Abstract

   In vitro-generated pluripotent stem cell (PSC)-derived Pax3-induced
   (iPax3) myogenic progenitors display an embryonic transcriptional
   signature, but upon engraftment, the profile of re-isolated iPax3
   donor-derived satellite cells changes toward similarity with postnatal
   satellite cells, suggesting that engrafted PSC-derived myogenic cells
   remodel their transcriptional signature upon interaction within the
   adult muscle environment. Here, we show that engrafted myogenic
   progenitors also remodel their metabolic state. Assessment of oxygen
   consumption revealed that exposure to the adult muscle environment
   promotes overt changes in mitochondrial bioenergetics, as shown by the
   substantial suppression of energy requirements in re-isolated iPax3
   donor-derived satellite cells compared to their in vitro-generated
   progenitors. Mass spectrometry-based metabolomic profiling further
   confirmed the relationship of engrafted iPax3 donor-derived cells to
   adult satellite cells. The fact that in vitro-generated myogenic
   progenitors remodel their bioenergetic signature upon in vivo exposure
   to the adult muscle environment may have important implications for
   therapeutic applications.

   Keywords: pluripotent stem cells, myogenic progenitors, metabolism, in
   vitro, in vivo, satellite cells

1. Introduction

   Homeostasis of the skeletal muscle is provided by muscle stem cells,
   also known as satellite cells. Upon injury, satellite cells become
   activated and give rise to proliferating myoblasts, which will
   differentiate into new myofibers and/or fuse to existing muscle fibers
   or to other myoblasts to repair muscle damage [[32]1,[33]2,[34]3]. A
   small subset of satellite cells retain the ability to self-renew, thus
   preserving the satellite cell reservoir [[35]3,[36]4]. While satellite
   cells have remarkable regenerative potential, their application on
   cell-based therapies for skeletal muscle disorders remains challenging
   due to limitations associated with ex vivo expansion [[37]2]. This is
   required since the number of satellite cells in a small muscle biopsy
   is not sufficient for cell-based therapy. A major caveat with ex vivo
   expansion is that once removed from the muscle environment and allowed
   to expand, satellite cells differentiate into myoblasts, which have
   limited regenerative potential [[38]2,[39]5,[40]6]. Because pluripotent
   stem cells (PSCs) can repeatedly produce large amounts of
   lineage-specific cell types, they represent an attractive source for
   the generation of muscle stem/early progenitor cells for therapeutic
   applications. We have reported that the conditional expression of the
   transcription factors Pax3 (iPax3) or Pax7 (iPax7) in differentiating
   PSCs enables the generation of large numbers of myogenic progenitors
   endowed with in vivo regenerative potential
   [[41]7,[42]8,[43]9,[44]10,[45]11]. Of interest is the fact that, while
   in vitro-generated PSC-derived myogenic progenitors display an
   embryonic transcriptional profile, this changes upon transplantation as
   engrafted re-isolated donor-derived satellite cells show a postnatal
   molecular signature [[46]12]. The majority of the genes found to be
   differentially expressed between in vitro-generated PSC-derived
   myogenic progenitors and adult bona fide satellite cells were no longer
   distinct in re-isolated donor-derived cells, including genes of the
   Notch pathway [[47]12], known to be important for satellite cell
   quiescence and function [[48]13,[49]14,[50]15].

   Metabolic pathways have also been reported to play an important role in
   satellite cell function during development and adult homeostasis, as
   well as upon stress [[51]16,[52]17,[53]18,[54]19]. However, the
   metabolic profile of in vitro-generated PSC-derived myogenic
   progenitors and subsequent post-transplant donor-derived satellite
   cells has never been investigated.

   To determine the metabolic status of PSC-derived myogenic progenitors
   and potential changes upon their in vivo exposure to the adult muscle
   environment, here we assessed transcriptional profiles pertinent to the
   metabolic state using published datasets [[55]12] and performed
   real-time metabolic flux analysis and mass spectrometry-based
   metabolomics in in vitro-generated iPax3 myogenic progenitors and
   post-transplant iPax3 donor-derived satellite cells side by side with
   adult satellite cells. Our results show stark changes in all parameters
   when comparing in vitro-generated myogenic progenitors to their in vivo
   post-transplant re-isolated counterparts and demonstrate that iPax3
   donor-derived satellite cells assume a metabolic profile similar to
   bona fide satellite cells.

