RESUMO
Microglial activation followed by recruitment of blood-borne macrophages into the central nervous system (CNS) aggravates neuroinflammation. Specifically, in multiple sclerosis (MS) as well as in experimental autoimmune encephalomyelitis (EAE), a rodent model of MS, activated microglia and macrophages (Mg/Mφ) promote proinflammatory responses and expand demyelination in the CNS. However, a potent therapeutic approach through the systemic route for regulating their functions has not yet been developed. Here, we demonstrate that a systemically injected DNA/RNA heteroduplex oligonucleotide (HDO), composed of an antisense oligonucleotide (ASO) and its complementary RNA, conjugated to cholesterol (Chol-HDO) distributed more efficiently to demyelinating lesions of the spinal cord in EAE mice with significant gene silencing than the parent ASO. Importantly, systemic administration of Cd40-targeting Chol-HDO improved clinical signs of EAE with significant downregulation of Cd40 in Mg/Mφ. Furthermore, we successfully identify that macrophage scavenger receptor 1 (MSR1) is responsible for the uptake of Chol-HDO by Mg/Mφ of EAE mice. Overall, our findings demonstrate the therapeutic potency of systemically administered Chol-HDO to regulate activated Mg/Mφ in neuroinflammation.
Assuntos
Encefalomielite Autoimune Experimental , Esclerose Múltipla , Animais , DNA/uso terapêutico , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/terapia , Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Microglia/patologia , Esclerose Múltipla/genética , Esclerose Múltipla/terapia , Oligonucleotídeos/uso terapêutico , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/uso terapêutico , RNARESUMO
Adipose stem and progenitor cells (ASPCs) have been isolated from humans and animals for use in regenerative medicine and therapy. However, knowledge of ASPCs in other species is limited. Particularly, ASPCs in livestock are expected to enhance the fat content and meat composition. In this study, we isolated bovine ASPCs using cell surface markers. Specifically, we focused on ASPC markers in humans and experimental animals, namely CD26, CD146, and CD54. Stromal vascular fraction cells from bovine fat were separated using flow cytometry before primary culture. We evaluated the self-renewal and adipogenic potential of each fraction. We identified four cell populations: CD26-CD146+CD54+, CD26-CD146+CD54-, CD26-CD146-, and CD26+CD146-. Among them, the CD26-CD146+ fraction, particularly CD54+, demonstrated the properties of preadipocytes (PreAs), characterized by slow proliferation and a high adipogenic capacity. In conclusion, we could collect and characterize possible PreAs as CD26-CD146+CD54+ or CD26-CD146+CD54-, which are expected for in vitro bovine adipogenic assays in the future.
Assuntos
Dipeptidil Peptidase 4 , Células-Tronco , Humanos , Bovinos , Animais , Dipeptidil Peptidase 4/metabolismo , Diferenciação Celular , Antígeno CD146/metabolismo , Células-Tronco/metabolismo , Citometria de Fluxo , Obesidade/metabolismo , Tecido Adiposo/metabolismoRESUMO
Myoepithelial (ME) cells in exocrine glands exhibit both epithelial and mesenchymal features, contributing to fluid secretion through contraction. However, the regulation mechanism of behind this unique phenotype in salivary glands remains unclear. We established a flow cytometry-based purification method using cell surface molecules, epithelial cell adhesion molecule (EpCAM) and alpha 6 integrin (CD49f), to characterize ME cells. EpCAM+CD49fhigh cells showed relatively high expression of ME cell-marker genes, such as alpha-smooth muscle actin (α-SMA). For lineage tracing and strict isolation, tdTomato+EpCAM+CD49fhigh-ME cells were obtained from myosin heavy chain 11 (Myh11) -CreERT2/tdTomato mice. Transcriptome analysis revealed that expression of genes involved in the epithelial-mesenchymal transition, including Snai2, were upregulated in the ME cell-enriched subset. Snai2 suppression in stable ME cells decreased α-SMA and increased Krt14 expression, suggesting that ME cell features may be controlled by the epithelial-mesenchymal balance regulated by Snai2. In contrast, ME cells showed reduced ME properties and expressed the ductal markers Krt18/19 under sphere culture conditions. Notch signaling was activated under sphere culture conditions; excessive activation of Notch signaling accelerated Krt18/19 expression, but reduced α-SMA and Snai2 expression, suggesting that the behavior of Snai2-expressing ME cells may be controlled by Notch signaling.
