Exogenously delivered iPSCs disrupt the natural repair response of endogenous MPCs after bone injury

Ethics statement

All animal studies were performed in accordance with the recommendations in the Canadian Council on Animal Care Guidelines. The reporting of this data in the manuscript follows the recommendations in the ARRIVE guidelines. The University of Calgary Health Sciences Animal Care Committee approved all animal protocols and surgical procedures used in this study (Ethics ID# AC16-0043).

iPSC generation

C57BL/6 embryos (day 12.5) were harvested, rinsed in Dulbecco’s phosphate buffered saline (DPBS), and macerated. After treatment with 0.25% trypsin, the resultant cell suspension was filtered to remove debris and plated in high glucose DMEM (Gibco), 10% FBS (Gibco), 1× MEM No-Essential Amino Acids (Gibco), 50 units/ml Penicillin–Streptomycin (Gibco). The resultant mouse embryonic fibroblasts (MEFs) were shipped Applied Biological Materials Inc. (Richmond, BC) for cellular reprogramming to produce iPSCs. MEFs were transduced with Lentiviruses SFFV-OCT4, -SOX2, and -KLF4. After one week, ESC-like colonies were transferred to fresh wells with feeders. Following two passages on feeder cells, mouse iPSCs were shipped to UCalgary. To incorporate GFP into the genome, CRISPR/Cas9 was used to insert a 6-kb transgene into the mouse safe harbor ROSA26 locus using homology-independent targeted integration (HITI) technique was used [136]. For each nucleofection reaction, CRISPR DNA 10 μl from 916 ng/μl and DNA 10 μl from 719 ng/μl Cas9 DNA construct mixed with 28.98 μl from 414 ng/μl GFP construct was used for iPSCs. All DNA constructs were dissolved in water. For each nucleofection reaction, constructs with three million iPSCs was used with nucleofection kit (mouse nucleofector kit, Lonza, Cat. No.VPH-1001) in program A-023 according to manufacturer’s instruction. Nucleofected iPSCs were grown on the gelatin and MEFs coated plates with ESC media for two days. Then FACS with SSEA1 marker was performed. iPSCs were digested enzymatically with 0.25% trypsin for 5 min in 37 °C. Single cells washed twice with cold DPBS and stained with conjugated SSEA1-PE with concentration of 1 μg/ml (Santa cruz: sc-21702 PE) for 15 min. Double positive for GFP and SSEA1 iPSCs were sorted into gelatin coated 96-well plates, one cell/well. Clones were sequenced to determine correct positioning and orientation of the transgene in the ROSA26 locus. To validate the cells remained as functional iPSCs, teratoma assays were undertaken in where 1.106 cells were transplanted subcutaneously into the dorsal area of SCID-beige mice. Samples were fixed, processed and embedded in paraffin. They were sectioned to a thickness of 10 μM and stained with Safranin O.

iPSC culture

Murine iPSCs were routinely cultured on mitotically inactivated mouse embryonic fibroblasts (MEFs) in T25 flasks (Fisher). Culture medium consisted of high glucose Dulbecco’s Modified Eagle Medium (DMEM, Lonza) supplemented with 1% non-essential amino acid, 1% Anti-Anti, 15% fetal bovine serum (FBS), and 0.1 mM β-mercaptoethanol (all Invitrogen). In order to maintain iPSC pluripotency, culture medium was supplemented with 1000 U/ml leukemia inhibitory factor (LIF). Cells were routinely passaged upon reaching approximately 80% confluence every third to fourth day and maintained in a humidified incubator with 5% CO2 at 37 °C. One passage before the cells were added to the collagen scaffold, they were cultured on gelatine (0.1%, Fisher) coated flasks to remove excess MEFs.

