Regenerating human skeletal muscle forms an emerging niche in vivo to support PAX7 cells

Skeletal muscle stem and progenitor cells including those derived from human pluripotent stem cells (hPSCs) offer an avenue towards personalized therapies and readily fuse to form human–mouse myofibres in vivo. However, skeletal muscle progenitor cells (SMPCs) inefficiently colonize chimeric stem cell niches and instead associate with human myofibres resembling foetal niches. We hypothesized competition with mouse satellite cells (SCs) prevented SMPC engraftment into the SC niche and thus generated an SC ablation mouse compatible with human engraftment. Single-nucleus RNA sequencing of SC-ablated mice identified the absence of a transient myofibre subtype during regeneration expressing Actc1. Similarly, ACTC1+ human myofibres supporting PAX7+ SMPCs increased in SC-ablated mice, and after re-injury we found SMPCs could now repopulate into chimeric niches. To demonstrate ACTC1+ myofibres are essential to supporting PAX7 SMPCs, we generated caspase-inducible ACTC1 depletion human pluripotent stem cells, and upon SMPC engraftment we found a 90% reduction in ACTC1+ myofibres and a 100-fold decrease in PAX7 cell numbers compared with non-induced controls. We used spatial RNA sequencing to identify key factors driving emerging human niche formation between ACTC1+ myofibres and PAX7+ SMPCs in vivo. This revealed that transient regenerating human myofibres are essential for emerging niche formation in vivo to support PAX7 SMPCs.

Skeletal muscle stem and progenitor cells including those derived from human pluripotent stem cells (hPSCs) offer an avenue towards personalized therapies and readily fuse to form human-mouse myofibres in vivo.However, skeletal muscle progenitor cells (SMPCs) inefficiently colonize chimeric stem cell niches and instead associate with human myofibres resembling foetal niches.We hypothesized competition with mouse satellite cells (SCs) prevented SMPC engraftment into the SC niche and thus generated an SC ablation mouse compatible with human engraftment.Single-nucleus RNA sequencing of SC-ablated mice identified the absence of a transient myofibre subtype during regeneration expressing Actc1.Similarly, ACTC1 + human myofibres supporting PAX7 + SMPCs increased in SC-ablated mice, and after re-injury we found SMPCs could now repopulate into chimeric niches.To demonstrate ACTC1 + myofibres are essential to supporting PAX7 SMPCs, we generated caspase-inducible ACTC1 depletion human pluripotent stem cells, and upon SMPC engraftment we found a 90% reduction in ACTC1 + myofibres and a 100-fold decrease in PAX7 cell numbers compared with non-induced controls.We used spatial RNA sequencing to identify key factors driving emerging human niche formation between ACTC1 + myofibres and PAX7 + SMPCs in vivo.This revealed that transient regenerating human myofibres are essential for emerging niche formation in vivo to support PAX7 SMPCs.
Skeletal muscle is one of the most regenerative organs due to the presence of an endogenous muscle stem cell called the satellite cell (SC) 1,2 .SCs are supported within anatomically defined specialized compartments, termed niches, that regulate the balance of self-renewal and differentiation, similar to other highly regenerative tissues [3][4][5] .The SC niche is composed of dynamic ligand and receptor signalling between the myofibre sarcolemma, SC and the laminin-rich basal lamina that enables SC quiescence 6,7 .Muscle injury disrupts the SC niche inducing a regeneration cascade beginning with rapid activation of SCs to proliferative myoblasts, which turn on fusion-capable genes to form Article https://doi.org/10.1038/s41556-023-01271-0 In the mouse embryo, it has been shown that hPSC-derived lineages are inefficient at forming niches within interspecies chimeras, but deletion of key niche genes can create a competitive niche to substantially extend hPSC donor chimerism 35 .Here we applied a similar concept by generating an immunocompromised inducible Pax7 SC ablation mouse model to study human SMPC niche formation in the absence of mouse SCs.In this model, we found an increase in human donor-derived PAX7 cells from both SCs and SMPCs but predominantly with human niches and not chimeric niches.However, upon re-injury in the Pax7-ablation model we now begin to see an increase in both human and chimeric niche occupancy demonstrating the emerging human myofibre niche supports PAX7 cells required for repopulation ability.Using Pax7-ablated and control mice as a model system to study new niche formation, we identified a transient regenerating ACTC1 + myofibre that is required for new niche formation during regeneration that is absent in Pax7-ablated mice.Spatial RNA profiling of engrafted stem cells and fibres identified key human skeletal muscle niche genes enabling ACTC1 + myofibre support of human PAX7 cells in vivo.We generated stable hPSC lines with a caspase 9-inducible kill switch to deplete ACTC1 + myofibres during engraftment, which we found abolished human PAX7 cells in vivo, demonstrating these ACTC1 + myofibres are a key component of the emerging niche needed to support human PAX7 hPSC SMPCs.In summary, this work identifies how emerging niches occur on newly regenerated myofibres in vivo and provides a new model for understanding early human stem cell niche formation using SMPCs.

