RESUMO
The challenge of treating corneal scarring through keratoplasties lies in the limited availability of donor tissue. Various studies have shown the therapeutic use of cultivated corneal stromal stem cells (CSSCs) to mitigate tissue inflammation and suppress fibrosis and scar tissue formation in preclinical corneal wound models. To develop CSSC therapy for clinical trials on patients with corneal scarring, it is necessary to generate clinical-grade CSSCs in compliant to Good Manufacturing Practice (GMP) regulations. This chapter elucidates human CSSC isolation, culture, and cryopreservation under GMP-compliant conditions. It underscores quality assessment encompassing morphological traits, expression of stemness markers, anti-inflammatory activity, and keratocyte differentiation potency.
Assuntos
Técnicas de Cultura de Células , Diferenciação Celular , Substância Própria , Humanos , Técnicas de Cultura de Células/métodos , Substância Própria/citologia , Separação Celular/métodos , Criopreservação/métodos , Células-Tronco/citologia , Células-Tronco/metabolismo , Células Cultivadas , Biomarcadores , Células Estromais/citologiaRESUMO
Glaucoma is one of the leading causes of irreversible blindness. Stem cell therapy has shown promise in the treatment of primary open-angle glaucoma in animal models. Stem cell-free therapy using stem cell-derived trophic factors might be in demand in patients with high-risk conditions or religious restrictions. In this chapter, we describe methods for trabecular meshwork stem cell (TMSC) cultivation, secretome harvesting, and protein isolation, as well as assays to ensure the health of TMSC post-secretome harvesting and for secretome periocular injection into mice for therapeutic purposes.
Assuntos
Células-Tronco , Malha Trabecular , Malha Trabecular/metabolismo , Malha Trabecular/citologia , Animais , Camundongos , Humanos , Células-Tronco/citologia , Células-Tronco/metabolismo , Regeneração , Glaucoma/terapia , Transplante de Células-Tronco/métodos , Secretoma , Modelos Animais de Doenças , Glaucoma de Ângulo Aberto/terapia , Células Cultivadas , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Técnicas de Cultura de Células/métodosRESUMO
Various strategies for replacing retinal neurons lost in degenerative diseases are under investigation, including stimulating the endogenous regenerative capacity of Müller Glia (MG) as injury-inducible retinal stem cells. Inherently regenerative species, such as zebrafish, have provided key insights into mechanisms regulating MG dedifferentiation to a stem-like state and the proliferation of MG and MG-derived progenitor cells (MGPCs). Interestingly, promoting MG/MGPC proliferation is not sufficient for regeneration, yet mechanistic studies are often focused on this measure. To fully account for the regenerative process, and facilitate screens for factors regulating cell regeneration, an assay for quantifying cell replacement is required. Accordingly, we adapted an automated reporter-assisted phenotypic screening platform to quantify the pace of cellular regeneration kinetics following selective cell ablation in larval zebrafish. Here, we detail a method for using this approach to identify chemicals and genes that control the rate of retinal cell regeneration following selective retinal cell ablation.
Assuntos
Peixe-Zebra , Animais , Retina/citologia , Retina/metabolismo , Fenótipo , Proliferação de Células , Regeneração , Células Ependimogliais/citologia , Células Ependimogliais/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Cinética , Regeneração Nervosa/fisiologiaRESUMO
OBJECTIVE: To investigate the influence of citric acid on the osteogenic and angiogenic potential of stem cells from apical papillae (SCAPs). MATERIALS AND METHODS: Stem cells from apical papillae were isolated from freshly extracted third permanent molars. These cells were treated with 20 and 100 µM citric acid. Alizarin red staining was used to evaluate mineral deposition. The secreted levels of vascular endothelial growth factor (VEGF) were assessed by ELISA on days 18, 24 and 28. Immunofluorescence analysis was performed to assess the expression of surface markers after exposure to 20 and 100 µM citric acid. RESULTS: Different mineralisation patterns were observed. Supplemented with citric acid, media showed more diffuse and less dense crystals. On day 18, most VEGF was secreted from the cells with no added citric acid. On day 24, there was a significant increase (p < 0.05) in the levels of VEGF secreted from cells treated with 20 µM citric acid. On day 28, cells from the control group did not secrete VEGF. There was a reduction in the levels of VEGF secreted by cells treated with 20 µM citric acid and a significant increase in the cells exposed to 100 µM citric acid (p < 0.05). CONCLUSION: Citric acid can promote the differentiation of SCAPs and angiogenesis.
