Isolation and characterization of mesenchymal stromal cells derived from human menstrual blood (MenSCs)
The menstrual blood samples were collected from three healthy females aged between 18-30 years. Donors provided a signed informed consent approved by the Ethics Committee of the Sede de Investigación Universitaria -SIU-, University of Antioquia, Medellín, Colombia. Menstrual blood (MenB) was collected by cup collection during the ﬁrst 3 days of menses. Briefly, menstrual blood samples were delivered into the laboratory and mixed with an equal volume of phosphate-buffered saline (PBS) containing 1 mM ethylenediamine tetra-acetic acid (EDTA), with 100 U/ml penicillin/streptomycin 0.25 mg/ml amphotericin B, and subject to cell lysis or standard Ficoll procedures within 24h as previously described by Liu et al., 2018 . After centrifugation, the cells suspending in a buffy coat were transferred into a new tube, washed in PBS twice, and suspended in growth medium (low-glucose DMEM medium supplemented with 10% FBS (Gibco, USA), 100 U/ml penicillin/streptomycin 0.25 mg/ml amphotericin B) and seeded into 25 cm2 plastic cell culture ﬂasks at 37°C with 5% humidiﬁed CO2. The medium was replaced every 3 days leaving behind the adherent cells that were growing as ﬁbroblastic cells in clusters. When the cells reached 80–90% conﬂuence (P0), the cells were detached by 0.25% trypsin/1 mM EDTA and sub-cultured to new ﬂasks by the ratio of 1:3. The isolated MenSCs were evaluated for the following characteristics: colony formation capacity (i.e., colonies with a typical adherent growth, colony-forming unit fibroblast activity, and spindle-shaped and fibroblast-like morphology); positivity for the mesenchymal-associated surface markers CD90, CD9, and CD73 (99% positive); and karyotype with normal shape, number, and distribution. The differentiation capacity into an osteoblast, chondrocyte, and adipocyte lineage of MenSCs, as well as the presence of neuronal precursors and astrocyte markers (e.g., NFL, b-TUB III, GFAP, S100b, TH, ChAT), was assessed according to Mendivil-Perez et al., 2016 and 2019 [12, 14].
Neurospheres (NSs) formation
MenSCs were seeded at a density of 2.5 x 104 cells/cm2 in a multi-well plate (Greinner-Bio-one, cat# 662102) using Fast-N-spheres medium (DMEN F-12 GIBCO®, cat#11330-032; supplemented with 2% B27® GIBCO® (cat #17504-044), 20 ng/ml basic fibroblast growth factor (bFGF, R&D Systems, Inc., MN), 20 ng/ml epidermal growth factor (EGF, Sigma cat#E9644), 1 µg/ml heparin sodium salt®, and 100 U/ml penicillin/streptomycin) for 0, 1 and 3 days according to .
Astrocyte-like Cells (ALCs) differentiation
For astrocyte differentiation, 1 x 103 MenSCs / cm2 were seeded in 25 cm2 culture flasks in regular culture medium (RCm, DMEM low glucose Sigma cat# D6046 media supplemented with 10% FBS) until reach 40% of confluence. Then, the medium was replaced and cells were incubated either in DMEM low glucose media supplemented with 2% FBS (minimal culture medium, thereafter MCm) or Astrocyte medium® (GIBCO®, cat#A1261301) for 0, 4, and 7 days.
Dopaminergic-like Neurons (DALNs) differentiation
The MenSCs were seeded at 1 x 103 MenSCs / cm2 in 25 cm2 culture flasks for 24h in regular culture medium (DMEM low glucose media supplemented with 10% FBS). Then, the medium was removed and cells were incubated either in MCm or dopaminergic differentiation medium (Neuroforsk medium, DMEM low glucose media supplemented with 2% FBS Forskolin (Sigma cat# F6886) 1mM final concentration) for 0, 4 and 7 days according to .
Cholinergic-Like Neurons (ChLNs) differentiation
The MenSCs were seeded at 3-5 x 103 cells/ cm2 in 25 cm2 culture flasks for 24h in regular culture medium. Then, the medium was removed and cells were incubated either in MCm or cholinergic differentiation medium (Cholinergic-N-Run medium containing DMEM/F-12 media 1:1 Nutrient Mixture Gibco (cat# 10565018, 10 ng/ mL), basic fibroblast growth factor (bFGF) recombinant human protein (Gibco cat# 13256029), 50 µg/ mL sodium heparine Hep (Sigma-Aldrich cat# H3393), 0.5 µM all-trans retinoic acid, 50 ng/ mL sonic hedgehog peptide (SHH, Sigma cat# SRP3156) and 1% FBS) at 37 °C for 0, 4 and 7 days.