SMPCs associate with human-only myofibres in vivo
We previously showed hPSC SMPCs can engraft and regenerate hundreds of dystrophin + myofibres in immunocompromised mouse models of Duchenne muscular dystrophy (mdx-NSG) following cardiotoxin (CTX) injury 20 .We have continued to improve myogenic potential by supplementing survival/maturation factors during directed differentiation of hPSCs in vitro, as well as reducing culture time of ERBB3 + NGFR + PAX7 + hPSC SMPCs before engraftment (Fig. 1a and Extended Data Fig. 1a-c).In vivo, we show that, while hPSC SMPCs fused with mouse skeletal muscle to form hundreds of multinucleated chimeric myofibres (maximum number 75-230 per cross sectional area (CSA), N = 7 mice), hPSC SMPCs also generated about twice as many human-only myofibres (maximum number 140-450 per CSA), which occupied large regions of engrafted skeletal muscle (Supplementary Video 1).Myofibre size as measured by Imaris software and exclusivity of human nuclei marked by human lamin AC were used to distinguish and quantify chimeric and human-only myofibres in transverse and longitudinal sections (Fig. 1a and Extended Data Fig. 2a).
An effective cell therapy must maintain the muscle stem cell pool.Yet so far retention of human PAX7 cells after transplantation of hPSC or foetal SMPCs has proven challenging.Thus, we were encouraged to find 20 or more human PAX7 + hPSC SMPCs within 0.15 mm 2 (20×).A total of 127 human PAX7 + cells were counted and categorized on the basis of proximity to human-only myofibres, chimeric myofibres, new skeletal myofibres, that will contain hundreds of myonuclei 8,9 .As new skeletal myofibres form, a subset of SCs re-enter the niche and revert from a proliferative to quiescent state, ensuring an SC pool for continual future regenerative events 10,11 .
The ability to establish new SC niches is essential for long-term cell therapies, in which transplanted muscle stem cells must balance the formation of new muscle fibres and maintain the stem cell pool 12,13 .More recently, small-molecule-mediated directed differentiation of human pluripotent stem cells (hPSCs) to skeletal muscle progenitor cells (SMPCs) has opened up the potential for personalized cell therapies and disease modelling for devastating muscle diseases from an in vitro cell source [14][15][16][17] including in concert with gene editing 18,19 .We have demonstrated that hPSC SMPCs fuse to form hundreds of new myofibres in vivo after gene editing 20 ; however, so far retention of human PAX7 cells after transplantation of hPSC SMPCs has proven challenging.Many reports show a high-magnification image of one or a few PAX7 cells following transplantation [20][21][22] , but to fully restore muscle function will probably require at least 8-15 engrafted PAX7 cells per 100 myofibres, similar to endogenous SC levels in adult 23,24 .Determining how transplanted SMPCs take up position in SC niches may enable support of PAX7 cells to improve long-term cell therapies for skeletal muscle diseases.The emerging SC niche (that is, the first myofibres that form upon engraftment that support PAX7 SMPCs) may also serve as an in vivo model to improve our basic understanding of human regeneration, similar to elegant studies of early development in mouse 25,26 .
Our laboratory generated a single-cell atlas of the human limb transcriptome from embryogenesis through adulthood, which determined the developmental identity of hPSC SMPCs in silico 27 .We found hPSC SMPCs most closely align with human SMPCs between weeks 7 and 12, a developmental time period that coincides with the transition from embryonic to foetal myogenesis, a period when foetal skeletal muscle is undergoing rapid growth to establish myofibres and foetal myoblasts are genetically maturing 28,29 .SMPC behaviour markedly differs from SCs including differences in mitotic activity 30 , receptor expression 31 and extracellular matrix (ECM) composition 32 .In mouse, SMPCs migrate across and take up position under the myofibre basal lamina to establish the first niches late in foetal development 33,34 , but the mechanisms by which human SMPCs form niches and transition from progenitor to SCs to enable SC maintenance during human development or after transplantation is unknown.
In this Article, we focused on the ability to retain PAX7 cells and reside in SC niches after transplantation in vivo.We used human foetal SMPCs and adult SCs to benchmark in vivo niche occupancy and repopulate muscle after re-injury, the hallmark assay for measuring SC self-renewing potential in vivo 12 .We found that transplanted cells across all developmental states and from hPSCs had the ability to form multinucleated chimeric (mouse-human) myofibres; however, unlike adult SCs, SMPCs did not enter the chimeric mouse niche efficiently.Instead, transplanted SMPCs formed large regions of human-only myofibres and PAX7 cells formed niches with these newly formed human-only myofibres.showing insets (rotated 90°) of chimeric and humanonly myofibre phenotypes.Human-specific antibodies lamin AC (red) mark human nuclei, and dystrophin (green) mark human myofibres, 4′,6-diamidino-2phenylindole (DAPI) (nuclei, blue).Scale bar, 200 μm.Right: Imaris was used to quantify myofibre cross-sectional area as one parameter of chimeric and humanonly myofibres.Scale bar, 100 μm.Graph shows mean ± standard deviation of engrafted human and human-mouse myofibres over the length of mouse TA muscle (N = 7).Histogram shows cross-sectional area of Imaris-identified myofibres using human dystrophin.b, 20× images were quantified for number of human PAX7 cells, chimeric myofibres, human-only myofibres, total human cells and mouse SCs (PAX7 green or dystrophin green depending on image; lamin AC and spectrin, red; DAPI, blue).Scale bar, 50 μm.To quantify location of PAX7 cells, numbers were then quantified using 20× images (0.15 mm 2 ) were separated into four regions: (A) fields of view containing >50 human-only myofibres, (B) fields of view containing >10 chimeric muscle myofibre, (C) mouse myofibres, interstitial space and epimysium, and (D) dense numbers of human cells but few fused myofibres.Graph shows mean ± standard deviation of six to ten images from each region, N = 3 mice were quantified; statistics were performed using one-way ANOVA with post-hoc Tukey test, *P < 0.05.interstitial space or regions with high numbers of human lamin AC cells but few myofibres (Fig. 1b).We found regions containing the greatest numbers of human-only myofibres also contained 98% of all engrafted PAX7 hPSC SMPCs.In contrast, regions containing only lamin AC, but few or no human-only myofibres, did not contain PAX7 cells.We also evaluated chimeric myofibre regions, and found these regions contained the other 2% of PAX7 cells (P < 0.05, Fig. 1b and Extended Data Fig. 2b).We also evaluated hPSC SMPC engraftment in another

