Methylation and epigenetic modification by 5’ azacytidine and valproic acid treatment increase stemness attributes in bone sarcoma cell lines

Bone sarcoma is an aggressive malignancy with high mortality rate. Despite recent advances, the prognosis is still extremely poor. Bone sarcomas contain a small cell population with stem cell like properties, referred to as cancer stem cells (CSCs) expressing CD133 (Tirino et al, 2009; 2011). The biological relevance and regulatory mechanism of CD133 expression are not yet understood. The aim of this study is to elucidate mechanisms regulating aberrant expression of CD133 and stemness phenotype. Saos-2, MG63 and BS15 cell lines were treated with 0,5 mM valproic acid (VPA) and 3μM 5’azacytidine (5-AZA) for 48 hours alone and in combination. CD133 and stemness markers expression including OCT4, Sox2 and Nanog were analyzed by flow cytometry and real-time PCR. Vimentin and osteocalcin levels were also tested. Sarcospheres formation rate was assessed as spheres number/seed single cell number. After treatment with 5-AZA or VPA, the expression level of CD133 mRNA as well as of protein was significantly increased in all three cell lines. Also OCT4, Sox2 and Nanog, stemness markers, and vimentin, mesenchymal marker resulted to be upregulated after treatment by real time-PCR. On the contrary, the expression level of osteocalcin remained similar before and after treatment. Interestingly, combined treatment with 5-AZA and VPA induced an increase of CD133 expression in a synergistic manner in all three cell lines. In addition, sarcospheres formation rate was increased after drug treatment compared to untreated cells. Also in this case, the drug combination lead to synergistic increase of formation rate of spheres. In conclusion, our results indicate that DNA methylation is an important determinant of CD133 and stemness profile in human bone sarcomas and this mechanism may be associated with histone deacetilase inhibition

Cancer stem cells in head and neck tumors: evidence for metastatic spread and treatment resistance

The major challenge in the management of patients with oral squamous cell carcinomas (OSCC) is the development of resistance to therapy leading to disseminated disease. Since cancer stem cells (CSC) have emerged as important players in OSCC metastasis, our objectives were to explore the implications of CSC in OSCC tumor progression, invasion and response to conventional therapies. Methods: A panel of well-characterized cell lines originated from the most common sites in the head and neck area was used. Cells were cultured as floating spheres or under normal adherent conditions and analyzed for CD44, ALDH, CD24, CD29, CD56 by flow cytometry, PCR arrays for genes related to stemness, metastasis and EMT . We also investigated sLeX expression, known to play a key-role in many cancers metastasis by promoting tumor cells binding to endothelial E-selectin. We analyzed the tumorigenic potential of OSCC cells by invasion assays and in vivo OSCC experimental models comparatively to CSC cells. Moreover resistance to cisplatin and radiation was assessed by annexin V/PI assay and by colony forming assay. Results: The highest levels of sLeX expression were found in cell lines originated from oral cavity (9%-47%) compared to other head and neck locations (0.1%-7%). Cells grown as spheres were 95-100% positive for sLeX compared to 10-40% of adherent counterpart. Although sLeX+ and sLeX- cells were both able to form spheres, sLeX+ spheres were predominant and larger. Flow cytometry and PCR arrays indicated that the spheres were highly enriched in CSC and metastatic markers. Consistently, the spheres showed increased invasive and tumorigenic potential, and resistance to conventional chemotherapy and radiations. Conclusion: these studies are the first to unveil a novel link between sLeX expression, stem cell formation and metastatic spread in OSCC, and provide supportive evidence for CSC resistance to treatment. Understanding the mechanisms of tumor invasion and metastasis will improve patient outcome and survival

Use of a 3D floating sphere culture system to maintain the neural crest-related properties of human dental pulp stem cells

