RESUMO
In vitro culture of bone marrow (BM) with Fms-like tyrosine kinase 3 ligand (Flt3L) is widely used to study development and function of type 1 conventional dendritic cells (cDC1). Hematopoietic stem cells (HSCs) and many progenitor populations that possess cDC1 potential in vivo do not express Flt3 and thus may not contribute to Flt3L-mediated cDC1 production in vitro. Here, we present a KitL/Flt3L protocol that recruits such HSCs and progenitors into the production of cDC1. Kit ligand (KitL) is used to expand HSCs and early progenitors lacking Flt3 expression into later stage where Flt3 is expressed. Following this initial KitL phase, a second Flt3L phase is used to support the final production of DCs. With this two-stage culture, we achieved approximately tenfold increased production of both cDC1 and cDC2 compared to Flt3L culture. cDC1 derived from this culture are similar to in vivo cDC1 in their dependence on IRF8, ability to produce IL-12, and induction of tumor regression in cDC1-deficient tumor-bearing mice. This KitL/Flt3L system for cDC1 production will be useful in further analysis of cDC1 that rely on in vitro generation from BM.
Assuntos
Células-Tronco Hematopoéticas , Fator de Células-Tronco , Camundongos , Animais , Medula Óssea , Células da Medula Óssea , Células DendríticasRESUMO
PURPOSE: Patients with polycystic ovarian morphology (PCOM) make up 20% cases for assisted reproductive technology (ART). Folliculogenesis is impaired in PCOS. Signaling molecules are involved in follicle development. Dysregulations of intrafollicular environment and signaling molecules are observed in PCOS. Granulosa cells (GCs) and oocytes secrete molecules into follicular fluid by exocytosis of SNAREs. The aim of this study is to evaluate vesicle transport and vesicle fusion proteins (SNAREs) in GCs from PCOS patients who have undergone IVF treatment. METHODS: Follicular fluids were collected from patients who undergo IVF/ICSI with the diagnosis of male factor (n = 10) and PCOS (n = 10) patients. GCs were separated and cultured. Each group of GCs was stimulated with FSH-hCG. The cells were examined under electron microscope. Immunofluorescent labeling was performed on cells for Stx6, SNAP25, StxBP1, FSHr, and KITL. Integrated density was analyzed from images of Stx6, SNAP25, StxBP1, FSHr, and KITL. RESULTS: Intercellular communication occurs by signal molecules; Stx6, SNAP25, and StxBP1 fusion proteins involved in exocytosis were decreased in the GCs of PCOS. There was no increase in in vitro stimulation with FSH-hCG either. In the electron microscope, it was observed that exocytosis of the vesicles was disrupted. CONCLUSIONS: Exocytosis and vesicular dynamics are among the basic physiological functions of human steroidogenic granulosa cells. Follicle development is necessary for production of competent oocytes and ovulation. Understanding the pathophysiology of PCOS at follicular level is important for disease management. According to our findings, deficits in vesicular dynamics of human granulosa cells in may be central to the treatment strategy for PCOS patients.
Assuntos
Síndrome do Ovário Policístico , Feminino , Humanos , Masculino , Células da Granulosa/metabolismo , Hormônio Foliculoestimulante/genética , Hormônio Foliculoestimulante/farmacologia , Hormônio Foliculoestimulante/metabolismo , Exocitose/genética , Comunicação CelularRESUMO
Oogenesis depends on close interactions between oocytes and granulosa cells. Abnormal signaling between these cell types can result in infertility. However, attempts to manipulate oocyte-granulosa cell interactions have had limited success, likely due to the blood-follicle barrier (BFB), which prevents the penetration of exogenous materials into ovarian follicles. Here, we used adenoviruses (AVs) to manipulate the oocyte-granulosa cell interactions. AVs penetrated the BFB and transduced granulosa cells through ovarian microinjection. Although AVs caused transient inflammation, they did not impair fertility in wild-type mice. Introduction of Kitl-expressing AVs into congenitally infertile KitlSl-t/KitlSl-t mutant mouse ovaries, which contained only primordial follicles because of a lack of Kitl expression, restored fertility through natural mating. The offspring showed no evidence of AV integration and exhibited normal genomic imprinting patterns for imprinted genes. These results demonstrate the usefulness of AVs for manipulating oogenesis and suggest the possibility of gene therapies for human female infertility.