Assuntos
Actinas , Cadeias Pesadas de Miosina , Camundongos , Animais , Integrina alfa6/metabolismo , Molécula de Adesão da Célula Epitelial/metabolismo , Actinas/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Células Epiteliais/metabolismo , Músculo Liso/metabolismo , Glândulas Salivares/metabolismo , Biomarcadores/metabolismoRESUMO
Human mesenchymal stem/stromal cells (hMSCs) have garnered enormous interest as a potential resource for cell-based therapies. However, the molecular mechanisms regulating senescence in hMSCs remain unclear. To elucidate these mechanisms, we performed gene expression profiling to compare clonal immature MSCs exhibiting multipotency with less potent MSCs. We found that the transcription factor Frizzled 5 (FZD5) is expressed specifically in immature hMSCs. The FZD5 cell surface antigen was also highly expressed in the primary MSC fraction (LNGFR+ THY-1+ ) and cultured MSCs. Treatment of cells with the FZD5 ligand WNT5A promoted their proliferation. Upon FZD5 knockdown, hMSCs exhibited markedly attenuated proliferation and differentiation ability. The observed increase in the levels of senescence markers suggested that FZD5 knockdown promotes cellular senescence by regulating the noncanonical Wnt pathway. Conversely, FZD5 overexpression delayed cell cycle arrest during the continued culture of hMSCs. These results indicated that the intrinsic activation of FZD5 plays an essential role in negatively regulating senescence in hMSCs and suggested that controlling FZD5 signaling offers the potential to regulate hMSC quality and improve the efficacy of cell-replacement therapies using hMSCs.
Assuntos
Diferenciação Celular/fisiologia , Senescência Celular/fisiologia , Receptores Frizzled/metabolismo , Células-Tronco Mesenquimais/metabolismo , Proliferação de Células/fisiologia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células Cultivadas , Humanos , Transplante de Células-Tronco Mesenquimais/métodosRESUMO
It is well known that the properties of hematopoietic stem/progenitor cells (HSCs), such as their self-renewal ability and multipotency, are maintained through interactions with mesenchymal stem/stromal cells (MSCs). MSCs are rare cells that are present in the bone marrow and are useful for clinical applications due to their functional ability. To obtain the necessary number of cells, MSCs must be cultured to expand, but this causes a remarkable decrease in stem cell properties, such as multipotency and proliferation ability. In this study, we show that the c-Mpl signal, which is related to the maintenance of hematopoietic stem cells, has an important effect on the proliferation and differentiation ability of MSCs. Utilizing a co-culture system comprising MSCs and HSCs, it is suggested that signaling from hematopoietic cells to MSCs supports cell proliferation. Interestingly, the enhanced proliferation ability of the HSCs was decreased in c-Mpl knock-out HSCs (c-Mpl-KO). In addition, the MSCs co-cultured with c-Mpl-KO HSCs had reduced MSC marker expression (PDGFRa and Sca-1) compared to the MSCs co-cultured with c-Mpl-wild-type HSCs. These results suggest that a hematopoietic-mesenchymal signal exists, and that the state of the HSCs is important for the stability of MSC properties.
