Novel Epigenetic Modifiers of Histones Presenting Potent Inhibitory Effects on Adenoid Cystic Carcinoma Stemness and Invasive Properties.

Adenoid cystic carcinoma (ACC) is a rare neoplasm known for its indolent clinical course, risk of perineural invasion, and late onset of distant metastasis. Due to the scarcity of samples and the tumor's rarity, progress in developing effective treatments has been historically limited. To tackle this issue, a high-throughput screening of epigenetic drugs was conducted to identify compounds capable of disrupting the invasive properties of the tumor and its cancer stem cells (CSCs). ACC cells were screened for changes in tumor viability, chromatin decondensation, Snail inhibition along tumor migration, and disruption of cancer stem cells. Seven compounds showed potential clinical interest, and further validation showed that Scriptaid emerged as a promising candidate for treating ACC invasion. Scriptaid demonstrated a favorable cellular toxicity index, effectively inhibited Snail expression, induced hyperacetylation of histone, reduced cell migration, and effectively disrupted tumorspheres. Additionally, LMK235 displayed encouraging results in four out of five validation assays, further highlighting its potential in combating tumor invasion in ACC. By targeting the invasive properties of the tumor and CSCs, Scriptaid and LMK235 hold promise as potential treatments for ACC, with the potential to improve patient outcomes and pave the way for further research in this critical area.

The rhythms of histones in regeneration: The epigenetic modifications determined by clock genes.

The evolutionary establishment of an internal biological clock is a primordial event tightly associated with a 24-h period. Changes in the circadian rhythm can affect cellular functions, including proliferation, DNA repair and redox state. Even isolated organs, tissues and cells can maintain an autonomous circadian rhythm. These cell-autonomous molecular mechanisms are driven by intracellular clock genes, such as BMAL1. Little is known about the role of core clock genes and epigenetic modifications in the skin. Our focus was to identify BMAL1-driven epigenetic modifications associated with gene transcription by mapping the acetylation landscape of histones in epithelial cells responding to injury. We explored the role of BMAL1 in epidermal wound and tissue regeneration using a loss-of-function approach in vivo. We worked with BMAL1 knockout mice and a contraction-resistance wound healing protocol, determining the histone modifications using specific antibodies to detect the acetylation levels of histones H3 and H4. We found significant differences in the acetylation levels of histones in both homeostatic and injured skin with deregulated BMAL1. The intact skin displayed varied acetylation levels of histones H3 and H4, including hyperacetylation of H3 Lys 9 (H3K9). The most pronounced changes were observed at the repair site, with notable alterations in the acetylation pattern of histone H4. These findings reveal the importance of histone modifications in response to injury and indicate that modulation of BMAL1 and its associated epigenetic events could be therapeutically harnessed to improve skin regeneration.

Feeling the Heat. Mapping the Epigenetic Modifications of Histone during Burn Wound Healing.

Burn injuries are observed throughout a wide range of ages, with over 1.1 million Americans suffering burns yearly, and half of these require hospitalization. Epigenetic modifications are fast-acting mechanisms that allow the human body to respond and adapt to environmental changes, including burn injuries. There is a lack of understanding of the epigenetic role during burn-induced tissue repair. Here, we characterize the histone modifications that follow burn injury, aiming at future pharmacological intervention using drugs capable of targeting epigenetic events. A clinically relevant porcine burn model was used to recapitulate the skin healing process after the burn. Isolated skin tissues at different time points were used to detect the acetylation levels of histones H3K27, H4K5, H4K8, and H4K12 as significant players of gene transcription using MetaXpress High-Content Imaging Analysis. We observed that the acetylation of histones is dynamically adjusted throughout healing, and its modifications are uniquely expressed according to the anatomical location and time of healing. We also observed that histone H4K5 is the most widely expressed during healing, followed by histone H3K27. We observed that histones expressed in intact skin tissue adjacent to the burn site could sense the burn injury by changing its histone acetylation pattern compared to control skin from uninjured and distant skin. Using a clinically relevant animal model, we have generated a comprehensive landscape of epigenetic modifications during burn healing. Our data will help us identify novel epi-drugs capable of manipulating histone modifications during healing to accelerate the healing process.

Disease resolution following treatment of peri-implant diseases: A systematic review.

