All procedures involving animals including animal housing, husbandry, and experiments were reviewed and approved by the University of Minnesota Institutional Animal Care and Use Committee with AAALAC accreditation according to protocols (#2201-39776A). Male and female wildtype BL6 (C57BL/6J, Jackson Laboratory, Bar Harbor, ME) and H2B-GFP mice (B6.Cg-Tg (HIST1H2BB/EGFP)1 Pa/J, Jackson Laboratory) at 3–5 months old were used to obtain MuSCs. Transplantation experiments were performed on both male and female 3–5 months old NSG-mdx^4cv mice as described previously (Arpke et al., 2013).

Hindlimb muscles were harvested and chopped into ∼2 mm pieces. The chopped muscle pieces were then incubated in a primary digestion buffer containing Dulbecco’s Minimum Essential Medium/High Glucose (DMEM, HyClone #SH30243.01, Logan, UT), 2 mg/mL Collagenase II (Gibco #17101-015, Gaithersburg, MD), and 1% penicillin/streptomycin (P/S) (Life Technologies #15140-122, Grand Island, NY) on a shaker at 250 rpm, 37 C for 1 h. Primary digestion was then halted by the addition of rinsing buffer consisting of Ham’s/F-10 medium (Caisson Labs #HFL01, Smithfield, UT), 10% horse serum (HyClone #SH30074.03), 1% HEPES buffer solution (Caisson Labs #HOL06), and 1% P/S and then centrifuged at 500 g for 10 min at 4°C. The tissues were then subjected to further enzymatic digestion consisting of rinsing buffer supplemented with 0.1 mg/mL Collagenase II and 0.5 mg/mL Dispase (Gibco #17105-041). This secondary digestion process continued for 30 min on a shaker at 250 rpm, 37°C. The digested tissues were then repeatedly drawn and released into a 10 mL syringe with a 16-gauge needle (4 times) followed by an 18-gauge needle (4 times) to facilitate additional dissociation. The resultant cellular suspension was filtered through a 100 μm cell strainer, spun down, resuspended in rinsing buffer, filtered through a 40 μm cell strainer, and then spun down again. Isolation of muscle stem cells from transplanted tibialis anterior muscles (see below) was performed similarly, except without passing through the 16-gauge needle nor the 100 μm cell strainer.

Isolated cells were incubated on ice for 1 h with fluorophore-conjugated antibodies for FACS (fluorescence-activated cell sorting). After 1 h, the cells were washed twice and resuspended in FACS buffer (PBS (HyClone #SH30256.01), 0.2% fetal bovine serum (FBS), and 1 μg/mL propidium iodide (PI, Sigma-Aldrich #P4170, St Louis, MO). The addition of PI into FACS Buffer served as a live/dead cell indicator, and only viable cells (PI⁻) were quantified. Cells were sorted into medium and kept on ice until they were cultured as described below. The antibodies utilized for sorting muscle stem cells (each at concentration of 0.5 μL per million cells) were PE-Cy7 anti-CD31 (BioLegend #102418, RRID: AB_830757, San Diego, CA), PE-Cy7 anti-CD45 (BioLegend #103114, RRID: AB_312979), APC anti-α7-Integrin (AbLab #67-0010-05, Vancouver, Canada), and PE anti-VCAM-1 (BioLegend #105714; RRID: AB_1134164). MuSCs are defined as CD31^– CD45^– α7-Integrin⁺ VCAM-1⁺ (α7-Integrin⁺ VCAM⁺). Cell analysis and sorting were executed using BD FACSAriaII instrument (BD Biosciences, San Diego, CA) operated with FACSDiva software (BD Biosciences). A four-way purity precision was employed for bulk sorting, while single-cell precision was implemented when sorting individual cells into 96-well plates for clonal analysis. FACS plots depicting the distribution of cellular populations were generated with FlowJo software (FLOWJO LLC, Ashland, OR).

FACS-sorted α7-Integrin⁺ VCAM⁺ cells were plated in 0.1% gelatin-coated wells and cultured in myogenic expansion medium (Ham’s/F-10, 20% FBS (Sigma-Aldrich #F0926), 10 ng/mL basic FGF (R&D Systems #233- FB/CF, Minneapolis, MN), 1% P/S, 2 mM Glutamax (Life Technologies #35050-061, Paisley, PA), and 0.1 mM β-mercaptoethanol) with or without LIF (1,000 units/mL, Sigma-Aldrich #ESG1107). Cells were passaged once they reached ∼60% confluency. For differentiation experiments, cells at 70%–80% confluency were switched to myogenic differentiation medium (DMEM, 4% horse serum, 2 mM Glutamax, 1 mM sodium pyruvate (Life Technologies #11360070), 1 μg/mL insulin (GeminiBio #800122, West Sacramento, CA), 1 μM dexamethasone (Hello Bio #HB2521, Princeton, NJ), and 1% P/S) for 3 days.