Assuntos
Ácido Cítrico , Células-Tronco , Fator A de Crescimento do Endotélio Vascular , Ácido Cítrico/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Humanos , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Papila Dentária/citologia , Papila Dentária/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Calcificação Fisiológica/efeitos dos fármacosRESUMO
Mesenchymal stem cell derived extracellular vesicles (MSC EVs) are paracrine modulators of macrophage function. Scientific research has primarily focused on the immunomodulatory and regenerative properties MSC EVs derived from bone marrow. The dental pulp is also a source for MSCs, and their anatomical location and evolutionary function has primed them to be potent immunomodulators. In this study, we demonstrate that extracellular vesicles derived from dental pulp stem cells (DPSC EVs) have pronounced immunomodulatory effect on primary macrophages by regulating the NFκb pathway. Notably, the anti-inflammatory activity of DPSC-EVs is enhanced following exposure to an inflammatory stimulus (LPS). These inhibitory effects were also observed in vivo. Sequencing of the naïve and LPS preconditioned DPSC-EVs and comparison with our published results from marrow MSC EVs revealed that Naïve and LPS preconditioned DPSC-EVs are enriched with anti-inflammatory miRNAs, particularly miR-320a-3p, which appears to be unique to DPSC-EVs and regulates the NFκb pathway. Overall, our findings highlight the immunomodulatory properties of DPSC-EVs and provide vital clues that can stimulate future research into miRNA-based EV engineering as well as therapeutic approaches to inflammation control and disease treatment.
Assuntos
Polpa Dentária , Vesículas Extracelulares , Imunomodulação , Inflamação , NF-kappa B , Polpa Dentária/citologia , Polpa Dentária/imunologia , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/imunologia , Humanos , Animais , Inflamação/imunologia , Inflamação/metabolismo , NF-kappa B/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , MicroRNAs/genética , Lipopolissacarídeos/farmacologia , Camundongos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/imunologia , Células Cultivadas , Transdução de Sinais , Células-Tronco/imunologia , Células-Tronco/metabolismo , MasculinoRESUMO
BACKGROUND: Hepatic progenitor cells serve not only as the origin of combined hepatocellular cholangiocarcinoma (cHCC-CCA) but are also responsible for malignancy recurrence after surgical resection. Nucleophosmin 1 (NPM1) has been implicated in cancer metastasis and poor prognosis. This study aimed to determine the expression of NPM1 by hepatic progenitor cells in cHCC-CCA and the effects of targeting NPM1 on hepatic progenitor cells and BEL-7402 cells with characteristics of both progenitor cells and cHCC-CCA. METHODS: First, NPM1 was detected by RTâPCR, western blotting, and double-immunofluorescence staining in cHCC-CCA tissues. NPM1 expression was subsequently analysed in rat hepatic progenitor cells cultured in vitro and in interleukin 6 (IL6)-treated cells. The effects and mechanism of NPM1 on hepatic progenitor cells were determined by knocking down NPM1 and performing RNA sequencing analysis. Finally, NSC348884, a small-molecule inhibitor that disrupts NPM1 dimer formation, was used to confirm the function of NPM1 in BEL-7402 cells. RESULTS: Both human hepatic progenitor cells in cHCC-CCA tissues and rat in vitro cultured hepatic progenitor cells highly expressed NPM1. IL6, a cytokine involved in the malignant transformation of hepatic progenitor cells, dose-dependently increased NPM1 and PCNA expression. Knocking down NPM1 reduced IL6R transcription (P < 0.0001) and inhibited the proliferation (P = 0.0065) of hepatic progenitor cells by suppressing the mTOR signalling pathway and activating the apoptosis pathway. Furthermore, knocking down NPM1 in hepatic progenitor cells resulted in more apoptotic cells (7.33 ± 0.09% vs. 3.76 ± 0.13%, P < 0.0001) but fewer apoptotic cells in the presence of NSC348884 (47.57 ± 0.49% vs. 63.40 ± 0.05%, P = 0.0008) than in the control cells, suggesting that low-NPM1-expressing cells are more resistant to NSC348884. In addition, NSC348884 induced the apoptosis of BEL-7402 cells with an IC50 of 2.77 µmol/L via the downregulation of the IL-6R and mTOR signalling pathways and inhibited the growth of BEL-7402 cells in a subcutaneous xenograft tumour model (P = 0.0457). CONCLUSIONS: Targeting NPM1 inhibits proliferation and induces apoptosis in hepatic progenitor cells and BEL-7402 cells, thus serving as a potential therapy for cHCC-CCA.
