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Therapies based on mesenchymal stromal cells (MSCs) have become one of the most significant advancements in veterinary regenerative medicine. The isolation of MSCs is usually performed by enzymatic digestion and requires variable times for cell expansion. In addition, these procedures need to be performed in specialized laboratory facilities. An alternative approach to in vitro-expanded MSC therapy is the use of microfragmented adipose tissue (microfat), which is a rich source of cells and growth factors from the stromal vascular fraction. Recent clinical studies support its safety and efficacy in the treatment of musculoskeletal disorders and wound healing. The aim of the present work was to characterize the microfragmented adipose tissue obtained by a new mechanical device, which provides sterile tissue that is ready for use in the clinic by the veterinarian, avoiding the need for specialized laboratory facilities. Microfat-derived MSCs were compared with enzymatically isolated MSCs in terms of their phenotypic characterization, growth rate and differentiation potential. Conditioned medium derived from microfat culture was evaluated for its ability to promote MSC vitality. No differences were observed between MSCs obtained through mechanical fragmentation and those derived from collagenase digestion of adipose tissue, suggesting that the device could serve as a practical source of microfragmented adipose tissue for use in veterinary clinics.

Background and rationale. Pleural mesothelioma (PM) is a rare and aggressive neoplasm that originates from the pleural mesothelium and whose onset is mainly linked to exposure to asbestos, which cannot be attacked with truly effective therapies with consequent poor prognosis. The rationale of this study is based on the use of mesenchymal stromal cells (MSCs) as a vehicle for chemotherapy drugs to be injected directly into the pathological site, such as the pleural cavity. Study design. The study involves the use of a conventional chemotherapeutic drug, Paclitaxel (PTX), which is widely used in the treatment of different types of solid tumors, including PM, although some limitations are related to pharmacokinetic aspects. The use of PTX-loaded MSCs to treat PM should provide several potential advantages over the systemically administered drug as reduced toxicity and increased concentration of active drug in the tumor-surrounding context. The PACLIMES trial explores the safety and toxicity of the local administration of Paclimes in chemonaive patients, candidates for pleurectomy. The secondary objective is to find the effective Paclimes dose for subsequent phase II studies and to observe and record the antitumor activity. Future direction . The experimental pre-clinical background and rationale are discussed as well.

Adipose tissue is mainly composed by adipocytes. Moreover, mesenchymal stromal/stem cells (MSCs), macrophages, endothelial cells, and extracellular matrix components are present. The variety of molecules as cytokines and growth factors of its structure very rich in blood vessel makes it also similar to a true endocrine organ that however needs still to be fully investigated. In our study, we used human lipoaspirate to obtain mechanically microfragmented fat (MiFAT) which was washed and then devitalized by freezing-thawing cycles. In our experiments, thawed MiFAT was used to stimulate cultures of MSCs from two different sources (adipose tissue and gingiva papilla) in comparison with a traditional stimulation in vitro obtained by culturing MSCs with adipogenic medium. MSCs stimulated with MiFAT showed a very early production of lipid droplets, after only 3 days, that correlated with an increased expression of adipokines. Furthermore, a significant upregulation of PPAR gamma 1 alpha coactivator (PPARGC1A) was observed with an overexpression of uncoupling protein 1 (UCP1) that suggest a pattern of differentiation compatible with the beige-brown fat., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2024 Valentina Coccè et al.)

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., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

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.

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 10 6 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.

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 ., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

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 + /PDGFRβ + /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.

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 IC 50 = 3.68 ± 0.69 µM; U373-MG IC 50 = 11.53 ± 0.16 µM; U138-MG IC 50 = 8.05 ± 0.23 µM) that was higher than that observed with the clinically approved CisPt (U87-MG IC 50 = 7.27 + 1.80 µM; U373-MG IC 50 = 22.69 ± 0.05 µM; U138-MG IC 50 = 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.

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.

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.

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., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

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.

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.

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.

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.

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.

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., (© 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

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., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

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, PDGFRβ 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.

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.

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., (Copyright © 2015. Published by Elsevier Ltd.)