2. Material and Methods

2.1. RNA-Sequencing and Microarray Analysis

   Bulk RNA-sequencing datasets from single-end libraries using the
   Illumina platform were downloaded from the gene expression omnibus
   (GEO) database using the Sequence Read Archive (SRA) toolkit for 3
   samples of mouse PSC-derived iPax3 myogenic progenitors and 3 samples
   of adult satellite cells [[56]12]. Additionally, Whole Mouse Genome
   Microarray 4x44K G4122F datasets for 3 samples of post-transplant
   re-isolated iPax3 donor-derived mononuclear cells (GFP+CD34+) and 3
   samples of adult satellite cells [[57]12] were downloaded using the
   GEOQuery [[58]20] package (v2.66.0) on R. Data alignment and comparison
   of expression were analyzed using the CHURP pipeline [[59]21] at the
   University of Minnesota Genomics Center (UMGC). We trimmed 1 × 100 bp
   FASTQ single-end reads for 6 samples (34.2 million reads average per
   sample) using Trimmomatic (v0.33) with the optional “-q” option
   enabled; 3 bp sliding-window trimming from 3′ end requiring minimum
   Q30. We performed quality control on raw sequence data for each sample
   with FastQC and read mapping with HISAT2 [[60]22] (v2.1.0) using the
   mouse genome (GRCh39.106) as reference. We performed gene
   quantification using Feature Counts for raw read counts. We identified
   differentially expressed genes using the EdgeR (negative binomial, R
   programming) feature in CLCGWB (Qiagen, Redwood City, CA, USA) using
   raw read counts. We filtered the generated list based on a minimum 2×
   absolute fold change and FDR-corrected p < 0.05. GSE analysis using the
   clusterProfiler [[61]23,[62]24] package (v4.6.0) on R was used to carry
   out Reactome [[63]25] and KEGG [[64]26] pathway analysis and gene
   ontology enrichment analysis. Pathways with an FDR q < 0.05 were
   considered to be significantly expressed. For the microarray data,
   pairwise comparisons, at the gene-level, were conducted using the limma
   [[65]27] package on R (v3.54.0).

2.2. Cell Culture

   For the studies described here, we used H2B-GFP-labelled inducible Pax3
   (iPax3) embryonic stem (ES) cells, which were differentiated as before
   [[66]12]. Briefly, ES cells were maintained in a combination of ES
   medium and 2 inhibitors (2i) medium (1:1). ES medium consists of
   KnockOut^TM DMEM (Invitrogen, Waltham, MA, USA) containing 15% FBS
   (Sigma, Burlington, MA, USA), 1% penicillin-streptomycin (Invitrogen),
   2 mM Glutamax (Gibco, Waltham, MA, USA), 0.1 mM non-essential amino
   acids (Gibco), and 0.1 mM β-mercaptoethanol (Gibco), whereas the 2i
   medium consists of neurobasal medium (Invitrogen) and DMEM F12 medium
   (Invitrogen) containing 0.5% N2 (Life Technologies, Carlsbad, CA, USA),
   0.5% B27 (Life Technologies), 0.05% BSA (Sigma), 1%
   penicillin-streptomycin, 150 µM monothioglycerol (MP Biomedicals, Santa
   Ana, CA, USA), 3 µM GSK3β inhibitor (CHIR 990217; Tocris, Minneapolis,
   MN, USA), 1 µM PD 0325901 (Cayman, Ann Arbor, MI, USA), and 1000 U/mL
   LIF (Millipore, Burlington, MA, USA). For differentiation into embryoid
   bodies (EB), we cultured ES cells in suspension at the concentration of
   40,000 cells/mL in culture medium containing IMDM (Invitrogen), 15% FBS
   (Sigma), 1% penicillin/streptomycin (Invitrogen), 2 mM GlutaMAX
   (Invitrogen), 50 μg/mL ascorbic acid (Sigma-Aldrich, Burlington, MA,
   USA), and 4.5 mM monothioglycerol (MP biomedicals). We induced Pax3
   expression by adding doxycycline (dox; Sigma-Aldrich D989) to cultures
   at day 3 of EB differentiation (final concentration 1 μg/mL). At day 5,
   EBs were sorted for the Vcam1+FLK1- cell fraction [[67]12]. Briefly,
   EBs were disaggregated, incubated with Fc block (1 μL/million cells; BD
   Biosciences, Franklin Lakes, NJ, USA) for 5 min, and then stained with
   Flk1-APC and Vcam1-biotin-conjugated antibodies (1 μL/million cells;
   e-Bioscience) for 60 min on ice, followed by 20 min incubation with
   streptavidin-PeCy7. Cells were washed twice with PBS and then
   resuspended in PBS containing 10% FBS and propidium iodide (PI) to
   exclude dead cells. Vcam1+FLK1- cells were sorted using a FACSAria II
   (BD Biosciences), plated on gelatin-coated dishes in the presence of EB
   differentiation media containing 1 μg/mL dox and 10 ng/mL mouse basic
   FGF (bFGF; PeproTech, Rocky Hill, NJ, USA; #100-18). After 3 passages,
   cells were used for transplantation studies.

