High Content And Functional Screening Method In A Physiologically Relevant Duchenne Or Becker Muscular Dystrophy Model

HIGH CONTENT AND FUNCTIONAL SCREENING METHOD IN A

PHYSIOLOGICALLY RELEVANT DUCHENNE OR BECKER MUSCULAR

DYSTROPHY MODEL FIELD OF THE INVENTION

The invention relates to a method for improving Duchenne or Becker muscular dystrophy myoblast differentiation into myotubes, and a method for compound screening in the field of Duchenne or Becker muscular dystrophy drug discovery. BACKGROUND OF THE INVENTION

Duchenne muscular dystrophy (DMD), and its more benign form Becker's muscular dystrophy, are the most common muscular dystrophies. They are caused by mutations in the dystrophin gene, located within the X chromosome. DMD results in the production of non-functional proteins or in the blocking of the overall dystrophin production. DMD is a severe and progressive muscle wasting disorder that appears in 2-3 years boys and leads to wheelchair dependency as well as premature death.

Dystrophin impairment in muscles from patient induces both an overall disruption of sarcomere organization and sarcolemma fragility. This weakness drastically leads to decrease myofibers protection during muscle contraction, inducing tissue degeneration and necrosis [Pasternak 1995].

Satellite cells are skeletal muscle specific stem cells which play a central role in tissue regeneration. When damages occur, quiescent satellite cells start differentiating, a process called myogenesis, to form replacing muscle tissue [Kuang 2007].

During myogenesis, satellite cells begin to proliferate and form myoblasts. A myoblast is a mononucleate cell type that, by fusion with other myoblasts (this is a part of a process being also called "differentiation"), gives rise to myotubes that maturate and later eventually develop into myofibers, the muscle subunits. Maturation of myotubes can easily be characterized through the presence and the striated organization of myosin heavy chain molecules within sarcomeres, and the location of nuclei at the center of the structure [Abmayr 2012].

In DMD pathological tissues, the satellite cells ability to differentiate is impaired due to premature aging [Schafer 2006], reducing tissue regeneration and subsequently inducing muscle atrophy and weakness.

To date few in vitro models were used in DMD related drug discovery, as compounds are mainly developed in long, expensive and poorly ethical animal testing. Successful high throughput and high content screening (respectively named HTS and HCS) campaigns are reviewed in [Gintjee 2014]. Most of them were realized by genetic approach, using luciferase or GFP gene reporters in human non-muscle cells or in murine muscle progenitors. However, these gene reporter systems were shown to generate off- target effects, largely decreasing the assay specificity [McElroy 2013]. Moreover, as compensatory effects occur during myogenesis, these systems do not guarantee the relevance of a target gene expression on the overall muscle physiology. Besides, currently used cellular models are far from human myofibers, and it remains difficult to extrapolate these results to the human muscle tissue.

Only one campaign was realized by using a phenotypic approach on human progenitors [Nierobisz 2013]. To date, no phenotypic approach was directly developed on differentiated human muscle subunits, i.e. the myotubes.

In addition, no in vitro DMD pathological model was successfully developed in a screening format, neither for HTS/HCS drug discovery by combining myotube morphological measurements though imaging, and functional analysis.

SUMMARY OF THE INVENTION

A goal of the invention is to provide a method that allows Duchenne or Becker muscular dystrophy myoblasts differentiation into myotubes, in order to enable compounds detection during HTS/HCS campaigns of DMD related drug discovery.

According to a first aspect, the invention provides a method for robustly allowing

DMD myoblast differentiation into myotubes and achieving a maturation level that reflects the in vivo situation during the later phases of myogenesis in DMD pathological condition.

