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1.
Nat Commun ; 12(1): 2594, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33972529

RESUMO

Adult neural stem cells (NSCs) must tightly regulate quiescence and proliferation. Single-cell analysis has suggested a continuum of cell states as NSCs exit quiescence. Here we capture and characterize in vitro primed quiescent NSCs and identify LRIG1 as an important regulator. We show that BMP-4 signaling induces a dormant non-cycling quiescent state (d-qNSCs), whereas combined BMP-4/FGF-2 signaling induces a distinct primed quiescent state poised for cell cycle re-entry. Primed quiescent NSCs (p-qNSCs) are defined by high levels of LRIG1 and CD9, as well as an interferon response signature, and can efficiently engraft into the adult subventricular zone (SVZ) niche. Genetic disruption of Lrig1 in vivo within the SVZ NSCs leads an enhanced proliferation. Mechanistically, LRIG1 primes quiescent NSCs for cell cycle re-entry and EGFR responsiveness by enabling EGFR protein levels to increase but limiting signaling activation. LRIG1 is therefore an important functional regulator of NSC exit from quiescence.


Assuntos
Células-Tronco Adultas/metabolismo , Ventrículos Laterais/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Células-Tronco Adultas/citologia , Células-Tronco Adultas/efeitos dos fármacos , Animais , Proteína Morfogenética Óssea 4/farmacologia , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células/genética , Proteínas de Ligação a DNA/metabolismo , Receptores ErbB/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Ontologia Genética , Imuno-Histoquímica , Interferons/farmacologia , Ventrículos Laterais/citologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Glicoproteínas de Membrana/genética , Camundongos , Proteínas do Tecido Nervoso/genética , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Proteômica , RNA-Seq , Regeneração/efeitos dos fármacos , Tetraspanina 29/metabolismo , Regulação para Cima
2.
Biochem Biophys Res Commun ; 554: 173-178, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-33798944

RESUMO

Neural crest-derived cells (NCDCs), a class of adult stem cells not restricted to embryonic tissues, are attractive tissue regenerative therapy candidates because of their ease of isolation, self-renewing properties, and multipotency. Although adult NCDCs can undergo osteogenic differentiation in vitro, whether they induce bone formation in vivo remains unclear. Previously, our group reported findings showing high amounts of NCDCs scattered throughout nasal concha tissues of adult mice. In the present study, NCDCs in nasal conchae labeled with enhanced green fluorescent protein (EGFP) were collected from adult P0-Cre/CAG-CAT-EGFP double transgenic mice, then cultured in serum-free medium to increase the number. Subsequently, NCDCs were harvested and suspended in type I atelocollagen gel, then an atelocollagen sponge was used as a scaffold for the cell suspension. Atelocollagen scaffolds with NCDCs were placed on bone defects created in a mouse calvarial bone defect model. Over the ensuing 12 weeks, micro-CT and histological analysis findings showed that mice with scaffolds containing NCDCs had slightly greater bone formation as compared to those with a scaffold alone. Furthermore, Raman spectroscopy revealed spectral properties of bone in mice that received scaffolds with NCDCs similar to those of native calvarial bone. Bone regeneration is important not only for gaining bone mass but also chemical properties. These results are the first to show the validity of biomolecule-free adult nasal concha-derived NCDCs for bone regeneration, including the chemical properties of regenerated bone tissue.


Assuntos
Células-Tronco Adultas/citologia , Regeneração Óssea/fisiologia , Crista Neural/citologia , Transplante de Células-Tronco/métodos , Conchas Nasais/citologia , Células-Tronco Adultas/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Crista Neural/metabolismo , Conchas Nasais/metabolismo
3.
Am J Physiol Gastrointest Liver Physiol ; 320(6): G1142-G1150, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33759566