Assuntos
Células-Tronco Mesenquimais , Células da Medula Óssea , Diferenciação Celular , Células Cultivadas , Técnicas de Cocultura , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Mesenquimais/metabolismoRESUMO
Bone metastatic lesions are classified as osteoblastic or osteolytic lesions. Prostate and breast cancer patients frequently exhibit osteoblastic-type and osteolytic-type bone metastasis, respectively. In metastatic lesions, tumor cells interact with many different cell types, including osteoblasts, osteoclasts, and mesenchymal stem cells, resulting in an osteoblastic or osteolytic phenotype. However, the mechanisms responsible for the modification of bone remodeling have not been fully elucidated. MicroRNAs (miRNAs) are transferred between cells via exosomes and serve as intercellular communication tools, and numerous studies have demonstrated that cancer-secreted miRNAs are capable of modifying the tumor microenvironment. Thus, cancer-secreted miRNAs can induce an osteoblastic or osteolytic phenotype in the bone metastatic microenvironment. In this study, we performed a comprehensive expression analysis of exosomal miRNAs secreted by several human cancer cell lines and identified eight types of human miRNAs that were highly expressed in exosomes from osteoblastic phenotype-inducing prostate cancer cell lines. One of these miRNAs, hsa-miR-940, significantly promoted the osteogenic differentiation of human mesenchymal stem cells in vitro by targeting ARHGAP1 and FAM134A Interestingly, although MDA-MB-231 breast cancer cells are commonly known as an osteolytic phenotype-inducing cancer cell line, the implantation of miR-940-overexpressing MDA-MB-231 cells induced extensive osteoblastic lesions in the resulting tumors by facilitating the osteogenic differentiation of host mesenchymal cells. Our results suggest that the phenotypes of bone metastases can be induced by miRNAs secreted by cancer cells in the bone microenvironment.
Assuntos
Neoplasias Ósseas/metabolismo , Neoplasias da Mama/patologia , Proteínas Ativadoras de GTPase/metabolismo , Proteínas de Membrana/metabolismo , MicroRNAs/metabolismo , Neoplasias da Próstata/metabolismo , Adenocarcinoma/metabolismo , Animais , Neoplasias Ósseas/secundário , Substitutos Ósseos , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Proteínas Ativadoras de GTPase/genética , Humanos , Masculino , Proteínas de Membrana/genética , Células-Tronco Mesenquimais , Camundongos , MicroRNAs/genética , Neoplasias Experimentais/metabolismoRESUMO
Cell adhesion between oligodendrocytes and neuronal axons is a critical step for myelination that enables the rapid propagation of action potential in the central nervous system. Here, we show that the transmembrane protein teneurin-4 plays a role in the cell adhesion required for the differentiation of oligodendrocytes. We found that teneurin-4 formed molecular complexes with all of the four teneurin family members and promoted cell-cell adhesion. Oligodendrocyte lineage cells attached to the recombinant extracellular domain of all the teneurins and formed well-branched cell processes. In an axon-mimicking nanofibers assay, nanofibers coated with the recombinant teneurin-4 extracellular domain increased the differentiation of oligodendrocytes. Our results show that teneurin-4 binds to all teneurins through their extracellular domain, which facilitates the oligodendrocyte-axon adhesion, and promotes oligodendrocyte differentiation via its homophilic interaction.
Assuntos
Adesão Celular , Diferenciação Celular , Espaço Extracelular/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Oligodendroglia/citologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oligodendroglia/metabolismo , Domínios Proteicos , Ratos , Ratos WistarRESUMO
Exocrine glands share a common morphology consisting of ductal, acinar, and basal/myoepithelial cells, but their functions and mechanisms of homeostasis differ among tissues. Salivary glands are an example of exocrine glands, and they have been reported to contain multipotent stem cells that differentiate into other tissues. In this study, we purified the salivary gland stem/progenitor cells of adult mouse salivary glands using the cell surface marker CD133 by flow cytometry. CD133+ cells possessed stem cell capacity, and the transplantation of CD133+ cells into the submandibular gland reconstituted gland structures, including functional acinar. CD133+ cells were sparsely distributed in the intercalated and exocrine ducts and expressed Sox9 at higher levels than CD133- cells. Moreover, we demonstrated that Sox9 was required for the stem cell properties CD133+ cells, including colony and sphere formation. Thus, the Sox9-related signaling may control the regeneration salivary glands.
