GB2575574A - Neuromuscular junction - Google Patents

Neuromuscular junction Download PDF

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Publication number
GB2575574A
GB2575574A GB1914656.2A GB201914656A GB2575574A GB 2575574 A GB2575574 A GB 2575574A GB 201914656 A GB201914656 A GB 201914656A GB 2575574 A GB2575574 A GB 2575574A
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United Kingdom
Prior art keywords
ipscs
cells
culturing
neuromuscular junction
media comprises
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GB1914656.2A
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GB2575574B (en
GB201914656D0 (en
Inventor
Sareen Dhruv
Mandefro Berhan
Kaus Anjoscha
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Cedars Sinai Medical Center
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Cedars Sinai Medical Center
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Priority claimed from US15/458,185 external-priority patent/US20170226478A1/en
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Publication of GB2575574A publication Critical patent/GB2575574A/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0658—Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/34—Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/105—Insulin-like growth factors [IGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/12—Hepatocyte growth factor [HGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2531/00—Microcarriers

Abstract

The invention relates to culturing motor neuron cells together with skeletal muscle cells in a fluidic device under conditions whereby the interaction of these cells mimic the structure and function of the neuromuscular junction (NMJ) providing a NMJ-on-chip. Good viability, formation of myo-fibers and function of skeletal muscle cells on fluidic chips allow for measurements of muscle cell contractions. Embodiments of motor neurons co-cultures with contractile myo-fibers are contemplated for use with modeling diseases affecting NMJ's, e.g. Amyotrophic lateral sclerosis (ALS).

Claims (25)

1. A method of generating myotubes, comprising: providing a quantity of induced pluripotent stem cells (iPSCs); culturing the iPSCs in the presence of a first induction media to generate mesoderm cells; further culturing mesoderm cells in the presence of a second induction media to generate myoblasts; and maturing the myoblasts into myotybes by culturing in the presence of a maturation media.
2. The method of claim 1, wherein the first induction media comprises CHIR99021 and/or LDN193189.
3. The method of claim 1, wherein the first induction media comprises bFGF (basic fibroblast growth factor).
4. The method of claim 1, wherein culturing the iPSCs in the presence of a first induction media comprises about 6, 7, 8, or 9 days.
5. The method of claim 1, wherein the second induction media comprises CHTR99021 and/or LDN193189.
6. The method of claim 1, wherein the second induction media comprises bFGF, HGF (hepatocyte growth factor) and/or IGF (insulin-like growth factor).
7. The method of claim 1, wherein further culturing mesoderm cells in the presence of a second induction media comprises about 2, 3, 4, or 5 days.
8. The method of claim 1, wherein the maturation media comprises serum replacement.
9. The method of claim 1, wherein the maturation media comprises HGF and/or IGF.
10. The method of claim 1, wherein maturing the myoblasts by culturing in the presence of a maturation media comprises about 25, 26, 27, 28, 29, 30, 31, 32, or 33 days.
11. The method of claim 1, wherein said myotubes form contractile tissue.
12. The method of claim 1, wherein said myotubes form polynucleated myo-fibers.
13. The method of claim 1, wherein culturing the iPSCS, further culturing mesoderm cells, and/or maturing the myoblasts is on a coated substrate.
14. The method of claim 13, wherein the coated substrate comprises one or more extracellular matrix proteins.
15. The method of claim 14, wherein the one or more extracellular matrix proteins comprises Matrigel.
16. The method of claim 14, wherein the one or more extracellular matrix proteins comprises laminin.
17. The method of claim 1, wherein the iPSCS are derived from a human.
18. The method of claim 1, wherein said human is diagnosed with a neuron disease and/or condition.
19. The method of claim 1, wherein said human is diagnosed with a muscle disease and/or condition.
20. A neuromuscular junction comprising: one or more neurons; and one or more muscle cells, wherein the one or more neurons and one or more muscle are fixed on a substrate, and the one or more neurons are capable of generating activation potential and/or inducing contraction in the one or more muscle cells.
21. The neuromuscular junction of claim 20, wherein the substrate comprises a surface of a microfluidic device.
22. The neuromuscular junction of claim 20, wherein the one or more neurons are differentiated from induced pluripotent stem cells (iPSCs).
23. The neuromuscular junction of claim 22, wherein said human is diagnosed with a neuron disease and/or condition.
24. The neuromuscular junction of claim 20, wherein the one or more muscle cells are differentiated from induced pluripotent stem cells (iPSCs).
25. The neuromuscular junction of claim 24, wherein said human is diagnosed with a muscle disease and/or condition.
GB1914656.2A 2017-03-14 2018-03-14 Neuromuscular junction Active GB2575574B (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US201762471273P 2017-03-14 2017-03-14
US15/458,185 US20170226478A1 (en) 2015-10-19 2017-03-14 Neuromuscular Junction: NMJ-ON-CHIP
PCT/US2018/022511 WO2018170180A1 (en) 2017-03-14 2018-03-14 Neuromuscular junction

