Extraocular muscle satellite cells are high performance myo-engines retaining efficient regenerative capacity in dystrophin deficiency

Abstract

Extraocular muscles (EOMs) are highly specialized skeletal muscles that originate from the head mesoderm and control eye movements. EOMs are uniquely spared in Duchenne muscular dystrophy and animal models of dystrophin deficiency. Specific traits of myogenic progenitors may be determinants of this preferential sparing, but very little is known about the myogenic cells in this muscle group. While satellite cells (SCs) have long been recognized as the main source of myogenic cells in adult muscle, most of the knowledge about these cells comes from the prototypic limb muscles. In this study, we show that EOMs, regardless of their distinctive Pax3-negative lineage origin, harbor SCs that share a common signature (Pax7(+), Ki67(-), Nestin-GFP(+), Myf5(nLacZ+), MyoD-positive lineage origin) with their limb and diaphragm somite-derived counterparts, but are remarkably endowed with a high proliferative potential as revealed in cell culture assays. Specifically, we demonstrate that in adult as well as in aging mice, EOM SCs possess a superior expansion capacity, contributing significantly more proliferating, differentiating and renewal progeny than their limb and diaphragm counterparts. These robust growth and renewal properties are maintained by EOM SCs isolated from dystrophin-null (mdx) mice, while SCs from muscles affected by dystrophin deficiency (i.e., limb and diaphragm) expand poorly in vitro. EOM SCs also retain higher performance in cell transplantation assays in which donor cells were engrafted into host mdx limb muscle. Collectively, our study provides a comprehensive picture of EOM myogenic progenitors, showing that while these cells share common hallmarks with the prototypic SCs in somite-derived muscles, they distinctively feature robust growth and renewal capacities that warrant the title of high performance myo-engines and promote consideration of their properties for developing new approaches in cell-based therapy to combat skeletal muscle wasting.

Keywords: Clonal growth; Cre/loxP; Duchenne muscular dystrophy; Engraftment; Extraocular muscles; FACS; Mdx(4cv); Myf5; MyoD; Myosin light chain 3F-nLacZ; Nestin-GFP; Pax3; Pax7; Renewal; Retractor bulbi; Satellite cells.

Fig. 1

In-situ detection, isolation and cell culture marker signature of EOM SCs from adult mice. (A) Isolated EOM myofibers from adult Nestin-GFP transgenic mice demonstrating co-expression of the SC marker Pax7 and transgenic Nestin-GFP along with DAPI⁺ nuclei. EOM myofibers vary in their dimensions and shown images illustrate “thick” (A1) and “narrow” (A2) specimens. Arrowheads point to examples of SCs defined based on triple-labeling for Pax7, Nestin-GFP and DAPI. (B) A representative flow cytometry profile of Nestin-GFP^high cells isolated from EOM by Pronase digestion. The plot shows GFP fluorescence (X-axis) vs. the side scatter parameter (Y-axis) among all G0–G1 cells. The % value indicates the frequency of the highlighted Nestin-GFP^high population out of the parent G0–G1 population analyzed. The side-scatter parameter, measuring cell granularity (internal complexity, Yablonka-Reuveni, 1988), has been used here to better distinguish the SC population. Additional cell sorting plots of SCs isolated from EOM vs. LIMB and DIA are shown in Fig. S3A and B. (C) Schematic of myogenic marker expression by EOM SCs and their progeny, showing the typical progression through proliferation, differentiation and renewal stages in primary culture (modified from Yablonka-Reuveni, 2011). SCs were sorted as shown in panel B and the expression of the characteristic myogenic markers was analyzed by immunostaining with commonly used antibodies (Danoviz and Yablonka-Reuveni, 2012; Shefer et al., 2006). (D–D‴) Pax7/sarcomeric myosin (MF20) immunostaining of day 14 cultures demonstrating Pax7⁺ renewal cells expressing the Nestin-GFP transgene and located in between the myotubes (MF20⁺). Nuclei of immunostained cultures are highlighted by DAPI counterstaining. Scale bars, 50 μm.