In accordance with the invention, the method comprises:

(i) providing a device comprising a substrate and at least one cell-adhesive pattern, wherein:

- said at least one cell-adhesive pattern has an elongated surface comprising a central region and two lateral regions extending from said central region in both directions along a longitudinal axis of the pattern with a contour discontinuity between the central region and each lateral region, - the ratio between the maximum width of the central region and the maximum width of the lateral regions is greater than or equal to 2, the length of the pattern being comprised between 100 and 1000 μηη and the maximum width of the pattern being comprised between 50 and 500 μηη, and

- the ratio between the length and the maximum width of the pattern is less than or equal to 4, (ii) depositing human primary myoblasts from Duchenne or Becker muscular dystrophy donor or group of donors or from a cell line, an isogenic cell line or derived stem cells recapitulating Duchenne or Becker muscular dystrophy on at least one cell-adhesive pattern of said device,

(iii) culturing said pathological myoblasts in a differentiation medium during a determined incubation time so as to promote cell differentiation into myotubes, and constrain elongation of the myotubes on the cell-adhesive pattern.

According to a preferred embodiment, said at least one cell-adhesive pattern consists of the partial superposition of three elliptical surfaces: a first elliptical surface defining the central region of the pattern and second and third elliptical surfaces having a major axis coinciding with the major axis of the first ellipse defining the lateral regions of the pattern, wherein the second and third elliptical surfaces intersect the first elliptical surface along their transversal axis.

Besides, said at least one cell-adhesive pattern may be symmetrical according to its longitudinal axis and to a transversal axis perpendicular to the longitudinal axis.

The substrate is advantageously a hard substrate such as glass.

Preferably, the surface area of said at least one cell-adhesive pattern is comprised between 5,000 and 500,000 μηι2.

Another object of the invention is a method for screening compounds driving changes in skeletal muscle cells from Duchenne or Becker muscular dystrophy donor or group of donors from a cell line, an isogenic cell line or derived stem cells recapitulating Duchenne or Becker muscular dystrophy, and in particular myotubes or myoblasts, comprising:

(i) providing a culture of myotubes from human primary myoblasts from Duchenne or Becker muscular dystrophy donor or group of donors or from a cell line, an isogenic cell line or derived stem cells recapitulating Duchenne or Becker muscular dystrophy, said culture being obtained by the method described above,

(ii) adding at least one compound to said culture,

(iii) after a determined incubation time of the myotubes with said compound, carrying out morphological, structural or functional readout of the myotubes to determine the effect of said compound on the myotubes.

According to an embodiment, said method comprises carrying out image analysis of the myotubes to measure morphological changes in skeletal muscle cells and in particular myotubes or myoblasts. Said measured morphological changes may comprise the maximal width of the myotubes after incubation with the compound and image analysis may comprise myotube image binarization, computation of a distance map of said myotubes and computation of the maximal width of each myotube from said distance map. The method may further comprise counting the myotubes having a maximal width greater than a predetermined value.According to an embodiment, in the field of drug discovery, the method is intended for identifying therapeutic compounds acting on atrophy or hypertrophy of skeletal muscle cells, and the image analysis is carried out to determine the effect of said compounds in terms of atrophic or hypertrophic properties.

According to an embodiment, said functional readouts comprise myotube maturation through the expression of specific biomarkers, including dystrophin expression.

According to an embodiment, said functional readouts comprise calcium release and resulting myotube contraction.

According to an embodiment, said functional readouts comprise myotube metabolism through the evaluation of glucose uptake.

According to an embodiment, said structural readouts comprise the integrity of the myotube plasma membrane through the evaluation of creatine kinase release.

DMD myoblasts robustly differentiate in myotubes by using the invention. Maturation of resulting DMD myotubes is found physiologically relevant to the in vivo pathological tissue. The present invention is the first in vitro DMD pathological model physiologically relevant providing enough material to perform cell-based assays. As myotube morphological and functional readouts are provided, the invention is a reliable tool for the detection of fine compounds effects on the physiology of DMD myotubes in a drug discovery compatible format.