Assuntos
Fatores de Transcrição SOX9/fisiologia , Células-Tronco/citologia , Glândula Submandibular/citologia , Antígeno AC133/análise , Adulto , Idoso , Animais , Autorrenovação Celular , Ensaio de Unidades Formadoras de Colônias , Feminino , Genes Reporter , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Ductos Salivares/citologia , Ductos Salivares/metabolismo , Transplante de Células-Tronco , Células-Tronco/metabolismo , Glândula Submandibular/metabolismoRESUMO
Pericytes are mesenchymal cells that surround the endothelial cells of small vessels in various organs. These cells express several markers, such as NG2, CD146, and PDGFRß, and play an important role in the stabilization and maturation of blood vessels. It was also recently revealed that like mesenchymal stem cells (MSCs), pericytes possess multilineage differentiation capacity, especially myogenic, adipogenic, and fibrogenic differentiation capacities. Although some previous studies have reported that pericytes also have osteogenic potential, the osteogenesis of pericytes can still be further elucidated. In the present study, we established novel methods for isolating and culturing primary murine pericytes. An immortalized pericyte line was also established. Multilineage induction of the pericyte line induced osteogenesis, adipogenesis, and chondrogenesis of the cells in vitro. In addition, pericytes that were injected into the fracture site of a bone fracture mouse model contributed to callus formation. Furthermore, in vivo pericyte-lineage-tracing studies demonstrated that endogenous pericytes also differentiate into osteoblasts and osteocytes and contribute to bone fracture healing as a cellular source of osteogenic cells. Pericytes can be a promising therapeutic candidate for treating bone fractures with a delayed union or nonunion as well as bone diseases causing bone defects.
Assuntos
Condrogênese , Consolidação da Fratura , Osteogênese , Pericitos/citologia , Cultura Primária de Células/métodos , Animais , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Condrócitos/citologia , Transplante de Células-Tronco Mesenquimais/métodos , Camundongos , Camundongos Endogâmicos BALB C , Osteoblastos/citologia , Pericitos/transplanteRESUMO
Satellite cells are maintained in an undifferentiated quiescent state, but during muscle regeneration they acquire an activated stage, and initiate to proliferate and differentiate as myoblasts. The transmembrane protein teneurin-4 (Ten-4) is specifically expressed in the quiescent satellite cells; however, its cellular and molecular functions remain unknown. We therefore aimed to elucidate the function of Ten-4 in muscle satellite cells. In the tibialis anterior (TA) muscle of Ten-4-deficient mice, the number and the size of myofibers, as well as the population of satellite cells, were reduced with/without induction of muscle regeneration. Furthermore, we found an accelerated activation of satellite cells in the regenerated Ten-4-deficient TA muscle. The cell culture analysis using primary satellite cells showed that Ten-4 suppressed the progression of myogenic differentiation. Together, our findings revealed that Ten-4 functions as a crucial player in maintaining the quiescence of muscle satellite cells.
