Skeletal muscle pericyte subtypes differ in their differentiation potential

Abstract

Neural progenitor cells have been proposed as a therapy for central nervous system disorders, including neurodegenerative diseases and trauma injuries, however their accessibility is a major limitation. We recently isolated Tuj1+ cells from skeletal muscle culture of Nestin-GFP transgenic mice however whether they form functional neurons in the brain is not yet known. Additionally, their isolation from nontransgenic species and identification of their ancestors is unknown. This gap of knowledge precludes us from studying their role as a valuable alternative to neural progenitors. Here, we identified two pericyte subtypes, type-1 and type-2, using a double transgenic Nestin-GFP/NG2-DsRed mouse and demonstrated that Nestin-GFP+/Tuj1+ cells derive from type-2 Nestin-GFP+/NG2-DsRed+/CD146+ pericytes located in the skeletal muscle interstitium. These cells are bipotential as they generate either Tuj1+ cells when cultured with muscle cells or become "classical" α-SMA+pericytes when cultured alone. In contrast, type-1 Nestin-GFP-/NG2-DsRed+/CD146+ pericytes generate α-SMA+pericytes but not Tuj1+ cells. Interestingly, type-2 pericyte derived Tuj1+ cells retain some pericytic markers (CD146+/PDGFRβ+/NG2+). Given the potential application of Nestin-GFP+/NG2-DsRed+/Tuj1+ cells for cell therapy, we found a surface marker, the nerve growth factor receptor, which is expressed exclusively in these cells and can be used to identify and isolate them from mixed cell populations in nontransgenic species for clinical purposes.

Figure 1. FDB-derived Nestin-GFP+/Tuj1+ cells, cultured for 7 days, express some pericytic markers

A. Antibodies against α-SMA and Connexin-43 (Cx43) recognized a population of pericytes but did not overlap with nestin-GFP+ neural cells, while other pericytic markers (CD146, PDGFRβ, and NG2) were found in both pericytes and nestin-GFP+/Tuj1+ cells. Nuclei were stained with Hoechst. B. Percent of cells expressing various cell markers in Nestin-GFP+ or Nestin-GFP- cells. Cells expressing α-SMA, Cx43, CD146, PDGFRβ, and NG2 in Nestin-GFP+ (yellow) or Nestin-GFP- (red) populations. Nestin-GFP+ cells that did not express the marker are in green. Markers are described in the x-axis. Note that Nestin-GFP+ neural cells lack some pericytic markers (α-SMA and Cx43), while expressing others (CD146, PDGFRβ, and NG2). NGFR (p75) identifies neural progenitors in 7 day-old FDB-derived cultures. NGFR was detected exclusively in nestin-GFP+/Tuj1+ cells. Representative immunocytochemistry of Nestin-GFP and NGFR fluorescence overlap is shown in A. NGFR overlapped with GFP which is expressed in the germline under the nestin control element in the Nestin-GFP transgenic mouse. C. Percent of cells sharing nestin-GFP and NGFR (p75) expression demonstrating that all NGFR overlapped with Nestin-GFP+ neural cells. No Nestin-GFP- cells expressed NGFR (p75) in culture. Data are mean ± s.e.m. (n=3). Scale bar = 100 μm.

Figure 2. FDB-derived DsRed+ cells from NG2-DsRed mice lose NG2-DsRed fluorescence with time in culture despite persistent NG2 protein expression

