The Transcriptional Program in the Response of Human Fibroblasts to Serum ( Iyer et al)

1. The Transcriptional Program inthe Response of HumanFibroblasts to Serum (Iyer et al) By: James Tirtorahardjo & Daphne Lin

2. Background Info • Fibroblasts important to tissue regeneration • Serum: Liquid portion of blood without blood cells and clotting factors • Exposure of fibroblasts to serum initiates a variety of wound healing processes

3. Fibroblast Function in Wound Repair • Fibroblasts are located outside the bloodstream • Extracellular matrix formation • Secretion of collagen and fibronectin • Formation of a wound matrix

4. Purpose • Discover why fibroblasts are important to tissue regeneration • To study the transition from G0 to proliferation • To identify differences in gene expression over time during wound repair. • To group genes into categories based on expression pattern and function • To show that cDNA microarrays are a viable method of expression profiling

5. Method • After culture, quiescent fibroblasts are deprived of serum for 48 hours • Then stimulated by solution of 10% FBS (fetal bovine serum) • Cells harvested for cDNA microarray at 12 different times

6. cDNA Synthesis • mRNA is harvested from cells via organic extraction • cDNA clones were identified via sequencing and comparison to a database

7. cDNA Microarray • Control cDNA made from G0 cells

8. Method • 8613 different genes surveyed • 4000 named human gene • 2000 genes from other organisms related to the named genes • 4000 other genes from the human transcript map • Half were unnamed ESTs (expressed sequence tags)

9. Results

10. Verification • Gradual change in gene expression over sequential time points showed that the observations were valid • Inclusion of duplicates • Three independent mRNA hybridizations used • PCR assay for gene expression

11. Analysis • Genes within cluster are related/perform similar functions • Ex: Cluster D contains cell proliferation genes

12. Fibroblast Reprogramming • Probably responsible for pulling fibroblast out of G0 phase and back into cell cycle • Reprogramming consists of turning off genes inducing quiescence and turning on genes that induce proliferation

13. Fibroblasts in Wound Healing • Genes with similar function have similar expression patterns

14. Significant Findings • Found 200 new genes • Other unknown genes can be placed into clusters based on expression pattern • cDNA microarray was shown to be an effective way to create an expression profile

15. Issues With the Method • Cannot perfectly replicate in vivo conditions • Cannot see exactly how the genes interact • Doesn’t tell the function of the individual genes (must be inferred) • Requires prior knowledge of the genes

16. Clinical Significance • Helps understand wound repair mechanisms • More info on G0 to proliferation transition • Identification of gene expression patterns for cell cycle

17. Future Studies • Testing fibroblast expression in response to other stimuli • Examining fibroblast expression in vivo wound repair response • Test serum response patterns in other cell types

18. Summary • cDNA hybridization to examine gene expression pattern in response to exposure to serum • Identified gene clusters responsible for various cell activities • Identified 200 new genes