Assuntos
Apoptose , Proliferação de Células , Proteínas Nucleares , Nucleofosmina , Transdução de Sinais , Células-Tronco , Serina-Treonina Quinases TOR , Humanos , Apoptose/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/genética , Proliferação de Células/efeitos dos fármacos , Animais , Transdução de Sinais/efeitos dos fármacos , Ratos , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Células-Tronco/metabolismo , Células-Tronco/citologia , Masculino , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/genética , Linhagem Celular Tumoral , CamundongosRESUMO
BACKGROUND: Understanding the role of cytokines in tooth development is critical for advancing dental tissue engineering. Fibroblast growth factor 9 (FGF9) is the only FGF consistently expressed throughout dental epithelial tissue, from the initiation of tooth bud formation to tooth maturation. However, mice lacking Fgf9 (Fgf9-/-) surprisingly show no obvious abnormalities in tooth development, suggesting potential compensation by other FGFs. Here we report findings from an Fgf9S99N mutation mouse model, a loss-of-function mutation with a dominant negative effect. Our study reveals that Fgf9 is crucial for dental epithelial stem cell (DESC) survival and enamel formation. METHODS: To dissect the role of Fgf9 in tooth development, we performed the micro-CT, histomorphological analysis and gene expression assay in mice and embryos with S99N mutation. In addition, we assessed the effect of FGF9 on the DESC survival and dental epithelial differentiation by DESC sphere formation assay and tooth explant culture. Cell/tissue culture methods, gene expression analysis, specific inhibitors, and antibody blockage analysis were employed to explore how Fgf9 regulates enamel differentiation and DESC survival through both direct and indirect mechanisms. RESULTS: The Fgf9S99N mutation in mice led to reduced ameloblasts, impaired enamel formation, and increased apoptosis in the cervical loop (CL). DESC sphere culture experiments revealed that FGF9 facilitated DESC survival via activating ERK/CREB signaling, without affecting cell proliferation. Furthermore, in vitro tissue culture experiments demonstrated that FGF9 promoted enamel formation in a manner dependent on the presence of mesenchyme. Interestingly, FGF9 stimulation inhibited enamel formation in isolated enamel epithelia and DESC spheres. Further investigation revealed that FGF9 supports DESC survival and promotes amelogenesis by stimulating the secretion of FGF3 and FGF10 in dental mesenchymal cells via the MAPK/ERK signaling pathway. CONCLUSIONS: Our study demonstrates that Fgf9 is essential for DESC survival and enamel formation. Fgf9 performs as a dual-directional regulator of the dental enamel epithelium, not only inhibiting DESC differentiation into ameloblasts to preserve the stemness of DESC, but also promoting ameloblast differentiation through epithelial-mesenchymal interactions.
Assuntos
Esmalte Dentário , Células Epiteliais , Fator 9 de Crescimento de Fibroblastos , Células-Tronco , Animais , Fator 9 de Crescimento de Fibroblastos/metabolismo , Fator 9 de Crescimento de Fibroblastos/genética , Camundongos , Esmalte Dentário/metabolismo , Células-Tronco/metabolismo , Células-Tronco/citologia , Células Epiteliais/metabolismo , Incisivo/metabolismo , Sobrevivência Celular , Diferenciação CelularRESUMO
BACKGROUND: The formation of stem cell clones enables close contact of stem cells inside. The gap junctions in such clone spheres establish a microenvironment that allows frequent intercellular communication to maintain self-renewal and functions of stem cells. Nevertheless, the essential gap junction protein for molecular signaling in clones is poorly known. METHODS: Primary human airway basal cells (hBCs) were isolated from brushing samples through bronchoscopy and then cultured. A tightly focused femtosecond laser was used to excite the local Ca2+ in an individual cell to initiate an internal Ca2+ wave in a clone to screen gap junction proteins. Immunoflourescence staining and clonogenicity assay were used to evaluate self-renewal and functions. RNA and protein levels were assessed by PCR and Western blot. Air-liquid interface assay was conducted to evaluate the differentiation potential. A Naphthalene injury mouse model was used to assess the regeneration potential. RESULTS: Herein, we identify Connexin 25 (Cx25) dominates intercellular Ca2+ communications in clones of hBCs in vitro to maintain the self-renewal and pluripotency of them. The self-renewal and in vitro differentiation functions and in vivo regeneration potential of hBCs in an airway damage model are both regulated by Cx25. The abnormal expression of Cx25 is validated in several diseases including IPF, Covid-19 and bronchiectasis. CONCLUSION: Cx25 is essential for hBC clones in maintaining self-renewal and functions of hBCs via gap junctions.
Assuntos
Conexinas , Regeneração , Humanos , Animais , Camundongos , Conexinas/metabolismo , Conexinas/genética , Diferenciação Celular , COVID-19/metabolismo , COVID-19/virologia , COVID-19/patologia , Junções Comunicantes/metabolismo , Autorrenovação Celular , Cálcio/metabolismo , Células Cultivadas , SARS-CoV-2/metabolismo , Masculino , Células-Tronco/metabolismo , Células-Tronco/citologiaRESUMO
Postprandial IL-1ß surges are predominant in the white adipose tissue (WAT), but its consequences are unknown. Here, we investigate the role of IL-1ß in WAT energy storage and show that adipocyte-specific deletion of IL-1 receptor 1 (IL1R1) has no metabolic consequences, whereas ubiquitous lack of IL1R1 reduces body weight, WAT mass, and adipocyte formation in mice. Among all major WAT-resident cell types, progenitors express the highest IL1R1 levels. In vitro, IL-1ß potently promotes adipogenesis in murine and human adipose-derived stem cells. This effect is exclusive to early-differentiation-stage cells, in which the adipogenic transcription factors C/EBPδ and C/EBPß are rapidly upregulated by IL-1ß and enriched near important adipogenic genes. The pro-adipogenic, but not pro-inflammatory effect of IL-1ß is potentiated by acute treatment and blocked by chronic exposure. Thus, we propose that transient postprandial IL-1ß surges regulate WAT remodeling by promoting adipogenesis, whereas chronically elevated IL-1ß levels in obesity blunts this physiological function.
