Myogenic potential of canine craniofacial satellite cells

Abstract

The skeletal fibers have different embryological origin; the extraocular and jaw-closer muscles develop from prechordal mesoderm while the limb and trunk muscles from somites. These different origins characterize also the adult muscle stem cells, known as satellite cells (SCs) and responsible for the fiber growth and regeneration. The physiological properties of presomitic SCs and their epigenetics are poorly studied despite their peculiar characteristics to preserve muscle integrity during chronic muscle degeneration. Here, we isolated SCs from canine somitic [somite-derived muscle (SDM): vastus lateralis, rectus abdominis, gluteus superficialis, biceps femoris, psoas] and presomitic [pre-somite-derived muscle (PSDM): lateral rectus, temporalis, and retractor bulbi] muscles as myogenic progenitor cells from young and old animals. In addition, SDM and PSDM-SCs were obtained also from golden retrievers affected by muscular dystrophy (GRMD). We characterized the lifespan, the myogenic potential and functions, and oxidative stress of both somitic and presomitic SCs with the aim to reveal differences with aging and between healthy and dystrophic animals. The different proliferation rate was consistent with higher telomerase activity in PSDM-SCs compared to SDM-SCs, although restricted at early passages. SDM-SCs express early (Pax7, MyoD) and late (myosin heavy chain, myogenin) myogenic markers differently from PSDM-SCs resulting in a more efficient and faster cell differentiation. Taken together, our results showed that PSDM-SCs elicit a stronger stem cell phenotype compared to SDM ones. Finally, myomiR expression profile reveals a unique epigenetic signature in GRMD SCs and miR-206, highly expressed in dystrophic SCs, seems to play a critical role in muscle degeneration. Thus, miR-206 could represent a potential target for novel therapeutic approaches.

Keywords: differentiation; dystrophic muscle; microRNA; presomitic and somitic satellite cells.

Figure 1

Characterization of canine satellite cells (SCs). (A,B) Phase contrast morphology of freshly isolated SCs (A) from somitic (SDM) and presomitic (PSDM) muscles biopsies; bar = 50 μm. SCs were cultured at low density to generate clones (B) and cells were maintained in culture until they stop to divide; bar = 250 μm. Growth curves of (C) SDM- and PSDM-SCs from young donors; (D) SDM- and PSDM-SCs from old donors; (E) SDM and PSDM-SCs from dystrophic dogs. Telomerase activity (F) in SCs collected at early and late passages from SDM and PSDM. Data are represented as mean ± SD.

Figure 2

Expression of myogenic markers in canine SCs. (A) Desmin positive (Desm⁺) SCs were quantified (B) for WT young SCs isolated from SDM and PSDM muscle biopsies; for WT old SCs isolated from SDM and PSDM; similarly the percentage of Desm⁺ SCs in SDM and PSDM isolated from GRMD dogs. (C,D) Examples of immunofluorescence analysis for Pax7 and MyoD of WT SDM-SCs (C) and PSDM-SCs (D). Quantifications of Pax7 and MyoD positive SCs (E) normalized to percentage of Des⁺ from WT SDM and PSDM are shown; in (F) are reported SCs from dystrophic dogs SDM and PSDM. Myogenin expression in SCs during differentiation was measured by quantitative real-time PCR (G). Myogenin is upregulated after 7 days of differentiation in all samples expect in GRDM SDM-SCs. Three independent experiments were performed in triplicates and statistically analyzed using Dunn’s Multiple Comparison Test. MyHC staining (H) was used to quantify the FI (I) of SCs isolated from SDM and PSDM of WT young; from SDM and PSDM of WT old and of GRMD SCs isolated from SDM and PSDM. Data are represented as mean ± SD and statistically analyzed using t-test; *p < 0.05. Bar = 50 μm.

Figure 3

Oxidant and calcium levels. Quantification of intracellular ROS levels by DCFH-DA assay expressed as a percentage respect to CTRL in SCs isolated from SDM (A,B) PSDM. (C) Quantification of $O_2^{-}$ by rate reduction of NBT. The OD 550 nm represents the quantity of formazan revealed spectrophotometrically. (D) Basal levels of [Ca2⁺]_i expressed in nanomole measured on undifferentiated living cells by video-imaging technique. Data are represented as mean ± SD and statistically analyzed using t-test; *p < 0.05; **p < 0.0001.

Figure 4

Relative miR-133a, miR-133b, miR-1, and miR-206, expression in canine SCs measured by quantitative real-time PCR. SCs were isolated from SDM and PSDM of young dogs. Note that miR-133a (A) and miR-133b (B) are upregulated only in PSDM-SCs after 7 days of differentiation (t7). miR-1 is upregulated only in PSDM-SCs (C) after 7 days of differentiation. At variance, miR-206 is highly expressed in PSDM-SCs during proliferation (D). The ubiquitously produced miR-16 was used as an internal control. Three independent experiments were performed in triplicates and statistically analyzed using ANOVA. Data are presented as mean ± SD; *p < 0.05.

Figure 5

Relative miR-133a, miR-133b, miR-1, and miR-206, expression in old canine PSDM-SCs measured by quantitative real-time PCR. In old PSDM-SCs, miR-133a (A) and miR-133b (B) are downregulated in both proliferating and differentiating cells. miR-1 and miR-206 regulations seem age-dependent, since old PSDM-SCs did not upregulate miR-1 during differentiation (C), while miR-206 is not highly expressed during proliferation (D). The ubiquitously produced miR-16 was used as an internal control. Three independent experiments were performed in triplicates and statistically analyzed using ANOVA; Data are presented as mean ± SD; *p < 0.05.

Figure 6

Relative miR-133a, miR-133b, miR-1, and miR-206 expression in GRMD SCs measured by quantitative real-time PCR. SCs were isolated from SDM and PSDM of GRMD dogs. Consistent with data obtained with WT canine SCs, miR-133a and miR-133b are highly expressed after 7 days of differentiation in PSDM-SCs. At variance, miR-133a and miR-133b are upregulated in SDM-SCs after 7 days of differentiation (A,B). Intriguingly, miR-1 and miR-206 are highly upregulated after 7 days of differentiation in PSDM-SCs (C,D). Three independent experiments were performed in triplicates and statistically analyzed using ANOVA; miR-16 was used as an internal control. Data are presented as mean ± SD; *p < 0.05.