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1.
Braz. j. biol ; 83: e246592, 2023. tab, graf
Article in English | LILACS, VETINDEX | ID: biblio-1339408

ABSTRACT

Abstract Mesenchymal stem cells (MSCs) have great potential for application in cell therapy and tissue engineering procedures because of their plasticity and capacity to differentiate into different cell types. Given the widespread use of MSCs, it is necessary to better understand some properties related to osteogenic differentiation, particularly those linked to biomaterials used in tissue engineering. The aim of this study was to develop an analysis method using FT-Raman spectroscopy for the identification and quantification of biochemical components present in conditioned culture media derived from MSCs with or without induction of osteogenic differentiation. All experiments were performed between passages 3 and 5. For this analysis, MSCs were cultured on scaffolds composed of bioresorbable poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and poly(ε-caprolactone) (PCL) polymers. MSCs (GIBCO®) were inoculated onto the pure polymers and 75:25 PHBV/PCL blend (dense and porous samples). The plate itself was used as control. The cells were maintained in DMEM (with low glucose) containing GlutaMAX® and 10% FBS at 37oC with 5% CO2 for 21 days. The conditioned culture media were collected and analyzed to probe for functional groups, as well as possible molecular variations associated with cell differentiation and metabolism. The method permitted to identify functional groups of specific molecules in the conditioned medium such as cholesterol, phosphatidylinositol, triglycerides, beta-subunit polypeptides, amide regions and hydrogen bonds of proteins, in addition to DNA expression. In the present study, FT-Raman spectroscopy exhibited limited resolution since different molecules can express similar or even the same stretching vibrations, a fact that makes analysis difficult. There were no variations in the readings between the samples studied. In conclusion, FT-Raman spectroscopy did not meet expectations under the conditions studied.


Resumo As células-tronco mesenquimais (MSCs) possuem grande potencial para aplicação em procedimentos terapêuticos ligados a terapia celular e engenharia de tecidos, considerando-se a plasticidade e capacidade de formação em diferentes tipos celulares por elas. Dada a abrangência no emprego das MSCs, há necessidade de se compreender melhor algumas propriedades relacionadas à diferenciação osteogênica, particularmente liga à biomateriais usados em engenharia de tecidos. Este projeto objetiva o desenvolvimento de uma metodologia de análise empregando-se a FT-Raman para identificação e quantificação de componentes bioquímicos presentes em meios de cultura condicionados por MSCs, com ou sem indução à diferenciação osteogênica. Todos os experimentos foram realizados entre as passagens 3 e 5. Para essas análises, as MSCs foram cultivadas sobre arcabouços de polímeros biorreabsorvíveis de poli (hidroxibutirato-co-hidroxivalerato) (PHBV) e o poli (ε-caprolactona) (PCL). As MSCs (GIBCO®) foram inoculadas nos polímeros puros e na mistura 75:25 de PHBV / PCL (amostras densas e porosas). As células foram mantidas em DMEM (com baixa glicose) contendo GlutaMAX® e 10% de SFB a 37oC com 5% de CO2 por 21 dias. A própria placa foi usada como controle. Os meios de cultura condicionados foram coletados e analisadas em FT-Raman para sondagem de grupos funcionais, bem como possíveis variações moleculares associadas com a diferenciação e metabolismo celular. Foi possível discernir grupos funcionais de moléculas específicas no meio condicionado, como colesterol, fosfatidilinositol, triglicerídeos, forma Beta de polipeptídeos, regiões de amida e ligações de hidrogênio de proteínas, além da expressão de DNA. Na presente avaliação, a FT-Raman apresentou como uma técnica de resolução limitada, uma vez que modos vibracionais de estiramento próximos ou mesmo iguais podem ser expressos por moléculas diferente, dificultando a análise. Não houve variações nas leituras entre as amostras estudadas, concluindo-se que a FT-Raman não atendeu às expectativas nas condições estudadas.


Subject(s)
Animals , Rats , Mesenchymal Stem Cells , Osteogenesis , Polyesters , Spectrum Analysis, Raman , Culture Media, Conditioned , Cell Proliferation , Tissue Scaffolds
2.
Rev. Círc. Argent. Odontol ; 80(231): 19-23, jul. 2022. ilus
Article in Spanish | LILACS | ID: biblio-1392286

ABSTRACT

En el campo de la odontología, prevalecen actualmente alternativas terapéuticas con una filosofía conservadora. Sin embargo, con el advenimiento de los tratamientos con células madre (CM), se amplían las posibilidades terapéuticas, que buscan la combinación y el equilibrio entre la intervención tradicional y las posibilidades de reposición de estructuras anatómicas dañadas, a través de la regeneración de tejidos utilizando células madre o sus derivados (AU)


In the dentistry field, therapeutic alternatives with a conservative philosophy currently prevail. However, with the advent of stem cell (SC) treatments, therapeutic possibilities are expanding, seeking a combination and balance between traditional intervention and the pos- sibility of replacing damaged anatomical structures through tissue regeneration, using stem cells or their derivatives (AU)


Subject(s)
Humans , Stem Cells , Tissue Engineering , Mesenchymal Stem Cells/physiology , Periodontal Ligament/physiology , Regeneration/physiology , Tooth/cytology , Tooth Germ/physiology , Biocompatible Materials/therapeutic use , Bone Regeneration/physiology , Dental Pulp/physiology , Tissue Scaffolds , COVID-19/therapy
3.
Rev. bras. ortop ; 57(2): 314-320, Mar.-Apr. 2022. graf
Article in English | LILACS | ID: biblio-1388003