In vivo niches from SMPCs resemble early foetal niches
Recent work from our lab has shown that directed differentiation of hPSCs generates embryonic or foetal-like skeletal muscle in vitro 27 and we hypothesized that engrafted hPSC SMPCs would produce similar immature skeletal muscle in vivo.To understand the developmental trajectory of transplanted PAX7 + hPSC SMPCs, we compared PAX7 cells and their niches with human skeletal muscle tissues from embryonic weeks 9-11, foetal weeks 16-20 and adult years 25-40 (Fig. 2a).We found embryonic and foetal myofibres were ∼30-fold smaller than adult myofibres, and this was similar to the small human-only myofibres observed after hPSC SMPC transplantation.PAX7 + cells in human embryonic week 10 tissues closely associated with the sarcolemma of embryonic myofibres that clustered together but lacked basal lamina expression of laminin.As myofibres matured from the embryonic to foetal stage, PAX7 + cells associated with the sarcolemma became encompassed by laminin, demonstrating the emergence of human niches (Fig. 2a).Here the interior sarcolemma lacked a laminin-rich basal lamina, but formed spectrin + cross bridges, which are a known hallmark of myogenic fusion 36 .Similarly, several engrafted hPSC myofibres could be found in regions undergoing potential fusion in myobundles marked by spectrin that were encompassed by laminin basal lamina.Both foetal week 20 tissues and engrafted hPSCs contained PAX7 + SMPCs within these myobundles (Fig. 2b).These data suggested hPSC SMPCs formed niches that more closely resembled a foetal week 20 phenotype upon engraftment in vivo.This was different than adult SC niches/myofibres, which are primarily in homeostatic state where all spectrin + sarcolemma was closely juxtaposed to a laminin-rich basal lamina.

SMPCs and SCs differ in niche occupancy in vivo
We next set out to compare niche acquisition ability of human muscle stem and progenitor cells.We performed fluorescence-activated cell sorting enrichment on embryonic/foetal SMPCs (weeks 8-20, Lin-ERBB3 + NGFR + ) or adult SCs (years 25-60, Lin-CD82 + NCAM + ) dissociated from primary human tissues and immediately transplanted human cells into mdx-NSG mice.These data were used to determine whether niche acquisition resembled the endogenous niches from which the stem and progenitor cells were derived.We first tested engraftment efficiency across a range of foetal SMPC numbers, from 10,000 to 500,000.Foetal SMPCs (weeks 17-20) produced >100 human myofibres per cross section when 500,000 foetal SMPCs were transplanted, but at lower densities, foetal SMPCs did not restore dystrophin efficiently and only 0-1 PAX7 cells remained at day 30 (Extended Data Fig. 3).Due to embryonic tissue size (weeks 8-10) we could only obtain 50-75,000 embryonic SMPCs, and these formed myofibres poorly upon engraftment and no PAX7 cells were found, which could have been related to survival or immaturity as seen in other stem cell systems 37 .In contrast, adult SCs fused to form dystrophin + myofibres and retained 5-20 PAX7 + SCs per cross section at 30 days in vivo with as few as 5,000 transplanted SCs (Fig. 2c and Extended Data Fig. 3).Furthermore, more than 80% of PAX7 + adult SCs were located in human-mouse chimeric myofibre niches 30 days post engraftment.Thus, chimeric niche acquisition is not a species compatibility issue (Fig. 2c).Foetal SMPC ability to colonize niches was intermediate between hPSC and adult SCs as we found that ∼20% foetal SMPCs were associated with small human-only myofibres and ∼30% were found inside chimeric myofibres.In contrast, 80% of PAX7 + hPSC SMPCs were associated within clusters of human-only myofibres and less than 5% were found within chimeric niches (Fig. 2c).These data suggest that both foetal and hPSC SMPCs can associate with human-only myofibres and ability for niche entry differs across development and adult.

SC-ablated mice enable human PAX7 retention
As it was unclear why human PAX7 + hPSC and foetal SMPCs were less able to form niches with chimeric myofibres, we tested two hypotheses related to niche entry and regeneration illustrated in Fig. 3a.We generated a new inducible Pax7 ablation dystrophin model by crossing dystrophic mdx-NSG mice to Pax7-Cre/Ert2 mice and Rosa-DTX mice.When these mice are treated with tamoxifen (TMX), expression of Pax7-driven Cre recombinase removes LoxP stop codons to induce expression of diphtheria toxin (DTX) resulting in SC death 38,39 (Fig. 3b).Thus, in this model, regeneration and niche formation would preferentially occur from transplanted human cells as competition with mouse SCs was removed.Pax7-DTX mice were evaluated for SC ablation following 7 days of TMX-infused chow, which we found was sufficient to ablate greater than 80% of endogenous Pax7 SCs relative to non-ablated controls (Fig. 3b).Following CTX-or BaCl 2 -induced injury of tibialis anterior muscles, Pax7 ablation resulted in decreased weight and cross-sectional area, demonstrating that Pax7 cells are required for muscle repair, but did not result in complete loss of all myofibres (37 mg versus 62 mg, P < 0.05) (Fig. 3b).We noted a subset of myofibres underwent hypertrophy after Pax7 ablation, growing up to four-fold larger than non-ablated controls, demonstrating myofibres are phenotypically influenced by loss of Pax7 SCs and their niches (P < 0.05, Fig. 3b and Extended Data Fig. 4a).
We next tested the ability of human foetal SMPCs to engraft and reside in the niche of Pax7-ablated mdx-NSG mice.We found a two-fold increase in PAX7 + foetal SMPCs 30 days post engraftment (P < 0.05, Fig. 3c).We evaluated the location of foetal PAX7 + SMPCs and surprisingly found these were less in mouse SC niches, and more associated with small human fibres upon mouse SC ablation (P < 0.05, Fig. 3d).While in some cases engrafted foetal SMPCs fused with mouse to form chimeric myofibres in mdx-NSG Pax7-ablated mice, we found foetal SMPCs predominately formed small human-only myofibres.Interestingly, adult SC engraftment in Pax7-DTX mice also followed a foetal  trajectory where adult SCs were more likely to be found near small human-only myofibres (Fig. 5a,b and Extended Data Figs.4b and 6c).
Together, these data point to the idea that, in the absence of mouse Pax7 SCs, human-only myofibres are left in a muscle microenvironment that promotes a developmental or regenerative-like paradigm that enables the support of engrafted PAX7 SMPCs.