A. Cells were grown for 10 days and fixed at days 3 and 10. More than half kept the intense DsRed fluorescence at day 3, but it was drastically reduced by day 10. B. Percent of DsRed+ and DsRed- cells at days 3 and 10 in the NG2 protein + population. Note the complete disappearance of DsRed fluorescence at day 10 in culture. Data are mean ± s.e.m. (n=3). FDB-derived NG2-DsRed cells isolated from NG2 transgenic mice show two cell populations in culture: Tuj1+/NGF receptor+ neural cells and α-SMA+ pericytes. C. Relative proportions of Tuj1, NGF receptor, and α-SMA in the NG2-DsRed+ population. Data are mean ± s.e.m. (n=3). Cells were grown for 3 days, fixed and stained for Tuj1 (class III β tubulin), NGF receptor (NGFR, p75), and α-SMA (D). Their corresponding NG2-DsRed fluorescence, Hoechst staining, and merged images are displayed. Note that Tuj1 and NGF receptor staining was positive in cells with neural morphology (neural cells) (yellow arrow), but negative in cells with fibroblast-like morphology of pericytes (yellow arrowhead); while α-SMA stained pericytes (white arrow), but did not stain cells with neural morphology (white arrowhead). Light blue arrows indicate Tuj1 and NGFR positive cells in which the DsRed fluorescence disappeared (A, B). (A) Scale bar = 100 μm, (D) Scale bar = 20 μm.

Figure 3. Skeletal muscle-derived NG2-DsRed+ and Nestin-GFP+ neural cells overlap in double transgenic Nestin-GFP/NG2-DsRed mice

FDB muscle-derived cells from Nestin-GFP/NG2-DsRed mice were grown for 3 days and fixed. A. Nestin-GFP and NG2-DsRed cells and their corresponding, Hoechst 33342, brightfield, and merge images are illustrated. The white arrow indicates Nestin-GFP+/ NG2-DsRed- cells; the yellow arrow, Nestin-GFP+/ NG2-DsRed+ cells; and arrowheads indicate Nestin-GFP-/ NG2-DsRed+ cells. GFP fluorescence is brighter in Nestin-GFP+/NG2-DsRed+ cells than /Nestin-GFP+/NG2-DsRed- cells, probably because, in the latter, it is disappearing with time in culture. Also, Nestin-GFP-/NG2-DsRed+ cells have fibroblast-like morphology, while the Nestin-GFP+/NG2-DsRed+ cells have more neural morphology with long, thin processes. B. Pie chart representing the fraction of Nestin-GFP+/NG2-DsRed- (green), Nestin-GFP+/NG2-DsRed+ (yellow), Nestin-GFP-/NG2-DsRed+ (red), and Nestin-GFP-/NG2-DsRed- (blue) cells. Circle area represents the total number of cells. Values are expressed as the mean percent (n = 5). C. Tuj1 (orange) is expressed exclusively in Nestin-GFP+/NG2-DsRed+ cells. D. α-SMA (orange) is expressed only in Nestin-GFP-/NG2-DsRed+ cells while it is absent in Nestin-GFP+/NG2-DsRed+ cells. Scale bars = 50 μm.

Figure 4. NG2-DsRed and Nestin-GFP expression by various cell populations in EDL muscle from Nestin-GFP/NG2-DsRed mice in vivo

A. Longitudinal section of EDL muscle from the double transgenic Nestin-GFP/NG2-DsRed/ mice. B. EDL muscle transverse sections. Yellow arrows indicate Nestin-GFP+/NG2-DsRed- cells; white arrows, Nestin-GFP-/NG2-DsRed+ cells; and arrowheads, cells positive for both markers. Scale bars = 20 μm.

