Isolation and expansion of BM-derived hMSCs
hMSCs were isolated, identified, and expanded from BM aspirates following previously described protocols [8, 9]. Briefly, BM aspirates (~6 ml) from the iliac crest of humans (up to 40 years old) were collected after obtaining written informed consent according to the Declaration of Helsinki and approval by the Lithuanian Bioethics Committee (No. 158200-14-741-257). BM donors were tested according to guidelines for the preparation of blood and blood products. Unprocessed bone marrow was seeded at a density of 12,000 mononuclear cells (MNC)/cm2 in T-75 flasks (NUNC, Wiesbaden, Germany) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10 % fetal bovine serum (FBS) at 37 °C in a humidified atmosphere of 5 % CO2. After 24 h, supernatant containing non-adherent cells was removed, cells were rinsed with phosphate-buffered saline (PBS) without Ca2+/Mg2+ (Biochrom, UK) and growth medium was added. Additional changes of growth medium were performed every 3 days. For further experiments, cells were used from passages 2–3. Only the MSCs that corresponded to all main MSC identification criteria were included in the study.
Transfection of hMSC with the mHCN2 gene
A full-length of mHCN2 cDNA, subcloned in a pIRES2-EGFP vector (BD Biosciences Clontech, San Jose, USA), was transfected into the hMSCs by electroporation using the Lonza 4D-Nucleofector™ system (Lonza, USA). hMSCs were transfected with 3 μg pIRES2-EGFP and pIRES2-mHCN2-EGFP vectors, and the efficiency of transfection was estimated by the number of green fluorescence-emitting cells (enhanced green fluorescence protein (EGFP) excitation 488 nm, emission 509 nm). The total number of green fluorescent cells 24–48 h after the incorporation of vectors revealed 40–50 % transfection efficiency. Transfected cells were cultured in hMSC growing medium (Poietics MSCGM, BioWhittaker, Lonza) at 37 °C in a 5 % CO2 humidified atmosphere. To select a pure population of mHCN2-expressing hMSCs, cells were exposed to 50 μM geneticin for an additional 5 days. Transfection efficiency was also confirmed by both quantitative real-time polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA).
Investigation of mHCN2 expression by RT-qPCR
pIRES-EGFP- and pIRES-mHCN2-EGFP-transfected hMSCs were lysed, and their RNA was extracted and purified using RNeasy Mini Spin columns (Qiagen, USA) according to the manufacturer’s instructions. RNA concentration and purity were evaluated with a SpectraMAX i3 spectrophotometer (Molecular Devices, USA). RNA samples were treated with DNase I (Thermo Scientific™) and reversely transcribed with the Maxima®First Strand cDNA Synthesis Kit (Thermo Scientific™) according to the manufacturer’s protocols. PCRs were performed using Maxima® Probe qPCR Master Mix (2×) (Thermo Scientific™) and Stratagene MX-3005P detection instrument (Agilent Technologies, USA). The TaqMan® Gene Expression Assays (Applied Biosystems, USA) for HCN2 (human, Hs00606903_m1), mHCN2 (mouse, Mm00468538_m1), and ACTB (housekeeping gene, Hs01060665_g1) were used for gene expression analysis and separation of endogenous human HCN2 from transfected mHCN2. Expression of the mHCN2 gene after transfection was compared with the level of the endogenous human HCN2 gene in hMSCs (Fig. 1a). All reactions were run in triplicate starting with a denaturation step for 10 min at 95 °C followed by 40 cycles of 15 s at 95 °C for denaturation and 60 s for annealing and extension. The gene expression ratio (pIRES-mHCN2 vs. non-transfected cells) was calculated using the 2-∆∆Ct equation. The efficiency of mHCN2 transfection was measured 5 days after the cell growth with 50 μM geneticin.
Evaluation of mHCN2 protein expression by ELISA
Cells were lysed using three cycles of freezing-thawing. Before making measurements, all samples were kept on ice. mHCN2 expression after hMSC transfection was measured using an ELISA kit for the estimation of mouse potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 2 (EIAab, cat. No.E15069m) following the manufacturer’s instructions. Absorbance was measured at 450 nm using a Spectramax plate reader. Three groups of cell lysates were investigated: pIRES-mHCN2-EGFP-expressing (positive control), pIRES-EGFP-transfected (control with transfection reagent), and non-transfected (negative control) hMSCs. The concentration of total protein in all tested groups was measured using the Bio-Rad DC Protein Kit according to the manufacturers’ instructions. Absorbance was read at 750 nm using a Spectramax plate reader. The final concentration of intracellular mHCN2 after transfection was expressed as ng/mg protein. The efficiency of mHCN2 protein expression in hMSCs was measured 5 days after cell growth with 50 μM geneticin.