Fig. 2

Growth analysis of LIMB, DIA and EOM SC primary cultures. SCs were isolated from adult Nestin-GFP mice as described in Figs. 1B and S3A and plated at 1000 cells per well in 24-well plates. (A–F) Representative phase images of day 5 and day 9 cultures. (G) The number of total nuclei per microscopic field (20 × objective) was counted based on DAPI staining of cultures fixed on days 5, 7, 10 and 14. Data are expressed as mean ± SEM. Per each culture day, one-way ANOVA analysis reveals a statistical difference (p<0.01) between the three muscle groups, except for DIA vs. EOM cultures on day 5 (p=0.36), and LIMB vs. DIA cultures on day 10 (p=0.05) and on day 14 (p=0.86). (H–J′) Representative fluorescent images of day 10 LIMB, DIA and EOM cultures double-immunostained for Pax7 and sarcomeric myosin (MF20), demonstrating the presence of Pax7⁺/Nestin-GFP⁺ renewal cells. (K) Quantification of differentiated and renewal cells on days 7, 10 and 14 of LIMB, DIA and EOM cultures immunostained for Pax7 and sarcomeric myosin (MF20). Scale bars, 100 μm (A–F) and (H–J′).

Fig. 3

Clonal analysis of LIMB, DIA and EOM SCs. SCs were isolated from adult Nestin-GFP mice as described in Figs. 1B and S3A. (A–D) Clonal cultures were fixed on day 10 and stained with DAPI for quantifying clone size according to the number of nuclei. (A and B) Examples of (A) low-magnification whole clone images (DAPI stained), and (B) high-magnification phase images demonstrating the typical morphology and cell density of average size LIMB, DIA and EOM clones. (C) Distribution of LIMB, DIA and EOM clones according to the number of nuclei per clone. Clones are clustered in bins in ascending order according to the number of nuclei per clone (x-axis) vs. the percentage of clones in each bin out of total clones analyzed (histograms, left y-axis). In the cumulative curves, each data point (round-shaped, right y-axis) shows the percentage of clones out of total clones analyzed that contain less than, or equal to (≤) the corresponding number of nuclei per clone size range indicated on the x-axis bins. (D) Clonal data analyzed in (C) are also illustrated as boxplots, depicting the quartile distribution, mean (black crosses) and outliers (red circles, right y-axis) for the number of nuclei per clone; whisker ranges and outliers were calculated as previously detailed by us in (Shefer et al., 2013). Asterisk denotes statistically significant difference in clone size between EOM vs. LIMB and DIA clones (p<0.01). (E) Representative phase and fluorescence images of day 21 clones established from LIMB, DIA and EOM SCs showing Nestin-GFP⁺ renewal cells with a typical higher manifestation in EOM clones. (F) Quantification of SC clones that harbor Nestin-GFP⁺ cells demonstrates that renewal cells are consistently more frequent in EOM clones. The relative clone size was also estimated by categorizing each clone as small, medium, large or X-large depending on whether the clone occupied a 20×, 10 ×, 4 × or several 4 × objective fields, respectively. On day 7, only EOM cultures displayed clones in the “Large” category. Then, by day 21, solely EOM harbored “X-Large” clones, while clones falling in the two smaller categories represented only 30% of total clones for EOM vs. 60% for LIMB cultures. Scale bars, 1 mm (A), 250 μm (B), 50 μm (E).

Fig. 4

Assessment of in-vivo proliferative activity of SCs. Unsorted cell preparations obtained after Pronase digestion of LIMB, DIA and EOM of adult (4–6 month old) and juvenile (3-week old) mice were subjected to cytocentrifugation, followed by Pax7/Ki67 double immunostaining combined with DAPI counter-staining. (A) Image shown is of a preparation from LIMB of juvenile mice, which contains the highest number of Ki67⁺/Pax7⁺ double-labeled cells. (B) SCs were identified based on Pax7 immunostaining and their proliferative activity was determined by calculating the percent of cells double-immunolabelled for Pax7 and Ki67 (Ki67⁺/Pax7⁺ cells) out of all Pax7⁺ cells. Preparations of spleen cells were used as a highly proliferative control for Ki67 immunolabeling (Ki67⁺ cells: 48.1% out of 7998 and 18.0% out of 4324 total nuclei, in juvenile and adult mice, respectively); these cells were also found negative for Pax7. Scale bar, 50 μm.

Fig. 5

H&E stained cross sections of tissue preparations from mdx^4cv mice. (A) TA, (B) DIA, and (C–F) periocular tissues from 1 year old mice. (C) Schematic of a periocular tissue preparation that comprises the EOMs, the retractor bulbi (RB) and the optic nerve (OptN), harvested together with the eyeball. The black dotted line indicates the level of the sections shown in: (D) periocular tissue (low magnification), (E) RB and (F) EOM (high magnification). Scale bars, 50 μm (A and B) and (E and F), 200 μm (D).