BRIEF DESCRIPTION OF THE DRAWINGS

Further features and advantages of the invention will be apparent from the following detailed description, referring to the appended drawings wherein:

- Figures 1A to 1 D illustrate a cell-adhesive pattern according to different embodiments of the invention;

- Figure 2 is a time course, with primary human myoblasts from healthy and DMD donor, showing an increase of the fusion index on patterns (mentioned as UFO) according to the invention compared to unpatterned surfaces (named NP), for a fixed time point;

- Figure 3A shows micrographies of myosin heavy chain and nuclei stainings of myotubes differentiated from myoblasts of healthy (left) and DMD (middle and right) donors cultured during 5 days on patterns according to the invention, in control conditions (DMD_CTRL, middle) and compounds treatment (DMDJGF-1 , right). Presence of dystrophin is shown on enlargments on the top right of the pictures. Figure 3B is a quantification of some relevant morphological parameters discriminating myotubes from healthy, and DMD donors cultured in control and IGF-1 conditions. Total nuclei number is a seeding control validating that the same number of cells were added on patterns in all conditions. Figure 3C shows the level of striated myotubes, using a myosin heavy chain staining, in each condition. Representative example of myotube striation from DMD donor are shown on enlargments on the right for control and IGF-1 treated conditions.

- Figures 4A and 4B are examples of functional readout quantification according to an embodiment of the invention. Figure 4A is a quantification of the creatine kinase activity in myotubes differentiated from myoblasts of healthy and DMD donors according to the invention. Results of treatments with prednizone and deflazacort palliative compounds are shown. Figure 4B is a quantification of glucose metabolism in myotubes differentiated from myoblasts of healthy and

DMD donors according to the invention. Results of treatments with prednizone and deflazacort are shown.

- Figure 5A show micrographs of myotubes on soft substrates of different Young's modulus and Figure 5B is a histogram showing the proportion of intact, shortened and collapsed myotubes at different instants of time in the differentiation medium.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The invention makes use of a device for culturing myoblasts that comprises a substrate and at least one cell-adhesive pattern designed to promote myoblast differentiation into myotubes on said substrate.

According to the invention, the cell-adhesive pattern has an elongated surface with a specific shape.

More precisely, the pattern comprises, on the one hand, a central region and, on the other hand, two lateral regions extending from said central region in both directions along a longitudinal axis of the pattern.

By "elongated" is meant that the maximum dimension of the pattern can be measured along said longitudinal axis and is greater than the maximum dimension of the pattern along a transversal axis perpendicular to the longitudinal axis.

The dimension along the longitudinal axis is thus called "length" and the dimension along the transversal axis is called "width".

The lateral regions are generally narrower (along the transversal axis) than the central region.

In addition, there is a contour discontinuity between the central region and each lateral region. In other words, at the junction between the central region and a lateral region, the tangent to the contour of the central region does not coincide with the tangent to the contour of the lateral region.

For example, the pattern may be considered as a combination of at least three geometrical surfaces that are partially superimposed in the central region of the pattern. Although not limited to this embodiment, the pattern is advantageously symmetrical with respect to both the longitudinal axis and the transversal axis.

According to an embodiment, each lateral region has a continuous contour.

Alternatively, at least one lateral region and/or the central region comprises itself a contour discontinuity.

In such an embodiment, the lateral region and/or central region may be considered as a combination of at least two geometrical surfaces that are partially superimposed.

When the lateral region is a combination of at least two geometrical surfaces, the maximum width of each surface decreases from the central region to the end of the pattern.

In a pattern according to the invention, the ratio between the maximum width of the central region and the maximum width of the lateral regions is greater than or equal to 2.

In addition, the ratio between the length and the maximum width of the pattern is less than or equal to 4.

Of course, this is a relative notion since the length of the pattern may be adjusted depending on the morphology required for resulting myotubes; this is why the dimensions of the pattern are not defined by numerical values but rather by ratios between different specific dimensions.

Typically, the length of the pattern may be comprised between 100 and 1000 μηη, the width of the pattern may be comprised between 50 and 500 μηη and the surface area of the pattern may be comprised between 5,000 and 500,000 μηι2.

Thanks to its shape and "finite" length (as compared to cell-adhesive strips that have a length much greater than myotubes length), the pattern allows confining the myotubes and thus constraining them so as to spatially guide their initiation and elongation.