Assuntos
Diferenciação Celular/genética , Proliferação de Células/genética , Proteínas de Membrana/genética , Regeneração/genética , Animais , Proteínas de Membrana/metabolismo , Camundongos , Músculo Esquelético/crescimento & desenvolvimento , Miofibrilas/metabolismo , Miofibrilas/patologia , Células Satélites de Músculo Esquelético/metabolismoRESUMO
Although meniscus defects and degeneration are strongly correlated with the later development of osteoarthritis, the promise of regenerative medicine strategies is to prevent and/or delay the disease's progression. Meniscal reconstruction has been shown in animal models with tendon grafting and transplantation of mesenchymal stem cells (MSCs); however, these procedures have not shown the same efficacy in clinical studies. Here, our aim was to investigate the ability of tendon grafts pretreated with exogenous synovial-derived MSCs to prevent cartilage degeneration in a rat partial meniscus defect model. We removed the anterior half of the medial meniscus and grafted autologous Achilles tendons with or without a 10-minute pretreatment of the tendon with synovial MSCs. The meniscus and surrounding cartilage were evaluated at 2, 4, and 8 weeks (n = 5). Tendon grafts increased meniscus size irrespective of synovial MSCs. Histological scores for regenerated menisci were better in the tendon + MSC group than in the other two groups at 4 and 8 weeks. Both macroscopic and histological scores for articular cartilage were significantly better in the tendon + MSC group at 8 weeks. Implanted synovial MSCs survived around the grafted tendon and native meniscus integration site by cell tracking assays with luciferase+, LacZ+, DiI+, and/or GFP+ synovial MSCs and/or GFP+ tendons. Flow cytometric analysis showed that transplanted synovial MSCs retained their MSC properties at 7 days and host synovial tissue also contained cells with MSC characteristics. Synovial MSCs promoted meniscus regeneration augmented by autologous Achilles tendon grafts and prevented cartilage degeneration in rats.
Assuntos
Tendão do Calcâneo/citologia , Diferenciação Celular/fisiologia , Células-Tronco Mesenquimais/citologia , Regeneração/fisiologia , Membrana Sinovial/citologia , Animais , Cartilagem Articular , Modelos Animais de Doenças , Masculino , Transplante de Células-Tronco Mesenquimais/métodos , Ratos , Medicina Regenerativa/métodosRESUMO
Repeated and dramatic pregnancy-induced uterine enlargement and remodeling throughout reproductive life suggests the existence of uterine smooth muscle stem/progenitor cells. The aim of this study was to isolate and characterize stem/progenitor-like cells from human myometrium through identification of specific surface markers. We here identify CD49f and CD34 as markers to permit selection of the stem/progenitor cell-like population from human myometrium and show that human CD45(-) CD31(-) glycophorin A(-) and CD49f(+) CD34(+) myometrial cells exhibit stem cell-like properties. These include side population phenotypes, an undifferentiated status, high colony-forming ability, multilineage differentiation into smooth muscle cells, osteoblasts, adipocytes, and chondrocytes, and in vivo myometrial tissue reconstitution following xenotransplantation. Furthermore, CD45(-) CD31(-) glycophorin A(-) and CD49f(+) CD34(+) myometrial cells proliferate under hypoxic conditions in vitro and, compared with the untreated nonpregnant myometrium, show greater expansion in the estrogen-treated nonpregnant myometrium and further in the pregnant myometrium in mice upon xenotransplantation. These results suggest that the newly identified myometrial stem/progenitor-like cells influenced by hypoxia and sex steroids may participate in pregnancy-induced uterine enlargement and remodeling, providing novel insights into human myometrial physiology.
Assuntos
Antígenos CD34/genética , Antígenos CD34/fisiologia , Integrina alfa6/genética , Integrina alfa6/fisiologia , Miométrio/metabolismo , Células-Tronco/fisiologia , Útero/fisiologia , Animais , Diferenciação Celular , Hipóxia Celular , Linhagem da Célula/genética , Feminino , Glicoforinas/biossíntese , Glicoforinas/genética , Células-Tronco Hematopoéticas , Humanos , Camundongos , Miométrio/citologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/biossíntese , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , GravidezRESUMO
BACKGROUND/PURPOSE: Despite technical advances in the surgical/medical care of anorectal malformation (ARM), persistent unsatisfactory postoperative bowel habit has been attributed to histopathologic abnormalities of the distal rectum/pouch (DRP) and hypoplasia of anal sphincter muscles (ASM). We used Sox10-Venus mice with ARM induced by all-trans retinoic acid (ATRA) to investigate neural crest cell (NCC) innervation in the DRP and ASM. METHOD: Pregnant Sox10-Venus mice were administered single doses of 50, 70, or 100 mg/kg of ATRA on embryonic day 8.5 (E8.5) then sacrificed on either E16.5 or E19.5. Bowel specimens comprising the anorectum were examined using fluorescence microscopy without immunohistochemical staining (FMIS). Anti-PGP9.5 was used to delineate ganglion cells and anti-SMA for smooth muscles. RESULTS: The appropriate dose of ATRA for inducing ARM was 50 mg/kg. Under FMIS, all ARM embryos (n = 5; all high type; 3 male:2 female) had less NCC innervation with thick Venus-positive nerve fibers in the DRP compared with normal embryos (n = 8); there was abnormal NCC innervation in the DRP and absent ASM in ARM mice. CONCLUSION: We are the first to delineate abnormal enteric nervous system innervation in the DRP of ARM mice without using immunohistochemical staining techniques thus allowing specimens to be examined without any distortion.
