In summary, we have developed a protocol that allows efficient isolation of MuSCs from fixed tissue, with comparable purity and yield to those of non-fixed muscles. Moreover, we show that the cells isolated from PFA-treated samples are efficiently fixed and, hence, safeguarded from the strong stimuli to which they are exposed during the dissociation procedure.

To ensure that the T0-SCs had been properly fixed and were biologically inert, we assessed their proliferation capacity by measuring incorporation of 5-ethynyl-2′-deoxyuridine (EdU), de novo RNA synthesis by the presence of the modified nucleotide ethynyl uridine (EU), and uptake of DAPI, a compound actively excluded from live cells. As expected for fixed cells, the T0-SCs did not incorporate EdU ( Figure 1 C), were transcriptionally inactive ( Figure 1 D), and were positive for DAPI ( Figures S1 C and S1D). Of note, the light scattering of T0-SCs was drastically different compared to the non-fixed T3-SCs, suggesting differences in the morphological complexity of the isolated cells. Indeed, MuSCs have a small cytoplasmic/nuclear ratio and in vivo are spindle shaped, with multiple branches emanating from their poles. Upon detachment from the myofibers, the stretched plasma membrane retracts to form small, round, uniform cells ( Figure 1 B, bottom left). Instead, when fixed, the dissociated MuSCs exhibit a complex morphology ( Figure 1 B, bottom right), possibly reflecting that found in vivo, as has been shown for fixed cardiomyocytes (). To confirm the differences in cell shape inferred from light scattering, we used the lectin marker WGA (wheat germ agglutinin) to stain cell membranes of T0- and T3-SCs ( Figure 1 E). Using an image-processing software (see Experimental Procedures ), we calculated the mean eccentricity of T0- and T3-SCs, a circle having an eccentricity of 0 and a straight line of 1. Interestingly, we found that in situ fixed T0-SCs have a mean eccentricity significantly greater than that of T3-SCs (0.80 ± 0.009 versus 0.40 ± 0.013), corroborating that in situ fixation retains the physiological spindle shape of MuSCs that is lost during the conventional dissociation protocol ( Figure 1 E).

In order to isolate quiescent MuSCs, muscles were fixed in situ by ice-cold 0.5% PFA before cell dissociation ( Figure 1 A; Supplemental In Situ Fixation Protocol Supplemental Experimental Procedures ). We refer to these fixed cells as time-zero stem cells (T0-SCs) and the cells isolated by the standard 3-hr-long protocol as T3-SCs. We applied both the standard and the in situ fixation protocols for the isolation of MuSCs from transgenic Tg:Pax7-nGFP mice, in which nuclear GFP is expressed in MuSCs () ( Figure 1 B). The purity of the isolated fixed cells was assessed by PAX7 staining and was similar to that of the cells isolated using the standard protocol (94% ± 3.1% and 92% ± 1.4% of PAX7cells for T3- and T0-SCs, respectively; Figure S1 A). Notably, equivalent numbers of GFPcells were isolated from fixed and fresh muscles, indicating that in situ fixation did not lead to loss of cells ( Figures 1 B and S1 B).

For the isolation of MuSCs using standard protocols, muscles are dissected, mechanically minced into 1- to 2-mm pieces, and subsequently incubated with proteolytic enzymes at 37°C for up to 2 hr to allow detachment of the myofiber bundles and dissociation of the MuSCs ( Figure 1 A). Following several steps of washes and filtrations, MuSCs are isolated by fluorescence-activated cell sorting (FACS), based on specific plasma membrane receptors or genetically expressed fluorescent markers ().

Data are reported as mean ± SD; n.d, not detected. Scale bars, 90 μm in (C), 20 μm in (D), 8 μm in (D, insets), and 10 μm in (E). See also Supplemental Information

(E) Morphology of T3 and T0 MuSCs immediately after the FACS. Cells were spun on Matrigel-coated slides, and membranes were stained with the lectin marker WGA (wheat germ agglutinin). Mean eccentricity of the cells was computationally calculated ( Experimental Procedures ).