The aim of this PRISMA-compliant systematic review was to analyze the evidence pertaining to disease resolution after treatment of peri-implant diseases (PROSPERO: CRD42022306999) with the following PICO question: what is the rate of disease resolution following non-surgical and surgical therapy for peri-implant diseases in adult human subjects? A literature search to identify studies that fulfilled a pre-established eligibility criteria was conducted. Data on primary therapeutic outcomes, including treatment success, rate of disease resolution and/or recurrence, as well as a variety of secondary outcomes was extracted and categorized. Fifty-five articles were included. Few studies investigated the efficacy of different non-surgical and surgical therapies to treat peri-implant diseases using a set of pre-defined criteria and with follow-up periods of at least one year. The definition of treatment success and outcomes of disease resolution differed considerably among the included studies. Treatment of peri-implant mucositis was most commonly reported to be successful in arresting disease progression for ≤60% of the cases, whereas most studies on peri-implantitis treatment reported disease resolution occurring in <50% of the fixtures. In conclusion, disease resolution is generally unpredictable and infrequently achieved after the treatment of peri-implant diseases. A great variety of definitions have been used to define treatment success. Notably, percentages of treatment success and disease resolution were generally underreported. The use of standardized parameters to evaluate disease resolution should be considered an integral component in future clinical studies.

Using PDX animal models to identify and stratify adenoid cystic carcinoma patients presenting an enhanced response to HDAC inhibitors.

Adenoid cystic carcinoma (ACC) patients face a highly infiltrative and metastatic disease characterized by poor survival rates and suboptimal response to available therapies. We have previously shown that sensitization of ACC tumors to chemotherapy using histone deacetylase inhibitors (HDACi) constitutes a promising therapeutic strategy to manage tumor growth. Here, we used patient-derived xenografts (PDX) from ACC tumors to evaluate the effects of in vivo administration of the HDAC inhibitor Entinostat combined with Cisplatin over tumor growth. RNA from PDX tumor samples receiving the proposed therapy were analyzed using NanoString technology to identify molecular signatures capable of predicting ACC response to the therapy. We also used an RNAseq dataset from 68 ACC patients to validate the molecular signature identified by the NanoString platform. We found that the administration of Entinostat combined with Cisplatin resulted in a potent tumor growth inhibition (TGI) ranging from 38% to 106% of the original tumor mass. Enhanced response to therapy is consistent with the reactivation of tumor suppressor genes, including SFRP1, and the downregulation of oncogenes like FGF8 and CCR7. Nanostring data from PDX tumors identified a genetic signature capable of predicting tumor response to therapy. We further stratified 68 ACC patients containing RNAseq data accordingly to the activity levels of the identified genetic signature. We found that 23% of all patients exhibit a genetic signature consistent with a high ACC tumor response rate to Entinostat and Cisplatin. Our study provides compelling preclinical data supporting the deployment of a powerful systemic anticancer therapy crafted and explicitly tested for ACC tumors.

Empowering the youth to choose non-traditional careers in research and academia.

BACKGROUD: The United States is facing a dramatic shortage of clinician-scientists within the domains of medicine and dentistry. POINT OF VIEW: In this perspective article, we stressed the problem involving the continuous shortage of specialized professionals capable of addressing complex basic sciences questions, while maintaining clinical relevance. Here we present a different perspective regarding the early engagement of young students to clinical sciences by teaming up with high schools across the United States, and to energize the debate on our current shortage of clinician-scientists.

Adenoid Cystic Carcinoma from the salivary and lacrimal glands and the breast: Different clinical outcomes to the same tumor.

Adenoid cystic carcinoma (ACC) is a biphasic malignant lesion that can develop at various anatomical sites. Salivary and lacrimal ACC lesions have a high risk of local invasion, metastasis, and poor prognosis. In more distant organs, such as the breast, ACC is a rarer and less aggressive lesion. One of the major predictors of mortality of ACC is perineural invasion, which can be seen in 30 % of breast lesions, 85% of salivary lesions, and almost 100 % of lacrimal gland tumors. The biological differences between these three ACC tumors are still poorly understood. We focused on the current understanding of the genetic variations observed on ACC tumors and prognostic differences associated with distinct anatomical sites. A special effort was made to present the currently available therapies alongside the emerging strategies under development.

Influence of epigenetics on periodontitis and peri-implantitis pathogenesis.