Figure 5. Cells expressing Nestin, NG2 and CD146 generate Tuj1+ cells in culture

Mononucleated cells, isolated from hindlimb muscles from young-adult Nestin-GFP/NG2-DsRed mice, were immediately sorted after enzymatic dissociation. (A) Number of neural cells (Nestin-GFP+/Tuj1+, with neural morphology) after 8 days in various culture conditions: Nestin+/NG2-, Nestin-/NG2+, Nestin+/NG2+, or Nestin-/NG2- cells cultured alone; and Nestin+/NG2-, Nestin-/NG2+, Nestin+/NG2+, or Nestin-/NG2- cells co-cultured with wild-type mouse FDB culture. The number of plated cells right after sorting was approximately 5,000 per dish in both conditions. Only Nestin-GFP+/Tuj1+ cells with 2 or more thin processes were counted. Data are mean ± s.e.m. (n=5). B. Representative image of Nestin-GFP+ neural cells derived from Nestin-GFP+/NG2-DsRed+ cells cultured with cells derived from wild-type FDB muscle co-stained with Tuj1 antibody. Notice that Nestin-GFP-/Tuj1+ cells shown in this image represent neural cells derived from wild-type cells. C and D. Mononuclear cells, isolated from hindlimb muscles from young-adult Nestin-GFP mice, were labeled with CD146-PerCP-Cy5.5-conjugated antibody and sorted as different cell populations. C. Representative dot plots showing CD146+ cells versus GFP fluorescence with gate set using unlabeled cells. D. Number of Tuj1+ cells after 8 days in various culture conditions: Nestin+/CD146-, Nestin-/CD146+, Nestin+/CD146+, or Nestin-/CD146- cells cultured alone; and Nestin+/CD146-, Nestin-/CD146+, Nestin+/CD146+, or Nestin-/CD146- cells co-cultured with medium from WT mouse FDB culture. The number of plated cells right after sorting was approximately 5,000 per dish in both conditions. Data are mean ± s.e.m. (n=5). E. Representative image of Nestin-GFP+ neural cells derived from Nestin-GFP+/CD146+ cells cultured with medium from WT mouse FDB culture co-stained with Tuj1 antibody. Scale bars = 50 μm.

Figure 6. Nestin-GFP+/NG2-DsRed+ and Nestin-GFP+/NG2-DsRed- cultures form α-SMA+ pericytes but not neural cells

Mononucleated cells, isolated from hindlimb muscles from young-adult Nestin-GFP/NG2-DsRed mice, were immediately sorted after enzymatic dissociation (Suppl. Fig. 4A, B). A. Representative images of staining for α-SMA marker (orange) staining of Nestin+/NG2-, Nestin-/NG2+, or Nestin+/NG2+ cells cultured after 8 days. Hoechst 33342, brightfield, and merge images are also shown. Scale bar = 100 μm. B. Number of pericytes (α-SMA+, with fibroblastic morphology) in Nestin+/NG2-, Nestin-/NG2+, or Nestin+/NG2+ cell population cultured for 8 days. The number of plated cells right after sorting was approximately 5,000 per dish. Data are mean ± s.e.m. (n=5).

Figure 7. A schematic representation of the two pericyte subtypes found in the skeletal muscle

Skeletal muscle is composed of bundles of multinucleated myofibers (in vivo). Each fiber carries a population of satellite cells (Pax7+/Nestin+/MyoD-) (red), which reside between the myofiber plasma membrane and the surrounding basal lamina. Two types of pericytes are present in the skeletal muscle interstitium: type 1 (Nestin-/NG2+/CD146+) (yellow) and type 2 (Nestin+/NG2+/CD146+) (green). (in vitro) After muscle dissociation into single cells and cultured for 7 days, these cells exhibit distinct differentiation potential: myoblasts (Pax7-/Nestin+/MyoD+) (red) and multinucleated myotubes (Nestin-/MyoD+/MHC+) (red) are derived from satellite cells (red); type 1 pericytes (yellow) remain as pericytes (Nestin-/NG2+/D146+/α-SMA+) (yellow, fibroblast-like aspect) when cultured alone or together with wild-type FDB culture; type 2 pericytes (green), similarly to type 1 pericytes, generate pericytes (yellow, fibroblast-like aspect) if cultured alone, but, in contrast to them, form potential neural progenitors (Nestin+/NG2+/CD146+/α-SMA-/Tuj1+) (green, thin processes and multipolar extensions) if co-cultured with wild-type FDB culture. Scale bar of image with FDB muscle = 500 μm. Scale bar of microscopic fluorescent images = 50 μm.