Human dental pulp is considered an interesting source of adult stem cells, due to the low-invasive isolation procedures, high content of stem cells and its peculiar embryological origin from neural crest. Based on our previous findings, a dental pulp stem cells sub-population, enriched for the expression of STRO-1, c-Kit, and CD34, showed a higher neural commitment. However, their biological properties were compromised when cells were cultured in adherent standard conditions. The aim of this study was to evaluate the ability of three dimensional floating spheres to preserve embryological and biological properties of this sub-population. In addition, the expression of the inwardly rectifying potassium channel Kir4.1, Fas and FasL was investigated in 3D-sphere derived hDPSCs. Our data showed that 3D sphere-derived hDPSCs maintained their fibroblast-like morphology, preserved stemness markers expression and proliferative capability. The expression of neural crest markers and Kir4.1 was observed in undifferentiated hDPSCs, furthermore this culture system also preserved hDPSCs differentiation potential. The expression of Fas and FasL was observed in undifferentiated hDPSCs derived from sphere culture and, noteworthy, FasL was maintained even after the neurogenic commitment was reached, with a significantly higher expression compared to osteogenic and myogenic commitments. These data demonstrate that 3D sphere culture provides a favorable micro-environment for neural crest-derived hDPSCs to preserve their biological properties

Hyaluronan-Based Gel Promotes Human Dental Pulp Stem Cells Bone Differentiation by Activating YAP/TAZ Pathway.

Background: Hyaluronans exist in different forms, accordingly with molecular weight and degree of crosslinking. Here, we tested the capability to induce osteogenic differentiation in hDPSCs (human dental pulp stem cells) of three hyaluronans forms: linear pharmaceutical-grade hyaluronans at high and (HHA) low molecular weight (LHA) and hybrid cooperative complexes (HCC), containing both sizes. Methods: hDPSCs were treated with HHA, LHA, HCC for 7, 14 and 21 days. The effects of hyaluronans on osteogenic differentiation were evaluated by qRT-PCR and WB of osteogenic markers and by Alizarin Red S staining. To identify the involved pathway, CD44 was analyzed by immunofluorescence, and YAP/TAZ expression was measured by qRT-PCR. Moreover, YAP/TAZ inhibitor-1 was used, and the loss of function of YAP/TAZ was evaluated by qRT-PCR, WB and immunofluorescence. Results: We showed that all hyaluronans improves osteogenesis. Among these, HCC is the main inducer of osteogenesis, along with overexpression of bone related markers and upregulating CD44. We also found that this biological process is subordinate to the activation of YAP/TAZ pathway. Conclusions: We found that HA's molecular weight can have a relevant impact on HA performance for bone regeneration, and we unveil a new molecular mechanism by which HA acts on stem cells. Keywords: YAP/TAZ pathway; dental pulp stem cells; hyaluronic acid; osteogenic differentiation

The role of autophagy in resistance to targeted therapies

Autophagy is a self-degradative cellular process, involved in stress response such as starvation, hypoxia, and oxidative stress. This mechanism balances macro-molecule recycling to regulate cell homeostasis. In cancer, autophagy play a role in the development and progression, while several studies describe it as one of the key processes in drug resistance. In the last years, in addition to standard anti-cancer treatments such as chemotherapies and irradiation, targeted therapy became one of the most adopted strategies in clinical practices, mainly due to high specificity and reduced side effects. However, similar to standard treatments, drug resistance is the main challenge in most patients. Here, we summarize recent studies that investigated the role of autophagy in drug resistance after targeted therapy in different types of cancers. We highlight positive results and limitations of pre-clinical and clinical studies in which autophagy inhibitors are used in combination with targeted therapies. Refereed/Peer-reviewe

Three dimensional sphere culture system enhances neural crest-related properties of a sub-population of human dental pulp stem cells expressing STRO-1, c-Kit and CD34 markers

Human dental pulp, a soft connective tissue contained within the pulp chamber of the tooth, is considered an interesting source of adult stem cells, due to the low-invasive procedures required for cell isolation, high content of stem cells and its peculiar embryological origin from neural crest [1-2]. Based on previous findings from our group, a dental pulp stem cells (hDPSCs) population sorted for the expression of STRO-1, c-Kit and CD34 showed a higher commitment towards neurogenic and glial lineages. Moreover, in standard culture conditions STRO-1+/c-Kit+/CD34+ hDPSCS, at late passages, underwent an arrest in cell proliferation and senescence occurred. To this regard, the aim of the present study was to evaluate the ability of three dimensional sphere structures to preserve the biological and stemness properties of this sub-population. In addition, the ability to differentiate towards neurogenic lineage as well as the expression of Fas ligand were investigated. Our data demonstrated that hDPSCs-derived spheres were able to maintain their fibroblast-like morphology and preserved the expression of the stemness markers and their proliferative capability. At late passages, only few cells derived from spheres were positive for β-Galactosidase activity. Interestingly, the expression of neural crest markers was maintained along the whole culture time and the neurogenic commitment was successfully achieved, as confirmed by confocal immunofluorescence and electrophysiological analyses. The expression of FasL, a key molecule for the modulation of immune response, was observed in undifferentiated hDPSCs derived from sphere culture and, surprisingly, it was maintained even after the neurogenic differentiation was reached, whereas after the induction towards osteogenic and myogenic lineages the expression of FasL significantly decreased (