Oral squamous cell carcinoma (OSCC) is an aggressive malignancy with high mortality rates. Major challenges for OSCC management include development of resistance to therapy and early formation of distant metastases. Cancer stem cells (CSCs) have emerged as important players in both pathologic mechanisms. Increased fucosylation activity and increased expression of fucosylated polysaccharides, such as Sialyl Lewis X (SLex), are associated with invasion and metastasis. However, the role of fucosylation in CSCs has not been elucidated yet. We used the spheroid culture technique to obtain a CSC-enriched population and compared orospheres with adherent cells. We found that orospheres expressed markers of CSCs and metastasis at higher levels, were more invasive and tumorigenic, and were more resistant to cisplatin/radiation than adherent counterparts. We found fucosyltransferases FUT3 and FUT6 highly up-regulated, increased SLex expression and increased adhesion by shear flow assays in orospheres. Inhibition of fucosylation negatively affected orospheres formation and invasion of oral CSCs. These results confirm that orospheres are enriched in CSCs and that fucosylation is of paramount importance for CSC invasion. In addition, SLex may play a key role in CSC metastasis. Thus, inhibition of fucosylation may be used to block CSCs and metastatic spread.

Neural crest cells, delaminating from the neural tube during migration, undergo an epithelial-mesenchymal transition and differentiate into several cell types strongly reinforcing the mesoderm of the craniofacial body area - giving rise to bone, cartilage and other tissues and cells of this human body area. Recent studies on craniomaxillofacial neural crest-derived cells have provided evidence for the tremendous plasticity of these cells. Actually, neural crest cells can respond and adapt to the environment in which they migrate and the cranial mesoderm plays an important role toward patterning the identity of the migrating neural crest cells. In our experience, neural crest-derived stem cells, such as dental pulp stem cells, can actively proliferate, repair bone and give rise to other tissues and cytotypes, including blood vessels, smooth muscle, adipocytes and melanocytes, highlighting that their use in tissue engineering is successful. In this review, we provide an overview of the main pathways involved in neural crest formation, delamination, migration and differentiation; and, in particular, we concentrate our attention on the translatability of the latest scientific progress. Here we try to suggest new ideas and strategies that are needed to fully develop the clinical use of these cells. This effort should involve both researchers/clinicians and improvements in good manufacturing practice procedures. It is important to address studies towards clinical application or take into consideration that studies must have an effective therapeutic prospect for humans. New approaches and ideas must be concentrated also toward stem cell recruitment and activation within the human body, overcoming the classical grafting.

Objectives: Stem cells have the ability to rescue and/or repair injured tissue. In humans, it is possible to isolate different types of stem cells from the body. Among these, dental pulp stem cells (DPSCs) are relatively easily obtainable and exhibit high plasticity and multipotential capabilities. In particular they represent a gold standard for neural-crest-derived bone reconstruction in humans and can be used for the repair of body defects in low-risk autologous therapeutic strategies., Sources: An electronic search was conducted on PubMed databases and supplemented with a manual study of relevant references., Results: All research described in this review highlight that DPSCs are mesenchymal stem cells that could be used in clinical applications. Unfortunately, very few clinical trials have been reported. Major obstacles imposed on researchers are hindering the translation of potentially effective therapies to the clinic. Both researchers and regulatory institutions need to develop a new approach to this problem, drawing up a new policy for good manufacturing practice (GMP) procedures. We strongly suggest that only general rules be standardized rather than everything. Importantly, this would not have an effect on the safety of patients, but may very well affect the results, which cannot be identical for all patients, due to physiological diversity in the biology of each patient. Alternatively, it would be important to study the role of specific molecules that recruit endogenous stem cells for tissue regeneration. In this way, the clinical use of stem cells could be successfully developed., Conclusions: DPSCs are mesenchymal stem cells that differentiate into different tissues, maintain their characteristics after cryopreservation, differentiate into bone-like tissues when loaded on scaffolds in animal models, and regenerate bone in human grafts. In summary, all data reported up to now should encourage the development of clinical procedures using DPSCs., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Bone defects are one of the most serious pathologies that need tissue regeneration therapies. Studies on mesenchymal stem cells are changing the way we treat bone diseases. MSCs have been used for the treatment of osteogenesis imperfecta, hypophosphatasia, osteonecrosis of the femoral head, osteoporosis, rheumatoid arthritis and osteoarthritis. In this context, it is becoming ever more clear that the future of therapies will be based on the use of stem cells. In this concise review, we highlight the importance of the use of MSCs in bone diseases, focusing on the role of histone deacetylases and Wnt pathways involved in osteogenesis. A better understanding of MSC biology and osteogenesis is needed in order to develop new and targeted therapeutic strategies for the treatment of bone diseases/disorders., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Adult mesenchymal stem cells, such as dental pulp stem cells, are of great interest for cell-based tissue engineering strategies because they can differentiate into a variety of tissue-specific cells, above all, into osteoblasts. In recent years, epigenetic studies on stem cells have indicated that specific histone alterations and modifying enzymes play essential roles in cell differentiation. However, although several studies have reported that valproic acid (VPA)-a selective inhibitor of histone deacetylases (HDAC)-enhances osteoblast differentiation, data on osteocalcin expression-a late-stage marker of differentiation-are limited. We therefore decided to study the effect of VPA on dental pulp stem cell differentiation. A low concentration of VPA did not reduce cell viability, proliferation, or cell cycle profile. However, it was sufficient to significantly enhance matrix mineralization by increasing osteopontin and bone sialoprotein expression. In contrast, osteocalcin levels were decreased, an effect induced at the transcriptional level, and were strongly correlated with inhibition of HDAC2. In fact, HDAC2 silencing with shRNA produced a similar effect to that of VPA treatment on the expression of osteoblast-related markers. We conclude that VPA does not induce terminal differentiation of osteoblasts, but stimulates the generation of less mature cells. Moreover, specific suppression of an individual HDAC by RNA interference could enhance only a single aspect of osteoblast differentiation, and thus produce selective effects., (© AlphaMed Press.)

