Mesenchymal Cells Retain the Specificity of Embryonal Origin During Osteogenic Differentiation.

Mesenchymal stem cells (MSCs) are widely used in therapy, but the differences between MSCs of various origins and their ability to undergo osteogenic differentiation and produce extracellular matrix are not fully understood. To address this, we conducted a comparative analysis of mesenchymal cell primary cultures from 6 human sources, including osteoblast-like cells from the adult femur, adipose-derived stem cells, Wharton's jelly-derived mesenchymal cells, gingival fibroblasts, dental pulp stem cells, and periodontal ligament stem cells. We analyzed these cells' secretome, proteome, and transcriptome under standard and osteogenic cultivation conditions. Despite the overall similarity in osteogenic differentiation, the cells maintain their embryonic specificity after isolation and differentiation in vitro. Furthermore, we propose classifying mesenchymal cells into 3 groups: dental stem cells of neural crest origin, mesenchymal stem cells, and fetal stem cells. Specifically, fetal stem cells have the most promising secretome for various applications, while mesenchymal stem cells have a specialized secretome optimal for extracellular matrix production. Nevertheless, mesenchymal cells from all sources secreted core bone extracellular matrix-associated proteins. In conclusion, our study illuminates the distinctive characteristics of mesenchymal stem cells from various sources, providing insights into their potential applications in regenerative medicine and enhancing our understanding of the inherent diversity of mesenchymal cells in vivo.

Pericentromeric Non-Coding DNA Transcription Is Associated with Niche Impairment in Patients with Ineffective or Partially Effective Multiple Myeloma Treatment.

Mesenchymal stromal cells (MSC) 'educated' by tumor cells are an essential component of the multiple myeloma (MM) tumor microenvironment (TME) involved in tumor progression. Transcription of tandemly repeated (TR) non-coding DNA is often activated in many tumors and is required for tumor progression and cancer cells genome reorganization. The aim of the work was to study functional properties including the TR DNA transcription profile of MSC from the hematopoietic niche of treated MM patients. Healthy donors (HD) and patients after bortezomib-based treatment (with partial or complete response, PoCR, and non-responders, NR) were enrolled in the study. Their trephine biopsies were examined histologically to evaluate the hematopoietic niche. MSC cultures obtained from the biopsies were used for evaluation of the proliferation rate, osteogenic differentiation, presence of tumor MSC markers, resistance to bortezomib, and pericentromeric TR DNA transcription level. The MSC 'education' by multiple myeloma cells was mimicked in co-culture experiments with or without bortezomib. The TR DNA transcription profile was accessed. The histological examination revealed the persistence of the tumor microenvironment (especially of the vasculature) in treated patients. In co-culture experiments, MSC of bortezomib-treated patients were more resistant to bortezomib and protected cancer MM cells of the RPMI8226 cell line more effectively than HD-MSC did. The MSC obtained from PoCR and NR samples differed in their functional properties (proliferation capacity, osteogenic potential, and cancer-associated fibroblasts markers). Transcriptome analysis revealed activation of the TR transcription in cells of non-hematopoietic origin from NR patients' bone marrow. The pericentromeric TR DNA of HS2/HS3 families was among the most upregulated in stromal MSC but not in cancer cells. The highest level of transcription was observed in NR-MSC. Transcription of HS2/HS3 was not detected in healthy donors MSC unless they were co-cultured with MM cancer cells and acquired cancer-associated phenotype. Treatment with TNFα downregulated HS2/HS3 transcription in MSC and upregulated in MM cells. Our results suggest that the hematopoietic niche retains the cancer-associated phenotype after treatment. Pericentromeric non-coding DNA transcription is associated with the MSC cancer-associated phenotype in patients with ineffective or partially effective multiple myeloma treatment.

The Long Linker Region of Telomere-Binding Protein TRF2 Is Responsible for Interactions with Lamins.