RESUMO

In recent years, organoids have become a novel in vitro method to study gastrointestinal organ development, physiology, and disease. An organoid, in short, may be defined as a miniaturized organ that can be grown from adult stem cells in vitro and studied at the microscopic level. Organoids have been used in multitudes of different ways to study the physiology of different human diseases including gastrointestinal cancers such as pancreatic cancer. The development of genome editing based on the bacterial defense mechanism clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has emerged as a laboratory tool that provides the opportunity to study the effects of specific genetic changes on organ development, physiology, and disease. The CRISPR/Cas9 approach can be combined with organoid technology including the use of induced pluripotent stem cell (iPSC)-derived and tissue-derived organoids. The goal of this review is to provide highlights on the development of organoid technology, and the use of this culture system to study the pathophysiology of specific mutations in the development of pancreatic and gastric cancers.NEW & NOTEWORTHY The goal of this review is not only to provide highlights on the development of organoid technology but also to subsequently use this information to study the pathophysiology of those specific mutations in the formation of malignant pancreatic and gastric cancer.


Assuntos
Células-Tronco Adultas/citologia , Edição de Genes , Células-Tronco Pluripotentes Induzidas/citologia , Organoides/citologia , Pâncreas/citologia , Animais , Sistemas CRISPR-Cas , Humanos
4.
J Vis Exp ; (168)2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33645580

RESUMO

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.


Assuntos
Células-Tronco Adultas/citologia , Técnicas de Cultura de Células/métodos , Fibras Musculares Esqueléticas/citologia , Células-Tronco/citologia , Animais , Células Cultivadas , Colagenases/metabolismo , Camundongos Endogâmicos C57BL , Desenvolvimento Muscular , RNA Interferente Pequeno/metabolismo , Regeneração , Fixação de Tecidos , Transfecção
5.
Int J Mol Sci ; 22(4)2021 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-33669748

RESUMO

Muse cells are adult stem cells that are present in the stroma of several organs and possess an enduring capacity to cope with endogenous and exogenous genotoxic stress. In cell therapy, the peculiar biological properties of Muse cells render them a possible natural alternative to mesenchymal stromal cells (MSCs) or to in vitro-generated pluripotent stem cells (iPSCs). Indeed, some studies have proved that Muse cells can survive in adverse microenvironments, such as those present in damaged/injured tissues. We performed an evaluation of Muse cells' proteome under basic conditions and followed oxidative stress treatment in order to identify ontologies, pathways, and networks that can be related to their enduring stress capacity. We executed the same analysis on iPSCs and MSCs, as a comparison. The Muse cells are enriched in several ontologies and pathways, such as endosomal vacuolar trafficking related to stress response, ubiquitin and proteasome degradation, and reactive oxygen scavenging. In Muse cells, the protein-protein interacting network has two key nodes with a high connectivity degree and betweenness: NFKB and CRKL. The protein NFKB is an almost-ubiquitous transcription factor related to many biological processes and can also have a role in protecting cells from apoptosis during exposure to a variety of stressors. CRKL is an adaptor protein and constitutes an integral part of the stress-activated protein kinase (SAPK) pathway. The identified pathways and networks are all involved in the quality control of cell components and may explain the stress resistance of Muse cells.


Assuntos
Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Proteoma/metabolismo , Proteômica , Estresse Fisiológico , Linhagem Celular , Dano ao DNA , Ontologia Genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Mapas de Interação de Proteínas , Transdução de Sinais
6.
Proc Natl Acad Sci U S A ; 118(4)2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33479180

RESUMO

An ability to safely harness the powerful regenerative potential of adult stem cells for clinical applications is critically dependent on a comprehensive understanding of the underlying mechanisms regulating their activity. Epithelial organoid cultures accurately recapitulate many features of in vivo stem cell-driven epithelial renewal, providing an excellent ex vivo platform for interrogation of key regulatory mechanisms. Here, we employed a genome-scale clustered, regularly interspaced, short palindromic repeats (CRISPR) knockout (KO) screening assay using mouse gastric epithelial organoids to identify modulators of Wnt-driven stem cell-dependent epithelial renewal in the gastric mucosa. In addition to known Wnt pathway regulators, such as Apc, we found that KO of Alk, Bclaf3, or Prkra supports the Wnt independent self-renewal of gastric epithelial cells ex vivo. In adult mice, expression of these factors is predominantly restricted to non-Lgr5-expressing stem cell zones above the gland base, implicating a critical role for these factors in suppressing self-renewal or promoting differentiation of gastric epithelia. Notably, we found that Alk inhibits Wnt signaling by phosphorylating the tyrosine of Gsk3ß, while Bclaf3 and Prkra suppress regenerating islet-derived (Reg) genes by regulating the expression of epithelial interleukins. Therefore, Alk, Bclaf3, and Prkra may suppress stemness/proliferation and function as novel regulators of gastric epithelial differentiation.