Scaffold preparation

The collagen-I scaffold was prepared according to previously published methods7,14,18. Bovine fibrillar collagen I (3 mg/ml, PureCol, Advanced Biomatrix) was polymerized as a 3D gel. Briefly, 80% v/v 3 mg/ml type-I collagen solution was mixed with a 1 million cells/ml iPSC suspension and 20% v/v beta-glycerol phosphate (βGP, Sigma Aldrich) dissolved in 5 × concentrated Dulbecco’s modified Eagle’s medium (DMEM)18. Subsequently, 15% FBS (Invitrogen), 1% non-essential amino acids, 1% Anti-Anti, and 0.1 mM β-mercaptoethanol (all Invitrogen) was added18. The collagen-I/iPSC construct was distributed into a 96-well plate with a volume of 100 μl per well. The polymerized scaffold was placed in an incubator at 37 °C and 5% CO2 to pre-differentiate for 5 days prior to in vivo implantation in the animal model. For the collagen-only scaffold control, the above protocol was also carried out, however, the iPSCs were not added. A general overview of the process is shown in Supplementary Fig. S1.

Real time polymerase chain reaction (RT-PCR)

The collagen-I scaffolds with iPSCs were collected at timepoints: 0, 1, 3, 5, 8, 11, 13, 15, 18 and 21 days post-seeding. Trizol (Invitrogen) was added and a 26 ½ gauge needled was used to lyse the collagen/cell matrix in the Trizol. RNA was isolated using the manufacture’s recommended protocol. cDNA was prepared using the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher). RT-PCR was performed on an ABI QuantStudio 6 using probes again Sp7 (Mm04209856_m1) and Bglap (Mm03413826_mH), Ibsp (Mm00492555_m1), Sox9 (Mm00448840_m1), Col2a1 (Mm01309565_m1), Oct4 (Mm03053917_g1), Nanog (Mm02019550_s1) with 18S (Mm03928990_g1) as a housekeeping control.

Viability assessment by flow cytometry

Cells were dissociated into single-cell suspensions with collagenase I treatment and subjected to flow cytometry using an Attune NXT and FlowJo software for analysis. At least ten thousand events were registered per sample, and analysis of whole cells was performed using appropriate scatter gates to avoid cellular debris and aggregates. Cells were stained using the Annexin-PI kit (ThermoFisher) following the manufactures instructions.

Animal models

MPC lineage tracing mice (Hic1cre-ERT2:ROSAtdTomato) on a C57BL/6 background were provided by Dr. T. Michael Underhill (University of British Columbia). Both males and females ranging 8–12 weeks old, were used in this study. The experimental design for the bone injury in intact and ovariectomy (OVX) mice is depicted in Supplementary Fig. S2.

In this mouse model the endogenous MPCs were permanently labelled with tdTomato post-tamoxifen induction. Mice were anesthetized and received intraperitoneal injections of the active isomer of tamoxifen ((Z)-4-Hydroxytamoxifen, Sigma) dissolved in sterilized sunflower oil. Mice were injected with 100 μl tamoxifen solution (100 mg/kg) once a day over 4 days, followed by a 1 week waiting period to allow for recombination.

One week after the last tamoxifen injection, a burr-hole (non-critical size) injury was performed. The burr-hole injury was performed following procedures previously described by Taiani et al.12,13,19, which was modified from methods previously published by Uusitalo et al.20 Mice were anesthetized with veterinary isoflurane and 0.1 ml of buprenorphine was administered subcutaneously prior to surgery. A high-speed microdrill (Fine Science Tools) was used to drill a 0.7 mm diameter hole into the medullary cavity (without damaging the opposite side) of the metaphysis of the proximal tibia19.

In the homeostatic fracture model, mice were divided into three groups: untreated control, empty collagen I construct, and collagen I + iPSCs. Immediately following the burr-hole injury, the skin of untreated control mice was stapled to close the incision site. For the empty collagen I and collagen I + iPSC treated mice, 100 µl of gel with (100,000 iPSCs per mouse) or without cells, was implanted into the burr-hole defect. The incision site was then closed with staples and mice were returned to their cages and allowed to weight-bear immediately following surgery.

