Preparation of lentivirus

Lentiviruses were prepared as previously described6,55. Twenty-four hours prior to transfection, 6 × 106 293FT cells (Life Technologies, R700-07) were seeded onto a 10 cm gelatin coated 10 cm dish. Following the manufacturer instructions, 10 µg DNA expression lentiviral plasmid (FUW-M2rtTA, pRRL-TetO-Pax7-IRES-EGFP, pRRL-MHCK7-GCaMP6, or pRRL-MHCK7-R-GECO) was co-transfected with 5 µg psPAX2 and 2 µg VSVg by Lipofectamine2000 (Thermo). The supernatant was collected 48–72 h post transfection, concentrated with Lenti-X concentrator (Clontech) at a 3:1 ratio (supernatant: concentrator) to 1/100 of the original volume, and stored at −80 °C for later use. Lentiviruses encoding Doxycycline-inducible Pax7/GFP were used to transduce undifferentiated hPSCs, while those encoding genetically encoded calcium indicators GCaMP656 or R-GECO51 were used to transduce iMPCs before formation of engineered muscle.

Myogenic differentiation of hPSCs into iMPCs

Human H9 (obtained from WiCell Institute), TRiPS, GM2525646, and Fucci (gift from Dr. Stephen Dalton47) pluripotent stem cell lines were maintained in feeder-free conditions in E8 medium (Stemcell Technologies) and were routinely tested for Mycoplasma contamination using commercially available kits (MycoAlert, Lonza). hPSC colonies were dissociated into single cells with Accutase (Stemcell Technologies) and seeded onto Matrigel (Corning) coated 6-well plates at a cell density of 1 × 103/cm2. Twenty-four hours post-plating, cells were infected with Tet-on lentivirus, the infected hPSCs were kept in E8 for expansion, then dissociated into single cells with Accutase and seeded onto matrigel coated 6-well plates in E8 supplemented with Y27632 (5 µM, Tocris) at 3.3 × 104 cells/cm2. The following day, E8 media was replaced with E6 media and cells were cultured for 2 days supplemented with CHIR99021 (10 µM, Selleck Chemical), after which CHIR99021 was removed and E6 media supplemented with 1 µg/mL Dox (Sigma) for 18 days until GFP+ induced myogenic progenitor cells (iMPCs) were sorted by FACS as described below. During differentiation of iMPCs, 10 ng/mL bFGF (R&D) was added starting at day 5 to enhance proliferation of GFP+ cells.

Flow cytometry analysis

Cells were dissociated with 0.25% Trypsin-EDTA, counted and washed with PBS, then resuspended in flow buffer (Supplementary Table 1) at a concentration of 2 × 106 to 1 × 107 cells/mL. To count cells expressing Tra-1-81 or CD56, anti-Tra-1-81 (Stemgent, 09-0011) or anti-CD56 (PE, R&D, FAB2408P) antibodies and isotype matched controls were applied according to manufacturer’s instructions and cells were analyzed using FACSCanto™ II flow cytometer (BD Biosciences) in Duke University Flow Cytometry Shared Resource. Cell population of interest was first gated for cell size and granularity, and then for the expression level of Tra-1-81 or CD56.

Sorting of iMPCs

At differentiation day 20, cells were dissociated with 0.25% Trypsin-EDTA (Thermo) and washed in neutralizing media (Supplementary Table 1). Detached cells were centrifuged at 300 g for 5 min, then resuspended in sorting solution (Supplementary Table 1) and filtered through 30 µM filter (SYSMEX) to remove clusters and debris. Single cell suspensions were kept on ice until sorting, with undifferentiated hPSCs used as negative control. Cells were sorted for GFP using MoFlo® Astrios™ cell sorter (Beckman Coulter) in Duke University Flow Cytometry Shared Resource.

Expansion of iMPCs

After sorting, GFP+ iMPCs were kept on ice in collecting solution (Supplementary Table 1), spun down at 300 g for 5 min, and resuspended in fresh E6 media supplemented with Y27632, Dox, and bFGF, then seeded at 4 × 104/cm2 in Matrigel-coated flasks. After 24–48 h of post sorting, cells were incubated in expansion media (EM, Supplementary Table 1), supplemented with Dox and bFGF, and passaged at a 1:3-1:6 ratio every 3-4 days after reaching 80% confluence.

2D differentiation of iMPCs

iMPCs were seeded at the density of 1 × 105/cm2 on Matrigel-coated dishes and after reaching 100% confluence, EM was washed out with PBS and switched to differentiation media (DM, Supplementary Table 1) that was changed every other day.

