Mice and Animal Care
This study was approved by the Institutional Animal Care and Use Committee (IACUC) of University of Illinois at Chicago, which is accredited by the American Association for the Accreditation of Laboratory Animal Care (AAALAC). All animals received humane care in compliance with the ‘Principles of Laboratory Animal Care’ formulated by the National Society for Medical Research and the ‘Guide for the Care and Use of Laboratory Animal Resources’ published by the US National Institutes of Health . Six to eight-week old mdx/scid mice - animal model for Duchenne Muscular Dystrophy (B10ScSn.Cg-Prkdcscid Dmdmdx/J, stock number 018018) with respective background wild type mice (C57BL/10ScSnJ, stock number 000476) were purchased from Jackson Laboratories for human DEC lines testing. Mice were housed in the Molecular Biology Research Building, an AAALAC-accredited animal facility, at University of Illinois at Chicago.
Human Myoblast Culture
Cryopreserved normal human myoblasts were purchased from Lonza Inc (Mapleton, IL, USA), while DMD-affected myoblast were purchased from Axol Bioscience Ltd. (Little Chesterford, UK). Myoblasts (MB) were cultured in standard conditions in specific Skeletal Muscle Cell Growth Medium-2 (SkGM-2 bulletkit, Lonza Inc, Mapleton, IL, USA) supplemented with human Epidermal Growth Factor (hEGF), 0.5 ml; Fetuin, 5.0 ml; Bovine Serum Albumin (BSA), 5.0 ml; Dexamethasone, 0.5 ml; Insulin, 5.0 ml; Gentamicin/Amphotericin B (GA), 0.5 ml. Culture medium was changed twice a week and upon reaching 60–70% confluence, myoblasts were harvested and passaged using mechanical and enzymatic dissociation methods of 5 min incubation with 0.25% trypsin EDTA (Gibco-ThermoFischer, Waltham, MA USA). Enzymatic activity for cell detachment was stopped with culture media supplemented with 10% FBS (fetal bovine serum, Hyclone, GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). Next, the cells were washed twice. Human MBs were harvested between passages 5–7, which is the optimal passage for ex vivo cell fusion procedure.
Cell Fusion Procedure
After harvesting and counting in 0.4% Trypan Blue staining solution (Gibco- ThermoFischer, Waltham, MA, USA), parent myoblast (MBN1 and MBN2 or MBN and MBDMD) were washed in serum-free DMEM culture media supplemented with antibiotics (1% Antibiotic–Antimycotic solution, Gibco- ThermoFischer, Waltham, MA, USA). Then, parent myoblasts (MBN1 and MBN2 or MBN and MBDMD) were fluorescently labeled using either PKH26 or PKH67 (Sigma, St. Louis, MO, USA) tracking membrane dyes according to the manufacturer’s instructions. Briefly, each parent cell pellet was suspended in 1 ml diluent C (Sigma, St. Louis, MO, USA) and 4 µl PKH dye was added to the 2 ml total volume. After 4-min room temperature incubation, the staining procedure was stopped with the addition of 1% BSA and consequent wash in culture media. Before the fusion procedure, parent cells were mixed and washed in serum-free DMEM basal media. After the pellet was mobilized, the fusion procedure was performed using 1.46 g/mL PEG solution (PEG 4000, EMD) containing 16% DMSO (Sigma, St. Louis, MO, USA) . The fused cells then were washed in complete culture media and transferred to PBS-based fluorescently activated cells sorting (FACS) buffer containing 5% HEPES, 1% EDTA and 5% FBS. Finally, cells presenting double (PKH26/PKH67) staining were selected via FACS (MoFlow Astrios, Beckman Coulter, San Jose, CA, USA) and used for further in vitro analysis or transplanted to recipient mdx/scid mice. A total of 6 cell fusions for each human DEC line (MBN1/MBN2 DEC and MBN/MBDMD DEC) were performed for in vitro assays and 8 fusions for each DEC line were performed for in vivo DEC cell delivery.