Similar to the experimental design of the homeostatic fracture healing model, Hic1cre-ERT2:ROSAtdTomato received intraperitoneal tamoxifen injections once per day for 4 days. One week after the last tamoxifen injection, mice received OVX surgery. Briefly, mice were anesthetized and given 0.1 ml of buprenorphine prior to surgery. Both ovaries were located and excised. One week after OVX surgery, burr-hole surgery was performed. The OVX mice were divided into three groups: OVX untreated control, OVX empty collagen I construct, and OVX and collagen I + iPSCs.

Histology and immunofluorescence

Tibiae were decalcified, processed and embedded in paraffin wax and cross-sectioned at 10 μm. Safranin-O/fast-green staining and immunofluorescence were performed. The specific markers used were: TdTomato, GFP, Anti-Mo/Rt Ki-67 eFluor 660 Clone SolA15 (Invitrogen, Ki67), Bone sialoprotein (BSP) Clone WVID1(9C5) (Developmental Studies Hybridoma Bank). Slides were treated with EverBrite™ Hardset Mounting Media with DAPI (Biotium), and imaged using the Zeiss Axioscan microscope.

Tissue cytometry

For quantitative analysis, the area of interest was acquired as digital greyscale images. Cells of a given phenotype were identified and quantitated using the TissueQuest software (TissueGnostics), with cut-off values determined relative to the negative controls (non-stained and secondary alone controls). Gating and quantification of single/double positive cells were undertaken using these thresholds.

X-ray microscopy (Xradia)

X-ray microscopy (XRM) imaging was employed on C57BL/6 mice (normal and OVX) to assess bone structure and callus formation within the injury area. The tibiae (including soft tissue and muscle) were harvested and fixed in 10% neutral buffered formalin (NBF) for 24 h. After 24 h, all soft tissue and muscle were removed from the bone. Samples were placed in 70% alcohol until XRM imaging. To prepare samples for XRM, the tibia was removed from the alcohol and secured using foam in an upright holder. PBS was used to ensure hydration during imaging. Three calcium hydroxyapatite (CHA) bone calibration phantoms (densities 50, 1000, and 1200 mg/cm3) were placed on top of a thin foam layer to sit flush and secured to the holder using tape. Low energy (40 kVp voltage, 3 W power) XRM scans were performed using a 4 × objective. The exposure time of each projection was 3 s, and 2001 projections were collected per rotation. Images were reconstructed to an isotropic voxel size of 4.9 μm. The raw data obtained from the XRM scan was processed using Amira software and custom SimpleITK scripts (v0.10.0, www.simpleitk.org/)21. All slices containing the burr hole defect were segmented, including the area up to where the cortical bone ends and all newly formed bone callus extending from the defect site (Supplementary Fig. S3). Regions of interest (ROIs) were placed in the CHA calibration phantoms avoiding edge artifacts using ITK-SNAP (v3.8.0, www.itksnap.org/)22. Average linear attenuation was computed in each ROI and a linear calibration equation was fit to calibrate the XRM images. For all calibrations, R2 > 99%. A density threshold of 800 mg/cm3 was used to segment fully mineralized tissue. Callus bone volume fracture (BV/TV) was computed as the number of fully mineralized voxels in the callus segmentation divided by the total number of voxels in the callus segmentation. Callus bone mineral density (BMD) was computed as the average density inside the callus segmentation. Callus renderings were generated using ParaView (5.7.0) software. Three filters were applied: (1) Threshold—at a minimum of 0.5, (2) Extract surface, and (3) Smooth—at 400 iterations.

Statistical analysis

All data was analyzed using GraphPad Prism 9. All data sets containing more than two experimental groups were analyzed using a one-way analysis of variance (ANOVA) with a 95% confidence interval (α = 0.05) with a Fisher’s LSD post-hoc test. Data sets containing only two groups were analyzed using a two-tailed unpaired parametric t-test with a 95% confidence interval (α = 0.05).

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