Muscle stem cell culture

Abstract

The present disclosure provides methods for culturing primary muscle stem cells, promoting self-renewal of primary muscle stem cells in cell culture, inhibiting the differentiation of primary muscle stem cells and increasing the clonogenicity of primary muscle stem cells, comprising culturing primary muscle stem cells in the presence of an inhibitor of mitochondrial maturation. Also provided are methods for promoting the differentiation of primary muscle stem cells, comprising culturing primary muscle stem cells in the presence of an promoter of mitochondrial maturation. The present disclosure also provides the use of inhibitors of mitochondrial maturation and promoters of mitochondrial maturation in such methods, and kits comprising the same.

Description

[0001] Muscle Stem Cell Culture

[0002] This application claims priority from GB 2315315.8 filed 6 October 2023, the contents and elements of which are herein incorporated by reference for all purposes.

[0003] Technical Field

[0004] The present disclosure relates to the field of molecular and cell biology. In particular, the present disclosure relates to cell culture of muscle stem cells.

[0005] Background

[0006] In skeletal muscle, quiescent muscle stem cells and committed myoblasts have different mitochondrial features and metabolic phenotypes2. It has previously been shown that age-selective apportioning of mitochondria epithelial stem-like cells can result in progeny cells with specific metabolic profiles that regulate cell fate34. The relationship between mitochondrial phenotypes and developmental cell fates of myogenic cells remains largely unexplored.

[0007] Rochard et al. J Biol Chem (2000) 275: 2733-2744 discloses the ability of chloramphenicol to inhibit differentiation of cells of an immortalised avian myoblast cell line, that had been induced (through culture under low serum conditions) to undergo terminal differentiation (see e.g. Abstract, Figure 3). Treatment of cells of the cell line with chloramphenicol is reported in Rochard et al. to reduce their proliferative capacity (see e.g. Figure 2).

[0008] Hamai et al. Cell Struct Funct (1997) 22(4): 421-431 discloses that tetracycline-treated cells of an immortalised mouse myoblast cell line (C2C12) display impaired myotube differentiation, and impaired induction of muscle creatine kinase (MCK) activity, following induction (through culture under low serum conditions) to undergo terminal differentiation. See e.g. ‘Cell Culture’ on page 422 and the first paragraph of ‘Results’ on page 423.

[0009] There remains an unmet need for the generation of muscle tissue with improved efficiency, e.g. in the context of production of cultivated meat, but also for use in regenerative medicine.

[0010] Summary

[0011] In a first aspect, the present disclosure provides a method for culturing muscle stem cells, comprising culturing muscle stem cells in which mitochondrial maturation is inhibited.

[0012] The present disclosure also provides a method for promoting the self-renewal of muscle stem cells in cell culture, comprising inhibiting mitochondrial maturation in muscle stem cells in cell culture.

[0013] The present disclosure also provides a method for inhibiting the differentiation of muscle stem cells, comprising inhibiting mitochondrial maturation in muscle stem cells in cell culture.

[0014] The present disclosure also provides a method for increasing the clonogenicity of muscle stem cells, comprising inhibiting mitochondrial maturation in muscle stem cells in cell culture. The present disclosure also provides a method for promoting the differentiation of muscle stem cells, comprising culturing muscle stem cells under conditions promoting mitochondrial maturation.

[0015] In some embodiments in accordance with the various aspects of the present disclosure, the method comprises culturing the muscle stem cells in the presence of an inhibitor of mitochondrial maturation.

[0016] The present disclosure also provides a method for culturing muscle stem cells, comprising:

[0017] (i) culturing muscle stem cells in which mitochondrial maturation is inhibited, and

[0018] (ii) subsequently releasing the muscle stem cells from inhibition of mitochondrial maturation.

[0019] The present disclosure also provides a method for producing muscle tissue, comprising:

[0020] (i) culturing muscle stem cells in which mitochondrial maturation is inhibited, and

[0021] (ii) subsequently releasing the muscle stem cells from inhibition of mitochondrial maturation, thereby promoting differentiation of muscle stem cells, and formation of muscle tissue.

[0022] In some embodiments, culturing muscle stem cells in which mitochondrial maturation is inhibited comprises culturing the muscle stem cells in the presence of an inhibitor of mitochondrial maturation.

[0023] In some embodiments, releasing the muscle stem cells from inhibition of mitochondrial maturation comprises promoting mitochondrial maturation.

[0024] The present disclosure also provides the use of an inhibitor of mitochondrial maturation to promote the self-renewal of muscle stem cells.

[0025] The present disclosure also provides the use of an inhibitor of mitochondrial maturation to inhibit the differentiation of muscle stem cells.

[0026] The present disclosure also provides the use of an inhibitor of mitochondrial maturation to increase the clonogenicity of a muscle stem cell.

[0027] In some embodiments in accordance with the various aspects of the present disclosure, the muscle stem cell is a primary muscle stem cell.

[0028] The present disclosure also provides a kit, comprising (i) an inhibitor of mitochondrial maturation, and (ii) a promoter of mitochondrial maturation.

[0029] In some embodiments in accordance with the various aspects of the present disclosure, the inhibitor of mitochondrial maturation is an inhibitor of mitochondrial translation.

[0030] In some embodiments, the inhibitor of mitochondrial translation is an inhibitor of bacterial protein synthesis. In some embodiments, the inhibitor of mitochondrial translation is selected from: chloramphenicol, thiamphenicol, florfenicol and actinonin.

[0031] Description

[0032] The present disclosure relates broadly to the manipulation of maturation of mitochondria of developmental precursors of muscle cells (e.g. adult muscle stem cells), to favour the production of certain myogenic cell types over others.

[0033] The present disclosure is based on the inventors’ finding that quiescent muscle stem cells (MuSCs) comprise mitochondria having an immature cristae- and electron transport chain (ETC)-deficient phenotype with a low oxygen consumption rate (OCR), characteristic of newly-formed mitochondria. By contrast, the mitochondria of myocytes, myotubes and myofibres have a more mature, cristae-dense and ETC-rich phenotype and a high OCR.

[0034] The spontaneous differentiation of primary muscle stem cells to their developmental successors is an obstacle to the large-scale culture of primary muscle stem cells. The inventors determined that it is possible to promote self-renewal of myoblasts over the differentiation to the developmental successors thereof ( / .e. myocytes, myotubes and myofibres) through inhibition of mitochondrial maturation. This effect was determined to be conserved between different species. The inventors moreover determined that biasing the developmental fate of muscle stem cells in this way is reversible, such that on release of cells from inhibition of mitochondrial maturation, they can progress to become differentiated muscle cells.

[0035] Finally, the inventors determined that manipulation of cellular metabolism of muscle stem cells to favour ATP production through oxidative phosphorylation over ATP production via glycolysis promotes mitochondrial maturation. They determined that by culturing cells under such conditions, it is possible to promote differentiation of muscle stem cells to their developmental successors, and thereby promote formation of muscle tissue with a high myogenic fusion index.

[0036] The present disclosure has wide-ranging applications to the generation of muscle tissue in vitro / ex vivo, e.g. in the context of the production of cultivated meat, and also for regenerative medicine.

[0037] Muscle stem cells and formation of muscle tissue

[0038] The present disclosure relates to muscle stem cells. Muscle stems cells (MuSCs) are skeletal muscle tissue-resident stem cells that support the long-term regenerative capability of skeletal muscle. The biology of muscle stem cells and their involvement in the formation of muscle tissue is described e.g. in Wang et al., J Cell Sci. (2014) 127(21): 4543-4548, Yin et al., Physiol Rev. (2013) 93(1): 23-67 and Yu et al., Stem Cells Int. (2021) 8884283, all of which are hereby incorporated by reference in their entirety.

[0039] Muscle stem cells (also referred to as muscle satellite cells or myosatellite cells) are found within muscle tissue in vivo outside of myofibres and underneath the basal lamina. They are capable of cell division to form further muscle stem cells, or differentiation to become skeletal muscle cells. Muscle stem cells may be characterised by expression of certain proteins. Markers of muscle stem cells include the transcription factors Pax7, Pax3 and Barx2, and emerin, the myogenic regulatory factor Myf5, and the cell surface proteins syndecan 3, syndecan 4, M-cadherin, CALCR, CXCR4, CAV1 , a7-integrin, p1 -integrin, NCAM1 , VCAM1 and CD34. Muscle stem cells are typically isolated by positive selection based on expression of a7-integrin and / or CD34, and negative selection for markers of hematopoietic and fibrogenic cell types using e.g. antibodies to CD45, CD11 b, CD31 and LY6A. Pax7 is the canonical biomarker for muscle stem cells, as it is specifically expressed in all quiescent muscle stem cells and activated satellite cells, across multiple species.

[0040] In some embodiments, a muscle stem cell according to the present disclosure is characterised by expression of one or more proteins selected from Pax7, Pax3, Myf5, a7-integrin, CD34, lamin A / C, emerin, Barx2, syndecan 3, syndecan 4, M-cadherin, CALCR, CXCR4, CAV1 , |31 -integrin, NCAM1 and VCAM1.

[0041] Herein, where a cell expresses a given protein it may be described as ‘positive’ for the given protein. By way of illustration, a cell that expresses Pax7 may be referred to as being ‘Pax7-positive’ or ‘Pax7+’. Conversely, where a cell substantially does not express a given protein, it may be described as ‘negative’ for the given protein.

[0042] In some embodiments, a muscle stem cell according to the present disclosure expresses Pax7 ( / .e. is Pax7+).

[0043] Muscle stem cells may be further subdivided into quiescent muscle stem cells and myoblasts. Quiescent muscle stem cells are activated in response to signalling molecules (e.g. IGF-1 , FGFs, HGF, PDGF, LIF, TNFa, IL-6) to become proliferative myoblasts.

[0044] Quiescent muscle stem cells are characterised by a mitotically quiescent ( / .e. Go), transcriptionally inactive phenotype and low metabolic activity. Morphologically they are characterised by a large nucleus- to-cytoplasm ratio, few organelles, a small nucleus and condensed interphase chromatin. Quiescent muscle stem cells express Pax7, express Myf5 at low levels, and do not express MyoD or myogenin.

[0045] Myoblasts (which are also referred to in the art as activated muscle stem cells, or myogenic precursor cells) are characterised by a proliferative phenotype. Like quiescent muscle stem cells, activated muscle stem cells express Pax7 and Myf5 (albeit with higher Myf5 expression than in quiescent muscle stem cells). Activated muscle stem cells do not express myogenin, but they do express MyoD. Thus, quiescent muscle stem cells and myoblasts can be distinguished on the basis of MyoD expression.

[0046] In some embodiments, a muscle stem cell according to the present disclosure is a quiescent muscle stem cell. In some embodiments, a muscle stem cell is Pax7+, Myf5+, MyoD- and myogenin'. In preferred embodiments, a muscle stem cell according to the present disclosure is a myoblast ( / .e. an activated muscle stem cell). In some embodiments, a muscle stem cell is Pax7+, Myf5+, MyoD+and myogenin-.

[0047] Myoblasts differentiate to become myocytes, which subsequently fuse to become multinucleated myotubes, which then mature to become myofibers. Myocytes have an elongate morphology and are characterised by the expression of myogenin and the absence of expression of Pax7. Accordingly, a myocyte according to the present disclosure may be myogenin* and Pax7_.

[0048] In some embodiments, a myocyte and / or a developmental successor thereof (e.g. myotube, myofiber) may be characterised by the expression of myogenin.

[0049] Muscle stem cells may further be characterised by the absence of expression of markers of myocytes, myotubes and / or myofibers. In some embodiments, a muscle stem cell according to the present disclosure does not display substantial expression of myogenin ( / .e. is myogenin ). In some embodiments, a muscle stem cell according to the present disclosure does not display substantial expression of myosin heavy chain (MyHC; i.e. is MyHC ).

[0050] In aspects and embodiments of the present disclosure, the muscle stem cells are primary cells. That is, in some embodiments, the muscle stem cells are / were isolated directly from living tissue (e.g. from tissue of a living subject). For example, the muscle stem cells may be / have been obtained from a biopsy of muscle tissue.

[0051] In some embodiments, the muscle stem cells are not cells of a cell line. In some embodiments, the muscle stem cells have not been / are not immortalised. In some embodiments, the muscle stem cells are not avian myoblast cells. In some embodiments, the muscle stem cells are not QM7 cells (i.e. cells of Cellosaurus Accession: CVCL_3450). In some embodiments, the muscle stem cells are not rodent myoblast cells. In some embodiments, the muscle stem cells are not murine myoblast cells. In some embodiments, the muscle stem cells are not mouse myoblast cells. In some embodiments, the muscle stem cells are not C2C12 cells (i.e. cells of Cellosaurus Accession: CVCL_0188).

[0052] In some embodiments, the muscle stem cells are not / have not been induced to differentiate. In some embodiments, the muscle stem cells are not / have not been induced to undergo terminal differentiation. In some embodiments, the muscle stem cells (e.g. myoblasts) are not / have not been induced to differentiate to become myocytes. In some embodiments, the muscle stem cells (e.g. myoblasts) are not / have not been cultured in cell culture medium for promoting their differentiation (e.g. to myocytes). In some embodiments, the muscle stem cells (e.g. myoblasts) are not / have not been cultured under low serum conditions. Herein, culture under ‘low’ serum conditions may refer to culture in cell culture medium comprising less than 10% (v / v) serum (e.g. mammalian serum, e.g. fetal calf serum), e.g. one of < 5%, < 4%, < 3%, < 2%, < 1%, < 0.5%, or less. In some embodiments, the muscle stem cells are human or non-human animal muscle stem cells. In some embodiments, the muscle stem cells are muscle stem cells from a non-human vertebrate. In some embodiments, the muscle stem cells are muscle stem cells from a non-human mammal.

[0053] In some embodiments, the muscle stem cells are muscle stem cells from a species of animal commonly consumed by humans or non-human animals (e.g. domesticated animals). In some embodiments, the muscle stem cells are muscle stem cells from a species of animal commonly raised in captivity for consumption by humans or non-human animals (e.g. domesticated animals).

[0054] In some embodiments, the muscle stem cells are muscle stem cells from a mammal, bovid, bison, water buffalo, cattle, yak, camelid, dromedary, llama, canid, dog, caprae, goat, cetacean, whale, cervid, deer, elk, moose, reindeer, chiroptera, bat, felid, cat, equid, donkey, horse, lagomorph, rabbit, macropodid, kangaroo, ove, sheep, primate, monkey, rodent, guinea pig, rat, suid, pig, boar, non-mammal, amphibian, frog, bird, chicken, duck, goose, turkey, quail, pigeon, guineafowl, ostrich, emu, peacock, fish, barramundi, carp, catfish, cod, seabream, dentex, flounder, halibut, meagre, milkfish, rabbitfish, salmon, seabass, bass, sturgeon, tilapia, trout, turbot, insect, cricket, grasshopper, worm, crustacean, crayfish, crab, lobster, shrimp, prawn, mollusc, oyster, scallop, mussel, snail, abalone, reptile, alligator, crocodile, turtle, snake or iguana.

[0055] In some embodiments, the muscle stem cells are muscle stem cells from pig, chicken, cattle, sheep, goat, turkey, duck, buffalo, goose, rabbit, fish, crustaceans, horse, reindeer, moose or bison.

[0056] In some embodiments, the muscle stem cells are human or non-human, non-avian animal muscle stem cells. In some embodiments, the muscle stem cells are muscle stem cells from a non-human, non-avian vertebrate. In some embodiments, the muscle stem cells are muscle stem cells from pig, cattle, sheep, goat, turkey, duck, buffalo, goose, rabbit, fish, crustaceans, horse, reindeer, moose or bison. In some embodiments, the muscle stem cells are not avian muscle stem cells. In some embodiments, the muscle stem cells are not from quail. In some embodiments, the muscle stem cells are not rodent muscle stem cells. In some embodiments, the muscle stem cells are not murine muscle stem cells. In some embodiments, the muscle stem cells are not mouse muscle stem cells.

[0057] Mitochondrial maturation

[0058] Aspects and embodiments of the present disclosure relate to mitochondrial maturation, and manipulation of mitochondrial maturation.

[0059] Mitochondrial biogenesis, maturation and function is described e.g. in Vega et al., Circ Res. (2015) 116(11):1820-34, Wilke, Membranes (Basel). (2021) 11 (7): 465 and Persad and Lopaschuk, Front Cell Dev Biol. (2022) 10: 886393, all of which are hereby incorporated by reference in their entirety.

[0060] Mitochondria are organelles found in almost all eukaryotic cells and are responsible for generating ATP through oxidative phosphorylation. The ETC is a series of protein complexes and electron carriers that transfer electrons from electron donors (such as NADH and FADH2) to electron acceptors (such as molecular oxygen) through a series of redox reactions. The ETC is formed of complex I (NADH:ubiquinone oxidoreductase), complex II (succinate dehydrogenase), ubiquinone (coenzyme Q), complex III (ubiquinol-cytochrome c oxidoreductase), cytochrome c, and complex IV (cytochrome c oxidase).

[0061] The constituent enzymes of the ETC assemble into respiratory supercomplexes, which are described e.g. in Vartak et al. Protein & Cell. (2013) 4(8): 582-590. A respiratory supercomplex comprising complexes I, III and IV of the ETC is sometimes referred to in the art as the respirasome (and may also be referred to herein as supercomplex l / lll / IV). Other respiratory supercomplexes that have been observed comprise complexes I and III (supercomplex l / lll), or complexes III and IV (supercomplex lll / IV).

[0062] The transfer of electrons along the ETC releases energy, which powers the transfer of protons (H+) across the inner mitochondrial membrane (from the mitochondrial matrix to the intermembrane space), creating an electrochemical proton gradient. The energy stored in this gradient is then harnessed by F1 / F0 ATP synthase (also located within the inner mitochondrial membrane), which couples the flow of protons back across the inner mitochondrial membrane ( / .e. from the intermembrane space to the mitochondrial matrix) to the synthesis of ATP from ADP and inorganic phosphate. This process is known as oxidative phosphorylation.

[0063] Cristae are key morphological features of mitochondria; they are tubular and lamella invaginations of the inner mitochondrial membrane that increase the available surface for ATP production. OPA1 , F1 / F0 ATP synthase and the MICOS complex play important roles in crista biogenesis and development. As used herein, ‘cristae density’ refers to the number of cristae per mitochondrial area (in pm2). Mitochondrial cristae density can be evaluated e.g. as described in Han et al., Nature (2023) 615: 712-719, which is hereby incorporated by reference in its entirety. Immature mitochondria have a lower cristae density than mature mitochondria. In some embodiments, immature mitochondria may have a cristae density <30 / pm2. In some embodiments, mature mitochondria may have a cristae density >30 / pm2.

[0064] Techniques for the analysis of ATP production are well known to the skilled person, and include bioluminescent ATP assays, which take advantage of the firefly luciferase enzymatic reaction, which uses ATP to generate photons of light.

[0065] Oxygen consumption rate (OCR) can be analysed e.g. as described in Plitzko and Loesgen, Bio Protoc. (2018) 8(10): e2850, which is hereby incorporated by reference in its entirety.

[0066] As used herein, ‘expression’ of a given polypeptide may refer to expression of nucleic acid encoding the polypeptide and / or expression of the polypeptide at the protein level. Evaluation of the expression of nucleic acid encoding a given polypeptide may comprise detecting and quantifying the level of nucleic acid (e.g. RNA) encoding the polypeptide. Techniques for evaluating expression of nucleic acid encoding a given polypeptide are well known to the skilled person, and include e.g. quantitative real-time PCR (qRT-PCR). Expression of a given polypeptide can be evaluated using any suitable technique for the detection and / or quantification of the relevant polypeptide. Such techniques include e.g. antibody-based methods, (for example flow cytometry, immunocytochemistry, western blot, ELISA), fluorescence microscopy and flow cytometry.