Assuntos
Adipócitos , Adipogenia , Tecido Adiposo Branco , Proteína beta Intensificadora de Ligação a CCAAT , Interleucina-1beta , Receptores Tipo I de Interleucina-1 , Adipogenia/efeitos dos fármacos , Adipogenia/genética , Animais , Interleucina-1beta/metabolismo , Humanos , Adipócitos/metabolismo , Adipócitos/citologia , Receptores Tipo I de Interleucina-1/metabolismo , Receptores Tipo I de Interleucina-1/genética , Camundongos , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/genética , Tecido Adiposo Branco/metabolismo , Tecido Adiposo Branco/citologia , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Proteína delta de Ligação ao Facilitador CCAAT/genética , Masculino , Camundongos Knockout , Células-Tronco/metabolismo , Células-Tronco/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Diferenciação Celular/efeitos dos fármacosRESUMO
BACKGROUND: Periodontitis is a dental disease characterized by inflammation of periodontal tissues and loss of the periodontal ligaments and alveolar bone. Exosomes are a class of extracellular vesicles that are involved in a variety of diseases by releasing active substances. In this study, we aimed to investigate the effect and mechanism of exosomes from M2 polarized macrophages (M2-exos) on osteogenic differentiation in human periodontal ligament stem cells (hPDLSCs). METHODS: M2-exos were isolated from IL-4-induced RAW264.7 cells (M2 macrophages) and then treated on hPDLSCs. Osteogenic differentiation was assessed by alkaline phosphatase (ALP) staining, alizarin red S (ARS) staining, measurement of osteogenic differentiation-related genes and proteins, and inflammation was evaluated by measuring the levels of inflammatory factors. The mechanism of M2-exo was confirmed through qPCR, western blot, ALP and ARS staining. RESULTS: Results suggested that M2-exo improved osteogenic differentiation and inhibited inflammation in LPS-induced hPDLSCs. CXCL12 expression was elevated in M2 macrophages, but decreased in LPS-induced hPDLSCs. Moreover, the effect of M2-exo on osteogenic differentiation and inflammation in LPS-induced hPDLSCs was reversed by CXCL12 knockdown. CONCLUSION: We demonstrated that M2-exo facilitated osteogenic differentiation and suppressed inflammation in LPS-induced hPDLSCs through promotion of CXCL12 expression. These results suggested the potential of M2-exo in the treatment of periodontitis, which may provide a new theoretical basis for M2-exo treatment of periodontitis.
Assuntos
Diferenciação Celular , Quimiocina CXCL12 , Exossomos , Inflamação , Macrófagos , Osteogênese , Ligamento Periodontal , Células-Tronco , Ligamento Periodontal/citologia , Ligamento Periodontal/metabolismo , Humanos , Exossomos/metabolismo , Macrófagos/metabolismo , Células-Tronco/metabolismo , Quimiocina CXCL12/metabolismo , Inflamação/metabolismo , Camundongos , Animais , Células Cultivadas , Periodontite/metabolismo , Células RAW 264.7RESUMO
OBJECTIVE: To investigate the biological regulatory function of Gremlin1 (GREM1) and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein eta (YWHAH) in dental pulp stem cells (DPSCs), and determine the underlying molecular mechanism involved. METHODS: Alkaline phosphatase (ALP) activity, alizarin red staining, scratch migration assays and in vitro and in vivo osteo-/dentinogenic marker detection of bone-like tissue generation in nude mice were used to assess osteo-/dentinogenic differentiation. Coimmunoprecipitation and polypeptide microarray assays were employed to detect the molecular mechanisms involved. RESULTS: The data revealed that knockdown of GREM1 promoted ALP activity, mineralisation in vitro and the expression of osteo-/dentinogenic differentiation markers and enhanced osteo-/ dentinogenesis of DPSCs in vivo. GREM1 bound to YWHAH in DPSCs, and the binding site was also identified. Knockdown of YWHAH suppressed the osteo-/dentinogenesis of DPSCs in vitro, and overexpression of YWHAH promoted the osteo-/dentinogenesis of DPSCs in vitro and in vivo. CONCLUSION: Taken together, the findings highlight the critical roles of GREM1-YWHAH in the osteo-/dentinogenesis of DPSCs.