One α7-Integrin⁺ VCAM⁺ cell per well was seeded via FACS into 0.1% gelatin-coated 96-well plates in myogenic clonal medium containing DMEM/F12 (Cellgro #15-090-CV, Manassas, VA), 20% FBS, 10% horse serum, 10 ng/mL basic FGF, 1% P/S, 2 mM Glutamax, and 0.5% chick embryo extract (US Biological #C3999, Salem, MA) with or without LIF (1,000 units/mL). Cells were incubated for 7 days undisturbed before analysis.

Two days prior to cell transplantation, the hindlimbs of recipient NSG-mdx^4cv mice were exposed to 1,200 cGy X-Ray irradiation. One day prior to transplantation, the recipients’ irradiated tibialis anterior (TA) muscles were injected with 15 μm of cardiotoxin (10 μM, Latoxan #L8102, France) to promote engraftment. For each recipient mouse, 40,000 donor cells were resuspended in PBS (total 10 μL) and injected into the TA muscle using a Hamilton syringe (Hamilton, Reno, NV). Recipients were anesthetized with ketamine (150 mg/kg, Dechra Veterinary Products, NDC: 59399-114-10, Overland Park, KS) and xylazine (10 mg/kg, Bimeda, NDC: 59399-111-50, Cambridge, Canada) intraperitoneally prior to each procedure. Transplanted TA muscles were harvested 4 weeks later for further analysis.

Harvested TA muscles were embedded in optimal cutting temperature (OCT) solution (Scigen #4586, Gardena, CA). The cryomold containing the specimens were snap-frozen on 2-methylbutane (Sigma-Aldrich #320404) pre-cooled with liquid nitrogen and stored at −80°C. Cryosections of 10 μm were cut on a Leica CM3050 S cryostat (Leica Microsystems, Buffalo Grove, IL) and collected onto glass slides for immunostaining.

For cultured cells, cells were fixed with 4% paraformaldehyde (Sigma-Aldrich #P6148) for 1 h. Cell membranes were permeabilized with 0.3% Triton X-100 (Sigma-Aldrich #X100) followed by blocking with 3% BSA (Thermo Fisher Scientific #BP1605, Waltham, MA). Cells were incubated overnight at 4°C with primary antibodies in 3% BSA. The next day, cells were incubated for 1 h at room temperature with secondary antibodies. Nuclei were stained with 4’, 6-diamidino-2-phenylindole (DAPI) (Life Technologies #D3571) for 10 min. For sections, samples were mounted with Immu-Mount (Thermo Fisher Scientific #9990402). Primary antibodies used were mouse anti-PAX7 (1:10, Developmental Studies Hybridoma Bank (DSHB) #PAX7, RRID: AB_395942, Iowa City, IA), mouse anti-myosin heavy chain (MHC) (1:20, DSHB #MF-20, RRID: AB_427788), mouse anti-MYOD1 (1:100, BD Pharmingen #554130, RRID: AB_395255, Franklin Lakes, NJ), rabbit anti-DYSTROPHIN (1:250, Abcam #ab15277; RRID: AB_301813, Cambridge, United Kingdom), and mouse anti-laminin (1:500, Sigma-Aldrich #L8271; RRID: AB_477162). Secondary antibodies (each 1:1,000) used were: goat anti-mouse IgG1 Alexa Fluor 555 (Life Technologies, #A21127, RRID: AB_2535769), goat anti-mouse IgG2b Alexa Fluor 647 (Life Technologies, #A21242, RRID: AB_ 2535811), goat anti-rabbit Alexa Fluor 555 (Life Technologies, #121429, RRID: AB_2535850), goat anti-mouse Alexa Fluor 647 (Life Technologies, #A21235, RRID: AB_2535804), and goat-anti mouse IgG2b Alexa Fluor 488 (Life Technologies, #121141, RRID: AB_ 2535778).

Fluorescent imagery was acquired utilizing a Zeiss AxioObserver Z1 inverted microscope paired with an AxioCamMR3 camera (Jena, Germany). Subsequent image processing and quantification procedures were executed with Fiji/ImageJ software (U.S. National Institute of Health, Bethesda, MD). Whole TA images were captured in tile mode and stitched together using ZEN software (Zeiss). Fiber engraftment is defined as the total cross-sectional area of DYSTROPHIN⁺ fibers over the total cross-sectional area of the whole TA, measured via Muscle2View, a CellProfiler pipeline (Sanz et al., 2019), with adjusted parameters.