[0067] In addition to ATP production, mitochondria are involved in a range of other processes, including calcium signaling, apoptosis, and the synthesis of certain amino acids, lipids, and nucleotides. Mitochondria also play a critical role in cellular metabolism and are involved in the breakdown of fatty acids and amino acids, as well as the regulation of glucose metabolism.

[0068] New mitochondria are formed by mitochondrial fission, whereby individual mitochondria divide into two smaller mitochondria. Immature mitochondria generally have a perinuclear subcellular localisation and a globular shape, while mature mitochondria are generally larger and have more elongate morphology. Immature mitochondria have fewer and less well-developed cristae than mature mitochondria. Immature mitochondria also comprise lower levels of the proteins of the ETC, lower levels of respiratory supercomplexes, lower levels of F1 / F0 ATP synthase, and therefore have lower capacity for ATP production and lower mitochondrial membrane potential (MMP) than mature mitochondria.

[0069] In some embodiments, immature mitochondria may be characterised herein by reference to characteristics of mitochondria having a cristae density <30 / pm2, ’n some embodiments, immature mitochondria may be characterised herein by reference to characteristics of the mitochondria of quiescent muscle stem cells (which have an immature phenotype).

[0070] In some embodiments, immature mitochondria may be characterised by one or more of: low cristae density (e.g. <30 / pm2); low number of cristae; globular morphology; low expression of one or more polypeptide(s) of the ETC; low expression of complex III; low level of a respiratory supercomplex (e.g. selected from supercomplex l / lll / IV, supercomplex l / lll and supercomplex lll / IV); low expression of F1 / F0 ATP synthase; low mitochondrial membrane potential; low ATP production; low oxygen consumption rate. In some embodiments, mature mitochondria may be characterised by one or more of: high cristae density (e.g. >30 / pm2); high number of cristae; elongate morphology; high expression of one or more polypeptide(s) of the ETC; high expression of complex III; high level of a respiratory supercomplex (e.g. selected from supercomplex l / lll / IV, supercomplex l / lll and supercomplex lll / IV); high expression of F1 / F0 ATP synthase; high mitochondrial membrane potential; high ATP production; high oxygen consumption rate.

[0071] It will be appreciated that the properties recited in the preceding paragraph are observed in the mitochondria, and not necessarily in cells comprising the mitochondria.

[0072] A ‘low’ number of cristae may be a number of cristae ( / .e. per mitochondrion) that is less than the average (e.g. mean) number of cristae of mitochondria having a cristae density >30 / pm2.A‘low’ number of cristae may be a number of cristae that is similar to the average (e.g. mean) number of cristae of mitochondria having a cristae density <30 / pm2, or of mitochondria of a quiescent muscle stem cell. A ‘high’ number of cristae may be a number of cristae ( / .e. per mitochondrion) that is greater than the average (e.g. mean) number of cristae of mitochondria having a cristae density <30 / pm2, or of mitochondria of a quiescent muscle stem cell. A ‘high’ number of cristae may be a number of cristae that is similar to the average (e.g. mean) number of cristae of mitochondria having a cristae density >30 / pm2. A number of cristae that is similar to a given reference number of cristae may be > 0.5 times and < 2 times, e.g. one of > 0.75 times and < 1 .5 times, > 0.8 times and < 1 .25 times, or > 0.9 times and < 1.1 times the reference number.

[0073] Similarly, ‘low’ expression of a polypeptide of the ETC, complex III, or F1 / F0 ATP synthase may be a level of expression that is less than the average (e.g. mean) level of expression of the relevant polypeptide by mitochondria having a cristae density >30 / pm2. ‘Low’ expression of a polypeptide of the ETC, complex III, or F1 / F0 ATP synthase may be a level of expression that is similar to the average (e.g. mean) level of expression of the relevant polypeptide by mitochondria having a cristae density <30 / pm2, or by mitochondria of a quiescent muscle stem cell. ‘High’ expression of a polypeptide of the ETC, complex III, or F1 / F0 ATP synthase may be a level of expression that is greater than the average (e.g. mean) level of expression of the relevant polypeptide by mitochondria having a cristae density <30 / pm2, or by mitochondria of a quiescent muscle stem cell. ‘High’ expression of a polypeptide of the ETC, complex III, or F1 / F0 ATP synthase may be a level of expression that is similar to the average (e.g. mean) level of expression of the relevant polypeptide by mitochondria having a cristae density >30 / pm2. A level of expression of a polypeptide that is ‘similar to’ a given reference level of expression may be > 0.5 times and < 2 times, e.g. one of > 0.75 times and < 1 .5 times, > 0.8 times and < 1 .25 times, or > 0.9 times and < 1.1 times the reference level.

[0074] Similarly, a ‘low’ level of a respiratory supercomplex may be a level that is less than the average (e.g. mean) level of the relevant respiratory supercomplex in mitochondria having a cristae density >30 / pm2. A ‘low’ level of a respiratory supercomplex may be a level that is similar to the average (e.g. mean) level of the respiratory supercomplex in mitochondria having a cristae density <30 / pm2, or in mitochondria of a quiescent muscle stem cell. A ‘high’ level of a respiratory supercomplex may be a level that is greater than the average (e.g. mean) level of the relevant respiratory supercomplex in mitochondria having a cristae density <30 / pm2, or in mitochondria of a quiescent muscle stem cell. A ‘high’ level of a respiratory supercomplex may be a level that is similar to the average (e.g. mean) level of the respiratory supercomplex in mitochondria having a cristae density >30 / pm2. A level of a respiratory supercomplex that is ‘similar to’ a given reference level may be > 0.5 times and < 2 times, e.g. one of > 0.75 times and < 1 .5 times, > 0.8 times and < 1 .25 times, or > 0.9 times and < 1.1 times the reference level.

[0075] Similarly, a ‘low’ mitochondrial membrane potential may be a mitochondrial membrane potential that is less than the average (e.g. mean) mitochondrial membrane potential of mitochondria having a cristae density >30 / pm2. A ‘low’ mitochondrial membrane potential may be a mitochondrial membrane potential that is similar to the average (e.g. mean) mitochondrial membrane potential of mitochondria having a cristae density <30 / pm2, or of mitochondria of a quiescent muscle stem cell. A ‘high’ mitochondrial membrane potential may be a mitochondrial membrane potential that is greater than the average (e.g. mean) mitochondrial membrane potential of mitochondria having a cristae density <30 / pm2, or of mitochondria of a quiescent muscle stem cell. A ‘high’ mitochondrial membrane potential may be a mitochondrial membrane potential that is similar to the average (e.g. mean) mitochondrial membrane potential of mitochondria having a cristae density >30 / pm2. A mitochondrial membrane potential that is ‘similar to’ a given reference value may be > 0.5 times and < 2 times, e.g. one of > 0.75 times and < 1 .5 times, > 0.8 times and < 1 .25 times, or > 0.9 times and < 1.1 times the reference value.

[0076] Similarly, ‘low’ ATP production may be a level of ATP production that is less than the average (e.g. mean) level of ATP production by mitochondria having a cristae density >30 / pm2. ‘Low’ ATP production may be a level of ATP production that is similar to the average (e.g. mean) level of ATP production by mitochondria having a cristae density <30 / pm2, or by mitochondria of a quiescent muscle stem cell. ‘High’ ATP production may be a level of ATP production that is greater than the average (e.g. mean) level of ATP production by mitochondria having a cristae density <30 / pm2, or by mitochondria of a quiescent muscle stem cell. ‘High’ ATP production may be a level of ATP production that is similar to the average (e.g. mean) level of ATP production by mitochondria having a cristae density >30 / pm2. A level of ATP production that is ‘similar to’ a given reference level may be > 0.5 times and < 2 times, e.g. one of > 0.75 times and < 1 .5 times, > 0.8 times and < 1 .25 times, or > 0.9 times and < 1 .1 times the reference level.

[0077] Similarly, a ‘low’ oxygen consumption rate may be an oxygen consumption rate that is less than the average (e.g. mean) oxygen consumption rate of mitochondria having a cristae density >30 / pm2. A ‘low’ oxygen consumption rate may be an oxygen consumption rate that is similar to the average (e.g. mean) oxygen consumption rate of mitochondria having a cristae density <30 / pm2, or of mitochondria of a quiescent muscle stem cell. A ‘high’ oxygen consumption rate may be an oxygen consumption rate that is greater than the average (e.g. mean) oxygen consumption rate of mitochondria having a cristae density <30 / pm2, or of mitochondria of a quiescent muscle stem cell. A ‘high’ oxygen consumption rate may be an oxygen consumption rate that is similar to the average (e.g. mean) oxygen consumption rate of mitochondria having a cristae density >30 / pm2. An oxygen consumption rate that is ‘similar to’ a given reference value may be > 0.5 times and < 2 times, e.g. one of > 0.75 times and < 1 .5 times, > 0.8 times and < 1 .25 times, or > 0.9 times and < 1.1 times the reference value.

[0078] Inhibiting mitochondrial maturation

[0079] Aspects and embodiments of the present disclosure relate to inhibition of mitochondrial maturation. Mitochondrial maturation refers to the development of immature mitochondria to mature mitochondria. Inhibition of mitochondrial maturation may also be referred to herein as reduction, attenuation, prevention, blocking or antagonism of mitochondrial maturation.

[0080] Inhibiting mitochondrial maturation may comprise inhibiting the nucleic acid and / or polypeptide expression of, and / or the activity of, a factor whose expression / activity is positively associated with mitochondrial maturation. In some embodiments, inhibition of mitochondrial maturation may comprise inhibiting the nucleic acid and / or polypeptide expression of, and / or the activity of, a factor whose expression / activity is a marker of a mature mitochondrion.

[0081] In some embodiments, inhibition of mitochondrial maturation may comprise inhibiting mitochondrial transcription and / or inhibiting mitochondrial translation. In some embodiments, mitochondria in which mitochondrial maturation has been inhibited may be characterised by one or more of the following, as compared to equivalent mitochondria (e.g. mitochondria from equivalent cells) in which mitochondrial maturation has not been inhibited: lower cristae density (e.g. <30 / pm2); lower number of cristae; lower number of respiratory supercomplexes; lower expression of one or more polypeptide(s) of the ETC; lower expression of F1 / F0 ATP synthase; lower mitochondrial membrane potential; lower ATP production; lower oxygen consumption rate.

[0082] Inhibition of mitochondrial maturation may be characterised by there being a greater number / proportion of immature mitochondria among the mitochondria of cells in which mitochondrial maturation has been inhibited (e.g. by treatment with an inhibitor of mitochondrial maturation), as compared to the number / proportion of immature mitochondria among mitochondria of equivalent cells, in the absence of such inhibition (e.g. in the absence of treatment with an inhibitor of mitochondrial maturation). Similarly, inhibition of mitochondrial maturation may be characterised by there being a lower number / proportion of mature mitochondria among the mitochondria of cells in which mitochondrial maturation has been inhibited (e.g. by treatment with an inhibitor of mitochondrial maturation), as compared to the number / proportion of mature mitochondria among mitochondria of equivalent cells, in the absence of such inhibition (e.g. in the absence of treatment with an inhibitor of mitochondrial maturation).

[0083] In some embodiments, inhibition of mitochondrial maturation may comprise reversal of mitochondrial maturation. That is, in some embodiments, inhibition of mitochondrial maturation comprises reversion of mitochondria having a mature phenotype to mitochondria having an immature phenotype.

[0084] In some embodiments, inhibition of mitochondrial maturation in accordance with the present disclosure employs an inhibitor of mitochondrial maturation.

[0085] An ‘inhibitor of mitochondrial maturation’ refers to any agent (e.g. any compound / molecule) capable of inhibiting mitochondrial maturation. That is, an inhibitor of mitochondrial maturation may be any agent that increases the number / proportion of immature mitochondria, and / or decreases the number / proportion of mature mitochondria, among the mitochondria of cells treated with the agent, as compared to the number / proportion of immature / mature mitochondria among mitochondria of equivalent cells in the absence of such treatment. Where an inhibitor of mitochondrial maturation is employed in connection with an aspect or embodiment of the present disclosure, it will be appreciated that the inhibitor of mitochondrial maturation is used at a concentration sufficient for inhibition of mitochondrial maturation to be observed.

[0086] An inhibitor of mitochondrial maturation according to the present disclosure may be identified by a method comprising: culturing a first population of cells in the presence of a putative inhibitor of mitochondrial maturation; culturing a second population of cells ( / .e. cells of the same type as the first population of cell) in the absence of the putative inhibitor of mitochondrial maturation, or in the presence of a suitable control agent known not to affect mitochondrial maturation; subsequently analysing the first and second populations of cells, after an appropriate period of time for an effect on mitochondrial maturation to be observed (e.g. 24, 36, 48 or 72 hours), to determine the number / proportion of immature and / or mature mitochondria within cells of the first and second populations of cells.

[0087] It will be appreciated that an inhibitor of mitochondrial maturation is an agent that yields a greater number / proportion of immature mitochondria and / or a lesser number / proportion of mature mitochondria within cells of the first population of cells, as compared to the number / proportion of immature / mature mitochondria in cells of the second population of cells.

[0088] Inhibitors of mitochondrial maturation according to the present disclosure include agents that inhibit the nucleic acid and / or polypeptide expression of, and / or the activity of, a factor whose expression / activity is positively associated with mitochondrial maturation. Inhibitors of mitochondrial maturation also include agents that inhibit the nucleic acid and / or polypeptide expression of, and / or the activity of, a factor whose expression / activity is a marker of a mature mitochondrion.

[0089] In some embodiments, an inhibitor of mitochondrial maturation according to the present disclosure displays one or more of the following: reduces the expression or activity of a polypeptide involved in cristae formation ( / .e. a polypeptide that is positively-associated with the formation of cristae); reduces the expression or activity of one or more polypeptide(s) of the ETC (e.g. reduces the expression or activity of one or more polypeptide(s) of complex III; e.g. reduces the expression or activity of Rieske iron-sulfur protein (RISP); reduces the expression or activity of F1 / F0 ATP synthase.

[0090] It will be appreciated that a given inhibitor of mitochondrial maturation may display more than one of the properties recited in the preceding paragraph. A putative inhibitor of mitochondrial maturation may be evaluated for the properties recited in the preceding paragraph using suitable assays. For example, the assays may be e.g. in vitro assays, optionally cell-based assays or cell-free assays.

[0091] Where assays are cell-based assays, they may comprise treating cells with a given agent in order to determine whether the agent displays one or more of the recited properties. Assays may comprise evaluating the recited properties following treatment of cells separately with a range of quantities / concentrations of a given agent (e.g. a dilution series).

[0092] Inhibitors of mitochondrial maturation that reduce the expression (e.g. nucleic acid and / or polypeptide expression) of a polypeptide involved in cristae formation / one or more polypeptide(s) of the ETC / F1 / F0 ATP synthase may reduce the level of RNA encoding the relevant polypeptide. Such inhibitors may be identified using assays comprising detecting and / or quantifying the level of RNA encoding the relevant polypeptide. Such assays may comprise quantifying RNA encoding the relevant polypeptide by RT- qPCR, northern blot, etc., which are techniques well known to the skilled person. The methods may employ primers and / or probes for the detection and / or quantification of RNA encoding the relevant polypeptide. Such assays may comprise contacting cells in in vitro culture with a putative inhibitor of mitochondrial maturation, and subsequently (e.g. after an appropriate period of time, i.e. a period of time sufficient for a change in the level of RNA encoding the relevant polypeptide to be observed) measuring the level of RNA encoding the relevant polypeptide. Such assays may further comprise comparing the level of RNA encoding the relevant polypeptide in cells treated with the putative inhibitor of mitochondrial maturation to the level of RNA encoding the relevant polypeptide detected in a control condition in which cells of the same type are subjected to the same conditions, except that instead of being treated with the putative inhibitor of mitochondrial maturation they are untreated, or otherwise treated with an appropriate control agent known not to affect the level of RNA encoding the relevant polypeptide.

[0093] Herein, ‘contacting’ cells with a given agent (e.g. a putative inhibitor of mitochondrial maturation) may comprise applying the agent to, and / or mixing the agent with, the cells. In some embodiments, the putative inhibitor of mitochondrial maturation is provided to the cells in combination with one or more further agents for facilitating introduction of the putative inhibitor of mitochondrial maturation into the cells, and / or for facilitating uptake of the putative inhibitor of mitochondrial maturation by the cells. For example, in embodiments wherein a putative inhibitor of mitochondrial maturation is, or is encoded by, one or more nucleic acids, the cells may be contacted with the nucleic acid(s) and an agent for facilitating introduction of the nucleic acid(s) into the cells, e.g. by transfection or transduction.

[0094] A reduction in the level of RNA encoding the relevant polypeptide ( / .e. a polypeptide involved in cristae formation, a polypeptide of the ETC, or F1 / F0 ATP synthase) may be a consequence of reduced transcription of DNA encoding the relevant polypeptide (e.g. as a consequence of reduced assembly and / or activity of factors required for transcription of DNA encoding the relevant polypeptide), increased degradation of RNA encoding the relevant polypeptide (e.g. as a consequence of RNA interference (RNAi)) and / or reduced stability of RNA encoding the relevant polypeptide.

[0095] Inhibitors of mitochondrial maturation that reduce the expression (e.g. nucleic acid and / or polypeptide expression) of a polypeptide involved in cristae formation / one or more polypeptide(s) of the ETC / F1 / F0 ATP synthase may reduce the level of the relevant polypeptide. Such inhibitors may be identified using assays comprising detecting and / or quantifying the level of the relevant polypeptide. Such assays may employ antibody or reporter-based methods (western blot, ELISA, immunohisto / cytochemistry, etc.). The methods may employ antibodies specific for the relevant polypeptide. Such assays may comprise contacting cells in in vitro culture with a putative inhibitor of mitochondrial maturation, and subsequently (e.g. after an appropriate period of time, i.e. a period of time sufficient for a change in the level of the relevant polypeptide to be observed) measuring the level of the relevant polypeptide. Such assays may further comprise comparing the level of the relevant polypeptide in cells treated with the putative inhibitor of mitochondrial maturation to the level of the relevant polypeptide detected in a control condition in which cells of the same type are subjected to the same conditions, except that instead of being treated with the putative inhibitor of mitochondrial maturation they are untreated, or otherwise treated with an appropriate control agent known not to affect the level of the relevant polypeptide.

[0096] A reduction in the level of the relevant polypeptide (i.e. a polypeptide involved in cristae formation, a polypeptide of the ETC, or F1 / F0 ATP synthase) may be a consequence of reduced translation of RNA encoding the relevant polypeptide, increased degradation of the relevant polypeptide (e.g. as a consequence of misfolding) and / or reduced stability of the relevant polypeptide. Inhibitors of mitochondrial maturation that reduce the activity of a polypeptide involved in cristae formation / one or more polypeptide(s) of the ETC / F1 / F0 ATP synthase may be identified using assays comprising detecting and / or quantifying the level of an activity of the relevant polypeptide. Such assays may comprise detecting and / or quantifying a correlate of the relevant activity. For example, detecting and / or quantifying the level of an activity of a polypeptide involved in cristae formation may comprise detecting and / or quantifying the number of cristae and / or cristae density. For example, detecting and / or quantifying the level of an activity of one or more polypeptide(s) of the ETC may comprise detecting and / or quantifying mitochondrial membrane potential. For example, detecting and / or quantifying the level of an activity of F1 / F0 ATP synthase may comprise detecting and / or quantifying the level of ATP. Such assays may comprise contacting cells in in vitro culture with a putative inhibitor of mitochondrial maturation, and subsequently (e.g. after an appropriate period of time, i.e. a period of time sufficient for a change in the level of the relevant activity to be observed) measuring the level of the relevant activity pf the relevant polypeptide. Such assays may further comprise comparing the level of the relevant activity in cells treated with the putative inhibitor of mitochondrial maturation to the level of the relevant activity detected in a control condition in which cells of the same type are subjected to the same conditions, except that instead of being treated with the putative inhibitor of mitochondrial maturation they are untreated, or otherwise treated with an appropriate control agent known not to affect the level of the relevant activity.

