SERUM-FREE MEDIUM FOR DIFFERENTIATION OF A PROGENITOR CELL

Abstract

The invention relates inter alia to a method for differentiating a muscle progenitor cell, comprising the step of: culturing a muscle progenitor cell in a serum-free medium for differentiating a muscle progenitor cell, wherein said serum-free medium comprises at least one differentiation inducer selected from the group consisting of a lysophosphatidic acid receptor 1 (LPAR1) agonist, a lysophosphatidic acid receptor 3 (LPAR3) agonist, an oxytocin receptor (OXTR) agonist, a glucagon receptor (GCGR) agonist, a lactate and a Notch signaling pathway inhibitor.

Claims

1. A method for differentiating a muscle progenitor cell, comprising the step of: culturing a muscle progenitor cell in a serum-free medium for differentiating a muscle progenitor cell, wherein said serum-free medium comprises at least one differentiation inducer selected from the group consisting of a lysophosphatidic acid receptor 1 (LPAR1) agonist, a lysophosphatidic acid receptor 3 (LPAR3) agonist, an oxytocin receptor (OXTR) agonist, a glucagon receptor (GCGR) agonist, a lactate and a Notch signaling pathway inhibitor.

2. The method according to claim 1, wherein said serum-free medium comprises (i) at least one differentiation inducer selected from the group consisting of a lysophosphatidic acid receptor 1 (LPAR1) agonist, a lysophosphatidic acid receptor 3 (LPAR3) agonist, an oxytocin receptor (OXTR) agonist, a glucagon receptor (GCGR) agonist and a lactate, and (ii) a Notch signaling pathway inhibitor.

3. The method according to claim 1, wherein said lysophosphatidic acid receptor 1 (LPAR1) agonist and/or said lysophosphatidic acid receptor 3 (LPAR3) agonist is a lysophosphatidic acid.

4. The method according to claim 1, wherein said oxytocin receptor (OXTR) agonist is oxytocin.

5. The method according to claim 1, wherein said glucagon receptor (GCGR) agonist is glucagon.

6. The method according to claim 1, wherein said Notch signaling pathway inhibitor is a gamma-secretase inhibitor.

7. The method according to claim 1, wherein said Notch signaling pathway inhibitor is a compound selected from the group consisting of DAPT, E2012, L685458, R04929097 and LY-411575.

8. The method according to claim 1, wherein said method is a method for proliferating a muscle progenitor cell followed by differentiating proliferated muscle progenitor cells, wherein said method further comprises, prior to differentiating said muscle progenitor cell, a step of: culturing a muscle progenitor cell in a serum-free medium for proliferating muscle progenitor cells, to thereby provide proliferated muscle progenitor cells.

9. The method according to claim 1, wherein said method for differentiating and/or said method for proliferating of a muscle progenitor cell followed by differentiating proliferated muscle progenitor cells, is an (entirely) serum-free method.

10. The method according to claim 1, wherein said muscle progenitor cell is a bovine muscle progenitor cell, preferably a bovine (myo)satellite cell.

11. The method according to claim 1, wherein said culturing of said muscle progenitor cell in said serum-free medium for differentiating is performed under conditions that allow for differentiation of said muscle progenitor cell into a myocyte, myotube and/or myofiber.

12. The method according to claim 11, further comprising the step of: incorporating said myocyte, myotube and/or myofiber into a meat product for human consumption, optionally in combination with adipocytes.

13. The method according to claim 1, wherein the serum-free medium for differentiating further comprises: (i) an epidermal growth factor (EGF) or a replacement thereof; (ii) an albumin or a replacement thereof; (iii) a source of glucose and/or a source of glutamine; (iv) a source of iron or an iron transporter; (v) ascorbic acid or a derivative thereof; (vi) sodium selenite; (v) ethanolamine; (vi) insulin; or (vii) sodium bicarbonate.

14-21. (canceled)

22. The method according to claim 1, wherein the serum-free medium for differentiating comprises: at least one differentiation inducer selected from the group consisting of a lysophosphatidic acid receptor 1 (LPAR1) agonist, a lysophosphatidic acid receptor 3 (LPAR3) agonist, an oxytocin receptor (OXTR) agonist, a glucagon receptor (GCGR) agonist and a lactate; an epidermal growth factor (EGF) or a replacement thereof; an albumin or a replacement thereof; a source of glucose and a source of glutamine; a source of iron or an iron transporter; ascorbic acid or a derivative thereof; sodium selenite; ethanolamine; insulin; and sodium bicarbonate; and optionally a Notch signaling pathway inhibitor.