As will be shown in the experimental results below, such a pattern having a wider central region and narrower lateral regions has the following effects on myotubes differentiation: due to its width, the central region is a surface where the cells that are seeded thereon may migrate freely. To the contrary, the narrower lateral regions provide an increased constraint on the cells (said constraint preferably increasing toward the ends of the pattern) and thus guide them along a preferred direction, which is the longitudinal axis of the pattern. The myoblast fusion and myotube elongation is thus controlled along said preferred direction.

Besides, the pattern can be imaged in its entirety by conventional imaging methods and thereby allows measuring the length of the myotubes grown thereon. Hence, the pattern provides additional information regarding the myotubes.

Figures 1 A to 1 D illustrate various embodiments of a cell-adhesive pattern according to the invention. In Figure 1A, the central region 2C of the pattern has a substantially elliptical shape with a maximum width Wc.

Each lateral region 2L has the shape of a portion of ellipse with a maximum width

WL.

The main axes of the ellipses that define the central region and the lateral regions coincide.

The junction between each lateral region and the central region is located approximately at the transversal axis of the ellipse defining the lateral region.

The length of the pattern is referred to as L.

In Figure 1 B, the central region 2C has substantially the shape of two joint ellipses.

The maximum width Wc of the central region is the maximum dimension of both ellipses along their transversal axis.

As in Figure 1A, each region 2L has the shape of a portion of ellipse with a maximum width WL.

The junction between each lateral region and the central region is located approximately at the transversal axis of the ellipse that defines the lateral region.

In Figure 1 C, the central region 2C has substantially the shape of an ellipse, as in Figure 2A.

Each lateral region 2L has the shape of two joint ellipses, wherein the ellipse closest to the central region has a greater width than the ellipse farthest from the central region.

In such case, the maximum width of each lateral region 2L is defined as being the maximum width of the wider ellipse.

The shape of the pattern may not be rounded as shown in Figures 1 A to 2C.

For example, as shown in Figure 1 D, the central region 2C of the pattern has a rectangular shape having a width Wc and each lateral region 2L has a rectangular shape having a width WL.

Of course, the embodiments illustrated in Figures 1A to 1 D are only examples and are not intended to limit the scope of the invention. Any other pattern complying with the above-described dimensional requirements is part of the invention.

Such a pattern is formed on a substrate suitable for screening such as a multi-well plate (e.g. a 96-well plate) (a pattern or a plurality of patterns being formed on the bottom surface of each well) so as to form a device for culturing and maturing myoblasts into myotubes and compatible with screening. While the pattern surface is cell-adhesive, the surrounding surface of the substrate is not cell-adhesive, or less cell-adhesive, so as to hinder myotube growth outside of the pattern.

According to an embodiment, the substrate is a hard substrate typically used for culturing cells, such as glass, silicone, plastics (e.g. polystyrene, polypropylene, polyethylene). By "hard" is meant here that Young's modulus of the substrate is greater than or equal to 1 MPa.

According to another embodiment, the substrate is a soft substrate. By "soft" is meant here that Young's modulus of the substrate is comprised between 5 and 15 kPa.

A Young's modulus around 10 kPa is considered to be the most appropriate for optimal differentiation and maturation [Engler 2008].

Such soft substrates include:

- synthetic hydrogels materials, such as poly(hydroxyethyl methacrylate) (PolyHEMA), polyacrylamide (PAA); polyethylene glycol (PEG), polyacrylic acid, Polyvinyl alcohol) (PVA), polyvinylpyrrolidone, polyimide, polyurethane, etc. and the hybrids of above mentioned materials and their derivatives;

- natural hydrogel materials, such as agarose, dextran, gelatin, matrigel, DNA, polyisocyanopeptides, etc;

- silicone materials,

Such soft materials are already used for culturing cells.

However, the applicant has noted that, although some scientific publications show that soft materials provide a good differentiation of myoblasts into myotubes, which has been obtained with a soft substrate consisting of an hydrogel deposited on a glass coverslip [Serena 2010], the application of a pattern on a soft substrate in a 96-well plate in view of screening does not provide the expected results. Indeed, myotubes newly formed on soft substrate start contracting after 3 days of differentiation and end up detaching from the substrate at Day4. This effect is shown in FIGS 5A and 5B.