Fabrication and differentiation of iSKM bundles

Three-dimensional engineered muscle tissues (iSKM bundles) were formed within polydimethylsiloxane (PDMS) molds containing two semi-cylindrical wells (7 mm long, 2 mm diameter), cast from 3D-machined Teflon masters, similar to our previously described methods6,37. PDMS molds were coated with 0.2% (w/v) pluronic (Invitrogen) for 1 h at room temperature to prevent hydrogel adhesion. Laser-cut Cerex® frames (9 × 9 mm2, 1 mm wide rim) positioned around the 2 wells served to anchor bundle ends and facilitate handling and implantation. Cell/hydrogel mixture (Supplementary Table 1) was injected into the PDMS wells and polymerized at 37 °C for 30 min. Formed iSKM bundles were kept on rocking platform in EM supplemented with 1 µg/mL Dox and 1.5 mg/mL 6-aminocaproic acid (ACA, Sigma) for 4 days. Media was then switched to DM supplemented with 2 mg/mL ACA and 50 µg/mL ascorbic acid (Sigma), with media changed daily.

Engineering of primary human myobundles

Native human skeletal muscle samples were obtained through standard needle biopsy or surgical waste from donors with informed consent under Duke University IRB approved protocols (Pro00048509 and Pro00012628). Muscle samples were minced and digested with 0.05% trypsin for 30 min at 37 °C. Isolated cells were centrifuged to remove residual enzyme and resuspended in PMM, then preplated for 2 h to reduce fibroblast fraction. After pre-plating, cells were seeded onto to a Matrigel (BD Biosciences) coated flask and expanded by passaging upon reaching 70% confluence. At passage 3 or 4, cells were detached from the flask and used to fabricate primary myobundles as described for iSKM bundles.

Force measurement

Contractile and passive force generation in iSKM bundles was assessed using a custom force measurement set-up as previously described6,57,58. Briefly, single iSKM bundles were transferred attached to frame in the bath with DM equilibrated at 37 °C. One end of the bundle was pinned to a fixed PDMS block and the other end was attached to a PDMS float connected with force transducer mounted on a motorized linear actuator (ThorLabs, Newton, NJ). The sides of the frame were cut to allow isometric measurement of contractile force and stretching of the bundles by the actuator. To assess the force–length relationship, iSKM bundle was stretched in 5% steps, then stimulated with a 40 V/cm, 10 ms long electrical pulse using a pair of platinum electrodes and the twitch force was recorded. At 20% stretch, 1 s long stimulations at 5, 10, 20 and 40 Hz were applied and the contractile force was recorded to assess the force–frequency relationship. Contractile force traces were analyzed for peak twitch or tetanus force, passive tension, time to peak twitch, and half relaxation time using a custom MATLAB program.

Immunofluorescence

Cells cultured in monolayers were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature and iSKM bundles were fixed in 2% paraformaldehyde in PBS overnight at 4 °C while rocking. Following fixation, samples were washed twice with PBS, and kept in PBS at 4 °C for up to 1 week. Before staining, samples were blocked in PBS with 5% chick serum and 0.5% Triton-X 100. The following primary antibodies were used for immunostaining of: Oct4 (Millipore, MAB4401, 1:200), Tra-1-81 (Stemgent, 09-0011, 1:100), T (R&D, AF2085, 1:200), Pax3 (R&D, MAB2457, 1:200), Myf5 (SCBT, sc-302, 1:100), MF20 (DSHB, 1:300), sarcomeric α-actinin (SAA, Sigma, a7811, 1:200), GFP (Thermo, A6455, 1:300), laminin (Abcam, Cambridge, MA, ab11575, 1:200), Dystrophin (Abcam, Cambridge, MA, ab15277, 1:100), CD31 (Abcam, Cambridge, MA, ab28364, 1:50), MyoD (BD Biosciences, 554130, 1:100), MyoG (SCBT, sc-576, 1:100), and Pax7 (DSHB, 1:100). Corresponding fluorescently labeled secondary antibodies (1:500), α-bungarotoxin (B13422, 1:200), and phalloidin (O7466, 1:300) (all from Thermo) were applied in blocking solution (Supplementary Table 1) for 1 h. Images were acquired using a Leica SP5 inverted confocal microscope and analyzed using LSM Image Software.

Transmission electron microscopy

iSKM bundles at 4 weeks of culture were fixed for 30 min in 0.1 M phosphate buffer (PB) containing 2% glutaraldehyde, then postfixed with 2% OsO 4 in PB. Samples were then sequentially dehydrated in 30, 50, 70, 95, 100% acetone, kept in acetone: epoxy (1: 1) overnight, and embedded in 100% epoxy. Ultrathin sections were collected on grids and stained with uranyl acetate before examination with a Philips CM12 transmission electron microscope operated at 120 kV. An XR60 camera system (Advanced Microscopy Techniques) was used for image acquisition.

