Umbilical cord blood MSC isolation and culture
UCB samples were collected from 45 full-term delivery cases with previous consent from the mothers according to the Institute's human ethical committee guidelines. The UCB samples collected were coded as hUCB-MSC SCB1 to SCB45. The samples used for mononuclear cell (MNC) isolation alone were coded as hUCB MNC01 to MNC03. Clinical parameters for the mother and baby were also noted for latter correlation with the yield of MSCs. The parameters recorded were the gestation period, UCB blood volume collected, and body weight of the baby (see Table S1 in Additional file 1).
UCB units were collected into 50 ml sterile tubes containing anticoagulant (acid citrate dextrose solution). All samples were processed within 4 to 6 hours from collection. Blood was diluted in 1× PBS in a 1:1 ratio and MNCs were isolated by density gradient centrifugation using Percoll gradient (GE Life Sciences, Piscataway, NJ, USA) . MNCs obtained after the Percoll gradient separation were washed twice in 1× PBS and resuspended with CD34 antibody-conjugated beads (Miltenyi Biotech, Cologne, Germany) and were incubated for 30 minutes at 4°C followed by selective depletion of CD34-positive cells. The CD34-depleted population was collected and further resuspended in proliferation medium consisting of Iscove's modified Dulbecco's medium (IMDM) (Gibco, Life Technologies, Carlsbad, CA, USA) with 20% FBS (Hyclone Laboratories, Logan, UT, USA), 20 ng/ml fibroblast growth factor (FGF)-2 (Chemicon, Millipore, Billerica, MA, USA), 2 mg/ml heparin (Sigma, St.Louis, MO, USA), 1% penicillin/streptomycin (Gibco) and was plated in T25 flasks at a density of 5 × 106 cells/ml. After 24 hours of incubation at 37°C with 5% CO2, the nonadherent cells were washed off and the attached cells were expanded further with medium being replenished every alternate day. All differentiation protocols were carried out with MSCs between passages 3 and 4. Immortalized hUCB-derived MSC line RCB 2080 procured from RIKEN (Tsukuba, Ibaraki, Japan) was cultured in the same medium and was used as a control. Cells were passaged upon reaching 70 to 80% confluence using 0.05% Trypsin-ethylenediamine tetraacetate (Gibco) and were replated or cryopreserved for subsequent experiments.
Immunophenotyping of human umbilical cord blood MSCs
Characterization of hUCB-MSCs was carried out by immunophenotyping using both MSC-positive and MSC-negative surface markers. Briefly, 60 to 80% confluent flasks of expanded MSCs were trypsinized, followed by washing with 1× PBS and fixed in 4% paraformaldehyde for 15 minutes at 4°C. Cells were then incubated with FITC/PE-conjugated CD73 (1:100; BD Pharmingen, San diego, CA, USA), CD44 (1:100; BD Pharmingen), CD45 (1:100; BD Pharmingen), CD105 (1:100; BD Pharmingen) and CD29 (1:100; BD Pharmingen) primary antibodies in the dark at 4°C for 1 hour and finally resuspended in 1× PBS containing 3% BSA for fluorescence-activated cell sorting analysis. To avoid nonspecificity and background staining, appropriate isotype secondary antibody controls (mouse IgG-Att488 and mouse IgG-PE) and cell-only controls were used. A total of 10,000 events were analyzed using a BD FACS Aria Flow Cytometer (BD Biosciences, San jos, CA, USA).
For immunostaining, hUCB-MSCs were grown on coverslips in 24-well plates for 48 hours for proliferation and for 5 to 12 days for differentiation. Immunofluorescence analysis of cells was carried out as described previously . Briefly, cells were fixed in 4% paraformaldehyde (Sigma), followed by blocking in 0.4% blocking solution for detecting nuclear antigens,0.2% blocking solution for cytoplasmic antigens and 0.1% blocking solution for surface antigens. The cells were then incubated with the following primary antibodies against β-III-tubulin (1:200; Chemicon), nestin (1:50; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, Iowa, USA), 5-bromo-2-deoxyuridine (BrdU) (1:100; Abcam Cambridge, UK), Sox2 (1:500; Abcam), Musashi (1:100; Chemicon), Vimentin (1:100; Developmental Studies Hybridoma Bank), CD105 (1:100; BD Pharmingen), CD29 (1:100; BD Pharmingen), CD44 (1:100; BD Pharmingen), CD73 (1:100; BD Pharmingen) and CD45 (1:100; BD Pharmingen). This incubation was followed by nuclear staining with 4',6-diamidino-2-phenylindole (DAPI) (Sigma).
