Dental Pulp-Derived Mesenchymal Stem Cells for Modeling Genetic Disorders

A subpopulation of mesenchymal stem cells, developmentally derived from multipotent neural crest cells that form multiple facial tissues, resides within the dental pulp of human teeth. These stem cells show high proliferative capacity in vitro and are multipotent, including adipogenic, myogenic, osteogenic, chondrogenic, and neurogenic potential. Teeth containing viable cells are harvested via minimally invasive procedures, based on various clinical diagnoses, but then usually discarded as medical waste, indicating the relatively low ethical considerations to reuse these cells for medical applications. Previous studies have demonstrated that stem cells derived from healthy subjects are an excellent source for cell-based medicine, tissue regeneration, and bioengineering. Furthermore, stem cells donated by patients affected by genetic disorders can serve as in vitro models of disease-specific genetic variants, indicating additional applications of these stem cells with high plasticity. This review discusses the benefits, limitations, and perspectives of patient-derived dental pulp stem cells as alternatives that may complement other excellent, yet incomplete stem cell models, such as induced pluripotent stem cells, together with our recent data.     

Proteomic Profiling of the First Human Dental Pulp Mesenchymal Stem/Stromal Cells from Carbonic Anhydrase II Deficiency Osteopetrosis Patients

Osteopetrosis is a hereditary disorder characterized by sclerotic, thick, weak, and brittle bone. The biological behavior of mesenchymal cells obtained from osteopetrosis patients has not been well-studied. Isolated mesenchymal stem/stromal cells from dental pulp (DP-MSSCs) of recently extracted deciduous teeth from osteopetrosis (OP) patients and healthy controls (HCs) were compared. We evaluated whether the dental pulp of OP patients has a population of MSSCs with similar multilineage differentiation capability to DP-MSSCs of healthy subjects. Stem/progenitor cells were characterized using immunohistochemistry, flow cytometry, and proteomics. Our DP-MSSCs were strongly positive for CD44, CD73, CD105, and CD90. DP-MSSCs obtained from HC subjects and OP patients showed similar patterns of proliferation and differentiation as well as gene expression. Proteomic analysis identified 1499 unique proteins with 94.3% similarity in global protein fingerprints of HCs and OP patients. Interestingly, we observed subtle differences in expressed proteins of osteopetrosis disease-related in pathways, including MAPK, ERK 1/2, PI3K, and integrin, rather than in the stem cell signaling network. Our findings of similar protein expression signatures in DP-MSSCs of HC and OP patients are of paramount interest, and further in vivo validation study is needed. There is the possibility that OP patients could have their exfoliating deciduous teeth banked for future use in regenerative dentistry.     

A Cell-Based Approach to Dental Pulp Regeneration Using Mesenchymal Stem Cells: A Scoping Review

Despite the recent explosion of investigations on dental pulp regeneration using various tissue engineering strategies, the translation of the findings from such studies into therapeutic applications has not been properly achieved. The purpose of this scoping review was to systematically review the efficacy of mesenchymal stem cell transplantation for dental pulp regeneration. A literature search was conducted using five electronic databases from their inception to January 2021 and supplemented by hand searches. A total of 17 studies, including two clinical trials and 15 animal studies using orthotopic pulp regeneration models, were included for the review. The risk of bias for the individual studies was assessed. This scoping review demonstrated that the regeneration of vascularized pulp-like tissue was achieved using the stem cell transplantation strategy in animal models. Autologous cell transplantation in two clinical studies also successfully regenerated vascularized vital tissue. Dental pulp stem cell subpopulations, such as mobilized dental pulp stem cells, injectable scaffolds such as atelocollagen, and a granulocyte-colony forming factor, were the most commonly used for pulp regeneration. The overall risk of bias was unclear for animal studies and was moderate or judged to raise some concerns for clinical studies. More high-quality clinical studies are needed to further determine the safety and efficacy of the stem cell transplantation strategy for dental pulp regeneration.     