[0097] In some embodiments, an inhibitor of mitochondrial maturation according to the present disclosure reduces the expression of a polypeptide involved in cristae formation / reduces the level of RNA encoding a polypeptide involved in cristae formation / reduces the level of a polypeptide involved in cristae formation / reduces the formation of a respiratory supercomplex / reduces the expression of one or more polypeptide(s) of the ETC (e.g. selected from a polypeptide of complex I, a polypeptide of complex II, a polypeptide of complex III, cytochrome c and a polypeptide of complex IV; e.g. a polypeptide of complex III; e.g. RISP) / reduces the level of RNA encoding one or more polypeptide(s) of the ETC (e.g. selected from a polypeptide of complex I, a polypeptide of complex II, a polypeptide of complex III, cytochrome c and a polypeptide of complex IV; e.g. a polypeptide of complex III; e.g. RISP) / reduces the level of one or more polypeptide(s) of the ETC (e.g. selected from a polypeptide of complex I, a polypeptide of complex II, a polypeptide of complex III, cytochrome c and a polypeptide of complex IV; e.g. a polypeptide of complex III; e.g. RISP) / reduces the expression of F1 / F0 ATP synthase / reduces the level of RNA encoding F1 / F0 ATP synthase / reduces the level of F1 / F0 ATP synthase / reduces the activity of a polypeptide involved in cristae formation / reduces the number of cristae / reduces cristae density / reduces the activity of one or more polypeptide(s) of the ETC (e.g. selected from a polypeptide of complex I, a polypeptide of complex II, a polypeptide of complex III, cytochrome c and a polypeptide of complex IV; e.g. a polypeptide of complex III; e.g. RISP) / reduces mitochondrial membrane potential / reduces the activity of F1 / F0 ATP synthase / reduces the level of ATP to less than 1 times, e.g. one of <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times, <0.05 times, or <0.01 times the level observed in the absence of the inhibitor of mitochondrial maturation, or in the presence of the same quantity of an appropriate control agent known not to possess such inhibitory activity, in a given assay. In some embodiments, an inhibitor of mitochondrial maturation according to the present disclosure reduces the expression of a polypeptide involved in cristae formation / reduces the level of RNA encoding a polypeptide involved in cristae formation / reduces the formation of a respiratory supercomplex / reduces the level of a polypeptide involved in cristae formation / reduces the expression of one or more polypeptide(s) of the ETC (e.g. selected from a polypeptide of complex I, a polypeptide of complex II, a polypeptide of complex III, cytochrome c and a polypeptide of complex IV; e.g. a polypeptide of complex III; e.g. RISP) / reduces the level of RNA encoding one or more polypeptide(s) of the ETC (e.g. selected from a polypeptide of complex I, a polypeptide of complex II, a polypeptide of complex III, cytochrome c and a polypeptide of complex IV; e.g. a polypeptide of complex III; e.g. RISP) / reduces the level of one or more polypeptide(s) of the ETC (e.g. selected from a polypeptide of complex I, a polypeptide of complex II, a polypeptide of complex III, cytochrome c and a polypeptide of complex IV; e.g. a polypeptide of complex III; e.g. RISP) / reduces the expression of F1 / F0 ATP synthase / reduces the level of RNA encoding F1 / F0 ATP synthase / reduces the level of F1 / F0 ATP synthase / reduces the activity of a polypeptide involved in cristae formation / reduces the number of cristae / reduces cristae density / reduces the activity of one or more polypeptide(s) of the ETC (e.g. selected from a polypeptide of complex I, a polypeptide of complex II, a polypeptide of complex III, cytochrome c and a polypeptide of complex IV; e.g. a polypeptide of complex lll) / reduces mitochondrial membrane potential / reduces the activity of F1 / F0 ATP synthase / reduces the level of ATP to less than 100%, e.g. one of <99%, <95%, <90%, <85%, <80%, <75%, <70%, <65%, <60%, <55%, <50%, <45%, <40%, <35%, <30%, <25%, <20%, <15%, <10%, <5%, or <1% of the level observed in the absence of the inhibitor of mitochondrial maturation, or in the presence of the same quantity of an appropriate control agent known not to possess such inhibitory activity, in a given assay.

[0098] Preferred levels of reduction in accordance with the preceding two paragraphs are reduction to <0.5 times / <50%, e.g. one of <0.4 times / <40%, <0.3 times / <30%, <0.2 times / <20%, <0.15 times / <15%, or <0.1 times / <10%.

[0099] In some embodiments, an inhibitor of mitochondrial maturation according to the present disclosure is a small molecule or an inhibitory nucleic acid.

[0100] As used herein, a ‘small molecule’ refers to a low molecular weight (< 1000 daltons, typically between ~300-700 daltons) organic compound. Small molecule inhibitors of mitochondrial maturation may be identified by screening libraries of such small molecules for the ability to inhibit mitochondrial maturation.

[0101] In some embodiments, a small molecule inhibitor of mitochondrial maturation may bind to and inhibit the activity of one or more factors whose activity is positively associated with mitochondrial maturation, and / or whose activity is a marker of a mature mitochondrion. In some embodiments, a small molecule inhibitor of mitochondrial maturation may bind to and inhibit the activity of a polypeptide involved in cristae formation. In some embodiments, a small molecule inhibitor of mitochondrial maturation may inhibit assembly of a respiratory supercomplex. In some embodiments, a small molecule inhibitor of mitochondrial maturation may bind to and inhibit the activity of one or more polypeptide(s) of the ETC (e.g. selected from a polypeptide of complex I, a polypeptide of complex II, a polypeptide of complex III, cytochrome c and a polypeptide of complex IV; e.g. a polypeptide of complex III; e.g. RISP). In some embodiments, a small molecule inhibitor of mitochondrial maturation may bind to and inhibit the activity of F1 / F0 ATP synthase.

[0102] Inhibitory nucleic acids according to the present disclosure may comprise or consist of DNA and / or RNA. Inhibitory nucleic acids may be single-stranded (e.g. in the case of antisense oligonucleotides (e.g. gapmers)). Inhibitory nucleic acids may be double-stranded or may comprise double-stranded region(s) (e.g. in the case of siRNA, shRNA, etc.). Inhibitory nucleic acids may comprise both double-stranded and single-stranded regions (e.g. in the case of shRNA and pre-miRNA molecules, which are double-stranded in the stem region of the hairpin structure, and single-stranded in the loop region of the hairpin structure).

[0103] In some embodiments, inhibitory nucleic acids according to the present disclosure may inhibit the expression of one or more factors whose expression and / or activity is positively associated with mitochondrial maturation, and / or whose expression and / or activity is a marker of a mature mitochondrion. In some embodiments, an inhibitory nucleic acid according to the present disclosure may inhibit the expression of a polypeptide involved in cristae formation. In some embodiments, an inhibitory nucleic acid according to the present disclosure may inhibit the expression of one or more polypeptide(s) of the ETC (e.g. selected from a polypeptide of complex I, a polypeptide of complex II, a polypeptide of complex III, cytochrome c and a polypeptide of complex IV; e.g. a polypeptide of complex III; e.g. RISP). In some embodiments, an inhibitory nucleic acid according to the present disclosure may inhibit the expression of F1 / F0 ATP synthase.

[0104] In some embodiments, an inhibitory nucleic acid according to the present disclosure may be, comprise or encode an antisense polynucleotide. An ‘antisense polynucleotide’ refers to a polyribonucleotide or polydeoxyribonucleotide that is complementary to at least a portion of a target nucleotide sequence (e.g. of RNA encoding the polypeptide whose expression is to be inhibited). Antisense polynucleotides according to the present disclosure are preferably single-stranded nucleic acids, and bind via complementary Watson-Crick base-pairing to a target nucleotide sequence. Complementary base-pairing may involve hydrogen bonding between complementary base pairs. Antisense polynucleotides may be provided as single-stranded molecules, as for example in the case of antisense oligonucleotides, or may be comprised in double-stranded molecular species, as for example in the case of siRNA, shRNA and pre-miRNA molecules.

[0105] In some embodiments, the antisense polynucleotide reduces / prevents transcription of nucleic acid comprising its target nucleotide sequence. In some embodiments, the antisense polynucleotide reduces / prevents association of factors required for normal transcription (e.g. enhancers, RNA polymerase) of nucleic acid comprising its target nucleotide sequence. In some embodiments, the antisense polynucleotide increases / potentiates degradation of nucleic acid comprising its target nucleotide sequence, e.g. through RNA interference. In some embodiments, the antisense polynucleotide reduces / prevents translation of nucleic acid comprising its target nucleotide sequence, e.g. through RNA interference or antisense degradation via RNase H activity. RNA interference is described e.g. in Agrawal et al., Microbiol. Mol. Bio. Rev. (2003) 67(4): 657-685 and Hu et al., Sig. Transduc. Tar. Ther. (2020) 5(101), both of which are hereby incorporated by reference in their entirety. Briefly, double-stranded RNA molecules are recognised by the argonaute component of the RNA-induced silencing complex (RISC). The double-stranded RNAs are separated into single strands and integrated into an active RISC, by the RISC-Loading Complex (RLC). The RISC-integrated strands bind to their target RNA through complementary base pairing, and depending on the identity of the RISC- integrated RNA and degree of complementarity to the target RNA, the RISC then either cleaves the target RNA resulting in its degradation, or otherwise blocks access of ribosomes thereby preventing its translation. RNAi based therapeutics have been approved for a number of indications (see e.g. Kim, Chonnam Med J. (2020) 56(2): 87-93).

[0106] In some embodiments, an inhibitory nucleic acid according to the present disclosure may be an siRNA, dsiRNA, miRNA, shRNA, pri-miRNA, pre-miRNA, saRNA or snoRNA or antisense oligonucleotide (e.g. a gapmer), comprising an antisense polynucleotide as described herein, or a nucleic acid encoding such a molecule.

[0107] In some embodiments, an inhibitor of mitochondrial maturation according to the present disclosure is an inhibitor of mitochondrial transcription and / or translation.

[0108] In some embodiments, an inhibitor of mitochondrial maturation according to the present disclosure is an inhibitor of mitochondrial translation. In the experimental examples of the present disclosure, the inventors demonstrate that inhibitors of mitochondrial translation such as chloramphenicol inhibit mitochondrial maturation.

[0109] In some embodiments, an inhibitor of mitochondrial maturation according to the present disclosure is not an inhibitor of mitochondrial translation. In some embodiments, an inhibitor of mitochondrial maturation is not an inhibitor of mitochondrial DNA replication. In some embodiments, an inhibitor of mitochondrial maturation is not an inhibitor of mitochondrial DNA transcription. In some embodiments, an inhibitor of mitochondrial maturation is not an inhibitor of mitochondrial DNA replication and / or transcription. In some embodiments, an inhibitor of mitochondrial maturation is not ethidium bromide. In some embodiments, an inhibitor of mitochondrial maturation is not an inhibitor of mitochondrial RNA polymerase. In some embodiments, an inhibitor of mitochondrial maturation is not rifampicin.

[0110] Herein, an ‘inhibitor of mitochondrial translation’ refers to any agent (e.g. any compound / molecule) capable of inhibiting translation in the mitochondria. That is, an inhibitor of mitochondrial translation inhibits translation of nucleic acid to a polypeptide in the mitochondria.

[0111] In preferred embodiments, an inhibitor of mitochondrial translation inhibits translation of nucleic acid to a polypeptide in the mitochondria, but does not substantially inhibit translation of nucleic acid to a polypeptide outside of the mitochondria of a eukaryotic cell. For example, in preferred embodiments, an inhibitor of mitochondrial translation does not substantially inhibit translation of nucleic acid to a polypeptide in the cytoplasm and / or in the rough endoplasmic reticulum of a eukaryotic cell. Inhibitors of mitochondrial translation contemplated in accordance with the present disclosure include agents that inhibit translation of nucleic acid to a polypeptide in prokaryotic cells.

[0112] In some embodiments, an inhibitor of mitochondrial translation according to the present disclosure displays one or more of the following: reduces the level of a polypeptide involved in cristae formation; reduces the level of one or more polypeptide(s) of the ETC; reduces the level of F1 / F0 ATP synthase.

[0113] Inhibitors of mitochondrial translation may be identified using assays comprising detecting and / or quantifying the level of one or more polypeptides that are translated in the mitochondria. For example, such assay may comprise detecting and / or quantifying the level of a polypeptide involved in cristae formation, or detecting and / or quantifying the level of one or more polypeptide(s) of the ETC (e.g. selected from a polypeptide of complex I, a polypeptide of complex II, a polypeptide of complex III, cytochrome c and a polypeptide of complex IV; e.g. a polypeptide of complex III; e.g. RISP), or detecting and / or quantifying the level of F1 / F0 ATP synthase. The methods may employ antibodies specific for the relevant polypeptide. Such assays may comprise contacting cells in in vitro culture with a putative inhibitor of mitochondrial translation, and subsequently (e.g. after an appropriate period of time, i.e. a period of time sufficient for a change in the level of the relevant polypeptide to be observed) measuring the level of the relevant polypeptide. Such assays may further comprise comparing the level of the relevant polypeptide in cells treated with the putative inhibitor of mitochondrial translation to the level of the relevant polypeptide detected in a control condition in which cells of the same type are subjected to the same conditions, except that instead of being treated with the putative inhibitor of mitochondrial translation they are untreated, or otherwise treated with an appropriate control agent known not to affect the level of the relevant polypeptide.

[0114] In some embodiments, an inhibitor of mitochondrial translation according to the present disclosure reduces the level of a polypeptide involved in cristae formation / reduces the level of one or more polypeptide(s) of the ETC (e.g. selected from a polypeptide of complex I, a polypeptide of complex II, a polypeptide of complex III, cytochrome c and a polypeptide of complex IV; e.g. a polypeptide of complex III; e.g. RISP) / reduces the level of F1 / F0 ATP synthase to less than 1 times, e.g. one of <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times, <0.05 times, or <0.01 times the level observed in the absence of the inhibitor of mitochondrial translation, or in the presence of the same quantity of an appropriate control agent known not to possess such inhibitory activity, in a given assay.

[0115] In some embodiments, an inhibitor of mitochondrial translation according to the present disclosure reduces the level of a polypeptide involved in cristae formation / reduces the level of one or more polypeptide(s) of the ETC (e.g. selected from a polypeptide of complex I, a polypeptide of complex II, a polypeptide of complex III, cytochrome c and a polypeptide of complex IV; e.g. a polypeptide of complex III; e.g. RISP) / reduces the level of F1 / F0 ATP synthase to less than 100%, e.g. one of <99%, <95%, <90%, <85%, <80%, <75%, <70%, <65%, <60%, <55%, <50%, <45%, <40%, <35%, <30%, <25%, <20%, <15%, <10%, <5%, or <1 % of the level observed in the absence of the inhibitor of mitochondrial translation, or in the presence of the same quantity of an appropriate control agent known not to possess such inhibitory activity, in a given assay.

[0116] Preferred levels of reduction in accordance with the preceding two paragraphs are reduction to <0.5 times / <50%, e.g. one of <0.4 times / <40%, <0.3 times / <30%, <0.2 times / <20%, <0.15 times / <15%, or <0.1 times / <10%.

[0117] In some embodiments, an inhibitor of mitochondrial translation according to the present disclosure is a small molecule or an inhibitory nucleic acid.

[0118] In some embodiments, an inhibitor of mitochondrial translation according to the present disclosure is an antibiotic. In some embodiments, an inhibitor of mitochondrial translation is a bacteriostatic.

[0119] In some embodiments, an inhibitor of mitochondrial translation is a bacterial protein synthesis inhibitor. Inhibitors of bacterial protein synthesis are described e.g. in Arenz and Wilson, Cold Spring Harb Perspect Med. (2016) 6(9): a025361 , which is hereby incorporated by reference in its entirety. Bacterial protein synthesis inhibitors include phenicols (e.g. chloramphenicol, thiamphenicol, florfenicol), lincosamides (e.g. clindamycin, lincomycin, pirlimycin), macrolides (e.g. azithromycin, clarithromycin, dirithromycin, erythromycin, carbomycin A, josamycin, kitasamycin, midecamycin, oleandomycin, spiramycin, troleandomycin, tylosin, roxithromycin) ketolides (e.g. telithromycin, cethromycin, solithromycin), oxazolidinones (e.g. linezolid, posizolid, tedizolid, radezolid, cycloserine, contezolid), spectinomycin, and tetracyclines (tetracycline, chlortetracycline, oxytetracycline, demeclocycline, lymecycline, meclocycline, methacycline, minocycline, rolitetracycline, doxycycline, tigecycline, eravacycline, sarecycline, omadacycline).

[0120] Phenicols, macrolides and ketolides inhibit protein chain elongation through binding to the centre of peptidyl transferase activity on the 23S rRNA domain of the 50S ribosomal subunit of prokaryotic ribosomes, thereby inhibiting peptidyl transferase activity. Lincosamides bind close to this same site on the 50S subunit and sterically inhibit peptidyl transferase activity. Oxazolidinones inhibit bacterial protein synthesis through inhibition of association of the N-formyl methionyl-tRNA with the prokaryotic ribosome. Spectinomycin and tetracyclines disrupt protein synthesis through binding to the 30S subunit of the prokaryotic ribosome, and inhibiting binding of aminoacyl tRNA to the RNA translation complex.

[0121] In some embodiments, an inhibitor of mitochondrial translation according to the present disclosure is a phenicol. In some embodiments, an inhibitor of mitochondrial translation is selected from chloramphenicol, thiamphenicol and florfenicol. In some embodiments, an inhibitor of mitochondrial translation is chloramphenicol.

[0122] In some embodiments, an inhibitor of mitochondrial translation according to the present disclosure is not a phenicol. In some embodiments, an inhibitor of mitochondrial translation is not selected from chloramphenicol, thiamphenicol and florfenicol. In some embodiments, an inhibitor of mitochondrial translation is not chloramphenicol. In some embodiments, an inhibitor of mitochondrial translation according to the present disclosure is not a tetracycline antibiotic. In some embodiments, an inhibitor of mitochondrial translation is not tetracycline, chlortetracycline, oxytetracycline, demeclocycline, lymecycline, meclocycline, methacycline, minocycline, rolitetracycline, doxycycline, tigecycline, eravacycline, sarecycline or omadacycline. In some embodiments, an inhibitor of mitochondrial translation is not tetracycline.

[0123] In some embodiments, an inhibitor of mitochondrial translation is an inhibitor of peptide deformylase. Peptide deformylase is an essential enzyme required for the removal of the formyl group at the N- terminus of nascent polypeptide chains in eubacteria. Inhibitors of peptide deformylase are described e.g. in Apfel et al., Antimicrob Agents Chemother. (2001) 45(4): 1058-1064, which is hereby incorporated by reference in its entirety. Such inhibitors include actinonin (described e.g. in Chen et al., Biochemistry (2000) 39:1256-1262), and Ro 66-0376 and Ro 66-6976 (described e.g. in Apfel et al., J Med Chem. (2000) 43:2324-2331).

[0124] In some embodiments, an inhibitor of peptide deformylase according to the present disclosure is selected from actinonin (also known as Ro 06-1467), Ro 66-0376 and Ro 66-6976. In some embodiments, an inhibitor of peptide deformylase is actinonin.