Assuntos
Células-Tronco Adultas/metabolismo , Quinase do Linfoma Anaplásico/genética , Células Epiteliais/metabolismo , Edição de Genes/métodos , Organoides/metabolismo , Proteínas de Ligação a RNA/genética , Via de Sinalização Wnt/genética , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Células-Tronco Adultas/citologia , Quinase do Linfoma Anaplásico/metabolismo , Animais , Sistemas CRISPR-Cas , Proliferação de Células , Células Epiteliais/citologia , Mucosa Gástrica/citologia , Mucosa Gástrica/metabolismo , Regulação da Expressão Gênica , Biblioteca Gênica , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Células HEK293 , Humanos , Interleucinas/genética , Interleucinas/metabolismo , Camundongos , Organoides/citologia , Proteínas de Ligação a RNA/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Estômago/citologia
7.
Biochem Biophys Res Commun ; 534: 337-342, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33250176

RESUMO

Naringenin (NAR) is a natural flavonoid which exerts extensive biological activity, including anti-oxidation, anti-inflammation, anti-cancer, immune regulation and so on. However, the effect and mechanism of NAR in the alveolar bone regeneration are still unclear, which limits its clinical use. Hence, we investigated the effects of NAR in the proliferation, osteogenic and endothelial differentiation of human periodontal ligament stem cells (hPDLSCs) and explore the possible mechanism. The results showed that the proper concentrations (100 nM-10 µM) of NAR can promote the proliferation rate, osteogenic and endothelial differentiation of hPDLSCs. And the 1 µM NAR had the best proliferation promoting effect, while the 10 µM NAR had the best ability of promoting osteogenic and endothelial differentiation. NAR also promoted the mRNA expression of SDF-1 in a concentration dependent manner in PDLSCs. After adding the selective CXCR4 antagonist AMD3100, the osteogenic effect of NAR on PDLSCs is slightly enhanced, while the endothelial differentiation effect of NAR on hPDLSCs is attenuated. In summary, these results indicated that NAR promoted the proliferation of hPDLSCs, and promoted endothelial differentiation of hPDLSCs via SDF-1 to activate SDF-1/CXCR4 signaling pathway. However, the mechanism of which SDF-1 related signaling pathway is activated by NAR to enhance the osteogenic differentiation of hPDLSCs still needs to be investigated.


Assuntos
Células-Tronco Adultas/citologia , Células-Tronco Adultas/efeitos dos fármacos , Flavanonas/farmacologia , Ligamento Periodontal/citologia , Ligamento Periodontal/efeitos dos fármacos , Células-Tronco Adultas/metabolismo , Regeneração Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Ligamento Periodontal/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores CXCR4/metabolismo , Transdução de Sinais/efeitos dos fármacos
8.
Methods Mol Biol ; 2185: 267-280, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33165854

RESUMO

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic stem cell transplantation but lack a matched donor. However, the limited number of HSCs within each UCB unit remains a major challenge for their use in regenerative medicine and HSC transplantation in adults. Efficient expansion of human HSCs in ex vivo cultures initiated with CD34+ cells isolated from UCBs can overcome this limitation. The method described here utilizes a deacetylase inhibitor, valproic acid (VPA), to rapidly expand to a high degree the numbers of functional HSCs and committed progenitors (HPCs). The expanded HSCs are capable of establishing both short-term and long-term multilineage hematopoietic reconstitution. This highly reproducible and simple protocol can be also applied to expansion of both HSCs and HPCs from different sources including the bone marrow and peripheral blood.