2.3. Mice and Cell Transplantation

   All animal experiments were carried out in strict accordance with
   protocols approved by the University of Minnesota Institutional Animal
   Care and Use Committee. Twelve-week-old male Pax7-ZsGreen reporter mice
   [[68]28] and six–eight-week-old male NOD-scid IL2Rg^null (NSG) mice
   (Jackson Laboratories, Bar Harbor, ME, USA) were used for satellite
   cell isolation (reference group) and transplantation studies,
   respectively. As previously described [[69]12], 2 days prior to
   intramuscular transplantation, both hind limbs were subjected to a 12
   Gy dose of local irradiation using an RS 2000 Biological Research
   Irradiator (Rad Source Technologies, Inc., Buford, GA, USA). One day
   before cell transplantation, we injured both TA muscles with 15 μL of
   cardiotoxin (10 μM in PBS, Sigma, Saint Louis, MO, USA). The next day,
   3 × 10^5 iPax3 myogenic progenitors were transplanted into injured
   muscles. Four weeks later, mice were euthanized for assessment of
   skeletal muscle engraftment and re-isolation of donor-derived satellite
   cells.

2.4. Satellite Cell Purification

   We purified post-transplant donor-derived satellite cells based on the
   expression of GFP (donor tracking) and the following satellite cell
   marker profile: CD31−/CD45−/Itga7+/CD34+ [[70]29]. For primary adult
   satellite cells, the ZsGreen+/CD31−/CD45−/Itga7+/CD34+ cell fraction
   was sorted.

2.5. Metabolic Flux Analysis

   Oxygen consumption rates (OCR) and extracellular acidification rates
   (ECAR) of iPax3 myogenic progenitors and donor-derived satellite cells,
   as well as bona fide satellite cells, were measured using the Seahorse
   XF96 Analyzer (Seahorse Bioscience, Billerica, MA, USA), as previously
   described [[71]18]. Cells were seeded onto matrigel-coated XF96 96-well
   microplates (for 24 h) at 10,000/well for in vitro iPax3 cells and
   re-isolated in vivo iPax3 cells and at 70,000/well for adult satellite
   cells. Following titration, optimal concentrations of respiratory
   modulators were chosen for these studies. Prior to the assay, we
   replaced the medium with 180 μL of 5 mM glucose-containing medium
   without sodium bicarbonate. Cells were pre-incubated for 1 h at 37 °C
   and 0% CO[2] prior to loading into a Seahorse Bioscience XF96
   extracellular flux analyzer. Over approximately 90 min, the ports of
   the cartridge containing the oxygen probes were loaded with the
   compounds to be injected during the assay and the cartridge was
   calibrated. ATP synthesis-linked O[2] consumption and proton
   leak-driven respiration were determined through the addition of
   oligomycin (1 μg/mL, Sigma, # O4876). After 3 measurement cycles, the
   uncoupler carbonyl cyanide 3-chlorophenylhydrazone (2 μM CCCP, Sigma, #
   C2759) was added to promote maximal respiratory capacity. After 3
   additional measurement cycles, we added rotenone (1 μM, Sigma, R8875)
   and antimycin A (1 μg/mL, Sigma, A8674) to inhibit complex I and
   complex III, respectively, therefore resulting in depletion of
   mitochondrial oxygen consumption.