FIG. 5A show micrographs (with an enlarged view in the top right corner) of myoblasts M grown on patterns P according to the invention, wherein the patterns have been formed on soft substrates having a Young's modulus of 2 kPa, 10 kPa and 40 kPa, respectively (from left to right), in a growth medium (top) and then in a differentiation medium (bottom). While the myoblasts M cover well the pattern surface in the growth medium, it can be noted that, in the differentiation medium, the myoblasts formed contracted myotubes M' that detach from the soft substrate, thus having a round shape that no longer covers the surface of the patterns P.

FIG. 5B shows the repartition of myotubes phenotypes at DayO (DO), Day3 (D3) and Day4 (D4) in the differentiation medium, for soft substrates having a Young's modulus of 2 kPa, 10 kPa and 40 kPa, respectively. The myotubes have been sorted into intact myotubes (left bar), shortened myotubes (middle bar) and collapsed myotubes (right bar), which no longer adhere to the pattern. While at DayO the major part of the myotubes is intact, the proportion of intact myotubes decreases with time and the myotubes progressively shorten and then collapse. Thus, the preferred substrate for a screening method is a hard substrate, preferably glass.

The patterns can be formed on such substrates by known techniques including:

- microcontact printing [Engler 2004],

- deep UV activation [Tseng 201 1 ],

- micropatterns transfer [Polio 2012],

- photochemical binding [Hahn 2006].

The induction of DMD myoblast differentiation into myotubes typically comprises the following steps. Although the description that follows is directed to DMD myoblasts, it is to be noted that the invention is also applicable to Becker muscular dystrophy myoblasts.

First, a device for culturing said DMD myoblasts and comprising a substrate (e.g. a hard substrate such as glass) and at least one cell-adhesive pattern as described above formed on said substrate is provided.

Then, DMD myoblasts are deposited on at least one cell-adhesive pattern.

The DMD myoblasts are incubated in a growth medium during a given growth time.

After this growth step, the cells are cultured in a differentiation medium during a determined incubation time so as to promote cell differentiation into myotubes and constrain elongation of the myotubes on the cell-adhesive pattern.

For example, for DMD myoblast culture, cells are cultured in growth medium during 24 hours; the seeding conditions were: 15.000 cells/well in 200 μΙ culture medium; the differentiation medium is DMEM/F12, 2% Horse Serum, 0.5% P/S and the incubation time is 5 days. (In the present text, the % used to define a composition refers conventionally to a weight in grams per 100 ml.)

At the end of this incubation time, the cells may be fixed and stained to reveal differentiated myotubes.

Figure 2 shows the evolution of the fusion index over the first 3 days of differentiation. These quantifications demonstrate that patterns according facilitate myoblast differentiation, and subsequently myotube formation, compared to a classic unpatterned culture device. We can clearly see that without patterns (NP) DMD myoblasts show a very low level (0.053) of differentiation after 3 days culture, providing a very poor quantity of material for a cell based assay. Inversely, the culture of DMD myoblasts on patterns according to the invention increases by 339% their differentiation (0.233) in myotubes.

The invention opens up an in vitro physiologically relevant approach of the DMD pathology to cell based assays and drug screening.

Figure 3A shows the representative shape of myotubes differentiated from myoblasts of healthy (left) and DMD (middle and right) donors cultured during 5 days on patterns according to the invention, in control conditions (DMD_CTRL, middle) and after compounds treatment (DMDJGF-1 , right). Immunostaining was realized against myosin heavy chain (in green for color image - in light grey in the appended black&white figures), dystrophin (in red for color image - in medium grey in the appended black&white figures, an enlargement is visible on the top right of the pictures) and nuclei (in blue for color image - in dark grey in the appended black&white figures). Images were acquired using an epifluorescence microscope (DMI6000, Leica). Regarding myotubes shape, one can clearly see that DMD myotubes are smaller and present a reduced differentiation compared to healthy ones. Drug treatment with IGF-1 , a reference compound known to increase myoblast differentiation, partially rescues myotubes formation. Figure 3B is a quantification of some relevant morphological parameters discriminating myotubes from healthy and DMD donors. This histogram shows that, for an equivalent cell number, myotube number is decreased by 66% comparing healthy and DMD donors. IGF-1 treatment rescues by 29% the number of myotubes. Fusion index, which quantify differentiation is decreased by 85% comparing heathy and DMD donors and IGF-1 rescue it by 25%. The width of myotubes is affected by a 48% decrease in DMD compared to heathy donor. IGF-1 slightly rescues 17% of the tube width.