[0125] Aspects and embodiments according to the present disclosure relate to cells (e.g. muscle stem cells, e.g. myoblasts) in which mitochondrial maturation is inhibited. It will be appreciated that ‘a cell in which mitochondrial maturation is inhibited’ is a cell experiencing inhibition of mitochondrial maturation. The cell may be experiencing inhibition of mitochondrial maturation as a consequence of treatment or modification to inhibit mitochondrial maturation, e.g. as described herein. Thus, in some embodiments, a cell in which mitochondrial maturation is inhibited is a cell in which mitochondrial maturation has been inhibited.

[0126] In some embodiments, mitochondrial maturation may be inhibited in a cell as a consequence of the action of an inhibitor of mitochondrial maturation according to the present disclosure. That is, the cell may have been contacted / treated with an inhibitor of mitochondrial maturation according to the present disclosure. For example, the cell may have been cultured in the presence of an inhibitor of mitochondrial maturation according to the present disclosure.

[0127] In some embodiments, mitochondrial maturation may be inhibited as a consequence of modification of a cell, and / or mitochondria of a cell, to inhibit mitochondrial maturation. That is, in some embodiments, a cell, and / or mitochondria of a cell, may comprise modification to inhibit mitochondrial maturation.

[0128] For example, a cell and / or mitochondria of a cell, may comprise modification to nucleic acid encoding a factor whose expression / activity is positively associated with mitochondrial maturation, and / or modification to nucleic acid encoding a factor whose expression / activity is a marker of a mature mitochondrion. The modification may inhibit / reduce / block / prevent expression of the relevant factor.

[0129] In some embodiments, a cell and / or mitochondria of a cell comprises modification to nucleic acid encoding a polypeptide involved in cristae formation, wherein the modification reduces expression of the polypeptide. In some embodiments, a cell and / or mitochondria of a cell comprises modification to nucleic acid encoding one or more polypeptide(s) of the ETC (e.g. selected from a polypeptide of complex I, a polypeptide of complex II, a polypeptide of complex III, cytochrome c and a polypeptide of complex IV; e.g. a polypeptide of complex III; e.g. RISP), wherein the modification reduces expression of the polypeptide(s). In some embodiments, a cell and / or mitochondria of a cell comprises modification to nucleic acid encoding F1 / F0 ATP synthase, wherein the modification reduces expression of the polypeptide.

[0130] In some embodiments, modification to nucleic acid encoding a given polypeptide that reduces expression of the polypeptide may introduce an insertion in, substitution of, deletion to, or larger-scale translocation / rearrangement of, the nucleotide sequence of the most common version of the nucleotide sequence of nucleic acid encoding the polypeptide ( / .e. the wildtype nucleotide sequence).

[0131] Site-specific nuclease (SSN) systems may be employed to reduce expression of a given polypeptide (e.g. a polypeptide involved in cristae formation, one or more polypeptide(s) of the ETC (e.g. selected from a polypeptide of complex I, a polypeptide of complex II, a polypeptide of complex III, cytochrome c and a polypeptide of complex IV; e.g. a polypeptide of complex III; e.g. RISP), or F1 / F0 ATP synthase).

[0132] Gene editing using SSNs is reviewed e.g. in Eid and Mahfouz, Exp Mol Med. (2016) 48(10): e265, which is hereby incorporated by reference in its entirety. Enzymes capable of creating site-specific double strand breaks (DSBs) can be engineered to introduce DSBs to target nucleic acid sequence(s) of interest. DSBs may be repaired by either error-prone non-homologous end-joining (NHEJ), in which the two ends of the break are rejoined, often with insertion or deletion of nucleotides. Alternatively, DSBs may be repaired by homology-directed repair (HDR), in which a DNA template with ends homologous to the break site is supplied and introduced at the site of the DSB. SSNs capable of being engineered to generate target nucleic acid sequence-specific DSBs include zinc-finger nucleases (ZFNs), transcription activatorlike effector nucleases (TALENs) and clustered regularly interspaced palindromic repeats / CRISPR- associated-9 (CRISPR / Cas9) systems.

[0133] ZFN systems are reviewed e.g. in Umov ef al., Nat Rev Genet. (2010) 11 (9):636-46, which is hereby incorporated by reference in its entirety. ZFNs comprise a programmable Zinc Finger DNA-binding domain and a DNA-cleaving domain (e.g. a Fok\ endonuclease domain). The DNA-binding domain may be identified by screening a Zinc Finger array capable of binding to the target nucleic acid sequence. TALEN systems are reviewed e.g. in Mahfouz et al., Plant Biotechnol J. (2014) 12(8): 1006-14, which is hereby incorporated by reference in its entirety. TALENs comprise a programmable DNA-binding TALE domain and a DNA-cleaving domain (e.g. a Fok\ endonuclease domain). TALEs comprise repeat domains consisting of repeats of 33-39 amino acids, which are identical except for two residues at positions 12 and 13 of each repeat which are repeat variable di-residues (RVDs). Each RVD determines binding of the repeat to a nucleotide in the target DNA sequence according to the following relationship: ‘HD’ binds to C, ‘Nl’ binds to A, ‘NG’ binds to T and ‘NN’ or ‘NK’ binds to G (Moscou and Bogdanove, Science (2009) 326(5959):1501 .). CRISPR / Cas9 and related systems e.g. CRISPR / Cpf1 , CRISPR / C2c1 , CRISPR / C2c2 and CRISPR / C2c3 are reviewed e.g. in Nakade et al., Bioengineered (2017) 8(3):265-273, which is hereby incorporated by reference in its entirety. These systems comprise an endonuclease (e.g. Cas9, Cpf1 etc.) and the single-guide RNA (sgRNA) molecule. The sgRNA can be engineered to target endonuclease activity to nucleic acid sequences of interest.

[0134] In preferred embodiments, inhibition of mitochondrial maturation is reversible. That is, in some embodiments the inhibition of mitochondrial maturation is non-permanent. The inhibition of mitochondrial maturation may be temporary.

[0135] In preferred embodiments it is possible for cells / mitochondria experiencing inhibition of mitochondrial maturation to be released from such inhibition, such that mitochondrial maturation is no longer inhibited. In preferred embodiments, inhibition of mitochondrial maturation is such that immature mitochondria in which mitochondrial maturation has been inhibited are able to subsequently mature to become mature mitochondria.

[0136] Releasing cells from inhibition of mitochondrial maturation

[0137] Aspects and embodiments of the present disclosure relate to releasing cells (e.g. muscle stem cells, e.g. myoblasts) from inhibition of mitochondrial maturation.

[0138] It will be appreciated that prior to releasing the cells from inhibition, the cells are experiencing inhibition of mitochondrial maturation. The cells may have been subjected to inhibition of mitochondrial maturation, e.g. as described herein. For example, the cells may have been contacted with an inhibitor of mitochondrial maturation as described herein above, or may have been modified to inhibit mitochondrial maturation as described herein above.

[0139] Releasing cells from inhibition of mitochondrial maturation may comprise treating the cells such that mitochondrial maturation is no longer inhibited, and / or modifying the cells such that mitochondrial maturation is no longer inhibited.

[0140] In some embodiments, releasing cells (e.g. muscle stem cells, e.g. myoblasts) from inhibition of mitochondrial maturation comprises ceasing / stopping (further) contacting / treatment of the cells with an inhibitor of mitochondrial maturation. For example, releasing cells from inhibition of mitochondrial maturation may comprise no longer contacting / treating cells with an inhibitor of mitochondrial maturation, i.e. no longer applying an inhibitor of mitochondrial maturation to the cells in culture. This may be the case in particular where the inhibitor of mitochondrial maturation is relatively unstable, is quickly degraded, has a relatively short half-life and / or is quickly metabolised by the cells. In such instances, simply not continuing to add the inhibitor of mitochondrial maturation to the cells in culture, and thereby not replenishing the inhibitor, may suffice to release the cells from inhibition of mitochondrial maturation. That is, degradation / metabolism of the inhibitor of mitochondrial maturation - in the absence of further addition of the inhibitor of mitochondrial maturation - may suffice to reduce the concentration of the inhibitor of mitochondrial maturation such that the cells are released from inhibition of mitochondrial maturation. In some embodiments, releasing cells (e.g. muscle stem cells, e.g. myoblasts) from inhibition of mitochondrial maturation comprises inhibiting the action of an inhibitor of mitochondrial maturation. In some embodiments, releasing cells from inhibition of mitochondrial maturation comprises depleting / removing the inhibitor of mitochondrial maturation from the cells in culture.

[0141] In some embodiments, releasing cells (e.g. muscle stem cells, e.g. myoblasts) from inhibition of mitochondrial maturation comprises reducing the concentration of the inhibitor of mitochondrial maturation in the cell culture. In some embodiments, releasing cells from inhibition of mitochondrial maturation comprises replacing cell culture medium comprising an inhibitor of mitochondrial maturation with cell culture medium lacking an inhibitor of mitochondrial maturation. For example, cell culture medium comprising an inhibitor of mitochondrial maturation may be removed from the cells in culture, and replaced with cell culture medium lacking the inhibitor of mitochondrial maturation.

[0142] In some embodiments, releasing cells (e.g. muscle stem cells, e.g. myoblasts) from inhibition of mitochondrial maturation comprises adding cell culture medium lacking the inhibitor of mitochondrial maturation to a cell culture comprising an inhibitor of mitochondrial maturation, such that the concentration of the inhibitor of mitochondrial maturation in the cell culture is reduced to a level such that cells of the culture are released from inhibition of mitochondrial maturation. That is, cell culture medium lacking the inhibitor of mitochondrial maturation may be added to the cell culture in a quantity sufficient to dilute the inhibitor of mitochondrial maturation to a concentration such that cells of the culture are released from inhibition of mitochondrial maturation.

[0143] In some embodiments, releasing cells (e.g. muscle stem cells, e.g. myoblasts) from inhibition of mitochondrial maturation may comprise contacting / treating cells that have been contacted / treated with an inhibitor of mitochondrial maturation with an antagonist of the inhibitor of mitochondrial maturation. An antagonist of a given inhibitor of mitochondrial maturation may be any agent (e.g. any compound / molecule) that is capable of reducing the activity of the inhibitor of mitochondrial maturation. In some embodiments, an antagonist of an inhibitor of mitochondrial maturation may be an agent that binds to and inactivates the inhibitor of mitochondrial maturation. In some embodiments, an antagonist of an inhibitor of mitochondrial maturation may be an agent that degrades or potentiates degradation of the inhibitor of mitochondrial maturation.

[0144] In some embodiments, releasing cells (e.g. muscle stem cells, e.g. myoblasts) from inhibition of mitochondrial maturation may comprise promoting mitochondrial maturation, e.g. as described hereinbelow.

[0145] Following release from inhibition of mitochondrial maturation, the mitochondria may mature, and may do so spontaneously. In some embodiments, following release from inhibition of mitochondrial maturation mitochondria may develop a morphology resembling that of mitochondria cultured for the same period of time in the absence of an inhibitor of mitochondrial maturation, but otherwise under equivalent conditions. In some embodiments, following release from inhibition of mitochondrial maturation, the mitochondria develop the phenotype of a mature mitochondrion, e.g. as described hereinabove.

[0146] Promoting mitochondrial maturation

[0147] Aspects and embodiments of the present disclosure relate to promotion of mitochondrial maturation. Promotion of mitochondrial maturation may also be referred to herein as potentiation, agonism or upregulation of mitochondrial maturation.

[0148] Promoting mitochondrial maturation may comprise promoting the nucleic acid and / or polypeptide expression of, and / or the activity of, a factor whose expression / activity is positively associated with mitochondrial maturation. In some embodiments, promotion of mitochondrial maturation may comprise promoting the nucleic acid and / or polypeptide expression of, and / or the activity of, a factor whose expression / activity is a marker of a mature mitochondrion.

[0149] In some embodiments, mitochondria in which mitochondrial maturation has been promoted may be characterised by one or more of the following, as compared to equivalent mitochondria (e.g. mitochondria from equivalent cells) in which mitochondrial maturation has not been promoted: higher cristae density (e.g. >30 / pm2);higher number of cristae; higher number of respiratory supercomplexes; higher expression of one or more polypeptide(s) of the ETC; higher expression of F1 / F0 ATP synthase; higher mitochondrial membrane potential; higher ATP production; higher oxygen consumption rate.

[0150] Promotion of mitochondrial maturation may be characterised by there being a greater number / proportion of mature mitochondria among the mitochondria of cells in which mitochondrial maturation has been promoted (e.g. by treatment with a promoter of mitochondrial maturation), as compared to the number / proportion of mature mitochondria among mitochondria of equivalent cells, in the absence of such promotion (e.g. in the absence of treatment with a promoter of mitochondrial maturation). Similarly, promotion of mitochondrial maturation may be characterised by there being a lower number / proportion of immature mitochondria among the mitochondria of cells in which mitochondrial maturation has been promoted (e.g. by treatment with a promoter of mitochondrial maturation), as compared to the number / proportion of immature mitochondria among mitochondria of equivalent cells, in the absence of such promotion (e.g. in the absence of treatment with a promoter of mitochondrial maturation).

[0151] In some embodiments, promotion of mitochondrial maturation in accordance with the present disclosure employs a promoter of mitochondrial maturation.

[0152] A ‘promoter of mitochondrial maturation’ refers to any agent (e.g. any compound / molecule) capable of promoting mitochondrial maturation. That is, a promoter of mitochondrial maturation may be any agent that increases the number / proportion of mature mitochondria, and / or decreases the number / proportion of immature mitochondria, among the mitochondria of cells treated with the agent, as compared to the number / proportion of immature / mature mitochondria among mitochondria of equivalent cells in the absence of such treatment. A promoter of mitochondrial maturation according to the present disclosure may be identified by a method comprising: culturing a first population of cells in the presence of a putative promoter of mitochondrial maturation; culturing a second population of cells ( / .e. cells of the same type as the first population of cell) in the absence of the putative promoter of mitochondrial maturation, or in the presence of a suitable control agent known not to affect mitochondrial maturation; subsequently analysing the first and second populations of cells, after an appropriate period of time for an effect on mitochondrial maturation to be observed (e.g. 24, 36, 48 or 72 hours), to determine the number / proportion of immature and / or mature mitochondria within cells of the first and second populations of cells.

[0153] It will be appreciated that a promoter of mitochondrial maturation is an agent that yields a greater number / proportion of mature mitochondria and / or a lesser number / proportion of immature mitochondria within cells of the first population of cells, as compared to the number / proportion of immature / mature mitochondria in cells of the second population of cells.

[0154] Promoters of mitochondrial maturation according to the present disclosure include agents that promote the nucleic acid and / or polypeptide expression of, and / or the activity of, a factor whose expression / activity is positively associated with mitochondrial maturation. Promoters of mitochondrial maturation also include agents that promote the nucleic acid and / or polypeptide expression of, and / or the activity of, a factor whose expression / activity is a marker of a mature mitochondrion.

[0155] In some embodiments, a promoter of mitochondrial maturation according to the present disclosure displays one or more of the following: increases the expression or activity of a polypeptide involved in cristae formation ( / .e. a polypeptide that is positively-associated with the formation of cristae); increases the expression or activity of one or more polypeptide(s) of the ETC; increases the expression or activity of F1 / F0 ATP synthase.

[0156] It will be appreciated that a given promoter of mitochondrial maturation may display more than one of the properties recited in the preceding paragraph. A putative promoter of mitochondrial maturation may be evaluated for the properties recited in the preceding paragraph using suitable assays. For example, the assays may be e.g. in vitro assays, optionally cell-based assays or cell-free assays.

[0157] Where assays are cell-based assays, they may comprise treating cells with a given agent in order to determine whether the agent displays one or more of the recited properties. Assays may comprise evaluating the recited properties following treatment of cells separately with a range of quantities / concentrations of a given agent (e.g. a dilution series).

[0158] Promoters of mitochondrial maturation that increase the expression (e.g. nucleic acid and / or polypeptide expression) of a polypeptide involved in cristae formation / one or more polypeptide(s) of the ETC / F1 / F0 ATP synthase may increase the level of RNA encoding the relevant polypeptide. Such promoters may be identified using assays comprising detecting and / or quantifying the level of RNA encoding the relevant polypeptide. Such assays may comprise quantifying RNA encoding the relevant polypeptide by RT- qPCR, northern blot, etc., which are techniques well known to the skilled person. The methods may employ primers and / or probes for the detection and / or quantification of RNA encoding the relevant polypeptide. Such assays may comprise contacting cells in in vitro culture with a putative promoter of mitochondrial maturation, and subsequently (e.g. after an appropriate period of time, i.e. a period of time sufficient for a change in the level of RNA encoding the relevant polypeptide to be observed) measuring the level of RNA encoding the relevant polypeptide. Such assays may further comprise comparing the level of RNA encoding the relevant polypeptide in cells treated with the putative promoter of mitochondrial maturation to the level of RNA encoding the relevant polypeptide detected in a control condition in which cells of the same type are subjected to the same conditions, except that instead of being treated with the putative promoter of mitochondrial maturation they are untreated, or otherwise treated with an appropriate control agent known not to affect the level of RNA encoding the relevant polypeptide.

[0159] In some embodiments, the putative promoter of mitochondrial maturation is provided to the cells in combination with one or more further agents for facilitating introduction of the putative promoter of mitochondrial maturation into the cells, and / or for facilitating uptake of the putative promoter of mitochondrial maturation by the cells. For example, in embodiments wherein a putative promoter of mitochondrial maturation is, or is encoded by, one or more nucleic acids, the cells may be contacted with the nucleic acid(s) and an agent for facilitating introduction of the nucleic acid(s) into the cells, e.g. by transfection or transduction.

[0160] An increase in the level of RNA encoding the relevant polypeptide (i.e. a polypeptide involved in cristae formation, a polypeptide of the ETC, or F1 / F0 ATP synthase) may be a consequence of increased transcription of DNA encoding the relevant polypeptide (e.g. as a consequence of increased assembly and / or activity of factors required fortranscription of DNA encoding the relevant polypeptide), reduced degradation of RNA encoding the relevant polypeptide and / or increased stability of RNA encoding the relevant polypeptide.

[0161] Promoters of mitochondrial maturation that increase the expression (e.g. nucleic acid and / or polypeptide expression) of a polypeptide involved in cristae formation / one or more polypeptide(s) of the ETC / F1 / F0 ATP synthase may increase the level of the relevant polypeptide. Such promoters may be identified using assays comprising detecting and / or quantifying the level of the relevant polypeptide. Such assays may employ antibody or reporter-based methods (western blot, ELISA, immunohisto / cytochemistry, etc.). The methods may employ antibodies specific for the relevant polypeptide. Such assays may comprise contacting cells in in vitro culture with a putative promoter of mitochondrial maturation, and subsequently (e.g. after an appropriate period of time, i.e. a period of time sufficient for a change in the level of the relevant polypeptide to be observed) measuring the level of the relevant polypeptide. Such assays may further comprise comparing the level of the relevant polypeptide in cells treated with the putative promoter of mitochondrial maturation to the level of the relevant polypeptide detected in a control condition in which cells of the same type are subjected to the same conditions, except that instead of being treated with the putative promoter of mitochondrial maturation they are untreated, or otherwise treated with an appropriate control agent known not to affect the level of the relevant polypeptide. An increase in the level of the relevant polypeptide ( / .e. a polypeptide involved in cristae formation, a polypeptide of the ETC, or F1 / F0 ATP synthase) may be a consequence of increased translation of RNA encoding the relevant polypeptide, reduced degradation of the relevant polypeptide and / or increased stability of the relevant polypeptide.

