MIAMI Cell Isolation
Whole bone marrow was obtained from the iliac crest of a 20 year old living male donor (Lonza Walkersville, Maryland; MIAMI #3515), and were handled and processed following the guidelines for informed consent set by the University of Miami School of Medicine Committee on the Use of Human Subjects in Research. As previously described , isolated whole bone marrow cells were plated at a constant density of 1 × 105 cells/cm2 in DMEM-low glucose media, containing 3% fetal bovine serum (FBS, Hyclone Waltham, MA, Lot#30039), 20 mM ascorbic acid (Fluka/Sigma St. Louis, MO, #49752), an essential fatty acid mixture (Sigma St. Louis, MO; 12.9 nM arachidonic acid, (#A9673), 1.12 μM cholesterol (#C3045), 290 nM DL-alpha tocopherol-acetate (#T3376), 85.9 nM myristic acid (#M3128), 69.4 nM oleic acid (#01383), 76.5 nM palmitic acid (#P5585), 77.1 nM palmitoleic acid (P9417) and 68.9 nM stearic acid (#S4751) (modified from ) and antibiotics (100 U/mL penicillin, 0.1 mg/mL streptomycin) (Gibco Carlsbad, CA, #15140) on 10 ng/ml fibronectin (Sigma St. Louis, MO, #F2518) coated flasks (Nunclone Rochester, NY). Whole bone marrow cells, containing adherent and non-adherent cells, were incubated at 37°C under hypoxic conditions (3% O2, 5% CO2 and 92% N2). Seven days later, half of the culture medium was replaced. Fourteen days after the initial plating, the non-adherent cells were removed. Pooled colonies of adherent cells were rinsed with PBS and plated at low density for expansion (100 cells/cm2) in 75 cm2 fibronectin coated flasks.
MIAMI Cell Culture Conditions
MIAMI cells were grown in expansion media consisting of DMEM-low glucose (as described above) in low oxygen conditions (3% O2, 5% CO2 and 92% N2). Media was changed every 2-3 days and the cells were detached and pelleted using trypsin (Gibco Carlsbad, CA, #25300) upon reaching ~60% confluency. Peleted cells were resuspended in media and plated in 10 ng/ml fibronectin (Sigma St Louis, MO, #F2518) coated flasks (Nunclon, Rochester, NY) at 100 cells/cm2. Prior to RNA isolation, adherent cells were rinsed 2× with PBS. MIAMI cells expanded for 3 passages were characterized using flow cytometry and were positive for; MHC1, CD29, CD81, CD90 and 50% positive for CD63, and negative for; MHC2, HLA-DR, CD49, CD109, CD54, CD56, CD36 (data not shown).
RS-1 Cell Culture Conditions
Human marrow stromal cells (hMSC, Donor#7081, 22yo male) were obtained from the laboratory of Dr. Darwin Prockop, Director, Texas A&M Health Science Center College of Medicine Institute for Regenerative Medicine. Bone marrow (BM) cells were isolated from human donors according to guidelines on the Use of Human Subjects in Research as described by all commercial vendors. The hMSC were cultured in Alpha-Minimum Essential medium (αMEM) with L-glutamine, but with no ribonucleosides or deoxyribonucleosides (Invitrogen/Gibco Carlsbad, CA, #12561-056), supplemented with 16.5% FBS (Hyclone Waltham, MA, #31752), 2 mM GlutaMAX (#35050) and antibiotics (Gibco Carlsbad, CA, #15140). To enrich for RS-1 cells, hMSC(P1) were plated at 37°C under normoxic conditions (21% O2, 5% CO2 and 74% N2) onto 10 ng/ml fibronectin (Sigma St. Louis, MO, #F2518) coated flasks (Nunclon Rochester, NY) overnight. The cells were detached using trypsin (Gibco Carlsbad, CA #25300) and seeded at 50 cells/cm2. RS-1 enriched hMSCs were detached at 30-40% confluency and re-plated at low density (50 cell/cm2) . RS-1 cells were harvested for RNA isolation at each passage. MSC derived RS-1 cells, passage 2, were positive for CD29, CD90, CD105 and CD73 as determined by Tulane University Center for Gene Therapy (hMSCs #7801). RS-1 cells derived in our facilities, passage 3, were positive for; MHC1, CD81, CD90, CD29 (20%), CD63 (45%) and negative for; MHC2, HLA-DR, CD49, CD109, CD54, CD56, CD36, as determined using flow cytometry analysis (data not shown).
