Mechanically Activated Adipose Tissue as a Source for Novel Therapies in Neurological Disease/Injury.

In this review, we describe a new avenue that involves the therapeutic use of human adipose tissue. In the past two decades, thousands of papers have described the potential clinical use of human fat and adipose tissue. Moreover, mesenchymal stem cells have been a source of great enthusiasm in clinical studies, and these have generated curiosity at academic levels. On the other hand, they have created considerable commercial business opportunities. High expectations have emerged for curing some recalcitrant diseases or reconstructing anatomically defective human body parts, but several concerns have been raised by generating criticism on the clinical practice that have not been substantiated by rigorous scientific evidence. However, in general, the consensus is that human adipose-derived mesenchymal stem cells inhibit the production of inflammatory cytokines and stimulate the production of anti-inflammatory cytokines. Here, we show that the application of a mechanical elliptical force for several minutes to human abdominal fat activates anti-inflammatory properties and gene-related expression. This may pave the way for new unexpected clinical developments.

A New Paclitaxel Formulation Based on Secretome Isolated from Mesenchymal Stem Cells Shows a Significant Cytotoxic Effect on Osteosarcoma Cell Lines.

BACKGROUND: Osteosarcoma (OS) represents a rare cancer with an unfavorable prognosis that needs innovative treatment. The aim was to isolate a secretome from mesenchymal stem cells (MSCs) that are treated with paclitaxel (PTX)-containing microvesicles as a drug delivery system and analyze its cytotoxic effects on OS cell lines (SJSA, MG63, and HOS). METHODS: Three batches of secretome (SECR-1, SECR-2, and SECR-3) were produced from three bone marrow (BM) MSCs samples treated for 24 h with 15 µg/mL of PTX or with a standard medium. The viability of the OS cell lines after 5 days of exposure to SECR-1-2-3 (pure and diluted to 1:2 and 1:4) was analyzed with an MTT assay. The same SECR batches were analyzed with high-performance liquid chromatography (HPLC) and with a nanoparticle tracking assay (NTA). RESULTS: A statistically significant decrease in the viability of all OS cell lines was observed after treatment with SECR-PTX 1-2-3 in a dose-response manner. The NTA analyses showed the presence of nanoparticles (NPs) with a mean size comparable to that of extracellular vesicles (EVs). The HPLC analyses detected the presence of PTX in minimal doses in all SECR batches. CONCLUSIONS: This proof-of-concept study showed that the conditioned medium isolated from MSCs loaded with PTX had a strong cytotoxic effect on OS cell lines, due to the presence of EV and PTX.

Mesenchymal stromal cells loaded with Paclitaxel (PacliMES) a potential new therapeutic approach on mesothelioma.

Malignant pleural mesothelioma is an asbestos-related tumor originating in mesothelial cells of the pleura that poorly responds to chemotherapeutic approaches. Adult mesenchymal stromal cells derived either from bone marrow or from adipose tissue may be considered a good model for cell-based therapy, a treatment which has experienced significant interest in recent years. The present study confirms that Paclitaxel is effective on mesothelioma cell proliferation in 2D and 3D in vitro cultures, and that 80,000 mesenchymal stromal cells loaded with Paclitaxel inhibit tumor growth at a higher extent than Paclitaxel alone. An in vivo approach to treat in situ mesothelioma xenografts using a minimal amount of 106 mesenchymal stromal cells loaded with Paclitaxel showed the same efficacy of a systemic administration of 10 mg/kg of Paclitaxel. These data strongly support drug delivery system by mesenchymal stromal cells as a useful approach against many solid tumors. We look with interest at the favourable opinion recently expressed by the Italian Drug Agency on the procedure for the preparation of mesenchymal stromal cells loaded with Paclitaxel in large-scale bioreactor systems and their storage until clinical use. This new Advanced Medicinal Therapy Product, already approved for a Phase I clinical trial on mesothelioma patients, could pave the way for mesenchymal stromal cells use as drug delivery system on other solid tumors for adjuvant therapy associated with surgery and radiotherapy.

Conditioned Medium of Mesenchymal Stromal Cells Loaded with Paclitaxel Is Effective in Preclinical Models of Triple-Negative Breast Cancer (TNBC).

