Musculoskeletal tissue regeneration by human non-embryonic stem cells

The aim of this thesis was to investigate the regenerative potential of alternative sources of stem cells, derived from human dental pulp (hDPSCs) and amniotic fluid (hAFSCs) and, specifically, to evaluate their capability to be committed towards osteogenic and myogenic lineages, for the eventual applicability of these stem cells to translational strategies in regenerative medicine of bone and skeletal muscle tissues. The in vitro bone production by stem cells may represent a radical breakthrough in the treatment of pathologies and traumas characterized by critical bone mass defects, with no medical or surgical solution. Human DPSCs and AFSCs were seeded and pre-differentiated on different scaffolds to test their capability to subsequently reach the osteogenic differentiation in vivo, in order to recover critical size bone defects. Fibroin scaffold resulted to be the best scaffold promoting mature bone formation and defect correction when combined to both hDPSCs and hAFSCs. This study also described a culture condition that might allow human DPSCs to be used for human cell therapy in compliance with good manufacturing practices (GMPs): the use of human serum (HS) promoted the expansion and the osteogenic differentiation of hDPSCs in vitro and, furthermore, allowed pre-differentiated hDPSCs to regenerate critical size bone defects in vivo. This thesis also showed that hDPSCs and hAFSCs can be differentiated towards the myogenic lineage in vitro, either when co-cultured with murine myoblasts and when differentiated alone after DNA demethylation treatment. Interestingly, when injected into dystrophic muscles of SCID/mdx mice - animal model of Duchenne Muscular Dystrophy (DMD) - hDPSCs and hAFSCs pre-differentiated after demethylating treatment were able to regenerate the skeletal muscle tissue and, particularly, to restore dystrophin expression. These observations suggest that human DPSCs and AFSCs might be eventually applied to translational strategies, in order to enhance the repair of injured skeletal muscles in DMD patients.

Transcriptional dynamics of the human DMD locus

The human DMD locus encodes dystrophin protein. Absence or reduced levels of dystrophin (DMD or BMD phenotype, respectively) lead to progressive muscle wasting. Little is known about the complex coordination of dystrophin expression and its transcriptional regulation is a field of intense interest. In this work we found that DMD locus harbours multiple long non coding RNAs which orchestrate and control transcription of muscle dystrophin mRNA isoforms. These lncRNAs are tissue-specific and highly expressed during myogenesis, suggesting a possible role in tissue-specific expression of DMD gene isoforms. Their forced ectopic expression in human muscle and neuronal cells leads to a specific and negative regulation of endogenous dystrophin full lenght isoforms. An intriguing aspect regarding the transcription of the DMD locus is the gene size (2.4Mb). The mechanism that ensures the complete synthesis of the primary transcript and the coordinated splicing of 79 exons is still completely unknown. By ChIP-on-chip analyses, we discovered novel regions never been involved before in the transcription regulation of the DMD locus. Specifically, we observed enrichments for Pol II, P-Ser2, P-Ser5, Ac-H3 and 2Me-H3K4 in an intronic region of 3Kb (approximately 21Kb) downstream of the end of DMD exon 52 and in a region of 4Kb spanning the DMD exon 62. Interestingly, this latter region and the TSS of Dp71 are strongly marked by 3Me-H3K36, an histone modification associated with the regulation of splicing process. Furthermore, we also observed strong presence of open chromatin marks (Ac-H3 and 2Me-H3K4) around intron 34 and the exon 45 without presence of RNA pol II. We speculate that these two regions may exert an enhancer-like function on Dp427m promoter, although further investigations are necessary. Finally, we investigated the nuclear-cytoplasmic compartmentalization of the muscular dystrophin mRNA and, specifically, we verified whether the exon skipping therapy could influence its cellular distribution.