ABSTRACT

Abstract Objective Our goal was to evaluate the modulation of the synovial fluid cells (SFC) from patients with and without osteoarthritis (OA) by bone morphogenetic protein 4 (BMP-4), Smad-3 and transforming growth factor beta (TGF-β). Methods Synovial fluid was collected from patients submitted to knee arthroscopy or replacement and were centrifuged to isolate cells from the fluid. Cells were cultured for 21 days and characterized as mesenchymal stem cells (MSCs) according to the criteria of the International Society of Cell Therapy. Then, we performed an [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (MTT) assay after exposing cells with and without OA to TGF-β, Smad3 and BMP-4 pathway inhibitors and to different concentrations of BMP4. Results Exposure to the TGF-β, Smad3 and BMP-4 inhibitors modifies the mitochondrial activity of the SFCs. The activity of the SFCs is modified by influences of increasing concentrations of BMP4, but there is no difference in cellular activity between patients with and without OA. Conclusion TGF-β, Smad3 and BMP-4 modulate the activity of SFCs from patients with and without knee OA.


Resumo Objetivo Nosso objetivo foi avaliar a modulação das células do líquido sinovial (SFCs, na sigla em inglês) de pacientes com e sem osteoartrite (OA) por proteína morfogenética óssea 4 (BMP-4), Smad3 e transformador do fator de crescimento β (TGF-β). Métodos O do líquido sinovial foi coletado de pacientes submetidos a artroscopia ou artroplastia do joelho, e centrifugados para isolar as células do liquido sinovial. As células foram cultivadas por 21 dias e caracterizadas como células-tronco mesenquimais (MSCs, na sigla em inglês) de acordo com os critérios da International Society of Cell Therapy. Em seguida, realizamos um ensaio de brometo de 3-4,5-dimetil-tiazol-2-il-2,5difeniltetrazólio (MTT) depois de expor células com e sem OA para TGF-β, inibidores de via Smad3 e BMP-4 e para diferentes concentrações de BMP-4. Resultados A exposição aos inibidores TGF-β, Smad3 e BMP-4 modifica a atividade mitocondrial das SFCs. A atividade das SFCs é modificada por influências sobre o aumento das concentrações de BMP-4, mas não há diferença na atividade celular entre pacientes com e sem OA. Conclusão TGF-β, Smad3 e BMP-4 modulam a atividade das SFCs de pacientes com e sem OA do joelho.


Subject(s)
Humans , Male , Female , Osteoarthritis , Synovial Fluid , Transforming Growth Factor beta1 , Mesenchymal Stem Cells
4.
NOVA publ. cient ; 20(3): [17], 2022 enero-junio. gráficos, mapas, tablas e ilustraciones
Article in Spanish | LILACS | ID: biblio-1397023

ABSTRACT

Resumen Introducción. Las células madre mesenquimales han generado interés en la ingeniería de tejidos, debido a sus propiedades proliferativas y capacidad de reparación de tejidos, sin embargo, para un trasplante exitoso, es necesario aumentar el número de células mediante un cultivo in-vitro. Durante este proceso la capacidad proliferativa disminuye, provocando cambios en la morfología y funcionalidad celular y afectando la viabilidad del cultivo, este estado se conoce como senescencia celular y como posibles causales, se ha considerado el estrés oxidativo y la falta de factores de crecimiento. Objetivos: Evaluar el efecto de FGF-2 sobre la senescencia de un cultivo de células madre mesenquimales aisladas de gelatina de Wharton y su papel en la regulación del estrés oxidativo. Metodología. Se añadieron dosis de 3,5 y 7,5 ng de FGF-2 al cultivo. Durante los pasajes 5 y 7, se estimó tanto la senescencia celular como la presencia de ROS (especies reactivas de oxígeno). Resultados.Se obtuvo en el pasaje 5, una diferencia significativa del 99,5% entre el control (+) con respecto a los tratamientos con FGF-2, sin embargo, en el pasaje 7 se observó un aumento en la producción de la enzima ß-galactosidasa y cambios morfológicos, confirmando un estado senescente en el cultivo en todos los tratamientos evaluados. Conclusión. Las dosis utilizadas en este estudio contribuyeron positivamente a disminuir el proceso senescente en el cultivo celular, además se determinó, que el FGF-2 puede prolongar el tiempo de cultivo, retardando parcialmente la concentración de especies reactivas de oxígeno


AbstractIntroduction. Mesenchymal stem cells have been generated interest in tissue engineering, due to their proliferative properties and tissue repair capacity, however, for a successful transplant process, it is necessary to increase the number of cells in a culture expansion process. During this process the proliferative capacity is limited, causing changes in cell morphology and functionality affecting the viability of the culture, this state is known as cell senescence. Oxidative stress and deregulation of growth factors are considered as reasons. Aims. To evaluate the effect of FGF-2 on the senescence of a mesenchymal stem cells culture isolated from Wharton ́s jelly and its role in the regulation of oxidative stress. Methodology: 3,5 and 7,5 ng doses of FGF-2 were added to the culture medium from passage 2, then the senescence of the culture was evaluated and the presence of reactive oxygen species was determined during passages 5 and 7. Results. We observed that in passage 5, there is a significant difference 99.5% between the control (+) concerning the FGF-2 treatments, however, in passage 7, an increase in the production of the enzyme ß-galactosidase was observed and changes in morphology such as: increase in size and elongated shape of the cell, confirming a senescent state on the culture in all the treatments evaluated. Conclusion. The doses used in this study contributed positively to decrease this process in a cell culture, also, the FGF- 2 can prolong the cultivation time, partially decreasing the concentration of reactive oxygen species