Triple-negative breast cancer (TNBC) is a very aggressive disease even in its early stages and is characterized by a severe prognosis. Neoadjuvant chemotherapy is one of the milestones of treatment, and paclitaxel (PTX) is among the most active drugs used in this setting. However, despite its efficacy, peripheral neuropathy occurs in approximately 20-25% of cases and represents the dose-limiting toxicity of this drug. New deliverable strategies to ameliorate drug delivery and reduce side effects are keenly awaited to improve patients' outcomes. Mesenchymal stromal cells (MSCs) have recently been demonstrated as promising drug delivery vectors for cancer treatment. The aim of the present preclinical study is to explore the possibility of a cell therapy approach based on the use of MSCs loaded with PTX to treat TNBC-affected patients. For this purpose, we in vitro evaluated the viability, migration and colony formation of two TNBC cell lines, namely, MDA-MB-231 and BT549, treated with MSC-PTX conditioned medium (MSC-CM PTX) in comparison with both CM of MSCs not loaded with PTX (CTRL) and free PTX. We observed stronger inhibitory effects on survival, migration and tumorigenicity for MSC-CM PTX than for CTRL and free PTX in TNBC cell lines. Further studies will provide more information about activity and potentially open the possibility of using this new drug delivery vector in the context of a clinical study.

Micro-fragmented Fat Inhibits the Progression of Human Mesothelioma Xenografts in Mice.

BACKGROUND: Malignant pleural mesothelioma is a pathology with no effective therapy and a poor prognosis. Our previous study demonstrated an in vitro inhibitory effect on mesothelioma cell lines of both the lysate and secretome of adipose tissue-derived Mesenchymal Stromal Cells. The inhibitory activity on tumor growth has been demonstrated also in vivo: five million Mesenchymal Stromal Cells, injected "in situ", produced a significant therapeutic efficacy against MSTO-211H xenograft equivalent to that observed after the systemic administration of paclitaxel. OBJECTIVE: The objective of this study is to evaluate the efficacy of low amount (half a million) Mesenchymal Stromal Cells and micro-fragmented adipose tissues (the biological tissue from which the Mesenchymal Stromal Cells were isolated) on mesothelioma cells growth. METHODS: Tumor cells growth inhibition was evaluated in vitro and in a xenograft model of mesothelioma. RESULTS: The inhibitory effect of micro-fragmented fat from adipose-tissue has been firstly confirmed in vitro on MSTO-211H cell growth. Then the efficacy against the growth of mesothelioma xenografts in mice of both micro-fragmented fat and low amount of Mesenchymal Stromal Cells has been evaluated. Our results confirmed that both Mesenchymal Stromal Cells and micro-fragmented fat, injected "in situ", did not stimulate mesothelioma cell growth. By contrast, micro-fragmented fat produced a significant inhibition of tumor growth and progression, comparable to that observed by the treatment with paclitaxel. Low amount of Mesenchymal Stromal Cells exerted only a little anticancer activity. CONCLUSION: Micro-fragmented fat inhibited mesothelioma cell proliferation in vitro and exerted a significant control of the mesothelioma xenograft growth in vivo.

CD146+ Pericytes Subset Isolated from Human Micro-Fragmented Fat Tissue Display a Strong Interaction with Endothelial Cells: A Potential Cell Target for Therapeutic Angiogenesis.

Pericytes (PCs) are mesenchymal stromal cells (MSCs) that function as support cells and play a role in tissue regeneration and, in particular, vascular homeostasis. PCs promote endothelial cells (ECs) survival which is critical for vessel stabilization, maturation, and remodeling. In this study, PCs were isolated from human micro-fragmented adipose tissue (MFAT) obtained from fat lipoaspirate and were characterized as NG2+/PDGFRß+/CD105+ cells. Here, we tested the fat-derived PCs for the dispensability of the CD146 marker with the aim of better understanding the role of these PC subpopulations on angiogenesis. Cells from both CD146-positive (CD146+) and negative (CD146-) populations were observed to interact with human umbilical vein ECs (HUVECs). In addition, fat-derived PCs were able to induce angiogenesis of ECs in spheroids assay; and conditioned medium (CM) from both PCs and fat tissue itself led to the proliferation of ECs, thereby marking their role in angiogenesis stimulation. However, we found that CD146+ cells were more responsive to PDGF-BB-stimulated migration, adhesion, and angiogenic interaction with ECs, possibly owing to their higher expression of NCAM/CD56 than the corresponding CD146- subpopulation. We conclude that in fat tissue, CD146-expressing cells may represent a more mature pericyte subpopulation that may have higher efficacy in controlling and stimulating vascular regeneration and stabilization than their CD146-negative counterpart.