Flow Cytometry and Confocal Microscopy Analysis for Confirmation of DEC Fusion
Following fusion procedure, samples of sorted double stained PKH67/PKH26 labeled DEC, as well as corresponding single stained controls (PKH67 labeled MBN1 and MBN, and PKH26 labeled MBN2 and MBDMD) and unstained controls were fixed in 4% paraformaldehyde for 15 min. and washed in PBS. Samples for flow cytometry were analyzed using Fortessa (Beckman Coulter, San Jose, CA, USA). For confocal microscopy cells were spun onto positively charged lysine coated microscope slides and counterstained with DAPI (Vector mounting media with DAPI). Cells were examined on Zeiss Meta confocal microscope and images captured and analyzed with ZEN software (USA, St. Louis, MO, USA).
PCR-STR and PCR-rSSOP DNA Profiling
DNA isolation was performed with DNeasy Blood and Tissue Isolation kit (Qiagen) according to manufacturer’s instructions. DNA samples of DEC and donor cells were typed using the polymerase chain reaction-reverse sequence-specific oligonucleotide probe (PCR-rSSOP) method using commercial kits (LABtype rSSO Typing Test, OLI) and polymerase chain reaction short-tandem repeat (PCR-STR). For the PCR-rSSOP, the sample DNA was subjected to PCR amplification (PE9700, Thermo cycler, Life technologies) in a 10 µL reaction volume, with the PCR run at 96 °C for 3 min, 96 °C for 20 s, 60 °C for 20 s, and 72 °C for 20 s, for 5 cycles, and 96 °C for 10 s, 60 °C for 15 s, and 72 °C for 20 s for 30 cycles followed by 72 °C for 10 min and stored at 4 °C. After amplification, the PCR products were denatured, and hybridized with the corresponding locus beads at 60 °C for 15 min, which were washed three times. Then, streptavidin-conjugated phycoerythrin (SAPE) was reacted with the products for 5 min at 60 °C, and following washing, the fluorescent products were detected using the Luminex 200 (Luminex, USA) and suspended in 60 µL wash buffer.
For the PCR-STR, 5 ng DNA was amplified using the ABI 3730xl DNA Analyzer with the Promega GenPrint 10 ® system (Promega, Madison, Wisconsin, USA). The 10 studied STR loci included: TH01, D21S11, D6S1043, D5S818, D13S317, D7S820, D16S539, CFS1PO, Amelogenin, vWA, TPOX. Amplification was conducted in a 25 µl reaction volume containing 5 µl of GenePrint Master Mix (Promega, Madison, Wisconsin, USA), 5 µL of GenePrint Primer Pair Mix (Promega, Madison, Wisconsin, USA), and DNA template. Appropriate negative and positive controls were used. The raw data were uploaded to GeneMapper® 5.0 analysis software (Applied Biosystems, Foster City, CA, USA) and allelic profile(s) were created according to analysis conditions supplied by Promega. The PCR conditions consisted of initial denaturation step at 96 °C for 5 min, followed by 30 cycles of 94 °C for 10 s, 59 °C for 1 min, and 72 °C for 30 s, with a final extension at 60 °C for 10 min and then a 4 °C incubation.
Quantification of Dystrophin Expression by Taqman Real-Time PCR
Total RNA was isolated from tissues using TRIzol reagent (LifeTechnologies) per manufacturer’s instructions. Concentration and quality of extracted total RNA was measured spectrophotometrically with NanoDrop® ND-1000. The ratio of sample absorbance A260/280 < 1.8 was considered an acceptable measure of RNA purity.
Total RNA in concentration of 600 ng/µl was reverse-transcribed to cDNA in a total volume of 20 µl, using High Capacity cDNA Reverse Transcription Kit (Applied Biosystem) according to the manufacturer’s instructions. The amount of cDNA synthesized in a single reaction was sufficient to PCR-amplify all interrogated genes.
Relative Quantification of Dystrophin Expression by Real-Time PCR
Quantitative assessment of dystrophin expression (Hs00758098_m1) was performed using the 7300 Real-Time PCR detection system with 7300 System SDS software (Applied Biosystem). Amplification was carried out in a total volume of 25 µl containing TaqMan Universal PCR Master Mix (2x), and Gene Expression Assay Mix (20x). The reactions were cycled 40 times using the following parameters: 50 °C for 2 min, 95 °C for 10 min, 95 °C for 30 s and 60 °C for 1 min. All PCR runs were performed in triplicate to achieve reproducibility. Expression of all examined genes was compared to endogenous controls of GAPDH (Hs99999905_m1, Applied Biosystem).
