Introduction

Future Directions

Methods

Results

TRANSCRIPTIONAL PROFILING OF HUMAN AMNIOTIC FLUID-DERIVED STEM CELLS - WHERE DO THEY RESIDE IN THE DEVELOPMENTAL CONTINUUM?

David Mack1, Kathryn Gallaher1 and Stephen J Walker1

1Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC, USA

Wake Forest Institute for REGENERATIVE MEDICINE

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Because of their fetal origin, it has been postulated that amniotic fluid-derived stem (AFS) cells lie phenotypically somewhere between embryonic (ES) and adult somatic stem cells. The expression of approximately 90 "stem cell-related" genes was recently measured in 22 clones of hAFS cells and compared to hES/iPS cells and adult bone marrow-derived mesenchymal stromal cells (MSCs). The analysis of the expression of this limited number of genes appears to corroborate that while AFS cells express a battery of MSC-like cell surface markers, they also share at least a partial gene expression profile of more pluripotent ES cells (like the expression of SSEA-4). Therefore, our goal was to take this analysis a step further by employing whole-genome microarray profiling to more fully characterize gene expression in these three cell types.

Total RNA was extracted from two human ES cell lines (H7 and H9), two BM-MSC lines (commercially available from Lonza and PromoCell) and two AFS cell lines isolated in house (A1 and H1). The quantity and purity of the extracted RNA was evaluated using a NanoDrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE) and its integrity measured using an Agilent Bioanalyzer. For microarray hybridizations, 1.5 ug of amplified cDNA from each sample was labeled with Arrayit 540 dye (Arrayit; Sunnyvale, CA) using the Arrayit Aminoallyl cDNA Labeling kit (Arrayit; Sunnyvale, CA) following the manufacturer’s protocol.  The amount and quality of the fluorescently labeled cRNA was assessed using a NanoDrop (ND-1000) spectrophotometer and an Agilent Bioanalyzer. Following manufacturer’s specifications, 2.0 ug of 540-labeled cRNA was hybridized to Human H25K Whole-Genome Microarrays (Arrayit; Sunyvale, CA) for 3.5 hrs, prior to washing and scanning. Data were extracted from scanned images using Arrayit’s Feature Extraction Software .

Background subtracted intensity data were generated for each transcript within each of the six samples. Following removal of transcripts for which the annotation was incomplete, there were ~16,000 data points for each sample. These were rank-ordered based on intensity, (from 1 to 65,000) and transcripts displaying an intensity of 200 or greater (background + 2SD) were used for further analysis. All genes displaying an intensity value of >200 were then evaluated in a Venn diagram to display genes that were: (a) expressed in all three cell types, (b) expressed exclusively in a single cell type or (c) expressed in two of the cell types but not the third.

Unsupervised principle component analysis revealed that the ES cell lines and the AFS cell lines cluster reasonably close together, whereas the MSC lines display a somewhat higher degree of variation. This result makes sense given that both the ES and AFS cell lines are clonal populations but the commercially available MSCs are mixed populations isolated solely on their adherence to plastic.

Functional AnnotationCommon to ES and AFS Cells (648 expressed transcripts)Term Count % P-Value

Phosphoprotein 418 66.9 6.4E-53Nucleus 304 48.6 1.4E-50Mitotic Cell Cycle 72 11.5 6.6E-35Cell Cycle Phase 80 12.8 7.9E-35Cell Cycle 107 17.1 2.6E-33Organelle Fission 55 8.8 1.1E-28Intracellular Non-Membrane-Bounded Organelle 184 29.4 1.3E-28Chromosome 71 11.4 3.8E-28Nuclear Lumen 126 20.2 7.1E-26Intracellular Organelle Lumen 138 22.1 8.4E-24

KEGG PathwaysCommon to ES and AFS Cells

Term Count % P-ValueCell Cycle 20 3.2 2.8E-8DNA Replication 9 1.4 2.0E-5Pyrimidine Metabolism 11 1.8 1.3E-3Homologous Recombination 6 1.0 2.2E-3Oocyte Meiosis 11 1.8 4.0E-3Pathways in Cancer 20 3.2 1.5E-2Small Cell Lung Cancer 8 1.3 2.3E-2Spliceosome 10 1.6 2.7E-2Mismatch Repair 4 0.6 4.2E-2RNA Degradation 6 1.0 4.3E-2Non-Homologous End-Joining 3 0.5 7.0E-2Prostate Cancer 7 1.1 8.1E-2Ubiquitin Mediated Proteolysis 9 1.4 9.4E-2

