Human muscle-derived CLEC14A-positive cells regenerate muscle independent of PAX7

Abstract

Skeletal muscle stem cells, called satellite cells and defined by the transcription factor PAX7, are responsible for postnatal muscle growth, homeostasis and regeneration. Attempts to utilize the regenerative potential of muscle stem cells for therapeutic purposes so far failed. We previously established the existence of human PAX7-positive cell colonies with high regenerative potential. We now identified PAX7-negative human muscle-derived cell colonies also positive for the myogenic markers desmin and MYF5. These include cells from a patient with a homozygous PAX7 c.86-1G > A mutation (PAX7null). Single cell and bulk transcriptome analysis show high intra- and inter-donor heterogeneity and reveal the endothelial cell marker CLEC14A to be highly expressed in PAX7null cells. All PAX7-negative cell populations, including PAX7null, form myofibers after transplantation into mice, and regenerate muscle after reinjury. Transplanted PAX7neg cells repopulate the satellite cell niche where they re-express PAX7, or, strikingly, CLEC14A. In conclusion, transplanted human cells do not depend on PAX7 for muscle regeneration.

Fig. 1. Characterization of human desmin-positive, PAX7- negative cell populations.

a Experimental design. Cell colonies grow out of human muscle fiber fragments (HMFF) within 3 weeks after hypothermic treatment. b Absence of PAX7 transcripts in PAX7null cells. The PAX7 c.86-1G > A mutation in PAX7null cells leads to deletion of exon 2 and a premature stop codon in exon 3. The PCR primers shown here recognize exons 4 and 5. PAX7neg-B cells are derived from donors with intact Pax7 gene and also do not express PAX7. c Expression of markers of myogenic and interstitial cells assessed by qPCR in PAX7null, PAX7neg, and PAX7pos cell colonies. All data were normalized to two reference genes and relativized to the mean of PAX7pos cells. The dotted line at twofold and 0.5-fold represents our threshold for differential expression. d Myogenic cells derived from HMFFs were stained for PAX7, desmin, and MYF5. PAX7-negative populations (PAX7null, PAX7neg) show no specific signal for PAX7 in immunofluorescence stainings. Cell lines are described in Supplementary Table 2. Scale bars: 20 µm.

Fig. 2. Single-cell transcriptomics reveals different gene expression pattern in PAX7pos, PAX7neg, and PAX7null myogenic cells.

a–c Two-dimensional tSNE representation of global gene expression relationships among individual myogenic cells from a PAX7-deficient donor (PAX7null) and from donor A (PAX7neg-A, PAX7pos-A). Each dot represents a cell. a tSNE plot colored by cell cluster. b tSNE plot colored by cell population. c Normalized gene expression levels of selected marker genes (red: high, gray: low). d Violin plots indicating gene expression levels and distributions of selected genes within cell clusters corresponding to panel a. Clusters 0, 5, and 7 correspond to proliferating cells (MKI67). PAX7null cells express strongly elevated levels of CLDN11, FHL1, COL6A3, and CLEC14A in clusters 0 and 1, and TFPI2 in clusters 0, 1, and 2.

Fig. 3. PAX7-negative cell populations can generate muscle.

a PAX7null, PAX7neg, and PAX7pos cells, used in transplantation experiments shown in b, indicate the same differentiation potential (skeletal myosin staining, Fi: Fusion index, Scale bars: 50 µm). b TA muscle sections from NOG mice demonstrate that 3 weeks after transplantation the PAX7null, PAX7neg, and PAX7pos human cell types contributed to muscle regeneration. Human anti-lamin A/C antibody (yellow) detects only human nuclei and anti-human spectrin antibody is specific for fibers of human origin (red). Nuclei in blue (Hoechst). Scale bar: 50 µm. c Quantification of experiments shown in b. Each dot represents one mouse grafted with the cells indicated below (n = 4 for PAX7null and n = 7 for PAX7neg). Shown are the values for the section with the highest number of human fibers for each mouse and the means. Only human muscle fibers with a diameter > 10 µm were counted. Mice with > 20 human fibers/section were additionally analyzed for PAX7 expression. The presence of PAX7-positive satellite cells is indicated by an open circle. The statistical differences between the groups were analyzed by two-tailed t tests, P > 0.05 = NS (not significant). d Repopulation of satellite cell niche after transplantation: In PAX7null transplants, the satellite cell niche is populated by CLEC14A-positive cells (red, upper row). After transplantation of PAX7neg- and PAX7pos cells, the satellite cell niche is populated by PAX7-positive cells (red, middle, and bottom row). Yellow, human lamin A/C; green, laminin; blue Hoechst. Scale bars: 10 µm (upper row) and 5 µm.

Fig. 4. CLEC14A is a marker for a human myogenic cell in satellite cell position.

a CLEC14A- staining of PAX7null, PAX7neg, PAX7pos cells. PAX7null cells are strongly positive for CLEC14A. Some PAX7neg cell populations from dornors with intact PAX7 gene are also CLEC14A-positive. Reconstitution of PAX7null cells with a lentiviral PAX7 vector changed the expression profile of CLEC14A and NCAM (bottom line). Red, PAX7; green, CLEC14A; blue, Hoechst. Bar 20 µm. b PAX7 and CLEC14A expression in PAX7neg cell colonies were mutual exclusive. Fifty-six cell populations from donors with intact PAX7 genes were analyzed for expression of PAX7 (green) and CLEC14A (red). Cell colonies are either positive for PAX7 or for CLEC14A. Some colonies contained both, cells that were either PAX7 or CLEC14A-positive. Staining for PAX7 and CLEC14A in “mixed” cell populations depicted mutually exclusive expression. Scale bar: 20 µm. c Gene expression in PAX7null cells selected by FACS-sorting (see also Supplementary Fig. 9) after lentiviral PAX7 transduction determined by qPCR 3, 6, and 12 days after DOX induction. All data were normalized to two reference genes and relativized to the mean of PAX7pos cell colonies. The dotted line at twofold and 0.5-fold represents the threshold for differential expression. d TA muscle section from a NOG mouse 3 weeks after transplantation of CLEC14Apos cells shows newly built human muscle fibers. Quantification reveals similar results as in Fig. 3b. Quantification as in 3c, but without PAX7 analysis. Each dot represents one mouse grafted with PAX7neg-CLEC14Apos cells (n = 8). Scale bar: 20 µm. e Staining for huCLEC14A, PAX3 and Syndecan-4 indicates cells in satellite cell position (arrows) in the muscle tissue of the PAX7null patient. Nuclei are stained with Hoechst (blue). About 8% of nuclei were positive for CLEC14A and localized in a satellite cell position (133 nuclei analyzed). Scale bars: 10 µm. f SmFISH for huCLEC14A in a frozen muscle section of a normal human donor. The red arrow indicates a nucleus in satellite cell position and the white one a endothelial cell nucleus. Scale bar: 20 µm.