Immunofluorescence Detection of Dystrophin In vitro
MBN1/MBN2 DEC and MBN/MBDMD DEC lines (n = 4 fusions/line) and parent myoblast lines (MBN1 and MBN2, MBN and MBDMD, n = 4/cell type) were cultured in a Skeletal Muscle Cell Growth Medium-2 on poly-L-lysin coated German glass coverslips (Corning Inc, New York, USA) placed in 6-well plates (Corning, New York, USA). At 1, 7, 14 and 21 days of culturing, cells were fixed with ice-cold acetone for 10 min, washed, and unspecific antibody binding was blocked with 10% normal goat serum. Mouse monoclonal anti-human anti-dystrophin primary antibody (MANDYS8, 1:200, ThermoFischer, Waltham, MA, USA) and goat anti-mouse AlexaFluor-467 conjugated secondary antibody (1:400, ThermoFisher, Waltham, MA, USA) were used for dystrophin detection. Nuclei were counterstained with DAPI (Vector Laboratories, Burlingame, CA). A Zeiss Meta confocal microscope with ZEN software was used for fluorescence signal detection and analysis.
Phenotype Analysis by Flow Cytometry
Myoblast phenotype markers were evaluated in both normal and DMD-affected parent myoblast populations (MBN1 and MBN2, MBN and MBDMD n = 4/line) as well as in fused DEC lines (MBN1/MBN2 DEC and MBN/MBDMD DEC) 12 h after fusion (n = 4/line). The following antibodies were used: anti-human antibodies against CD34, CD90, CD45 and CD56 (BD Biosience, San Jose, CA, USA). Fluorescence detection was performed by flow cytometry (Beckman Coulter Gallios, San Jose, CA, USA) and results were analyzed by FlowJo software (FlowJo, LLC, Ashland, Oregon, USA).
Proliferation of parent cells (MBN and MBDMD, n = 3/cell type) before cell fusion procedure and fused DEC lines (MBN1/MBN2 DEC and MBN/MBDMD DEC; n = 3 fusions/line) was assessed by flow cytometry up to 21 day. Parent MB and DEC populations were prepared at a single-cell suspension to be labeled. Cells were washed two times with PBS to remove any serum. Cells were suspended in pre-warmed PBS. A 5 µM solution of cell proliferation dye eFluor™ 670 (eBioscience- ThermoFischer, Waltham, MA, USA) in PBS was used for labeling cells. After incubation for 10 min at 37 °C in the dark, labeling was stopped by adding 5 volumes of cold complete media (SKGM, containing 20% FBS) followed by incubation on ice for 5 min. Cells were then washed three times and cultured on 6-well plates for 1, 3, 6, 13, 17 and 21 days. Cells were harvested with a 5-min 0.25% EDTA-trypsin (Gibco- ThermoFischer, Waltham, MA, USA) incubation and fixed with 4% paraformaldehyde. Freshly stained and fixed cells were used as a negative (non-proliferating cells) control. Samples were analyzed by flow cytometery (Beckham Coulter Gallios, San Jose, CA). Detected fluorescence of eFluor™ 670 of each sample was presented as independent histograms. Fluorescence detected of PKH26 and PKH67 pre-fusion staining of DEC was measured as well, and shift of fluorescence mean values was correlated with the post-fusion eFluor™ 670 fluorescence.
Myogenic Differentiation of DEC
To confirm myogenic differentiation potential of DEC, freshly fused DEC lines (MBN1/MBN2 DEC and MBN/MBDMD DEC, n = 4 fusions/ line) and control normal myoblasts (MBN) were cultured on German glass coverslips (ThermoFischer, Waltham, MA, USA) in serum-free Myogenic Differentiation Media (PromoCell, USA) supplemented with 10 µg/ml insulin to induce myogenic differentiation for 7 days. To assess dystrophin and SMHC co-expression, cells were fixed with ice-cold acetone and unspecific antibody binding was blocked with 10% normal goat serum. Rabbit polyclonal anti-fast myosin skeletal heavy chain antibody (1:200, Abcam, Cambridge, MA, USA) and mouse monoclonal anti-dystrophin antibody (MANDYS8, 1:200, ThermoFischer, Waltham, MA, USA) were used for primary detection of dystrophin and myosin heavy chain. For α-sarcoglycan, β-sarcoglycan and desmin detection, cells were fixed with 4% paraformaldehyde and non-specific antibody binding blocked with 5% normal goat serum. Primary detection was obtained with the sarcoglycan complex antibodies rabbit anti α-sarcoglycan (1:50, DSHB Hybridoma Product IVD3(1)A9, University of Iowa, USA) and rabbit polyclonal anti-β-sarcoglycan (1:50, Novus Biologicals, Littleton, CO, USA). Rabbit polyclonal anti-desmin (1:100, Invitrogen, ThermoFischer, Waltham, MA, USA) was used for primary detection of desmin.