In Vitro Activity of Monofunctional Pt-II Complex Based on 8-Aminoquinoline against Human Glioblastoma.

A new cationic Pt(II) complex bearing 8-aminoquinoline as chelating ligand (called Pt-8AQ) was evaluated against two human carcinomas, one mesothelioma, and three glioblastoma cell lines. The in vitro comparison to the clinically approved CisPt showed a minor activity of Pt-8AQ against carcinoma and mesothelioma, whereas a significant activity of Pt-8AQ was observed on the proliferation of the three glioblastoma cell lines (U87-MG IC50 = 3.68 ± 0.69 µM; U373-MG IC50 = 11.53 ± 0.16 µM; U138-MG IC50 = 8.05 ± 0.23 µM) that was higher than that observed with the clinically approved CisPt (U87-MG IC50 = 7.27 + 1.80 µM; U373-MG IC50 = 22.69 ± 0.05 µM; U138-MG IC50 = 32.1 ± 4.44 µM). Cell cycle analysis proved that Pt-8AQ significantly affected the cell cycle pattern by increasing the apoptotic cells represented by the sub G0/G1 region related with a downregulation of p53 and Bcl-2. Moreover, an NMR investigation of Pt-8AQ interaction with 9-EtG, GSH, and Mets7 excluded DNA as the main target, suggesting a novel mechanism of action. Our study demonstrated the high stability of Pt-8AQ after incubation at 37 °C and a significant antineoplastic activity on glioblastomas. These features also make Pt-8AQ a good candidate for developing a new selective advanced cell chemotherapy approach in combination with MSCs.

Single-Shot Local Injection of Microfragmented Fat Tissue Loaded with Paclitaxel Induces Potent Growth Inhibition of Hepatocellular Carcinoma in Nude Mice.

Hepatocellular carcinoma (HCC) is poorly beneficiated by intravenous chemotherapy due to inadequate availability of drugs at the tumor site. We previously demonstrated that human micro-fragmented adipose tissue (MFAT) and its devitalized counterpart (DMFAT) could be effective natural scaffolds to deliver Paclitaxel (PTX) to tumors in both in vitro and in vivo tests, affecting cancer growth relapse. Here we tested the efficacy of DMFAT-PTX in a well-established HCC in nude mice. MFAT-PTX and DMFAT-PTX preparations were tested for anti-cancer activity in 2D and 3D assays using Hep-3B tumor cells. The efficacy of DMFAT-PTX was evaluated after a single-shot subcutaneous injection near a Hep-3B growing tumor by assessing tumor volumes, apoptosis rate, and drug pharmacokinetics in an in vivo model. Potent antiproliferative activity was seen in both in vitro 2D and 3D tests. Mice treated with DMFAT-PTX (10 mg/kg) produced potent Hep-3B growth inhibition with 33% complete tumor regressions. All treated animals experienced tumor ulceration at the site of DMFAT-PTX injection, which healed spontaneously. Lowering the drug concentration (5 mg/kg) prevented the formation of ulcers, maintaining statistically significant efficacy. Histology revealed a higher number of apoptotic cancer cells intratumorally, suggesting prolonged presence of PTX that was confirmed by the pharmacokinetic analysis. DMFAT may be a potent and valid new tool for local chemotherapy of HCC in an advanced stage of progression, also suggesting potential effectiveness in other human primary inoperable cancers.

Inhibition of Human Malignant Pleural Mesothelioma Growth by Mesenchymal Stromal Cells.