Titanium Functionalized with Polylysine Homopolymers: In Vitro Enhancement of Cells Growth

In oral implantology, the success and persistence of dental implants over time are guaranteed by the bone formation around the implant fixture and by the integrity of the peri-implant mucosa seal, which adheres to the abutment and becomes a barrier that hinders bacterial penetration and colonization close to the outer parts of the implant. Research is constantly engaged in looking for substances to coat the titanium surface that guarantees the formation and persistence of the peri-implant bone, as well as the integrity of the mucous perimeter surrounding the implant crown. The present study aimed to evaluate in vitro the effects of a titanium surface coated with polylysine homopolymers on the cell growth of dental pulp stem cells and keratinocytes to establish the potential clinical application. The results reported an increase in cell growth for both cellular types cultured with polylysine-coated titanium compared to cultures without titanium and those without coating. These preliminary data suggest the usefulness of polylysine coating not only for enhancing osteoinduction but also to speed the post-surgery mucosal healings, guarantee appropriate peri-implant epithelial seals, and protect the fixture against bacterial penetration, which is responsible for compromising the implant survival

Human DPSCs fabricate vascularized woven bone tissue : a new tool in bone tissue engineering

Human dental pulp stem cells (hDPSCs) are mesenchymal stem cells that have been successfully used in human bone tissue engineering. To establish whether these cells can lead to a bone tissue ready to be grafted, we checked DPSCs for their osteogenic and angiogenic differentiation capabilities with the specific aim of obtaining a new tool for bone transplantation. Therefore, hDPSCs were specifically selected from the stromal-vascular dental pulp fraction, using appropriate markers, and cultured. Growth curves, expression of bone-related markers, calcification and angiogenesis as well as an in vivo transplantation assay were performed. We found that hDPSCs proliferate, differentiate into osteoblasts and express high levels of angiogenic genes, such as vascular endothelial growth factor and platelet-derived growth factor A. Human DPSCs, after 40 days of culture, give rise to a 3D structure resembling a woven fibrous bone. These woven bone (WB) samples were analysed using classic histology and synchrotron-based, X-ray phase-contrast microtomography and holotomography. WB showed histological and attractive physical qualities of bone with few areas of mineralization and neovessels. Such WB, when transplanted into rats, was remodelled into vascularized bone tissue. Taken together, our data lead to the assumption that WB samples, fabricated by DPSCs, constitute a noteworthy tool and do not need the use of scaffolds, and therefore they are ready for customized regeneration

Human DPSCs fabricate vascularized woven bone tissue: a new tool in bone tissue engineering

Human dental pulp stem cells (hDPSCs) are mesenchymal stem cells that have been successfully used in human bone tissue engineering. To establish whether these cells can lead to a bone tissue ready to be grafted, we checked DPSCs for their osteogenic and angiogenic differentiation capabilities with the specific aim of obtaining a new tool for bone transplantation. Therefore, hDPSCs were specifically selected from the stromal-vascular dental pulp fraction, using appropriate markers, and cultured. Growth curves, expression of bone-related markers, calcification and angiogenesis as well as an in vivo transplantation assay were performed. We found that hDPSCs proliferate, differentiate into osteoblasts and express high levels of angiogenic genes, such as vascular endothelial growth factor and platelet-derived growth factor A. Human DPSCs, after 40 days of culture, give rise to a 3D structure resembling a woven fibrous bone. These woven bone (WB) samples were analysed using classic histology and synchrotron-based, X-ray phase-contrast microtomography and holotomography. WB showed histological and attractive physical qualities of bone with few areas of mineralization and neovessels. Such WB, when transplanted into rats, was remodelled into vascularized bone tissue. Taken together, our data lead to the assumption that WB samples, fabricated by DPSCs, constitute a noteworthy tool and do not need the use of scaffolds, and therefore they are ready for customized regeneration