Goat anti-rabbit IgG Alexa Fluor 488 (1:500, Molecular Probes, ThermoFischer, Waltham, MA, USA) and goat anti-mouse IgG2a Alexa Fluor 647 (1:500, ThermoFisher, Waltham, MA, USA) fluorescently conjugated secondary antibodies were used for corresponding primary antibody visualization. Appropriate negative tissue controls and isotype controls were implemented in the experiments. Fluorescence images were captured on Zeiss Meta confocal microscope and fluorescence.
Transplantation of DEC
DEC were counted and washed in sterile DPBS twice and transferred in 60µ l total volume PBS to tuberculin syringe with 27G needle (ThermoFischer, Waltham, MA, USA). Mice were anesthetized with 1.5% isoflurane inhalation and the skin on the left posterior calf was shaved and aseptically prepared. Based on a standard circle shaped template, six microinjections (10µ l/injection, total volume 60 µl) were delivered into the gastrocnemius muscle (GM). Animals were allowed to recover in a heated environment and promptly returned to the colony.
The following experimental groups were performed after randomization of age matched 6–8 weeks old mdx/scid recipients for follow-up of 7 and 90 days post-transplant: vehicle treatment (n = 12, 60µ l DPBS), not fused MBN from each of the donors (n = 9, 0.25 × 106 /donor- total 0.5 × 106 in 60 µl DPBS), fused MBN1/MBN2 DEC (n = 9, 0.5 × 106 in 60µ l DPBS) and fused MBN/MBDMD DEC (n = 9, 0.5 × 106 in 60µ l DPBS).
Animals’ follow-up consisted of in vivo muscle strength tests (grip strength and wire hanging) twice a week and on day 7 and day 90 in situ, and ex vivo muscle strength tests were performed.
Histological and Immunofluorescence Analysis
Formalin fixed and paraffin embedded gastrocnemius muscles (GM) were cut at 5-micron sections. Sections were stained with hematoxylin-eosin to analyze muscle structure and to quantify the central nucleated regenerating fibers. Five standardized regions of three non-serial cross-sections of n = 6 animals/ group were analyzed and fibers with centrally positioned nuclei were counted and normalized to total nuclei number.
OCT embedded frozen GM muscle was cut with a cryotome (ThermoFisher, Waltham, MA, USA) at 4-micron section. Cross-sections were fixed with ice-cold acetone. Immunoblocking was performed with 10% normal goat serum in 1% BSA. Dystrophin expression was detected using a primary rabbit polyclonal anti-human anti-dystrophin antibody (ThermoFischer, Waltham, MA, USA) in combination with goat anti-rabbit Alexa Fluor (AF) 488 conjugated secondary antibody (ThermoFischer, Waltham, MA, USA). Nuclei were counterstained with DAPI (Vector). A Zeiss Meta confocal microscope with ZEN software (Carl Zeiss, Oberkochen, Germany) was used for fluorescence signal detection and analysis. The number of dystrophin-positive muscle fibers in five standardized regions of each cross-section were counted and normalized to total nuclei numbers; three, non-serial cross-sections were quantified in each animal (n = 3/group at day 7 and n = 6/group at day 90). The total number of dystrophin-positive fibers was normalized to the number of total nuclei. This quantification method was chosen over normalization to the total fiber count as fiber morphology and specifically the diameter varied over time between experimental groups due to the DMD pathology progression or potential therapeutic effect. The co-localization of HLA-ABC and dystrophin signal was detected using anti-human mouse anti-HLA class I (1:200, Abcam, Cambridge, MA,USA) in combination with goat anti-mouse AF467 conjugated secondary antibody (1:400, Abcam Cambridge, MA, USA). The co-localization of HLA-ABC and skeletal myosin heavy chain expression was detected using anti-human anti-HLA class I (1:200, Abcam, Cambridge, MA, USA) in combination with goat AF467 conjugated secondary antibody (1:400, Abcam Cambridge, MA, USA) and rabbit polyclonal anti-myosin heavy chain antibody (1:200, Abcam, Cambridge, MA, USA) as primary and goat anti-rabbit Alexa Fluor 647 (1:500, Molecular Probes, ThermoFischer, Waltham, MA, USA) conjugated secondary antibody. Nuclei were counterstained with DAPI. A Zeiss Meta confocal microscope with ZEN software (Carl Zeiss, Oberkochen, Germany) was used to detect fluorescence signal. Quantification of dystrophin positive revertant fibers was performed counting 50 adjacent fibers in 4 non-consecutive cross-sections of each animal (total 200 fibers) of a 0.05 mm2 surface. Results were expressed in percentages.