(D) Nascent RNA synthesis in FACS-isolated T3-SCs and T0-SCs cultured for 48 hr and incubated with labeled ethynyl uridine (EU) ribonucleoside for 2 hr; n = 3. Average of 80 nuclei counted per sample, 100% EU + and EU − for T3-SC and T0-SC, respectively. Dotted line delineates cell’s nucleus.

(C) Proliferation of FACS-isolated T3-SCs and T0-SCs cultured for 48 hr and stained with EdU (24-hr chase); n = 3. EdU + cells: 0% for T0-SCs and 74% ± 0.03% for T3-SCs.

(B) FACS profiles of non-fixed (T3-SC) and fixed (T0-SC) GFP + MuSCs from Tg:Pax7-nGFP muscle preparations. Sorted GFP + cells are marked as red dots in all plots. SSC, side scatter, FSC, forward scatter. Values on the plots indicate mean percentage of sorted GFP + cells of the total number of events, excluding small SSC/FSC and doublets; n = 5.

(A) Graphical scheme of the in situ fixation protocol for MuSCs isolation and comparison to the standard protocol. A detailed description of the protocols is available in the Supplemental Information . BM, basement membrane; T0-SC, time-zero/quiescent MuSC; T3-SC, time 3 hr/activated MuSC.

Having developed a method to isolate a highly pure population of in situ fixed MuSCs, we then sought to analyze its transcriptional profile. First, however, a series of experiments was performed to measure the putative effects of PFA fixation on mRNA quality and composition. As shown in Figure 2 A, fresh muscles from Tg:Pax7-nGFP mice were digested, and GFPMuSCs were isolated by FACS. Subsequently, half of the sorted MuSCs were immediately lysed, and the other half were fixed in 0.5% PFA before being lysed for RNA extraction. Of note, RNA extraction from fixed cells was optimized ( Experimental Procedures ), as standard protocols were not suitable (data not shown). We found that the RNA quality was not compromised by fixation, as the RNA integrity numbers (RINs) were similar between the fixed and non-fixed T3-SCs ( Figure 2 B). Moreover, RNA yields were comparable between fixed and non-fixed T3-SCs ( Figure 2 C). In order to extend our comparison, the extracted RNA was subjected to next-generation RNA sequencing (RNA-seq). Analysis of the distribution of the sequencing reads to all genes showed no 5′ or 3′ bias for the fixed or the non-fixed T3-SCs, further indicating that the fixation protocol did not interfere with RNA integrity ( Figure 2 D). Also, the mRNA composition of fixed and live T3-SCs transcriptomes were highly similar, as only 3 differentially expressed genes were identified ( Figure 2 E). Finally, the simple error ratio estimate (SERE) statistic () and principal-component analysis demonstrated high similarity between live and fixed T3-SCs ( Figures 2 F, S2 A, and S2B). In conclusion, the quality and composition of the total RNA extracted from 0.5% PFA-fixated MuSCs is comparable to those from non-fixed cells and suitable for transcriptomic analysis.

n.s, not significant. Data are reported as mean ± SD. See also Figure S2

(F) Simple error ratio estimate (SERE) coefficient for the three sets of paired live and fixed T3-SC RNA-seq samples (pairs A, B, and C). For comparison, a matrix table with the SERE coefficients between all the samples used in this study is shown in Figure S2 A.

(E) MA plot of live versus fixed T3-SCs RNA-seq data. Only three significantly deregulated transcripts (FDR < 0.05) were identified and are highlighted in red; n = 3.

(C) Mean of total RNA recovered per cell in live and fixed T3-SCs preparations, calculated with the Bioanalyzer 2100.

(A) Graphical scheme of the procedure for the isolation of non-fixed (Live T3-SC) and fixed MuSCs (Fixed T3-SC). Following the standard protocol, half of the FACS-isolated MuSCs were fixed in 0.5% PFA for 30 min, and total RNA was extracted and compared to that of live T3-SCs.