Myofibres that support SC niches are lost upon SC ablation
While it is well known that Pax7 cells are essential for myofibre formation [39][40][41] , it is less clear how the SC niche and regeneration are affected by the absence of Pax7 cells.Therefore, we set out to identify which genes or populations were missing in the myofibres of Pax7-ablated mice.Mouse SC niches are re-established in parallel with new myofibre formation 7-10 days post-injury (dpi) 10,42 .Thus we selected this timepoint post injury to study niche formation, which we confirmed by embryonic myosin heavy chain (eMyHC) expression and increased mouse Pax7 SCs in mdx-NSG mice 8 dpi, and these cell types were both lost in mdx-NSG Pax7-DTX mice (Fig. 4a).We performed single-nucleus RNA sequencing (snRNA-seq) on Pax7-ablated muscle compared with non-ablated and regenerating muscle 8 dpi, and used recently published datasets as a starting point to identify new and known populations in our ablation models [43][44][45] .In total 14,300 nuclei were sequenced, enabling identification of several population shifts between regenerating, non-ablated and Pax7-ablated myofibres (Fig. 4b, deposited as GSE241368).Seurat analysis identified a total of 21 populations including 10 myonuclear and 11 mononuclear populations (Supplementary Table 1).We found a two-fold influx in mononucleated cells in regenerating mdx-NSG mice 8 dpi, which included immune, stromal and myogenic populations (Fig. 4c).In contrast, Pax7 ablation had a blunted influx of mononucleated cells 8 dpi.Macrophages and Itga7 + Vim + myoblasts (MB.2, described by ref. 46) were the only mononuclear populations to increase in Pax7-ablated mice.Seurat analysis identified a Pax7 + Myod1 − SC population (SC.1) which as expected was eliminated in Pax7 ablated muscle (0.1% ablated versus 5.6% non-ablated).In non-ablated muscle, Myod1 + Ki67 + myoblasts (MB.1, 0.6% versus 4.5%) and Myog + Myh3 + myocytes (MC, 1.3% versus 4.2%) increased 8 dpi compared with no injury, respectively, but upon Pax7 ablation the regenerative response of myoblasts (0.7% versus 2.6%) and myocytes (0.6% versus 1.4%) was diminished 8 dpi, validating that regeneration was absent following Pax7 ablation (Fig. 4c).
Since Actc1 was the highest-enriched gene in IIB.3 myonuclei, we confirmed with immunostaining that Actc1 protein expression overlaps with timing of SC niche establishment during mouse muscle regeneration (Extended Data Fig. 6a).In contrast, while embryonic myosin protein is co-expressed on Actc1 + myofibres, the Myh3 RNA expression is gone at 8 dpi.Thus, Actc1 expression may be longer lasting than Myh3 and may mark a myofibre important for supporting new SC niche formation.Our snRNA-seq analysis identified 134 upregulated genes expressed by Actc1 + (IIB.3)myofibres, which included genes important for sarcomere formation, nucleotide and lipid metabolism, mitochondrial energetics and calcium regulation (for example, Tnnt3, Eya4, Prkag3, Ampd1 and Casq1).These myofibres also expressed membrane-associated gene signatures related to Notch, Integrin and Jak/Stat signalling (Fig. 4d, Extended Data Fig. 5 and Supplementary Table 2).We used CellChat analysis 47 to analyse ligand-receptor niche interactions, which identified increased laminin signalling between Actc1 + myofibres and mouse Pax7 + SCs compared with other myofibre populations (Fig. 4d).

Article
https://doi.org/10.1038/s41556-023-01271-0we evaluated if ACTC1 shares common functions with the human-only myofibres supporting PAX7 + SMPC engraftment.ACTC1 is also known to be expressed during foetal skeletal muscle development 48 , which further supported the likelihood that ACTC1 would be important in the emerging niches formed by hPSC SMPCs.Indeed, we found that human-only myofibres from hPSC and foetal SMPCs strongly expressed ACTC1 following engraftment, but in myofibres that we identified as chimeric, ACTC1 was not expressed at 30 days (Fig. 5a  and Extended Data Fig. 6b).To further demonstrate that ACTC1 was expressed by human-only myofibres, but not chimeric myofibres, we took advantage of mouse-specific myosin expression Myh4 in tibialis anterior (TA), which human skeletal muscle does not express.
Staining confirmed the presence of chimeric myofibres (lamin AC + spectrin + Myh4 + ) and human-only myofibres (ACTC1 + Myh4 − ), in which the smallest human myofibres had the greatest ACTC1 expression (Fig. 5b).ACTC1 + human myofibres were more prominent when engrafted in mdx-NSG Pax7-DTX mice, and moreover adult SCs now predominately formed ACTC1 + human-only myofibres in Pax7-ablated mice, suggesting that, when mouse regeneration is impeded, human SC niche formation initially occurs with regenerating human-only myofibres (Extended Data Fig. 6c).Since we found that SC niche formation is predominantly occurring near human-only fibres in mdx-NSG Pax7-DTX mice we set out to evaluate whether these niches were functional.The hallmark in vivo assay for evaluating true muscle stem cell potential is the re-injury assay, in which PAX7 cells must regenerate new myofibres as well as maintain the stem cell pool 12 .After 30 days, engrafted muscles are re-injured using CTX, which damages myofibres but preserves PAX7 cell regeneration ability 49 .As controls for re-injury, day 30 and uninjured day 60 engrafted tissues are used.We found human adult SC and foetal SMPCs were able to form dystrophin + myofibres and retain some PAX7 + cells at all timepoints, but engraftment dynamics were very different across mouse models (Fig. 5c and Extended Data Fig. 7a).In mdx-NSG mice, adult SCs where able to sustain PAX7 + SC numbers in chimeric niches following injury, but foetal PAX7 + SMPCs decreased by >2-fold, and the ratio of PAX7 foetal SMPCs in chimeric niches decreased by 43% (Fig. 5c).After re-injury, 1 in 3 transplanted adult SCs expressed PAX7, whereas only 1 in 15 foetal SMPCs expressed PAX7 (Extended Data Fig. 7b).In contrast, in the mdx-NSG Pax7-DTX mice, we found after re-injury there was a notable increase in the ability of foetal SMPCs to repopulate both human-only and chimeric niches (Fig. 5c).We also found several instances where two or more PAX7 + foetal SMPCs were within 5 μm of each other, which is suggestive of daughter foetal SMPCs derived from a common parent following re-injury (Fig. 5c).These data show that the increase in ACTC1 + human-only myofibres in the mdx-NSG Pax7-DTX mice can support SC niche formation and repopulation after re-injury.