Mesenchymal stem cell (MSC) therapy holds promise for treating diseases and tissue repair. Regeneration of skeletal muscle tissue that is lost during pathological muscle degeneration or after injuries is sustained by the production of new myofibers. Human Adipose stem cells (ASCs) have been reported to regenerate muscle fibers and reconstitute the pericytic cell pool after myogenic differentiation in vitro. Our aim was to evaluate the differentiation potential of constructs made from a new cross-linked hyaluronic acid (XHA) scaffold on which different sorted subpopulations of ASCs were loaded. Thirty days after engraftment in mice, we found that NG2(+) ASCs underwent a complete myogenic differentiation, fabricating a human skeletal muscle tissue, while NG2(-) ASCs merely formed a human adipose tissue. Myogenic differentiation was confirmed by the expression of MyoD, MF20, laminin, and lamin A/C by immunofluorescence and/or RT-PCR. In contrast, adipose differentiation was confirmed by the expression of adiponectin, Glut-4, and PPAR-γ. Both tissues formed expressed Class I HLA, confirming their human origin and excluding any contamination by murine cells. In conclusion, our study provides novel evidence that NG2(+) ASCs loaded on XHA scaffolds are able to fabricate a human skeletal muscle tissue in vivo without the need of a myogenic pre-differentiation step in vitro. We emphasize the translational significance of our findings for human skeletal muscle regeneration., (Copyright © 2013 Wiley Periodicals, Inc.)

Stem cell based therapies for the repair and regeneration of various tissues are of great interest for a high number of diseases. Adult stem cells, instead, are more available, abundant and harvested with minimally invasive procedures. In particular, mesenchymal stem cells (MSCs) are multi-potent progenitors, able to differentiate into bone, cartilage, and adipose tissues. Human adult adipose tissue seems to be the most abundant source of MSCs and, due to its easy accessibility; it is able to give a considerable amount of stem cells. In this study, we selected MSCs co-expressing CD34 and CD90 from adipose tissue. This stem cell population displayed higher proliferative capacity than CD34(-) CD90(-) cells and was able to differentiate in vitro into adipocytes (PPARγ(+) and adiponectin(+)) and endothelial cells (CD31(+) VEGF(+) Flk1(+)). In addition, in methylcellulose without VEGF, it formed a vascular network. The aim of this study was to investigate differentiation potential of human adipose CD34(+) /CD90(+) stem cells loaded onto commercial collagen sponges already used in clinical practice (Gingistat) both in vitro and in vivo. The results of this study clearly demonstrate that human adult adipose and loose connective tissues can be obtained in vivo, highlighting that CD34(+) /CD90 ASCs are extremely useful for regenerative medicine., (Copyright © 2012 Wiley Periodicals, Inc.)

Mesenchymal stem cells deriving from dental pulp differentiate into osteoblasts capable of producing bone. In previous studies, we extensively demonstrated that, when seeded on collagen I scaffolds, these cells can be conveniently used for the repair of human mandible defects. Here, we assess the stability and quality of the regenerated bone and vessel network 3 years after the grafting intervention, with conventional procedures and in-line holotomography, an advanced phase-imaging method using synchrotron radiation that offers improved sensitivity toward low-absorbing structures. We found that the regenerated tissue from the graft sites was composed of a fully compact bone with a higher matrix density than control human alveolar spongy bone from the same patient. Thus, the regenerated bone, being entirely compact, is completely different from normal alveolar bone. Although the bone regenerated at the graft sites is not of the proper type found in the mandible, it does seem to have a positive clinical impact. In fact, it creates steadier mandibles, may well increase implant stability, and, additionally, may improve resistance to mechanical, physical, chemical, and pharmacological agents.

