Human msc nucleofector kit

One million hMSCs were transfected with 10 μg DNA using the 4D-Nucleofector™ System together with Human MSC Nucleofector® Kit (Lonza) on high-efficiency settings. Following nucleofection the cells were plated in DMEM/ps with 10% FBS overnight to recover before passaging onto PAM gels in DMEM/ps with 0.5% FBS. Images for analysis were collected on a LSM710 Zeiss confocal microscope equipped with a 40X water immersion objective (Zeiss, Jena, GER) using a heated stage at 37 °C with 5% CO₂ atmosphere. CFP and FRET channels were recorded using a 458 nm laser line and collecting the emissions in the donor (BP 470e490 nm) and acceptor (BP 530e590 nm) emission regions, respectively. In addition, crosstalk and YFP channels were recorded using the 514 nm laser line for excitation and collecting the emission in the donor and acceptor emission regions. Images were acquired by sequential acquisition. For FRET measurements, a modified version of the FRET emission ratio was used to calculate this parameter on a pixel-by-pixel basis, as described previously^{14 (link)}. The FRET index was calculated for every image as the average [FRET/Donor] emission ratio for selected regions of interest. Greater than 20 cells were tested per condition and repeated using multiple MSC donors.

The RANKL specific gRNA construct (pNV-RANKL/KO) is based on the parenteral gRNA-Cas9-2A-GFP vector, which is transiently expressing gRNA and Cas9 and enables GFP selection of transfected MSCs, was purchased from abm (Richmond, Canada). Three vectors were purchased with the following gRNA sequences: pNV-RANKL1/KO—5’-CAGGAATTACAACATATCGT-3’ (472221110290), pNV-RANKL2/KO—5’-CAGCGATGGTGGATGGCTCA-3’ (472221110390), pNV-RANKL3/KO—5’-TTAATAGTGAGATGAGCAAA-3’ (472221110490). pmaxGFP (purchased from Lonza, Human MSC Nucleofector^® Kit) was used to control transfection efficiency. Plasmids were propagated by transforming Escherichia Coli (Mix&Go! Competent Cells—DH5 Alpha, Zymo Research) using standard procedures. The plasmids were purified using the ZymoPURE™ II Plasmid Midiprep Kit (Zymo Research) according to the manufacturer’s instructions. The plasmid concentration was measured with the NanoDrop™ One. The plasmids were stored at—20°C.

To knock down IDO, the ON-TARGETplus SMART-pool small interfering RNA (siRNA) (L-010337-01-0005; GE Dharmacon, Lafayette, CO, USA) was used. The sequences were as follows: Human IDO1, NM_002164, sense, 5′-UCACCAAAUCCACGAUCAUUU-3′, antisense, 5′-PUAUGCGAAGAACACUGAAAUU-3′; sense, 5′-UUUCAGUGUUCUUCGCAUAUU-3′, antisense, 5′-PUAUGCGAAGAACACUGAAAUU-3′; sense, 5′-GUAUGAAGGGUUCUGGGAAUU-3′, antisense, 5′-PUUCCCAGAACCCUUCAUACUU-3′; and sense, 5′-GAACGGGACACUUUGCUAAUU-3′, antisense, 5′-PUUAGCAAAGUGUCCCGUUCUU-3′. The ON-TARGETplus siCONTROL Nontargeting Pool (D-001810-10-05; GE Dharmacon) was used as the negative control. siRNAs were transfected into MSCs by the Amaxa Nucleofector II device (Lonza) and the Human MSC Nucleofector kit (Lonza). Briefly, aliquots of 5 × 10⁵ MSCs were re-suspended in 100 μl of the Nucleofector solution in the presence of 1.5 μg of IDO or control siRNA and then electroporated by the U-23 program. Cells were immediately transferred into 37 °C pre-warmed complete culture medium and plated onto six-well dishes. After 72 h from transfection, cells were harvested and lysed for the immunoblotting measurement of IDO levels, while the supernatants were recovered for the assessment of IDO activity and to be used in functional assays.