Spatial analysis identifies key emerging niche pathways
To better define the ACTC1 + niche supporting human PAX7 cells, we optimized spatial RNA sequencing (RNA-seq) and evaluated the niche in both long-term engraftment and after re-injury (Fig. 6a).To do this, we used the Nanostring GeoMx platform, which enables cell type and location specific analysis of gene expression at single antibody resolution.GeoMx allows for region of interest (ROI) selection and segmentation on PAX7 cells and ACTC1 + and ACTC1 − myofibres (Supplementary Tables 3 and 4).In brief, we engrafted foetal SMPCs for up to 4 months and induced an injury in a subset of engrafted mice midway at 2 months.We then mounted flash-frozen muscle sections on microscope slides, hybridized a whole transcriptome human RNA oligo library onto the muscle, and stained with morphology markers ACTC1, PAX7, lamin AC and spectrin to identify human muscle engrafted in mouse.After ROI selection and segmentation, the RNA oligos are photocleaved and collected for sequencing (N = 44 ROIs, Extended Data Fig. 10, deposited as GSE243875).Principal component analysis (PCA) found distinct clustering of cell types including ACTC1 + and ACTC1 − myofibres (Fig. 6b), and our data confirmed a robust repopulation ability by foetal SMPCs in mdx-NSG Pax7-DTX mice that was sustained through 4 months (Fig. 6c).At the time of re-injury, we identified unique gene signatures in the ACTC1 + myofibres, which included increased embryonic and foetal myosins, ECM development and cell adhesion.In contrast the ACTC1 − myofibres had higher expression of metabolism genes and maturation genes.We additionally performed DEG analysis of PAX7 foetal SMPCs after repopulation versus PAX7 foetal SMPCs that remained over a 4-month time period (Supplementary Table 5).Foetal PAX7 SMPCs that had undergone repopulation expressed many genes that could be unique candidates associated with new niche formation including NOTCH4, ITGB1, FGFR1 and MEGF10 as well as stem cell maintenance, chromatin organization and negative regulation of cell proliferation (Fig. 6c,d).We then used CellChat analysis to predict receptor-ligand interactions between the ACTC1 + myofibres and the PAX7 foetal SMPCs identified potential candidates associated with emerging niches including robust association of DAG1 on PAX7 cells and LAMA5 as well as LAMC1 on ACTC1-positive myofibres.Interestingly, THBS4 was a top candidate on ACTC1 + myofibres interacting strongly with receptors on PAX7 cells including integrins and with other myofibres through CD36, a regulator of lipid metabolism and fatty acid uptake (Fig. 6d).Thus, spatial analysis revealed several potential interacting pathways supporting emerging niche formation in vivo in addition to ACTC1.

ACTC1 + myofibres are essential for PAX7 in emerging niches
To definitively prove that ACTC1 myofibres are critical for supporting PAX7 cells, we followed recent approaches that have introduced an inducible suicide gene in hPSCs 50,51 .HPSCs are uniquely able to be genetically manipulated while retaining pluripotency and ability to differentiate into downstream lineages.We engineered an inducible apoptosis system that included a FKBP12 linked to a caspase 9 (iCasp9) and a self-cleaving T2A peptide immediately following a homology arm of the last 3′ base pair in the ACTC1 gene (Extended Data Fig. 8a).Treatment with the bio-inert small molecule AP1903 induces cross-linking of the drug-binding domains of this chimeric protein, which in turn dimerizes caspase 9 and activates the downstream executioner caspase 3 molecule, resulting in gene-specific cellular apoptosis 52 (Fig. 7a).To determine the optimal conditions for inserting iCasp9 by homology-directed repair, we tested 17 conditions in H9 and H1 hPSC lines including 3 guide RNA (gRNA) targets, lipo-STEM and nucleofection, and compared homology-directed repair protocols described by Skarnes et al., to the protocol provided by Lonza (ref.53 and Extended Data Fig. 8b).We confirmed the presence of inserted iCasp9 in both H9 and H1 lines following nucleofection (Fig. 7a) and proceeded to statistical tests with Benjamini-Hochberg (BH) correction P < 0.05.Bar graphs show Gene Ontology of selected pathways upregulated by ACTC1 + and ACTC1 − myofibres.Bottom left: PAX7 in the re-injury group and regions used for selecting single PAX7 cells for sequencing.d, Volcano plot of human foetal PAX7 + SMPCs after re-injury or no-injury 120 days after engraftment.Red dots represent differentially expressed genes, and selected key genes are shown.Graphs show Gene Ontology of foetal PAX7 + SMPCs undergoing repopulation after re-injury of selected pathways related to the SC niche comparisons between two groups.For comparing different cell types or treatments to generate volcano plot data, LMM statistical tests with BH correction were performed.Neg.reg., negative regulation.Right: CellChat analysis show predicted sender (bottom) and receiver (top) ligand and receptor interactions between foetal PAX7 + SMPCs and ACTC1 + and ACTC1 − myofibres from spatial RNA-seq of engrafted tissues.Re-injury single-cell cloning in 384 wells.Of these, 108 clones survived, and we found 2 clones that contained a heterozygous insertion for iCasp9.We performed sequencing to confirm an exact match for our engineered iCasp9 vector within the ACTC1 locus (Extended Data Fig. 8c).
We next performed directed differentiation of ACTC1-iCasp9 hPSCs to generate SMPCs for engraftment.We used Pax7-ablated mdx-D2 NSG Pax7-DTX mice as these mice readily enable human-only myofibre formation.Two weeks following engraftment, mice were treated with AP1903 (10 μM in 100 μl saline) or control (0.1% dimethyl sulfoxide in saline) every two weeks for 90 days.Similar to previous results, in non-treated mice we found hundreds of hPSC myofibres that were able to engraft over approximately 6 mm 3 of mouse TA, and we counted up to 90 PAX7 + hPSC SMPCs associated with hPSC myofibres within a 1.5-mm 2 region of tissue (Fig. 7b and Extended Data Fig. 9a).AP1903 treatment induced a ∼90% reduction in hPSC myofibres, as we found fewer than 20 myofibres per cross section in treated mice.Remarkably the apoptosis of ACTC1 + myofibres resulted in an almost complete elimination of PAX7 cells in the AP1903-treated mice.We counted a total of 269 human PAX7 cells in five regions of control mice and only 5 PAX7 cells within the same engrafted regions of AP1903-treated mice (Fig. 7b,c).However, in the AP1903-treated groups there were hundreds of remaining lamin AC + cells that were neither myofibres nor PAX7 + .
We further validated the iCasp9 system using GeoMx spatial sequencing on engrafted hPSC muscle cells with and without AP1903 treatment (ROI, N = 59).Spatial analysis showed an abundance of cell death-related pathways including several upregulated caspase genes in the AP1903-treated muscles but not in control muscles (Fig. 7c,d and Supplementary Table 6).We compared the remaining hPSC lamin AC + cells (called hPSC PAX7 − ) with hPSC PAX7 + cells, and these expressed genes enriched in non-stem cell or non-myogenic related cell fate change including POSTN, CDH11 and PDGFRA, suggesting that in the absence of human myofibres these cells change fate to other mesoderm lineages, whereas the PAX7 + cells expressed key stem cell genes such as ITGA7, NOTCH3 and CDH15 (Extended Data Fig. 9 and Supplementary Table 7).To better define how the hPSC SMPCs are interacting with the emerging fibres we compared the PAX7 cells with the hPSC myofibres in vivo again using spatial RNA-seq.We also discovered that key stem cell signalling and adhesion pathways are turned on demonstrating maintenance of stem cell fate in this model when ACTC1-positive fibres are present, indicating support of human PAX7 cells by these emerging niches (Fig. 7d).To verify this, we performed CellChat analysis and found that hPSC myofibres send several signals through embryonic laminins and delta ligands (LAMA1 and DLK1) that are predicted to interact with receptors (NOTCH3, FGFR4, DAG1, ITGA7 and CD44) on the hPSC PAX7 + SMPCs (Fig. 7e and Supplementary Table 8).These identified key stemness pathways are turned on, which could be key to supporting hPSC PAX7 cells in emerging niches.