Subject(s)
Humans , Mesenchymal Stem Cells , Fibroblast Growth Factor 2 , Intercellular Signaling Peptides and Proteins , Wharton Jelly
5.
Article in Chinese | WPRIM | ID: wpr-928762

ABSTRACT

Mesenchymal stem cell (MSC) is widely used in cell therapy because of its high proliferative and multi directional differentiation potential as well as its low immunogenicity. The transplantation of MSC can help the repair of the injured organs, however, the MSC transplanted to the local organs are affected by oxidative stress and lead to premature aging or apoptosis. Heme oxygenase 1 (HO1) is a key ratelimiting enzyme in the process of heme metabolism, which has the functions of antiinflammation, antioxidation, antiapoptosis, antiaging, reducing cell damage and promoting angiogenesis. Induced high expression of HO1 in MSC could increase the ability of MSC against oxidative stress injury, delay the senescence and apoptosis of MSC, and alleviate cell injury. In this reviews, the research progress of HO1 on antioxidative stress injury of MSC.


Subject(s)
Apoptosis , Cell Differentiation , Heme Oxygenase-1/metabolism , Humans , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells , Oxidative Stress
6.
Article in Chinese | WPRIM | ID: wpr-928732

ABSTRACT

OBJECTIVE@#To investigate the effect of acute myeloid leukemia cells in leukemia-microenvironment on proliferation and apoptosis of bone marrow-derived mesenchymal stromal cells (BM-MSC).@*METHODS@#Acute myeloid leukemia (AML) murine models overexpressing MLL-AF9 were established. The number of BM-MSC of wild type (WT) and AML-derived mice were analyzed by flow cytometry. Morphology and growth differences between WT and AML-derived BM-MSC were analyzed by inverted fluorescence microscope. Proliferation and apoptosis of BM-MSC between these two groups were detected by Brdu and Annexin V/PI.@*RESULTS@#Compared with WT-derived BM-MSC, the number and proliferation rate of AML-derived BM-MSC significantly increased (P<0.01, P<0.001), while apoptosis rate decreased (P<0.05). When cultured in vitro, BM-MSC grew faster under conditional medium.@*CONCLUSION@#AML cells can promote proliferation and inhibit apoptosis of BM-MSC.


Subject(s)
Animals , Apoptosis , Bone Marrow , Bone Marrow Cells , Cell Proliferation , Humans , Leukemia, Myeloid, Acute , Mesenchymal Stem Cells , Mice , Tumor Microenvironment
7.
Article in Chinese | WPRIM | ID: wpr-928713

ABSTRACT

Acute lymphoblastic leukemia (ALL) is a kind of the most common hematopoietic malignancy, its recurrence and drug resistance are closely related to the bone marrow microenvironment. Bone marrow stromal cell (BMSC) is an important part of the bone marrow microenvironment and their interaction with leukemia cells cannot be ignored. BMSC participates in and regulate signaling pathways related to proliferation or apoptosis of ALL cells by secretes cytokines or extracellular matrix proteins, thus affecting the survival of ALL cells. In this review, the research advance of several signaling pathways of the interaction between BMSC and ALL cells was summarized briefly.


Subject(s)
Apoptosis , Bone Marrow , Bone Marrow Cells , Humans , Mesenchymal Stem Cells , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Stromal Cells , Tumor Microenvironment
8.
Article in Chinese | WPRIM | ID: wpr-928711

ABSTRACT

In recent years, studies have found that mitochondrial transfer between leukemic cells and different types of cells in their bone marrow microenvironment, especially mesenchymal stem cells, plays a key role in the occurrence, development and drug resistance of hematological malignant tumors. This paper mainly introduces the role and latest research progress of mitochondrial transfer in acute and chronic myeloid leukemia, acute lymphoblastic leukemia and multiple myeloma, and briefly describes the mechanism of drug resistance caused by mitochondrial transfer in leukemic cells during chemotherapy. The aim is to provide a new idea and theoretical basis for using intercellular mitochondrial transfer as a potential therapeutic target.


Subject(s)
Bone Marrow , Hematologic Neoplasms/metabolism , Humans , Mesenchymal Stem Cells , Mitochondria , Multiple Myeloma/metabolism , Tumor Microenvironment
9.
Article in English | WPRIM | ID: wpr-928575

ABSTRACT

Bronchopulmonary dysplasia (BPD) is a chronic lung disease due to impaired pulmonary development and is one of the main causes of respiratory failure in preterm infants. Preterm infants with BPD have significantly higher complication and mortality rates than those without BPD. At present, comprehensive management is the main intervention method for BPD, including reasonable respiratory and circulatory support, appropriate enteral nutrition and parenteral nutrition, application of caffeine/glucocorticoids/surfactants, and out-of-hospital management after discharge. The continuous advances in stem cell medicine in recent years provide new ideas for the treatment of BPD. Various pre-clinical trials have confirmed that stem cell therapy can effectively prevent lung injury and promote lung growth and damage repair. This article performs a comprehensive analysis of the mechanism of mesenchymal stem cells in the treatment of BPD, so as to provide a basis for clinical applications.