[0162] Promoters of mitochondrial maturation that increase the activity of a polypeptide involved in cristae formation / one or more polypeptide(s) of the ETC / F1 / F0 ATP synthase may be identified using assays comprising detecting and / or quantifying the level of an activity of the relevant polypeptide. Such assays may comprise detecting and / or quantifying a correlate of the relevant activity. For example, detecting and / or quantifying the level of an activity of a polypeptide involved in cristae formation may comprise detecting and / or quantifying the number of cristae and / or cristae density. For example, detecting and / or quantifying the level of an activity of one or more polypeptide(s) of the ETC may comprise detecting and / or quantifying mitochondrial membrane potential. For example, detecting and / or quantifying the level of an activity of F1 / F0 ATP synthase may comprise detecting and / or quantifying the level of ATP. Such assays may comprise contacting cells in in vitro culture with a putative promoter of mitochondrial maturation, and subsequently (e.g. after an appropriate period of time, i.e. a period of time sufficient for a change in the level of the relevant activity to be observed) measuring the level of the relevant activity of the relevant polypeptide. Such assays may further comprise comparing the level of the relevant activity in cells treated with the putative promoter of mitochondrial maturation to the level of the relevant activity detected in a control condition in which cells of the same type are subjected to the same conditions, except that instead of being treated with the putative promoter of mitochondrial maturation they are untreated, or otherwise treated with an appropriate control agent known not to affect the level of the relevant activity.

[0163] In some embodiments, a promoter of mitochondrial maturation according to the present disclosure increases the expression of a polypeptide involved in cristae formation / increases the formation of a respiratory supercomplex / increases the level of RNA encoding a polypeptide involved in cristae formation / increases the level of a polypeptide involved in cristae formation / increases the expression of one or more polypeptide(s) of the ETC (e.g. selected from a polypeptide of complex I, a polypeptide of complex II, a polypeptide of complex III, cytochrome c and a polypeptide of complex IV; e.g. a polypeptide of complex III; e.g. RISP) / increases the level of RNA encoding one or more polypeptide(s) of the ETC (e.g. selected from a polypeptide of complex I, a polypeptide of complex II, a polypeptide of complex III, cytochrome c and a polypeptide of complex IV; e.g. a polypeptide of complex III; e.g. RISP) / increases the level of one or more polypeptide(s) of the ETC (e.g. selected from a polypeptide of complex I, a polypeptide of complex II, a polypeptide of complex III, cytochrome c and a polypeptide of complex IV; e.g. a polypeptide of complex III; e.g. RISP) / increases the expression of F1 / F0 ATP synthase / increases the level of RNA encoding F1 / F0 ATP synthase / increases the level of F1 / F0 ATP synthase / increases the activity of a polypeptide involved in cristae formation / increases the number of cristae / increases cristae density / increases the activity of one or more polypeptide(s) of the ETC (e.g. selected from a polypeptide of complex I, a polypeptide of complex II, a polypeptide of complex III, cytochrome c and a polypeptide of complex IV; e.g. a polypeptide of complex III; e.g. RISP) / increases mitochondrial membrane potential / increases the activity of F1 / F0 ATP synthase / increases the level of ATP to greater than 1 times, e.g. one of >2 times, >3 times, >4 times, >5 times, >10 times, >20 times, >50 times, >100 times, >500 times or >1000 times the level observed in the absence of the promoter of mitochondrial maturation, or in the presence of the same quantity of an appropriate control agent known not to possess such mitochondrial maturation promoting activity, in a given assay.

[0164] In some embodiments, a promoter of mitochondrial maturation according to the present disclosure is a promoter of mitochondrial translation.

[0165] Herein, a ‘promoter of mitochondrial translation’ refers to any agent (e.g. any compound / molecule) capable of promoting translation in the mitochondria. That is, a promoter of mitochondrial translation promotes translation of nucleic acid to a polypeptide in the mitochondria.

[0166] In some embodiments, a promoter of mitochondrial translation according to the present disclosure displays one or more of the following: increases the level of a polypeptide involved in cristae formation; increases the level of one or more polypeptide(s) of the ETC; increases the level of F1 / F0 ATP synthase.

[0167] Promoters of mitochondrial translation may be identified using assays comprising detecting and / or quantifying the level of one or more polypeptides that are translated in the mitochondria. For example, such assays may comprise detecting and / or quantifying the level of a polypeptide involved in cristae formation, or detecting and / or quantifying the level of one or more polypeptide(s) of the ETC (e.g. selected from a polypeptide of complex I, a polypeptide of complex II, a polypeptide of complex III, cytochrome c and a polypeptide of complex IV; e.g. a polypeptide of complex III; e.g. RISP), or detecting and / or quantifying the level of F1 / F0 ATP synthase. The methods may employ antibodies specific for the relevant polypeptide. Such assays may comprise contacting cells in in vitro culture with a putative promoter of mitochondrial translation, and subsequently (e.g. after an appropriate period of time, i.e. a period of time sufficient for a change in the level of the relevant polypeptide to be observed) measuring the level of the relevant polypeptide. Such assays may further comprise comparing the level of the relevant polypeptide in cells treated with the putative promoter of mitochondrial translation to the level of the relevant polypeptide detected in a control condition in which cells of the same type are subjected to the same conditions, except that instead of being treated with the putative promoter of mitochondrial translation they are untreated, or otherwise treated with an appropriate control agent known not to affect the level of the relevant polypeptide.

[0168] In some embodiments, a promoter of mitochondrial translation according to the present disclosure increases the level of a polypeptide involved in cristae formation / increases the level of one or more polypeptide(s) of the ETC (e.g. selected from a polypeptide of complex I, a polypeptide of complex II, a polypeptide of complex III, cytochrome c and a polypeptide of complex IV; e.g. a polypeptide of complex III; e.g. RISP) / increases the level of F1 / F0 ATP synthase to greater than 1 times, e.g. one of >2 times, >3 times, >4 times, >5 times, >10 times, >20 times, >50 times, >100 times, >500 times or >1000 times the level observed in the absence of the promoter of mitochondrial maturation, or in the presence of the same quantity of an appropriate control agent known not to possess such mitochondrial translation promoting activity, in a given assay. In some embodiments, promoting mitochondrial maturation in accordance with the present disclosure comprises subjecting cells to glucose deprivation and / or endoplasmic reticulum (ER) stress. Glucose deprivation and ER stress have recently been demonstrated to promote the formation of cristae, respiratory supercomplex formation and ATP production, through activation of protein kinase R (PKR)-like ER kinase (PERK) - see Balsa et al., Mol Cell. (2019) 74(5): 877-890. e6, which is hereby incorporated by reference in its entirety.

[0169] Cells may be subjected to glucose deprivation by culture in cell culture medium comprising low levels of glucose, or by culture in cell culture medium lacking glucose. In some embodiments, cells may be subjected to glucose deprivation by culture in cell culture medium comprising galactose instead of glucose.

[0170] In some embodiments, promoting mitochondrial maturation comprises manipulating cellular metabolism to favour ATP production through oxidative phosphorylation over ATP production through glycolysis.

[0171] In some embodiments, promoting mitochondrial maturation in accordance with the present disclosure comprises culturing cells under conditions in which glycolysis is inhibited. In some embodiments, promoting mitochondrial maturation in accordance with the present disclosure comprises culturing cells under conditions in which oxidative phosphorylation is promoted. In some embodiments, promoting mitochondrial maturation in accordance with the present disclosure comprises culturing cells under conditions in which glycolysis is inhibited and oxidative phosphorylation is promoted.

[0172] In some embodiments, promoting mitochondrial maturation in accordance with the present disclosure comprises culturing cells in cell culture medium comprising galactose. In some embodiments, promoting mitochondrial maturation in accordance with the present disclosure comprises culturing cells in cell culture medium substantially lacking glucose. In some embodiments, promoting mitochondrial maturation comprises culturing cells in cell culture medium comprising galactose and substantially lacking glucose.

[0173] Cells cultured in cell culture medium comprising galactose and lacking glucose are forced to rely on oxidative phosphorylation in the mitochondria for the generation of ATP, rather than glycolysis in the cytoplasm. In some embodiments, promoting mitochondrial maturation in accordance with the present disclosure comprises subjecting cells to conditions promoting ATP production through oxidative phosphorylation and / or subjecting cells to conditions inhibiting ATP production through glycolysis.

[0174] In some embodiments, a promoter of mitochondrial maturation according to the present disclosure promotes oxidative phosphorylation over glycolysis, in a cell. In some embodiments, a promoter of mitochondrial maturation according to the present disclosure inhibits glycolysis. In some embodiments, a promoter of mitochondrial maturation according to the present disclosure promotes oxidative phosphorylation. In some embodiments, a promoter of mitochondrial maturation according to the present disclosure inhibits glycolysis and promotes oxidative phosphorylation. In some embodiments, a promoter of mitochondrial maturation according to the present disclosure is a promoter of PERK. A promoter of PERK may also be referred to as an activator, agonist or upregulator of PERK. In some embodiments, a promoter of PERK may upregulate the nucleic acid and / or polypeptide expression of, and / or the activity of, PERK. Promoters of PERK include DHBDC and CCT020312 (see Balsa et al., Mol Cell. (2019) 74(5): 877-890. e6).

[0175] Methods and uses of the disclosure

[0176] The present disclosure provides methods for promoting self-renewal of muscle stem cells (e.g. myoblasts) in cell culture, comprising inhibiting mitochondrial maturation in muscle stem cells in cell culture. The present disclosure also provides the use of an inhibitor of mitochondrial maturation to promote the self-renewal of muscle stem cells (e.g. myoblasts).

[0177] It will be appreciated that ‘self-renewal’ refers to the production of progeny cells of the same cell type as the parental cell. The progeny cells preferably possess the same phenotype as the parental cell. Accordingly, ‘self-renewal’ of a muscle stem cell refers to the generation of muscle stem cells from a muscle stem cell.

[0178] The present disclosure also provides methods for inhibiting the differentiation of muscle stem cells (e.g. myoblasts) in cell culture, comprising inhibiting mitochondrial maturation in muscle stem cells in cell culture. In some embodiments, the methods are for inhibiting the differentiation of muscle stem cells (e.g. myoblasts) to myocytes and / or developmental successors thereof (e.g. myotubes, myofibers). The present disclosure also provides the use of an inhibitor of mitochondrial maturation to inhibit the differentiation of muscle stem cells (e.g. myoblasts), e.g. to myocytes and / or developmental successors thereof (e.g. myotubes, myofibers).

[0179] In some embodiments, the methods are for inhibiting the spontaneous differentiation of muscle stem cells. Spontaneous differentiation of primary muscle stem cells to their developmental successors presents a challenge for the expansion of primary muscle stem cell populations in cell culture, and the inventors have determined that such spontaneous differentiation of primary muscle stem cells can be inhibited through inhibition of mitochondrial maturation.

[0180] Numbers / proportions of cells of given cell types within a heterogeneous population of cells can be evaluated using methods well known to the skilled person.

[0181] In some embodiments, culture of muscle stem cells (e.g. myoblasts) in which mitochondrial maturation is inhibited (e.g. as consequence of treatment with an inhibitor of mitochondrial maturation) in accordance with the present disclosure yields a greater number / proportion of muscle stem cells (e.g. myoblasts) among cells of the culture (after a given period of time), as compared to the number / proportion of such cells among cells of a culture of cells of the same type in which mitochondrial maturation is not inhibited, but otherwise cultured under the same conditions and for the same period of time. In some embodiments, culture of muscle stem cells (e.g. myoblasts) in which mitochondrial maturation is inhibited (e.g. as consequence of treatment with an inhibitor of mitochondrial maturation) in accordance with the present disclosure yields a lower number / proportion of myocytes and / or developmental successors thereof (e.g. myotubes, myofibers) among cells of the culture (after a given period of time), as compared to the number / proportion of such cells among cells of a culture of cells of the same type in which mitochondrial maturation is not inhibited, but otherwise cultured under the same conditions and for the same period of time.

[0182] Culture of muscle stem cells in accordance with methods of the present disclosure preferably yields cells retaining sternness. Nucleic acid and / or polypeptide expression of Pax7 by muscle stem cells may be a marker of sternness. In some embodiments, muscle stem cells experiencing inhibition of mitochondrial maturation express Pax7. In some embodiments, cells obtained by culture of muscle stem cells in accordance with methods of the present disclosure express Pax7.

[0183] Accordingly, the present disclosure provides methods for culturing muscle stem cells (e.g. myoblasts) while retaining sternness (e.g. retaining expression of Pax7), comprising inhibiting mitochondrial maturation in muscle stem cells in cell culture. The present disclosure also provides the use of an inhibitor of mitochondrial maturation in a method for culturing muscle stem cells (e.g. myoblasts) while retaining sternness (e.g. retaining expression of Pax7).

[0184] Culture of muscle stem cells in accordance with methods of the present disclosure preferably yields cells retaining their proliferative capacity.

[0185] In some embodiments, cells obtained by culture of muscle stem cells in accordance with methods of the present disclosure have similar proliferative capacity as the proliferative capacity of muscle stem cells not cultured under such conditions. For example, cells obtained by culture of muscle stem cells in accordance with methods of the present disclosure may have similar proliferative capacity as muscle stem cells prior to such culture.

[0186] The proliferative capacity of a given cell / population of cells may be evaluated e.g. by culturing the cells in vitro, and determining the number / proportion of cells in the culture after a given period of time. In some embodiments, muscle stem cells (e.g. myoblasts) obtained by a method according to the present disclosure which are then further cultured in vitro under given conditions yield a number / proportion of cells that is > 0.5 times and < 2 times, e.g. one of > 0.75 times and < 1 .5 times, > 0.8 times and < 1 .25 times, or > 0.9 times and < 1.1 times the number / proportion of cells obtained by the culture under the same given conditions of equivalent muscle stem cells (e.g. myoblasts) that have not been obtained by a method according to the present disclosure.

[0187] In some embodiments, culture of muscle stem cells (e.g. myoblasts) in which mitochondrial maturation is inhibited (e.g. as consequence of treatment with an inhibitor of mitochondrial maturation) in accordance with the present disclosure yields a greater number / proportion of cells expressing Pax7 among cells of the culture (after a given period of time), as compared to the number / proportion of such cells among cells of a culture of cells of the same type in which mitochondrial maturation is not inhibited, but otherwise cultured under the same conditions and for the same period of time.

[0188] In some embodiments, culture of muscle stem cells (e.g. myoblasts) in which mitochondrial maturation is inhibited (e.g. as consequence of treatment with an inhibitor of mitochondrial maturation) in accordance with the present disclosure yields a lower number / proportion of cells lacking expression of Pax7 among cells of the culture (after a given period of time), as compared to the number / proportion of such cells among cells of a culture of cells of the same type in which mitochondrial maturation is not inhibited, but otherwise cultured under the same conditions and for the same period of time.

[0189] Culture of muscle stem cells in accordance with methods of the present disclosure preferably yields cells having increased clonogenicity. Clonogenicity refers to the ability of a given cell to divide and grow into a colony of cloned cells. Methods for evaluating clonogenicity are well known to the skilled person, and are described e.g. in Franken et al., Nature Protocols (2006) 1 :2315-2319, which is hereby incorporated by reference in its entirety.

[0190] Accordingly, the present disclosure also provides methods for increasing the clonogenicity of muscle stem cells (e.g. myoblasts), comprising inhibiting mitochondrial maturation in muscle stem cells in cell culture. The present disclosure also provides the use of an inhibitor of mitochondrial maturation to increase clonogenicity of a muscle stem cell (e.g. a myoblast).

[0191] In some embodiments, muscle stem cells experiencing inhibition of mitochondrial maturation have increased clonogenicity as compared to equivalent cells not experiencing such inhibition of mitochondrial maturation. In some embodiments, muscle stem cells cultured in the presence of an inhibitor of mitochondrial maturation have increased clonogenicity as compared to equivalent cells not cultured in the presence of an inhibitor of mitochondrial maturation. In some embodiments, cells obtained by culture of muscle stem cells in accordance with methods of the present disclosure have increased clonogenicity.

[0192] In some embodiments, culture of muscle stem cells (e.g. myoblasts) in which mitochondrial maturation is inhibited (e.g. as consequence of treatment with an inhibitor of mitochondrial maturation) in accordance with the present disclosure yields cells having increased clonogenicity (after a given period of time), as compared to cells of a culture of cells of the same type in which mitochondrial maturation is not inhibited, but otherwise cultured under the same conditions and for the same period of time.

[0193] The present disclosure provides methods comprising culturing muscle stem cells (e.g. myoblasts).

[0194] In some embodiments, the methods comprise obtaining or isolating muscle stem cells (e.g. myoblasts). Muscle stem cells may be obtained / isolated from muscle tissue. The muscle stem cells may be obtained / isolated from a muscle tissue biopsy from a live subject, or may be obtained from muscle tissue of a dead (e.g. slaughtered non-human animal) subject. In some embodiments, obtaining or isolating muscle stem cells comprises mechanical and / or chemical (e.g. enzymatic) processing of muscle tissue to obtain a single cell suspension, or small clusters of cells.

[0195] In some embodiments, the methods comprise enriching muscle stem cells from a heterogeneous population of cells obtained / isolated from muscle tissue. In some embodiments, muscle stem cells may be isolated / separated from other cell types by cell sorting techniques (e.g. MACS, FACS), on the basis of expression of a muscle stem cell marker. In some embodiments, a muscle stem cells may be enriched / isolated / separated from other cell types on the basis of positive selection for a7 integrin expression.

[0196] In some embodiments, the methods comprise depleting cell types other than muscle stem cells from a heterogeneous population of cells obtained / isolated from muscle tissue. In some embodiments, cell types other than muscle stem cells may be depleted by negative selection using cell sorting techniques (e.g. MACS, FACS), on the basis of expression of markers of cells other than muscle stem cells.

[0197] In some embodiments, muscle stem cells may be obtained / isolated essentially as described in Example 1 .2 herein.

[0198] The methods comprise culturing muscle stem cells (e.g. myoblasts) to increase their number.

[0199] Suitable culture conditions for the culture of muscle stem cells / myoblasts will be apparent to a person skilled in the art. Suitable conditions are described e.g. in Choi et al., Compr Rev Food Sci Food Saf. (2021) 20(1):429-457 and Kadim et al., J Integ. Agriculture (2015) 14(2): 222-233, both of which are hereby incorporated by reference in their entirety.

[0200] The cells may be cultured in cell culture medium comprising amino acids, vitamins, inorganic salts and sugars. In some embodiments, the cell culture medium comprises amino acids selected from: L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cystine / L-cysteine, L-glutamic acid, L-glutamine, glycine, L- histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine. In some embodiments, the cell culture medium comprises vitamins and / or vitaminoids selected from: D-biotin, choline chloride, D-calcium pantothenate, folic acid, myoinositol, niacinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, and vitamin B12. In some embodiments, the cell culture medium comprises further components selected from: calcium chloride, hypoxanthine, ferric nitrate, linoleic acid, putrescine hydrochloride, sodium pyruvate, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, sodium phosphate monobasic, thioctic acid and thymidine. In some embodiments, the cell culture medium comprises D-glucose.

[0201] In some embodiments, the cell culture medium comprises growth factors. In some embodiments, the cell culture medium comprises insulin-like growth factor (IGF), fibroblast growth factors (e.g. FGF2), and / or transforming growth factor-p (TGF-p). Growth factors may be provided in the form of one or more recombinantly-produced growth factors, or in the form of a cell culture medium additive comprising growth factors. Commonly used cell culture medium additives comprising growth factors include e.g. fetal bovine serum (FBS). However, in some embodiments, it may be desirable not to employ animal-derived growth factor-containing cell culture medium additives such as FBS.

[0202] In some embodiments, the cell culture medium comprises one or more antibiotics to prevent bacterial contamination, e.g. penicillin-streptomycin.