Discussion
The ability to efficiently support human PAX7 cells after engraftment remains a major roadblock to translating cell therapies for skeletal muscle.We demonstrate that the formation of human-only myofibres following transplantation is a key source of niche emergence from transplanted human cells, which has previously been overlooked.Human-only myofibres have a 50-fold better ability to support transplanted PAX7 cells and can be identified by expression of the foetal skeletal muscle actin isoform ACTC1.The ACTC1 + hPSC myofibres form niches that phenocopy late-stage foetal myogenesis, which enables the generation of human niches that support PAX7 cells from hPSCs.To demonstrate that ACTC1 + myofibres are key to supporting stemness from PAX7 + hPSC SMPCs, we developed iCasp9 suicide hPSC lines where we took advantage of the power of hPSCs with genetic engineering to evaluate this question.We demonstrate that ablation of the ACTC1 myofibres results in substantial PAX7 loss.Using multiple functional studies, we conclude that transplanted human SMPC/SCs form niches using skeletal muscle regeneration pathways and do not appear to 'home' to empty niches like the haematopoietic stem cell system 54 .We show reciprocation between the myofibre and stem cell; when mouse Pax7 + SCs are ablated, the regenerative Actc1 + myofibres are lost, and conversely when ACTC1 + emerging myofibres are eliminated, the PAX7 hPSC SMPCs are lost.We also have identified unique gene signatures including ligands and receptors found on foetal and hPSC myofibres that may support de novo niche formation and could improve our ability to translate human PAX7 cells for cell and regenerative therapies.
Our new engraftment model specifically eliminated mouse SCs to evaluate SMPC niche formation in the absence of SCs.Interestingly, in Pax7-ablated mice we found increased numbers of PAX7 + hPSC and foetal SMPCs, but rather than residing in empty chimeric mouse niches, SMPCs increased association with ACTC1 + Myh4 − human-only myofibres at 30 days.We also found that PAX7 + foetal SMPCs and adult SCs now predominantly formed niches with ACTC1 + human myofibres in Pax7-ablated mice, suggesting that in this environment PAX7 SMPCs/ SCs prefer association with regenerative myofibres even though our ablation and injury model still contained hundreds of mouse myofibres (Extended Data Fig. 4c).We show that foetal and hPSC SMPCs can maintain PAX7 + expression for several months in vivo when associated with ACTC1 + myofibres, and when re-injury is applied after 60 days foetal PAX7 + SMPCs are now better able to engraft into both human-only and chimeric niches, suggesting association with these niches enables an increase in functional maturation.
Our approach is a more precise ablation model compared with long-standing practices in the field, which has removed competition with the endogenous SCs by irradiation aiming to increase niche occupancy from engrafted donor human or mouse cells.Muscle irradiation studies are confounded by precise dosing and timing regimens required, and high doses of radiation reduce donor mouse-to-mouse engraftment [55][56][57] .In our hands and others, irradiation did not improve human muscle cell engraftment into mouse 58 .Further, irradiation damages cells in the microenvironment, which orchestrates muscle regeneration and supports SC niche formation [59][60][61] , and studies have not fully investigated the extent to which radiation regimens affect the myofibre regenerative response.We also cannot rule out that the formation of human-only myofibres will be increased close to the injection