Subject(s)
Bronchopulmonary Dysplasia/prevention & control , Enteral Nutrition , Humans , Infant, Newborn , Infant, Premature , Lung , Mesenchymal Stem Cells
10.
Article in Chinese | WPRIM | ID: wpr-928326

ABSTRACT

OBJECTIVE@#To observe the effects of Taohong Siwu Decoction(, THSWD) on the mesenchymal stem cells(MSCs) migration, homing number and cytokine expression in callus during the early process of fracture healing, and to explore the mechanism of THSWD on accelerationg fracture healing by regulating the homing of MSCs in rats.@*METHODS@#A rat model of right femoral shaft open fracture was established. Thirty-two 5-week-old male Sprague-Dawley rats, weighting 110 to 130 g, were divided into control group, low-dose group, medium-dose group and high-dose group by using random number table. Distilled water was given to the control group, and the other groups were given Taohong Siwu Decoction. The rats were gavaged twice a day for 5 consecutive days after surgery. Bone volume/tissue volume(BV/TV) and bone mineral density(BMD) were observed using micro-computed tomography (micro-CT) at 21 days after surgery. At 5 days post-fracture, peripheral blood MSCs from THSWD treated and untreated rats were cultured in vitro. Subsequently, the migration ability of MSCs was observed by cell migration assay. The number of MSCs homing to the callus at the early stage of fracture (5 d) was detected by Immunohistochemistry (IHC). Protein chip was used to detect the expression of cytokines in callus.@*RESULTS@#Micro-CT results showed that BV/TV was higher in the high-dose group than in the medium-dose group (P=0.032), and higher in the medium-dose group than in the low-dose group(P=0.041), with no difference between the control and low-dose group (P=0.651). In addition, there was no difference in BMD between low-dose group and the model group (P=0.671), and lower in the low-dose group than in the medium-dose group(P=0.018), and the medium-dose group was lower than the high-dose group(P=0.008). Cell migration assay showed that THSWD promotes enhanced the migration ability of peripheral blood MSCs. IHC assay revealed that CD45-, CD90+, CD29+ MSCs significantly increased in bone callus after THSWD intervention compared with the control group. Protein chip showed that THSWD promoted the upregulation of CINC-1(×2.91), CINC-3(×1.59), LIX(×1.5), Thymus Chemokine (×2.55), VEGF (×1.22) and the down-regulation of TIMP-1 (×2.98).@*CONCLUSION@#THSWD, a representative formula of "promoting blood circulation and removing blood stasis", can significantly accelerate fracture healing, and its mechanism may be related to enhancing the migration ability of peripheral blood MSCs and up-regulating CINC-1, CINC-3, LIX, Thymus Chemokine, VEGF and down-regulating TIMP-1 in bone callus, which promotes the peripheral blood MSCs homing in the early stage of fracture.


Subject(s)
Animals , Drugs, Chinese Herbal , Fracture Healing , Fractures, Bone/drug therapy , Humans , Male , Mesenchymal Stem Cells , Rats , Rats, Sprague-Dawley , Tissue Inhibitor of Metalloproteinase-1/pharmacology , Vascular Endothelial Growth Factor A , X-Ray Microtomography
11.
Chinese Journal of Biotechnology ; (12): 1183-1196, 2022.
Article in Chinese | WPRIM | ID: wpr-927773

ABSTRACT

Mesenchymal stem cells (MSCs) have broad application potentials in regenerative medicine and translational medicine. Obtaining large quantities of primary-cultured MSCs and select the most suitable cell origin for targeted diseases are critical to research. To select the most suitable seed cells of MSCs from different origins for clinical treatment and research, biological characteristics of MSCs from human umbilical cord and placenta were compared. These include cell morphology, surface marker expression, differentiation and karyotype. Transcriptome sequencing of four MSCs from fetus were performed and the results were analyzed from the perspective of proliferation and cytokine secretion. The results revealed that MSCs from umbilical cord (UC), amniotic membrane (AM), chorionic membrane (CM), chorionic villi (CV) and deciduae (DC) met the minimum standards of the International Society of Cell Therapy (ISCT) in 2006 and had the general characteristics of stem cells. Karyotype analysis showed that MSCs derived from UC, AM, CM and CV were all from fetus except that the DC-MSCs were from mother. Transcriptome sequencing analysis showed that hMSCs from umbilical cord and placenta had similar gene expression patterns, while different expression patterns were observed in specific genes involved in cell cycle, cell division, cell death, cell growth and development. These genes play important roles in transcriptional regulation, DNA repair, DNA replication and chromosome stability, which were momentous components of cellular or subcellular fraction movement, cell communication, cell tissue protrusions, cytokine secretion and hormone metabolism. Transcriptome sequencing analysis explained the differences in biological characteristics among MSCs from different sources, while verification experiments based on the transcriptome sequencing results showed that the proliferation and cytokine secretion capabilities of MSCs from different sources were significantly different. In all, UC-MSCs and CV-MSCs with stronger proliferation and higher levels of paracrine factors secretion may show their respective advantages in treating diseases.


Subject(s)
Cell Differentiation , Female , Fetus , Humans , Mesenchymal Stem Cells , Placenta , Pregnancy , Umbilical Cord
12.
São Paulo; s.n; s.n; 2022. 116 p. tab, graf.
Thesis in English | LILACS | ID: biblio-1378343