BACKGROUND: Malignant Pleural Mesothelioma (MPM) is an aggressive tumor that has a significant incidence related to asbestos exposure with no effective therapy and poor prognosis. The role of mesenchymal stromal cells (MSCs) in cancer is controversial due to their opposite effects on tumor growth and in particular, only a few data are reported on MSCs and MPM. METHODS: We investigated the in vitro efficacy of adipose tissue-derived MSCs, their lysates and secretome against different MPM cell lines. After large-scale production of MSCs in a bioreactor, their efficacy was also evaluated on a human MPM xenograft in mice. RESULTS: MSCs, their lysate and secretome inhibited MPM cell proliferation in vitro with S or G0/G1 arrest of the cell cycle, respectively. MSC lysate induced cell death by apoptosis. The efficacy of MSC was confirmed in vivo by a significant inhibition of tumor growth, similar to that produced by systemic administration of paclitaxel. Interestingly, no tumor progression was observed after the last MSC treatment, while tumors started to grow again after stopping chemotherapeutic treatment. CONCLUSIONS: These data demonstrated for the first time that MSCs, both through paracrine and cell-to-cell interaction mechanisms, induced a significant inhibition of human mesothelioma growth. Since the prognosis for MPM patients is poor and the options of care are limited to chemotherapy, MSCs could provide a potential new therapeutic approach for this malignancy.

Paclitaxel Priming of TRAIL Expressing Mesenchymal Stromal Cells (MSCs-TRAIL) Increases Antitumor Efficacy of Their Secretome.

BACKGROUND: Adipose tissue derived MSCs engineered with the tumor necrosis factor-related apoptosis-inducing ligand protein (MSCs-TRAIL) have a significant anticancer activity. MSCs, without any genetic modifications, exposed to high doses of chemotherapeutic agents are able to uptake the drug and release it in amount affecting tumor proliferation. The purpose of this study was to verify the ability of MSCs-TRAIL to uptake and release paclitaxel (PTX) by providing an increased antitumor efficacy. METHODS: MSCs and MSCs-TRAIL were tested for their sensitivity to Paclitaxel (PTX) by MTT assay and the cells were loaded with PTX according to a standardized procedure. The secretome was analysed by HPLC for the presence of PTX, microarray assay for soluble TRAIL (s-TRAIL) and tested for in vitro anticancer activity. RESULTS: MSCs-TRAIL were resistant to PTX and able to incorporate and then release the drug. The secretion of s-TRAIL by PTX loaded MSCs-TRAIL was not inhibited and the PTX delivery together with s-TRAIL secretion resulted into an increased antitumor efficacy of cell secretoma as tested in vitro on human pancreatic carcinoma (CFPAC-1) and glioblastoma (U87-MG). CONCLUSIONS: Our result is the first demonstration of the possible merging of two new MSCs therapy approaches based on genetic manipulation and drug delivery. If confirmed in vivo, this could potentiate the efficacy of MSCs-TRAIL and strongly contribute to reduce the toxicity due to the systemic treatment of PTX.

Automated Large-Scale Production of Paclitaxel Loaded Mesenchymal Stromal Cells for Cell Therapy Applications.

Mesenchymal stromal cells (MSCs) prepared as advanced therapies medicinal products (ATMPs) have been widely used for the treatment of different diseases. The latest developments concern the possibility to use MSCs as carrier of molecules, including chemotherapeutic drugs. Taking advantage of their intrinsic homing feature, MSCs may improve drugs localization in the disease area. However, for cell therapy applications, a significant number of MSCs loaded with the drug is required. We here investigate the possibility to produce a large amount of Good Manufacturing Practice (GMP)-compliant MSCs loaded with the chemotherapeutic drug Paclitaxel (MSCs-PTX), using a closed bioreactor system. Cells were obtained starting from 13 adipose tissue lipoaspirates. All samples were characterized in terms of number/viability, morphology, growth kinetics, and immunophenotype. The ability of MSCs to internalize PTX as well as the antiproliferative activity of the MSCs-PTX in vitro was also assessed. The results demonstrate that our approach allows a large scale expansion of cells within a week; the MSCs-PTX, despite a different morphology from MSCs, displayed the typical features of MSCs in terms of viability, adhesion capacity, and phenotype. In addition, MSCs showed the ability to internalize PTX and finally to kill cancer cells, inhibiting the proliferation of tumor lines in vitro. In summary our results demonstrate for the first time that it is possible to obtain, in a short time, large amounts of MSCs loaded with PTX to be used in clinical trials for the treatment of patients with oncological diseases.