Periodontitis is a disease characterized by tooth-associated microbial biofilms that drive chronic inflammation and destruction of periodontal-supporting tissues. In some individuals, disease progression can lead to tooth loss. A similar condition can occur around dental implants in the form of peri-implantitis. The immune response to bacterial challenges is not only influenced by genetic factors, but also by environmental factors. Epigenetics involves the study of gene function independent of changes to the DNA sequence and its associated proteins, and represents a critical link between genetic and environmental factors. Epigenetic modifications have been shown to contribute to the progression of several diseases, including chronic inflammatory diseases like periodontitis and peri-implantitis. This review aims to present the latest findings on epigenetic influences on periodontitis and to discuss potential mechanisms that may influence peri-implantitis, given the paucity of information currently available.

Histone Modification on Parathyroid Tumors: A Review of Epigenetics.

Parathyroid tumors are very prevalent conditions among endocrine tumors, being the second most common behind thyroid tumors. Secondary hyperplasia can occur beyond benign and malignant neoplasia in parathyroid glands. Adenomas are the leading cause of hyperparathyroidism, while carcinomas represent less than 1% of the cases. Tumor suppressor gene mutations such as MEN1 and CDC73 were demonstrated to be involved in tumor development in both familiar and sporadic types; however, the epigenetic features of the parathyroid tumors are still a little-explored subject. We present a review of epigenetic mechanisms related to parathyroid tumors, emphasizing advances in histone modification and its perspective of becoming a promising area in parathyroid tumor research.

From Tissue Physoxia to Cancer Hypoxia, Cost-Effective Methods to Study Tissue-Specific O2 Levels in Cellular Biology.

The human body is endowed with an extraordinary ability to maintain different oxygen levels in various tissues and organs. The maintenance of physiological levels of oxygen is known as physoxia. The development of hypoxic conditions plays an important role in the biology of several pathologies, including cancer. In vitro studies using normal and neoplastic cells require that culture conditions be carried out under appropriate oxygen levels, either physoxic or hypoxic conditions. Such requirements are difficult to widely implement in laboratory practice, mainly due to the high costs of specialized equipment. In this work, we present and characterize a cost-effective method to culture cells under a range of oxygen levels using deoxidizing pouches. Our results show that physoxic and hypoxic levels using deoxidizing absorbers can be achieved either by implementing a gradual change in oxygen levels or by a regimen of acute depletion of oxygen. This approach triggers the activation of an epithelial-mesenchymal transition in cancer cells while stimulating the expression of HIF-1α. Culturing cancer cells with deoxidizing agent pouches revealed PI3K oncogenic pathway exacerbations compared to tumor cells growing under atmospheric levels of oxygen. Similar to the PI3K signaling disturbance, we also observed augmented oxidative stress and superoxide levels and increased cell cycle arrest. Most interestingly, the culture of cancer cells under hypoxia resulted in the accumulation of cancer stem cells in a time-dependent manner. Overall, we present an attractive, cost-effective method of culturing cells under appropriate physoxic or hypoxic conditions that is easily implementable in any wet laboratory equipped with cell culture tools.

Periodontal disease affects oral cancer progression in a surrogate animal model for tobacco exposure.

For decades, the link between poor oral hygiene and the increased prevalence of oral cancer has been suggested. Most recently, emerging evidence has suggested that chronic inflammatory diseases from the oral cavity (e.g., periodontal disease), to some extent, play a role in the development of oral squamous cell carcinoma (OSCC). The present study aimed to explore the direct impact of biofilm­induced periodontitis in the carcinogenesis process using a tobacco surrogate animal model for oral cancer. A total of 42 Wistar rats were distributed into four experimental groups: Control group, periodontitis (Perio) group, 4­nitroquinoline 1­oxide (4­NQO) group and 4NQO/Perio group. Periodontitis was stimulated by placing a ligature subgingivally, while oral carcinogenesis was induced by systemic administration of 4NQO in the drinking water for 20 weeks. It was observed that the Perio, 4NQO and 4NQO/Perio groups presented with significantly higher alveolar bone loss compared with that in the control group. Furthermore, all groups receiving 4NQO developed lesions on the dorsal surface of the tongue; however, the 4NQO/Perio group presented larger lesions compared with the 4NQO group. There was also a modest overall increase in the number of epithelial dysplasia and OSCC lesions in the 4NQO/Perio group. Notably, abnormal focal activation of cellular differentiation (cytokeratin 10­positive cells) that extended near the basal cell layer of the mucosa was observed in rats receiving 4NQO alone, but was absent in rats receiving 4NQO and presenting with periodontal disease. Altogether, the presence of periodontitis combined with 4NQO administration augmented tumor size in the current rat model and tampered with the protective mechanisms of the cellular differentiation of epithelial cells.