Assuntos
Diferenciação Celular , Polpa Dentária , Peptídeos e Proteínas de Sinalização Intercelular , Osteogênese , Células-Tronco , Animais , Humanos , Camundongos , Proteínas 14-3-3/metabolismo , Proteínas 14-3-3/genética , Células Cultivadas , Polpa Dentária/citologia , Polpa Dentária/metabolismo , Dentinogênese/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos Nus , Osteogênese/genética , Células-Tronco/metabolismoRESUMO
Honeybees (Apis mellifera) are a keystone species for managed pollination and the production of hive products. Eusociality in honeybees leads to much of the reproduction in a hive driven by the queen. Queen bees have two large active ovaries that can produce large numbers of eggs if conditions are appropriate. These ovaries are also active throughout the long lives of these insects, up to 5â years in some cases. Recent studies have indicated that the germline precursors of the adult honeybee queen ovary are organized into 8-cell clusters, joined together by a polyfusome; a cytoplasmic bridge. To understand the origin of these clusters, and trace the development of the honeybee queen ovary, we examined the cell types and regionalization of the developing larval and pupal queen ovaries. We used established (nanos and castor), and novel (odd skipped) gene expression markers to determine regions of the developing ovary. Primordial germline cells develop in the honeybee embryo and are organized into ovary structures before the embryo hatches. The ovary is regionalized by larval stage 3 into terminal filaments and germaria. At this stage, clusters of germline cells in the germaria are joined by fusomes and are dividing synchronously. The origin of the 8-cell clusters in the adult germarium is therefore during larval stages. On emergence, the queen ovary has terminal filaments and germaria but has not yet developed any vitellaria, which are produced after the queen embarks on a nuptial flight. The lack of germaria, and the storing of germline progenitors as clusters, may be adaptions for queen bees to endure the metabolic demands of a nuptial flight, as well as rapidly lay large numbers of eggs to establish a hive.
Assuntos
Células Germinativas , Larva , Ovário , Animais , Abelhas/fisiologia , Ovário/citologia , Feminino , Células Germinativas/citologia , Células Germinativas/metabolismo , Larva/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
Periodontitis, a common oral disease characterized by the progressive infiltration of bacteria, is a leading cause of adult tooth loss. Periodontal stem cells (PDLSCs) possess good selfrenewal and multipotential differentiation abilities to maintain the integrity of periodontal support structure and repair defects. The present study aimed to analyze the roles of Wnt7B and frizzled4 (FZD4) in the osteogenic differentiation and macrophage polarization during periodontitis using an in vitro cell model. First, Wnt7B expression in the periodontitisaffected gingival tissue of patients and lipopolysaccharide (LPS)stimulated PDLSCs was assessed using the GSE23586 dataset and western blot analysis, respectively. In Wnt7Boverexpressing PDLSCs exposed to LPS, the capacity of osteogenic differentiation was evaluated by detecting alkaline phosphatase activity, the level of Alizarin Red S staining and the expression of genes related to osteogenic differentiation. Subsequently, conditioned medium from PDLSCs overexpressing Wnt7B was used for M0 macrophage culture. The expression of CD86 and INOS was examined using immunofluorescence staining and western blot analysis. In addition, reverse transcriptionquantitative PCR was employed to examine the expression of TNFα, IL6 and IL1ß in macrophages. The binding between Wnt7B and FZD4 was estimated using coimmunoprecipitation. In addition, FZD4 was silenced to perform the rescue experiments to elucidate the regulatory mechanism between Wnt7B and FZD4. The results demonstrated a decreased expression of Wnt7B in periodontitisaffected gingival tissue and in LPSexposed PDLSCs. Wnt7B overexpression promoted the osteogenic differentiation of LPSexposed PDLSCs and suppressed the M1 polarization of macrophages. Additionally, Wnt7B bound to FZD4 and upregulated FZD4 expression. FZD4 silencing reversed the effects of Wnt7B overexpression on the osteogenic differentiation in LPSexposed PDLSCs and the M1 polarization of macrophages. In summary, Wnt7B plays an antiperiodontitis role by binding FZD4 to strengthen the osteogenic differentiation of LPSstimulated PDLSCs and suppress the M1 polarization of macrophages.