Article
https://doi.org/10.1038/s41556-023-01271-0site, and therefore would increase the probability of local association with human PAX7 cells.However, this finding is also interesting and could inform on cell numbers needed to support PAX7 SMPCs upon engraftment and the need for multiple injections per muscle.
To understand the mechanisms of failed regeneration and niche formation in Pax7-ablated mice, we performed snRNA-seq in Pax7-ablated and control mice with and without injury.Similar to work from several groups [39][40][41] , we show that loss of Pax7 cells led to severe muscle regeneration defects in acute-injury models.Of the remaining myofibres in the Pax7-ablated mouse muscle, our sequencing data find increased numbers of highly glycolytic Car3 + myofibres, but most striking was the reduction of the regenerative Actc1 + mouse myofibres marked by several genes involved with sarcomere assembly (Tmod1, Myhpb3 and Tnnt3), which suggests that niche formation occurs in parallel with construction of sarcomeres.Notable niche genes identified in the Actc1 + myofibres that interact with laminin include Itgb1pb2, which binds integrins expressed by SCs during niche formation 62 , and Tmem38a, which regulates heterochromatin and repositions select genes to the nuclear periphery of the myofibre, which happens to include key regulators of SC migration such as Vcam1.It also brings in Nidogen, a gene involved in repositioning of laminin binding and that we found expressed by human foetal and hPSC myofibres in vivo 63 .
Although it is well understood how mouse regeneration occurs after injury, this work develops a human model to study regeneration leading to new emerging niche formation in vivo.We took advantage of spatial RNA-seq to investigate differentially expressed genes between ACTC1 + and ACTC1 − myofibres and ligand-receptor pairs interacting with genes expressed on foetal SMPCs.One of the unique identified pathways expressed on engrafted ACTC1 + foetal myofibres was Thrombospondin 4 (THBS4), which enhances attachment of dystrophin-glycoprotein and integrin complex to stabilize the muscle membranes 64 and is predicted to interact with ITGB1 expressed on foetal SMPCs.We also performed spatial RNA-seq on engrafted hPSC SMPCs and myofibres, and identified key in vivo niche genes including CD44 expressed by PAX7+ hPSC SMPCs which is activated during regeneration and foetal development 65 , and is predicted to interact with Collagen 6 expressed by hPSC myofibres, which promotes autonomous niche formation through ECM production 66 .Other key identified interactions in our analysis that may also be critical to supporting PAX7 + SMPCs include multiple NOTCH signalling pathways that are sending and receiving in both directions from hPSC SMPCs and myofibres.We also found Dystroglycan 1 (DAG1) on hPSC SMPCs interacting with laminin 111 (LAMA1) expressed by hPSC myofibres.One caveat to the use of GeoMx for spatial RNA-seq is the close, but not perfect, precision of morphology marker segmentation, and we do find some muscle genes in the PAX7 SMPC datasets due to their close spatial proximity demonstrating further validations will be required.To avoid this, the datasets we focused on comparing hPSC SMPCs with myofibres to identify enriched genes such as CD82, FGFR4 and CERCAM, and these may represent key targets to modulate for improved support of PAX7 cell engraftment in future studies.
The significance for identification of the ACTC1 + myofibres for initiating niche formation could have both basic and therapeutic applications.Many labs have highlighted that both regenerating adult myofibres and developing foetal myofibres support PAX7 cell numbers at orders of magnitude greater per unit area than adult myofibres in homeostasis 33,67 .It would be interesting to determine whether myofibres in homeostasis could support additional PAX7 cells if stimulated by myofibre genes involved in emerging SC niche formation.Actc1 has previously been shown to be highly expressed in foetal skeletal muscle 68 , and overexpression of Actc1 has been used to rescue defects in skeletal actin myopathies that increased the expression of regenerating markers such as embryonic myosin 69 .Similarly, we found mouse SCs during regeneration also clustered near newly formed Actc1 + myofibres.Looking to other stem cell niche systems such as dental pulp 70 and hair follicle 71 , stem cell density in the niche is far greater than in skeletal muscle, suggesting the upper limit to stem cell density in niches found in biological systems could be increased.Regulation of the mouse Pax7 cell number set point has been demonstrated via Wnt4 knockdown 72 , and increasing the SC number set point could have exciting implications for muscle regeneration and establishing niches from hPSCs.
Our results highlight that SMPCs that do not associate with human myofibres lose PAX7 expression.Loss of stemness also occurs for mouse SCs when displaced from the myofibre niche 73 .Thus, myofibres are important to support the stem cell state.In the absence of myofibres in Myf5/MyoD double-null mice, satellite cells can adopt mesenchymal or alternative somatic lineages after injury, but the double-null satellite cells were retained when myofibres were present and uninjured.This observation suggests that myofibres contribute to maintaining the identity of satellite cells having the ability to adapt non-myogenic cells 74,75 .Similarly, using iCasp9 in human ACTC1 myofibres, we found that hPSC SMPCs adopted alternative cell fates when not associated with myofibres (Extended Data Fig. 9).PAX7 − lamin AC + cells expressed high levels of mesenchymal (PDGFRA and POSTN) and mesodermal (CDH11) genes.Both the lack of niche association and the immaturity of hPSC SMPCs may also result in increased plasticity, similar to work showing that foetal SMPCs have plasticity to form both bone/muscle without niches 27,76 .
The in vivo microenvironment has been shown to improve maturation of mouse embryonic stem cells following overexpression of Pax3 or Pax7 (iPAX) 77 and hPSC SMPCs in vivo 78 , but all are largely foetal and not equivalent to quiescent adult SCs.Recent work has also shown that maturation can be enhanced by epigenetic modulation in other stem cell systems 79,80 .We evaluated hPSC SMPCs in vivo compared with engrafted foetal or adult SCs and tissues, and our work suggests that hPSC SMPCs do mature in vivo from an embryonic week 7-12 to a later foetal week 17-20 stage, but do not fully mature to quiescent adult SCs.How human SMPCs become quiescent SCs in the niche is an exciting direction that could require lineage tracing to follow SC and niche dynamics over time in vivo.Interestingly, we find that the myofibre populations between engrafted foetal and hPSC myofibres and foetal tissues are more dissimilar than are the PAX7 + cells (Extended Data Fig. 10), and thus the myofibres may be one of the most important targets for PAX7 + SMPC maturation, including approaches to enhance innervation.
In summary, we developed a new muscle stem cell ablation mouse model compatible with engraftment and demonstrated that emerging niche formation from hPSCs forms similar to the process of regeneration.This work has global implications for cell therapies and provides a new model system to study how to support human regeneration and niche emergence.Further, this new system could provide a platform to screen for new regulators of niche formation and the key regulators of stem cell-niche interactions supporting PAX7 cells in vivo.

