PTHrP Sequence and Minicircle (mc) Vector production
We utilized the sequence of PTHrP 1–34 and 107–139 described by Toshiyuki et.al. To construct the therapeutic minicircle, PTHrP 1–34 + 107–139 (282 bp) sequences were sub-cloned into the parental plasmid pMC. The CMV-MCS-EF1-GFP-SV40-Poly A was purchased from SBI (System Biosciences, # MN501A-1). The mc Mock and mcPTHrP 1–34 + 107–139 were synthesized as described by Park and Kay et.al. 15–16 The mc DNA vector was isolated using the Nucelobond Xtra Midi kit (Macherey-Nagel, # 740410.100). We confirmed the successful generation of sequences containing mc PTHrP 1–34 + 107–139 with Bam HI and Xbal via gel imaging.
Hek 293t Cell Culture And Minicircle Vector Transfection
Human embryonic kidney (HEK293T) cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific, #11965-092) supplemented with 7.5% fetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin (Thermo Fisher Scientific,#15240096). HEK293T cells were transfected with the minicircle vectors (Mock, PTHrP1-34 + 107–139) using a Lipofectamine 2000 reagent (Thermo Fisher Scientific, #11668019) following the manufacturer’s instructions. The expression of GFP and PTHrP antibodies (Novusbio, #3H1-5G8) in the cells was assessed via immunofluorescence staining under microscopy. The gene expression of the inserted sequence of PTHrP 1–34 + 107–139 was confirmed by RT-PCR in the cell lysate.
Hmscs Cell Culture And Minicircle Vector Transfection
Bone marrow-derived human mesenchymal stem cells (MSCs) were purchased from The Catholic Institute of Cell Therapy, South Korea. MSCs were maintained in DMEM (Thermo Fisher Scientific, #11965-092) supplemented with 20% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin (Thermo Fisher Scientific, #15240096). At passage 5, MSCs (1 × 106 cells) were transfected with minicircle vectors for microporation using the Neon transfection system (Thermo Fisher Scientific, #MPK5000S). Several conditions were tested for the microporation of minicircle vector transfection. Microporation at 1400 pulse voltage, 20 pulse width, and 2 pulse no was used for the transfection of MSCs at passage 5. The immunophenotypes of the non-transfected MSCs and mc PTHrP 1–34 + 107–139 MSCs at passage 5 were determined using CD 34, CD45, CD73 and 105 (BD Biosciences) using flow cytometric analysis.
Animal Experiment And Group Allocation
C57BL/6 female mice (Orient bio Inc, Korea) aged 12 weeks with a mass range of 17 to 23 g were ovariectomized to induce postmenopausal osteoporosis. Mice were randomized to the following groups: sham (health control) group (n = 3), OVX (disease control) group (n = 6), mc PTHrP 1–34 + 107–139 (n = 5), MSCs (n = 4), and eMSCs (mc PTHrP 1–34 + 107–139 MSCs, n = 6). Animal experiment procedures were reviewed and approved by the Animal Studies Committee of the School of Medicine, The Catholic University of Korea (IACUC approval No.CUMC-2017-0250-03).
Delivery And Detection Of Mc Pthrp 1–34 + 107–139 In Vivo
The plasmid mc PTHrP 1–34 + 107–139 DNA vector was delivered hydrodynamically using intravenous injection into the tail vein. Mice were injected with 40 µg of 1.8 mL PBS three times each week for 3 weeks at 4 weeks post-OVX. The eMSCs (1 × 106 cells) were resuspended in 150 µL of PBS. Cells were injected twice at 4 and 6 weeks, post-OVX via intraperitoneal injection. mc PTHrP 1–34 + 107–139 was delivered to C57BL6 female mice for 12 weeks, into organs such as spleen, kidney and liver at 3, 7, 15 and 37 days after the injection. After co-staining GFP-positive cells with PTHrP (Novusbio) antibody, the homogenizes tissues were extracted with TRIzol solution. RT-PCR was carried out using inserted PTHrP 1–34 + 107–139 sequence primers based on gel images.
Micro Ct Analysis
At 10 weeks post-OVX, the distal femurs were imaged at a scanning voxel size of 13.85 µm with a high-resolution microtomographic system (Sky Scan 1173). Using the SkyScan of Nrecon (ver. 220.127.116.11) reconstruction program, the bone microarchitectural parameters in the trabecular bone regions were calculated to determine the trabecular-specific surface area (BS/BV) based on the trabecular bone microarchitecture for trabecular thickness (Tb.Th), trabecular bone pattern factor (Tb.Pf), and structure model index (SMI).
Measurement Of Bone Turnover Markers In Serum
Blood samples were collected at 10 weeks post-OVX. To evaluate bone remodeling markers in the serum, the bone formation marker N-terminal propeptide of type 1 procollagen (PINP) and the bone resorption marker C-terminal telopeptide of type 1 collagen (CTX-1) were measured using enzyme immune-assays (EIA). PINP and CTX-1 concentrations were evaluated via Rat/Mouse PINP EIA (Immunodiagnostic Systems, # AC-33F1) and RatLaps™ EIA (Immunodiagnostic Systems,# AC-06F1).
All results were expressed as mean ± SEM in the figures and the legends. The results were analyzed using Kruskal-Wallis using SPSS program (IBM Corporation). Inter-group comparisons were done using Mann-Whitney analysis. A P value of less than 0.05 was considered statistically significant.