[0203] In some embodiments, the cell culture medium is a cell culture medium suitable for the culture of mammalian cells. Such cell culture media include Dulbecco's Modified Eagle Medium (DMEM), F-12 medium and DMEM / F12 medium.

[0204] In preferred embodiments, the cell culture medium is suitable cell culture medium for the culture of cells for the production of a product for consumption, e.g. by humans.

[0205] It will be appreciated that the cells are cultured under suitable environmental conditions. The cells may be cultured at 35.5°C to 37.5°C, e.g. at one of about 35.5°C, about 36°C, about 36.5°C, about 37°C, or about 37.5°C. The cells may be cultured in 4% to 10% CO2, e.g. 5% to 8% CO2. The cells may be cultured e.g. at >90% humidity, e.g. about 95% humidity. The cells may be cultured without agitation, or with agitation. Agitation may be at 75 rpm to 175 rpm, e.g. 90 rpm to 130 rpm, e.g. about 110 rpm. The pH of the cell culture may be between 6.8 to 7.4, e.g. one of about 6.8, 6.9, 7.0, 7.1 , 7.2, 7.3 or 7.4. The pH of the cell culture may be between 7.2 and 7.4.

[0206] Cell culture may be performed in a bioreactor provided with an appropriate supply of nutrients, air / oxygen and / or growth factors. Bioreactors may monitor and control environmental conditions such as pH, oxygen, flow rates into and out of, and agitation within the vessel such that optimum conditions are provided for the cells being cultured.

[0207] The culture may be a continuous culture, with a continuous flow of cell culture medium into, and a continuous flow of cultured cells from, the cell culture vessel. In some embodiments, the culture may be a batch culture, employing a closed system and a finite amount of cell culture medium. In some embodiments, the culture may be a fed-batch culture, in which cell culture medium is supplied to the cell culture vessel during the culture, but wherein unlike a continuous culture, material is not removed from the cell culture vessel during the course of cell culture.

[0208] In some embodiments, the methods comprise adding one or more components to the cell culture during the period of culture. In some embodiments, the methods comprise adding cell culture medium to the cell culture during the period of culture. In some embodiments, the methods comprise adding nutrients to the cell culture during the period of culture. In some embodiments, the methods comprise adding amino acids to the cell culture during the period of culture.

[0209] In some embodiments, muscle stem cells may be cultured essentially as described in Example 1 .2 herein. In some embodiments, the methods comprise differentiating muscle stem cells (e.g. myoblasts), e.g. to myocytes and / or developmental successors thereof (e.g. myotubes, myofibers). In some embodiments, differentiating muscle stem cells (e.g. myoblasts), e.g. to myocytes and / or developmental successors thereof (e.g. myotubes, myofibers) comprises culturing the muscle stem cells in cell culture medium comprising one or more factors inducing differentiation to such cell types. For example, in some embodiments differentiating muscle stem cells (e.g. myoblasts), e.g. to myocytes and / or developmental successors thereof (e.g. myotubes, myofibers) comprises culturing cells in cell culture medium comprising insulin, transferrin and / or selenium.

[0210] In some embodiments, the methods comprise culturing cells under conditions suitable for formation of muscle tissue. In some embodiments, the methods comprise culturing myotubes / myofibers (and / or developmental precursors thereof) on a scaffold. A scaffold may be made of any suitable material, and may facilitate the three-dimensional organisation of myotubes / myofibers to form a super-cellular structure resembling that of muscle tissue. The scaffold may be biodegradable. The scaffold may be edible.

[0211] In some embodiments, the methods comprise providing electrical and / or mechanical stimulation to muscle tissue produced by the methods of the present disclosure. Such electrical stimulation induces muscle contraction, while the mechanical stimulation helps to improve the texture and overall quality of the muscle tissue.

[0212] It is contemplated to employ muscle tissue and populations of cells produced by the methods of the present disclosure in the production of products for ingestion. Accordingly, the present disclosure provides the use of muscle tissue and / or a population of cells obtained or obtainable by a method according to the present disclosure in the production of a product for ingestion. In some embodiments, the method according to the present disclosure is a method for producing cultivated meat. In some embodiments, the methods comprise processing muscle tissue to form a product for ingestion.

[0213] It is also contemplated to employ muscle tissue and populations of cells produced by the methods of the present disclosure in methods of medical treatment and prophylaxis, and also cosmetic applications. Accordingly, the present disclosure provides the use of muscle tissue and / or a population of cells obtained or obtainable by a method according to the present disclosure in a method of medical treatment or prophylaxis, or a cosmetic method. For example, the muscle tissue may be used as a tissue graft. In accordance with such embodiments, the muscle stem cells may be human muscle stem cells. In some embodiments the muscle stem cells are obtained / de rived from the subject to which the graft is to be provided ( / .e. the graft is an autograft).

[0214] Kits

[0215] The present disclosure also provides kits of parts.

[0216] In some aspects and embodiments, a kit of parts according to the present disclosure comprises (i) an inhibitor of mitochondrial maturation, and (ii) a promoter of mitochondrial maturation. Kits of parts according to the present disclosure may comprise a predetermined quantity of articles according to (i) and / or (ii), as described in the preceding paragraph. In some embodiments, articles according to (i) and / or (ii) are provided in containers (e.g. in vials or bottles). The kit may provide articles according to (i) and / or (ii) together with instructions (e.g. a protocol) as to how to employ them in accordance with a method described herein.

[0217] In some embodiments, the kit of parts further comprises reagents, buffers and / or standards required for execution of a method according to the present disclosure. Kits according to the present disclosure may include instructions for use, e.g. in the form of an instruction booklet or leaflet. The instructions may include a protocol for performing any one or more of the methods described herein.

[0218] The manufacture of kits of parts according to the present disclosure preferably follows standard procedures which are known to the person skilled in the art.

[0219] ***

[0220] The present disclosure includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

[0221] The section headings used herein are for organisational purposes only and are not to be construed as limiting the subject matter described.

[0222] Aspects and embodiments of the present disclosure will now be illustrated, by way of example, with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0223] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word ‘comprise,’ and variations such as ‘comprises’ and ‘comprising,’ will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0224] It must be noted that, as used in the specification and the appended claims, the singular forms ‘a,’ ‘an,’ and ‘the’ include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from ‘about’ one particular value, and / or to ‘about’ another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent ‘about,’ it will be understood that the particular value forms another embodiment.

[0225] Methods described herein may preferably be performed in vitro. The term ‘in vitro' is intended to encompass procedures performed with cells in culture whereas the term ‘in vivo’ is intended to encompass procedures with / on intact multi-cellular organisms. Brief Description of the Figures

[0226] Embodiments and experiments illustrating the principles of the present disclosure will now be discussed with reference to the accompanying figures.

[0227] Figure 1a. Shows satellite cells on extensor dig ito rum longus (EDL) myofiber from pax7CreERT2:R26Cox8aDendramouse showing differential mitochondrial content. Scale bar = 6 pm.

[0228] Figure 1b. TEM analysis of mitochondria from Quiescent, freshly isolated MuSCs and myoblast at 72h activation. Scale bar = 1 pm.

[0229] Figure 1c. Quantification of cristae density (calculated as number of cristae / mitochondrial area) from TEM of MuSCs at different time points of ex vivo activation (Freshly isolated (Fl), 24h, 48h N=3; 36h N=2;72h N=3; >200 mitochondria / timepoint). One-way ANOVA with Tukey’s multiple comparisons post hoc test were performed. P values are shown in relevant panels; P-values < 0.05 were considered significant.

[0230] Figure 1d. Measurement of oxygen consumption rate (OCR) during ex vivo activation of MuSCs in steady state for 90h from plating (N=4).

[0231] Figure 1e. (Top) Schematic showing temporal labelling strategy to chronologically label mitochondria from Fl MuSCs from pax7CreERT2:R26Cox8aDendramodel. (Bottom) Representative immunostaining of Hsp60 to label total mitochondria (left panel) and HPG incorporation (right panel), visualised with fluorescent azide by click it reaction in MuSCs at 36H (upper panel) and 72H (lower panel) of activation.

[0232] Figure 1f. Quantitative assessment of mitochondrial translation in HPG + CHI treated MuSCs at different time points of activation (24H, 36H, 48H, 72H). Relative fluorescent intensities normalised to total mitochondrial content (HSP60 intensity) from 50-250 segmented mitochondrial domains (N=3) are shown from each time point. One-way ANOVA with Tukey’s multiple comparisons post hoc test were performed. P values are shown in relevant panels; P-values < 0.05 were considered significant.

[0233] Figure 1g. (Top) Schematic showing temporal labelling strategy to chronologically label mitochondria from Fl MuSCs from pax7c'eERT2:p26SPAPO:rip25model. (Bottom) (Old mitochondria: Sir647; New mitochondria: TMR-star). Freshly-isolated satellite cells from pax7CreERT2:p26SNAPomp25mice were labelled with a flourescent snap substrate (Sir647), to visualise mitochondria. 24h after ex vivo activation, newly- synthesised mitochondria were visualised with a different fluorescent snap substrate (TMR-star).

[0234] Figure 1h. Correlation analysis of Pax7 level of MuSCs to mitochondrial biogenesis, quantified as New / Old mitochondria at 24h activation.

[0235] Figure 1i. (Top) strategy for age-specific mitochondria labelling for first cell division following MuSC activation (N = 41 cells pairs). (Bottom left) Immunostaining microscopy images of sister cell pairs, showing staining for tubulin and Pax7 (scale bar 2 pm). (Bottom right) Correlation analysis of age- selective segregation of mitochondria during anaphase of first mitotic cycle following MuSC activation. Each data point represents a sister cell of a division pair (n = 41 cell pairs) with age of the apportioned mitochondria represented as % inherited from mother cell.

[0236] Figure 1j. (Top) Schematic showing experimental strategy. (Bottom) Fluorescence microscopy illustrating age-selective asymmetric apportioning of mitochondrial domains in a dividing cell pair at 72h activation (scale bar = 2 pm).

[0237] Figure 1 k. Quantification of age-selective apportioning of mitochondria between daughter cells of divided cells pairs at 72h activation. Each data point represents a sister cell of a division pair (n = 56 cell pairs) with age selectively apportioned mitochondria represented as % inherited from mother cell.

[0238] Figure 2a. (Top) Schematic showing strategy for age-specific mitochondria labelling. (Bottom, panels 1-3) Correlative Light and Electron Microscopy (CLEM) analysis of daughter cell pairs that have age- selectively apportioned mitochondria. (Bottom, panel 4) 3D rendered segmented Z-stacks from serial section TEM of mitochondria from asymmetric cell pairs.

[0239] Figure 2b. Quantification of CLEM data, showing mitochondrial properties in old vs young mitochondria. Data analysed using non-parametric Mann-Whitney U test. P values are shown in relevant panels and P- values < 0.05 were considered significant.

[0240] Figure 2c. (Top) Schematic showing strategy to isolate proliferating cells with differential enrichment of mitochondrial age classes for FACS analysis. (Bottom left and middle) FACS plots. Cells with diluted CellTrace violet dye are considered proliferating. The 10% of cells most enriched for the old mitochondrial SNAP label and 10% of cells most depleted for the old mito SNAP label were sorted. (Bottom right) Representative images of a dividing cell pair (OldMitoHi and OldMitoLo cell, scale bar = 10 pm).

[0241] Figure 2d. OldMitoHi and OldMitoLo cells were immunostained for Pax7 and MyoD and quantified as percentage of sorted cell population, analysed immediately post sorting. Data analysed using one-way ANOVA with Tukey’s multiple comparisons post hoc test. P values are shown in relevant panels; P-values

[0242] < 0.05 were considered significant.

[0243] Figure 2e. Immunostaining for Pax7 and Myosin Heavy Chain (MyHC), 48h post sorting. Nuclei stained with Hoechst. Scale bar = 50 pm.

[0244] Figure 2f. Differentiation of OldMitoHi vs OldMitoLo cells, as measured by myogenic fusion index. N = 14. Data analysed using two-tailed unpaired Student’s t-test. P values are shown in relevant panels; P-values

[0245] < 0.05 were considered significant.

[0246] Figure 2g. Differentiation of OldMitoHi vs OldMitoLo cells, as measured by sternness (percentage of Pax7+ cells). N = 8. Data analysed using two-tailed unpaired Student’s t-test. P values are shown in relevant panels; P-values < 0.05 were considered significant. Figure 2h. Schematic showing experimental strategy for Figures 2e-g.

[0247] Figure 3a. (Top) Schematic showing experimental strategy for Figure 3a and b. (Bottom) Fluorescence microscopy illustrating mitochondrial structural changes in control and Cap50 (chloramphenicol 50 pg / mL) treated MuSCs at 72hr activation.

[0248] Figure 3b. (Left) TEM analysis showing differences in cristae density in control and Cap50-treated MuSCs at 72h activation. (Right) Quantification of cristae density observed in control and Cap50 treated MuSCs at 72h activation. Data analysed using two-tailed unpaired Student’s t-test. P values are shown in relevant panels; P-values < 0.05 were considered significant.

[0249] Figure 3c. Mitochondrial membrane potential of unstained, control (40h) (72h), and Cap50-treated (72h) MuSCs.

[0250] Figure 3d. Quantification of OCR at 24h and 72h activation in control and Cap50-treated MuSCs. Data analysed using one-way ANOVA with Tukey’s multiple comparisons post hoc test. P values are shown in relevant panels; P-values < 0.05 were considered significant.

[0251] Figure 3e. Quantification of the extracellular acidification rate (ECAR)ZOCR at 72h activation in control and Cap50-treated MuSCs. Data analysed using two-tailed unpaired Student’s t-test. P values are shown in relevant panels; P-values < 0.05 were considered significant.

[0252] Figure 3f. qRT-PCR analysis of the expression of myogenin in control and Cap50-treated cells at 24h, 48h, and 72h of ex vivo activation.

[0253] Figure 3g. (Left) Control and Cap50 treated cells stained with EdU and Hoechst , and immunostained for Myogenin and Pax7. (Right) Quantification showing percentage of EdU positive cells at 5 days activation. Data analysed using two-tailed unpaired Student’s t-test. P values are shown in relevant panels; P-values < 0.05 were considered significant.

[0254] Figure 3h. Snapshot of timelapse imaging showing cell division kinetics in control and Cap50-treated MuSCs. Demonstrates the lack of cell differentiation in Cap50-treated cells (bottom panel) compared to control (top panel) after 90h activation.

[0255] Figure 3i. Shows change in cell number over activation time in control (upper line) and Cap50-treated (lower line) MuSCs. Data analysed using two-tailed unpaired Student’s t-test. P values are shown in relevant panels; P-values < 0.05 were considered significant.

[0256] Figure 3j. Trajectory inference of scRNA-seq data of Cap50-treated cells on de Micheli trajectory based on pseudotime progression. Primary cells (including Cap50-treated cells) highlighted on the trajectory (lower panel). Figure 4a. Analysis of the metabolomics of U-[13C]-glucose to measure carbon flux in control (Cont), Cap50-treated (Cap), and rescue (cells released from mitochondrial translation inhibition, Res) cells, using isotope labelling. Data presented as mean ± SD. Data analysed using one-way ANOVA with Tukey’s multiple comparisons post hoc test. P values are shown in relevant panels; P values < 0.05 were considered significant.

[0257] Figure 4b. Analysis of the metabolomics of U-[13C]-glutamine to measure carbon flux in control (Cont), Cap50-treated (Cap), and rescue (Res) cells. Data presented as mean ± SD. Data analysed using oneway ANOVA with Tukey’s multiple comparisons post hoc test. P values are shown in relevant panels; P values < 0.05 were considered significant.

[0258] Figure 4c. Shows the ratio of NADH / NAD in control (Con), Cap50-treated (Cap50), and rescue (R24h) cells. Data presented as mean ± SD. Data analysed using one-way ANOVA with Tukey’s multiple comparisons post hoc test. P values are shown in relevant panels; P values < 0.05 were considered significant.

[0259] Figure 4d. qRT-PCR analysis for the expression of Pax7, MyoD, and Myogenin in control (Cont), Cap50- treated (Cap), and rescue (Res) cells. Data presented as mean ± SD. Data analysed using one-way ANOVA with Tukey’s multiple comparisons post hoc test. P values are shown in relevant panels; P values < 0.05 were considered significant.

[0260] Figure 4e. Control, Cap50-treated and rescue cells immunostained for MyHC. Nuclei stained with Hoechst.

[0261] Figure 4f. Schematic showing experimental strategy for testing cells in control, Cap50-treated, and galactose-treated conditions over 40-70h activation.

[0262] Figure 4g. OCR measurements overtime of activation (40-70h) in Control, Cap50-treated, and Galactose-treated cells.

[0263] Figure 4h. qRT-PCR analysis for the expression of myogenin in control, Cap50-treated (MitoTB), galactose-treated, and galactose + Cap50-treated (Gal + MitoTB) cells, at 60h activation.

[0264] Figure 4i. Control, Cap50-treated, and Galactose-treated cells immunostained for Pax7 and MyHC at 4 days activation. Nuclei stained with Hoechst.

[0265] Figure 4j. Shows the differentiation potential, as measured by myogenic fusion index, of control (Con), Cap50-treated (Cap50), and galactose-treated (Gal) cells. Data presented as mean ± SD. Data analysed using one-way ANOVA with Tukey’s multiple comparisons post hoc test. P values are shown in relevant panels; P values < 0.05 were considered significant. Figure 4k. Differentiation of control (Con), Cap50-treated (Cap50) and galactose-treated (Gal) cells, as measured by sternness (Pax7 intensity). Cells were immunostained for nuclei (Hoechst) and Pax7. Pax7 intensity was measured using Tonga software as described previously (https: / / www.frontiersin.org / articles / 10.3389 / fcomp.2022.777458 / full). To quantify Pax7 positive cells, nuclei were segmented using the Hoechst channel and a threshold was set to determine positive Pax7 staining. Data presented as mean ± SD. Data analysed using one-way ANOVA with Tukey’s multiple comparisons post hoc test. P values are shown in relevant panels; P values < 0.05 were considered significant.

[0266] Figure 5a. Schematic showing experimental strategy for muscle injury and in vivo, intramuscular SNAP labelling strategy in pax-7CreERT2:R26SNAPomp25mice.

[0267] Figure 5b. Immunostaining of regenerating muscle at day 3 post-injury for OldMito and Laminin. Nuclei stained with Hoechst. (Top left) Uninjured muscle with SNAP-labelled satellite cell.

[0268] Figure 5c. Immunostaining of regenerating muscle at day 5 post-injury for OldMito, Pax7 and MyHC. Nuclei stained with Hoechst. Pax7 cells contiguous to nascent myofiber (dotted boundary) have less OldMito label.

[0269] Figure 5d. (Left) Quantification of OldMito SNAP label intensity between Pax7 cells contiguous and noncontiguous to nascent myofiber. (Right) Representative images of cells immunostained with OldMito, Pax7, and Hsp60, showing the Pax7 classes. Data presented as mean ± SD. Data analysed using, two- tailed unpaired Student’s t-test. P values are shown in relevant panels; P-values < 0.05 were considered significant.

[0270] Figure 5e. Immunostaining of regenerating muscle at day 5 post-injury for OldMito, Pax7, Uqcfsl and Hsp60.

[0271] Figure 5f. Differential expression of Uqcrfs1 / Hsp60 in Pax7+ cells from day 5 post-injury (D5PI).

[0272] Figure 5g. (Right) Differential expression of Uqcrfs1 / Hsp60 in muscle satellite cells and myonuclei. (Left) Representative images of cells immunostained with Pax7, Hsp60 and Uqcrfsl .

[0273] Figure 6a. Fluorescence microscopy analysis of satellite cells on EDL myofiber from Pax7CreERT2:R26SNAPomp25mouse showing mitochondrial localisation to cellular projections (white arrow head). Scale bar = 3 pm.