Assuntos
Infertilidade Feminina , Camundongos , Feminino , Animais , Humanos , Infertilidade Feminina/genética , Infertilidade Feminina/terapia , Infertilidade Feminina/metabolismo , Adenoviridae/genética , Folículo Ovariano/metabolismo , Células da Granulosa/metabolismo , Oócitos/metabolismo , Fertilidade/genéticaRESUMO
Homeostasis and function of limbal epithelial stem cells (LESCs) rely on the limbal niche, which, if dysfunctional, leads to limbal epithelial stem cell deficiency (LSCD) and impaired vision. Hence, recovery of niche function is a principal therapeutic goal in LSCD, but the molecular mechanisms of limbal niche homeostasis are still largely unknown. Here, we report that the neural crest transcription factor SOX10, which is expressed in neural crest-derived limbal niche cells (LNCs), is required for LNCs to promote survival of LESCs both in vivo and in vitro. In fact, using mice with a Sox10 mutation and in vitro coculture experiments, we show that SOX10 in LNCs stimulates the production of KIT ligand (KITL), which in turn activates in LESCs the KIT-AKT signalling pathway that protects the cells against activated CASPASE 3-associated cell death. These results suggest that SOX10 and the KITL/KIT-AKT pathway play key roles in limbal niche homeostasis and LESC survival. These findings provide molecular insights into limbal niche function and may point to rational approaches for therapeutic interventions in LSCD.
Assuntos
Células Epiteliais/citologia , Limbo da Córnea/citologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Fatores de Transcrição SOXE/metabolismo , Fator de Células-Tronco/metabolismo , Nicho de Células-Tronco , Células-Tronco/metabolismo , Animais , Sobrevivência Celular , Células Epiteliais/metabolismo , Camundongos , Comunicação Parácrina , Transdução de SinaisRESUMO
In mammals, progressive activation of primordial follicles is essential for maintenance of the reproductive lifespan. Several reports have demonstrated that mitogen-activated protein kinases 3 and 1 (MAPK3/1)-mammalian target of rapamycin complex 1 (mTORC1) signaling in pre-granulosa cells promotes primordial follicle activation by increasing KIT ligand (KITL) expression and then stimulating phosphatidylinositol 3 kinase signaling in oocytes. However, the mechanism of mTORC1 signaling in the promotion of KITL expression is unclear. Immunofluorescence staining results showed that phosphorylated cyclic AMP response element-binding protein (CREB) was mainly expressed in pre-granulosa cells. The CREB inhibitor KG-501 and CREB knockdown by Creb siRNA significantly suppressed primordial follicle activation, reduced pre-granulosa cell proliferation and dramatically increased oocyte apoptosis. Western blotting results demonstrated that both the MAPK3/1 inhibitor U0126 and mTORC1 inhibitor rapamycin significantly decreased the levels of phosphorylated CREB, indicating that MAPK3/1-mTORC1 signaling is required for CREB activation. Furthermore, CREB could bind to the Kitl promoter region, and KG-501 significantly decreased the expression levels of KITL. In addition, KG-501 and CREB knockdown significantly decreased the levels of phosphorylated Akt, leading to a reduced number of oocytes with Foxo3a nuclear export. KG-501 also inhibited bpV (HOpic)-stimulated primordial follicle activation. Taken together, the results show that CREB is required for MAPK3/1-mTORC1 signaling-promoted KITL expression followed by the activation of primordial follicles.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Folículo Ovariano/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos , Camundongos Endogâmicos ICR , Naftóis/farmacologia , Organofosfatos/farmacologia , Folículo Ovariano/efeitos dos fármacos , Fosforilação , Transdução de Sinais/genética , Fator de Células-Tronco/antagonistas & inibidores , Fator de Células-Tronco/metabolismo , Técnicas de Cultura de Tecidos , Compostos de Vanádio/antagonistas & inibidores , Compostos de Vanádio/farmacologiaRESUMO