The IL-6R and Bmi-1 axis controls self-renewal and chemoresistance of head and neck cancer stem cells.

Despite major progress in elucidating the pathobiology of head and neck squamous cell carcinoma (HNSCC), the high frequency of disease relapse correlates with unacceptably deficient patient survival. We previously showed that cancer stem-like cells (CSCs) drive tumorigenesis and progression of HNSCC. Although CSCs constitute only 2-5% of total tumor cells, CSCs contribute to tumor progression by virtue of their high tumorigenic potential and their resistance to chemo-, radio-, and immunotherapy. Not only are CSCs resistant to therapy, but cytotoxic agents actually enhance cancer stemness by activating transcription of pluripotency factors and by inducing expression of Bmi-1, a master regulator of stem cell self-renewal. We hypothesized therapeutic inhibition of interleukin-6 receptor (IL-6R) suppresses Bmi-1 to overcome intrinsic chemoresistance of CSCs. We observed that high Bmi-1 expression correlates with decreased (p = 0.04) recurrence-free survival time in HNSCC patients (n = 216). Blockade of IL-6R by lentiviral knockdown or pharmacologic inhibition with a humanized monoclonal antibody (Tocilizumab) is sufficient to inhibit Bmi-1 expression, secondary sphere formation, and to decrease the CSC fraction even in Cisplatin-resistant HNSCC cells. IL-6R inhibition with Tocilizumab abrogates Cisplatin-mediated increase in CSC fraction and induction of Bmi-1 in patient-derived xenograft (PDX) models of HNSCC. Notably, Tocilizumab inhibits Bmi-1 and suppresses growth of xenograft tumors generated with Cisplatin-resistant HNSCC cells. Altogether, these studies demonstrate that therapeutic blockade of IL-6R suppresses Bmi-1 function and inhibits cancer stemness. These results suggest therapeutic inhibition of IL-6R might be a viable strategy to overcome the CSC-mediated chemoresistance typically observed in HNSCC patients.

Synergistic efficacy of combined EGFR and HDAC inhibitors overcomes tolerance to EGFR monotherapy in salivary mucoepidermoid carcinoma.

OBJECTIVES: Mucoepidermoid carcinoma (MEC) is the most common type of salivary gland malignancy. Advanced or high-grade MECs are refractory to chemotherapy, often leading to tumor recurrence/metastasis and abysmal ~35% 5-year survival. Causal links have been established between Epithelial Growth Factor Receptor (EGFR) activation and poor outcome. Herein we investigated the therapeutic efficacy of EGFR inhibition against MEC using in vitro pre-clinical models. MATERIALS AND METHODS: Five human MEC cell lines were used in cell viability, cytotoxicity, apoptosis, cell cycle, 2D-clonogenicity, and 3D-spheroid formation assays following treatment with Erlotinib (EGFR inhibitor), SAHA (Histone Deacetylase inhibitor; HDAC) and CUDC-101 (dual EGFR-HDAC inhibitor). Effects on MEC cancer stem cells were evaluated using flow cytometry. Gene expression and pathway regulation were evaluated via qPCR and Western blot, respectively. RESULTS: MEC cells enter a quiescent, non-proliferative yet rapidly reversible drug tolerant state upon EGFR inhibition. Despite robust suppression of MEC cell proliferation, no discernable apoptosis is detected. Combination of EGFR and HDAC inhibitors exhibits synergistic effects, exerting ~5-fold more potent cell cytotoxicity compared to HDAC or EGFR monotherapy. CUDC-101, a single molecule with dual EGFR-HDAC inhibitor moieties, exerts irreversible and potent cytotoxic activity against MEC cells and blunts MEC cancer stem-cell tumorigenicity. CONCLUSION: MEC cells are intrinsically tolerant to EGFR inhibition. Combining EGFR and HDAC inhibitors exerts synergistic and potent cytotoxic effects, suggesting that EGFR inhibitors still hold significant promise against MEC. Future studies are needed to assess the applicability and efficacy of dual EGFR-HDAC inhibitors for the clinical management of MEC.

Metformin-loaded nanospheres-laden photocrosslinkable gelatin hydrogel for bone tissue engineering.