Assuntos
Células-Tronco Adultas/metabolismo , Técnicas de Cultura de Células , Células-Tronco Hematopoéticas/metabolismo , Ácido Valproico/farmacologia , Células-Tronco Adultas/citologia , Células Cultivadas , Células-Tronco Hematopoéticas/citologia , Humanos
9.
Int J Mol Sci ; 21(24)2020 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-33322051

RESUMO

Lower back pain is a leading cause of disability worldwide. The recovery of nucleus pulposus (NP) progenitor cells (NPPCs) from the intervertebral disc (IVD) holds high promise for future cell therapy. NPPCs are positive for the angiopoietin-1 receptor (Tie2) and possess stemness capacity. However, the limited Tie2+ NPC yield has been a challenge for their use in cell-based therapy for regenerative medicine. In this study, we attempted to expand NPPCs from the whole NP cell population by spheroid-formation assay. Flow cytometry was used to quantify the percentage of NPPCs with Tie2-antibody in human primary NP cells (NPCs). Cell proliferation was assessed using the population doublings level (PDL) measurement. Synthesis and presence of extracellular matrix (ECM) from NPC spheroids were confirmed by quantitative Polymerase Chain Reaction (qPCR), immunostaining, and microscopy. Compared with monolayer, the spheroid-formation assay enriched the percentage of Tie2+ in NPCs' population from ~10% to ~36%. Moreover, the spheroid-formation assay also inhibited the proliferation of the Tie2- NPCs with nearly no PDL. After one additional passage (P) using the spheroid-formation assay, NPC spheroids presented a Tie2+ percentage even further by ~10% in the NPC population. Our study concludes that the use of a spheroid culture system could be successfully applied to the culture and expansion of tissue-specific progenitors.


Assuntos
Células-Tronco Adultas/citologia , Proliferação de Células , Núcleo Pulposo/citologia , Receptor TIE-2/metabolismo , Esferoides Celulares/citologia , Adulto , Células-Tronco Adultas/metabolismo , Células-Tronco Adultas/fisiologia , Células Cultivadas , Proteínas da Matriz Extracelular/metabolismo , Feminino , Humanos , Masculino , Cultura Primária de Células/métodos , Receptor TIE-2/genética , Esferoides Celulares/metabolismo , Esferoides Celulares/fisiologia
10.
Cell Death Dis ; 11(12): 1075, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33323934

RESUMO

Mesenchymal stem cell (MSC)-based therapy has emerged as a novel strategy to treat many degenerative diseases. Accumulating evidence shows that the function of MSCs declines with age, thus limiting their regenerative capacity. Nonetheless, the underlying mechanisms that control MSC ageing are not well understood. We show that compared with bone marrow-MSCs (BM-MSCs) isolated from young and aged samples, NADH dehydrogenase (ubiquinone) iron-sulfur protein 6 (Ndufs6) is depressed in aged MSCs. Similar to that of Ndufs6 knockout (Ndufs6-/-) mice, MSCs exhibited a reduced self-renewal and differentiation capacity with a tendency to senescence in the presence of an increased p53/p21 level. Downregulation of Ndufs6 by siRNA also accelerated progression of wild-type BM-MSCs to an aged state. In contrast, replenishment of Ndufs6 in Ndufs6-/--BM-MSCs significantly rejuvenated senescent cells and restored their proliferative ability. Compared with BM-MSCs, Ndufs6-/--BM-MSCs displayed increased intracellular and mitochondrial reactive oxygen species (ROS), and decreased mitochondrial membrane potential. Treatment of Ndufs6-/--BM-MSCs with mitochondrial ROS inhibitor Mito-TEMPO notably reversed the cellular senescence and reduced the increased p53/p21 level. We provide direct evidence that impairment of mitochondrial Ndufs6 is a putative accelerator of adult stem cell ageing that is associated with excessive ROS accumulation and upregulation of p53/p21. It also indicates that manipulation of mitochondrial function is critical and can effectively protect adult stem cells against senescence.