2.6. qRT-PCR

   Cells were harvested using TRIzolTM (Invitrogen). RNA was extracted
   using the PurelinkTM RNA Mini kit (Invitrogen) and reverse transcribed
   using the Superscript^® VILOTM cDNA synthesis kit (Invitrogen). TaqMan
   probes for Pax7, Sdhd, Cox6b2, Atp5c1, and Gapdh were used (Applied
   Biosystems, Waltham, MA, USA).

2.7. Immunofluorescence

   TA muscles were embedded in Tissue-Tek O.C.T. (Sakura, Finetek Japan.
   Co., Ltd., Tokyo, Japan) and snap frozen on isopentane pre-cooled with
   liquid nitrogen. Cryosections of 14 µm were collected on glass slides
   and preserved at −80 °C. Prior to staining, muscles cryosections were
   rehydrated with PBS for 5 min at room temperature (RT), fixed with 4%
   PFA for 30 min at RT, washed with PBS, permeabilized 15 min at RT with
   0.3% Triton X100 (Sigma) in PBS, washed again with PBS, blocked for 30
   min with 3% BSA (Sigma), and incubated overnight at 4 °C with primary
   antibodies for GFP (chicken 1:500; Abcam, Waltham, MA, USA; #13970) and
   dystrophin (mouse 1:20; Leica, Nanterre Cedex, France; DYS1-CE). The
   next day, cryosections were rinsed with PBS and incubated with goat
   anti-chicken (1:500; Thermo Fisher, # A-11039) and goat anti-mouse
   (1:500; Thermo Fisher; #A-21235) secondary antibodies for 1 h at RT.
   Following three PBS washes, slides were dried and mounted with Prolong
   Gold with DAPI (Invitrogen).

2.8. Metabolomics Sample Preparation and UHPLC-MS Analysis

   In preparation for sample submission, cells were counted, pelleted, and
   stored at −80 °C. Prior to LC-MS analysis, samples were placed on ice
   and re-suspended with methanol:acetonitrile:water (5:3:2) at a
   concentration of 2 million cells per ml. Suspensions were vortexed
   continuously for 30 min at 4 °C. Insoluble material was removed via
   centrifugation at 10,000× g for 10 min at 4 °C. Isolated supernatants
   were dried under vacuum and resuspended in 15% methanol containing 0.1%
   formic acid.

   UHPLC-MS analyses were performed as previously described
   [[72]30,[73]31]. Briefly, the analytical platform employs a Vanquish
   UHPLC system (Thermo Fisher Scientific, San Jose, CA, USA) coupled
   online to a Q Exactive mass spectrometer (Thermo Fisher Scientific).
   Polar extracts (2 µL injections) were resolved over a Kinetex C18
   column, 2.1 × 150 mm, 1.7 µm particle size (Phenomenex, Torrance, CA,
   USA), equipped with a guard column (SecurityGuard^TM
   Ultracartridge—UHPLC C18 for 2.1 mm ID Columns—AJO-8782—Phenomenex,
   Torrance, CA, USA) using an aqueous phase (A) of water and 0.1% formic
   acid and a mobile phase (B) of acetonitrile and 0.1% formic acid for
   positive ion polarity mode, and an aqueous phase (A) of
   water:acetonitrile (95:5) with 1 mM ammonium acetate and a mobile phase
   (B) of acetonitrile:water (95:5) with 1 mM ammonium acetate for
   negative ion polarity mode. The Q Exactive mass spectrometer (Thermo
   Fisher Scientific) was operated independently in positive or negative
   ion mode, scanning in Full MS mode (2 μscans) from 60 to 900 m/z at
   70,000 resolution, with 4 kV spray voltage, 45 sheath gas, 15 auxiliary
   gas, AGC target = 3 × 10^6, and maximum IT = 200 ms. Samples were
   analyzed in randomized order with a technical mixture injected after
   every 10 samples to qualify instrument performance. Calibration was
   performed prior to analysis using the Pierce^TM Positive and Negative
   Ion Calibration Solutions (Thermo Fisher Scientific). Acquired data
   were converted from raw to mzXML file format using Mass Matrix
   (Cleveland, OH, USA). Metabolite assignments were performed using
   accurate intact mass (sub-10 ppm), isotopologue distributions, and
   retention time/spectral comparison to an in-house standard compound
   library (MSMLS, IROA Technologies, NJ, USA) using MAVEN (Princeton, NJ,
   USA).

