Isolation of Wharton Jelly (WJ)-MSC spheres and evaluation of their growth factor secretion capability
WJ-MSCs were subjected to suspension culturing conditions to form WJ-MSC spheres as described in our previous study [15]. Briefly, the WJ-MSCs were separated from fresh umbilical cord samples after the necessary consent forms were filed by parents. The WJ-MSCs were seeded in Poly-HEMA-coated plates and incubated under hypoxic condition containing 5% O2 (New Brunswick, Germany) to support the forming of spheres. Next, the formed spheres were cultured in serum-free media for 48 hours. The collected supernatant was concentrated using Vivaspin® 500 (Sartorius, Germany) with centrifugal force. Concentrations of growth factors including Angs, Wnts, SCF, and Flt3-L were measured using ELISA method (MyBioSource, USA) according to the manufacturer's protocol.
Isolation and cultivation of HSCs
After the necessary consent forms were filed, HSCs were isolated from fresh umbilical cord blood using ficoll gradient protocol as described previously [31]. CD34+ cells were positively selected using magnetic-activated cell sorting (MACS) according to the kit instructions (Miltenyi Biotec, Germany). The purity of CD34+ cells and their CD38, CD90, and CD45RA expression levels were analyzed using flow cytometry (Life Technologies Attune Nxtm, USA). The used antibodies and fluorochromes are as follows: CD34 APC/PE, CD38 FITC, CD45RA PE, and CD90 FITC (BD, Biosciences, Germany). The cells were mixed with serum-free stem span media (Stem Cell Technologies, USA) containing a cytokine cocktail (Flt3-L, TPO, and SCF) and then were seeded on culture plates. The culture medium was refreshed twice per week. This group of HSCs (expanded in cytokines without feeder layer condition) was named “Cyto”.
Co-culturing HSCs with adherent MSCs or MSC spheres under hypoxia or normoxia
MSC spheres were transferred into culture plates and the mixture of HSCs with stem span medium containing cytokines (named Sph-MSC+ Cyto) or without cytokines (named Sph-MSC) were added to the plates. Co-culturing HSCs with adherent monolayer MSCs (named MSC) was considered as another experimental group. All of the mentioned culture plates were incubated at 37°C for 10 days (20% O2 and 5% CO2). The plates containing MSC spheres with HSCs without cytokine addition and the plates containing adherent MSC with HSCs incubated under reduced O2 pressure (5% O2) to induce hypoxia (named Sph-MSC+ Hyp and MSC+ Hyp, respectively) were considered as other experimental groups. The culture medium was exchanged twice per week. After 10 days, MSC spheres were separated from HSCs using filters and TNC counting was performed using trypan blue staining and hemocytometer . Then the number of CD34+/CD38- cells was also calculated based on the TNC counting and their defined percentage by flow cytometry. The fold change in TNC and CD34+/CD38- cell number was calculated by comparing with the initial number of the seeded cells.
CFU-assay
On the 10th day of the experiment, the expanded cells of Cyto, MSC, MSC+ Hyp, Sph-MSC, Sph-MSC+ Hyp, and Sph-MSC+ Cyto groups were cultivated in iscove’s MDM medium (Sigma, Germany) containing 1% methylcellulose, and they were incubated for 14 days. Clusters containing 50 or more cells were counted to calculate CFU capacity. Granulocyte macrophage-colony forming units (CFU-GM), erythroid burst/colony-forming units (BFU-E/CFU-E), and granulocyte erythrocyte monocyte megakaryocyte-colony forming units (CFU-GEMM) were scored and counted in triplicate by laboratory experts. The CFU fold change of expanded cells was estimated by comparing it with the CFU of fresh unexpanded cells.
LTC-IC-assay
LTC-IC assay was conducted on an irradiated feeder. The expanded CD34+ cells were seeded in MyelocultMyelocM5300 (Stem Cell Technologies, USA) and the medium was changed twice a week. After 6 weeks, the viability of harvested cells was determined using trypan blue exclusion dye, and two thousand cells were subjected to CFU-assay according to the previously mentioned procedure. The enumerated colonies were considered as LTC-IC. The LTC-IC fold change of the expanded cells was estimated in comparison to the LTC-IC of fresh unexpanded cells.
Real-time reverse transcription polymerase chain reaction (qRT–PCR)
qRT-PCR was performed to measure the expression levels of NS and Nfix as self-renewal genes as well as CXCR4 and VLA-4 as homing genes using Syber green dye. Briefly, 6 µl of Syber green master mix (Takara, Japan), 1 µl of synthesized cDNA (BIONEER, South Korea), and 10 µmol/l of specific primers were mixed and the total volume was adjusted to 12µl. The primer sequences were as follows: CXCR4 F: 5′-CTATTGAACCCCATCCTGCT-3′ R: 5′- TCCACGATGAATGCTCGCTTT-3′, VLA-4 F: 5′-ATGTTGCGCATGTTCTACTG -3′ R: 5′-AGCCTTCCACATAACATATGAG-3′, NS F: 5′-CTGATGCTGGAGTTGGATGC -3′ R: 5′- CATTGATCACGTTGAAGGC-3′, Nfix F: 5′-CATTCTTGTCGCCGTCCTG-3′ R: 5′-TCCGTTCCGCAAGCATCAC-3′ and Β-actin F: 5′-GATACCGCAAATGTGACACG-3′ R: 5′-GGGCTCACAGGAACAGTTCT-3′. qRT-PCR was performed using the LightCycler® 96 System (Roche, Bavaria, Germany) in triplicate. The specificity of PCR amplification was analyzed by melting curve. The relative expression of the mentioned genes in comparison with the expression of β-actin was determined using the 2 (-∆∆CT) method. PCR conditions were as follows: initial denaturation at 95°C for 15min followed by 40 cycles of 20s at 95°C, 60s at 60°C for VLA-4, NS, Nfix, and β-actin, and 63°C for CXCR4.
Immunophenotyping for EPCR, CXCR4, and VLA-4 expression
After 10 days of HSCs expansion, the expression of EPCR, CXCR4, and VLA-4 was assayed using flow cytometer (Life Technologies Attune Nxtm, USA). The cells were incubated and stained with an optimized amount of related antibodies at room temperature for 30 minutes in the dark. Then, the cells were checked by the flow cytometer instrument.
Western blot analysis
Total protein of expanded HSCs was extracted using lysis buffer (Roche, Germany). After electrophoresis of the protein samples on 12% SDS-polyacrylamide gel, they were transblotted onto polyvinylidene fluoride membrane (Roche, Germany) using semi-dry blotter (Bio-Rad, USA) in an optimized voltage for an appropriate duration. Then, the membranes were incubated with anti-NS (Abcam, UK), anti-Nfix (Merck, Germany) and anti- β-actin (Sigma, USA) antibodies at optimized conditions (for anti-NS antibody: 1:3000 dilution, 3 hours at room temperature with shaking, for anti-Nfix antibody: 1:5000 dilution, 3 hours at room temperature with shaking, and for anti-β-actin antibody: 1:2000, 2 hours at room temperature with shaking). After immersing the membranes in the diluted (1: 1000) secondary antibody (Abcam, UK), ECL substrate solution was poured on the membranes. Furthermore, images were captured using a gel doc imager (Bio-Rad, USA) and the protein expression levels were semi-quantified using Image Lab software.
Statistical analysis
Parametric ANOVA test was used for data analysis and the p-value of P<0.05 was considered significant. The experiments were carried out in duplicate in 3 independent experiments.