Assuntos
Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Senescência Celular , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , NADH Desidrogenase/metabolismo , Células-Tronco Adultas/ultraestrutura , Animais , Células da Medula Óssea/metabolismo , Diferenciação Celular , Regulação para Baixo/genética , Técnicas de Silenciamento de Genes , Humanos , Células-Tronco Mesenquimais/ultraestrutura , Camundongos Endogâmicos C57BL , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/genética , NADH Desidrogenase/genética , Subunidades Proteicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
11.
Proc Natl Acad Sci U S A ; 117(49): 31448-31458, 2020 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-33229571

RESUMO

Adult neural stem cells (NSC) serve as a reservoir for brain plasticity and origin for certain gliomas. Lineage tracing and genomic approaches have portrayed complex underlying heterogeneity within the major anatomical location for NSC, the subventricular zone (SVZ). To gain a comprehensive profile of NSC heterogeneity, we utilized a well-validated stem/progenitor-specific reporter transgene in concert with single-cell RNA sequencing to achieve unbiased analysis of SVZ cells from infancy to advanced age. The magnitude and high specificity of the resulting transcriptional datasets allow precise identification of the varied cell types embedded in the SVZ including specialized parenchymal cells (neurons, glia, microglia) and noncentral nervous system cells (endothelial, immune). Initial mining of the data delineates four quiescent NSC and three progenitor-cell subpopulations formed in a linear progression. Further evidence indicates that distinct stem and progenitor populations reside in different regions of the SVZ. As stem/progenitor populations progress from neonatal to advanced age, they acquire a deficiency in transition from quiescence to proliferation. Further data mining identifies stage-specific biological processes, transcription factor networks, and cell-surface markers for investigation of cellular identities, lineage relationships, and key regulatory pathways in adult NSC maintenance and neurogenesis.


Assuntos
Envelhecimento/genética , Linhagem da Célula , Ventrículos Laterais/anatomia & histologia , Ventrículos Laterais/citologia , Nicho de Células-Tronco/genética , Transcriptoma/genética , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Animais , Biomarcadores/metabolismo , Linhagem da Célula/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Transgenes
12.
Sci Rep ; 10(1): 18921, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33144601

RESUMO

The role of miRNAs in intestinal lipid metabolism is poorly described. The small intestine is constantly exposed to high amounts of dietary lipids, and it is under conditions of stress that the functions of miRNAs become especially pronounced. Approaches consisting in either a chronic exposure to cholesterol and triglyceride rich diets (for several days or weeks) or an acute lipid challenge were employed in the search for intestinal miRNAs with a potential role in lipid metabolism regulation. According to our results, changes in miRNA expression in response to fat ingestion are dependent on factors such as time upon exposure, gender and small intestine section. Classic and recent intestinal in vitro models (i.e. differentiated Caco-2 cells and murine organoids) partially mirror miRNA modulation in response to lipid challenges in vivo. Moreover, intestinal miRNAs might play a role in triglyceride absorption and produce changes in lipid accumulation in intestinal tissues as seen in a generated intestinal Dicer1-deletion murine model. Overall, despite some variability between the different experimental cohorts and in vitro models, results show that some miRNAs analysed here are modulated in response to dietary lipids, hence likely to participate in the regulation of lipid metabolism, and call for further research.


Assuntos
Gorduras na Dieta/farmacologia , Intestinos/efeitos dos fármacos , MicroRNAs/genética , Organoides/efeitos dos fármacos , Células-Tronco Adultas/química , Células-Tronco Adultas/citologia , Células-Tronco Adultas/efeitos dos fármacos , Animais , Células CACO-2 , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , RNA Helicases DEAD-box/genética , Feminino , Deleção de Genes , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Intestinos/química , Intestinos/citologia , Metabolismo dos Lipídeos , Masculino , Camundongos , Organoides/química , Organoides/citologia , Ribonuclease III/genética , Análise de Sequência de RNA , Caracteres Sexuais , Fatores de Tempo
13.
PLoS One ; 15(10): e0239601, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33112876