Assuntos
Diferenciação Celular , Receptores Frizzled , Lipopolissacarídeos , Macrófagos , Osteogênese , Ligamento Periodontal , Células-Tronco , Proteínas Wnt , Humanos , Receptores Frizzled/metabolismo , Receptores Frizzled/genética , Osteogênese/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Ligamento Periodontal/citologia , Ligamento Periodontal/metabolismo , Proteínas Wnt/metabolismo , Proteínas Wnt/genética , Células-Tronco/metabolismo , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Periodontite/metabolismo , Periodontite/patologia , Células Cultivadas , Adulto , Ligação ProteicaRESUMO
OBJECTIVE: This study was designed to evaluate TFAP2A-AS1 expression in the dental pulp of teeth with or without pulpitis and to determine the function of TFAP2A-AS1 in pulp cells. METHODS: GSE92681 was analyzed to filter out differentially expressed lncRNAs. Pulp samples from teeth with pulpitis and healthy teeth (control) were examined using real-time (RT) quantitative polymerase chain reaction (qPCR). Human dental pulp stem cells (hDPSCs) were cultured in a specific medium for osteogenic induction, or treated with lipopolysaccharide (LPS) to simulate inflammation. The viability and apoptosis of human DPSCs (hDPSCs) were determined by XTT assay and apoptosis detection kit. Inflammation was induced by LPS and assessed by measuring the expression and release of inflammatory cytokines after TFAP2A-AS1 knockdown. Osteogenic differentiation of hDPSCs was investigated by determining expression levels of osteogenic markers and alkaline phosphatase (ALP) activity after TFAP2A-AS1 overexpression. The downstream microRNA (miRNA) was predicted. Dual-luciferase reporter was used to confirm the binding between miR-32-5p and TFAP2A-AS1. RESULTS: The expression of TFAP2A-AS1 was evaluated in inflamed pulp using RT-qPCR. TFAP2A-AS1 had a discriminatory ability for healthy individuals and patients with pulpitis. The expression of TFAP2A-AS1 decreased upon the osteogenic differentiation of hDPSCs, and increased upon the LPS induction. TFAP2A-AS1 can reverse the osteogenic differentiation of hDPSCs, as evidenced by decreased levels of dentine sialophosphoprotein, dentin matrix protein-1, and ALP activity. TFAP2A-AS1 knockdown can promote cell proliferation of hDPSCs and relieve LPS-induced inflammation, as evidenced by decreased levels of TNF-α, IL-1ß, and IL-6. miR-32-5p was identified as a downstream miRNA of TFAP2A-AS1. CONCLUSION: This study demonstrated the expression and potential function of TFAP2A-AS1 in the human dental pulp. TFAP2A-AS1 can inhibit odontogenic differentiation but promote inflammation in pulp cells.
Assuntos
Polpa Dentária , MicroRNAs , Pulpite , RNA Longo não Codificante , Fator de Transcrição AP-2 , Humanos , MicroRNAs/genética , RNA Longo não Codificante/genética , Polpa Dentária/metabolismo , Polpa Dentária/patologia , Pulpite/metabolismo , Pulpite/genética , Pulpite/patologia , Fator de Transcrição AP-2/metabolismo , Fator de Transcrição AP-2/genética , Diferenciação Celular/genética , Osteogênese/genética , Apoptose/genética , Regulação da Expressão Gênica , Células Cultivadas , Lipopolissacarídeos , Células-Tronco/metabolismoRESUMO
We previously showed that inhibition of glycolysis in cutaneous squamous cell carcinoma (SCC)-initiating cells had no effect on tumorigenesis, despite the perceived requirement of the Warburg effect, which was thought to drive carcinogenesis. Instead, these SCCs were metabolically flexible and sustained growth through glutaminolysis, another metabolic process frequently implicated to fuel tumorigenesis in various cancers. Here, we focused on glutaminolysis and genetically blocked this process through glutaminase (GLS) deletion in SCC cells of origin. Genetic deletion of GLS had little effect on tumorigenesis due to the up-regulated lactate consumption and utilization for the TCA cycle, providing further evidence of metabolic flexibility. We went on to show that posttranscriptional regulation of nutrient transporters appears to mediate metabolic flexibility in this SCC model. To define the limits of this flexibility, we genetically blocked both glycolysis and glutaminolysis simultaneously and found the abrogation of both of these carbon utilization pathways was enough to prevent both papilloma and frank carcinoma.
Assuntos
Carcinoma de Células Escamosas , Glutaminase , Glicólise , Folículo Piloso , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/genética , Animais , Folículo Piloso/metabolismo , Glutaminase/metabolismo , Glutaminase/genética , Camundongos , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/genética , Células-Tronco/metabolismo , Glutamina/metabolismo , Humanos , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/genética , Carcinogênese/metabolismo , Carcinogênese/genéticaRESUMO