2.9. Statistical Analysis

   GraphPad Prism v9 (GraphPad Software, LLC) was used to perform one-way
   and two-way ANOVA with post hoc correction for multiple comparisons
   using the Bonferroni–Holm or Tukey tests. p values < 0.05 were
   considered statistically significant. Multivariate analyses of
   metabolomics data including principal component analysis, hierarchical
   clustering analysis, ANOVA, and pathway enrichment analysis were
   performed using Metaboanalyst 5.0 [[74]32].

3. Results

3.1. Transcriptional Landscape of Metabolic Pathways in iPax3 Myogenic
Progenitors

   To determine the transcriptional profile pertinent to the metabolic
   state of in vitro-generated PSC-derived Pax3-induced (iPax3) myogenic
   progenitors in comparison to adult Pax7^+ satellite cells, we
   interrogated metabolic genes in our published datasets [[75]12]. By
   combining the annotations from the Reactome pathway [[76]25] and the
   Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway [[77]26]
   databases, we identified 986 shared genes related to metabolic pathways
   ([78]Figure 1A). Pairwise comparisons between in vitro-generated iPax3
   myogenic progenitors (hereafter referred to as in vitro iPax3) and
   adult satellite cells revealed 360 differentially expressed genes
   (DEGs) mapping onto metabolic pathways, with the majority (77%) of the
   DEGs being significantly underexpressed in in vitro iPax3 cells
   compared to adult satellite cells ([79]Figure 1B). This analysis showed
   that in vitro iPax3 cells display significant overexpression of genes
   involved in mitochondrial respiration, such as mitochondrial-encoded
   cytochrome C oxidase III (mt-Co3), mitochondrial membrane ATP synthase
   (Atp5pb), and succinate dehydrogenase complex (Sdhd), relative to adult
   satellite cells ([80]Figure 1C). In contrast, they display
   significantly lower expression of genes involved in fatty acid and
   lipid metabolism, such as lipin 3 (Lpin3), hydroxysteroid 11-β
   dehydrogenase (Hsd11b1), and neuraminidase 2 (Neu2), relative to adult
   satellite cells ([81]Figure 1C). Linear dimensionality reduction via
   principal component analysis (PCA) unveiled stark differences between
   in vitro iPax3 cells and adult satellite cells ([82]Figure 1D).
   Unsupervised hierarchical clustering analysis of the 360 metabolic DEGs
   revealed two main clusters ([83]Supplementary Figure S1A). Gene
   ontology (GO) analysis of these two clusters showed highly enriched
   genes related to lipid metabolic process, carbohydrate derivative
   metabolic process, and membrane lipid metabolic process (cluster 1,
   [84]Supplementary Figure S1B) as well as oxidation-reduction process,
   tricarboxylic acid (TCA) metabolic process, and respiratory electron
   transport chain (cluster 2, [85]Supplementary Figure S1C).

Figure 1.

   [86]Figure 1
   [87]Open in a new tab

   Transcriptional profile of metabolic programs in murine iPax3 myogenic
   progenitors compared to adult satellite cells. (A) Venn diagram of
   genes annotated to metabolic pathways with the Reactome and KEGG
   databases. (B) Venn diagram showing overlap between differentially
   expressed genes (DEGs) after pairwise comparisons and genes annotated
   to metabolic pathways (top), as well as a breakdown of upregulated
   (brown) and downregulated (blue) DEGs (bottom). (C) Volcano plot for
   986 metabolic genes. (D) Principal component analysis (PCA) of bulk
   RNA-seq in in vitro iPax3 (orange) and satellite cells (blue). (E,F)
   Representative gene expression levels of in vitro iPax3 myogenic
   progenitors (orange) vs. satellite cells (blue) across metabolic
   pathways enriched in cluster 1 (E) and cluster 2 (F). (G) PCA of
   microarray in in vivo iPax3 myogenic progenitors (brown) and adult
   satellite cells (blue). (H,I) Representative gene expression levels of
   in vivo iPax3 myogenic progenitors (brown) vs. satellite cells (blue)
   across metabolic pathways previously enriched in cluster 1 (H) and
   cluster 2 (I). Studies represent three biological samples per group.
   **** p < 0.0001 by ANOVA with Bonferroni correction. Abbreviations:
   Tricarboxylic acid (TCA), Electron Transport Chain (ETC).