RESUMO

APC mutations drive human colorectal cancer (CRC) development. A major contributing factor is colonic stem cell (SC) overpopulation. But, the mechanism has not been fully identified. A possible mechanism is the dysregulation of neuroendocrine cell (NEC) maturation by APC mutations because SCs and NECs both reside together in the colonic crypt SC niche where SCs mature into NECs. So, we hypothesized that sequential inactivation of APC alleles in human colonic crypts leads to progressively delayed maturation of SCs into NECs and overpopulation of SCs. Accordingly, we used quantitative immunohistochemical mapping to measure indices and proportions of SCs and NECs in human colon tissues (normal, adenomatous, malignant), which have different APC-zygosity states. In normal crypts, many cells staining for the colonic SC marker ALDH1 co-stained for chromogranin-A (CGA) and other NEC markers. In contrast, in APC-mutant tissues from familial adenomatous polyposis (FAP) patients, the proportion of ALDH+ SCs progressively increased while NECs markedly decreased. To explain how these cell populations change in FAP tissues, we used mathematical modelling to identify kinetic mechanisms. Computational analyses indicated that APC mutations lead to: 1) decreased maturation of ALDH+ SCs into progenitor NECs (not progenitor NECs into mature NECs); 2) diminished feedback signaling by mature NECs. Biological experiments using human CRC cell lines to test model predictions showed that mature GLP-2R+ and SSTR1+ NECs produce, via their signaling peptides, opposing effects on rates of NEC maturation via feedback regulation of progenitor NECs. However, decrease in this feedback signaling wouldn't explain the delayed maturation because both progenitor and mature NECs are depleted in CRCs. So the mechanism for delayed maturation must explain how APC mutation causes the ALDH+ SCs to remain immature. Given that ALDH is a key component of the retinoic acid (RA) signaling pathway, that other components of the RA pathway are selectively expressed in ALDH+ SCs, and that exogenous RA ligands can induce ALDH+ cancer SCs to mature into NECs, RA signaling must be attenuated in ALDH+ SCs in CRC. Thus, attenuation of RA signaling explains why ALDH+ SCs remain immature in APC mutant tissues. Since APC mutation causes increased WNT signaling in FAP and we found that sequential inactivation of APC in FAP patient tissues leads to progressively delayed maturation of colonic ALDH+ SCs, the hypothesis is developed that human CRC evolves due to an imbalance between WNT and RA signaling.


Assuntos
Transformação Celular Neoplásica/genética , Colo/citologia , Colo/metabolismo , Neoplasias Colorretais/genética , Genes APC , Peptídeo 2 Semelhante ao Glucagon/metabolismo , Mutação , Somatostatina/metabolismo , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/metabolismo , Polipose Adenomatosa do Colo/patologia , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Família Aldeído Desidrogenase 1/metabolismo , Animais , Biomarcadores/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Cromogranina A/metabolismo , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Retroalimentação Fisiológica , Receptor do Peptídeo Semelhante ao Glucagon 2/metabolismo , Células HCT116 , Células HT29 , Humanos , Camundongos , Modelos Genéticos , Células Neuroendócrinas/citologia , Células Neuroendócrinas/metabolismo , Receptores de Somatostatina/metabolismo , Transdução de Sinais , Nicho de Células-Tronco , Tretinoína/metabolismo , Via de Sinalização Wnt
14.
Curr Protoc Immunol ; 130(1): e106, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32940424

RESUMO

Human intestinal organoids derived from adult stem cells are miniature ex vivo versions of the human intestinal epithelium. Intestinal organoids are useful tools for the study of intestinal physiology as well as many disease conditions. These organoids present numerous advantages compared to immortalized cell lines, but working with them requires dedicated techniques. The protocols described in this article provide a basic guide to establishment and maintenance of human intestinal organoids derived from small intestine and colon biopsies. Additionally, this article provides an overview of several downstream applications of human intestinal organoids. © 2020 The Authors. Basic Protocol 1: Establishment of human small intestine and colon organoid cultures from fresh biopsies Basic Protocol 2: Mechanical splitting, passage, and expansion of human intestinal organoids Alternate Protocol: Differentiation of human intestinal organoids Basic Protocol 3: Cryopreservation and thawing of human intestinal organoids Basic Protocol 4: Immunofluorescence staining of human intestinal organoids Basic Protocol 5: Generation of single-cell clonal intestinal organoid cultures Support Protocol 1: Production of Wnt3A conditioned medium Support Protocol 2: Production of Rspo1 conditioned medium Support Protocol 3: Extraction of RNA from intestinal organoid cultures.