Publications (3)

Publication Number Publication Date
GB201914656D0 GB201914656D0 (en) 2019-11-27
GB2575574A true GB2575574A (en) 2020-01-15
GB2575574B GB2575574B (en) 2022-08-17

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Family Applications (1)

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GB1914656.2A Active GB2575574B (en) 2017-03-14 2018-03-14 Neuromuscular junction

Country Status (5)

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AU (1) AU2018236273A1 (en)
CA (1) CA3056089A1 (en)
GB (1) GB2575574B (en)
SG (1) SG11201908358PA (en)
WO (1) WO2018170180A1 (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11414648B2 (en) 2017-03-24 2022-08-16 Cedars-Sinai Medical Center Methods and compositions for production of fallopian tube epithelium
US11473061B2 (en) 2016-02-01 2022-10-18 Cedars-Sinai Medical Center Systems and methods for growth of intestinal cells in microfluidic devices
US11767513B2 (en) 2017-03-14 2023-09-26 Cedars-Sinai Medical Center Neuromuscular junction
US11913022B2 (en) 2017-01-25 2024-02-27 Cedars-Sinai Medical Center In vitro induction of mammary-like differentiation from human pluripotent stem cells

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP7033095B2 (en) * 2019-03-04 2022-03-09 日清食品ホールディングス株式会社 Three-dimensional muscle tissue and its manufacturing method
DE102019127604A1 (en) * 2019-10-14 2021-04-15 Georg-August-Universität Göttingen Stiftung Öffentlichen Rechts, Universitätsmedizin Production of skeletal muscle cells and tissue from pluripotent stem cells
US20220202872A1 (en) * 2020-07-10 2022-06-30 Max-Delbrück-Centrum Für Molekulare Medizin In Der Helmholtz-Gemeinschaft Method for generating a three-dimensional neuromuscular organoid in vitro
CN112877282A (en) * 2021-02-09 2021-06-01 南通大学 Method for culturing primary neuromuscular junction in vitro

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Boyer et al, more than a bystander: the contributions of intrinsic skeletal muscle defects in motor neuron diseases. Frontiers in Physiology 18/12/2013, Vol.4, article 356, pages 1-12 *
Chal et al, differentiation of pluripotent stem cells to muscle fiber to model Duchenne muscular dystrophy. Nature Biotechnology 3/8/15, vol. 3, No.9, pgs 962-969 *
Hughes et al, Matrigel: A complex protein mixture required for optimal growth of cell culture proteomics 2010, Vol. 10, pages 1886-1890 *
Jang et al, JAK-STAT pathway and myogenic differentiation, JAKSTAT 01/04/2013, vol2, No. 2, pages 1-6 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11473061B2 (en) 2016-02-01 2022-10-18 Cedars-Sinai Medical Center Systems and methods for growth of intestinal cells in microfluidic devices
US11913022B2 (en) 2017-01-25 2024-02-27 Cedars-Sinai Medical Center In vitro induction of mammary-like differentiation from human pluripotent stem cells
US11767513B2 (en) 2017-03-14 2023-09-26 Cedars-Sinai Medical Center Neuromuscular junction
US11414648B2 (en) 2017-03-24 2022-08-16 Cedars-Sinai Medical Center Methods and compositions for production of fallopian tube epithelium

Also Published As

Publication number Publication date
GB2575574B (en) 2022-08-17
GB201914656D0 (en) 2019-11-27
AU2018236273A1 (en) 2019-10-31
WO2018170180A1 (en) 2018-09-20
SG11201908358PA (en) 2019-10-30
CA3056089A1 (en) 2018-09-20

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