Figure 3C shows that myotubes formed from healthy and DMD myoblasts are equally striated, revealing comparable maturation levels.

Taken together, these data show that by using dedicated patterns according to the invention, IGF-1 partially rescues myotube formation from DMD donors with a similar maturity compared to the ones from the healthy donor.

Figure 4A shows that creatine kinase is largely decreased (- 83%) in DMD myotubes compared to healthy ones, confirming the failure of the plasma membrane the restrain this enzyme within mitochondria. Treatment with prednizone and deflazacort, both palliative compounds used in DMD treatment, increases creatine kinase activity in healthy (respectively 7% and 29%) and DMD myotubes (respectively 26% and 31 %) compared to the controls.

Figure 4B shows that glucose metabolism is reduced (-41 %) in DMD myotubes compared to healthy ones. Prednizone and deflazacort treatments have no effect on this functional readout in both DMD and healthy condition.

This validates that myotubes are functionally affected by drug treatment and that functional readouts can be used to discriminate healthy to DMD myotubes, and mostly in drug discovery for the detection of new compounds recuing DMD myotube physiology.

By allowing DMD myoblast differentiation and maturation into myotubes, patterns according to the invention support the identification of compounds that modulate DMD myotube formation and outgrowth. Fully developed in a HTS/HCS format, method according to the invention allows new drugs detection during screening campaigns dedicated to the DMD pathology. This opens up the first in vitro phenotypic approach to drug discovery on differentiated muscle subunits from donors suffering from DMD.

Cell based assays

One advantage of the invention is its compatibility with HCS cell based assays in the field of DMD drug discovery and compounds screening.

In particular, the invention provides a method for screening compounds driving changes in skeletal muscle cells and in particular myotubes or myoblasts derived from DMD donors.

The invention also provides a method for identifying compounds regulating skeletal muscle differentiation, maturation, atrophy and hypertrophy.

The invention also provides a method for studying molecular mechanisms regulating changes in skeletal muscle cells and in particular myotubes or myoblasts derived from DMD donors.

Patterns according to the invention can be used in DMD drug discovery to characterize compounds mode of action on forming myotubes.

In this regard, the invention provides a method for identifying therapeutic compounds acting on skeletal muscle cells and in particular myotubes or myoblasts derived from DMD donors based on structural or phenotypic (morphology, structure, orientation,...) or functional readouts. Functional readouts include for example impact on myotube contractility metabolism and plasma membrane porosity, that can be evaluated through glucose uptake and creatine kinase release, respectively. Another example of functional readout is the ability of myotube to contract after calcium release within the cytoplasm.

In addition to myoblasts taken from donors suffering from DMD, cell lines, isogenic cell lines, stem cells derived myoblasts recapitulating DMD or Becker pathology (including IPs derived cells, ES cells), can be used with the present method to identifiy candidates that ultimately will lead to the discovery of new drugs having curative or palliative effects on the targeted pathology: for example compounds providing hypertrophy or increasing dystrophin or utrophin expression in myoblasts taken from patient suffering from DMD.

By "curative effects" is meant compounds treatment restoring a native dystrophin expression.

By "palliative effects" is meant compounds improving patient quality of life, for example by reinforcing and stabilising muscles, reducing inflammation, decreasing surgeries.

The screening method typically comprises the following steps. Such a screening method is carried out in vitro.

A device comprising cell-adhesive patterns as described above is provided.