Assuntos
Células-Tronco Adultas/citologia , Técnicas de Cultura de Células , Mucosa Intestinal/citologia , Organoides/citologia , Organoides/metabolismo , Técnicas de Cultura de Tecidos , Células-Tronco Adultas/metabolismo , Biomarcadores , Biópsia , Diferenciação Celular , Separação Celular/métodos , Células Cultivadas , Colo/citologia , Criopreservação/métodos , Meios de Cultivo Condicionados , Imunofluorescência , Humanos , Imuno-Histoquímica , Imunofenotipagem , Intestino Delgado/citologia
15.
Mol Biol Rep ; 47(10): 8347-8352, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32997309

RESUMO

Previous works characterized a novel cell population from adult human peripheral blood, designated peripheral blood insulin-producing cells (PB-IPC). PB-IPC displayed the pluripotent potential of differentiations after the treatment with platelet-derived mitochondria and gave rise to three germ layer-derived cells such as the mitochondrion-induced CD34+ hematopoietic stem cells (HSC)-like cells (miCD34+ HSC). To determine the molecular mechanism underlying the differentiation of miCD34+ cells, mechanistic studies established that MitoTracker Deep Red-labeled mitochondria could enter into the PB-IPC in a dose-dependent manner. Blocking Notch signaling pathway with a γ-secretase inhibitor, DAPT, markedly inhibited the proliferation of PB-IPC and improved the differentiation of miCD34+ HSC. Additionally, treatment with platelet-derived mitochondria can reprogram the differentiation of PB-IPC into miCD34+ HSC through inhibition of the Notch/HEY2 signaling pathway, as demonstrated by blocking experiments with HEY2 small interfering RNA (siRNA). The data indicated that Notch signaling pathway contributes to the miCD34+ HSC differentiation, thus advancing our understanding of the mitochondrial reprogramming and the potential treatment of human hematopoietic disease.


Assuntos
Células-Tronco Adultas/metabolismo , Antígenos CD34/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Plaquetas/metabolismo , Diferenciação Celular , Células-Tronco Hematopoéticas/metabolismo , Insulina/metabolismo , Mitocôndrias/metabolismo , Receptores Notch/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Adulto , Células-Tronco Adultas/citologia , Células-Tronco Hematopoéticas/citologia , Humanos
16.
Biochem Biophys Res Commun ; 530(1): 209-214, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32828287

RESUMO

More than two decades after the discovery of adult neurogenesis in humans, researchers still struggle to elucidate the underlying transcriptional and post-transcriptional mechanisms. RNA interference is a crucially important process in the central nervous system, and its role in adult neurogenesis is poorly understood. In this work, we address the role of Dicer-dependent microRNA biogenesis in neuronal differentiation of adult neural stem cells within the subventricular zone of the mouse brain. Loss of the Dicer1 gene in the tailless (Tlx)-positive cells did not cause the decline in their numbers, but severely affected differentiation. Thus, our findings identify yet another phenomenon associated with microRNA pathway deregulation in adult neural stem cells which might be of relevance both for neuroscience and clinical practice.