23. The method according to claim 1, wherein all components in said serum-free medium for differentiating and/or proliferating are animal-free.

24. A serum-free medium for differentiating a muscle progenitor cell, wherein said medium is as defined in claim 1.

25. A composition comprising a serum-free medium for differentiating as defined in claim 24 and a muscle progenitor cell and/or a partially or terminally differentiated cell such as a myocyte, myotube and/or myofiber.

26. The serum-free medium according to claim 24, wherein said muscle progenitor cell is a bovine muscle progenitor cell.

27. A culture of myocytes, myotubes and/or myofibers obtainable by a method according to claim 1.

28. A meat product, comprising myocytes, myotubes and/or myofibers obtainable by a method according to claim 1.

29. The culture according to claim 27, wherein said culture is devoid of serum components such as fetal bovine serum components.

30. The composition according to claim 25, wherein said muscle progenitor cell is a bovine muscle progenitor cell.

31. The meat product according to claim 28, wherein said culture or said meat product is devoid of serum components such as fetal bovine serum components.

32. The meat product of claim 28 further comprising adipocytes

33. The meat product of claim 32 wherein the adipocytes are bovine adipocytes.

Description

FIGURE LEGENDS

[0133] FIG. 1 shows myogenic differentiation of bovine muscle progenitor cells into myocytes in a serum-free myogenic differentiation medium as disclosed herein containing the agonists (differentiation inducers) of the identified overexpressed receptors or lactate. Before myogenic differentiation, the cells were expanded in a serum-free proliferation medium.

[0134] FIG. 2 shows myogenic differentiation of bovine muscle progenitor cells into myocytes in a serum-free myogenic differentiation medium as disclosed herein, whereby said agonists (differentiation inducers) of the identified overexpressed receptors or lactate where added in a range of concentrations. In FIG. 2, LPA refers to lysophosphatidic acid, OT refers to oxytocin and GCG refers to glucagon.

[0135] FIG. 3.

Notch signaling inhibits myogenic differentiation in a subpopulation of satellite cells, which can be improved by the addition of an inhibitor of Notch signaling such as a gamma secretase inhibitor like DAPT. More specifically, FIG. 3 shows myogenic differentiation of bovine muscle progenitor cells into myocytes in a serum-free myogenic differentiation medium with the addition of DAPT in a range of concentration (1 M, 5 uM and 10 uM) in comparison to the control serum-free myogenic differentiation medium in which DAPT is absent.

EXAMPLES

Example 1. RNA Expression During Myogenic Differentiation

Materials and Methods

Myogenic Differentiation

[0136] Bovine satellite cells were differentiated on Matrigel coated flasks by seeding 510.sup.5 cells/cm.sup.2 in growth medium containing 20% bovine serum. After 24 h, differentiation was induced by decreasing serum concentration from 20% to 2%.

RNA Isolation

[0137] RNA lysates were harvested from tissue culture samples by directly adding TRK lysis buffer onto the samples after removing culture media and washing with PBS. The RNA was purified by using the Omega MicroElute Total RNA Kit (Bio-Rad) following the supplier's protocol for tissue culture. RNA concentrations were determined by nanospectrometry.

[0138] Pre-sequencing quality control was performed using a bioanalyzer. The library was prepared using the TruSeq stranded mRNA kit (Illumina) and sequenced with 12 samples per run on a high-output 75 bp NextSeq flowcell. On average, 37.0*10{circumflex over ()}6 (7.3*10{circumflex over ()}6) of aligned reads per samples were obtained.

Read Alignment and Quantification

[0139] The single-end reads were aligned to the reference genome tau9 Bos_taurus.ARS-UCD1.2.98.gtf using STAR aligner (Dobin et al., Bioinformatics 29, 15-21 (2013)) and assigned to genes using FeatureCounts function of the Rsubread package (Liao et al., Bioinforma. Oxf. Engl., 30:923-930 (2014)). On average, 78.86% (1.04%) of reads were uniquely assigned to genes.

Quality Control and Normalization

[0140] Next, a DGEList-object was created using the obtained count matrix and gene meta information from the Btaurus_gene_ensembl data set (Yates et al., Ensembl 2020. Nucleic Acids Res. 48, D682-D688 (2020); Robinson et al., Bioinforma. Oxf. Engl., 26, 139-140 (2010)). Low-abundance genes (min total count 15 below, expressed in at least 3 of 4 replicates) were removed and normalization factors were calculated using the trimmed-mean of M-values (TMM) method in the NormFactor function of edgeR (Robinson et al., Genome Biol., 11, R25 (2010); Anders et al., Genome Biol. 11, R106 (2010)). Finally, counts per million (cpm) and reads-per-kilobase per million were computed based on the normalized library sizes.