MSC Cell Culture Conditions
Human mesenchymal stem cells (MSC) derived from the iliac crest were purchased from Lonza (Walkersville, Maryland (PT-2501: 21yo female)). Bone marrow (BM) cells were isolated from human donors according to guidelines on the Use of Human Subjects in Research as described by all commercial vendors. The MSC were plated at 6,000 cells/cm2 in DMEM-high glucose media (Gibco Carlsbad, CA, #31053) supplemented with 15% FBS (Hyclone Waltham, MA, #30039), ascorbic acid, antibiotics and essential fatty acids (as described above), and expanded at 21% O2, 5% CO2 and 92% N2. The entire culture media was changed every 3-4 days and the cells were detached and replated every 7 days . MSC purchased from Lonza were positive for CD105, CD166, CD29, CD44, and negative for; CD14, CD34 and CD45, as determined by flow cytometry (Lonza Wlkersville, Maryland (Document # TS-PT-212-8 06/09).
Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) treatment of MIAMI cells was performed using 20 ng/mL each of EGF (#AF-100-15) and bFGF (Peprotech Rocky Hill, NJ, #AF-100-18B) alone or in combination. The pre-treated cells were detached using trypsin and replated after day 5, followed by a second 5 day pretreatment period. Pre-treated cells were grown in expansion media under expansion conditions (3% O2, 5% CO2 and 92% N2). Media was changed every 2-3 days and the cells were split using trypsin (Gibco Carlsbad, CA, #25300) upon reaching ~60% confluency.
For endothelial differentiation, MIAMI cells were plated at 20,000 cells/cm2 in 6 well plates (Nunclone Rochester, NY) in DMEM-low glucose media, containing 100 μM Ascorbic Acid, antibiotics, essential fatty acids, angiogenic growth factor cocktail [Sigma St. Louis, MO: 10 ng/ml bFGF, 10 ng/ml EGF, 10 ng/ml IGF; R&D Systems, Inc. Minneapolis, MN: 100 ng/ml VEGF], 100 nM Hydrocortisone, in atmosphere of 21% O2, 5% CO2 and incubated at 37°C for 21 days, with media changes every 5 days. Cells were harvested at day 10 and 21 and evaluated by RT-qPCR for the endothelial marker CD 31.
Total RNA Sample Preparation and cDNA Synthesis
MIAMI cells were detached (Trypsin) and centrifuged to form a cell pellet. RNA was isolated using the RNAqueous® -4PCR kit (Ambion Austin, TX, #AM1914) according to manufacturer's directions. Total RNA was quantified on the Nanodrop ND-1000 Spectrophotometer (Nanodrop Wilmington, DE). Reverse transcription of 2 μg total RNA to cDNA was done with random hexamer primers using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems Foster City, CA, #4368814). The cDNA was diluted 1:20 (Nuclease-Free Water: Gibco#10977-015) to a final cDNA concentration of 5 ng/μl, aliqoted, and stored at -20°C until next use. Only RNA with a 260/280 ratio between 1.9-2.0 was used for PCR analysis.
Quantitative real-time RT-PCR (RT-qPCR)
Quantitative real-time PCR (RT-qPCR) was done using 10 μl of 1:20 diluted cDNA (50 ng) on the Mx3005P Multiplex Quantitative PCR System (Stratagene#401513) using RT-qPCR SYBR GREEN Reagents (Brilliant® II SYBR® Green QPCR Master Mix, Agilent Technologies) with ROX reference dye. Forward and reverse primer pairs were reconstituted in Nuclease Free Water (Gibco#10977-015). A 2 μM stock solution containing both forward and reverse primer pairs was mixed and stored at -20°C. A final concentration of 160 nM forward and reverse primer pairs was used for each RT-qPCR reaction. The cycling conditions were as follows: an initial 95°C for 10 minutes, followed by 40 cycles of 95°C for 30 sec, 58°C for 30 sec, 72°C for 15 sec. MxPro-Mx3005P v4.10 software was used to determine the CP for each amplification reaction. Results were exported to Microsoft Excel for analysis.
Analysis of RT-qPCR data
All of the corresponding RT-qPCR data was analyzed using the ΔΔCP method [13
] and normalized against one negative control, and two reference genes (housekeeping genes).
The crossing point (CP) is defined as the point at which the fluorescence rises appreciably above the background fluorescence. The 'Fit Point Method' was used by the Mx3005P software to determine the CP for each reaction. The control sample was set to a value of "1" in all cases and error bars in the respective figures are displayed as standard deviation. The number of independent experiments is designated as "N" with 2-3 individual data points collected per experiment.