ABSTRACT

Stem cells are undifferentiated cells that can be distinguished from others by their ability to self-renew and to differentiate into new specific cell types. Mesenchymal stem cells (MSC) are adult stem cells that can be obtained from different sources, such as adipose tissue, bone marrow, dental pulp, and umbilical cord. They can either replicate, originating new identical cells, or differentiate into cells of mesodermal origin and from other germ layers. MSC have been studied as new tools for regenerative therapy. Although encouraging results have been demonstrated, MSC-based therapies still face a great barrier: the difficulty of isolating these cells from heterogeneous environments. MSC are currently characterized by immunolabelling through a set of multiple surface membrane markers, including CD29, CD73, CD90 and CD105, which are also expressed by other cell types. Hence, the present work aimed to identify new specific biomarkers for the characterization of human MSC using DNA aptamers produced by the SELEX (Systematic Evolution of Ligands by EXponential Enrichment) technique. Our results showed that MSC from different origins bound to DNA candidate aptamers, that is, DNA or RNA oligonucleotides selected from random libraries that bind specifically to biological targets. Aptamer-bound MSC could be isolated by fluorescenceactivated cell sorting (FACS) procedures, enhancing the induction of differentiation into specific phenotypes (chondrocytes, osteocytes and adipocytes) when compared to the whole MSC population. Flow cytometry analyses revealed that candidate aptamers bound to 50% of the MSC population from dental pulp and did not present significant binding rates to human fibroblasts or lymphocytes, both used as negative control. Moreover, immunofluorescence images and confocal analyses revealed staining of MSC by aptamers localized in the surfacemembrane of these cells. The results also showed internal staining of human monocytes by our investigated aptamers. A non-specific control aptamer (CNTR APT) obtained from the random pool was then utilized to compare the specificity of the aptamers bound to the analyzed non-apoptotic cells, showing no staining for MSC. However, 40% of the monocytes bound to the CNTR APT. Normalized data based on the cells bound to candidate aptamers compared to those bound to the CNTR APT, revealed a 10 to 16-fold higher binding rate for MSC against 2-fold for monocytes. Despite its low specificity, monocyte-aptamer binding occurs probably due to the expression of shared markers with MSC, since monocytes are derived from hematopoietic stem cells and are important for the immune system ability to internalize/phagocyte external molecules. Given that, we performed a pull-down assay followed by mass spectrometry analysis to detect which MSC-specific protein or other target epitope not coexpressed by monocytes or the CNTR APT would bind to the candidate aptamer. Distinguishing between MSC and monocyte epitopes is important, as both cells are involved in immunomodulatory effects after MSC transplantations. ADAM17 was found to be a target of the APT10, emerging as a possible biomarker of MSC, since its involvement in the inhibition of the TGF signaling cascade, which is responsible for the differentiation of MSC. Thus, MSC with a higher stemness profile should overexpress the protein ADAM17, which presents a catalytic site with affinity to APT10. Another target of Apt 10 is VAMP3, belonging to a transmembrane protein complex that is involved in endocytosis and exocytosis processes during immune and inflammatory responses. Overall, proteins identified as targets of APT10 may be cell surface MSC biomarkers, with importance for MSC-based cell and immune therapies


Células tronco são células indiferenciadas que podem ser distinguidas de outros tipos celulares por meio da habilidade de se auto renovarem e de se diferenciarem em novos tipos celulares. Células tronco mesenquimais (MSC) são células tronco adultas encontradas em diferentes tecidos como tecido adiposo, polpa de dente e cordão umbilical. Estas células podem se autodividir em células idênticas ou se diferenciarem em células de origem mesodermal. Estas células têm sido estudadas em novas aplicações que envolvem terapia regenerativas. Embora resultados encorajadores tenham sido demonstrados, terapias que utilizam MSC ainda encontram uma grande barreira: a dificuldade no isolamento destas células a partir de um ambiente heterogêneo. MSC são caracterizadas por populações positivas em ensaios de imunomarcação para os epítopos membranares CD29, CD73, CD90 e CD105, presentes também em outros tipos celulares. Assim, o presente trabalho tem o objetivo de identificar novos biomarcadores de MSC de origem humana, utilizando aptâmeros de DNA produzidos pela técnica SELEX (Systematic Evolution of Ligands by EXponential Enrichment) como ferramenta. Nossos resultados mostraram que MSC de diferentes origens ligam-se a aptâmeros (oligonucleotídeos de DNA ou RNA que atuam como ligantes específicos de alvos moleculares) de DNA candidatos que atuam no isolamento de MSC por meio da técnica FACS de separação celular, promovendo uma maior indução de diferenciação em células específicas (condrócitos, osteócitos e adipócitos) comparada com a população total de MSC. Análises de citometria de fluxo mostraram que os aptâmeros candidatos se ligam a 50% das MSC de polpa de dente e não apresentam taxa de ligação significante para fibroblastos e linfócitos de origem humana - utilizados como controles negativo. Além domais, imagens de imunofluorescência e confocal mostraram ligação na superfície da membrana de MSC e a marcação interna de monócitos a estes aptâmeros. Portanto, um aptâmero controle (CNTR APT) foi utilizado para comparar a especificidade dos aptâmeros ligados a células viáveis, mostrando a não ligação deste aptâmero a MSC. Porém, 40% da população de monócitos ligou-se ao CNTR APT. Uma normalização baseada na comparação entre as taxas de ligação entre células ligadas com aptâmeros candidatos e o aptâmero controle gerou uma taxa de especificidade entre 10-16 vezes maior para MSC contra 2,5 vezes para os monócitos. Deste modo, embora os resultados tenham mostrado uma taxa de ligação entre monócitos e aptâmeros, as MSC ligadas aos aptâmeros candidatos possuem uma maior taxa de especificidade devido a uma maior presença de antígenos que são expressos em ambas as células. Um ensaio de Pull Down seguido de espectrometria de massas foi utilizado para a identificação de biomarcadores que se ligariam aos aptâmeros candidatos, e que não seriam co-expressos por monócitos e por antígenos ligados ao aptâmero controle. Deste modo, a proteína ADAM17 foi identificada nas amostras de APT10 ligadas às MSC. Tal proteína está relacionada à inibição de uma cascata de sinalização da família de proteínas TGF, responsável pela diferenciação de MSC. Assim, MSC com maior potencial tronco deveriam expressar ADAM17 em maior quantidade. Tal proteína apresenta um sítio catalítico que demonstra interagir com o APT10, de acordo com predição Docking entre proteína e DNA. Foi identificada também, a proteína VAMP3, que pertence a um complexo proteico transmembranar responsável pelos processos de endocitose e exocitose, e que podem ter um papel importante na liberação de citocinas e outras moléculas relacionadas às respostas imune e inflamatórias. Deste modo, o APT10 identificou proteínas importantes que devem estar relacionas com a melhora de imunoterapias que utilizam MSC


