Study design
The study made use of human BMSCs collected from healthy subjects. In order to determine the best concentration of HPGF-C18 for BMSCs growth passage 2 BMSCs were cultured in various concentrations of HPGF-C18 and compared to BMSCs cultured in 20% FBS, the concentration that is used in our laboratory for the production of BMSCs following GMPs [22]. After the best concentration of HPGF-C18 was identified, bone marrow aspirates were obtained from 5 healthy subjects and BMSCs were isolated and cultured for 4 passages, the cellular expansion, surface markers, and immunosuppressive activities were compared between HPGF-C18 cultured BMSCs and 20% FBS cultured BMSCs. We also measured a variety of cytokines and growth factors in the supernatant and compared the gene expression profiles and microRNA profiles of the BMSCs cultured under both conditions. These studies were approved by an NHLBI committee on the use of human subjects in research.
HPGF-C18
HPGF-C18 was prepared from 52 outdated units of apheresis platelets each of which contained approximately 4 × 1011 platelets (GwoWei Technology Co, Ltd., Taipei, Taiwan). The pooled platelets were solvent/detergent (S/D)-treated (1% tri-n-butyl phosphate and 1% Triton X-45), extracted with oil, purified by C18 hydrophobic interaction chromatography and sterile filtered as previously described [23].
Determination of the optimal HPGF-C18 concentration for BMSC growth
Cryopreserved passage 2 BMSCs were cultured to determine the best concentration of HPGF-C18 for BMSC growth. These passage 2 BMSCs were isolated from marrow aspirates of 3 healthy subjects using media supplemented in 20% FBS and were cryopreserved in 5% DMSO and 6% HES and stored in the vapor phase of liquid nitrogen. The passage 2 BMSCs were thawed, washed and suspended in alpha MEM with 2 mM glutamine (Lonza, Walkersville, MD), supplemented with 10 µg/mL Gentamicin and 20% lot-selected FBS (Hyclone, Thermo Fischer Scientific, Waltham, MA) or 5, 10, 15 and 20% HPGF-C18 and were seeded on T75 flasks at a density of 3000 cells/cm2. The cells were cultured and harvested with 5 mL TrypLE Express (Invitrogen, Life Technologies) when they reached 70–80% confluence and were re-seeded on T75 flasks at a density of 3000 cells/cm2 for expansion. Passage 4 cells were harvested and surface marker of passage 4 BMSCs was compared. The number of BMSCs at passage 3 and 4 was manually counted and the population doubling (PD) for each passage was calculated.
Culture of BMSCs from marrow aspirates
The marrow aspiration and BMSC culture were performed according to standard operating procedures (SOP) established in our laboratory [22]. After obtaining informed consent, marrow was collected from the posterior iliac crest of 5 healthy donors. A total of 5–10 mL of marrow was collected in Bone Marrow Prep Syringes (Pharmacy Department, NIH, Bethesda, MD) and then washed with 2.5 × volume of HBSS (Lonza, Walkersville, MD). A single cell suspension was made with BMSC culture media (BMSC CM) [alpha MEM with 2 mM glutamine (Lonza), supplemented with 20% lot-selected FBS (Hyclone, Thermo Fischer Scientific, Waltham, MA) or 10% HPGF-C18 and 10 µg/mL gentamicin] and was plated at a density of 2 × 105/cm2 in T-75 flasks (Corning Life Sciences, Corning, NY) and incubated at 37 °C in 5% CO2. Non-adherent cells were removed after 24 h; the media was changed every 3 days until the colonies reached 70–80% confluence.
The primary BMSCs were washed with 10 mL HBSS twice and lifted with 5 mL TrypLE Express (Invitrogen, Life Technologies, Grand Island, NY), the cells were then centrifuged at 406×g for 10 min and the cell number was counted. The cells harvested at this stage were designated as Passage 1. The BMSCs were then seeded on plastic surface at a density of 3000 cells/cm2, cultured and harvested as described above when they reached 70–80% confluence.
Passage 4 cells were harvested for evaluation of surface marker expression, suppression on the proliferation of mixed lymphocytes, global gene expression profiling, and microRNA expression analysis. Cytokine and growth factor levels were measured in the supernatant of passage 4 BMSCs. The number of BMSCs at passage 3 and 4 was manually counted and the population doublings (PDs) for each passage was calculated. Cumulative PDs were calculated in relation to the number of cells at the first passage.
Primary colony-forming efficiency (CFE) enumeration
Bone marrow aspirates were diluted in culture media with 20% FBS or 10% HPGF respectively, and then plated at a density of 1 × 105 per T25 flask and cultured for 13 days without changing culture medium. The colonies were fixed with methanol for 30 min and stained with saturated methyl-violet water solution for 20 min. Colonies were observed under low magnitude light microscope field (25×). Colonies containing 50 or more cells of fibroblastic morphology were counted, and CFE (number of BMSC colonies/plating nucleated cells) was calculated.
Surface marker expression
BMSC surface markers were analyzed by flow cytometry. The cells were incubated with antibodies CD90-FITC, CD73-PE, CD146-PE, CD106-APC, CD45-FITC, CD14-PE, CD19-FITC, CD34-APC, HLA-DR-APC (BD Bioscience, San Diego, CA), CD11b-FITC and CD105-APC (eBioscience, San Diego, CA) for 20 min at 4 °C, washed on Lyse Wash Assistant (LWA, BD Bioscience) and acquired 30,000 events on a FACSCanto (BD Bioscience). The data were analyzed using FACSDiva 6.0 software (BD Bioscience).