Western blotting (WB) analysis
Cells treated with DALNs, ChLNs, NSs, and ALCs differentiation medium for 0, 4, and 7 days were detached with 0.25% trypsin and lysed in 50 mM Tris-HCl, pH 8.0, with 150 mM sodium chloride, 1.0% Igepal CA-630 (NP-40), and 0.1% sodium dodecyl sulfate and a protease inhibitor cocktail (Sigma-Aldrich). All lysates were (quantified using the bicinchoninic acid assay; Thermo Scientific cat # 23225) and 30 μg of proteins were loaded onto 12% electrophoresis gels and transferred onto nitrocellulose membranes (Hybond-ECL, Amersham Biosciences) at 270 mA for 90 min using an electrophoretic transfer system (BIO-RAD). The membranes were incubated overnight at 4 °C with monoclonal/ polyclonal antibodies against sex-determining region Y)-box 2 (SOX2 cat# PA1-094, Thermo), anti-Nestin (Thermo, cat #MA1 5841), dopamine transporter (DAT cat # PA1-4656), tyrosine hydroxylase (TH, cat# AB152, Millipore), glial fibrillary acidic protein (GFAP, cat# sc6170, Santa Cruz), S100β (cat# 676604, Biolegend), β-tubulin III (β-TUB III cat# G712A, Promega), microtubule-associated protein 2 (MAP2, MA1-25044, Invitrogen), Neurofilament-L (NF-L, cat# 125044, Thermo), vesicular acetylcholine transporter (VAChT, cat# SAB4200559, Sigma-Aldrich) and choline acetyltransferase (ChAT, cat# AB144P, Millipore) primary antibodies (1:5000). Anti-actin antibody (cat #MAB1501, Millipore; 1:1000) was used as expression control. Secondary infrared antibodies (goat anti-rabbit IRDye® 680RD, cat #926-68071; donkey anti-goat IRDye ® 680RD, cat # 926-68074; and goat anti-mouse IRDye ® 800CW, cat #926-32270; LICORBiosciences) 1:1000 was used for Western blotting analysis and data were acquired by using Odyssey software.
Immunofluorescence (IMF) analysis
For immunofluorescence analysis of neural and astrocytes markers, cells treated with MCm, Neuroforsk, Gibco® Astrocyte Medium (Liao et al., 2016; Mytych et al., 2015; Rao et al., 2015) or Ch-N-Rm medium for 0, 4 and 7 days and Neurospheres Fast medium for 0, 24 and 72 hours were fixed with paraformaldehyde for 20 min, followed by Triton X-100 (0.1%) permeabilization and 5% bovine serum albumin (BSA) blockage. Cells were then incubated overnight with primary antibodies against DAT, TH, GFAP, S100β, b-TUB III, MAP2, NFL, VAChT, and ChAT proteins (1:500). After exhaustive rinsing, we incubated the cells with secondary fluorescent antibodies (DyLight 488 and 595 donkey anti-rabbit and -goat and -mouse, Cat DI 2488 and DI 1094, respectively) 1:500. The nuclei were stained with Hoechst 33342 (1 µM, life technologies) and images were acquired on a Floyd Cells Imaging Station microscope.
Flow cytometry (FC) analysis of astrocytic, dopaminergic, and cholinergic markers
Flow cytometry acquisition was used to determine the percentage of GFAP/S100β, DAT/TH, ChAT/ VAChT double-positive cells, according to previous reports (Moghaddam et al., 2017, Tcw, J et al., 2017, Fathi et al., 2018). Cells treated with MCm, Neuroforsk, astrocyte, or Ch-N-Rm medium at days 0, 4, and 7 were detached with 0.25% trypsin- EDTA 1mM and fixed in suspension with paraformaldehyde (overnight). After washing, cells were simultaneously incubated with GFAP, S100β, DAT, TH, ChAT, and VAChT primary antibodies (1:500) at 4°C overnight. Cell suspensions were washed and incubated with DyeLight 594 donkey anti-goat and DyeLight 488 donkey anti-rabbit antibodies (1:500). Finally, cells were washed and re-suspended in PBS for analysis on a Canto cytometer (Beckman coulter). Ten thousand events were acquired and the acquisition analysis was performed using FlowJo 7.6.2 Data Analysis Software. Positive staining was defined as the fluorescence emission that exceeded levels of the population stained with the negative control (only secondary antibodies staining).
Intracellular calcium imaging
The cytoplasmic Ca2+ concentration ([Ca2+]i) was measured. Briefly, DALNs, ChLNs, and ALCs cultured in Neuroforsk, Ch-N-Rm, and Astrocyte medium respectively for 0, 4, and 7 days were transferred to a bath solution (NBS; in mM: 137 NaCl, 5 KCl 2.5 CaCl2, 1 MgCl2, 10 HEPES, pH 7.3, and 22 glucose) containing a Ca2+ sensitive indicator (2 µM Fluo3-AM, an acetoxymethyl ester form of the fluorescent dye Fluo-3; Thermo Fisher Scientific Cat F1242) for 30 min at room temperature and then washed five times. The intracellular Ca2+ transients were evoked by dopamine-hydrochloride (DA, 1mM final), acetylcholinesterase (ACh, 1 mM final), and glutamate (GLUT, 100 µL final). The amplitudes of the Ca2+ related fluorescence transients were expressed relative to the resting fluorescence (ΔF/ F) and were calculated by the formula ΔF/ F= (Fmax-Frest)/(Frest-Fbg) according to Pap et al., 2009 . The Image J program (https://imagej.net/) was used for the calculation of the fluorescence intensities as previously published by Pap et al., 2009 and Mendivil-Perez et al., 2019 [11, 12].
Statistical analyses were conducted using the Student-t analysis or one-way ANOVA followed by Bonferroni posthoc comparison calculated with the GraphPad Prism 6 scientific software (GraphPad, Software, Inc. La Jolla, CA, U.S.A.). Statistical significance was accepted at *p<0.05; **p<0.01; ***p<0.001.