Glucose-6-phosphate dehydrogenase blockade potentiates tyrosine kinase inhibitor effect on breast cancer cells through autophagy perturbation.

BACKGROUND: Glucose-6-phospate dehydrogenase (G6PD) is the limiting enzyme of the pentose phosphate pathway (PPP) correlated to cancer progression and drug resistance. We previously showed that G6PD inhibition leads to Endoplasmic Reticulum (ER) stress often associated to autophagy deregulation. The latter can be induced by target-based agents such as Lapatinib, an anti-HER2 tyrosine kinase inhibitor (TKI) largely used in breast cancer treatment. METHODS: Here we investigate whether G6PD inhibition causes autophagy alteration, which can potentiate Lapatinib effect on cancer cells. Immunofluorescence and flow cytometry for LC3B and lysosomes tracker were used to study autophagy in cells treated with lapatinib and/or G6PD inhibitors (polydatin). Immunoblots for LC3B and p62 were performed to confirm autophagy flux analyses together with puncta and colocalization studies. We generated a cell line overexpressing G6PD and performed synergism studies on cell growth inhibition induced by Lapatinib and Polydatin using the median effect by Chou-Talay. Synergism studies were additionally validated with apoptosis analysis by annexin V/PI staining in the presence or absence of autophagy blockers. RESULTS: We found that the inhibition of G6PD induced endoplasmic reticulum stress, which was responsible for the deregulation of autophagy flux. Indeed, G6PD blockade caused a consistent increase of autophagosomes formation independently from mTOR status. Cells engineered to overexpress G6PD became resilient to autophagy and resistant to lapatinib. On the other hand, G6PD inhibition synergistically increased lapatinib-induced cytotoxic effect on cancer cells, while autophagy blockade abolished this effect. Finally, in silico studies showed a significant correlation between G6PD expression and tumour relapse/resistance in patients. CONCLUSIONS: These results point out that autophagy and PPP are crucial players in TKI resistance, and highlight a peculiar vulnerability of breast cancer cells, where impairment of metabolic pathways and autophagy could be used to reinforce TKI efficacy in cancer treatment.

Cytoplasmic Interactions between the Glucocorticoid Receptor and HDAC2 Regulate Osteocalcin Expression in VPA-Treated MSCs.

Epigenetic regulation has been considered an important mechanism for influencing stem cell differentiation. In particular, histone deacetylases (HDACs) have been shown to play a role in the osteoblast differentiation of mesenchymal stem cells (MSCs). In this study, the effect of the HDAC inhibitor, valproic acid (VPA), on bone formation in vivo by MSCs was determined. Surprisingly, VPA treatment, unlike other HDAC inhibitors, produced a well-organized lamellar bone tissue when MSCs⁻collagen sponge constructs were implanted subcutaneously into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice, although a decrease of osteocalcin (OC) expression was observed. Consequently, we decided to investigate the molecular mechanisms by which VPA exerts such effects on MSCs. We identified the glucocorticoid receptor (GR) as being responsible for that downregulation, and suggested a correlation between GR and HDAC2 inhibition after VPA treatment, as evidenced by HDAC2 knockdown. Furthermore, using co-immunoprecipitation analysis, we showed for the first time in the cytoplasm, binding between GR and HDAC2. Additionally, chromatin immunoprecipitation (ChIP) assays confirmed the role of GR in OC downregulation, showing recruitment of GR to the nGRE element in the OC promoter. In conclusion, our results highlight the existence of a cross-talk between GR and HDAC2, providing a mechanistic explanation for the influence of the HDAC inhibitor (namely VPA) on osteogenic differentiation in MSCs. Our findings open new directions in targeted therapies, and offer new insights into the regulation of MSC fate determination.

HDAC2 depletion promotes osteosarcoma's stemness both in vitro and in vivo: a study on a putative new target for CSCs directed therapy.