Dimensionality Reduction and Differential Expression Analysis

[0141] Principal component analysis was performed using the 500 most variable genes based on the variance of RPKMs. Differential expression analysis was performed for each gene between each day of differentiation by empirical eBayes moderation towards a common value with a lfc threshold of log(1.2) (McCarthy et al., Nucleic Acids Res. 40, 4288-4297 (2012); Ritchie et al., Nucleic Acids Res. 43, e47 (2015)). Genes were considered differentially expressed (DE) above a log-FC cutoff of 1 and a FDR below 5% and visualized in respective volcano plots. A heatmap showing the z-values of the 1000 most differentially expressed genes between DO and D1 was constructed in which samples were clustered using the Ward's minimum variance method with euclidean distances (Ward, J. Am. Stat. Assoc. 58, 236-244 (1963)). Finally, over-represented gene ontology (GO) terms were computed for both upregulated and downregulated DE genes (Ashburner et al., The Gene Ontology Consortium. Nat. Genet. 25, 25-29 (2000); The Gene Ontology Consortium, Nucleic Acids Res. 47, D330-D338 (2019)).

Results

[0142] The RNA sequencing experiments provided for the identification of receptors that are upregulated in the early phase of myogenic differentiation, which allows for the identification of myogenic differentiation inducers. Table 1 identifies, by analysis of RNA sequencing data obtained during early phase (day0/day1) myogenic differentiation under serum-containing conditions, upregulated genes coding for membrane receptors, with an indication of respective agonists to be used as inducers under serum-free conditions.

TABLE-US-00001 TABLE 1 Identification of differentiation inducers from RNAseq during serum-containing differentiation. Ratio Day 0/Day 1 Upregulation (log.sub.2)* (Ratio) Gene Name Agonist 4.693 25.9 LPAR1 lyso- lyso- phosphatidic phosphatidic acid receptor 1 acid 3.559 11.8 LPAR3 lyso- lyso- phosphatidic phosphatidic acid receptor 3 acid 3.429 22.6 OXTR oxytocin oxytocin receptor 2.051 4.1 GCGR glucagon glucagon receptor *a negative value means upregulation on day 1 compared with day 0.

Example 2. Production of a Serum-Free Medium of the Invention

[0143] An exemplary serum-free medium for differentiation of a progenitor cell as disclosed herein was prepared as follows.

[0144] The DMEM/F12 1:1 or 10:1 or 1:0 or alpha-MEM or M199 medium was supplemented with albumin (recombinant human albumin from Richcore) at 0.5 mg/ml, insulin (recombinant human insulin, 10-365 from Peprotech) at 19.4 ug/ml, transferrin (recombinant human transferrin, 10-366 from Peprotech) at 10.7 ug/ml, sodium selenite (S5261 from SigmaAldrich) at 0.014 ug/ml, Ethanolamine from Sigma Aldrich cat nr E9508 at 4 ug/ml, ascorbic acid (L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, A8960 from SigmaAldrich) at 115.22 ug/ml, EGF (recombinant human EGF, AF-100-15 from Peprotech) at 10 ng/ml and one of the following inducers: lactate (Sodium L-lactate, 71718 from SigmaAldrich) at 10 mM, LPA (Oleoyl-L--lysophosphatidic acid sodium salt, L7260 from SigmaAldrich) at 5 uM, oxytocin (06379 from SigmaAldrich) at 50 nM, or glucagon (G2044 from SigmaAldrich) at 1 uM.