Transient transfection of β-catenin siRNA, RelA/p65 siRNA (Stealth RNAi duplex siRNA, Invitrogen) or control siRNA (SiRNA-A, Santa Cruz Biothech., Inc.) into human MSCs or KM101 cells was performed by electroporation with the Human MSC Nucleofector Kit (Lonza) according to the manufacturer’s instruction and as we described44 (link). In briefly, cells were harvested by trypsinization, and resuspended one million cells in 100 μL of human MSC nucleofector solution with 100 pmole of β-catenin, p65 or control siRNA. Electroporation was performed in Nucleofector^TM II with program U-23 provided by Lonza/Amaxa Biosystems. Immediately after electroporation, MSCs were transferred into 6 or 24-well plates or 60 mm dishes in MEM-α with 10% FBS-HI and KM101 cells were transferred into 60 mm dishes in IMEM with 10% FBS-HI. After confluence, cells were used for Western blot assays (60 mm dishes) and SA-β-Gal staining (6-well plates) or the medium was changed into osteogenic medium for 7 days to analyze osteogenic marker ALP enzyme activity (24-well plates).

The hMSC were transfected with green fluorescent protein (GFP)-carrying plasmid by electroporation using the human MSC nucleofector kit (Lonza, Walkersville, US). GFP-labeled hMSC were seeded on plastic plate or nano-fiber for 24 hours then used for treatment. Three days later, collected the tissues including ankle, spleen, LN, lung, liver and kidney. The hMSC were detected by GFP and human beta actin (ACTB) expression using PCR or anti-GFP immunohistochemistry staining (rabbit polyclonal anti-GFP antibody, at 1∶50 dilution (ab6556, Abcam, Cambridge, UK)). HRP-conjugated goat anti-rabbit secondary antibody was used and the antigens were visualized using a DAB substrate.
For PCR, the primer sequences and conditions were as follows: GFP, 5′- AGGACAGCGTGATCTTCACC- 3′ (forward) and 5′-CTTGAAGTGCATGTGGCTGT -3′ (reverse) (TM, melting temperature; 55°C, 35 cycles); human ACTB, 5′-AGCGAGCATCCCCCAAAGTT -3′ (forward) and 5′-GGGCACGAAGGCTCATCATT-3′ (reverse) (TM, melting temperature; 55°C, 35 cycles). The ladder for PCR electrophoresis is OneSTEP Marker 4 (Wako, Osaka, Japan). 2% agarose gel was used for electrophoresis. The suitable range of the ladder is 72 to1,353 bps. The size of GFP is 153 bps. Three times of biological replicates were used and each independent experiment was performed by triplicates.

Passage 3 to 4 equine BMSCs (n = 3) were nucleofected with the fluorescent-protein tagged adhesion markers paxillin mEmerald and vinculin mApple using a human MSC nucleofector kit (Lonza, Basel, Switzerland). Cells were passaged at 48 h post-nucleofection and plated onto 12-well fibronectin-coated glass plates at a concentration of 2 × 10³ cells/cm² in serum-free media (Dulbecco’s modified Eagles’ media, 1000 mg/L glucose; 1 ng/mL bFGF; 25 mM HEPES; 100 units/mL penicillin-streptomycin). BMSCs were plated in the presence and absence of 100 mM β-lactose (Santa Cruz Biotechnology Inc., Dallas, TX, USA). At 8 h post-plating, glass plates were fixed in 4% paraformaldehyde for 10 minutes, rinsed in PBS, followed by actin staining with phalloidin Alexa647 and nuclear staining with DAPI for 15 minutes, followed by several PBS rinses. Cells were imaged with a × 100, NA 1.3 objective on an Olympus IX83 microscope equipped with a Yokagawa X1 spinning disk confocal scanning unit and an Andor Ultra 897 EMCCD camera. Images were overlaid in Adobe Photoshop CS5 (Adobe Systems Inc., San Jose, CA, USA).