Cells at 70%–80% confluency were harvested for gene expression analysis. Total RNA was extracted using Quick-RNA Miniprep Kit (Zymo Research #R1055, Orange, CA). Genomic DNA removal and reverse transcription (RT) were performed using Verso cDNA Synthesis Kit (Thermo Fisher Scientific #AB1453A). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed in triplicates using TB Premix Ex Taq II (Takara Bio, #RR390A, Japan) in a QuantStudio 6 Flex Real-Time PCR System using QuantStudio Real-Time PCR Software (both Applied Biosystems, Waltham, MA). TaqMan probes used were: Pax7: Mm00834079_m1, Klf5: Mm00456521_m1, Myf5: Mm00435125_m1, Myod1: Mm00440387_m1, Myog: Mm00446194_m1, and Gapdh: Mm99999915_g1 (all Thermo Fisher Scientific). The ΔΔCt method was utilized to calculate gene expression relative to that of the housekeeping gene Gapdh in control samples.

The ATP assay was performed on LIF-treated and untreated MuSCs using the CellTiter-Glo Luminescent Cell Viability Assay kit (Promega #G7572, Madison, WI) according to the manufacturer’s manual. Luminescence was measured using a Cytation 3 plate reader (BioTek, Winooski, VT).

The RNA-seq dataset in Figure 1 was assessed from GEO: GSE182508. Data are presented as mean ± SEM. Differences between two groups were assessed using Student’s t-test. Differences among three or more groups were assessed by ANOVA with Tukey’s post hoc tests. p values <0.05 were considered significant (*: p < 0.05, **: p < 0.01, and ***: p < 0.001). GraphPad Prism (GraphPad Software, La Jolla, CA) was used to perform statistical analyses.

We previously reported that in vivo PSC differentiation from both mouse and human PSCs produced skeletal myogenic progenitors that were both engraftable and expandable (Chan et al., 2018; Xie et al., 2021; Pappas et al., 2022; Xie et al., 2023). These in vivo differentiated skeletal myogenic progenitors had excellent muscle regeneration potency upon transplantation and could produce new fibers to a similar extent as adult MuSCs (Chan et al., 2018; Xie et al., 2021). Remarkably, in vivo differentiated skeletal myogenic progenitors could be cultured and expanded in vitro over several passages while still retaining their outstanding engraftability (Xie et al., 2021; Xie et al., 2023). This contrasts with adult MuSCs whose engraftment potential abruptly diminished upon a few days in culture (Montarras et al., 2005; Sacco et al., 2008; Xie et al., 2021). We speculated that factors that regulate engraftment might have their expression level correlating to engraftability. In other words, cell populations with better engraftment would express pro-engraftment factors at higher levels, and vice versa. Therefore, in a previous study, we performed an RNA-seq analysis together with a transplantation assay to evaluate the relationship between gene expression and fiber engraftment on 4 skeletal myogenic populations with various degrees of engraftability: fresh MuSCs (M_F), cultured MuSCs (M_C), fresh in vivo differentiated skeletal myogenic progenitors (I_F), and cultured in vivo differentiated skeletal myogenic progenitors (I_C) (Figure 1A) (Xie et al., 2021). We first calculated the Pearson correlation coefficient between gene expression and engraftment and identified genes with a positive correlation of r > 0.9 (Figure 1B). We were particularly interested in factors involved in signaling transduction pathways because their activities would be more readily modulated by commercially available agonists and inhibitors. Using these criteria, we identified Lifr (LIFR): highly expressed in I_F and M_F (both highly engraftable), moderately expressed in I_C (moderately engraftable), and minimally expressed in M_C (least engraftable) (Figures 1B,C).

LIFR is a receptor of LIF and the LIF-LIFR pathway has been shown to regulate MuSC biology (Bower et al., 1995; Kami et al., 2000; White et al., 2001; Spangenburg and Booth, 2002). When MuSCs were isolated from the hind limb muscles of adult mice and cultured in vitro for 4 days, the skeletal muscle-specific transcription factor MYOD1 emerged with a concurrent downregulation of the MuSC factor PAX7 (Figures 2A,B). This corresponds to the spontaneous differentiation of MuSCs into a PAX7^– MYOD1⁺ myoblast stage in vitro, where their engraftment potential is severely limited (Olguin and Olwin, 2004; Zammit et al., 2004; Sacco et al., 2008). Interestingly, LIF (1,000 units/mL) upregulated PAX7 while producing minimal effect on MYOD1 (Figures 2A,B). These observations were also supported by gene expression analysis in which MuSCs cultured with LIF exhibited elevated levels of Pax7, while Klf5, Myf5, Myod1 and Myog expression remained unchanged (Figure 2C).

