Cell culture
Human nasal inferior turbinate stem cells (ITSCs) were obtained via minimally invasive surgical procedure according to Hauser and colleagues taking into account of local and international guidelines [26]. All experimental procedures were ethically approved by the ethics board of the medical faculty of the University of Münster (No. 2012–015-f-S). Experiments were conducted using ITSCs from 3 male and 3 female donors. Cells were cultivated in TC25 flasks (Sarstedt, Nürnbrecht, Germany) containing preheated (37°C) stem cell medium consisting of DMEM F12 mixed with 0.1 mg/ml penicillin/streptomycin (Sigma Aldrich, Taufkirchen, Germany), 200 mM L-glutamine (Sigma Aldrich), 20ng/ml FGF2 (Peprotech, Rocky Hill, USA), 20 ng/ml EGF (Peprotech), 6% B-27™ supplement (50X) (Thermofisher Scientific, Waltham, USA) with additional 10% human blood plasma (BP) (Institute for Laboratory- and Transfusion- Medicine, Heart- and Diabetes-Centre NRW, Bad Oeyenhausen, Germany) in a humidified incubator (Binder, Tuttlingen, Germany) with hypoxic atmosphere containing 5% O2 and 5% CO2 at 37°C. The cells were fed every 2 days with fresh stem cell medium, until the matrix was digested completely. For passaging, the medium was removed and adherent cells were washed with 1x Phosphate-buffered saline (PBS) (Sigma Aldrich). After removal of PBS, 3ml of collagenase solution containing 40 mg collagenase powder type A (Worthington Biochemical Corporation, Lakewood New Jersey, USA), 49 ml PBS and 50 µl of 3M calcium chloride (labmade) was transferred to the flasks for 20–30 minutes until the cells were detached for further processes.
Pharmacological Treatment Of Itscs
After standard cultivation described above, 5x104 Cells were seeded on roughened cover glasses and cultured until all cells were completely adherent. Subsequently, 0.3, 0.5, 1, 2.5, 5 and 10 U/ml EPO (Sigma Aldrich) was added to the cells followed by incubation for 30 minutes at 37°C. The medium was completely removed, followed by immunocytochemical staining (see below).
Neuronal Differentiation
For neuronal differentiation, cells of 3 male and 3 female donors were cultivated in DMEM high glucose (Sigma Aldrich) containing 200 mM L-glutamine and 10% fetal calf serum (Sigma Aldrich) at a density of 5x104 cells/well in a 12-well plate on top of roughened cover glasses. After 2 days, 1 µM dexamethasone (Sigma Aldrich), 2 µM insulin (Sigma Aldrich), 500 µM 3-isobutyl-1-methylxanthine (Sigma Aldrich), 200 M indomethacin (Sigma Aldrich) and 200 µM ethanol were added to the medium to induce neuronal differentiation (neuronal induction medium, NIM). In addition, the test group was supplemented with 1 U/ml EPO. Medium exchange and EPO treatment was performed every 2–3 days. 9 days later, cells were additionally induced adding 0.5 µM retinoic acid (Sigma Aldrich) and 1x N-2 supplement (Gibco, Darmstadt, Germany). Afterwards, half of the medium was removed and replaced with pre-warmed NIM containing 1x N-2 supplement. Neuronal differentiation of ITSCs was performed for 1, 3, 6 and 10 weeks.
Camera Lucida Tracing
For determining neuronal morphology and calculating axon length and neurite counts, images of neuronal differentiated ITSCs were analyzed in CorelDRAW 2021 version 23.1.0.389 (Core Corporation, Ottawa, Canada). The outline of the cell body was traced and the neuron was transferred to an adjusted Sholl ring, where the axonal length can be measured.
RT-PCR
PCR was performed using Taq DNA Polymerase with TermoPol® Buffer (New England Biolabs, F.a.M, Germany) according to manufacturers guidelines with the following primers for EPO-R (GAA GTA GTG CTC CTA GAC GCC, CCT CGT AGC GGA TGT GAG AC) and GAPDH (CAT GAG AAG TAT GAC AAC AGC CT, AGT CCT TCC ACG ATA CCA AAG T).
Immunocytochemistry
ITSCs were seeded on roughened cover glasses with a density of 5x104 Cells. After cultivation or differentiation, the cells were fixated by applying 4% paraformaldehyde (labmade) in 1x PBS for 15 minutes at room temperature (RT). Blocking and permeabilization was subsequently done using 5% goat serum (Dianova, Hamburg, Germany) or 1% bovine serum albumin (BSA) (Dianova) in 1x PBS and 0,02% Triton X-100 (Sigma Aldrich) for 30 minutes at RT. Afterwards, primary antibodies against RELA (mouse, 200,301,065, 1:5000, Rockland, PA. USA), RELB (rabbit, D7D7W, 1:500, Cell Signaling Technology, Danvers, USA), c-Rel (rabbit, 4727, 1:500, Cell Signaling Technology), vGlut-II (rabbit, 07-1402-I, 1:300, Merck Millipore, Burlington, USA), NF200 (mouse, SAB3200747, 1:200, Sigma Aldrich), MAP2 (mouse, sC-390543, 1:500, Santa Cruz, Dallas, USA) and synaptophysin (rabbit, ab32127, 1:500, Abcam, Cambridge, UK) were incubated for 1h at RT. Secondary fluorochrome-conjugated antibodies (Alexa 488 anti-rabbit, Alexa 555 anti-mouse, Alexa 555 anti-rabbit, 1:300, Life Technologies, Germany) were incubated for 1h at RT under the exclusion of light, followed by DAPI nuclear counterstaining (1 µg/ml in 1xPBS: Sigma Aldrich) for 10 minutes at RT. Fluorescence microscopy was performed using the confocal laser scanning microscope LSM 780 (Carl Zeiss, Jena, Germany). Randomly placed pictures were analyzed using ImageJ [34]. Nuclear fluorescence intensity was determined using the “measure” function in ImageJ on the nuclei of the cells.
Statistical Analysis
Data were evaluated with at least 3 biological replicates and were statistically verified with the Software Prism V8.4.3 (GraphPad Software, Inc., San Diego, CA, USA) using the Mann-Whitney test. Significance value of p < 0.05 was considered as statistically significant. The presented data shows the means ± standard deviation (SD).