   In agreement with previous observations of freshly isolated quiescent
   satellite cells [[88]33], our analysis of Pax7^+ satellite cells
   revealed significant high expression of signaling pathways involved in
   lipid-, phospholipid-, and carbohydrate-metabolism ([89]Figure 1E and
   [90]Supplementary Figure S1D). On the other hand, in vitro iPax3 cells
   display enhanced expression of signaling pathways related to glucose
   metabolism, TCA cycle, and the respiratory electron transport
   ([91]Figure 1F and [92]Supplementary Figure S1E). These data suggest
   that in vitro-generated PSC-derived iPax3 myogenic progenitors have a
   predominant oxidative phosphorylation and glycolytic metabolic state,
   opposed to the predominant fatty acid-based metabolic state observed in
   adult satellite cells.

   To understand how the in vivo environment might influence the
   transcriptional signature of these metabolic pathways, we performed
   similar analyses using published datasets of re-isolated iPax3
   donor-derived satellite cells [[93]12] (hereafter referred to as in
   vivo iPax3). PCA comparing in vivo iPax3 to adult satellite cells
   showed that these two cell populations shared a highly related
   signature ([94]Figure 1G). Indeed, of the 360 DEGs found between in
   vitro iPax3 and satellite cells, only 3 (<1%) remained differentially
   expressed after exposure to the in vivo environment. This apparent
   overlap in the transcriptional profile between in vivo iPax3 and adult
   satellite cells prompted us to examine the signaling pathways
   previously identified as differentially expressed ([95]Figure 1E,F).
   Here, we no longer found any statistical differences in expression of
   genes represented in metabolic pathways ([96]Figure 1H,I,
   [97]Supplementary Table S1). Using the adult satellite cells as the
   reference group, we looked at the fold change in gene expression for
   the top 50 DEGs in the in vitro iPax3 group compared to the in vivo
   iPax3 group ([98]Supplementary Figure S1F). Here, we found that the
   relative gene expression for those genes changed towards zero after
   exposure to the in vivo environment, indicating a level of expression
   that is closer to the adult satellite cells. These data suggest that in
   vitro-generated iPax3 myogenic progenitors undergo transcriptional
   changes consistent with switching of metabolic pathways when exposed to
   the adult in vivo environment that are closely similar to that of bona
   fide satellite cells.