Subject(s)
Stem Cells , Biomarkers/analysis , SELEX Aptamer Technique/instrumentation , Mesenchymal Stem Cells/classification , ADAM17 Protein/pharmacology , Patient Isolation , Mass Spectrometry/methods , Staining and Labeling/methods , Transplantation/adverse effects , Umbilical Cord , DNA/agonists , Transforming Growth Factors/agonists , Cell Separation/instrumentation , Cytokines/adverse effects , Adipocytes/metabolism , Chondrocytes/classification , Scientists for Health and Research for Development , Adult Stem Cells/classification , Fibroblasts/chemistry , Flow Cytometry/instrumentation , Germ Layers , Antigens/adverse effects
13.
São Paulo; s.n; s.n; 2022. 62 p. graf.
Thesis in Portuguese | LILACS | ID: biblio-1378655

ABSTRACT

As Células-Tronco Mesenquimais (CTMs), são células multipotentes, presentes em diversos tecidos, sendo bastante estudada devido sua capacidade imunorregulatória por meio da liberação de fatores solúveis. Fatores estes que atuam sobre as funções de células do sistema imunitário. Simultaneamente, estudos indicam que os compostos flavonoides, em destaque a Delfinidina, presente em alguns frutos e flores, possuem atuação anti-inflamatória e inibitória sobre células do sistema imunitário. Todavia, são escassos os estudos em relação entre a capacidade imunorregulatória da CTM e a influência da Delfinidina, sendo este o objetivo deste estudo. Inicialmente, a Delfinidina 3-O-ß-D-glicosídeo foi escolhido, devido a sua maior estabilidade e a dose de 50 µM foi selecionada após análise por citometria de fluxo que mostrou aumento da fase proliferativa do ciclo celular. Posteriormente ao realizar análise da produção de fatores solúveis pelas CTM, os resultados mostraram aumento da produção de IL-10, TGF-ß e Oxido nítrico pelas CTM tratadas com Delfinidina. Bem como, diminuição da expressão de p-NF-κB/NF-κB pelas CTMs tratadas com Delfinidina, quando avaliadas por Wersten Blot. Adicionalmente, para analisar a Delfinidina sobre os efeitos imunorregulatórios da CTM sob macrófagos (RAW 264.7), célula esta, importante no sistema imune inato. Foram realizadas culturas condicionadas, com posterior análise da produção de fatores solúveis, os resultados mostraram aumento da produção de IL-10, e diminuição da produção de TNF-α, IL-1α e IL-12 pelos macrófagos, nas culturas condicionadas. Assim como, diminuição da expressão do fator p-NF-κB/NF-κB pelos macrófagos nas culturas condicionadas, quando avaliadas por Wersten Blot. Ademais, ao analisar a atividade metabólica dos macrófagos por ensaio de MTT, os resultados mostraram que as culturas condicionadas e a Delfinidina per si foi capaz de diminuir a atividade metabólica, sem alterar os efeitos anti-inflamatórios sobre a célula. Em síntese, a Delfinidina mostrou acentuar a atuação imunorregulatória da CTM sobre a linhagem macrofágica, célula esta, de grande importância para o sistema imune inato


Mesenchymal Stem Cells (MSCs) are multipotent cells present in various tissues, being widely studied due to their immunoregulatory capacity through the release of soluble factors. These factors act on the functions of cells of the immune system. Simultaneously, studies indicate that flavonoid compounds, especially Delphinidin, present in some fruits and flowers, have anti inflammatory and inhibitory effects on immune system cells. However, there are few studies on the relationship between the immunoregulatory capacity of MSC and the influence of Delphinidin, which is the objective of this study. Initially, Delphinidin 3-O-ß-D-glycoside was chosen due to its greater stability and the 50 µM dose was selected after analysis by flow cytometry which showed an increase in the proliferative phase of the cell cycle. Subsequently, when analyzing the production of soluble factors by MSCs, the results showed an increase in the production of IL-10, TGF-ß and nitric oxide by MSCs treated with Delphinidin. As well as decreased expression of p-NF-κB/NF-κB by MSCs treated with Delphinidin, when evaluated by Wersten Blot. Additionally, to analyze Delphinidin on the immunoregulatory effects of MSC on macrophages (RAW 264.7), this cell is important in the innate immune system. Conditioned cultures were performed, with subsequent analysis of the production of soluble factors, the results showed an increase in the production of IL-10, and a decrease in the production of TNF-α, IL-1α and IL-12 by macrophages, in the conditioned cultures. As well as decreased expression of p-NF-κB/NF-κB factor by macrophages in conditioned cultures, when evaluated by Wersten Blot. Furthermore, when analyzing the metabolic activity of macrophages by MTT assay, the results showed that conditioned cultures and Delphinidin itself was able to decrease the metabolic activity, without altering the anti-inflammatory effects on the cell. In summary, Delphinidin has shown to enhance the immunoregulatory action of MSC on the macrophage lineage, a cell that is of great importance for the innate immune system


Subject(s)
Flavonoids/analysis , Immune System , Transforming Growth Factors , Interleukin-1/adverse effects , Interleukin-10/adverse effects , Mesenchymal Stem Cells/classification , Flow Cytometry/instrumentation , Anti-Inflammatory Agents/administration & dosage
14.
Article in Chinese | WPRIM | ID: wpr-936312