In this study, we established an in vitro exposure model of murine ovarian granulosa cells to observe the effect of Cd on alternative splicing of the kitl pre-mRNA and subsequently to explore the role of kitl gene expression regulation-related miRNAs through miRNA prediction, miRNA chip, bioinformatics and real-time quantitative polymerase chain reaction analyses. Our results showed that the kitl1/kitl2 mRNA ratio was significantly different (P < 0.05) at different dosages and times. The miRNA chip analysis showed that the miRNA expression profiles for the Cd treatment were significantly changed, and the expression of 29 miRNAs involved in alternative splicing of the kitl pre-mRNA was changed. The gene ontology analysis showed that the target gene functions of these 29 miRNAs were mainly enriched in the biological processes of cell metabolism regulation, post-transcriptional regulation of mRNA, interleukin-6-mediated signal transduction, cell cycle, cell proliferation, differentiation and migration. The pathway enrichment analysis showed that the target genes of the differentially expressed miRNAs were mainly enriched in the Ras signaling pathway, the Rap1 signaling pathway, the Foxo signaling pathway, the Hippo signaling pathway, the MAPK signaling pathway and the carcinogenic pathway. Polymerase chain reaction verification results showed that compared to the control group, the variation trends in the expression of mmu-miR-27a-3p, mmu-miR-34b-5p, mmu-miR-297a-3p, mmu-miR-129-5p and mmu-miR-107-3p in the 4 hour 10 µm Cd treatment group were basically the same as that of the chip result. Our results indicate that Cd exposure can affect alternative splicing of the kitl pre-mRNA in ovarian granulosa cells, and miRNAs play regulatory roles in the alternative splicing of kitl.
Assuntos
Processamento Alternativo/efeitos dos fármacos , Cádmio/toxicidade , Células da Granulosa/efeitos dos fármacos , MicroRNAs/genética , Precursores de RNA/genética , Fator de Células-Tronco/genética , Animais , Células Cultivadas , Feminino , Regulação da Expressão Gênica , Ontologia Genética , Células da Granulosa/metabolismo , Camundongos Endogâmicos ICR , Cultura Primária de CélulasRESUMO
The spleen plays critical roles in immunity and also provides a permissive microenvironment for hematopoiesis. Previous studies have reported that the TALE-class homeodomain transcription factor Pbx1 is essential in hematopoietic stem and progenitor cells (HSPCs) for stem cell maintenance and progenitor expansion. However, the role of Pbx1 in the hematopoietic niche has not been investigated. Here we explored the effects that genetic perturbation of the splenic mesenchymal niche has on hematopoiesis upon loss of members of the Pbx family of homeoproteins. Splenic mesenchyme-specific inactivation of Pbx1 (SKO) on a Pbx2- or Pbx3-deficient genetic background (DKO) resulted in abnormal development of the spleen, which is dysmorphic and severely hypoplastic. This phenotype, in turn, affected the number of HSPCs in the fetal and adult spleen at steady state, as well as markedly impairing the kinetics of hematopoietic regeneration in adult mice after sub-lethal and lethal myelosuppressive irradiation. Spleens of mice with compound Pyx deficiency 8 days following sublethal irradiation displayed significant downregulation of multiple cytokine-encoding genes, including KitL/SCF, Cxcl12/SDF-1, IL-3, IL-4, GM-CSF/Csf2 IL-10, and Igf-1, compared with controls. KitL/SCF and Cxcl12/SDF-1 were recently shown to play key roles in the splenic niche in response to various haematopoietic stresses such as myeloablation, blood loss, or pregnancy. Our results demonstrate that, in addition to their intrinsic roles in HSPCs, non-cell autonomous functions of Pbx factors within the splenic niche contribute to the regulation of hematopoiesis, at least in part via the control of KitL/SCF and Cxcl12/SDF-1. Furthermore, our study establishes that abnormal spleen development and hypoplasia have deleterious effects on the efficiency of hematopoietic recovery after bone marrow injury.