Primary tumors are responsible for 10% of cancer deaths. In most cases, the main cause of mortality is the formation of metastases. Accumulating evidence suggests that a subpopulation of tumor cells with distinct stem-like properties is responsible for tumor initiation, invasive growth, and metastasis formation. This population is defined as cancer stem cells (CSCs). Existing therapies have enhanced the length of survival after diagnosis of cancer but have completely failed in terms of recovery. CSCs appear to be resistant to chemotherapy, may remain quiescent for extended periods, and have affinity for hypoxic environments. The CSCs can be identified and isolated by different methodologies, including isolation by CSC-specific cell surface marker expression, detection of side population phenotype by Hoechst 33342 exclusion, assessment of their ability to grow as floating spheres, and aldehyde dehydrogenase (ALDH) activity assay. None of the methods mentioned are exclusively used to isolate the solid tumor CSCs, highlighting the imperative to delineate more specific markers or to use combinatorial markers and methodologies. This review provides an overview of the main characteristics and approaches used to identify, isolate, and characterize CSCs from solid tumors.

Dental pulp stem cells (DPSCs) can be found within the "cell rich zone" of the dental pulp. Their embryonic origin, from neural crests, explains their multipotency. Up to now, it has been demonstrated that these cells are capable of producing bone tissue, both in vitro and in vivo, as well as a simil-dentin tissue, in vitro. In addition, it has been reported that these cells differentiate into adipocytes, endotheliocytes, melanocytes, neurons, and glial cells and can be easily cryopreserved and stored for long periods of time and retain their multipotency and bone-producing capacity. Moreover, recent attention has been focused on tissue engineering and on the properties of these cells: several scaffolds have been used to promote 3D tissue formation and studies have demonstrated that DPSCs show good adherence and bone tissue formation on microconcavity surface textures. In addition, adult bone tissue with good vascularization has been obtained in grafts. Interestingly, they seem to possess immunoprivileges as they can be grafted into allogenic tissues and seem to exert anti-inflammatory abilities, like many other mesenchymal stem cells. Their recent use in clinical trials for bone repair enforces the notion that DPSCs can be used successfully in patients. Therefore, their isolation, selection, differentiation, and banking are of great importance. The isolation technique used in most laboratories is based on the use of flow cytometry with cell sorter termed FACS (fluorescent activated cell sorter). It is now important to obtain new methods/protocols to select and isolate stem cells without staining by fluorescent markers or use of magnetic beads. These new procedures should be based on biophysical differences among the different cell populations in order to obtain interesting peculiarities for implementation in biomedical/clinical laboratories. It is emphasized that the new methods must address simplicity and short times of preparation and use of samples, complete sterility of cells, the potential disposable, low cost and complete maintenance of the viability, and integrity of the cells with real-time response for subsequent applications in the biomedical/clinical/surgical fields.

The study and investigation of cancer stem cells (CSCs) or tumour initiating cells (TICs) have received enormous attention over the 10 years. CSCs are rare, quiescent and capable of self-renewing and maintaining tumour growth and heterogeneity. Better understanding of CSCs will no doubt lead to a new era of both basic and clinical cancer research, reclassification of human tumours and development of novel therapeutic strategies. Therefore, the biological properties of CSCs, the relevance of CSCs to cancer therapy and methodologies to identify them are essential in order to address real and efficacious therapeutic strategies to eradicate the cancer. Here, we describe the main protocols to identify CSCs starting from primary tumours including glioblastoma, sarcoma, lung and breast cancers.

Dental pulp stem cells (DPSCs), originating from neural crests, can be found within dental pulp. Up to now, it has been demonstrated that these cells are capable of producing bone tissue, both in vitro and in vivo and differentiate into adipocytes, endotheliocytes, melanocytes, neurons, glial cells, and can be easily cryopreserved and stored. Moreover, recent attention has been focused on tissue engineering and on the properties of these cells. In addition, adult bone tissue with good vascularisation has been obtained in grafts. The latest use in clinical trials for bone repair enforces the notion that DPSCs can be used successfully in patients. Therefore, their isolation, selection, differentiation and banking is of great importance. The isolation and detection techniques used in most laboratories are based on the use of antibodies revealed by flow-cytometers with cell sorter termed FACS (fluorescent activated cell sorter). In this report, we focus our attention on the main procedures used in the selection of DPSCs by flow cytometry, cell culture, freezing/thawing, cell cycle evaluation, histochemistry/immunofluorescence and differentiation of DPSCs. In addition, new methods/protocols to select and isolate stem cells without staining by fluorescent markers for implementation in biomedical/clinical laboratories are discuss. We emphasize that the new methods must address simplicity and short times of preparation and use of samples, complete sterility of cells, the potential disposable, low cost and complete maintenance of the viability and integrity of the cells with real-time response for subsequent applications in the biomedical/clinical/surgical fields.

