was then cut into pieces smaller than 0.1 cm 3 , digested with 0.2% collagenase I (RD 3442‐050‐01) at 37°C for 1 hr, filtered through a 100 mm strainer, before resuspending 500 g of cells in complete medium for 3 min. Next, the sample was transferred to a 10‐cm dish incubated at 37°C and 5% CO 2 (Spinazzola & Gussoni, 2017). FASTKD2 was knocked down in human‐derived primary muscle cells by transient transfection of stealth RNAi duplex constructs (5′‐ GAGCTATCTGCTAAGACAA‐3′) using lipofectamine RNAiMAX (Thermo Fisher Scientific).…”

Section: Methodsmentioning

confidence: 99%

Mutations inFASTKD2are associated with mitochondrial disease with multi‐OXPHOS deficiency

Wei

et al. 2020

Human Mutation

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Mutations in FASTKD2, a mitochondrial RNA binding protein, have been associated with mitochondrial encephalomyopathy with isolated complex IV deficiency. However, deficiencies related to other oxidative phosphorylation system (OXPHOS) complexes have not been reported. Here, we identified three novel FASTKD2 mutations, c.808_809insTTTCAGTTTTG, homoplasmic mutation c.868C>T, and heteroplasmic mutation c.1859delT/c.868C>T, in patients with mitochondrial encephalomyopathy.Cell-based complementation assay revealed that these three FASTKD2 mutations were pathogenic. Mitochondrial functional analysis revealed that mutations in FASTKD2 impaired the mitochondrial function in patient-derived lymphocytes due to the deficiency in multi-OXPHOS complexes, whereas mitochondrial complex II remained unaffected. Consistent results were also found in human primary muscle cell and zebrafish with knockdown of FASTKD2. Furthermore, we discovered that FASTKD2 mutation is not inherently associated with epileptic seizures, optic atrophy, and loss of visual function. Alternatively, a patient with FASTKD2 mutation can show sinus tachycardia and hypertrophic cardiomyopathy, which was partially confirmed in zebrafish with knockdown of FASTKD2. In conclusion, both in vivo and in vitro studies suggest that loss of function mutation in FASTKD2 is responsible for multi-OXPHOS complexes deficiency, and FASTKD2-associated mitochondrial disease has a high degree of clinical heterogenicity.FASTKD2, metabolic genetic diseases, mitochondrial disease, OXPHOS complex Xiujuan Wei, Miaomiao Du, and Dongxiao Li contributed equally to this study.

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“…Patient‐derived myoblasts were isolated from biopsy tissue obtained from 1406‐1 (proband) and 1234‐1 (control) following a standard protocol (Spinazzola & Gussoni, ). The myoblasts were cultured in Skeletal Muscle Cell Growth Medium (PromoCell) with 20% fetal bovine serum (Sigma), 1% GlutaMAX TM (Life Technologies), and 1% Penicillin–Streptomycin (Caisson Laboratories).…”

Section: Methodsmentioning

confidence: 99%

Identification of a pathogenic mutation in ATP2A1 via in silico analysis of exome data for cryptic aberrant splice sites

Bruels

Mendoza

et al. 2019

Molec Gen & Gen Med

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Background Pathogenic mutations causing aberrant splicing are often difficult to detect. Standard variant analysis of next‐generation sequence (NGS) data focuses on canonical splice sites. Noncanonical splice sites are more difficult to ascertain. Methods We developed a bioinformatics pipeline that screens existing NGS data for potentially aberrant novel essential splice sites (PANESS) and performed a pilot study on a family with a myotonic disorder. Further analyses were performed via qRT‐PCR, immunoblotting, and immunohistochemistry. RNAi knockdown studies were performed in Drosophila to model the gene deficiency. Results The PANESS pipeline identified a homozygous ATP2A1 variant (NC_000016.9:g.28905928G>A; NM_004320.4:c.1287G>A:p.(Glu429=)) that was predicted to cause the omission of exon 11. Aberrant splicing of ATP2A1 was confirmed via qRT‐PCR, and abnormal expression of the protein product sarcoplasmic/endoplasmic reticulum Ca ++ ATPase 1 (SERCA1) was demonstrated in quadriceps femoris tissue from the proband. Ubiquitous knockdown of SERCA led to lethality in Drosophila, as did knockdown targeting differentiating or fusing myoblasts. Conclusions This study confirms the potential of novel in silico algorithms to detect cryptic mutations in existing NGS data; expands the phenotypic spectrum of ATP2A1 mutations beyond classic Brody myopathy; and suggests that genetic testing of ATP2A1 should be considered in patients with clinical myotonia.

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Isolation of Primary Human Skeletal Muscle Cells

Cited by 38 publications

References 7 publications

Skeletal Muscle Constructs Engineered from Human Embryonic Stem Cell Derived Myogenic Progenitors Exhibit Enhanced Contractile Forces When Differentiated in a Medium Containing EGM‐2 Supplements

Skeletal Muscle Constructs Engineered from Human Embryonic Stem Cell Derived Myogenic Progenitors Exhibit Enhanced Contractile Forces When Differentiated in a Medium Containing EGM‐2 Supplements

Mutations inFASTKD2are associated with mitochondrial disease with multi‐OXPHOS deficiency

Identification of a pathogenic mutation in ATP2A1 via in silico analysis of exome data for cryptic aberrant splice sites

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