A Hyaluronan-Based Scaffold for the in Vitro Construction of Dental Pulp-Like Tissue

Dental pulp tissue supports the vitality of the tooth, but it is particularly vulnerable to external insults, such as mechanical trauma, chemical irritation or microbial invasion, which can lead to tissue necrosis. In the present work, we present an endodontic regeneration method based on the use of a tridimensional (3D) hyaluronan scaffold and human dental pulp stem cells (DPSCs) to produce a functional dental pulp-like tissue in vitro. An enriched population of DPSCs was seeded onto hyaluronan-based non-woven meshes in the presence of differentiation factors to induce the commitment of stem cells to neuronal, glial, endothelial and osteogenic phenotypes. In vitro experiments, among which were gene expression profiling and immunofluorescence (IF) staining, proved the commitment of DPSCs to the main components of dental pulp tissue. In particular, the hyaluronan-DPSCs construct showed a dental pulp-like morphology consisting of several specialized cells growing inside the hyaluronan fibers. Furthermore, these constructs were implanted into rat calvarial critical-size defects. Histological analyses and gene expression profiling performed on hyaluronan-DPSCs grafts showed the regeneration of osteodentin-like tissue. Altogether, these data suggest the regenerative potential of the hyaluronan-DPSC engineered tissue.     

Concurrent Expression of Oct4 and Nanog Maintains Mesenchymal Stem-Like Property of Human Dental Pulp Cells

Human dental pulp stem cells (DPSCs), unique mesenchymal stem cells (MSCs) type, exhibit the characteristics of self-renewal and multi-lineage differentiation capacity. Oct4 and Nanog are pluripotent genes. The aim of this study was to determine the physiological functions of Oct4 and Nanog expression in DPSCs. Herein, we determined the critical role of an Oct4/Nanog axis modulating MSCs properties of DPSCs by lentiviral-mediated co-overexpression or co-knockdown of Oct4/Nanog in DPSCs. MSCs properties including osteogenic/chondrogenic/adipogenic induction differentiation was assayed for expression of osteogenic/chondrogenic/adipogenic markers by quantitative real-time RT-PCR analysis. Initially, we observed that the expression profile of Oct4 and Nanog in dental pulp cells, which exerted properties of MSCs, was significantly up-regulated compared to that of STRO-1⁻CD146⁻ dental pulp cells. Down-regulation of Oct4 and Nanog co-expression significantly reduced the cell proliferation, osteogenic differentiation capability, STRO-1, CD146, and Alkaline phosphatase (ALP) activity of DPSCs. In contrast, co-overexpression of Oct4 and Nanog enhanced the expression level of STRO-1 and CD146, proliferation rate and osteogenic/chondrogenic/adipogenic induction differentiation capability, and expression of osteogenic/chondrogenic/adipogenic induction differentiation markers. Our results suggest that Oct4-Nanog signaling is a regulatory switch to maintain properties in DPSCs.     

Wharton’s Jelly-Derived Mesenchymal Stem Cells: Phenotypic Characterization and Optimizing Their Therapeutic Potential for Clinical Applications

Wharton’s jelly (WJ) is a gelatinous tissue within the umbilical cord that contains myofibroblast-like stromal cells. A unique cell population of WJ that has been suggested as displaying the stemness phenotype is the mesenchymal stromal cells (MSCs). Because MSCs’ stemness and immune properties appear to be more robustly expressed and functional which are more comparable with fetal than adult-derived MSCs, MSCs harvested from the “young” WJ are considered much more proliferative, immunosuppressive, and even therapeutically active stem cells than those isolated from older, adult tissue sources such as the bone marrow or adipose. The present review discusses the phenotypic characteristics, therapeutic applications, and optimization of experimental protocols for WJ-derived stem cells. MSCs derived from WJ display promising transplantable features, including ease of sourcing, in vitro expandability, differentiation abilities, immune-evasion and immune-regulation capacities. Accumulating evidence demonstrates that WJ-derived stem cells possess many potential advantages as transplantable cells for treatment of various diseases (e.g., cancer, chronic liver disease, cardiovascular diseases, nerve, cartilage and tendon injury). Additional studies are warranted to translate the use of WJ-derived stem cells for clinical applications.     