This study aimed to identify, isolate, and characterize cancer stem cells from human primary sarcomas. We performed cytometric analyses for stemness and differentiation antigens, including CD29, CD34, CD44, CD90, CD117, and CD133, on 21 human primary sarcomas on the day of surgery. From sarcoma biopsies, we obtained 2 chondrosarcoma-stabilized cell lines and 2 osteosarcoma stabilized cell lines, on which sphere formation, side population profile, stemness gene expression, and in vivo and in vitro assays were performed. All samples expressed the CD133, CD44, and CD29 markers. Therefore, we selected a CD133(+) subpopulation from stabilized cell lines that displayed the capacity to grow as sarcospheres able to initiate and sustain tumor growth in nonobese diabetic/severe combined (NOD/SCID) mice, to express stemness genes, including OCT3/4, Nanog, Sox2, and Nestin, and to differentiate into mesenchymal lineages, such as osteoblasts and adipocytes. Our findings show the existence of cancer stem cells in human primary bone sarcomas and highlight CD133 as a pivotal marker for identification of these cells. This may be of primary importance in the development of new therapeutic strategies and new prognostic procedures against these highly aggressive and metastatic tumors.

The aim of this study was to evaluate the behavior of human Dental Pulp Stem Cells (DPSCs), as well as human osteoblasts, when challenged on a Biocoral scaffold, which is a porous natural hydroxyapatite. For this purpose, human DPSCs were seeded onto a three-dimensional (3D) Biocoral scaffold or on flask surface (control). Either normal or rotative (3D) cultures were performed. Scanning electron microscopic analyses, at 8, 24 and 48 h of culture showed that cells did not adhere on the external surface, but moved into the cavities inside the Biocoral structure. After 7, 15 and 30 days of culture, morphological and molecular analyses suggested that the Biocoral scaffold leads DPSCs to hook into the cavities where these cells quickly start to secrete the extra cellular matrix (ECM) and differentiate into osteoblasts. Control human osteoblasts also moved into the internal cavities where they secreted the ECM. Histological sections revealed a diffuse bone formation inside the Biocoral samples seeded with DPSCs or human osteoblasts, where the original scaffold and the new secreted biomaterial were completely integrated and cells were found within the remaining cavities. In addition, RT-PCR analyses showed a significant increase of osteoblast-related gene expression and, above all, of those genes highly expressed in mineralized tissues, including osteocalcin, OPN and BSP. Furthermore, the effects on the interaction between osteogenesis and angiogenesis were observed and substantiated by ELISA assays. Taken together, our results provide clear evidence that DPSCs differentiated into osteoblasts, forming a biocomplex made of Biocoral, ECM and differentiated cells.

During human embryonic development, odontogenic tissues, deriving from the neural crest, remain undifferentiated until the adult age. This study was aimed at characterising the cells of the follicle enveloping the dental germ, due to its direct origin from neural crests. Sixty dental follicles were collected from patients aged 18 to 45 years. This research has clarified that dental follicles, if extracted in a very early stage, when dental roots did not start to be formed, contain a lineage of cells, characterised by a high degree of plasticity in comparison with other adult stem cell populations. In particular, we found that these cells share the following features with ES: (i) high levels of embryonic stem cell markers (CD90, TRA1-60, TRA1-81, OCT-4, CD133, and SSEA-4); (ii) mRNA transcripts for Nanog and Rex-1; (iii) broader potency, being able to differentiate in cell types of all three germ layer, including smooth and skeletal muscle, osteoblasts, neurons, glial cells, and adipocytes; (iv) high levels of telomerase activity; (v) ability to form embryoid bodies; (vi) ability, after injection in murine blastocysts, to be localised within the inner cell mass; (vii) no teratoma formation after injection; (viii) in vivo tissue formation after transplantation. Our results demonstrate that these cells represent a very easy accessible and extraordinary source of pluripotent cells and point out the fact that they own the cardinal feature of embryonic stem cells.