Biology subjects

Cucchiarini, M.

Publications and source records attributed to Cucchiarini, M..

3 recordsLinked to original sources

bioRxiv2026

Gene amplification during differentiation of mesenchymal stem cells towards chondrocytes

For decades gene amplifications were described as an attribute of tumor cells and as a physiological mechanism to increase gene copy numbers for the higher protein demand during development of amphibians and flies. An increasing number of publications describe gene amplifications in normal mammalian cells during differentiation. Many amplified genes detected in tumor cells overlap with amplified genes detected during stem cell differentiation. Since stem cells have a valuable potency in regenerative therapies and since cartilage regeneration is a highly demanded therapeutic strategy, we investigated gene amplification dynamics during chondrogenic differentiation of human mesenchymal stem cells (hMSCs). Using quantitative PCR, we analyzed copy number changes for genes previously implicated in differentiation as well as genes amplified in chondrosarcoma including CDK4, MDM2, AGAP2, CPT1B, SHANK3, TRIB1, and MYC. Amplifications were transient and stage-specific: CDK4, CPT1B, and SHANK3 exhibited the highest copy number increases at day 2, followed by a gradual decline by day 7, while AGAP2 and MDM2 increased later in differentiation. Laser microdissection of toluidine blue-stained areas revealed heterogeneity in amplification patterns: CDK4 amplification was prominent in areas lacking or showing moderate proteoglycan deposition; CPT1B amplification occurred in regions with absent, moderate, or intense proteoglycan deposition; and SHANK3 amplification was restricted to areas with intense proteoglycan deposition. Notably, regions with the strongest proteoglycan staining exhibited no gene amplification, suggesting that gene amplification is an early, transient event that diminishes as differentiation progresses. These findings highlight gene amplification as a mechanism during chondrogenesis, potentially critical for early differentiation stages and genome stability in mature cells.

Developmental Biology↗

bioRxiv2026

Physiological re-replication during human stem cell differentiation

During defined developmental windows in Drosophila, controlled re-replication generates physiological gene amplification. Although gene amplification has also been observed during human stem cell differentiation, re-replication in human cells has largely been linked to tumor-associated genome instability. Here, we demonstrate that re-replication likewise operates as a physiological mechanism in human stem cells. Using Rerep-Seq and DNA fiber-combing, we identify distinct phases of re-replication during the differentiation of human myoblasts into myotubes and during the lineage commitment of mesenchymal stem cells toward adipogenic, osteogenic, chondrogenic, and neuronal fates. In all differentiation systems examined, re-replication occurred within defined temporal windows. FACS-isolated re-replicating cells exhibited elevated gene expression using RNA-Seq specifically within re-replicated genomic regions. Moreover, re-replicated DNA was detected as extranuclear DNA. These findings support a model in which cells that do not undergo re-replication, and thus avoid increased chromosomal instability, may nonetheless boost the expression of differentiation-relevant genes by acquiring re-replicated DNA released from neighboring re-replicating cells. We propose that human stem cells exploit an evolutionarily conserved re-replication mechanism to transiently increase gene copy number and thereby meet the heightened protein demands associated with differentiation.

genomics↗

bioRxiv2019

Single-Cell RNA-Seq Reveals Novel Mitochondria-related Musculoskeletal Cell Populations during Adult Axolotl Limb Regeneration Process

While the capacity to regenerate tissues or limbs is limited in mammals including humans, unlike us, axolotls are able to regrow entire limbs and major organs. The wound blastema have been extensively studied in limb regeneration. However, due to the inadequate characterization and coordination of cell subpopulations involved in the regeneration process, it hinders the discovery of the key clue for human limb regeneration. In this study, we applied unbiased large-scale single-cell RNA sequencing to classify cells throughout the adult axolotl limb regeneration process. We computationally identified 7 clusters in regenerating limbs, including the novel regeneration-specific mitochondria-related cluster supporting regeneration through energy providing and the COL2+ cluster contributing to regeneration through cell-cell interactions signals. We also discovered the dedifferentiation and re-differentiation of the COL1+/COL2+ cellular subpopulation and uncovered a COL2-mitochondria sub-cluster supporting the musculoskeletal system regeneration. On the basis of these findings, we reconstructed the dynamic single-cell transcriptome atlas of adult axolotl limb regenerative process, and identified the novel regenerative mitochondria-related musculoskeletal populations, which yielded deeper insights into the crucial interactions between cell clusters within the regenerative microenvironment.

genomics↗