Assuntos
Hematopoese Extramedular , Proteínas de Homeodomínio/fisiologia , Fatores de Transcrição/fisiologia , Animais , Citocinas/metabolismo , Camundongos , Camundongos Knockout , Fator de Transcrição 1 de Leucemia de Células Pré-B , Baço/fisiologia , Estresse FisiológicoRESUMO
Oocytes and granulosa cells closely interact with each other during follicular development, and a lack of appropriate signaling between them results in infertility. Attempts to manipulate oocyte microenvironment have been impeded by the impermeability of the blood-follicle barrier (BFB). To establish a strategy for manipulating oogenesis, we use adeno-associated viruses (AAVs), which have a unique ability of transcytosis. Microinjecting of AAVs into the ovarian stroma penetrates the BFB and achieves long-term gene expression. Introduction of an AAV carrying the mouse Kitl gene restores oogenesis in congenitally infertile KitlSl-t/KitlSl-t mutant mouse ovaries, which lack Kitl expression but contain only primordial follicles. Healthy offspring without AAV integration are born by natural mating. Therefore, AAV-mediated gene delivery not only provides a means for studying oocyte-granulosa interactions through the manipulation of the oocyte microenvironment but could also be a powerful method to treat female infertility resulting from somatic cell defects.
Assuntos
Infertilidade Feminina , Ovário , Animais , Dependovirus/genética , Feminino , Fertilidade/genética , Humanos , Infertilidade Feminina/genética , Camundongos , Folículo OvarianoRESUMO
Hematopoietic stem cells (HSCs) and skeletal stem cells (SSCs) cohabit in the bone marrow. KITL (C-KIT ligand) from LEPR+ adult bone marrow stromal cells is pivotal for HSC maintenance. In contrast, it remains unclear whether KITL/C-KIT signaling also regulates SSCs. Here, we lineage traced C-KIT+ cells and found that C-KIT was expressed by fetal, but not postnatal skeletal progenitors. Fetal C-KIT+ cells gave rise to 20% of LEPR+ stromal cells in adult bone marrow, forming nearly half of all osteoblasts. Disruption of mTOR signaling in fetal C-KIT+ cells impaired bone formation. Notably, conditional deletion of Kitl from PRX1+ fetal bone marrow stromal cells, but not LEPR+ adult bone marrow stromal cells, significantly increased bone formation. Thus, our work identified C-KIT+ skeletal progenitors as an important source of bones formed during development.
Assuntos
Osso e Ossos/citologia , Feto/citologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Células-Tronco/citologia , Adipócitos/metabolismo , Animais , Animais Recém-Nascidos , Desenvolvimento Ósseo , Células da Medula Óssea/metabolismo , Linhagem da Célula , Condrócitos/citologia , Condrócitos/metabolismo , Deleção de Genes , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese , Transdução de Sinais , Fator de Células-Tronco/metabolismo , Transcriptoma/genéticaRESUMO
In the testis, KIT ligand (KITL, also called stem cell factor) is expressed by Sertoli cells and its receptor (c-kit, KIT) is expressed by spermatogonia and Leydig cells. Although KITL-KIT signaling is critical for the spermatogenesis, its roles in Leydig cell development during puberty are not clear. In the present study, we investigated effects of KITL on stem Leydig cell proliferation and differentiation. Using an in vitro culture system of seminiferous tubules from Leydig cell-depleted testis, we found that KITL increased the proliferation activity of putative stem Leydig cells at higher concentration (10 and 100 ng/ml). Low concentration (1 ng/ml) of KITL significantly induced the differentiation of stem Leydig cells via increasing the expression level of steroidogenic acute regulatory protein (Star). In contrast, higher concentration (100 ng/ml) of KITL inhibited the differentiation of stem Leydig cells via inhibiting the steroidogenic enzyme (Cyp11a1, Cyp17a1, and Hsd17b3) expression levels. We cultured rat progenitor Leydig cells with KITL for 48 h and did not find any influence of KITL on the proliferation and androgen production of these cells. In conclusion, KITL is a growth factor that regulates the development of the stem Leydig cell.