Significant Transcriptional Alterations Are Induced during the MuSC Isolation Protocol

Comparison of the T0-, T3-, and T5-SC transcriptomes demonstrated that the standard MuSC isolation procedure induced significant transcriptional changes. Strikingly, a large number of genes showed strong transcript inductions during the isolation (2,822 T3/T0 upregulated genes with a fold change [FC] > 2 and a false discovery rate [FDR] < 0.05), whereas the changes were moderate between the T3 and T5 time points (222 T5/T3 upregulated genes; FC > 2, and FDR < 0.05) ( Figures 3 A, 3B, and S3 B; Table S1 ). Of interest, a large number of transcripts was downregulated during the isolation process (4,840 T3/T0 downregulated genes; FC > 2, and FDR < 0.05), suggesting that RNA degradation mechanisms, in addition to attenuation of transcription, may operate at the early phases of MuSC activation.

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Dong Z. Blocking activator protein-1 activity, but not activating retinoic acid response element, is required for the antitumor promotion effect of retinoic acid. The comparison between the T0 and T3 transcriptomes identified MuSC activation-induced genes already described, validating our approach but also revealing that these changes occur earlier than previously estimated. Within just 3 hr, the downstream targets of Notch signaling Hes1 and HeyL were decreased by 5- and 8-fold, respectively, consistent with what has been reported for in-vivo-activated MuSCs at 20 hr post-injury (). Similarly, the hallmark MuSC gene Pax7, whose encoded protein marks both stem and progenitor muscle cells, was found 10-fold downregulated in T3-SCs, supporting a quiescence-specific function of this paired-type homeodomain transcription factor. Instead, expression of the muscle regulatory factor Myod was rapidly upregulated 10-fold in T3-SCs. As shown in Figure 3 D, the dissociation procedure also induced the expression of early response genes, which are known to be transiently activated in response to a variety of stimuli (). Among these, we found members of the Immediate early response Ier-2, -3, -5, and -5l and Early growth response genes Egr-1, -2, and -3. Also, in the T3-SCs, which correspond to the MuSCs isolated to date by the standard protocols, we found an over-representation of a number of oncogenes, like c-myc and Maff and also members of the AP-1 transcription factor family, like c-Fos, c-Jun, and others ( Figure 3 D). To investigate the functional role of AP-1 in the early activation of quiescent MuSCs, we incubated freshly isolated cells in the presence of the pathway inhibitor SR-11302 (). We found that SR-11302 resulted in a 3-fold decrease of proliferating cells after 48 hr in culture, indicating that AP-1 is involved in the exit of MuSCs from quiescence ( Figures S3 C and S3D).

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et al. Epigenetic stress responses induce muscle stem-cell ageing by Hoxa9 developmental signals. The in-situ-fixed MuSC transcriptome enabled us to identify potentially important quiescence factors that were previously missed, due to their sharp downregulation during the isolation process. Of interest, we uncovered a substantial number of zinc finger protein-encoding genes (223 in total [FC > 2, and FDR < 0.05], of which 143 had at least one transcriptional repression Krüppel Associated Box (KRAB) domain and 182 had at least one DNA-binding zinc finger C2H2 type [ZNF-C2H2] motif). Also, we identified a large group of Hox-a, -b, and -c genes and, further, confirmed them as quiescence specific. We validated by immunofluorescence the differential expression of HOXA9, which we found present in quiescent but absent in activated MuSCs following muscle injury ( Figure 3 E). Our observation suggests a role of HOXA9 in quiescence, extending a recent study that found HOXA9 absent in freshly isolated and activated MuSCs ().

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et al. A module of negative feedback regulators defines growth factor signaling. Paradoxically, several proto-oncogenes, including the AP-1 members, have been proposed as quiescence enriched, based on comparisons between freshly isolated and tissue-cultured or in-vivo-activated and even proliferating MuSCs (). In fact, on average, 21% of the microarray-based quiescent signatures reported in the literature (transcripts enriched in freshly isolated MuSCs, FC > 5) correspond to genes that we found as specific for the T3-SC transcripts ( Figure 3 F) and, hence, are not quiescence-related genes but, instead, consist of early-activation- and isolation-induced artifacts. Of note, the fact that these genes were found at higher levels in the freshly isolated cells than in the activated and/or proliferating cells indicates that they could constitute transiently and highly induced transcripts, a characteristic feature of early-response genes ().