Muscle Strength Evaluation
Wire Hanging and Grip Strength Test
Mice motor function was monitored up to 90-day endpoint; wire hanging test and modified grip strength test were performed twice a week on alternate days. The order of animal test performance was randomly assigned.
The wire hanging test was performed a maximum of three consecutive times to prevent animal training for negative performance and the wire hanging time was measured. Although this muscle force evaluation was not specific for the GM, it provided supportive information regarding the general muscle strength of the DEC injected vs. control animals. A modified grip strength test for posterior limbs [26, 27] was used to measure GM-specific force. Briefly, the hook of grip meter (Digital Force Gauge, HL-50) was placed to touch the mouse toes. Upon the presence of grip, the hook was pulled repeated 10 times and the average maximum peak was used for further analysis.
In Situ Muscle Force Test
In situ muscle force measurements were performed at 90-days endpoint (n = 4/group). In situ muscle force measurements were performed under isoflurane anesthesia. The Achilles tendon was dissected and tied with silk to a force transducer. The sciatic nerve was isolated and stimulated with a bipolar wire electrode. Muscle force was measured after optimal voltage and length were determined. Fatigue was measured after 10 min of submaximal tetanic stimulation as described previously . The GM was kept moist during the whole procedure by continuous drip of Krebs–Henseleit solution (in mM: 130 NaCl, 5 KCl, 1 CaCl2, 1.1KH2PO4, 0.85 MgSO4, 0.6 MgCl2, 25 HEPES, 25 NaCO3, 11 glucose bubbled with 95% oxygen and 5% carbon dioxide). The impact of the drip did not introduce mechanical artifacts. Optimal passive tension was determined by stimulating the sciatic 6 s. The passive tension was increased every 3 twitches until the maximum force was recorded. Optimal voltage was re-determined after each test. When the optimal voltage changed, the data from the previous test were discarded and the test was repeated. To ensure proper voltage, a set of twitches was elicited beginning at 1.0 V with a 1-ms pulse every 3 s with gradual increases in voltage until maximum force was obtained. The voltage used for the experiments was 1.2 times the optimal voltage determined and was usually 2.0 V. After optimal voltage and length were determined, the nerve was stimulated every 3 s with 1-ms pulses for 10 repetitions. The amplitude of the twitches and the rates of force generation and relaxation were measured. Twitches were repeated throughout the test to verify that the optimal voltage and passive tension were maintained.
A 300-ms, 50-HZ burst of stimulation was applied to the nerve every 3 s for 10 min Fatigue is reported as the minimum force, usually at 10 min, as a fraction of the reference force. The reference force was recorded from the second contraction. In these measurements, a smaller number means greater fatigue. Potentiation was reported as the maximal force, usually within the first 40 s of the test, as a percent of reference force.
Ex Vivo Muscle Force Test
After euthanasia, the contractile and passive properties of the GM were measured ex vivo using the Aurora Scientific in vitro muscle test system . After whole GM dissection including the Achilles tendon, GMs were placed in warmed (37 °C) Krebs–Henseleit solution in a Radnoti glass chamber tissue bath. The Achilles tendon and proximal pole of the muscles were attached to the force transducer with silk ties. Muscle force was measured after establishing optimal length through a standardized stimuli pattern until reaching maximal wave and maximal strain.
Data are expressed as mean ± SD. OriginPro 2017 software was used to perform statistical analysis. Student T-test and one-way ANOVA with Tukey post-hoc test for pairwise comparisons were used to define statistical significance. P values were considered significant below 0.05.