Assuntos
Anormalidades Múltiplas/induzido quimicamente , Anormalidades Múltiplas/patologia , Canal Anal/anormalidades , Anus Imperfurado/induzido quimicamente , Anus Imperfurado/patologia , Intestinos/patologia , Crista Neural/inervação , Crista Neural/patologia , Reto/anormalidades , Anormalidades Múltiplas/embriologia , Canal Anal/embriologia , Canal Anal/patologia , Animais , Malformações Anorretais , Anus Imperfurado/embriologia , Modelos Animais de Doenças , Feminino , Intestinos/embriologia , Masculino , Camundongos , Microscopia de Fluorescência , Reto/embriologia , Reto/patologia , TretinoínaRESUMO
Mesenchymal stem/stromal cells (MSCs) are distributed in various tissues and are used in clinical applications as a source of transplanted cells because of their easy harvestability. Although MSCs express numerous cell-surface antigens, single-cell analyses have revealed a highly heterogeneous cell population depending on the original tissue and donor conditions, including age and interindividual differences. This heterogeneity leads to differences in their functions, such as multipotency and immunomodulatory effects, making it challenging to effectively treat targeted diseases. The therapeutic efficacy of MSCs is controversial and depends on the implantation site. Thus, there is no established recipe for the transplantation of MSCs (including the type of disease, type of origin, method of cell culture, form of transplanted cells, and site of delivery). Our recent preclinical study identified appropriate MSCs and their suitable transplantation routes in a mouse model of inflammatory bowel disease (IBD). Three-dimensional (3D) cultures of MSCs have been demonstrated to enhance their properties and sustain engraftment at the lesion site. In this note, we explore the methods of MSC transplantation for treating IBDs, especially Crohn's disease, from clinical trials published over the past decade. Given the functional changes in MSCs in 3D culture, we also investigate the clinical trials using 3D constructs of MSCs and explore suitable diseases that might benefit from this approach. Furthermore, we discuss the advantages of the prospective isolation of MSCs in terms of interindividual variability. This note highlights the need to define the method of MSC transplantation, including interindividual variability, the culture period, and the transplantation route.
RESUMO
The demand for stem cell-based cultured meat as an alternative protein source is increasing in response to global food scarcity. However, the definition of quality controls, including appropriate growth factors and cell characteristics, remains incomplete. Cluster of differentiation (CD) 29 is ubiquitously expressed in bovine muscle tissue and is a marker of progenitor cells in cultured meat. However, CD29+ cells are naturally heterogeneous, and this quality control issue must be resolved. In this study, the aim was to identify the subpopulation of the CD29+ cell population with potential utility in cultured meat production. The CD29+ cell population exhibited heterogeneity, discernible through the CD44 and CD344 markers. CD29+CD44-CD344- cells displayed the ability for long-term culture, demonstrating high adipogenic potential and substantial lipid droplet accumulation, even within 3D cultures. Conversely, CD29+CD44+ cells exhibited rapid proliferation but were not viable for prolonged culture. Using cells suitable for adipocyte and muscle differentiation, we successfully designed meat buds, especially those rich in fat. Collectively, the identification and comprehension of distinct cell populations within bovine tissues contribute to quality control predictions in meat production. They also aid in establishing a stable and reliable cultured meat production technique.