Quantitative RT–PCR

Native human skeletal muscle samples were obtained through standard needle biopsy or surgical waste from donors with informed consent under Duke University IRB approved protocols (Pro00048509 and Pro00012628). Total RNA from 2D cells, 3D engineered muscle bundles, and native human muscle was isolated using either RNeasy Plus Mini Kit (QIAGEN) or Aurum Total RNA Mini Kit (Bio-Rad), then reverse-transcribed by iScript cDNA Synthesis Kit (Bio-Rad). Quantitative RT-PCR for muscle related genes was performed with iTaq Universal SYBR Green Supermix (Bio-Rad) according to manufacturer’s instructions. Primer information can be found in Supplementary Table 2.

Implantation of iSKM bundles in dorsal window chambers

All animal experiments were approved by the Duke University IACUC. NSG or nude mice (male, 6–10wk of age; 28–34 g) were anesthetized by intraperitoneal (IP) injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). After removing the hair on the back, the dorsal skin was attached to a temporary “C-frame” at the center of the back. The skin was perforated in three locations to accommodate the screws of the chamber, and a circular region (~12 mm) of the forward-facing skin (i.e., cutis, subcutis, retractor and panniculus carnosis muscles, and associated fascia) was dissected away to accommodate the chamber. The forward and rearward pieces of the titanium dorsal skinfold chamber were assembled together from opposite sides of the skin, and a Cerex® frame with double-bundle constructs was laid perpendicular (verified under microscope) to the intact panniculus carnosis muscle of the rearward-facing skin, providing a source of microvessels for vascularization. A sterile cover glass was placed over the window and engineered tissue while superfusing with sterile saline solution. The chamber was then secured with suture and the “C-frame” was removed. Post-operatively, the mouse was injected subcutaneously with buprenorphine (1 mg/kg) painkiller and let to recover on a heating pad.

Implantation of iSKM bundles into hindlimb muscle

Adult NSG mice (male, 6–8wk of age, 25–35 g), were anesthetized by IP injection of Ketamine (100 mg/kg) and Xylazine (5 mg/kg). The hindlimb hair was clipped, skin was decontaminated with 0.5% chlorhexidine/70% ethanol, and a 1.5 cm skin incision was made parallel to the tibia. Micro-dissecting forceps was used to separate tibialis anterior (TA) muscle along a 1 cm-long midline and iSKM bundle was inserted in the muscle separation. Bundle ends and mid-point were sutured to the TA muscle, the skin was closed, and buprenorphine (1 mg/kg) was applied for post-operative pain management.

Intravital imaging of blood vessels

Intravital recordings in dorsal window chambers were performed in anesthetized mice on days 3, 6, 9, and 15 post-implantation (PI), as previously described37. Mice were anesthetized by nose cone inhalation of isoflurane and positioned on a heating pad under a microscope objective. Hyperspectral brightfield image sequences (10 nm increments from 500 to 600 nm) were captured at ×2.5 and ×5 magnification using a tunable filter (Cambridge Research & Instrumentation, Inc.) and a DVC camera (ThorLabs), as previously described59, then converted to hemoglobin saturation maps, and processed to binary blood vessel density images (Supplementary Fig. 14) for quantification of blood vessel density (BVD).

Intravital recording of spontaneous Ca2+ transients

Intravital recording of spontaneous Ca2+ transients in dorsal window chambers was performed immediately after vessel imaging with mice still anesthetized. GCaMP6 signals in implanted bundles were recorded through a FITC-filter using a fast fluorescent camera (Andor; at 16 µm spatial and 20 ms temporal resolution).

In vitro and ex vivo measurements of Ca2+ transients

Electrically-stimulated Ca2+ transients were recorded from engineered muscle bundles after 1, 2, 4 week of in vitro culture and from muscle explants 7–15 days post implantation, as previously described6,37. In vitro cultured iSKM bundles and excised dorsal skins and TA muscles with engrafted bundle implants were transferred into a custom chamber mounted on an upright fluorescence microscope (Leica M165 FC, for TA muscle explants) or an inverted fluorescence microscope (Nikon TE2000-U, for window chamber explants), placed in 37 °C differentiation media (DM, Supplementary Table 1), and electrically stimulated (10 ms pulse, 3 V/mm). Resulting GCaMP6 (510–560 nm bandpass emission filter) or R-GECO (590-660 nm bandpass emission filter) signals were recorded using a fast EMCCD camera (Andor iXon 860; 24 µm spatial and 20 ms temporal resolution). Amplitudes of Ca2+ transients were determined using the Solis software (Andor) by averaging relative fluorescence intensity (ΔF/F) from each bundle.

Statistical analysis

Experimental data are reported as mean ± SEM. Statistical significances were evaluated by one-way ANOVA with Tukey–Kramer HSD test using JMP Pro software. P−value < 0.05 was considered statistically significant. Sample sizes for in vitro experiments were determined based on variance of previously reported measurements6. Sample sizes for implantation experiments were determined, in part, based on cost and animal availability. No randomization of animal groups was done. No blinding of animal experiments was done.

Data availability

All data supporting the results of these studies are available within the paper and Supplemental Materials.