Assuntos
Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/metabolismo , Androgênios/biossíntese , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Fator de Células-Tronco/farmacologiaRESUMO
BACKGROUND: After severe wounding, hair follicles were known to be regenerated de novo along with the re-epithelialization. However, the regenerated hairs lack pigmentation. OBJECTIVE: We aimed to find out the condition to regenerate pigmented hairs after severe wounding. METHODS: De novo hair regeneration was observed during the re-epithelialization process after the full thickness excision of dorsal skin. Hair pigmentation mechanism was assessed by the modulation of Wnt and Kit signalings. RESULTS: Stable regeneration of pigmented hairs was demonstrated when a wound was created to the mice during the anagen stage of the hair cycle. A significant increase in the number of melanocyte stem cells in the postnatal 1st anagen interfollicular skin of 5-week-old mice was observed. An increase of Wnt7a of the keratinocytes was observed in the skin at this stage, which may direct melanocyte stem cells to produce pigmented hairs in the regenerating follicles. This was supported by the finding that transgenic mice expressing the melanocyte stimulatory factor Kitl in their skin promoted the regeneration of pigmented hairs irrespective of the stage of the hair cycle. CONCLUSION: Our results provide a new insight into the intimate regulation process between two follicular stem cell systems, keratinocyte stem cells and melanocyte stem cells, during de novo hair regeneration after wounding.
Assuntos
Folículo Piloso/fisiologia , Cabelo/fisiologia , Pigmentação , Regeneração/fisiologia , Pele/metabolismo , Proteínas Wnt/metabolismo , Animais , Humanos , Melanócitos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-kit/metabolismo , Reepitelização , Células-Tronco/citologia , CicatrizaçãoRESUMO
Chronic myeloid leukemia (CML) can be contextualized as a disease of unregulated self-renewal of stem cells which exist in a quiescent state and are instructed to differentiate and mobilize to circulation under pathologic circumstances leading to tumor invasion and metastasis. Here we found that matrix metalloproteinase-9 (MMP-9), induced by TGF-ß1, upregulated s-KitL and s-ICAM-1, permitting the transfer of c-kit(+) hematopoietic stem cells (HSCs) from the quiescent to proliferative niche in CML. Further study showed that this MMP-9 production was raised by CML specific BCR/ABL(+) oncogene mediated TGF-ß1. Besides, phosphatidylinositol-3 kinase (PI3K)/Akt/nuclear factor (NF)-κB signaling pathway was evidenced to govern this stem cell recruitment in CML pathogenesis. Overall, our observations defined a novel critical role for TGF-ß1 induced PI3K/Akt/NF-κB signaling pathway in the recruitment of the malignant cells in CML by releasing s-KitL and s-ICAM-1 and this was through a distinct PI3K/Akt/NF-κB signaling pathway.
Assuntos
Movimento Celular/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Transdução de Sinais/fisiologia , Nicho de Células-Tronco , Adolescente , Adulto , Western Blotting , Medula Óssea/metabolismo , Medula Óssea/patologia , Células Cultivadas , Técnicas de Cocultura , Ensaio de Desvio de Mobilidade Eletroforética , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Células-Tronco Hematopoéticas/patologia , Humanos , Imunoprecipitação , Hibridização in Situ Fluorescente , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Fator de Crescimento Transformador beta1/metabolismo , Adulto JovemRESUMO
We have recently demonstrated that a DNA vaccine targeting membrane-bound KIT ligand (KITL) inhibits tumor growth by interfering with vessel stabilization/permeability and by disrupting the recruitment of inflammatory cells and regulatory T cells, the latter being an essential mechanism by which tumors resist available treatments. Combining KITL-targeting vaccines with conventional chemotherapy might avert drug resistance and improve the efficacy of standard-of-care therapeutic interventions.
RESUMO
We previously reported that insulin-like growth factor 1 (IGF-1) protects cochlear hair cells against aminoglycosides through activation of the PI3K/Akt and MEK/ERK pathways in supporting cells. In this study, we found that IGF-1 up-regulated the expression levels of Gap43 and Ntn1 as measured using cDNA microarray analysis and qRT-PCR. Using inhibitors of the PI3K/Akt and MEK/ERK pathways, we reveal that both pathways are involved in the up-regulation of Gap43 and Ntn1 expression. Moreover the time window of Gap43 and Ntn1 transcription was limited to within 12h after IGF-1 treatment, indicating that downstream gene expression was tightly controlled by IGF-1.