BACKGROUND: Cancer stem cells (CSCs) play a key role in cancer initiation, progression and chemoresistance. Epigenetic alterations have been identified as prominent factors that contribute to the CSCs phenotype. Here, we investigated the effects of the HDAC inhibitor valproic acid (VPA) and the demethylating agent, 5'azacytidine (DAC) on the stem phenotype of MG63 and Saos2 osteosarcoma cell lines. METHODS: Saos2 and MG63 cells were treated with DAC and VPA, alone and in combination. Untreated and treated cells were examined for stemness phenotype by cytometry and real-time PCR. Sarcospheres and colonies formation were also evaluated. Moreover, histone modification and methylation were tested by flow cytomery and western blotting. HDAC2 depleted cells were examined for stemness phenotype and their ability to generate tumors in NOD/SCID IL2R-gamma-0 (NSG) mice. HDAC2 expression on human osteosarcoma tissues was evaluated. RESULTS: We found that DAC and VPA induce an increased expression of stem markers including CD133, OCT4, SOX2 and NANOG, and an increased ability in sarcospheres and colonies formation efficiency. Interestingly, we showed that DAC and VPA treatment decreased repressive histone markers, while increased the active ones. These histone modifications were also associated with an increase of acetylation of histones H3, a decrease of DNA global methylation, HDAC2 and DNMT3a. Furthermore, HDAC2 silenced-MG63 and Saos2 cells acquired a stem phenotype, and promoted in vivo tumorigenesis. In human osteosarcoma tissues, HDAC2 was strongly expressed in nucleus. CONCLUSIONS: Collectively, our results suggest that VPA and DAC induce an expansion of osteosarcoma CSCs, and we report for the first time that HDAC2 is a key factor regulating both CSCs phenotype and in vivo cancer growth. In conclusion, we have identified HDAC2 as a potential therapeutic target in human osteosarcoma treatment.

A new inhibitor of glucose-6-phosphate dehydrogenase blocks pentose phosphate pathway and suppresses malignant proliferation and metastasis in vivo.

Pentose phosphate pathway (PPP) is a major glucose metabolism pathway, which has a fundamental role in cancer growth and metastasis. Even though PPP blockade has been pointed out as a very promising strategy against cancer, effective anti-PPP agents are not still available in the clinical setting. Here we demonstrate that the natural molecule polydatin inhibits glucose-6-phosphate dehydrogenase (G6PD), the key enzyme of PPP. Polydatin blocks G6PD causing accumulation of reactive oxygen species and strong increase of endoplasmic reticulum stress. These effects are followed by cell cycle block in S phase, an about 50% of apoptosis, and 60% inhibition of invasion in vitro. Accordingly, in an orthotopic metastatic model of tongue cancer, 100 mg/kg polydatin induced an about 30% tumor size reduction with an about 80% inhibition of lymph node metastases and 50% reduction of lymph node size (p < 0.005). Polydatin is not toxic in animals up to a dose of 200 mg/kg and a phase II clinical trial shows that it is also well tolerated in humans (40 mg twice a day for 90 days). Thus, polydatin may be used as a reliable tool to limit human cancer growth and metastatic spread.

Concise Review: Cancer Cells, Cancer Stem Cells, and Mesenchymal Stem Cells: Influence in Cancer Development.

Tumors are composed of different types of cancer cells that contribute to tumor heterogeneity. Among these populations of cells, cancer stem cells (CSCs) play an important role in cancer initiation and progression. Like their stem cells counterpart, CSCs are also characterized by self-renewal and the capacity to differentiate. A particular population of CSCs is constituted by mesenchymal stem cells (MSCs) that differentiate into cells of mesodermal characteristics. Several studies have reported the potential pro-or anti-tumorigenic influence of MSCs on tumor initiation and progression. In fact, MSCs are recruited to the site of wound healing to repair damaged tissues, an event that is also associated with tumorigenesis. In other cases, resident or migrating MSCs can favor tumor angiogenesis and increase tumor aggressiveness. This interplay between MSCs and cancer cells is fundamental for cancerogenesis, progression, and metastasis. Therefore, an interesting topic is the relationship between cancer cells, CSCs, and MSCs, since contrasting reports about their respective influences have been reported. In this review, we discuss recent findings related to conflicting results on the influence of normal and CSCs in cancer development. The understanding of the role of MSCs in cancer is also important in cancer management. Stem Cells Translational Medicine 2017;6:2115-2125.