Example 3. Myogenic Differentiation of Progenitor Cells

Materials and Methods

[0145] Bovine muscle progenitor cells were isolated from a bovine muscle tissue (Bos taurus) and sorted based on their positive expression of CD29 as previously described (Ding et al., Sci. Rep., 17(8): 10808 (2018)). Muscle progenitor cells were propagated in a serum-free proliferation medium for at least 8 population doublings prior to differentiation. Briefly, the cells were seeded at a density of 5000 cells/cm.sup.2 in a serum-free proliferation medium (albumin (5 mg/ml), somatotropin (2 ng/ml), L-Ascorbic acid 2-phosphate (50 g/ml), hydrocortisone (36 ng/ml), -linolenic acid (1 g/ml), insulin (10 g/ml), transferrin (5.5 g/ml), sodium selenite (0.0067 g/ml), ethanolamine (2 g/ml), L-alanyl-L-glutamine or glutamine (2 mM), IL-6 (5 ng/ml), FGF2 also referred to as bFGF (10 ng/ml), IGF1 (100 ng/ml), VEGF (10 ng/ml), HGF (5 ng/ml), PDGF-BB (10 ng/ml) and DMEM/F12 basal medium) in an appropriate collagen-coated cell culture vessel. The cells were passaged upon reaching 90% confluency by rinsing once with phosphate buffer saline (PBS, 20012027 from ThermoFischer Scientific) followed by the addition of trypsin (25200072 from ThermoFischer Scientific). Once the cells were detached, trypsin was neutralised by the addition of trypsin inhibitor from Glycine max (T6522 from Sigma Aldrich), the cells collected into PBS and centrifuged at 350 g. The supernatant was aspirated and the cell pellet resuspended in said serum-free proliferation medium. The cells were plated in said serum-free proliferation medium onto a Matrigel Matrix (356230 from Corning)-coated cell culture vessel at a density of 37500 cells/cm.sup.2. Differentiation was induced by changing the serum-free proliferation medium to the serum-free differentiation media as indicated in Example 2 (with DMEM/F12 1:1 as basal) 24 h later.

Results

[0146] It was observed that induction of serum-free myogenic differentiation could be achieved in cells previously proliferated in serum-free proliferation medium by incorporating agonists of the identified overexpressed receptors or lactate as myogenic differentiation inducers into a serum-free differentiation medium having different basal formulations, as indicated (FIG. 1). It was further established that a range of concentrations of said differentiation inducers can successfully be employed to induce said myogenic differentiation (FIG. 2).

Example 4. Upregulation of Notch Signaling Receptors During Myogenic Differentiation

[0147] As an add-on to Example 1, it was discovered that notch signaling receptors NOTCH2 and NOTCH3 are upregulated in a subset of satellite cells during myogenic differentiation (Table 2).

TABLE-US-00002 TABLE 2 Differentially expressed genes in the Notch pathway Ratio Day 0/Day 1 Upregulation Pathway (log2) (Ratio) Gene Name Inhibitor 0.906 1.87 NOTCH2 notch receptor 2 DAPT 4.156 17.8 NOTCH3 notch receptor 3 DAPT

Example 5. Myogenic Differentiation of Progenitor Cells with or without Notch Pathway Inhibition

Materials and Methods

[0148] Myogenic progenitor cells as disclosed in Example 2 were plated in Matrigel-coated (1:200 in PBS) plates at a seeding density of 40 k cells/cm{circumflex over ()}2 and cultured for 24 h in a serum-free proliferation medium as disclosed in Example 2. Myogenic differentiation was induced by adding an exemplary serum-free differentiation medium (Table 3) in the presence and absence of DAPT (gamma-Secretase inhibitor #ab120633, abcam) at 0 M (absence of DAPT; control medium), 1 M, 5 M and 10 M. Cells were imaged with brightfield microscopy and the differentiation phenotypes were compared.

TABLE-US-00003 TABLE 3 Exemplary serum-free differentiation medium of the invention DMEM (Gibco catalog number 22320- 022) + pyruvate, glutamax, glucose ITSE (insulin-transferrin-sodium 2% selenite-ethanolamine) Sodium Bicarbonate 6.5 mM MEM AA solution 50x (Gibco catalog 0.5%.sup. number 11130051) Soy Hydrolysates (Sigma Aldrich - 1% 58903C) PSA (Penicillin Streptomycin 1% Amphotericin) Lactate (inducer) 10 mM Vitamin C 40 M Albumin 0.5 mg/ml EGF 10 ng/ml

Results

[0149] It was observed that Notch signaling inhibits myogenic differentiation in a subpopulation of bovine satellite cells, which can be improved by the addition of DAPT, a gamma secretase inhibitor. FIG. 3 shows improved myogenic differentiation of bovine muscle progenitor cells into myocytes in a serum-free myogenic differentiation medium with the addition of DAPT in a range of concentration (1 uM, 5 uM and 10 uM) as compared to the control medium which does not include DAPT. It was observed that the addition of the -secretase inhibitor DAPT during myogenic differentiation increases the number of cells that fuse into myotubes. DAPT was shown to improve differentiation at various concentrations ranging between 1 and 10 uM.