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Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/s41556-023-01271-0.

Fig. 3 |
Fig. 3 | Pax7-ablation model enables evaluation of human stem cell niche formation in the absence of mouse Pax7 cell competition.a, Cartoon depicting hypotheses for SMPC association with human-only myofibres in the Pax7-ablation model (Pax7-ablation (DTX) mdx-NSG).Top: the occupied niche hypothesis predicts SMPCs cannot home to chimeric niches occupied by endogenous satellite cells, and the regenerative niche hypothesis predicts mouse SCs take up position in chimeric niches during regeneration.Bottom: engraftable Pax7 ablation mouse shows expected results for occupied niche and regenerative niche hypotheses.b, Cartoon of ablation mouse model generation and Pax7 cell numbers (white boxes) in Pax7-ablated and non-ablated control mice after 7 days of TMX treatment (resulting in Pax7 ablation or DTX), mean ± standard deviation, N = 5 mice, t-test *P = 0.0003.Tibialis anterior muscles of Pax7-ablated mice are atrophic; some individual myofibres of Pax7-ablated muscle undergo hypertrophy as measured by haematoxylin and eosin staining.c, Human foetal SMPCs increase PAX7 + numbers in Pax7-ablation mice.Representative images show co-staining of human nuclei (red), PAX7 cells (green) and 4′,6-diamidino-2-phenylindole (DAPI).White boxes indicate human PAX7 cells, and yellow boxes indicate mouse Pax7 SCs.Mean ± standard deviation of human PAX7 cells are quantified from Pax7-ablated and non-ablated mice, N = 5 per group, t-test, *P < 0.0004.d, Location of foetal PAX7 + cells are quantified 30 days post engraftment.Scale bar, 50 μm.Inset shows that human PAX7 foetal SMPCs are associated with small human-only myofibres relative to non-ablated controls, N = 5 per group, t-test, *P = 0.034.

Fig. 5 |
Fig.5| ACTC1 is expressed by human-only myofibres and increased in Pax7ablated mice, enabling improved SMPC repopulation after injury.a, SMPC engraftment after 30 days show that human-only myofibres, but not chimeric myofibres express ACTC1 (green).N = 4 engrafted tissues.Scale bar, 100 μm.b, Human-only myofibres, but not chimeric myofibres, express regenerative signature as shown by ACTC1.SC niches form near small regenerating myofibres, which enables PAX7 cells to remain.Scale bar, 100 μm.Mouse TA express high levels of Myh4, which is not expressed by human, enabling further identification of human-only and chimeric myofibres.ACTC1 expression is reduced in chimeric Myh4 + spectrin + lamin AC + myofibres.c, Cartoon of repopulation experiments is shown.Top: representative images show fibre size in no injury or after re-injury (dystrophin (green), lamin AC/spectrin (red) or PAX7 (green).Insets show PAX7 location near other PAX7 foetal SMPCs.Fibre size is quantified using Imaris in non-injured or re-injured muscle.Bottom: graphs quantify mean ± standard deviation repopulation ability after re-injury, and dots represent individual images used for quantification; 10-15 images per sample, N = 3 adult, N = 4 foetal biological replicates per timepoint.PAX7 + SMPC/SCs and total human nuclei are quantified from the same image sets.t-test, *P < 0.05 between PAX7 + SMPC/ SCs in chimeric or human-only myofibre niches.Data show that foetal SMPCs cannot repopulate after re-injury, but adult SCs can repopulate in mdx-NSG mice; however, in Pax7-ablated mdx-NSG mice in re-injury (D60 + RI) foetal SMPCs can repopulate both chimeric and human-only myofibres.t-test, *P < 0.006.

Fig. 7 |
Fig. 7 | ACTC1 is essential for emerging niche formation and support of hPSC PAX7 SMPCs.a, Cartoon showing iCaspase9 vector (iCasp9) insertion immediately in 3′ of ACTC1 locus of hPSCs.AP1903 causes iCasp9 activation and gene-specific cell death.PCR products show insertion of iCasp9 across two hPSC lines (H1 and H9).b, Top: Pax7-DTX (ablated) Mdx-D2-NSG mice were treated with TMX, and hPSC SMPCs transplanted into Pax7-ablated tissues.Mice were treated six times every two weeks with AP1903 over 90 days, N = 5 cross sections.Left: images taken on the GeoMx of 90-day engrafted hPSC SMPCs show regions of transplantation with or without AP1903 treatment.Zoom shows morphology markers and masked ROI overlays.Single PAX7 cells and myofibres were collected for spatial RNA-seq.c, Graph show mean ± standard deviation of PAX7 counts from cross-sectional area and a decrease in AP1903-treated Articlehttps://doi.org/10.1038/s41556-023-01271-0
Table shows total number of ROIs used for spatial RNA seq from week 20 fetal tissues (N = 22), engrafted week 20 fetal SMPCs in mouse TA (N = 42), and engrafted hPSC SMPCs in mouse (N = 59).ROIs are categorized by morphology markers at time of collection.b.PCA plot comparing spatial RNA seq three samples (week 20 fetal tissues, hPSC transplant, and fetal transplant) and two groups (PAX7+ SMPCs, and myofibers).As fetal engrafted myofibers contained ACTC1+ and ACTC1− myofibers, these further separated.Plot show that PAX7 cells across tissues align more closely than do myofibers across tissues.