ABSTRACT

OBJECTIVE@#To investigate the changes in autophagy of mesenchymal stem cells (MSCs) from patients with ankylosing spondylitis and explore the mechanism for decreased autophagy in ASMSCs.@*METHODS@#MSCs collected from 14 patients with AS (ASMSCs) and from 15 healthy donors (HDMSCs) were cultured in the absence or presence of 25 ng/mL TNF-α for 6 h. Autophagy of the cells was determined by immunofluorescence staining of GFP-LC3B, and the results were confirmed by detecting the protein expressions of autophagy markers LC3 II/LC3 I and P62. The mRNA expressions of the related genes were detected using qRT-PCR, and the protein expressions of the autophagy markers and signaling pathway-related molecules were determined with Western blotting. TG100713 was used to block the PI3K/AKT/mTOR signal pathway, and its effect on autophagy of ASMSCs was evaluated.@*RESULTS@#ASMSCs showed significantly weaker GFP-LC3B puncta staining and lower protein expression levels of LC3 II/LC3 I but higher levels of P62 protein (P < 0.05), indicating a decreased autophagy capacity as compared with HDMSCs. TNF-α-induced ASMSCs showed significantly higher protein expressions of p-PI3K/ PI3K, p-AKT/AKT and p-mTOR/mTOR than HDMSCs (P < 0.05), suggesting hyperactivation of the PI3K/AKT/mTOR signaling pathway in ASMSCs. Blocking PI3K/AKT/mTOR signaling with TG100713 eliminated the difference in TNF-α-induced autophagy between HDMSCs and ASMSCs.@*CONCLUSION@#In patients with AS, hyperactivation of the PI3K/AKT/mTOR signaling pathway results in decreased autophagy of the MSCs and potentially contributes to chronic inflammation.


Subject(s)
Autophagy , Humans , Mesenchymal Stem Cells/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Spondylitis, Ankylosing , TOR Serine-Threonine Kinases/metabolism , Tumor Necrosis Factor-alpha/metabolism
15.
Article in Chinese | WPRIM | ID: wpr-936192

ABSTRACT

Objective: To investigate the effects of adipose-derived mesenchymal stem cells (ADMSCs) on proliferation and hormone secretion of parathyroid cells in votro. Methods: The parathyroid cells and ADMSCs were obtained from 10 SD rats by cell separation and culture. The phenotype of P3 generation for ADMSCs was detected by flow cytometry. The co-culture of parathyroid cells and ADMSCs was conducted in the ratios of 2∶1, 1∶1, 1∶2 and 1∶5, respectively. The level of parathyroid hormone in cell supernatant was determined. The results were compared with the parathyroid hormone in the supernatant of parathyroid cells cultured separately in the corresponding number. The effects of ADMSCs on the hormone secretion of parathyroid cells were evaluated. SPSS 11.0 software was used for statistical analysis. Results: The primary culture of either parathyroid cells or ADMSCs and the co-culture of these cells in vitro were performed successfully, and the in vitro culture of different proportions of the two cells showed different effects on parathyroid hormone secretion. The co-culture of parathyroid cells and ADMSCs, especially in the ratio of 1∶5, facilitated the secretion of parathyroid hormone ((1.3±0.0) vs. (0.8±0.1), (1.3±0.0) vs. (0.9±0.0), (1.7±0.5) vs. (0.9±0.0), (1.7±0.0) vs. (1.2±0.2))ng/L with t value of 25.46, 64.30, 3.32, 7.16, P<0.05 on the 2nd, 4th, 6th and 8th days respectively. Secondly, when the ratio was 1∶2, the PTH level showed an upward trend. Conclusion: Parathyroid cells and ADMSCs can be co-cultured in vitro, facilitating the secretion of parathyroid hormone under the appropriate cell proportion such as the ratio of by parathyroid cells to ADMSCs at 1∶5.


Subject(s)
Adipose Tissue , Animals , Coculture Techniques , Mesenchymal Stem Cells , Rats , Rats, Sprague-Dawley
16.
Article in Chinese | WPRIM | ID: wpr-936154

ABSTRACT

OBJECTIVE@#To preliminarily investigate the role of long non-coding RNA (lncRNA) MIR4697 host gene (MIR4697HG) in regulating the adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).@*METHODS@#For adipogenic differentiation, BMSCs were induced in adipogenic media for 10 days. The mRNA expression levels of lncRNA MIR4697HG and adipogenic marker genes including peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhanced binding protein α (CEBP/α) and adiponectin (ADIPQ) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) at different time points (0, 1, 2, 3, 5, 7, 10 days). The MIR4697HG stable knockdown-BMSC cell line was generated by infection of MIR4697HG shRNA-containing lentiviruses. To avoid off-target effect, two target sequences (shMIR4697HG-1, shMIR4697HG-2) were designed. And then cells were induced to differentiate in adipogenic medium. Oil red O staining, Western blot and qRT-PCR were used to detect the effect of MIR4697HG knockdown on adipogenic differentiation of BMSCs.@*RESULTS@#The mRNA expression level of MIR4697HG was significantly increased during adipogenic differentiation (P < 0.01), and adipogenic differentiation of BMSCs was evidenced by upregulated mRNA levels of specific adipogenesis-related genes including PPARγ, CEBP/α and ADIPQ. Observed by fluorescence microscopy, more than 90% transfected target cells expressed green fluorescent protein successfully after shMIR4697HG-1 group, shMIR4697HG-2 group and shNC group transfection for 72 h. And the transfection efficiency of MIR4697HG examined by qRT-PCR was above 60%. Then the BMSCs were treated with adipogenic media for 7 days and showed that the mRNA expression levels of adipogenesis-related genes including PPARγ, CEBP/α and ADIPQ were significantly decreased in the MIR4697HG knockdown group (P < 0.01), while the expression levels of PPARγ and CEBP/α proteins were decreased remarkably as well (P < 0.01). Consistently, MIR4697HG knockdown BMSCs formed less lipid droplets compared with the control BMSCs, which further demonstrated that MIR4697HG knockdown inhibited adipogenic differentiation of BMSCs.@*CONCLUSION@#lncRNA MIR4697HG played a crucial role in regulating the adipogenic differentiation of BMSCs, and MIR4697HG knockdown significantly inhibited the adipogenic differentiation of BMSCs. These data may suggest that lncRNA MIR4697HG could serve as a therapeutic potential target for the aberrant adipogenic differentiation-associated disorders including osteoporosis.