3.2. Metabolic Profile of In Vitro and In Vivo iPax3 Cells

   To determine whether the transcriptional switch of metabolic pathways
   is accompanied by functional changes, we next characterized the
   bioenergetic profiles of in vitro and in vivo iPax3 cells, as well as
   of bona fide adult satellite cells as a reference ([99]Figure 2A). For
   these studies, we utilized in vitro-generated iPax3 myogenic
   progenitors, as previously described for transcriptomic studies
   [[100]12]. For in vivo re-isolation, GFP-labelled myogenic progenitors
   were transplanted into pre-injured tibialis anterior (TA) muscles of
   immunocompromised NSG mice ([101]Figure 2A). Upon confirmation of
   myofiber engraftment ([102]Figure S2A), donor-derived satellite cells,
   identified as GFP+Lin-Int7+CD34+ [[103]29], were purified ([104]Figure
   S2B). Satellite cells were isolated from Pax7-ZsGreen mice [[105]33]
   using ZsGreen in combination with the aforementioned satellite cell
   markers ([106]Figure S2C). As expected, Pax7 expression was detected in
   bona fide satellite cells as well as in iPax3 donor-derived satellite
   cells, but not in iPax3 cells cultured in vitro ([107]Figure S2D). We
   then examined these three cell populations for bioenergetics using the
   seahorse mitochondrial stress test, which allowed for measurements of
   oxygen consumption rate (OCR) and extracellular acidification rate
   (ECAR), indicators of aerobic respiration and glycolytic flux,
   respectively. This revealed that, under baseline conditions, in vitro
   iPax3 progenitor cells display enhanced OCR, but this decreases
   significantly upon engraftment as in vivo iPax3 cells show low rates of
   mitochondrial respiration, comparable to levels of adult satellite
   cells ([108]Figure 2B,C). Similar findings were obtained with
   ATP-linked to OCR ([109]Figure 2D). In addition, mitochondrial H^+
   (proton) leak, representing the remaining basal respiration not coupled
   to ATP, was high in in vitro iPax3 cells but decreased once these cells
   were exposed to the in vivo environment ([110]Figure 2E). Likewise, the
   maximal oxygen consumption rate, measured by adding the uncoupler CCCP,
   was also higher in in vitro Pax3 cells when compared to in vivo iPax3
   and satellite cells ([111]Figure 2F). The spare respiratory capacity
   was elevated in in vitro iPax3 cells and much lower in both
   donor-derived iPax3 and bona fide satellite cell fractions ([112]Figure
   2G). However, the reserve capacity was clearly still present
   ([113]Figure 2G and [114]Supplementary Figure S2E), indicating that
   basal respiratory levels are not limited by overall respiratory
   capacity.

Figure 2.

   [115]Figure 2
   [116]Open in a new tab

   Bioenergetics of PSC-derived myogenic progenitors and re-isolated
   donor-derived satellite cells. (A) Outline of experiments. (B) Typical
   traces of real-time OCR before and after the addition of inhibitors to
   derive several parameters of mitochondrial respiration in Pax3 in
   vitro, iPax3 in vivo, and adult satellite cells (SC). (C) Initially,
   basal O[2] consumption OCR was measured, from which basal cellular
   respiration can be derived by subtracting non-mitochondrial
   respiration. (D) ATP-linked represent the difference in OCR before and
   after Oligomycin. (E) H+ leak-linked OCR in Pax3 in vitro, iPax3 in
   vivo, and adult SC represent the difference in OCR after Oligomycin
   injection and Antimycin A and Rotenone. (F) Maximal OCR was determined
   via addition of a mitochondrial uncoupler CCCP that stimulates maximal
   respiration by mimicking a physiological energy demand, leading to an
   increase in oxygen consumption. (G) Spare respiratory capacity OCR
   reflects the difference between basal and maximal respiratory rate, and
   this capacity was determined by measuring OCR after treatment with
   oligomycin and CCCP. (H) Real-time whole-cell extracellular
   acidification rate (ECAR) is an indicator of the rate of acid efflux
   formed during glycolytic energy metabolism used to generate ATP. (I)
   ECAR quantification in iPax3 in vitro, iPax3 in vivo, and adult SC. (J)
   Non-mitochondrial respiration OCR has been observed at low levels in
   iPax3 in vivo and Adult SC. (K) Graphs shows gene expression for Sdhd,
   Cox6b2, Atp5c1, and gapdh. Results are normalized to Actb. Data are
   presented as mean ± SEM (n = three biological samples per group). * p <
   0.05, ** p < 0.01, and *** p < 0.001 from one-way ANOVA followed by
   post hoc Tukey.

   The inhibition of mitochondrial respiration is often accompanied by an
   increase in fermentation of glucose to lactate and changes in
   concentration of hydrogen ions, which are accompanied by a drop in the
   pH levels. However, our data revealed that the ECAR in in vivo iPax3,
   as well as adult satellite cells ([117]Figure 2H,I and [118]Figure
   S2F), did not change, suggesting that purified skeletal muscle stem
   cell populations indeed have lower ATP production and do not undergo a
   shift towards acidification. Moreover, we observed that both these
   satellite cell fractions present lower rates of non-mitochondrial
   respiration ([119]Figure 2J), suggesting that a subset of cellular
   enzymes continue to consume oxygen after addition of rotenone and
   antimycin A ([120]Supplementary Figure S2E).

   Gene expression studies confirmed these metabolic pathway preferences,