According to a preferred embodiment, the patterns are the patterns of Figure 1A. Myoblasts are deposited on the cell-adhesive patterns. Myoblasts can be obtained from a human donor suffering from a DMD or Becker related lack of dystrophin (including in frame mutations, nonsense mutations, and mutations amenable to exon skipping), from a cell line, an isogenic cell line, derived stem cells (including IPs, ES) recapitulating DMD or Becker pathology. Obtaining the myoblasts is a preliminary step that is not included in the present invention.

The myoblasts are cultured in a differentiation medium so as to promote their differentiation into myotubes.

At a given time, at least one compound is added to the cell culture. The addition of the compound may be carried out at the beginning of myoblasts culture, during incubation of the myoblasts or once the myotubes are mature. The skilled person is able to select the appropriate time of addition of the compound depending on the goal of the assay.

After a determined incubation time of the myotubes with said compound, morphological, structural and/or functional readouts are carried out to determine the effect of said compound on the myotubes. The incubation time depends on the experimental protocol and on the cell type. This incubation time is typically comprised between 2 and 15 days, preferably between 2 and 6 days for human myotubes.

The reproducibility of the morphological parameters of the myotubes between patterns allows determining characteristic features of the myotubes in view of quantifying the effect of the tested compound. Indeed, the effect of the compound can be assessed by measuring morphological changes, such as size, diameter, thickness or width, induced by atrophy or hypertrophy in skeletal muscle cells and in particular myotubes or myoblasts.

In order to characterize compounds mode of action on the myotube model (including myoblasts differentiation, myotube maturation, myotube hypertrophy, myotube atrophy, or cell viability), automated image segmentation and analysis methods has been developed by the inventors using the Acapella software library (PerkinElmer).

First, customized segmentations of myotubes and nuclei are performed.

Then, objects are analyzed to extract basic parameters such as myotube count, nuclei count, myotube morphology (including their length, width, area, and orientation), fusion index (through the percentage of nuclei contained within myotubes). Taking said parameters into account, aberrant myotubes are removed.

Finally, an advanced descriptor is measured, called "maximal width readout", which is the highest value of the internal distance map within remaining myotubes.

By "distance map" is meant the result of an image process involving "distance transform" operation realized after myotube image binarization, which assigns to each pixel the Euclidean distance to the nearest object border point. Distance maps are known in the art and will not need further description. Figure 3B is a representative result of the image processing comparing myotube formation from heathy and DMD donor, with or without IGF-1 treatment, in therm of tube number, fusion index, tube width and the total number of nuclei.

In order to estimate the compatibility of the invention with cell based assay compounds screening, Z'-factors were calculated for each readout. As it was up to 0.4 for "fusion index" and "tube width" readouts, between myotubes from heathy and DMD origins, patterns according to the invention allow a robust segragation of the two populations. Moreover, IGF-1 effect on DMD myotube shows a Z'-factor of 0.2 compared to the untreated condition. This validates the invention as compatible with HCS drug discovery for compounds detection to cure DMD.

REFERENCES

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[Kuang 2007] S Kuang et al, Asymmetric self-renewal and commitment of satellite stem cells in muscle. Cell 129, 999-1010 (2007)

[Abmayr 2012] S.M. Abmayr et al, Myoblast fusion: lessons from flies and mice, Development 139, 641 -656 (2012)

[Schafer 2006] R Schafer et al, Age dependence of the human skeletal muscle stem cell in forming muscle tissue, Artif. Organs 30, 130-140 (2006)

[Gintjee 2014] TJ Gintjee et al, High throughput screening in duchenne muscular dystrophy: from drug discovery to functional genomics, Biology (Basel) 3, 752-780 (2014)

[McElroy 2013] SP McElroy et al, A lack of premature termination codon read- through efficacy of PTC124 (Ataluren) in a diverse array of reporter assays, PLoS Biol. 1 1 , e1001593 (2013)

[Nierobisz 2013] LS Nierobisz et al, High-content screening of human primary muscle satellite cells for new therapies for muscular atrophy/dystrophy, Curr. Chem.