We further evaluated how LIF influenced the colony-forming ability of individual MuSCs. Single α7-Integrin⁺ VCAM⁺ MuSCs were FACS-sorted and cultured for 7 days under conditions that supported both cell self-renewal and cell differentiation, i.e., not biasing towards self-renewal or differentiation (Yablonka-Reuveni, 2004; Day et al., 2010; Ippolito et al., 2012; Xie et al., 2021). Although LIF had minimal impact on the capability of single MuSCs to form myogenic colonies per se, LIF-treated MuSCs tended to develop into larger colonies (Figures 2D,E).

Under pro-differentiation conditions, isolated MuSCs gradually developed into MHC⁺ multi-nucleated myotubes (Figure 2F). Nevertheless, a small population of MuSCs did not differentiate but remained PAX7⁺, known as “reserve cells” (Olguin and Olwin, 2004; Day et al., 2010). Interestingly, we observed an abundance of PAX7⁺ “reserve cells” in LIF-treated MuSC undergoing differentiation (Figure 2F). The higher level of PAX7 in MuSCs treated with LIF under both maintenance and differentiation conditions suggested that LIF might promote cultured MuSCs to remain at the stem cell stage, and thereby making them more likely to be engraftable.

The above experiments were performed on MuSCs cultured for a relatively short period of time (e.g., 4 days) and without passaging. We next evaluated the effect of LIF on MuSCs that have been cultured for 12 days over 3 passages. We did not observe a significant difference in cell growth between control and LIF-treated MuSCs, although the latter tended to grow slower (Figure 2G). We also found minimal effects of LIF on MuSC growth in an ATP assay that determined the number of viable cells based on ATP quantification (Figure 2H). Interestingly, LIF promoted the α7-Integrin⁺ VCAM⁺ population (markers of MuSCs) in day 12/passage 3 MuSCs at the expense of the α7-Integrin⁺ VCAM^–population (markers of myoblasts) (Figure 2I). Moreover, similarly to the effect on the unpassaged MuSCs, LIF upregulated Pax7 gene expression in passage 3 MuSCs (Figure 2J, top left panel). In addition, LIF-treated passage 3 MuSCs exhibited higher levels of Myod1 with lower levels of Myog than untreated controls (Figure 2J, bottom panels). The levels of Klf5 and Myf5 remained unchanged (Figure 2J, top middle and right panels). Altogether, these findings suggested that LIF promoted the maintenance of cultured MuSCs at a stem cell stage.

The above results encouraged us to evaluate whether LIF could improve the engraftment potential of cultured MuSCs in a transplantation assay (Figure 3A). We first obtained MuSCs from the hind limb muscles of wildtype BL6 mice by FACS using established markers: CD31^– CD45^– (i.e., non-endothelial and non-hematopoietic, respectively) α7-Integrin⁺ VCAM⁺. We subsequently cultured isolated α7-Integrin⁺ VCAM⁺ cells in myogenic medium with or without LIF (1,000 units/mL) for 12 days over 3 passages. Untreated and LIF-treated passage 3 MuSC cultures were then transplanted at 40,000 cells/muscle into the TA muscles of NSG-mdx^4Cv mice, a DMD model we have previously used to evaluate cell transplantation success (Arpke et al., 2013; Chan et al., 2018; Xie et al., 2021; Xie and Chan, 2023; Xie et al., 2023). Four weeks later, the transplanted muscles were harvested for fiber engraftment evaluation, as defined by donor-derived DYSTROPHIN⁺ fibers (recipient mice lack dystrophin in their muscles). As expected, untreated cultures engrafted poorly (3.5% ± 0.8%, n = 8). Remarkably, LIF treatment significantly increased the ability of passage 3 MuSCs to develop into DYSTROPHIN⁺ fibers upon transplantation (18.2% ± 3.2%, n = 8, p < 0.001) (Figures 3B,C).

We next evaluated whether LIF-treated MuSC cultures could repopulate the endogenous MuSC pool. For this experiment, we used H2B-GFP BL6 mice as donors to obtain α7-Integrin⁺ VCAM⁺ MuSCs. These mice constitutively express GFP in their nuclei and thereby allow distinction between donor (GFP⁺) and recipient (GFP^–) cells upon transplantation. FACS-sorted GFP⁺ MuSCs were subsequently cultured with or without LIF (1,000 units/mL) for 12 days over 3 passages for transplantation. Four weeks after transplantation, we observed a significant contribution of donor-derived cells in the MuSC compartment (GFP⁺ α7-Integrin⁺ VCAM⁺) (Figures 3D,E). Therefore, these results confirmed the role of LIF-LIFR signaling in regulating the engraftment of cultured MuSCs to develop into muscle fibers and repopulate the endogenous MuSC niche.