How bone marrow-derived human mesenchymal stem cells respond to poorly crystalline apatite coated orthopedic and dental titanium implants

Due to the delayed and weak bone-implant integration in dental and orthopedic devices, there have been several attempts to enhance implant–bone interactions for rapid osseointegration. In this paper, the interactions of human bone marrow-derived stromal (mesenchymal) stem cells (hMSCs) with uncoated and coated titanium alloy implants with poorly crystalline apatite are studied. First the configuration and chemical composition of the apatite coatings and their deposition progress in different experimental conditions are investigated and discussed. Then, hMSCs are cultured on different substrates and cell attachment and proliferation are monitored and evaluated for different time intervals. Although the uncoated and coated substrates indicate good cell attachment, the differences in proliferation and morphology of the cells spread over the coated samples are significant. It is concluded that the coated samples improve the capability for accepting the cells in three-dimensional and slender shapes. The migration of hMSCs on both substrates are discussed. As such cell migration is directly associated to the osteoconduction, the findings confirm the hypothesis of enhancement in bone formation on the surface of biomimetically poorly crystalline apatite coated titanium implants. This in vitro study demonstrates that the coated samples are nontoxic and biocompatible enough for ongoing osteogenic studies in bone or dental defects in animal models in vivo.     

Potential Spermatogenesis Recovery with Bone Marrow Mesenchymal Stem Cells in an Azoospermic Rat Model

Non-obstructive azoospermia is the most challenging type of male infertility. Stem cell based therapy provides the potential to enhance the recovery of spermatogenesis following cancer therapy. Bone marrow-derived mesenchymal stem cells (BMSCs) possess the potential to differentiate or trans-differentiate into multi-lineage cells, secrete paracrine factors to recruit the resident stem cells to participate in tissue regeneration, or fuse with the local cells in the affected region. In this study, we tested whether spermatogenically-induced BMSCs can restore spermatogenesis after administration of an anticancer drug. Allogeneic BMSCs were co-cultured in conditioned media derived from cultured testicular Sertoli cells in vitro, and then induced stem cells were transplanted into the seminiferous tubules of a busulfan-induced azoospermatic rat model for 8 weeks. The in vitro induced BMSCs exhibited specific spermatogonic gene and protein markers, and after implantation the donor cells survived and located at the basement membranes of the recipient seminiferous tubules, in accordance with what are considered the unique biological characteristics of spermatogenic stem cells. Molecular markers of spermatogonial stem cells and spermatogonia (Vasa, Stella, SMAD1, Dazl, GCNF, HSP90α, integrinβ1, and c-kit) were expressed in the recipient testis tissue. No tumor mass, immune response, or inflammatory reaction developed. In conclusion, BMSCs might provide the potential to trans-differentiate into spermatogenic-like-cells, enhancing endogenous fertility recovery. The present study indicates that BMSCs might offer alternative treatment for the patients with azoospermatic infertility after cancer chemotherapy.     

Potential Effect of CD271 on Human Mesenchymal Stromal Cell Proliferation and Differentiation

The Low-Affinity Nerve Growth Factor Receptor (LNGFR), also known as CD271, is a member of the tumor necrosis factor receptor superfamily. The CD271 cell surface marker defines a subset of multipotential mesenchymal stromal cells and may be used to isolate and enrich cells derived from bone marrow aspirate. In this study, we compare the proliferative and differentiation potentials of CD271⁺ and CD271⁻ mesenchymal stromal cells. Mesenchymal stromal cells were isolated from bone marrow aspirate and adipose tissue by plastic adherence and positive selection. The proliferation and differentiation potentials of CD271⁺ and CD271⁻ mesenchymal stromal cells were assessed by inducing osteogenic, adipogenic and chondrogenic in vitro differentiation. Compared to CD271⁺, CD271⁻ mesenchymal stromal cells showed a lower proliferation rate and a decreased ability to give rise to osteocytes, adipocytes and chondrocytes. Furthermore, we observed that CD271⁺ mesenchymal stromal cells isolated from adipose tissue displayed a higher efficiency of proliferation and trilineage differentiation compared to CD271⁺ mesenchymal stromal cells isolated from bone marrow samples, although the CD271 expression levels were comparable. In conclusion, these data show that both the presence of CD271 antigen and the source of mesenchymal stromal cells represent important factors in determining the ability of the cells to proliferate and differentiate.     