[0274] Figure 6b. TEM image of satellite cells on myofiber. Enlarged inset shows representative mitochondria.

[0275] Figure 6c. TEM analysis showing the difference in cristae width between freshly isolated and activated (48h) MuSCs. Figure 6d. (Left) Immunostaining of freshly isolated (Fl) MuSCs for Hsp60, Ndufa9, Uqcrfsl and ATP5a, Nuclei stained with Hoechst. Scale bar = 4 pm. (Right) Quantification of ETC complex subunits. Data analysed using one-way ANOVA with Tukey’s multiple comparisons post hoc test. Data are presented as means ± SD. P values are shown in relevant panels; P-values < 0.05 were considered significant.

[0276] Figure 6e. Schematic depicting CRE mediated recombination driving expression of Rosa26-SNAPOmp25reporter line.

[0277] Figure 6f. Fluorescence microscopy showing chronological labelling of mitochondria at 24h ex vivo activation in pax7CreERT2:R26SNAPomp25model (Old mitochondria: Sir647; New mitochondria: Tmr star; (nuclear -Pax7 immunostaining). Freshly-isolated satellite cells from pax7CreERT2:R26SNAPomp25mice were labelled with a flourescent snap substrate (Sir647), to visualise mitochondria. 24h after ex vivo activation, newly-synthesised mitochondria were visualised with a different fluorescent snap substrate (TMR-star).

[0278] Figure 6g. (Top) Schematic showing experimental strategy. (Bottom) Fluorescence microscopy showing tubulin and symmetric and asymmetric distribution of old mitochondria.

[0279] Figure 6h. (Top) Schematic showing experimental strategy. (Bottom) Snapshots taken during timelapse imaging of asymmetric mitochondrial apportioning in dividing cells. White arrows point to midbody.

[0280] Figure 6i. SNAP label intensity of old (upper liner) and young (lower line) mitochondria overtime in dividing cells.

[0281] Figure 7a. Correlative Light and Electron Microscopy (CLEM) images of MuSCs that have intracellular age-selective segregation of mitochondria (left panel scale bar = 10 pm).

[0282] Figure 7b. Correlative Light and Electron Microscopy (CLEM) images of MuSCs that have selective enrichment of old and young mitochondrial Omp25 snap label (left panel scale bar = 10 pm; right panel scale bar = 1 pm).

[0283] Figure 7c. 3D rendered segmented Z-stacks from serial section TEM of mitochondria from asymmetric cell pairs.

[0284] Figure 7d. Scheme showing mitochondrial types that were used to classify mitochondrial age class.

[0285] Figure 7e. (Top) Schematic showing strategy to isolate cells with differential enrichment of mitochondrial age class in proliferating cells. Cells were labelled with CellTrace violet (Ctv) 20h before FACS sort. At 72h, proliferating cells were sorted, and Ctv dilution analysed. (Bottom) FACS plots.

[0286] Figure 7f. (Left) Fluorescence microscopy showing the clonogenic capacity of sorted OldMitoHi and OldMitoLo cells 10 days post sort. Nuclei stained with Hoescht. Scale bar = 20 pm. (Right) Quantification. Data presented as mean ± SD. Data analysed using two-tailed unpaired Student’s t-test. P values are shown in relevant panels; P-values < 0.05 were considered significant.

[0287] Figure 7g. Immunostaining of Old Mito Hi and OldMitoLo cells for Pax7 and myogenin. Nuclei stained with Hoechst. Scale bar = 20 pm.

[0288] Figure 7h. Percentage of stem (Pax7) vs differentiated (myogenin) cells in sorted OldMitoHi and OldMitoLo clones. Data presented as mean ± SD. Data analysed using two-tailed unpaired Student’s t- test. P values are shown in relevant panels; P-values < 0.05 were considered significant.

[0289] Figure 8a. Blue-Native (BN)-PAGE quantification of mitochondrial respiratory chain subunits in Control, Cap50-treated, and Rescue groups in C2C12 myoblast cell lines. Using antibodies against NDUFA9 (Cl), SDHA (CH), UQCRC2 subunit (Cl II), and MTCO1 (CIV). N = 2 per group.

[0290] Figure 8b. OCR measurement of control (upper line) and Cap50-treated (lower line) C2C12 cells.

[0291] Figure 8c. Fluorescence microscopy showing mitochondrial morphology of control (networked) and cap- treated (fragmented) cells in MuSCs from pax7CreERT2:R26Pox8aDendramouse.

[0292] Figure 8d. (Top) Schematic showing experimental strategy. (Bottom) Fluorescence microscopy showing EDL myofiber in control and Cap50-treated cells cultured ex vivo for 72h, immunostained for Pax7 and myogenin. Nuclei stained with Hoechst.

[0293] Figure 8e. Shows a TSNE projection of scRNAseq dataset of control, Cap50-treated and rescue (r4h, r8h, r16h) data sets (total of 20,000 cells). TSNE projection visualizes variation in single-cell transcriptomes.

[0294] Figure 8f. Shows cell type predictions from de Micheli trajectory as reference data based on the pseudotime progression of these cells.

[0295] Figure 8g. Violin plot graphs showing the differences in expression of muscle progenitor markers and contractile genes in control and Cap50-treated cells.

[0296] Figure 8h. Percentage enrichment of isotopologues of metabolites in U13C-Glutamine tracing in control, Cap50-treated, and rescue (R24h) cells.

[0297] Figure 9a. Cristae density measurements in control, cap-treated, and rescue cells (at time points 4h and 10h). Data are presented as means ± SD. Data analysed using one-way ANOVA with Tukey’s multiple comparisons post hoc test. P values are shown in relevant panels; P-values < 0.05 were considered significant. Figure 9b. Relative enrichment of isotopologues of aKG (m+3 / m+2 and m+4 / m+2, m+5 / m+2) from U13C- Pyruvate tracing in control and galactose treated cells are shown (N = 2). Data analysed using one-way ANOVA with Tukey’s multiple comparisons post hoc test.

[0298] Figure 10a. Schematic showing muscle injury and in vivo, intramuscular, SNAP labelling strategy in pQ CreERT2 ■ 2Q °x a en ramic©

[0299] Figure 10b. Immunostaining of an asymmetrically dividing cell, 5 days post injury.

[0300] Figure 10c. CLEM images of in vivo activated MuSCs, 24h after activation.

[0301] Figure 10d. Immunostaining of regenerating muscle at day 5 post injury showing non-continuous old mitochondria label, Pax7, Uqcrfsl , and Hsp60.

[0302] Figure 10e. Correlation analysis of OldMito SNAP intensity compared to Uqcrfsl intensity in Pax7+ cells day 5 post injury.

[0303] Figure 11a. Representative phase contrast images of control and Cap50-treated ovine myoblasts in growth media. Multinucleated myotubes are outlined by white dotted lines. Scale bar = 100pm.

[0304] Figure 11b. Proportion of myoblasts in long-term continuous culture of myoblasts from bovine, ovine and porcine sources. Multinucleated myotubes resulting from spontaneous myoblast differentiation are excluded from quantification.

[0305] Figure 12a. Schematic showing experimental set-up for bovine, ovine and porcine myoblast immunohistochemical analysis. Myoblasts from bovine, ovine and porcine cell culture (passage 3) were seeded at equal density of 0.3 x 106 / ml cells and cultured in growth media (control) or Cap50 (test). Cells were analysed 48 hours after seeding.

[0306] Figure 12b. Immunostaining of myoblasts from bovine, ovine and porcine sources for Desmin and Hoechst 48 hours after seeding in control and Cap50 conditions. Magnified views of Cap50 samples show staining for proliferation marker Ki67. Scale bar = 10pm

[0307] Figure 12c. Percentage of differentiated cells in control and Cap50-treated samples from bovine, ovine and porcine sources. Measurements from 10-15 Regions of Interest (ROIs) are used for each species. Data presented as mean ± SD. Data analysed using one-way ANOVA with Tukey’s multiple comparisons post hoc test. P values are shown in relevant panels; P values < 0.05 were considered significant.

[0308] Examples Example 1 : Material and Methods

[0309] 1.1 Animals and mouse breeding

[0310] All mice were housed according to institutional guidelines in IVC cages with 12 hours light-dark cycle with standard chow diet and water ad libitum. Animal housing and experiments were done in accordance with Finnish National Animal Experimentation Board with ethical approval. To generate the Pax7CreERT2;ROSA26LSL-SNAPOMP25 (Pax7CreERT2:R26SNAPOmP25) line, 8-12 week-old Gt(ROSA)26Sortm1(CAG'SNAP')Pka(MGI:6466976) mice were crossed with 10-16 week-old B6. Cg- pax7tmi(cre / ERT2)Gaka / j (Murphy et al., 2011) (Strain #:017763, Jackson Laboratory) mice. To generate Pax7CreERT2; ROSA26cox8aDendraline, PhAM floxed B6;129S-Gt(ROSA)26Sortm1 (CAG- COX8A / Dendra2)Dcc / J (Strain #:018385, Jackson Laboratory) mice were crossed with B6.Cg-

[0311] 1 .2 Satellite cell isolation and culture

[0312] Muscle stem cells (MuSCs) were isolated from the hindlimb muscles of mice of wildtype or reporter mouse lines using standard isolation protocols with modifications. Briefly, muscles were dissected and digested with Collagenase and Dispase. Single cell suspensions prepared from dissociated tissue samples were centrifuged and filtered using cell strainer.

[0313] Lineage-negative non-stem cells were depleted, and a7-integrin+ MuSCs were isolated and enriched using Magnetic-Activated Cell Sorting (MACS). Cells were cultured in Advanced-DMEM:F12 (Life Technologies, #12634028) supplemented with 2 ng / mL FGF2 (Novus Biologicals, #NBP2-3492), 2 mM L- Glutamine, 1% penicillin / streptomycin and 10% Fetal Bovine Serum (FBS), on Matrigel-coated dishes (Corning, #354234). For inhibiting mitochondrial translation, chloramphenicol (50-100 pg / mL) (Sigma, #C3175) was added to the standard growth media, with daily media changes. In order to promote mitochondrial maturation, galactose (Sigma) 10 mM was added to DMEM, no Glucose media (Thermo Fischer, #11966025), supplemented with 10% FBS.

[0314] Single myofibers were isolated from extensor digitorum longus (EDL) muscles. The EDL muscle was exposed by removing the skin, connective tissue, and overlaying tibialis anterior (TA) muscle, after which the EDL was removed by cutting its proximal and distal tendons. Myofibers were then enzymatically dissociated by, immediately after isolation, incubating the EDL in pre-warmed isolation media containing 0.2% collagenase D (cat. #11088882001 Sigma-Aldrich) in a water bath at 37°C, for 45-60 min.

[0315] Mechanical trituration was performed to further dissociate individual myofibers, and dissociated single myofibers were manually collected under a dissection microscope using modified Pasteur pipettes and placed on horse serum-coated culture dishes containing growth media. Prior to staining and confocal imaging, viable myofibers were individually collected on horse serum-coated 35 mm glass-bottom dishes (cat. #P35G-1 ,5-14-C, Mattek).

[0316] 1 .3 SNAP substrate Labeling

[0317] SNAP-Cell substrate stock solutions were prepared according to manufacturer’s instructions. The labeling solution was prepared by diluting the stock solution in complete cell culture media to a working concentration of 1 .5 pM for SNAP-Cell Sir647 SNAP-Cell TMR-star and SNAP-Cell Oregon Green or 4.5 pM for SNAP-Cell Block.

[0318] MuSCs at specific timepoints of activation were labeled with cell permeable SNAP-Cell substrates (NEB) diluted in culture media for 30 minutes followed by washes with 1X Dulbecco-PBS with 0.2% BSA. The media is again replaced after 30 minutes to completely remove any unbound substrates. For in vivo labeling, cell permeable SNAP substrates (NEB, see above) were dissolved in 6 pL DMSO and diluted with saline (0.9% NaCI) to final concentration at the desired volume. For in vivo labeling of mitochondria in satellite cells : 5 pL of labeling solution (5-15 nmols SNAP-Cell 647-Sir (S9102S) or 5-15 nmols SNAP- Cell TMR-Star (S9105S) was injected intramuscularly into the TA of pax7CreERT2:R26SNAPCmp25mice immediately after the BaCI2 induced chemical injury.

[0319] 1 .4 Transmission electron microscopy (TEM)

[0320] Cells were plated and cultured on EM grade glass coverslips (thickness #1) coated with a thin layer of Matrigel and fixed with 2% glutaraldehyde (cat. #16300, Electron Microscopy Sciences) in 0.1 M Sodium Cacodylate buffer (cat. #11650, Electron Microscopy Sciences) for 20 minutes at room temperature. Cells were postfixed with 1% osmium tetroxide, dehydrated in ethanol, and embedded in Epon resin. Ultrathin 60 nm sections were cut with an ultramicrotome, placed on grids, and post-stained with uranyl acetate and lead citrate. Sections were imaged using Jeol JEM-1400 (Jeol Ltd., Tokyo, Japan) electron microscope equipped with a Gatan Orius SC 1000B bottom mounted CCD-camera. Images of cells and mitochondria were acquired with a magnification of 200X-400X and 4000X-10,000X, respectively.

[0321] 1 .5 Correlative Light and Electron Microscopy (CLEM)

[0322] Cells were plated and cultured on grided 35 mm glass-bottom dishes (cat. #P35G-0.170-14-C, MatTek) coated with a thin layer of Matrigel. After mitochondrial labelling, cells were fixed for electron microscopy as described in Ding et al., J Biol Chem. (2012) 287(50):42373-8, and nuclei were counterstained with 1 pg / mL Hoechst stain. Fluorescent z-stack images of cells and organelles were obtained by confocal microscopy using 63x magnification and 0.2 pm z-step size, after which Leica LAS X navigator was used to locate grid coordinates corresponding to the ROI. Grid coordinates were used to mark the ROI for epon embedding, after which samples were processed for electron microscopy and imaged as described in Ding et al., J Biol Chem. (2012) 287(50):42373-8. For alignment of images, corresponding planes from TEM sections and confocal z-stack images were manually chosen based on calculated section depth and visible landmarks. Images were imported into ImageJ plugin BigWarp, and an affine transformation was performed using 4 correlating landmark points such as mitochondria, nuclei, or the cell membrane. Transformed TEM images were exported and merged with the corresponding confocal composite plane as an individual channel.

[0323] 1.6 Cell-IQ

[0324] Cells were plated in duplicates (around 30,000 cells / well) on a 12-well plate coated with Matrigel. Samples were placed the following day in a high content screener (Cell-IQ) with 5% CO2 at 37°C. 5 regions / well were selected and images were obtained at 1 h intervals with phase contrast using Nikon 10x objective. Cells were imaged for 90h, with daily media changes for both control and chloramphenicol (CAP)-treated samples. Images were exported to Cell-IQ analyzer, and cell counts were automatically calculated using a trained classifier that distinguished between cells, debris and fused myotubes. The efficiency of the classification and cell identification was manually evaluated (visually) from each well.

[0325] 1.7 Muscle injury

[0326] Under isoflurane anesthesia, chemical injury was performed by intramuscular (IM) injection of 10 pL of barium chloride (1 .2% in sterile, demineralized water) (Sigma Aldrich, MO, USA) in the Tibialis Anterior (TA) muscle, after skin incision and muscle exposition. The skin was then sutured with 4.0 suture string and mice returned to cage after injury. Mice were given analgesics: Buprenorphine (0.05 mg / Kg body weight) before the procedure followed by Caprofen (2 mg / Kg body weight) after the procedure.

[0327] 1 .8 OCR measurement using Resipher

[0328] 10,000 -20,000 freshly isolated MuScs were seeded into wells of Matrigel-coated Falcon, Flat-Bottom 96- Well Microplate. The cells were allowed to attach, and experimentation was started 24h after plating. On the day of experimentation, fresh culture medium was changed and media supplemented with mitochondrial translation inhibitor (Chloramphenicol) or Galactose (10 mM) and the Resipher oxygen sensing lid (Lucid Scientific) was attached. Live oxygen consumption rate was measured for 80-90 hours with periodic media change. Data were analyzed on Resipher web application (Lucid Scientific).

[0329] 1 .9 Blue-Native PAGE

[0330] Mitochondrial lysate samples from 2x106C2C12 cells and the 4-15% gradient-native gel were prepared as described in Konovalova, J Vis Exp. (2019) 144. A total of 20 pg of protein for each sample were run on gradient gel, and proteins were transferred to a PVDF membrane (Trans-Blot Turbo Mini 0.2 pm PVDF Transfer Packs #1704156, BioRad) using the Trans-Blot Turbo system (BioRad) at 25 V and 1 .0 A for 30 min. Blots were blocked in 5% BSA / TBST for 1 h at RT, and then incubated with primary antibody overnight at 4°C, and HRP-conjugated secondary antibody for 1 h at RT. Blots were washed three times for 10 mins with TBST (TBS, pH 7.4 with 0.1% Tween-20) between antibody incubations. Final images were developed with Pierce™ECL (#32106, Themrofisher) using Chemidoc XRS system (Bio-Rad).

[0331] 1.10 Labelling Mitochondrial Translation

[0332] Mitochondrial translation was measured by labeling newly-synthesized mitochondrial-encoded peptides using a click chemistry-based approach, as described in Yousefi et al., EMBO Rep. (2021) 22(4):e51635, with some modifications. Briefly, MuSCs were transferred to methionine-free medium (Thermo Fisher Catalog Number: 21013024). Cytosolic translation was stopped using Cycloheximide (Chx, 50 pg / ml, sigma) for 10 min. 500 pM of l-Homopropargylglycine (HPG, Thermo Fisher) was then added for 30 min. Cells were then transferred to buffer A (containing 10 mM HEPES, 10 mM NaCI, 5 mM KCI, 300 mM sucrose) + 0.015% digitonin, for 2 min at room temperature, followed by 1 min in buffer A (without digitonin). Coverslips were fixed using 4% PFA in PBS for 10 min at room temperature and were then further processed for click chemistry. 1.11 TMRM imaging

[0333] For analysis of tetramethylrhodamine, methyl ester (TMRM) fluorescence intensity in mitochondrial domains, cells were plated on MatTek glass-bottom dishes. After Snap-labelling, cells were treated with TMRM (20 nM in culture medium) and incubated for 30 min at 37°C. Cells were given a fresh media change and imaged AT LEICA stellaris confocal microscope. For uantifications, the sum of the intensity projections of the cells was determined, and domains of the mitochondrial network were arbitrarily outlined using Snap-labels as reference. Integrated fluorescence intensities for Snap-labels and TMRM were measured using FIJI software. The old-to-new-label ratio for individual mitochondrial domains was calculated and plotted against mean fluorescence intensity (MFI) for TMRM.

[0334] Example 2: Temporal control of Age-selective apportioning of mitochondria in muscle stem cells Quiescent satellite cells are reported to have low mitochondrial content, but limited data is available on their dynamics. Recently, mitochondrial fission-fusion processes have been shown to be important for muscle stem cell dynamics. Intriguingly, analysis of satellite cells on myofibers in pax7CreET2:R26Cox8aDendrareporter mice (in which mitochondria of satellite cells are specifically labelled), revealed an interconnected tubular network with remarkable heterogeneity in mitochondrial content (Fig. 1a). Apart from perinuclear localisation2, mitochondria were also localised to cytoplasmic projections of satellite cells spanning the myofiber (Fig. 6a). Further, ultrastructure analysis of guiescent, freshly-isolated muscle stem cells (MuSCs) revealed that their mitochondria have an immature morphology, with wide, disorganised cristae as compared to activated MuSCs (Fig. 1 b, Figs. 6b, 6c), and also lower cristae density (which are features of ETC-deficient mitochondria56).