Fig. 6

EOM SCs from dystrophin-null mdx^4cv mice retain performance superiority. LIMB, DIA and EOM SCs were isolated from Nestin-GFP (wt) and mdx^4cv/Nestin-GFP (mdx) 6 month old mice by flow cytometry according to Nestin-GFP expression combined with an antigen-based sorting approach (Fig. S3B), which permitted assaying the level of CD45⁺ cells as a reference for the inflammatory process associated with dystrophinopathy. (A) For each muscle group, the number of total nuclei was counted based on DAPI staining of cultures fixed on days 3–7. Data are expressed as mean (±SEM) number of nuclei per 10 microscopic fields (20 × objective) and not per single field as in Fig. 2G due to the low number of cells in mdx LIMB and DIA cultures. Within each muscle group, asterisks denote statistically significant differences (p<0.03, one-way ANOVA) for wt vs. mdx cultures per each culture day; notably, there are no statistical differences between EOM wt vs. mdx at any of the culture days. (B) Representative images of day 14 cultures of LIMB, DIA and EOM depict the meager growth of mdx cultures from LIMB and DIA SCs, while also highlighting the outperformance of EOM SCs within the wt groups. Among the mdx cultures, only those from EOM developed Nestin-GFP⁺ renewal cells. Scale bar, 100 μm. (C) Clonal cultures of LIMB, DIA and EOM SCs isolated from wt and mdx mice were quantified on day 10 in the same manner as in Fig. 3A–D. Clones are plotted individually based on total number of DAPI⁺ nuclei per clone. One-way ANOVA analysis reveals a statistical difference (p<0.03, asterisks) between wt and mdx clones within LIMB and DIA, but not within EOM (p=0.13). A statistical difference (p<0.03) was also noted between EOM clones vs. LIMB and DIA regardless of mdx or wt origin. (D) Summary of additional relevant data from the analysis of the sort profiles, and the primary and clonal cultures. The percentage of CD45⁺ cells was determined by using a distinctive fluorochrome (PE-Cy7 for anti-CD45 vs. APC for anti-CD31 and anti-Sca1) when gating out the CD31⁺, CD45⁺, and Sca1⁺ cells. For all panels, asterisks denote statistically significant differences (p<0.03).

Fig. 7

X-gal staining and PCR analysis demonstrating higher engraftment efficiency of EOM vs. LIMB SCs following intra-muscular transplantation into the TA muscles of host Rag1^−/−/mdx^5cv mice. (A) EOM and LIMB SCs were isolated from double transgenic Nestin-GFP/MLC3F-nLacZ mice (4–5 month old, 3 donors per experiment) as described in Figs. 1B and S3A and injected into the TAs of Rag1^−/−/mdx^5cv host mice (8–9 week old, 3–4 hosts per experiment). For each host animal, the TA from one leg was injected with EOM donor SCs, while the TA from the contralateral leg was injected with LIMB donor SCs. The level of donor-derived contribution in host muscles was then determined 3-weeks post transplantation. (B) Images of X-gal stained host TAs from one representative mouse, demonstrating the expression of the MLC3F-nLacZ reporter (from transplanted donor SCs) in myofiber nuclei. TAs were first cut longitudinally into 2 halves before fixation and were further trimmed after X-gal reaction to obtain more in-depth view and better imaging of the sites containing LacZ positive nuclei. Scale bars, for each injected TA from left to right, 0.5 mm, 1 mm and 0.1 mm. (C) Examples of PCR products used to quantify the level of donor-derived genomic material (LacZ and GFP) in comparison to the level of the reference genes contributed by host and donor material (Cyp1A1 and H-ras). (D) Each donor gene product (LacZ and GFP) was first normalized with each of the reference gene products (Cyp1A1 and H-ras), resulting in a total of 4 normalized values (2 per each donor gene). The EOM/LIMB ratio was then calculated for each of these normalized values. Given that within the same mouse these ratios are comparable regardless of the donor or reference gene used, final results are presented per each mouse as the average of these ratios. Black histograms denote mice demonstrating a higher engraftment of EOM SCs (~1.8–5.6 fold over LIMB SCs engraftment), while gray histograms denote mice with no apparent distinctions between EOM and LIMB SC engraftment. For both X-gal staining and PCR analysis, controls with vehicle only injected TAs were found negative for donor-derived signal, ensuring the reliability of detection.