Characterization and Evaluation of Neuronal Trans-Differentiation with Electrophysiological Properties of Mesenchymal Stem Cells Isolated from Porcine Endometrium

Endometrial stromal cells (EMSCs) obtained from porcine uterus (n = 6) were positive for mesenchymal stem cell markers (CD29, CD44 and CD90), and negative for epithelial marker CD9 and hematopoietic markers CD34, CD45 analyzed by flow cytometry. Further the cells were positive for expression of mesenchymal markers, CD105, CD140b, and CD144 by PCR. Pluripotent markers OCT4, SOX2, and NANOG were positively expressed in EMSCs analyzed by Western blotting and PCR. Further, differentiation into adipocytes and osteocytes was confirmed by cytochemical staining and lineage specific gene expression by quantitative realtime-PCR. Adipocyte (FABP, LPL, AP2) and osteocyte specific genes (ON, BG, RUNX2) in differentiated EMSCs showed significant (p < 0.05) increase in expression compared to undifferentiated control cells. Neurogenic transdifferentiation of EMSCs exhibited distinctive dendritic morphology with axon projections and neuronal specific genes, NFM, NGF, MBP, NES, B3T and MAP2 and proteins, B3T, NFM, NGF, and TRKA were positively expressed in neuronal differentiated cells. Functional analysis of neuronal differentiated EMSCs displayed voltage-dependence and kinetics for transient outward K⁺ currents (I_to), at holding potential of −80 mV, Na⁺ currents and during current clamp, neuronal differentiated EMSCs was more negative than that of control EMSCs. Porcine EMSCs is a suitable model for studying molecular mechanism of transdifferentiation, assessment of electrophysiological properties and their efficiency during in vivo transplantation.     

Polycystin-1 Enhances Stemmness Potential of Umbilical Cord Blood-Derived Mesenchymal Stem Cells

Polycystic Kidney Disease (PKD) is a disorder that affects the kidneys and other organs, and its major forms are encoded by polycystin-1 (PC1) and polycystin-2 (PC2), as PKD1 and PKD2. It is located sandwiched inside and outside cell membranes and interacts with other cells. This protein is most active in kidney cells before birth, and PC1 and PC2 work together to help regulate cell proliferation, cell migration, and interactions with other cells. The molecular relationship and the function between PKD1 and cancer is well known, such as increased or decreased cell proliferation and promoting or suppressing cell migration depending on the cancer cell type specifically. However, its function in stem cells has not been revealed. Therefore, in this study, we investigated the biological function of PC1 and umbilical cord blood-derived mesenchymal stem cell (UCB-MSC). Furthermore, we assessed how it affects cell migration, proliferation, and the viability of cells when expressed in the PKD1 gene. In addition, we confirmed in an ex vivo artificial tooth model generated by the three-dimension printing technique that the ability to differentiate into osteocytes improved according to the expression level of the stemness markers when PKD1 was expressed. This study is the first report to examine the biological function of PKD1 in UCB-MSC. This gene may be capable of enhancing differentiation ability and maintaining long-term stemness for the therapeutic use of stem cells.     

Alteration of 3D Matrix Stiffness Regulates Viscoelasticity of Human Mesenchymal Stem Cells

Human mesenchymal stem cells (hMSCs) possess potential of bone formation and were proposed as ideal material against osteoporosis. Although interrogation of directing effect on lineage specification by physical cues has been proposed, how mechanical stimulation impacts intracellular viscoelasticity during osteogenesis remained enigmatic. Cyto-friendly 3D matrix was prepared with polyacrylamide and conjugated fibronectin. The hMSCs were injected with fluorescent beads and chemically-induced toward osteogenesis. The mechanical properties were assessed using video particle tracking microrheology. Inverted epifluorescence microscope was exploited to capture the Brownian trajectory of hMSCs. Mean square displacement was calculated and transformed into intracellular viscoelasticity. Two different stiffness of microspheres (12 kPa, 1 kPa) were established. A total of 45 cells were assessed. hMSCs possessed equivalent mechanical traits initially in the first week, while cells cultured in rigid matrix displayed significant elevation over elastic (G′) and viscous moduli (G″) on day 7 (p < 0.01) and 14 (p < 0.01). However, after two weeks, soft niches no longer stiffened hMSCs, whereas the effect by rigid substrates was consistently during the entire differentiation course. Stiffness of matrix impacted the viscoelasticity of hMSCs. Detailed recognition of how microenvironment impacts mechanical properties and differentiation of hMSCs will facilitate the advancement of tissue engineering and regenerative medicine.     