BACKGROUND: The human endometrium, lining the inner uterus, regenerates over 400 times uniquely during a woman's reproductive life. Endometrial stem cells (eSCs) enrich the tissue, resulting in a dense vascular network, significant angiogenic potential, and effective regeneration power. Being of natural angiogenic properties and proven effective in the treatment of vascular disorders, eSCs can be considered safe, reliable, and superior to other post-natal stem cells. Cluster of Differentiation 146 (CD146) has emerged as a pivotal marker associated with pericytes and endothelial cells for promoting angiogenesis. Endometrial cells with high CD146 expression could proliferate and differentiate into multiple lineages. This study will explore the role of CD146 in eSCs, focusing on the potential to boost the angiogenic and regenerative functions of the cells. The novelty of this study lies in the investigation of CD146 on eSC function, which may open new possibilities for eSC-based therapy in regenerative medicine and vascular disorders. METHODS: The study involved obtaining endometrial biopsies from active reproducing women to isolate and cultivate eSCs. eSCs were assessed for growth factor secretion pattern, characterized for their mesenchymal properties. Finally, eSCs were tested for their angiogenic potential by angiogenic gene expression profile and in-ovo chick embryo model. As aimed, to check the role of CD146 in eSC angiogenesis, CD146+ cells were magnetically sorted and cultured. The sorted cells underwent various analyses, including flowcytometry to identify mesenchymal markers and human growth factor panel to analyze growth factor secretion profiles The study evaluated the angiogenic potential of CD146 + cells using functional assays, including ring formation, endothelial differentiation, and wound scratch assays, to evaluate cell migration and healing capabilities. Molecular insights were obtained through chemokine and cytokine investigations In-ovo Chick model assay was conducted to check the angiogenic potential and evaluated through macroscopic as well as through immunohistochemistry. RESULT: Endometrial stem cells (eSCs) were successfully isolated using a combination of mechanical and enzymatic digestion, followed by culturing in complete DMEM media. The secretion profile of eSCs revealed significant production of various angiogenic growth factors, including Granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), Vascular endothelial growth factor (VEGF), Fibroblast growth factors (FGF), and Platelet derived growth factor AA (PDGF-AA). The angiogenic gene profile indicated upregulation of several angiogenic genes in eSCs. The mesenchymal nature of eSCs was demonstrated through surface marker analysis (Cluster of differentiation 73, Cluster of differentiation 90, Cluster of differentiation 105) and trilineage differentiation. The in-ovo chick model confirmed the angiogenic potential of eSCs. CD146+ cells, isolated via magnetic sorting, exhibited enhanced angiogenic potential. These cells secreted significant levels of angiogenic growth factors such as VEGF. In Matrigel assays, CD146+ cells formed endothelial ring structures more rapidly and persistently than unsorted eSCs. Semi-quantitative PCR confirmed their endothelial differentiation. CD146+ cells express various angiogenic chemokines such as CXCL5, CXCL8, CCL3, and CCL20 and cytokines such as GM-CSF, Interleukin-1ß (IL-1ß), Interleukin-6 (IL-6), PDGF AA/BB, Epidermal growth factor (EGF), Endothelin 1, Angiopoietin. In-ovo chick model assay showed that CD146+ cells had superior angiogenesis, with more nodes, junctions, and segments compared to eSCs and controls. Immunohistochemistry confirmed increased expression of endothelial markers (Cluster of differentiation 31, VEGF, Vascular associated protein (VAP), Von Willebrand factor (vWF) in CD146+ cells. CONCLUSION: The study highlights the angiogenic potential of endometrial stem cells, particularly the CD146+ cell population. These cells promote angiogenesis, secreting growth factors and forming stable blood vessel structures. CD146+ cells have higher expression levels of VEGF and TGF-α, key factors in angiogenesis. This suggests CD146+ eSCs may be promising for therapeutic applications in vascular diseases requiring angiogenesis. Further research is needed.
Assuntos
Antígeno CD146 , Endométrio , Neovascularização Fisiológica , Feminino , Antígeno CD146/metabolismo , Antígeno CD146/genética , Endométrio/metabolismo , Endométrio/citologia , Endométrio/irrigação sanguínea , Humanos , Animais , Diferenciação Celular , Células-Tronco/metabolismo , Células-Tronco/citologia , Embrião de Galinha , Células Cultivadas , AdultoRESUMO
BACKGROUND: The transplantation of periodontal ligament stem cells (PDLSCs) has been shown to enhance periodontal regeneration in animal models and clinical trials. However, it is not known whether PDLSCs are antibacterial and whether this affects oral microbiota and periodontal regeneration. METHODS: We isolated human PDLSCs from periodontal ligament of extracted teeth. Rats' periodontal fenestration defects were prepared, and treated with PDLSC injections (Cell group), using saline injections (Saline group) as the control. The oral microbiota was explored by 16 S rDNA sequencing and compared with that before surgery (PRE group). The antibacterial property of PDLSCs and its underlying mechanism were tested in vitro. RESULTS: Microbiome analyses reveal a decreased biodiversity, a changed community structure, and downregulated community functions of the oral microbiome in the Saline group. PDLSCs injections enhance periodontal regeneration, reverse the decrease in diversity, and increase the abundance of non-pathogenic bacterial Bifidobacterium sp. and Lactobacillus sp., making the oral microbiome similar to that of the PRE group. In vitro, PDLSCs inhibit the growth of Staphylococcus aureus, Escherichia coli, and Fusobacterium nucleatum. The main mechanism of action is postulated to involve production of the cationic antimicrobial peptide LL-37. CONCLUSIONS: Our findings reveal that PDLSC injections enhance periodontal regeneration and regulate the oral microbiome to foster an oral cavity microenvironment conducive to symbiotic microbiota associated with health.