The aim of this investigation was to engineer metformin (MF)-loaded mesoporous silica nanospheres (MSNs)-laden gelatin methacryloyl (GelMA) photocrosslinkable hydrogels and test their effects on the mechanical properties, swelling ratio, drug release, cytocompatibility, and osteogenic differentiation of stem cells from human exfoliated deciduous teeth (SHEDs). As-received and carboxylated MSNs (MSNs-COOH) were characterized by scanning and transmission electron microscopies (SEM and TEM), as well as Fourier-transform infrared spectroscopy (FTIR) prior to hydrogel modification. MF-MSNs-COOH were obtained by loading MF into MSNs at a 1:1 mass ratio. Upon MSNs-COOH laden-hydrogels fabrication, the mechanical properties, swelling ratio and MF release were evaluated. SHEDs were seeded on the hydrogels and cytocompatibility was examined. The effects of the MF-MSNs-COOH/GelMA on the osteogenic differentiation of SHEDs were measured by ALP activity, Alizarin Red assay, and Real-time PCR. Statistics were performed using one-way ANOVA (α = 0.05). Morphological (SEM and TEM) analyses of pristine and carboxylated MSNs revealed a mean particle size of 200 nm and 218 nm, respectively. Importantly, an intrinsic nanoporous structure was noticed. Incorporation of MSNs-COOH at 1.5 mg/mL in GelMA led to the highest compressive modulus and swelling ratio. The addition of MSNs-COOH (up to 3 mg/mL) in GelMA did not impact cell viability. The presence of MF in MSNs-COOH/GelMA significantly promoted cell proliferation. Significant upregulation of osteogenic-related genes (except OCN) were seen for modified (MSNs-COOH and MF-MSNs-COOH) hydrogels when compared to GelMA. Altogether, the engineered MF-MSNs-COOH/GelMA shows great promise in craniomaxillofacial applications as an injectable, cell-free and bioactive therapeutics for bone regeneration.

Immunohistological composition of peri-implantitis affected tissue around ceramic implants-A pilot study.

BACKGROUND: Aim of the pilot study was the histologic classification of the inflamed peri-implant soft tissue around ceramic implants (CI) in comparison with titanium implants (TI). METHODS: Peri-implant tissue were retrieved from 15 patients (aged 34 to 88 years, seven males/eight females) with severe peri-implantitis (eight CI, seven TI). The peri-implant soft tissue samples were retrieved from the sites during scheduled removal of the implant and prepared for immunohistochemical analysis. Monoclonal antibodies (targeting CD3, CD20, CD138, and CD68) were used to identify T- and B-cells, plasma cells and macrophages. Quantitative assessment was performed by one histologically trained investigator. Linear mixed regression models were used. RESULTS: A similar numerical distribution of the cell population was found in peri-implantitis around CI compared with TI. CD3 (TI, 17% to 85% versus CI, 20% to 70% of total cell number) and CD138 (TI, 1% to 73% versus CI, 12% to 69% of total cell number) were predominantly expressed. Notably, patient-individual differences of numerical cell distribution were detected. Co-localization of B- and T-lymphocytes was observed. CONCLUSIONS: Peri-implantitis around CI in comparison with TI seems to have a similar histological appearance. Differences in cellular composition of peri-implantitis lesions might also depend on the patient's specific immune status and not only on the material used.

Photobiomodulation therapy drives massive epigenetic histone modifications, stem cells mobilization and accelerated epithelial healing.

Emerging evidence indicates the clinical benefits of photobiomodulation therapy (PBMT) in the management of skin and mucosal wounds. Here, we decided to explore the effects of different regiments of PBMT on epithelial cells and stem cells, and the potential implications over the epigenetic circuitry during healing. Scratch-wound migration, immunofluorescence (anti-acetyl-Histone H3, anti-acetyl-CBP/p300 and anti-BMI1), nuclear morphometry and western blotting (anti-Phospho-S6, anti-methyl-CpG binding domain protein 2 [MBD2]) were performed. Epithelial stem cells were identified by the aldehyde dehydrogenase enzymatic levels and sphere-forming assay. We observed that PBMT-induced accelerated epithelial migration and chromatin relaxation along with increased levels of histones acetylation, the transcription cofactors CBP/p300 and mammalian target of rapamycin. We further observed a reduction of the transcription repression-associated protein MBD2 and a reduced number of epithelial stem cells and spheres. In this study, we showed that PBMT could induce epigenetic modifications of epithelial cells and control stem cell fate, leading to an accelerated healing phenotype.