Temporomandibular Joint Osteoarthritis: Regenerative Treatment by a Stem Cell Containing Advanced Therapy Medicinal Product (ATMP)—An In Vivo Animal Trial

Temporomandibular joint osteoarthritis (TMJ-OA) is a chronic degenerative disease that is often characterized by progressive impairment of the temporomandibular functional unit. The aim of this randomized controlled animal trial was a comparative analysis regarding the chondroregenerative potency of intra-articular stem/stromal cell therapy. Four weeks after combined mechanical and biochemical osteoarthritis induction in 28 rabbits, therapy was initiated by a single intra-articular injection, randomized into the following groups: Group 1: AB Serum (ABS); Group 2: Hyaluronic acid (HA); Group 3: Mesenchymal stromal cells (STx.); Group 4: Mesenchymal stromal cells in hyaluronic acid (HA + STx.). After another 4 weeks, the animals were euthanized, followed by histological examination of the removed joints. The histological analysis showed a significant increase in cartilage thickness in the stromal cell treated groups (HA + STx. vs. ABS, p = 0.028; HA + ST.x vs. HA, p = 0.042; STx. vs. ABS, p = 0.036). Scanning electron microscopy detected a similar heterogeneity of mineralization and tissue porosity in the subchondral zone in all groups. The single intra-articular injection of a stem cell containing, GMP-compliant advanced therapy medicinal product for the treatment of iatrogen induced osteoarthritis of the temporomandibular joint shows a chondroregenerative effect.     

Comparison of Osteogenic Potentials of Dental Pulp and Bone Marrow Mesenchymal Stem Cells Using the New Cell Transplantation Platform,CellSaic, in a Rat Congenital Cleft-Jaw Model

Scaffolds stimulate cell proliferation and differentiation and play major roles in providing growth and nutrition factors in the repair of bone defects. We used the recombinant peptide Cellnest™ to prepare the three-dimensional stem cell complex, CellSaic, and evaluated whether CellSaic containing rat dental pulp stem cells (rDPSCs) was better than that containing rat bone marrow stem cells (rBMSCs). rDPSC-CellSaic or rBMSC-CellSaic, cultured with or without osteogenic induction medium, formed the experimental and control groups, respectively. Osteoblast differentiation was evaluated in vitro and transplanted into a rat model with a congenital jaw fracture. Specimens were collected and evaluated by microradiology and histological analysis. In the experimental group, the amount of calcium deposits, expression levels of bone-related genes (RUNX2, ALP, BSP, and COL1), and volume of mineralized tissue, were significantly higher than those in the control group (p < 0.05). Both differentiated and undifferentiated rDPSC-CellSaic and only the differentiated rBMSC-CellSaic could induce the formation of new bone tissue. Overall, rBMSC-CellSaic and rDPSC-CellSaic made with Cellnest™ as a scaffold, provide excellent support for promoting bone regeneration in rat mandibular congenital defects. Additionally, rDPSC-CellSaic seems a better source for craniofacial bone defect repair than rBMSC-CellSaic, suggesting the possibility of using DPSCs in bone tissue regenerative therapy.     

Menstrual Blood-Derived Endometrial Stem Cells’ Impact for the Treatment Perspective of Female Infertility

When looking for the causes and treatments of infertility, much attention is paid to one of the reproductive tissues—the endometrium. Therefore, endometrial stem cells are an attractive target for infertility studies in women of unexplained origin. Menstrual blood stem cells (MenSCs) are morphologically and functionally similar to cells derived directly from the endometrium; with dual expression of mesenchymal and embryonic cell markers, they proliferate and regenerate better than bone marrow mesenchymal stem cells. In addition, menstrual blood stem cells are extracted in a non-invasive and painless manner. In our study, we analyzed the characteristics and the potential for decidualization of menstrual blood stem cells isolated from healthy volunteers and women diagnosed with infertility. We demonstrated that MenSCs express CD44, CD166, CD16, CD15, BMSC, CD56, CD13 and HLA-ABC surface markers, have proliferative properties, and after induction of menstrual stem cell differentiation into epithelial direction, expression of genes related to decidualization (PRL, ESR, IGFBP and FOXO1) and angiogenesis (HIF1, VEGFR2 and VEGFR3) increased. Additionally, the p53, p21, H3K27me3 and HyperAcH4 proteins’ expression increased during MenSCs decidualization, they secrete proteins that are involved in the regulation of the actin cytoskeleton, estrogen and relaxin signaling pathways and the management of inflammatory processes. Our findings reveal the potential use of MenSCs for the treatment of reproductive disorders.     

