Genome-wide expression profiling of stem cells sub-populations characterized by new membrane markers identified with an immunoproteomics approachStem cells-based therapies represent an emerging therapeutic approach to treat a variety of degenerative, neoplastic and genetic diseases. This project aims at characterizing Hematopoietic Stem Cells (HSC) and Mesenchymal Stem Cells (MSC) subpopulations identified by the expression of new surface markers.
Multipotent stem cells, such as HSC and MSC, are present in the bone marrow and in the cord blood. Stem cells subpopulations with different commitments may be phenotypically different. Then, it is likely that they express membrane proteins unknown so far. The identification of stem cells subpopulations with a defined commitment represents an appealing opportunity in the field of stem cells-based therapies. This project proposes to use a library of polyclonal antisera, specific for membrane proteins so far uncharacterized, to meet this need. The library was obtained from an “in silico” selection of about 3000 human genes potentially encoding membrane proteins with unknown function and tissue distribution. The corresponding recombinant proteins have been produced in E.coli and used to immunized mice or rabbits. The antisera obtained from the immunized animals will be used to analyze by flow cytometry the expression of new HSC or MSC membrane proteins. The antisera identifying a HSC or MSC subset will be used to sort the positive subpopulations in order to evaluate its fate by “in vitro” differentiation assays. The functional characterization of HSC and MSC subsets will be completed by the gene expression profile and miRNA expression profile evaluation to define the molecular basis of stemness vs. commitment.
In conclusion, the final goal of this project is to develop a functional genomics and transcriptomics integrated platform to identify phenotypic and molecular “signatures” of HSC and MSC subsets with a specific commitment. This achievement will significantly improve the stem cell-based therapeutic approaches.
Our plan is proposed to associate functional analysis of stem cells populations with the global profile of their miRNA and genes expression, using "Human DNA Microarrays". The microarray approach allows to simultaneously analyze the expression of all human genes. The gene expression data will be analyze with "clustering-analysis" software and other bioinformatics tools, with the purpose to:
i) Obtain a general transcription profile map of differentially regulated genes groups in stem cells expressing markers of interest. Genes differentially regulated will be sub-sequentially located on the chromosome.
ii) Perform direct sequencing of single specific disclosed genes, in order to define new molecular variants like sources of phenotypic variability.
iii) Investigate the gene regulation with RNA-mediated interference experiments, mechanism for RNA-guided regulation in which double-stranded ribonucleic acid inhibits the expression of genes with complementary nucleotide sequences.
iv) Define microRNA (miRNA) expression profiles in stem cell sub-populations, in order to generate a global differentially expressed genes picture with both the transcriptome results and differentially expressed miRNAs database.
In conclusion, the proposed project will develop an innovative platform encompassing multitask targets focus on proteomics and gene expression, in order to disclose new phenotypic and molecular markers related to stem cell sub-populations characterized for a well-defined differentiation commitment. This achievement will increase the knowledge of stem cell biology with a significant improvement of stem cells-based therapeutic approaches.