Microarray Analysis of Genes Regulated during Induction of Osteogenesis by Human Lim Mineralization Protein-3 (LMP-3) in Human Mesenchymal Stem Cells

Wanda Lattanzi, Camilla Bernardini, Enrico Pola, Fabrizio Michetti, Andrea Gambotto, Paul D. Robbins

Risultato della ricerca: Contributo in libroContributo a convegno

Abstract

INTRODUCTION: Gene transfer of osteoinductive agents is being developed for a variety of orthopaedic applications includine spine fusion and treatment of non-union fractions and craniofacial defects. Human Lim Mineralization Protein-3 (hLMP-3) is an osteoinductive agent coded by one of the three LMP gene splice variants. We have previously demonstrated that LMP-3 upregulates bone-specific genes in murine fibroblasts and human mesenchymal stem cells (hMSCs) following adenoviral gene transfer and can induce bone formation in murine models, similar to BMPs, following in vivo and ex vivo delivery approaches. Recently, microarray analysis has been used to identify new early regulators of BMPs-induced osteoblast differentiation of multipotent mesenchymal. The aim of this study is to examine the gene expression profile of hMSCs expressing hLMP3 in order to elucidate the molecular mechanisms involved in LMP3-induced osteogenesis. METHODS: hMSCs were transduced with 1000 MOI (particles per cell) of a defective adenoviral vector carrying either the hLMP3 codon-optimized sequenze (AdLMP3) or the green fluorescent protein (AdEGFP). Cells transduced with an empty adenoviral vector (Ad5) and untransduced cells served as additional controls. mRNA was isolated from cells 24 hrs post-transduction and transgene expression was assessed by RT-PCR using sequence-specific primers. RNA was then reverse-transcripted, labeled and amplified for microarray expression analysis. Replicate experiments were performed in order to increase singificance and control reproducibility. The microarray analysis was performed by a specific software based on GC-RMA algorythms for normalization and filtering. Analysis of variance was performed on filtered data by means of t-Test. RESULTS: 189 out of 8793 genes spotted on the array were modulated inLMP3- transduced cells by more than 1.2 fold change. Crossed normalization against Ad5- and mock-transduced cells data showed a smaller set of genes (25) exhibiting significant (p<0.01) differential expression in AD.LMP3 transduced cells (Table). These transcripts are represented predominantly by genes involved in cell cycle control (SAP18), chromatin remodeling and transcriptional regulation (SMARCC2, TADA2L), post-transductional processing (TRAM2), signal transduction and metabolic pathways (AGPAT1, FDFT1), along with tissue-specific genes (PRKAB2, MAP1B, CNTNAP1). CONCLUSION: This study has identified a subset of genes regulated by LMP-3 in human MSC that potentially are involved inducing stem cell commitment and osteogenesis.
Lingua originaleEnglish
Titolo della pubblicazione ospite9th American Society of Gene Therapy annual meeting
PagineS128-S129
Numero di pagine2
Stato di pubblicazionePubblicato - 2006
Evento9th Annual Meeting of the ASGT - Baltimore, Maryland (USA)
Durata: 31 mag 20064 giu 2006

Convegno

Convegno9th Annual Meeting of the ASGT
CittàBaltimore, Maryland (USA)
Periodo31/5/064/6/06

Keywords

  • Lim Mineralization Protein-3
  • gene expression
  • mesenchymal stem cells
  • microarray

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