Assuntos
Proliferação de Células , MicroRNAs/genética , Células-Tronco Neurais/citologia , Neurogênese , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Animais , Células Cultivadas , RNA Helicases DEAD-box/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Ventrículos Laterais/citologia , Ventrículos Laterais/metabolismo , Masculino , Camundongos , Células-Tronco Neurais/metabolismo , Ribonuclease III/genética , Transcriptoma
17.
Biochem Biophys Res Commun ; 530(1): 222-229, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32828290

RESUMO

Efficiency of the induction protocol is crucial for the generation of insulin-producing cells (IPCs) from human dental pulp stem cells (hDPSCs). Here, we established the integrative induction protocol by merging genetic manipulation technique with our previous published 3-step induction protocol aiming to enhance the pancreatic progenitor commitment and production yield. We found that the overexpression of PDX1 following with 3-step induction protocol were able to generate the 3-dimensional (3D) colony structure of pancreatic progenitors (PPs) with the beneficial trends of pancreatic endoderm commitment and production yield, while other protocols using the prolong maintenance of PDX1-overexpressed hDPSCs and the PDX1 overexpression after definitive endoderm induction were unable to generate and sustain the 3D structure of the colonies. Further Notch signaling manipulation by DAPT treatment showed lesser degree of positive effects on progenitor commitment and production yield. Although the generated PPs from the integrative protocol expressed pancreatic mRNA markers along with pro-insulin and insulin proteins, they still contained the defective glucose-responsive C-peptide secretion. Only basal secreted C-peptide level was observed. In summary, the integrative induction protocol potentially enhanced the PP generation with high colony production yield and could serve as an efficient platform for further hDPSC-derived IPC production and maturation.


Assuntos
Células-Tronco Adultas/citologia , Polpa Dentária/citologia , Células Secretoras de Insulina/citologia , Pâncreas/citologia , Células-Tronco Adultas/metabolismo , Peptídeo C/metabolismo , Técnicas de Cultura de Células , Células Cultivadas , Polpa Dentária/metabolismo , Glucose/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , Pâncreas/metabolismo , Transativadores/genética , Transativadores/metabolismo , Regulação para Cima
18.
Nat Commun ; 11(1): 4275, 2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32848155

RESUMO

New neurons are generated in adult mammals. Adult hippocampal neurogenesis is considered to play an important role in cognition and mental health. The number and properties of newly born neurons are regulatable by a broad range of physiological and pathological conditions. To begin to understand the underlying cellular mechanisms and functional relevance of adult neurogenesis, many studies rely on quantification of adult-born neurons. However, lack of standardized methods to quantify new neurons is impeding research reproducibility across laboratories. Here, we review the importance of stereology, and propose why and how it should be applied to the study of adult neurogenesis.


Assuntos
Encéfalo/citologia , Encéfalo/fisiologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/fisiologia , Neurogênese/fisiologia , Adulto , Células-Tronco Adultas/citologia , Células-Tronco Adultas/fisiologia , Animais , Giro Denteado/citologia , Giro Denteado/fisiologia , Humanos , Modelos Neurológicos , Plasticidade Neuronal
20.
Blood ; 136(20): 2296-2307, 2020 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-32766876

RESUMO

The exact localization of hematopoietic stem cells (HSCs) in their native bone marrow (BM) microenvironment remains controversial, because multiple cell types have been reported to physically associate with HSCs. In this study, we comprehensively quantified HSC localization with up to 4 simultaneous (9 total) BM components in 152 full-bone sections from different bone types and 3 HSC reporter lines. We found adult femoral α-catulin-GFP+ or Mds1GFP/+Flt3Cre HSCs proximal to sinusoids, Cxcl12 stroma, megakaryocytes, and different combinations of those populations, but not proximal to bone, adipocyte, periarteriolar, or Schwann cells. Despite microanatomical differences in femurs and sterna, their adult α-catulin-GFP+ HSCs had similar distributions. Importantly, their microenvironmental localizations were not different from those of random dots, reflecting the relative abundance of imaged BM populations rather than active enrichment. Despite their functional heterogeneity, dormant label-retaining (LR) and non-LR hematopoietic stem and progenitor cells both had indistinguishable localization from α-catulin-GFP+ HSCs. In contrast, cycling juvenile BM HSCs preferentially located close to Cxcl12 stroma and farther from sinusoids/megakaryocytes. We expect our study to help resolve existing confusion regarding the exact localization of different HSC types, their physical association with described BM populations, and their tissue-wide combinations.


Assuntos
Células-Tronco Adultas/citologia , Células-Tronco Hematopoéticas/citologia , Nicho de Células-Tronco , Animais , Camundongos
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