Functional AnnotationCommon to MSC and AFS Cells (890 expressed transcripts)

Term Count % P-ValueGolgi Apparatus 80 9.2 8.3E-8Vesicle-Mediated Transport 64 7.4 1.3E-6Skeletal System Development 44 5.1 8.2E-7Phosphoprotein 405 46.6 2.2E-7Blood Vessel Development 35 4 5.4E-5Endoplasmic Reticulum 86 9.9 9.1E-5Negative Regulation of Phosphorylation 14 1.6 5.7E-5

Regulation of I-kappaB Kinase/NF-kappaB Cascade 20 2.3 5.7E-5Negative Regulation of Phosphate Metabolic Process 14 1.6 9.4E-5Regulation of Protein Kinase Cascade 32 3.7 1.2E-5Focal Adhesion 29 3.3 5.9E-5Regulation of Cell Growth 28 3.2 1.6E-4Positive Regulation of Signal Transduction 36 4.1 1.6E-4Cell Motion 49 5.6 1.5E-4Identical Protein Binding 58 6.7 9.1E-4

KEGG PathwaysCommon to MSC and AFS Cells

Term Count % P-ValueFocal Adhesion 29 3.3 5.9E-5MAPK Signaling Pathway 29 3.3 7.3E-3ECM-Receptor Interaction 13 1.5 3.6E-2Apoptosis 13 1.5 3.7E-2Lysosome 15 1.7 4.8E-2Wnt Signaling Pathway 17 2.0 6.8E-2Endocytosis 19 2.2 7.8E-2RIG-I-like Receptor Signaling Pathway 10 1.2 1.3E-1Cytosolic DNA-Sensing Pathway 8 0.9 2.2E-1Regulation of Actin Cytoskeleton 19 2.2 2.2E-1Axon Guidance 13 1.5 2.6E-1TGF-beta Signaling Pathway 10 1.2 2.6E-1Pathways in Cancer 25 2.9 2.8E-1Adherens Junction 9 1.0 2.9E-1p53 Signaling Pathway 8 0.9 3.6E-1

Calcium Signaling Pathway 15 1.7 3.6E-1GnRH Signaling Pathway 10 1.2 3.5E-1Melanogenesis 10 1.2 3.3E-1Cytokine-Cytokine Receptor Interaction 19 2.2 4.6E-1

A Venn diagram generated from a comparison of ~7000 of the most highly expressed genes from each cell type (ES vs. AFS vs. MSC) showed that 65% of the transcripts are expressed in all three. AFS cells and MSCs shared an additional 890 expressed transcripts, bringing their transcriptional similarity up to approximately 78%.

The original hypothesis that AFS cells are more similar to ES cells than are MSCs was supported by these data. AFS and ES cells share an additional 648 common transcripts whereas MSCs and ES cells only share an additional 70 transcripts.

Using data from the Venn diagram, the list of (890) genes that were expressed exclusively in MSCs and AFS cells was imported into Ingenutiy (IPA) software. Here we performed gene ontology and pathway analysis to assess the signaling and metabolic pathways, molecular networks, and biological processes that are most significantly represented in a both MSCs and AFS, but not ES, cells. The same analysis was then performed using the list of genes (648) common and exclusive to AFS and ES cells. The gene ontology (functional annotation) for each comparison is displayed in the tables on the left, and the pathway analysis results are in the tables to the right.

Conclusions

RNA and DNA derived from the six cells lines assayed in this pilot study will be used to take the characterization further. Through microRNA and epigenetic (promoter methylation) profiling we should better able to describe molecular features that define functional aspects of each of these three cell types.

The gene expression data generated in this study suggest that: (1) gene expression between individual MSC cell lines is more variable than gene expression in either AFS or ES cell lines and, (2) AFS cells share more common transcript expression with ES cells than do MSCs.