[0335] Immunostaining of freshly-isolated MuSCs for ETC complex subunits revealed high expression of Ndufa9 (Complex I) in the mitochondria, but limited mitochondrial expression of the catalytic subunit Ugcrfsl (Complex III), which is instead seguestered to the cytoplasm. Ugcrfsl (Rieske-lron sulphur protein, Risp) is central to ETC stability and Super Complex (SC) formation7. Additionally, low expression of F1 / F0 ATP synthase (Atp5a; Complex V) in guiescent MuSCs reflects the lower ETC coupling capacity of these cells (Fig. 6d).

[0336] Previous studies have shown that MuSC activation from a guiescent to a proliferative state is accompanied by a metabolic shift to glycolysis, despite an increase in mitochondrial content89. Examination of MuSCs at multiple time points of activation revealed that mitochondrial biogenesis upon activation is not followed by an immediate structural maturation, as evidenced by lower cristae density (Fig. 1c) and lower basal respiration levels (Oxygen Consumption Rate, OCR) in proliferating myoblasts (Fig. 1d). Mitochondrial structural maturation peaked after 60-72h of activation, which is also reflected in the OCR readings that show a dramatic increase as cells undergo myogenic fusion at 80h of ex vivo activation (Fig. 1d).

[0337] Mitochondrial translation has previously been shown to determine the rate of mitochondrial oxidative phosphorylation, and is reguired for cristae formation (Aibara et al., Elife (2020) 9:e58362). Mitochondrial translation was monitored across MuSC activation time points, using the alkyne-containing non-canonical amino acid L-homopropargylglycine (HPG) as described in Yousefi et al., EMBO Rep. (2021) 22(4):e51635 (see Example 1.10). At early activation timepoints, a lower level of mitochondrial translation per mitochondrial domain was observed. However, by 72 h the level of mitochondrial translation per mitochondrial domain had increased significantly, and coincided with the increase in cristae density and OCR at this time point (Fig. 1e, Fig. 1f).

[0338] Taken together, these data suggest that mitochondria of freshly-isolated MuSCs are immature, and show gradual structural and functional maturation as they transition from quiescence to proliferating myoblasts.

[0339] To further investigate the new findings of temporal control and pace of mitochondrial maturation in muscle stem cells and cell fate decisions in these cells, a Cre-inducible reporter mouse model (pax7CreERT2R26SNAPOnnp25) was developed, which upon tamoxifen treatment expresses Omp25-SNAP tag to outer mitochondrial membrane in satellite cells (Fig. 6e). This permits tracking of mitochondria by chronological age from quiescence to activation. Within 24h of activation, many domains of newly-formed mitochondria were clearly distinguishable from the old (mitochondria of the freshly isolated MuSCs) (Fig. 1g). This indicates that mitochondrial biogenesis is an immediate response to quiescent MuSC cell activation, and interestingly this was independent of the level of Pax7 expression in the cells (Fig. 1 h, Fig. 6f). These data suggest that early mitochondrial dynamics in activated stem cells are independent of the myogenic transcriptional program.

[0340] Next, using a SNAP labelling strategy34, including the unlabelled substrate (block) that effectively separates mitochondria temporally, the inventors analysed how age-separated mitochondria are allocated to daughter cells as MuSCs underwent first mitotic entry after activation (30-36h). Analysing cell pairs in division revealed no age-selective segregation of domains, and mitochondrial apportioning was symmetric between daughter cells (Fig. 1 i). However, as the window of analysis was shifted further down the lineage progression (72h), while keeping the labelling interval constant, myogenic progenitors in division strikingly segregated old (36h) and young (5h) mitochondria (Fig. 1j), and cell division followed age-selective apportioning of mitochondria between daughter cells (20%) (Fig. 1 k, Figs. 6g, 6h, 6i). The window of asymmetric apportioning of mitochondria corresponded strikingly well with the functional maturation timeline of mitochondria in the myogenic population, further indicating temporal control of mitochondrial dynamics in these cells.

[0341] Example 3: Chronologically separated mitochondria show distinct ultrastructure and determine cell fate of inherited cells

[0342] To further assess how chronologically distinct mitochondria differ in dividing cells, state-of-the-art Correlative Light and Electron Microscopy (CLEM)10was employed to analyse the ultrastructure of old and young mitochondria in progenitors and asymmetrically dividing cells (Fig. 2a). CLEM combined with 3D segmentation analysis of asymmetrically divided sister cells and progenitors revealed that old mitochondria have mature morphology, with organised cristae and higher cristae density, whereas young mitochondria have an immature morphology, with less dense and wider cristae (Figs. 2a, 2b, Figs. 7a-7d). Cristae are sites of active mitochondrial oxidative phosphorylation and indicative of the respirational capacity of mitochondria. The inventors next investigated whether structural differences between mitochondria correlated with functional differences between old and young mitochondria. The mitochondrial membrane potential (MMP) of live cells was analyzed by staining with TMRM, as described in Example 1.11. Mitochondrial domains having higher proportions of old mitochondria were found to have higher MMP, confirming that mitochondrial domains enriched for old mitochondria are structurally and functionally more mature. These data agree with previous observations that older mitochondria are enriched for ETC complex proteins relative to younger mitochondria in epithelial stem-like cells4.

[0343] Next, to examine the functional consequence of age-selective mitochondrial apportioning in myogenic population, proliferating cells (Cell tracer violet diluted) were FACS-isolated at 72h, based on their enrichment (OldMitoHi) or depletion (OldMitoLo) of old mitochondria (Fig. 2c, Fig. 7e). Sorted cells that differ in old mitochondrial enrichment were similar in myogenic cell state (Pax7+Myod+) (Fig. 2d). Importantly, OldMitoHi cells readily (48h, post sort) differentiated to MyHC+ myotubes whereas OldMitoLo cells retained sternness (Pax7+) with higher clonogenic capacity (Fig. 7f) and delayed differentiation (Figs. 2e, 2f, 2g, Figs. 7f-7h). Taken together, these data establish that chronologically separated mitochondria differ in their maturation, and that myogenic cells have differential cell fates that correlate with the functional maturity of their inherited mitochondria.

[0344] Example 4: Mitochondrial maturation and functional ETC is dispensable to retain self- renewal potential of Muscle stem cells

[0345] The inventors’ findings that freshly-isolated (Fl) MuSCs have immature mitochondria and that temporal, asymmetric apportioning determines cell fate based on the inherited mitochondria, provide tantalising possibility that mitochondrial function may act as a semi-autonomous regulator of myogenic fate decisions. To investigate this further, the inventors first blocked mitochondrial maturation in activated satellite cells. Mitochondrial DNA encodes for 13 subunits essential for ETC assembly and function. Blocking mitochondrial translation using a pharmacological inhibitor would prevent maturation of ETC and respiratory complex assembly, with minimum interference to general translation11. Indeed, mitochondria isolated from myoblast cell line C2C12 treated with chloramphenicol 50pg / ml (Cap50) displayed reduced super complex (SC) assembly (SC1 , SC2, SC3) as revealed by BN-PAGE, and reduced OCR (Figs. 8a, 8b).

[0346] Next, freshly-isolated MuSCs were treated with Cap50 and cultured ex vivo; this allowed the inventors to impede mitochondrial maturation and retain their status as close as possible to that of the freshly-isolated MuSCs. Blocking mitochondrial translation did not prevent mitochondrial biogenesis in these cells, but prevented increase in cristae density (Figs. 3a, 3b) and fusion (Fig. 8c), and resulted in lower mitochondrial membrane potential (Fig. 3c), mimicking the phenotype of young mitochondria (see Fig. 2c). Remarkably, these cells retained OCR values at the same levels as that of the freshly-isolated MuSCs even at 72h of activation (Fig. 3d) but remained glycolytic with high ECAR / OCR levels (Fig. 3e). The Cap50-treated cells retained expression of myogenic factors (e.g. Pax7) but failed to progress to terminal differentiation, with no / reduced myogenin expression (Figs. 3f, 3g, Fig. 8d). Strikingly, these cells retained high proliferative capacity, and sustained as a constantly self-renewing population of Pax7+ progenitors (Figs. 3h-3i). To further confirm that mitochondrial modulation in these cells had not shifted them in the myogenic lineage, scRNA-seq data from Cap50-treated and Control cells was mapped to a pseudotime trajectory12of muscle regeneration. Both samples mapped to MuSCs at day 5 of regenerating muscle and did not differ in their locations along the trajectory (Fig. 3j, Figs. 8e-8f). However, Cap50- treated MuSCs displayed low expression of major muscle contractile proteins (Tpm2, Ttn, Tnnt3; Fig. 8g), indicating that the cells are within the myogenic trajectory but fail to proceed to differentiation.

[0347] Interestingly, proliferating Cap50-treated MuSCs were found to have a higher NADH / NAD redox ratio than control cells not treated with Cap50, and cells treated with Cap50 and subsequently released from blockade of mitochondrial translation (Fig. 4c). The inventors next investigated the metabolic status of cells having immature mitochondria. Evaluation of carbon flux by analysis of the metabolomics of U-[13C]- glucose revealed that Cap50-treated cells having immature mitochondria showed higher glycolytic flux, as evidenced by a high ratio of labelled (m+3) Lactate / Pyruvate, as well as higher shunt of glucose-derived carbon to the Pentose Phosphate Pathway (PPP). Interestingly, Cap50-treated cells displayed minimal shunt of glucose-derived carbon to the tricarboxylic acid (TCA) cycle (Fig. 4a, Fig. 8i), which was restored to control levels in cells released from mito-translation block (Res), also resulted in increased cristae density at 10h from rescue (Fig. 9a). The lack of glucose shunt to TCA cycle was interesting, and Cap50- treated cells also displayed upregulation of glutamine transporters (Slc1a5 and Slc7a5), suggesting a possible anaplerotic contribution of glutamine to generate TCA intermediates in these cells (Fig. 8g). Indeed, metabolic tracing with U-[13C]-glutamine revealed that these cells employ active glutaminolysis to maintain levels of TCA intermediates. Importantly, the cells showed high levels of m+4 succinate and m+5 2-hydroxyglutarate (2-HG) over 2-alpha-ketoglutarate (2-aKG; Fig. 4b, Fig. 8h); succinate and 2-HG are known to inhibit activity of 2-oxoglutarate-dependent dioxygenases, including DNA and histone demethylases.13

[0348] Indeed, active demethylation of myogenin by Tet2 has been shown to be important for its expression and muscle differentiation14 15. Releasing cells from mitochondrial translation block (Res) increased cristae density and restored cellular metabolism to control levels (Fig. 4a, 4b). The restoration of metabolism followed rapid progression of the cells to differentiation with high myogenin expression (Fig. 4d), and rescued cells underwent myoblast fusion to form MyHC+ primary myotubes (Fig. 4e).

[0349] Taken together, these data show that a functional ETC is not required for quiescent satellite cell activation and myoblast expansion. Also, the immature mitochondrial phenotype (that mimics that of young mitochondria) remodels metabolism and bolsters self-renewal of MuSCs over differentiation.

[0350] Example 5: Mitochondrial functional heterogeneity drives asymmetric apportioning

[0351] Having established that mature ETCs are dispensable to retain sternness of MuSCs, the inventors next investigated whether advanced maturation of ETC can skew myogenic progenitors to a more differentiated fate, thereby mimicking the old mitochondrial phenotype.

[0352] To achieve this, cells were shifted from glucose to galactose media, which is known to enhance SC assembly and mitochondrial respiration16. The shift to galactose media was accompanied by an immediate increase in flux of TCA cycle, as revealed by U-[13C]-pyruvate tracing (Fig. 9b) and an increase in basal OCR within 10h of treatment (Fig. 4g). This was followed by 15-20-fold upregulation in myogenin expression in these cells (Fig. 4h). The upregulation of myogenin transcript was significantly abrogated when cells cultured in the galactose media were treated with Cap50, indicating that increased differentiation potential of these cells was indeed due to expedited ETC maturation (Fig. 4h). Cells cultured in galactose medium also displayed accelerated differentiation to form myotubes as compared to control cells cultured in glucose media (Fig. 4i). Myogenic Fusion index was increased (Fig. 4j) considerably, with reduced Pax7 expression (Fig. 4k) in cells showing reduced sternness and increased differentiation followed by enhanced mitochondrial function. Collectively, the data demonstrate that mitochondrial function can act as a temporal regulator to define the pace of myogenic lineage trajectory of activated satellite cells.

[0353] Example 6: Apportioning off old mitochondria primes myogenic progenitors to satellite cell fate during injury regeneration

[0354] MuSCs are mitotically quiescent during homeostasis but undergo active proliferation in response to muscle injury17. To investigate whether the mitochondrial dynamics observed during ex vivo activation are recapitulated during in vivo muscle regeneration, Tibialis Anterior (TA) muscle of Pax7CreERT2 :R26SNAPOmp25 mice was subjected to barium chloride (BaChj-induced experimental muscle injury18. To label the mitochondria of MuSCs just before activation, SNAP substrate was injected immediately after BaCh administration (Fig. 5a). Similar to ex vivo data, CLEM Analysis of activated MuSCs 24h post injury revealed limited maturation of mitochondrial morphology (Fig. 9a). Analysis of regenerating muscle at day 3 post-injury showed symmetric distribution of old mitochondria, with myoblasts lining the ghost fibre to initiate repair (Fig. 5b). By day 5 post-injury, nascent myofibers with centrally-located nuclei were visible, and juxtaposed to this regenerated muscle were self-renewed Pax7+ cells. Analysis of muscle at day 5 post-injury revealed that differentiated MyHC+ fibers had the highest enrichment of old mitochondria whereas Pax7+ cells were depleted of old mitochondria (Fig. 5c). Interestingly, Pax7+ cells noncontiguous to nascent myofiber had higher enrichment of old mitochondria, suggesting that Pax7- expressing cells associated to newly-regenerated muscle has apportioned off their old mitochondria (Fig. 5d, Fig. 10b). Further analysis of Pax7+ cells in day 5 post-injury muscle showed that a major subset of these cells had low Uqcrfsl (complex III) levels over total mitochondria (Hsp60) (Figs. 5e, 5f, Figs. 10d, 10e) - reminiscent of quiescent MuSCs (Fig. 5g). Taken together these results show that age-selective apportioning of mitochondria is a plausible mechanism for muscle stem cells to generate daughter cells with opposing metabolic demands, i.e. cells with immature mitochondria for self-renewal, vs. cells with mature mitochondria and a functional ETC to differentiate to myofibers.

[0355] Example 7: Inhibition of mitochondrial maturation prevents myoblast differentiation in porcine, bovine, and ovine cells

[0356] To investigate whether blocking mitochondrial maturation inhibits muscle stem cell differentiation and bolsters self-renewal in alternative species, the inventors also explored this effect using bovine, ovine and porcine myoblasts.

[0357] Isolated myoblasts from each species were separately treated with Cap50 and cultured ex v / vo; this allowed the inventors to impede mitochondrial maturation and retain their status as close as possible to that of the freshly-isolated myoblasts. Initial analyses of the cultured cells showed that Cap50-treated samples consistently demonstrated reduced numbers of multinucleated myoblasts compared to control samples. Representative phase contrast images of control and Cap50-treated ovine myoblasts in growth media are shown in Fig. 11a.

[0358] Moreover, control samples demonstrated high levels of spontaneous differentiation of myoblasts into multinucleated myotubes during long-term continuous culture, while all Cap50-treated samples had vastly reduced levels of differentiation, each retaining high proportions of undifferentiated myoblasts. The Cap50-treated cells exhibit a high proliferative capacity and can be continuously expanded in culture over multiple passages (Fig. 11 b).

[0359] Primary porcine, bovine and ovine myoblasts were also subjected to immunohistochemical analysis. Myoblasts of each species were seeded at equal density of 0.3 x 106 / ml cells and cultured in growth media (control samples; C) or media containing Cap50 (test samples; Cap). Cells were analysed 48 hours after seeding. A schematic of the experimental set-up is shown in Figure 12a.

[0360] Immunostaining of myoblasts from each species for Desmin (an abundant muscle-specific intermediate filament) and Hoechst (a DNA stain) showed that control samples exhibit strong differentiation with fused myotubes (Desmin+), while Cap50-treated cells demonstrated no visible myotube formation (see Fig. 12b). A significant decrease in myoblast differentiation in Cap50-treated samples compared to control samples is confirmed in Fig. 12c.

[0361] Staining for Ki67 (a proliferative marker) additionally demonstrated that that Cap50-treated cells from each species retained high proliferative capacity and sustained as a constantly self-renewing population (Fig. 12b, magnified views).

[0362] Thus, treatment of bovine, ovine and porcine myoblasts with an inhibitor of mitochondrial translation (Cap50) was found to strongly inhibit cellular differentiation, while preserving a high proliferative capacity.

[0363] Together, these data suggest the relationship between mitochondrial maturation and muscle stem cell differentiation is conserved between diverse species, and that blocking mitochondrial maturation is an effective strategy for inhibiting differentiation of muscle stem cells from a wide variety of different species.

[0364] References

[0365] A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. The entirety of each of these references is incorporated herein.

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Claims

Claims:1 . A method for culturing primary muscle stem cells, comprising culturing primary muscle stem cells in the presence of an inhibitor of mitochondrial maturation.

2. A method for promoting self-renewal of primary muscle stem cells in cell culture, comprising culturing primary muscle stem cells in the presence of an inhibitor of mitochondrial maturation.

3. A method for inhibiting the differentiation of primary muscle stem cells, comprising culturing primary muscle stem cells in the presence of an inhibitor of mitochondrial maturation.

4. A method for increasing the clonogenicity of primary muscle stem cells, comprising culturing primary muscle stem cells in the presence of an inhibitor of mitochondrial maturation.

5. A method for promoting the differentiation of primary muscle stem cells, comprising culturing primary muscle stem cells in the presence of a promoter of mitochondrial maturation.

6. A method for culturing primary muscle stem cells, comprising:(i) culturing primary muscle stem cells in the presence of an inhibitor of mitochondrial maturation, and(ii) subsequently releasing the primary muscle stem cells from inhibition of mitochondrial maturation.

7. A method for producing muscle tissue, comprising:(i) culturing primary muscle stem cells in the presence of an inhibitor of mitochondrial maturation, and(ii) subsequently releasing the primary muscle stem cells from inhibition of mitochondrial maturation, thereby promoting differentiation of the primary muscle stem cells, and formation of muscle tissue.

8. The method according to claim 6 or claim 7, wherein releasing the primary muscle stem cells from inhibition of mitochondrial maturation comprises culturing the primary muscle stem cells in the presence of a promoter of mitochondrial maturation.

9. Use of an inhibitor of mitochondrial maturation to promote the self-renewal of primary muscle stem cells.

10. Use of an inhibitor of mitochondrial maturation to inhibit the differentiation of primary muscle stem cells.11 . Use of an inhibitor of mitochondrial maturation to increase the clonogenicity of a primary muscle stem cell.

12. Use of a promoter of mitochondrial maturation to promote the differentiation of primary muscle stem cells.

13. A kit, comprising (i) an inhibitor of mitochondrial maturation, and (ii) a promoter of mitochondrial maturation.

14. The method according to any one of claims 1 to 4 or 6 to 8, the use according to any one of claims 9 to 11 , or the kit according to claim 13, wherein the inhibitor of mitochondrial maturation is an inhibitor of mitochondrial translation.

15. The method according to claim 14, the use according to claim 14, or the kit according to claim 14, wherein the inhibitor of mitochondrial translation is an inhibitor of bacterial protein synthesis.

16. The method according to claim 14 or claim 15, the use according to claim 14 or claim 15, or the kit according to claim 14 or claim 15, wherein the inhibitor of mitochondrial translation is selected from: chloramphenicol, thiamphenicol, florfenicol and actinonin.

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