Regeneration of Articular Cartilage in Lizard Knee from Resident Stem/Progenitor Cells

The epiphysis of femur and tibia in the lizard Podarcis muralis can extensively regenerate after injury. The process involves the articular cartilage and metaphyseal (growth) plate after damage. The secondary ossification center present between the articular cartilage and the growth plate is replaced by cartilaginous epiphyses after about one month of regeneration at high temperature. The present study analyzes the origin of the chondrogenic cells from putative stem cells located in the growing centers of the epiphyses. The study is carried out using immunocytochemistry for the detection of 5BrdU-labeled long retaining cells and for the localization of telomerase, an enzyme that indicates stemness. The observations show that putative stem cells retaining 5BrdU and positive for telomerase are present in the superficial articular cartilage and metaphyseal growth plate located in the epiphyses. This observation suggests that these areas represent stem cell niches lasting for most of the lifetime of lizards. In healthy long bones of adult lizards, the addition of new chondrocytes from the stem cells population in the articular cartilage and the metaphyseal growth plate likely allows for slow, continuous longitudinal growth. When the knee is injured in the adult lizard, new populations of chondrocytes actively producing chondroitin sulfate proteoglycan are derived from these stem cells to allow for the formation of completely new cartilaginous epiphyses, possibly anticipating the re-formation of secondary centers in later stages. The study suggests that in this lizard species, the regenerative ability of the epiphyses is a pre-adaptation to the regeneration of the articular cartilage.     

Biomedical Application of Low Molecular Weight Heparin/Protamine Nano/Micro Particles as Cell- and Growth Factor-Carriers and Coating Matrix

Low molecular weight heparin (LMWH)/protamine (P) nano/micro particles (N/MPs) (LMWH/P N/MPs) were applied as carriers for heparin-binding growth factors (GFs) and for adhesive cells including adipose-derived stromal cells (ADSCs) and bone marrow-derived mesenchymal stem cells (BMSCs). A mixture of LMWH and P yields a dispersion of N/MPs (100 nm–3 μm in diameter). LMWH/P N/MPs can be immobilized onto cell surfaces or extracellular matrix, control the release, activate GFs and protect various GFs. Furthermore, LMWH/P N/MPs can also bind to adhesive cell surfaces, inducing cells and LMWH/P N/MPs-aggregate formation. Those aggregates substantially promoted cellular viability, and induced vascularization and fibrous tissue formation in vivo. The LMWH/P N/MPs, in combination with ADSCs or BMSCs, are effective cell-carriers and are potential promising novel therapeutic agents for inducing vascularization and fibrous tissue formation in ischemic disease by transplantation of the ADSCs and LMWH/P N/MPs-aggregates. LMWH/P N/MPs can also bind to tissue culture plates and adsorb exogenous GFs or GFs from those cells. The LMWH/P N/MPs-coated matrix in the presence of GFs may provide novel biomaterials that can control cellular activity such as growth and differentiation. Furthermore, three-dimensional (3D) cultures of cells including ADSCs and BMSCs using plasma-medium gel with LMWH/P N/MPs exhibited efficient cell proliferation. Thus, LMWH/P N/MPs are an adequate carrier both for GFs and for stromal cells such as ADSCs and BMSCs, and are a functional coating matrix for their cultures.     

Cell Fusion of Mesenchymal Stem/Stromal Cells and Breast Cancer Cells Leads to the Formation of Hybrid Cells Exhibiting Diverse and Individual (Stem Cell) Characteristics

Cancer is one of the most common diseases worldwide, and treatment bears many challenges such as drug and radioresistance and formation of metastases. These difficulties are due to tumor heterogeneity, which has many origins. One may be cell fusion, a process that is relevant in both physiological (e.g., wound healing) and pathophysiological (cancer and viral infection) processes. In this study, we examined if cell fusion between mesenchymal stem/stromal cells (MSCs) and breast cancer (BC) cells occurs and if newly generated hybrid cells may exhibit cancer stem/initiating cell (CS/IC) characteristics. Therefore, several methods such as mammosphere assay, AldeRed assay, flow cytometry (CD24, CD44, CD104) and Western blot analysis (of epithelial to mesenchymal transition markers such as SNAIL, SLUG and Twist) were applied. In short, four different hybrid clones, verified by short tandem repeat (STR) analysis, were analyzed; each expressed an individual phenotype that seemed not to be explicitly related to either a more stem cell or cancer cell phenotype. These results show that cancer cells and MSCs are able to fuse spontaneously in vitro, thereby giving rise to hybrid cells with new properties, which likely indicate that cell fusion may be a trigger for tumor heterogeneity.