Can propranolol act as a chemopreventive agent during oral carcinogenesis? An experimental animal study.

The multistep process of oral carcinogenesis provides a biological rationale for the use of chemoprevention in individuals at increased risk of developing oral cancer. We aimed to determine if low doses of propranolol can prevent the development of oral cancer using a tobacco-relevant and p53-associated animal model of cancer initiation. Twenty-six Wistar rats were randomly allocated into two groups, vehicle, and propranolol. All animals received 4-nitroquinoline N-oxide (4NQO) at 25 ppm diluted in the drinking water for 20 weeks. Animals from the propranolol group received propranolol (0.1 mg/kg) 5 days per week by gavage for 18 weeks. The clinical analysis was performed by measuring the area of the lesion and assessment of scores based on lesion appearance (papule; plaque; nodule or ulcerated). Histopathological analysis was performed to determine the presence of epithelial dysplasia or invasive squamous cell carcinoma (SCC). The average lesion area in 4NQO + vehicle and in 4NQO + propranolol groups were 0.20 and 0.28 mm2, respectively (P = 0.53). The percentage of cases clinically graded as papules, thick plaques, nodular areas, and ulcerated lesions was similar between groups (P = 0.94). Histopathological diagnosis also did not differ between groups (P = 0.65), with 54.5 and 70% of cases being diagnosed as SCC in 4NQO and in 4NQO + propranolol groups, respectively. In conclusion, daily doses propranolol at 0.1 mg/kg were not as effective as a chemopreventive therapy in an animal model of 4NQO-induced carcinogenesis.

Head and neck cancer patient-derived xenograft models - A systematic review.

BACKGROUND: Patient-derived xenograft (PDX) involve the direct surgical transfer of fresh human tumor samples to immunodeficient mice. This systematic review aimed to identify publications of head and neck cancer PDX (HNC-PDX) models, describing the main methodological characteristics and outcomes. METHODS: An electronic search was undertaken in four databases, including publications having used HNC-PDX. Data were analyzed descriptively. RESULTS: 63 articles were yielded. The nude mouse was one most commonly animal model used (38.8 %), and squamous cell carcinoma accounted for the majority of HNC-PDX (80.6 %). Tumors were mostly implanted in the flank (86.3 %), and the latency period ranged from 30 to 401 days. The successful rate ranged from 17 % to 100 %. Different drugs and pathways were identified. CONCLUSION: HNC-PDX appears to significantly recapitulate the morphology of the original HNC and represents a valuable method in translational research for the assessment of the in vivo effect of novel therapies for HNC.

SCF/C-Kit Signaling Induces Self-Renewal of Dental Pulp Stem Cells.

INTRODUCTION: The maintenance of a stem cell pool is imperative to enable healing processes in the dental pulp tissue throughout life. As such, knowing mechanisms underlying stem cell self-renewal is critical to understand pulp pathophysiology and pulp regeneration. The purpose of this study was to evaluate the impact of stem cell factor (SCF) signaling through its receptor tyrosine kinase (c-Kit) on the self-renewal of human dental pulp stem cells (hDPSCs). METHODS: The hDPSCs were stably transduced with lentiviral vectors expressing shRNA-c-Kit or vector control. The impact of the SCF/c-Kit axis on hDPSC self-renewal was evaluated by using a pulpsphere assay in low attachment conditions and by evaluating the expression of polycomb complex protein Bmi-1 (master regulator of self-renewal) by Western blot and flow cytometry. RESULTS: The c-Kit-silenced hDPSCs formed fewer pulpspheres when compared with hDPSCs transduced with control vector (P < .05). Evaluation of pulpsphere morphology revealed the presence of 3 distinct sphere types, ie, holospheres, merospheres, and paraspheres. Although c-Kit silencing decreased the number of holospheres compared with control cells (P < .05), it had no effect on the number of merospheres and paraspheres. Recombinant human stem cell factor (rhSCF) increased the number of holospheres (P < .05) and induced dose-dependent Bmi-1 expression in hDPSCs. As expected, the inductive capacity of rhSCF on Bmi-1 expression and fraction of Bmi-1-positive cells was inhibited when we silenced c-Kit in hDPSCs. CONCLUSIONS: These results unveiled the role of SCF/c-Kit signaling on the self-renewal of hDPSCs and suggested that this pathway enables long-term maintenance of stem cell pools in human dental pulps.