Cells were examined for epifluorescence following incubation with appropriate secondary antibody conjugated to Cy3/FITC (1:400; Jackson Immunoresearch, Philadelphia, PA, USA) in an upright fluorescent microscope (Olympus BX61, Olympus, Tokyo, Japan) and images were captured using a cooled CCD camera (Andor 885; Andor Technologies, Belfast, UK). For quantification of the percentage of cells expressing a specific marker in any given experiment, the number of positive cells of the whole population was determined relative to the total number of 4',6-diamidino-2-phenylindole-stained cells. The specificity of nestin, Sox2 and Musashi antibodies were determined using embryonic day 14 cortical neuronal cultures (see Figure S1 in Additional file 2); similarly, nonspecific binding of flurochrome-conjugated secondary antibodies was excluded by incubating the secondary antibody alone with MSC cultures (see Figure S2 in Additional file 3).
5-Bromo-2-deoxyuridine incorporation and proliferation assay
BrdU incorporation for proliferation analysis was performed as previously described . Briefly, 10 μM BrdU (Sigma) was added along with culture medium for proliferation assays. Cells were fixed in 4% paraformaldehyde at 4°C for 15 minutes followed by treatment with 2 N HCl for 45 minutes and with 0.1 M boric acid for 10 minutes to expose the DNA for BrdU immunostaining. The treated cells were incubated with primary anti-BrdU antibody (1:100; Abcam) overnight at 4°C followed by 1 hour of incubation at room temperature with goat anti-rat FITC-conjugated secondary antibody.
RT-PCR analysis was carried out as previously described . Briefly, total RNA was isolated from unexpanded hUCB-MNCs, proliferating UCB-MSCs and differentiating UCB-MSCs using an RNA isolation kit (Qiagen RNAeasy kit, Qiagen, Valencia, CA, USA). Then ~2 μg RNA was transcribed into cDNA using random hexamers (Promega Corporation, Madison, WI, USA) and Superscript RT (Invitrogen, Carlsbad, CA, USA). Specific transcripts were amplified with gene-specific forward and reverse primers (see Table S2 in Additional file 4) in a Robocycler Gradient 96 thermocycler (Stratagene, Stratagene, La Jolla, CA, USA). The housekeeping gene β-actin was used as control to normalize the gene-specific expression. PCR products were separated by gel electrophoresis on 1.8% agarose gel in 1× Tris-acetate-ethylenediamine tetraacetate buffer and visualized by ethidium bromide staining, and images were captured using a Biorad Gel documentation system.
Neuronal differentiation of human umbilical cord blood MSCs
We used two different protocols for neuronal differentiation of hUCB-MSCs. The first was a four-step induction protocol consisting of exposure to a series of growth factors that push the MSCs towards a neuronal lineage . Briefly, the cells were exposed for 3 days to Step-1 medium consisting of IMDM supplemented with FGF-2 (5 ng/ml), retinoic acid (0.5 μM) and 1 mM 2-mercaptoethanol, followed by 3 days in Step-2 medium consisting of IMDM supplemented with 1 mM cAMP and 100 μM AsA. The cells were further exposed to Step-3 medium consisting of IMDM supplemented with 10 μM hydrocortisone and 1 mM cAMP, followed again by 3 days in Step-4 medium consisting of IMDM supplemented with 20 ng/ml αFGF, 10 ng/ml Shh, 10 ng/ml brain-derived neurotrophic factor, 10 ng/ml nerve growth factor, 25 ng/ml vitronectin, 100 μM AsA, 0.1 mM 3-isobutyl-1-methylxanthine, 10 μM forskolin and 20 nM phorbol myristate acetate. For the second protocol involving direct neuronal differentiation, we used normal neuronal differentiation medium containing neurobasal medium supplemented with FGF-2 (10 ng/ml) and 1% FBS for 5 days.
Data from all the experiments are represented as the mean ± standard deviation of triplicates (n = 3) from three different experiments. Statistical significance between groups was calculated by independent Student t test assuming equal variance. P < 0.05 was considered statistically significant.