Assuntos
Carne in vitro , Carne , Animais , Bovinos , Células-Tronco , Adipócitos , Controle de QualidadeRESUMO
BACKGROUND: Severe peripheral nerve damage always requires surgical treatment. Autologous nerve transplantation is a standard treatment, but it is not sufficient due to length limitations and extended surgical time. Even with the available artificial nerves, there is still large room for improvement in their therapeutic effects. Novel treatments for peripheral nerve injury are greatly expected. METHODS: Using a specialized microfluidic device, we generated artificial neurite bundles from human iPSC-derived motor and sensory nerve organoids. We developed a new technology to isolate cell-free neurite bundles from spheroids. Transplantation therapy was carried out for large nerve defects in rat sciatic nerve with novel artificial nerve conduit filled with lineally assembled sets of human neurite bundles. Quantitative comparisons were performed over time to search for the artificial nerve with the therapeutic effect, evaluating the recovery of motor and sensory functions and histological regeneration. In addition, a multidimensional unbiased gene expression profiling was carried out by using next-generation sequencing. RESULT: After transplantation, the neurite bundle-derived artificial nerves exerted significant therapeutic effects, both functionally and histologically. Remarkably, therapeutic efficacy was achieved without immunosuppression, even in xenotransplantation. Transplanted neurite bundles fully dissolved after several weeks, with no tumor formation or cell proliferation, confirming their biosafety. Posttransplant gene expression analysis highlighted the immune system's role in recovery. CONCLUSION: The combination of newly developed microfluidic devices and iPSC technology enables the preparation of artificial nerves from organoid-derived neurite bundles in advance for future treatment of peripheral nerve injury patients. A promising, safe, and effective peripheral nerve treatment is now ready for clinical application.
RESUMO
Myeloid cells, which originate from hematopoietic stem/progenitor cells (HSPCs), play a crucial role in mitigating infections. This study aimed to explore the impact of mesenchymal stem/stromal cells (MSCs) on the differentiation of HSPCs and progenitors through the C-C motif chemokine CCL2/CCR2 signaling pathway. Murine MSCs, identified as PDGFRα+Sca-1+ cells (PαS cells), were found to secrete CCL2, particularly in response to lipopolysaccharide stimulation. MSC-secreted CCL2 promoted the differentiation of granulocyte/macrophage progenitors into the myeloid lineage. MSC-derived CCL2 plays an important role in the early phase of myeloid cell differentiation in vivo. Single-cell RNA sequencing analysis confirmed that CCL2-mediated cell fate determination was also observed in human bone marrow cells. These findings provide valuable insights for investigating the in vivo effects of MSC transplantation.
Assuntos
Quimiocina CCL2 , Células-Tronco Mesenquimais , Animais , Humanos , Camundongos , Diferenciação Celular , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Receptores CCR2/genética , Receptores CCR2/metabolismo , Transdução de SinaisRESUMO
The epithelial-mesenchymal transition (EMT) contributes to the malignant progression of cancer cells including acquisition of the ability to undergo metastasis. However, whereas EMT-related transcription factors (EMT-TF) are known to play an important role in the malignant progression of epithelial tumors, their role in mesenchymal tumors remains largely unknown. We show that expression of the gene for Twist2 is downregulated in human osteosarcoma and correlates inversely with tumorigenic potential in mouse osteosarcoma. Forced expression of Twist2 in highly tumorigenic murine osteosarcoma cells induced a slight inhibition of cell growth in vitro but markedly suppressed tumor formation in vivo. Conversely, knockdown of Twist2 in osteosarcoma cells with a low tumorigenic potential promoted tumor formation in vivo, suggesting that Twist2 functions as a tumor suppressor in osteosarcoma cells. Furthermore, Twist2 induced expression of fibulin-5, which has been reported as a tumor suppressor. Medium conditioned by mouse osteosarcoma cells overexpressing Twist2 inhibited expression of the MMP9 gene as well as invasion in mouse embryonic fibroblasts, and forced expression of Twist2 in osteosarcoma cells suppressed MMP9 gene expression in tumor tissue. Data from the present study suggest that Twist2 inhibits formation of a microenvironment conducive to tumor growth and thereby attenuates tumorigenesis in osteosarcoma.