Patch-clamp and dye transfer measurements
For electrophysiological recordings, glass coverslips with hMSCs were transferred to the experimental chamber with constant flow-through perfusion, and mounted on the stage of an inverted microscope (Olympus IX81). Junctional conductance between hMSCs (abutted or connected through tunneling tubes (TT)) was measured using the dual whole-cell patch-clamp technique. Cells 1 and 2 of a cell pair were voltage clamped independently with the patch-clamp amplifier (MultiClamp 700B; Molecular Devices, Inc., USA) at the same holding potential (V1 = V2). Voltages and currents were digitized using the Digidata 1440A data acquisition system (Molecular Devices, Inc.) and acquired and analyzed using pClamp 10 software (Molecular Devices, Inc.). By stepping the voltage in cell 1 (ΔV1) and keeping the other constant, junctional current was measured as the change in current in the unstepped cell 2, Ij = ΔI2. Thus, gj was obtained from the ratio –Ij/ΔV1, where ΔV1 is equal to transjunctional voltage (Vj), and a negative sign indicates that the junctional current measured in cell 2 is oppositely oriented to that measured in cell 1.
To examine whether cells residing on the opposite sides of Kapton® scaffold can couple through 3 μm diameter pores, non-transfected hMSCs were seeded on one side of the scaffold and 24 h later the mHCN2-transfected cells were seeded on the other side of the scaffold. After the attachment of transfected cells, DAPI dye (20 μM) was injected through the patch pipette into the mHCN2-transfected hMSC, and its transfer to the non-transfected cells residing on the other side of the scaffold was monitored by time lapse imaging at 37 °C in a humidified atmosphere of 5 % CO2 using an incubation system (INUBG2E-ONICS; Tokai Hit, Shizuoka-ken, Japan) with an incubator mounted on the stage of the microscope equipped with an Orca-R2 cooled digital camera (Hamamatsu Photonics K.K., Japan), fluorescence excitation system MT10 (Olympus Life Science Europa Gmbh, Hamburg, Germany), and XCELLENCE software (Olympus Soft Imaging Solutions Gmbh, München, Germany).
Patch pipettes were pulled from borosilicate glass capillary tubes with filaments. To minimize the effect of series resistance on the measurements of gj [10], we maintained pipette resistances below 3 milli-ohms. Patch pipettes were pulled from borosilicate glass capillary tubes with filaments. Experiments were performed at room temperature in Krebs-Ringer solution (mM): NaCl, 140; KCl, 4; CaCl2, 2; MgCl2, 1; glucose, 5; pyruvate, 2; HEPES, 5 (pH = 7.4). Patch pipettes were filled with internal solution (mM): KCl, 130; Na aspartate, 10; MgATP, 3; MgCl2, 1; CaCl2, 0.2; EGTA, 2; HEPES, 5 (pH = 7.3). Patch-clamp measurements were performed 48–72 h after transfection.
Cell viability-apoptosis assay
The number of viable cells, type of cell, and cell death, and stage of apoptosis of mHCN2-expressing hMSCs after selection with geneticin were analyzed using the Muse® Annexin V & dead cell assay (Merck Millipore) following the manufacturer’s instructions. This assay allows quantitative identification of live, early and late apoptotic, and dead cells by measuring the intensity of cell fluorescence. Briefly, cells transfected with the mHCN2 gene, only, with the pIRES vector, and non-transfected hMSCs were harvested with trypsin-EDTA and suspended in DMEM containing 10 % fetal calf serum. Cell suspension (100 μl) and Muse™ annexin V & dead cell reagent (100 μl; Annexin V and 7-AAD) were thoroughly mixed. Samples were incubated for 20 min in the dark. The Muse™ Cell Analyzer (Merc Millipore) was used to measure cell fluorescence. Cells were separated into groups according to the intensity of green (Annexin; early and late apoptotic cells) and red (7-AA; dead cells) fluorescence. Cell viability was tested 5 days after the transfected cell growth with 50 μM geneticin. Non-transfected cells were grown in parallel for the same time period.
