Marcella La Noce

Cancer stem cells in solid tumors: an overview and new approaches for their isolation and characterization

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.—Tirino, V., Desiderio, V., Paino, F., De Rosa, A., Papaccio, F., La Noce, M., Laino, L., De Francesco, F., Papaccio, G. Cancer stem cells in solid tumors: an overview and new approaches for their isolation and characterization. FASEB J. 27, 13–24 (2013). www.fasebj.org

Dental pulp stem cells: State of the art and suggestions for a true translation of research into therapy

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.

Histone Deacetylase Inhibition with Valproic Acid Downregulates Osteocalcin Gene Expression in Human Dental Pulp Stem Cells and Osteoblasts: Evidence for HDAC2 Involvement

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. Stem Cells 2014;32:279–289

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.

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.

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, 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.

Hybrid Complexes of High and Low Molecular Weight Hyaluronans Highly Enhance HASCs Differentiation: Implication for Facial Bioremodelling

Background/Aims: Adipose-derived Stem Cells (ASCs) are used in Regenerative Medicine, including fat grafting, recovery from local tissue ischemia and scar remodeling. The aim of this study was to evaluate hyaluronan based gel effects on ASCs differentiation and proliferation. Methods: Comparative analyses using high (H) and low (L) molecular weight hyaluronans (HA), hyaluronan hybrid cooperative complexes (HCCs), and high and medium cross-linked hyaluronan based dermal fillers were performed. Human ASCs were characterized by flow cytometry using CD90, CD34, CD105, CD29, CD31, CD45 and CD14 markers. Then, cells were treated for 7, 14 and 21 days with hyaluronans. Adipogenic differentiation was evaluated using Oil red-O staining and expression of leptin, PPAR-γ, LPL and adiponectin using qRT-PCR. Adiponectin was analyzed by immunofluorescence, PPAR-γ and adiponectin were analyzed using western blotting. ELISA assays for adiponectin and leptin were performed. Results: HCCs highly affected ASCs differentiation by up-regulating adipogenic genes and related proteins, that were also secreted in the culture medium. H-HA and L-HA induced a lower level of ASCs differentiation. Conclusion: HCCs-based formulations clearly enhance adipogenic differentiation and proliferation, when compared with linear HA and cross-linked hyaluronans. Injection of HCCs in subdermal fat compartment may recruit and differentiate stem cells in adipocytes, and considerably improving fat tissue renewal.

NZ-GMP Approved Serum Improve hDPSC Osteogenic Commitment and Increase Angiogenic Factor Expression

Human dental pulp stem cells (hDPSCs), selected from the stromal-vascular fraction of dental pulp, are ecto-mesenchymal stem cells deriving from neural crests, successfully used in human bone tissue engineering. For their use in human therapy GMP procedures are required. For instance, the use of fetal bovine serum (FBS) is strongly discouraged in clinical practice due to its high risk of prions and other infections for human health. Alternatively, clinical grade sera have been suggested, including the New Zealand FBS (NZ-FBS). Therefore, the aim of this study was to evaluate the behavior of hDPSCs expanded in culture medium containing NZ-FBS. Since it was widely demonstrated hDPSCs display relevant capabilities to differentiate into osteogenic and angiogenic lineages, we performed a comparative study to assess if these features are also retained by cultivating the cells with a safer serum never tested on this cell line. hDPSCs were grown using NZ-FBS and conventional (C-FBS) for 7, 14, and 21 days, in both 2D and 3D cultures. Growth curves, expression of bone-related markers, calcification and angiogenesis were evaluated. NZ-FBS induced significant cell growth with respect to C-FBS and promoted an earlier increase expression of osteogenic markers, in particular of those involved in the formation of mineralized matrix (BSP and OPN) within 14 days. In addition, hDPSCs cultured in presence of NZ-FBS were found to produce higher mRNA levels of the angiogenic factors, such as VEGF and PDGFA. Taken together, our results highlight that hDPSCs proliferate, enhance their osteogenic commitment and increase angiogenic factors in NZ-FBS containing medium. These features have also been found when hDPSC were seeded on the clinical-grade collagen I scaffold (Bio-Gide®), leading to the conclusion that for human therapy some procedures and above all the use of GMP-approved materials have no negative impact.

Molecular Profiling of Human Primary Chondrosarcoma-Derived Spheres Reveals Specific and Target Genes Involved in Multidrug Resistance and Metastasis

Chondrosarcoma is a malignant bone tumor that accounts for approximately 25% of all bone neoplasms. The “Cancer Stem Cell” (CSC) hypothesis states that tumor contains a cell subpopulation with stemness features. Sphere cultures are routinely used for self-renewal assays and to select CSCs. Our purpose was to investigate the gene profile of chondrospheres and identify target genes for chondrosarcoma treatment. Whole-genome microarray was used to compare the gene expression of floating spheres with that of adherent counterparts derived from a human primary chondrosarcoma. In addition, CD133, OCT4, SOX2, and collagen type II markers were tested both with Real-Time PCR and flow cytometry, and cell cycle analysis and resistance to cisplatin treatment were performed. Microarray analyses revealed that 1405 genes were found differentially expressed, of which 629 genes were upregulated and 776 down-regulated in chondrospheres, with a 2-fold cut-off threshold. Restricting analyses with a 3-fold cut-off threshold, the number of up-regulated and down-regulated probes was 251 and 302, respectively. The most highly up-regulated genes were involved in stemness, multidrug resistance, cell cycle, apoptosis regulation, migration, motility, and invasion. Furthermore, chondrospheres expressed CD133, OCT3/4, and SOX2, and showed a remarkable resistance to cisplatin-induced apoptosis compared with their adherent counterpart. In conclusion, this study highlights that: (i) the molecular profile of chondrospheres identifies genes that are potential targets for chondrosarcoma treatment and (ii) chondrospheres are strongly resistant to cisplatin treatment.

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.