Telomere-binding factor 2 (TRF2) is part of the shelterin protein complex found at chromosome ends. Lamin A/C interacts with TRF2 and influences telomere position. TRF2 has an intrinsically disordered region between the ordered dimerization and DNA-binding domains. This domain is referred to as the long linker region of TRF2, or udTRF2. We suggest that udTRF2 might be involved in the interaction between TRF2 and lamins. The recombinant protein corresponding to the udTRF2 region along with polyclonal antibodies against this region were used in co-immunoprecipitation with purified lamina and nuclear extracts. Co-immunoprecipitation followed by Western blots and mass spectrometry indicated that udTRF2 interacts with lamins, preferably lamins A/C. The interaction did not involve any lamin-associated proteins, was not dependent on the post-translation modification of lamins, nor did it require their higher-order assembly. Besides lamins, a number of other udTRF2-interacting proteins were identified by mass spectrometry, including several heterogeneous nuclear ribonucleoproteins (hnRNP A2/B1, hnRNPA1, hnRNP A3, hnRNP K, hnRNP L, hnRNP M), splicing factors (SFPQ, NONO, SRSF1, and others), helicases (DDX5, DHX9, and Eif4a3l1), topoisomerase I, and heat shock protein 71, amongst others. Some of the identified interactors are known to be involved in telomere biology; the roles of the others remain to be investigated. Thus, the long linker region of TRF2 (udTRF2) is a regulatory domain responsible for the association between TRF2 and lamins and is involved in interactions with other proteins.

Comparative Analysis of Dental Pulp and Periodontal Stem Cells: Differences in Morphology, Functionality, Osteogenic Differentiation and Proteome.

Dental stem cells are heterogeneous in their properties. Despite their common origin from neural crest stem cells, they have different functional capacities and biological functions due to niche influence. In this study, we assessed the differences between dental pulp stem cells (DPSC) and periodontal ligament stem cells (PDLSC) in their pluripotency and neuroepithelial markers transcription, morphological and functional features, osteoblast/odontoblast differentiation and proteomic profile during osteogenic differentiation. The data were collected in paired observations: two cell cultures, DPSC and PDLSC, were obtained from each donor. Both populations had the mesenchymal stem cells surface marker set exposed on their membranes but differed in Nestin (a marker of neuroectodermal origin) expression, morphology, and proliferation rate. OCT4 mRNA was revealed in DPSC and PDLSC, while OCT4 protein was present in the nuclei of DPSC only. However, transcription of OCT4 mRNA was 1000-10,000-fold lower in dental stem cells than in blastocysts. DPSC proliferated at a slower rate and have a shape closer to polygonal but they responded better to osteogenic stimuli as compared to PDLSC. RUNX2 mRNA was detected by qPCR in both types of dental stem cells but RUNX2 protein was detected by LC-MS/MS shotgun proteomics only in PDLSC suggesting the posttranscriptional regulation. DSPP and DMP1, marker genes of odontoblastic type of osteogenic differentiation, were transcribed in DPSC but not in PDLSC samples. Our results prove that DPSC and PDLSC are different in their biology and therapeutic potential: DPSC are a good candidate for osteogenic or odontogenic bone-replacement cell-seeded medicines, while fast proliferating PDLSC are a prospective candidate for other cell products.

Fibrin Glue Implants Seeded with Dental Pulp and Periodontal Ligament Stem Cells for the Repair of Periodontal Bone Defects: A Preclinical Study.

A technology to create a cell-seeded fibrin-based implant matching the size and shape of bone defect is required to create an anatomical implant. The aim of the study was to develop a technology of cell-seeded fibrin gel implant creation that has the same shape and size as the bone defect at the site of implantation. Using computed tomography (CT) images, molds representing bone defects were created by 3D printing. The form was filled with fibrin glue and human dental pulp stem cells (DPSC). The viability, set of surface markers and osteogenic differentiation of DPSC grown in fibrin gel along with the clot retraction time were evaluated. In mice, an alveolar bone defect was created. The defect was filled with fibrin gel seeded with mouse DPSC. After 28 days, the bone repair was analyzed with cone beam CT and by histological examination. The proliferation rate, set of surface antigens and osteogenic potential of cells grown inside the scaffold and in 2D conditions did not differ. In mice, both cell-free and mouse DPSC-seeded implants increased the bone tissue volume and vascularization. In mice with cell-seeded gel implants, the bone remodeling process was more prominent than in animals with a cell-free implant. The technology of 3D-printed forms for molding implants can be used to prepare implants using components that are not suitable for 3D printing.