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CLAIMS

1 . Method for screening compounds driving changes in skeletal muscle cells from Duchenne or Becker muscular dystrophy donor or group of donors from a cell line, an isogenic cell line or derived stem cells recapitulating Duchenne or Becker muscular dystrophy, and in particular myotubes or myoblasts, comprising:

(i) providing a culture of myotubes from human primary myoblasts from Duchenne or Becker muscular dystrophy donor or group of donors or from a cell line, an isogenic cell line or derived stem cells recapitulating Duchenne or Becker muscular dystrophy, said culture being obtained by the following method,

a) providing a device comprising a substrate (1 ) and at least one cell- adhesive pattern (2), wherein:

- said at least one cell-adhesive pattern (2) has an elongated surface comprising a central region (2C) and two lateral regions (2L) extending from said central region in both directions along a longitudinal axis of the pattern with a contour discontinuity between the central region (2C) and each lateral region (2L),

- the ratio between the maximum width (Wc) of the central region (2C) and the maximum width (WL) of the lateral regions (2L) is greater than or equal to 2, the length (L) of the pattern being comprised between 100 and 1000 μηη and the maximum width (Wc) of the pattern being comprised between 50 and 500 μηι, and

- the ratio between the length (L) and the maximum width (Wc) of the pattern (2) is less than or equal to 4,

b) depositing human primary myoblasts from Duchenne or Becker muscular dystrophy donor or group of donors or from a cell line, an isogenic cell line or derived stem cells recapitulating Duchenne or Becker muscular dystrophy on at least one cell-adhesive pattern of said device,

c) culturing said pathological myoblasts in a differentiation medium during a determined incubation time so as to promote cell differentiation into myotubes, and constrain elongation of the myotubes on the cell-adhesive pattern;

(ii) adding at least one compound to said culture,

(iii) after a determined incubation time of the myotubes with said compound, carrying out morphological, structural or functional readout of the myotubes to determine the effect of said compound on the myotubes.

2. Method according to claim 1 , wherein said at least one cell-adhesive pattern consists of the partial superposition of three elliptical surfaces: a first elliptical surface defining the central region of the pattern and second and third elliptical surfaces having a major axis coinciding with the major axis of the first ellipse defining the lateral regions of the pattern, wherein the second and third elliptical surfaces intersect the first elliptical surface along their transversal axis.

3. Method according to claim 1 or claim 2, wherein said at least one cell- adhesive pattern is symmetrical according to its longitudinal axis (X) and to a transversal axis (Y) perpendicular to the longitudinal axis.

4. Method according to one of claims 1 to 3, wherein the substrate is a hard substrate. 5. Method according to one of claims 1 to 4, wherein the surface area of said at least one cell-adhesive pattern is comprised between 5,000 and 500,000 μηι2.

6. Method according to one of claims 1 to 5, comprising carrying out image analysis of the myotubes to measure morphological changes in skeletal muscle cells and in particular myotubes or myoblasts.

7. Method according to one of claims 1 to 6, wherein said functional readouts comprise myotube maturation through the expression of specific biomarkers, including dystrophin expression.

8. Method according to one of claims 1 to 7, wherein said functional readouts comprise calcium release and resulting myotube contraction.

9. Method according to one of claims 1 to 8, wherein said functional readouts comprise myotube metabolism through the evaluation of glucose uptake.

10. Method according to one of claims 1 to 9, wherein said structural readouts comprise the integrity of the myotube plasma membrane through the evaluation of creatine kinase release.

1 1 . Method according to claim 6, wherein said measured morphological changes comprise the maximal width of the myotubes after incubation with the compound and wherein image analysis comprises myotube image binarization, computation of a distance map of said myotubes and computation of the maximal width of each myotube from said distance map.

12. Method according to claim 1 1 further comprising counting the myotubes having a maximal width greater than a predetermined value.

13. Method according to claim 6, in the field of drug discovery, for identifying therapeutic compounds acting on atrophy or hypertrophy of skeletal muscle cells, and the image analysis is carried out to determine the effect of said compounds in terms of atrophic or hypertrophic properties.

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