Multipotent human stromal cells isolated from cord blood, term placenta and adult bone marrow show distinct differences in gene expression pattern

Multipotent mesenchymal stromal cells derived from human placenta (pMSCs), and unrestricted somatic stem cells (USSCs) derived from cord blood share many properties with human bone marrow-derived mesenchymal stromal cells (bmMSCs) and are currently in clinical trials for a wide range of clinical settings. Here we present gene expression profiles of human cord blood-derived unrestricted somatic stem cells (USSCs), human placental-derived mesenchymal stem cells (hpMSCs), and human bone marrow-derived mesenchymal stromal cells (bmMSCs), all derived from four different donors. The microarray data are available on the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession number E-TABM-880. Additionally, the data has been integrated into a public portal, www.stemformatics.org. Our data provide a resource for understanding the differences in MSCs derived from different tissues.

Multipotent mesenchymal stromal cells derived from human placenta (pMSCs), and unrestricted somatic stem cells (USSCs) derived from cord blood share many properties with human bone marrow-derived mesenchymal stromal cells (bmMSCs) and are currently in clinical trials for a wide range of clinical settings. Here we present gene expression profiles of human cord blood-derived unrestricted somatic stem cells (USSCs), human placental-derived mesenchymal stem cells (hpMSCs), and human bone marrow-derived mesenchymal stromal cells (bmMSCs), all derived from four different donors. The microarray data are available on the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession number E-TABM-880. Additionally, the data has been integrated into a public portal, www.stemformatics.org. Our data provide a resource for understanding the differences in MSCs derived from different tissues. Experimental design, materials and methods

Isolation and culture of cells USSCs
Cord blood was collected with informed consent from healthy mothers undergoing elective Caesarean section. The protocol was approved by the University of Melbourne and the Royal Women's Hospital Human Ethics Review Committees. A USSC population was successfully generated as described by Kögler et al., 2004. The phenotype of this population has previously been published [1,2]. Cells from passages 5-8 were used in this study.

bmMSCs and pMSCs
Human bone marrow was obtained from healthy donors after informed consent. Placentas were obtained from healthy mothers during routine elective Caesarean section births at or near term. Full informed consent was obtained. The protocols used to obtain bone marrow and

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Genomics Data j o u r n a l h o m e p a g e : h t t p : / / w w w . j o u r n a l s . e l s e v i e r . c o m / g e n o m i c s -d a t a / placental MSCs have been described [3,4]. Cells from passages 4-6 were used in this study. Protocols for the isolation and use of each population were approved by the Mater Health Services Human Ethics Review Committee, Brisbane.

Differentiation assays
Osteogenic lineage Cells were cultured for 21 days in medium containing 10-7 M dexamethasone, 10 mM glycerol-2-phosphate disodium salt (Sigma) and 50 μg/ml ascorbic acid 2 phosphate (Sigma). Cells were fixed for 10 min with 70% ice-cold ethanol at 4°C and stained with 1% Alizarin Red S (Sigma) in distilled water, pH 4.2. After cells were washed in distilled water and a final wash with PBS (Ca 2+ and Mg 2+ free), images were captured using a Leica DMIRB inverted microscope and AxioVision 4.2 software (Carl Zeiss AG, www.zeiss.com).

RNA extractions
Total RNA was extracted at passages 4-6 from human bmMSCs, pMSCs and USSCs, using 4 separate donors for each. RNA was extracted using a Qiagen RNeasy kit (www.qiagen.com). All RNA preparations were quantified using a Nanodrop spectrophotometer (Thermo Scientific) and quality was accessed using an Agilent 2100 Bioanalyser (RNA Nano chips). The RNA integrity number ranged between 9.9 and 10, demonstrating high quality starting material.

Sample labeling and scanning
Five hundred nanograms of RNA was amplified using the Ambion Illumina RNA amplification kit with biotin UTP labeling (Ambion, Inc), including a 4 h in vitro transcription using T7 RNA polymerase. A total of 750 ng of cRNA was hybridized to human whole-genome Illumina Human-Refseq8 v2 BeadChips (Illumina, Inc.). Slides were scanned on an Illumina Beadstation and bead summarization was performed  Data normalization and filtering Data were exported from BeadStudio with no additional processing, and imported to R/BioConductor using the readBead function from the BeadExplorer package. Background adjustment and quantile normalization was performed using function: bg.adjust and normalize.quantiles. Genes were initially filtered using Illumina® detection p-value. A gene/probe was included in QC assessment if it had a detection pvalue ≥ 0.99 all four donor samples within the tissue source.

Chemokine receptor display
The surface and intracellular chemokine displays of bmMSCs and pMSCs were very similar. USSCs differed from bmMSCs and pMSCs by showing positive staining for the presence of intracellular CCR8, CCR10 and CCR11 (Table 2).

Mesodermal differentiation assays
Typical MSC morphology was confirmed in culture (Fig. 1), and the mesodermal differentiation potential of each of the three cell populations was assessed in vitro to determine their multipotency ability. This was analyzed according to the cells' ability to differentiate into osteocytes and adipocytes. All three undifferentiated populations showed marked ability to differentiate to the osteogenic lineage. bmMSCs showed stronger differentiation to the adipogenic pathway that pMSCs and USSCs, each of which showed only a slight degree of adipogenesis (Fig. 1).

Gene expression quality
Normalization reduced the between array variation (Fig. 2). Principle component analysis was performed to demonstrate the difference between the MSC sources and the reproducibility of the replicate donors. When plotting on the first two components, the samples clustered based on their tissue source, but some donor variation was apparent (Fig. 2a). All replicates were tightly clustered except for one USSC sample (Chip ID: 4294077038_D; Sample ID: USSC4). However, this sample was still highly correlated to the other USSC donors (Average Pearson of 0.89) compared to 0.95 for the bmMSCs and pMSCs. When plotting components two and three the samples clustered based on their tissue source (Fig. 2b).  C. Heazlewood isolated the bone marrow MSCs and the placental MSCs and generated the flow cytometry data and mesodermal differentiation data. A. Mackay-Sim, C. Wells and K. Atkinson co-funded the study. C. Wells supervised the microarray data acquisition and the bioinformatics analysis and contributed to the writing of the manuscript. K. Atkinson wrote the initial draft and final version of the manuscript. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Funding was also provided by the Australian Stem Cell Centre, The Heart Foundation, The Mater Foundation and Inner Wheel Australia. CH and KK were recipients of PhD scholarships from The University of Queensland and the Queensland University of Technology respectively. The authors would also like to thank Robert Williamson for his enthusiastic support of this study.