Eight genes were tested for normalization [beta-actin (ACTB, NM_001101), beta-2-microglobulin (B2M, NM_004048), eukaryotic translational elongation factor 1 alpha (EF1α, NM_001402), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, NM_002046), Hypoxanthine phosphoribosyltransferase 1 (HPRT1, NM_000194), ribosomal protein L13a (RPL13a, NM_01242), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide variant 1 & 2 (YWHAZ, NM_003406 & NM_145690), and ubiquitin C (UBC, NM_021009)]. A list of primer pair sequences used are in Table# 1.
Determination of Primer Pair Efficiency
The determination of each genes' primer pair efficiency (E) for RT-qPCR was calculated using this equation: E = 10^(-1/m) . The slope (m) was calculated by plotting the cycle number crossing point (CP) calculated during the exponential phase of the amplification plot (PxPro-Mx3005P v4.10 software) against the total cDNA concentration. Concentrations of cDNA ranged from 50-1 ng per reaction. The percent efficiency (%E) was also calculated: %E = (E-1)*100. N = 4 (2-3 data points per experiment) (Additional file # 1).
Construction of species-specific primer pairs
In order to create species-specific primer pairs that detect only human mRNA sequences or only rat mRNA sequences within a human-rat cDNA library, the corresponding human and rat mRNA sequences must have a unique region of at least 60 bp or more. Using the human and corresponding rat FASTA mRNA sequences for EF1α, RPL13a and YWHAZ, we used Blast-n http://blast.ncbi.nlm.nih.gov/Blast.cgi to compare the sequences. EF1α had 99% sequence coverage (100% identity) between the human and rat mRNA sequences. RPL13a had 57% sequence coverage (87% identity) and YWHAZ transcript variants 1 and 2 had 92% - 63% sequence coverage (100% identity). Therefore, RPL13a and YWHAZ both were candidate human species-specific normalization genes while EF1α did not have a region containing a unique sequence (≥60 bp) in order to create primer pairs. Human species-specific primer pairs were constructed for the 2 normalization genes; RPL13a and YWHAZ and for 3 target genes; stanniocalcin-1 (STC-1), tumor necrosis factor, alpha-induced protein 6 (TSG6), and latent transforming growth factor binding protein 2 (LTBP2). NCBI Primer-BLAST http://www.ncbi.nlm.nih.gov/tools/primer-blast/ was used for primer pair sequence construction  using the species-unique mRNA sequences (FASTA format). Gradient PCR was used to determine optimum annealing temperature. All human and rat specific primer pairs were validated with RT-qPCR using cDNA from human MIAMI cells H3515(3) or rat hippocampal organotypic cultures either separately or in combination. All primer pairs produced 1 species-specific amplicon, with minimum off-target amplification. This was determined by the melting curve of each amplification reaction (Additional File # 2) and agarose gel electrophoresis (data not shown). Approximately 3-5 primer pairs were tested per human or rat species-specific normalization or target gene. All RT-qPCR results were normalized against a negative control, and the 2 normalization genes hRPL13a and hYWHAZ (human), or rRPL13a (rat). Using this same method rat specific primer pairs were also constructed for RPL13a, IGF1, IGFBP3, and IGFBP5.
Model of ex vivo global cerebral ischemia for cross-species RT-qPCR analysis
All animal experiments were performed according to approved guidelines established by the University of Miami IACUC. The rat hippocampal organotypic slice preparation has been described in detail [19, 24]. Briefly, 400 μm brain slices were obtained from rat pups of either sex between postnatal days 9 and 10. Slices were cultured for two weeks in a medium consisting of 25% heat inactivated horse serum, 50% minimal essential medium, and 25% Hank's balanced salt solution, 5.5 mg/mL D-glucose and 1 mmol/L glutamine. For ischemia we used an established model consisting of combined oxygen and glucose deprivation ([19, 24]) during 40 mins. For OGD, oxygen is replaced with nitrogen and glucose with sucrose. MIAMI cells were pre-treated with bFGF and EGF (7 days: 50 ng/ml) prior to injection in the CA1 region of the hippocampus (7,500 cells/μl per injection (3 injections)). One hour after OGD induction and 24 hours after OGD total RNA was isolated from rat hippocampal organotypic slice cultures (described in ) with or without injected MIAMI. As described previously, 2 μg of total RNA was used for cDNA synthesis. RT-qPCR analysis was done using 5 μl of undiluted cDNA. Human and rat specific primer pairs are designated by (h) and (r) respectively (Table# 2: human specific primer pairs are designated by (*)).
Only data sets containing N ≥ 3 independent experiments (2-3 samples per condition per experiment) were used for statistical analysis. A One-way ANOVA followed by Tukey's post-hoc analysis was used to calculate statistical significance between conditions using GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego, CA, http://www.graphpad.com. All error bars represent standard deviation.