Measurements of cell proliferation and cell cycle
Proliferation measurements were performed using the CCK-8 kit (Dojindo Molecular Technologies, USA) according to the manufacturer's instructions. Briefly, after selection with geneticin, mHCN2- and pIRES-transfected hMSCs were grown for 5 days in 12-well plates. Similar numbers (25 × 103) of non-transfected hMSCs were seeded in 12-well plates and allowed to attach for 24 h. Then, the number of proliferating cells in each cell group was measured every 24 h by adding a required volume of CCK-8 reagent with subsequent incubation for 3 h and measurement of absorption at 450 nm. Proliferation rates were measured for 72 h. Non-transfected hMSCs were grown and analyzed in parallel to the transfected ones.
Cell cycle was also measured 72 h after cell growth following the manufacturer’s instructions. Briefly, mHCN2- and pIRES-transfected, and non-transfected hMSCs were harvested with trypsin-EDTA. Cells were suspended in growth media containing 10 % fetal calf serum; 200 μl cells were added to each tube, centrifuged at 300 × g for 5 min and washed once with PBS. Then cells were suspended in 200 μl ice cold 70 % ethanol and incubated for 3 h at −20 °C. Cells were centrifuged at 300 × g for 5 min and washed once with PBS; 200 μl Muse™ Cell Cycle Reagent was added to each tube and incubated for 30 min at room temperature in the dark. The cell cycle reagent was a mixture of propidium iodide (PI) and RNAse A in respective proportions subsequently intercalating nuclear DNA. The assay allows identification and measurement of the percentage of cells in each cell cycle phase (G0/G1, S, and G2/M) according to the intensity of PI-based red fluorescence. The DNA Muse™ Cell Analyzer program was used to evaluate the results.
Investigation of transcription factors by RT-qPCR
After selection with geneticin, pIRES-EGFP, pIRES-mHCN2-EGFP-transfected, and non-transfected hMSCs were lysed and RNA was extracted as described above. RNA was reversely transcribed with the RT2 First Strand Kit (Qiagen, USA) according to the manufacturer’s protocols. PCRs were performed using RT2 SYBR Green ROX qPCR Mastermix (Qiagen, USA) and the Stratagene MX-3005P detection instrument (Agilent Technologies). The Human Transcription Factors RT2 Profiler™ PCR Array (Qiagen, PAHS-075Z) was used to screen gene expression changes. Samples of three independent transfection experiments were investigated. Raw data were analyzed by the GeneGlobe Data Analysis Center platform (available online: http://www.qiagen.com/fo/shop/genes-and-pathways/data-analysis-center-overview-page/; Qiagen, USA). For the normalization of gene expression, the geometric mean of four threshold cycles (Ct) of reference genes was used. Gene expression ratio was calculated using the 2-∆∆Ct equation. Data were statistically significant at p < 0.05. The expression of transcription factors in transfected cells was measured after cell growth with 50 μM geneticin for 5 days and compared with non-transfected hMSCs.
Manufacture of Kapton® scaffold
Pores were micromachined in a commercially available 12.7-μm thickness polyimide Kapton® HN (DuPont, USA) film using FemtoLab workstation (Workshop of Photonics) and second harmonic (515 nm) of Yb:KGW femtosecond laser Pharos (Light Conversion). The diameters of the resulting pores were 1–3 μm, as determined by scanning electron microscope (Quanta 200 FEG) [11] under the low-vacuum mode.
Immunocytochemistry
To detect the growth of mHCN2-transfected hMSCs and their possible transmigration through the Kapton® scaffold, 4 × 104 non-transfected hMSC were labeled with PKH26 (red fluorescent cell linker kit; Sigma-Aldrich) following the manufacturer’s instructions and seeded on one side of a scaffold. The next day, 4 × 104 mHCN2-transfected hMSCs (green fluorescence of EGFP) were seeded on the other side of the scaffold, which was then fixed on a specially designed cell crown holder. After 24 h of cell growth in media at 37 °C in a humidified atmosphere with 5 % CO2, cell were washed and fixed with 4 % paraformaldehyde and mounted in the Vectashield (Vector Labs, USA) containing DAPI for visualization of the nuclei. All samples were analyzed using a Leica TCS SP8 confocal microscope.
Statistical analyses
All statistical analyses were performed using the SPSS package (version 19.0 for Windows; SPSS Inc., Chicago, IL, USA) and considered to be significant at the 5 % level. Differences between transfected and non-transfected cells were tested by analysis of variance (ANOVA) and Student’s t test. Data are presented as means ± SD.
Ethical approval
All hMSC isolation procedures were approved by the Ethics Committee of Vilnius Regional Biomedical Research, Lithuania (No. 158200-14-741-257). All volunteers gave written consent and agreed with the investigational procedure of BM.