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 adipose stem cell differentiation is highly affected by cancer cells both in vitro and in vivo: implication for autologous fat grafting.

Recent studies showed that mesenchymal stem cells derived from adipose tissue can promote tumour progression, raising some concerns regarding their use in regenerative medicine. In this context, we co-cultured either SAOS2 osteosarcoma or MCF7 breast cancer cells with human adipose stem cells (hASCs), in order to evaluate potential effects of cancer cells on hASCs differentiation, in vitro and in vivo. In this study we observed that both SAOS2 and MCF7 cell lines induced an increase in hASCs proliferation, compared to hASCs alone, but, surprisingly, neither changes in the expression of CD90, CD29, CD324 and vimentin, nor variations in the Twist and Slug mRNAs were detectable. Noteworthy, SAOS2 and MCF7 cells induced in hASCs an upregulation of CD34 expression and stemness genes, including OCT3/4, Nanog, Sox2 and leptin, and a decrease in angiogenic factors, including CD31, PDGFα, PDGFRα, PDGFRß and VEGF. SMAD and pSMAD2/3 increased only in hASCs alone. After 21 days of co-culture, hASCs differentiated both in adipocytes and endothelial cells. Moreover, co-injection of MCF7 cells with hASCs led to the formation of a highly vascularized tumour. Taken together our findings suggest that mesenchymal stem cells, under tumour cell induction, do not differentiate in vitro or facilitate the angiogenesis of the tumour in vivo, thus opening interesting new scenarios in the relationship between cancer and stem cells. These findings may also lead to greater caution, when managing autologous fat grafts in cancer patients.

Changing Paradigms in Cranio-Facial Regeneration: Current and New Strategies for the Activation of Endogenous Stem Cells.

Craniofacial area represent a unique district of human body characterized by a very high complexity of tissues, innervation and vascularization, and being deputed to many fundamental function such as eating, speech, expression of emotions, delivery of sensations such as taste, sight, and earing. For this reasons, tissue loss in this area following trauma or for example oncologic resection, have a tremendous impact on patients' quality of life. In the last 20 years regenerative medicine has emerged as one of the most promising approach to solve problem related to trauma, tissue loss, organ failure etc. One of the most powerful tools to be used for tissue regeneration is represented by stem cells, which have been successfully implanted in different tissue/organs with exciting results. Nevertheless, both autologous and allogeneic stem cell transplantation raise many practical and ethical concerns that make this approach very difficult to apply in clinical practice. For this reason different cell free approaches have been developed aiming to the mobilization, recruitment, and activation of endogenous stem cells into the injury site avoiding exogenous cells implant but instead stimulating patients' own stem cells to repair the lesion. To this aim many strategies have been used including functionalized bioscaffold, controlled release of stem cell chemoattractants, growth factors, BMPs, Platelet-Rich-Plasma, and other new strategies such as ultrasound wave and laser are just being proposed. Here we review all the current and new strategies used for activation and mobilization of endogenous stem cells in the regeneration of craniofacial tissue.

Surface biocompatibility of differently textured titanium implants with mesenchymal stem cells.

OBJECTIVE: The major challenge for contemporary dentistry is restoration of missing teeth; currently, dental implantation is the treatment of choice in this circumstance. In the present study, we assessed the interaction between implants and Dental Pulp Stem Cells (DPSCs) in vitro by means of 3D cell culture in order to better simulate physiological conditions. METHODS: Sorted CD34+ DPSCs were seeded onto dental implants having either a rough surface (TriVent) or one coated with a ceramic layer mimicking native bone (TiUnite). We evaluated preservation of DPSC viability during osteogenic differentiation by an MTT assay and compared mineralized matrix deposition with SEM analysis and histological staining; temporal expression of osteogenic markers was evaluated by RT-PCR and ELISA. RESULTS: Both surfaces are equally biocompatible, preserve DPSC viability, stimulate osteogenic differentiation, and increase the production of VEGF. A slight difference was observed between the two surfaces concerning the speed of DPSC differentiation. SIGNIFICANCE: Our study of the two implant surfaces suggests that TriVent, with its roughness, is capable of promoting cell differentiation a bit earlier than the TiUnite surface, although the latter promotes greater cell proliferation.