Assuntos
Neoplasias Ósseas/genética , Genes Supressores de Tumor , Osteossarcoma/genética , Proteínas Repressoras/genética , Proteína 1 Relacionada a Twist/genética , Animais , Neoplasias Ósseas/metabolismo , Carcinogênese/genética , Carcinogênese/metabolismo , Diferenciação Celular/genética , Linhagem Celular Tumoral , Regulação para Baixo , Transição Epitelial-Mesenquimal/genética , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Feminino , Fibroblastos/metabolismo , Humanos , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Osteossarcoma/metabolismo , Proteínas Repressoras/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Regulação para CimaRESUMO
Cancer-associated fibroblasts contribute to cancer progression that is caused by epithelial-mesenchymal transition (EMT). Recently, mesenchymal stem cells (MSCs) were found to be the major candidate involved in the development of tumor-promoting cancer stroma. Here we report that α-smooth muscle actin-positive myofibroblast-like cells originating from MSCs contribute to inducing EMT in side population cells of pancreatic cancer. More importantly, MSC-derived myofibroblasts function to maintain tumor-initiating stem cell-like characteristics, including augmenting expression levels of various stemness-associated genes, enhancing sphere- forming activity, promoting tumor formation in a mouse xenograft model, and showing resistance to anticancer drugs. Furthermore, both γ-secretase inhibitor and siRNA directed against Jagged-1 attenuated MSC-associated E-cadherin suppression and sphere formation in pancreatic cancer side population cells. Thus, our results suggest that MSC-derived myofibroblasts play important roles in regulating EMT and tumor-initiating stem cell-like properties of pancreatic cancer cells through an intermediating Notch signal.
Assuntos
Transição Epitelial-Mesenquimal/fisiologia , Células-Tronco Mesenquimais/patologia , Neoplasias Pancreáticas/patologia , Actinas/genética , Actinas/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular Tumoral , Progressão da Doença , Transição Epitelial-Mesenquimal/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína Jagged-1 , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , RNA Interferente Pequeno/genética , Receptores Notch/genética , Receptores Notch/metabolismo , Proteínas Serrate-Jagged , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Stem cell-based therapy has been proposed as a promising strategy for regenerating tissues lost through incurable diseases. Side population (SP) cells have been identified as putative stem cells in various organs. To examine therapeutic potential of SP cells in hypofunction of exocrine glands, SP cells isolated from mouse exocrine glands, namely, lacrimal and salivary glands, were transplanted into mice with irradiation-induced hypofunction of the respective glands. The secretions from both glands in the recipient mice were restored within 2 months of transplantation, although the transplanted cells were only sparsely distributed and produced no outgrowths. Consistent with this, most SP cells were shown to be CD31-positive endothelial-like cells. In addition, we clarified that endothelial cell-derived clusterin, a secretory protein, was an essential factor for SP cell-mediated recovery of the hypofunctioning glands because SP cells isolated from salivary glands of clusterin-deficient mice had no therapeutic potential, whereas lentiviral transduction of clusterin restored the hypofunction. In vitro and in vivo studies showed that clusterin had an ability to directly inhibit oxidative stress and oxidative stress-induced cell damage. Thus, endothelial cell-derived clusterin possibly inhibit oxidative stress-induced hypofunction of these glands.