Assuntos
Microbiota , Ligamento Periodontal , Regeneração , Células-Tronco , Ligamento Periodontal/citologia , Humanos , Células-Tronco/metabolismo , Células-Tronco/citologia , Animais , Ratos , Masculino , Antibacterianos/farmacologia , Ratos Sprague-Dawley , Transplante de Células-Tronco/métodos , Boca/microbiologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , CatelicidinasRESUMO
The primary objective of omics in space with focus on the human organism is to characterize and quantify biological factors that alter structure, morphology, function, and dynamics of human cells exposed to microgravity. This review discusses exciting data regarding genomics, transcriptomics, epigenomics, metabolomics, and proteomics of human cells and individuals in space, as well as cells cultured under simulated microgravity. The NASA Twins Study significantly heightened interest in applying omics technologies and bioinformatics in space and terrestrial environments. Here, we present the available publications in this field with a focus on specialized cells and stem cells exposed to real and simulated microgravity conditions. We summarize current knowledge of the following topics: (i) omics studies on stem cells, (ii) omics studies on benign specialized different cell types of the human organism, (iii) discussing the advantages of this knowledge for space commercialization and exploration, and (iv) summarizing the emerging opportunities for translational regenerative medicine for space travelers and human patients on Earth.
Assuntos
Genômica , Metabolômica , Células-Tronco , Ausência de Peso , Humanos , Células-Tronco/metabolismo , Células-Tronco/citologia , Genômica/métodos , Metabolômica/métodos , Proteômica/métodos , Epigenômica/métodos , Voo Espacial , Simulação de Ausência de Peso , AnimaisRESUMO
To accurately study human organ function and disease 'in the dish', it is necessary to develop reliable cell-based models that closely track human physiology. Our interest lay with the liver, which is the largest solid organ in the body. The liver is a multifunctional and highly regenerative organ; however, severe liver damage can have dire consequences for human health. A common cause of liver damage is adverse reactions to prescription drugs. Therefore, the development of predictive liver models that capture human drug metabolism patterns is required to optimise the drug development process. In our study, we aimed to identify compounds that could improve the metabolic function of stem cell-derived liver tissue. Therefore, we screened a compound library to identify additives that improved the maturity of in vitro-engineered human tissue, with the rationale that by taking such an approach, we would be able to fine-tune neonatal and adult cytochrome P450 metabolic function in stem cell-derived liver tissue.
Assuntos
Sistema Enzimático do Citocromo P-450 , Fígado , Humanos , Sistema Enzimático do Citocromo P-450/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/química , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Engenharia Tecidual/métodos , Células-Tronco/metabolismo , Células-Tronco/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/efeitos dos fármacosRESUMO
BACKGROUND: Human adipose-derived stromal/stem cells (hASCs) play important roles in regenerative medicine and numerous inflammatory diseases. However, their cellular heterogeneity limits the effectiveness of treatment. Understanding the distinct subtypes of hASCs and their phenotypic implications will enable the selection of appropriate subpopulations for targeted approaches in regenerative medicine or inflammatory diseases. METHODS: hASC subtypes expressing dipeptidyl peptidase-4 (DPP4) were identified via fluorescence-activated cell sorting (FACS) analysis. DPP4 expression was knocked down in DPP4+ hASCs via DPP4 siRNA. The capacity for proliferation, hepatocyte differentiation, inflammatory factor secretion and T-cell functionality regulation of hASCs from DPP4-, DPP4+, and control siRNA-treated DPP4+ hASCs and DPP4 siRNA-treated DPP4+ hASCs were assessed. RESULTS: DPP4+ hASCs and control siRNA-treated DPP4+ hASCs presented a lower proliferative capacity but greater hepatocyte differentiation capacity than DPP4- hASCs and DPP4 siRNA-treated DPP4+ hASCs. Both DPP4+ hASCs and DPP4- hASCs secreted high levels of vascular endothelial growth factor-A (VEGF-A), monocyte chemoattractant protein-1 (MCP-1), and interleukin 6 (IL-6), whereas the levels of other factors, including matrix metalloproteinase (MMP)-1, eotaxin-3, fractalkine (FKN, CX3CL1), growth-related oncogene-alpha (GRO-alpha, CXCL1), monokine induced by interferon-gamma (MIG), macrophage inflammatory protein (MIP)-1beta, and macrophage colony-stimulating factor (M-CSF), were significantly greater in the supernatants of DPP4+ hASCs than in those of DPP4- hASCs. Exposure to hASC subtypes and their conditioned media triggered changes in the secreted cytokine profiles of T cells from healthy donors. The percentage of functional T cells that secreted factors such as MIP-1beta and IL-8 increased when these cells were cocultured with DPP4+ hASCs. The percentage of polyfunctional CD8+ T cells that secreted multiple factors, such as IL-17A, tumour necrosis factor alpha (TNF-α) and TNF-ß, decreased when these cells were cocultured with supernatants derived from DPP4+ hASCs. CONCLUSIONS: DPP4 may regulate proliferation, hepatocyte differentiation, inflammatory cytokine secretion and T-cell functionality of hASCs. These data provide a key foundation for understanding the important role of hASC subpopulations in the regulation of T cells, which may be helpful for future immune activation studies and allow them to be customized for clinical application.