Cytokine and growth factor analysis of BMSC culture supernatant
The cytokine and growth factor concentrations in BMSC culture supernatant were evaluated using SearchLight Protein Array Analysis (Aushon Biosystems, Billerica, MA). Culture supernatant was collected, centrifuged for 10 min at 1400 rpm to remove cell debris and then stored at − 80 °C. The supernatants of BMSC from 5 healthy donors were evaluated for IL4, IL6, IL8, IL10, KGF, LIF, PEDF, TGFB1, FGF2, HGF, PDGFBB, VEGF, SDF1, PLGF, ANG2, and Endoglin.
Mixed lymphocyte reaction (MLR)
The immunosuppressive properties of BMSCs were compared using MLR assay (SAIC-Frederic, Frederic, MD). Ficoll-separated peripheral blood mononuclear cells were plated in 96-well plates at 1 × 105 responders per well. Responders were co-cultured with 2500 cGy irradiated stimulator peripheral blood mononuclear cells at a concentration of 1 × 105 cells per well. BMSCs cultured by either 10% HPGF-C18 or 20% FBS were added at concentrations of 1 × 104 and 4 × 104 cells/well. Culture plates were incubated for 6 days in a humidified 5% CO2 incubator at 37 °C. On the day of harvest, 0.5 μ Ci of 3H-thymidine (3H-TdR) was added to each well for 4 h with lymphocyte proliferation measured using a liquid scintillation counter. The effect of BMSCs on MLR was calculated as the percentage of the suppression compared with the proliferative response of the positive control without BMSC, where the positive control was set to 0% suppression. The experiments were performed three times for each variable described.
Microarray gene expression analysis
Total RNA was extracted using miRNeasy Mini Kit (Qiagen, Hilden, Germany) and assessed using Nano Drop 2000 (Thermo Scientific, Wilmington, DE). Microarray expression experiments were performed on 4 × 44 K Whole Human Genome Microarray (Agilent technologies, Santa Clara, CA, USA) according to our protocols [24]. Generally, 0.5 µg of BMSC RNA was labeled with Cyanine 5-CTP and Universal Human Reference RNA (Stratagene, Santa Clara, CA, USA) was labeled with Cyanine 3-CTP using a Quick Amp Labeling kit (Agilent). After purification, 825 ng of labeled cRNA from BMSC and reference RNA was pooled, fragmented and then hybridized on 4 × 44 K microarrays for 17 h at 65 °C. Images of the arrays were acquired using a microarray scanner G2505B (Agilent technologies) and image analysis was performed using feature extraction software version 9.5 (Agilent Technologies). The Agilent GE2-v5_95 protocol was applied using default settings.
RT-qPCR analysis of gene expression
For verification of the gene expression profiling results, RT-qPCR gene expression analysis was performed using custom made PCR arrays (Qiagen). The data analysis was conducted using the ΔΔCt method. HPRT1 was used as housekeeping gene and its Ct values were used to normalize the data. The normalized ΔCt for each gene of interest (GOI) was calculated by deducting the averaged Ct of HPRT1 from the Ct of each GOI: ΔCt = (CtGOI − CtHPRT1). The ΔΔCt for each GOI was calculated by deducting the average ΔCt of GOI in the HPGF group from the ΔCt of each GOI in the FBS group: ΔΔCt = average ΔCt (HPGF group)—average ΔCt (FBS group). The fold-change of each GOI in HPGF group compared to the FBS group was calculated as: fold-change = 2(−ΔΔCt).
MicroRNA expression analysis
The expression of MicroRNAs was measured by using the Human miRNome miScript miRNA PCR Array (Qiagen) following the manufacturer’s instructions. In brief, 1 µg of RNA was reverse-transcribed with miScript reverse transcription (RT) master mix for 60 min at 37 °C, then heated at 95 °C for 5 min and diluted with water. The diluted RT product was loaded into miRNA PCR Array for amplification using the following parameters, 95 °C for 1 min, then 40 cycles of amplification: 94 °C for 15 s, 55 °C for 30 s, 70 °C for 30 s, followed by the dissociation curve stage. The data analysis was conducted using ΔΔCt method. RNU6-2 was used to normalize the data. The normalized ΔCt for each miRNA of interest (MOI) was calculated by deducting the averaged Ct of RNU6-2 from the Ct of each MOI: ΔCt = (CtMOI − CtRNU6-2). The ΔΔCt for each MOI was calculated by deducting the average ΔCt of MOI in the HPGF group from the ΔCt of each MOI in the FBS group: ΔΔCt = average ΔCt (HPGF group)—average ΔCt (FBS group). The fold-change of each MOI compared to the FBS group was calculated as: fold-change = 2(−ΔΔCt).
Data processing and statistical analyses
Global gene expression analysis was performed according to a standard procedure. Raw data was uploaded into mAdb database (http://madb.nci.nih.gov/) and then imported into BRB-ArrayTools [25] (http://linus.nci.nih.gov/BRB-ArrayTools.html). Tests for differences between HPGF-C18 and FBS were conducted for individual genes using paired two-sided t tests, considering P values of < 0.001 as significant. In a class prediction model, genes significantly different between the classes (HPGF-C18 vs FBS) at 0.001 significance level were used for class prediction, and the leave-one-out cross-validation method was used to compute misclassification rate. The Benjamini and Hochberg method was used to estimate the false discovery rate. Gene set enrichment analysis (GSEA) was conducted by following the instructions of Broad Institute [26] (http://www.broadinstitute.org/gsea/index.jsp).