Subject(s)
Adipogenesis/genetics , Bone Marrow Cells/metabolism , Cell Differentiation , Cells, Cultured , Mesenchymal Stem Cells , Osteogenesis , PPAR gamma/pharmacology , RNA, Long Noncoding/genetics , RNA, Messenger/metabolism
17.
Frontiers of Medicine ; (4): 56-82, 2022.
Article in English | WPRIM | ID: wpr-929195

ABSTRACT

Contributing to organ formation and tissue regeneration, extracellular matrix (ECM) constituents provide tissue with three-dimensional (3D) structural integrity and cellular-function regulation. Containing the crucial traits of the cellular microenvironment, ECM substitutes mediate cell-matrix interactions to prompt stem-cell proliferation and differentiation for 3D organoid construction in vitro or tissue regeneration in vivo. However, these ECMs are often applied generically and have yet to be extensively developed for specific cell types in 3D cultures. Cultured cells also produce rich ECM, particularly stromal cells. Cellular ECM improves 3D culture development in vitro and tissue remodeling during wound healing after implantation into the host as well. Gaining better insight into ECM derived from either tissue or cells that regulate 3D tissue reconstruction or organ regeneration helps us to select, produce, and implant the most suitable ECM and thus promote 3D organoid culture and tissue remodeling for in vivo regeneration. Overall, the decellularization methodologies and tissue/cell-derived ECM as scaffolds or cellular-growth supplements used in cell propagation and differentiation for 3D tissue culture in vitro are discussed. Moreover, current preclinical applications by which ECM components modulate the wound-healing process are reviewed.


Subject(s)
Cell Differentiation , Cell Proliferation , Decellularized Extracellular Matrix , Extracellular Matrix/metabolism , Humans , Mesenchymal Stem Cells , Tissue Engineering/methods , Tissue Scaffolds/chemistry
18.
Article in English | WPRIM | ID: wpr-929152

ABSTRACT

Aging of craniofacial skeleton significantly impairs the repair and regeneration of trauma-induced bony defects, and complicates dental treatment outcomes. Age-related alveolar bone loss could be attributed to decreased progenitor pool through senescence, imbalance in bone metabolism and bone-fat ratio. Mesenchymal stem cells isolated from oral bones (OMSCs) have distinct lineage propensities and characteristics compared to MSCs from long bones, and are more suited for craniofacial regeneration. However, the effect of epigenetic modifications regulating OMSC differentiation and senescence in aging has not yet been investigated. In this study, we found that the histone demethylase KDM4B plays an essential role in regulating the osteogenesis of OMSCs and oral bone aging. Loss of KDM4B in OMSCs leads to inhibition of osteogenesis. Moreover, KDM4B loss promoted adipogenesis and OMSC senescence which further impairs bone-fat balance in the mandible. Together, our data suggest that KDM4B may underpin the molecular mechanisms of OMSC fate determination and alveolar bone homeostasis in skeletal aging, and present as a promising therapeutic target for addressing craniofacial skeletal defects associated with age-related deteriorations.


Subject(s)
Aging , Cell Differentiation , Facial Bones/physiology , Humans , Jumonji Domain-Containing Histone Demethylases/genetics , Mesenchymal Stem Cells/cytology , Osteogenesis , Osteoporosis
19.
Article in English | WPRIM | ID: wpr-929141

ABSTRACT

The high neurogenic potential of dental and oral-derived stem cells due to their embryonic neural crest origin, coupled with their ready accessibility and easy isolation from clinical waste, make these ideal cell sources for neuroregeneration therapy. Nevertheless, these cells also have high propensity to differentiate into the osteo-odontogenic lineage. One strategy to enhance neurogenesis of these cells may be to recapitulate the natural physiological electrical microenvironment of neural tissues via electroactive or electroconductive tissue engineering scaffolds. Nevertheless, to date, there had been hardly any such studies on these cells. Most relevant scientific information comes from neurogenesis of other mesenchymal stem/stromal cell lineages (particularly bone marrow and adipose tissue) cultured on electroactive and electroconductive scaffolds, which will therefore be the focus of this review. Although there are larger number of similar studies on neural cell lines (i.e. PC12), neural stem/progenitor cells, and pluripotent stem cells, the scientific data from such studies are much less relevant and less translatable to dental and oral-derived stem cells, which are of the mesenchymal lineage. Much extrapolation work is needed to validate that electroactive and electroconductive scaffolds can indeed promote neurogenesis of dental and oral-derived stem cells, which would thus facilitate clinical applications in neuroregeneration therapy.


Subject(s)
Cell Differentiation , Mesenchymal Stem Cells/metabolism , Neural Stem Cells/metabolism , Neurogenesis , Tissue Scaffolds
20.
Article in English | WPRIM | ID: wpr-929133

ABSTRACT

Neural crest-derived mesenchymal stem cells (MSCs) are known to play an essential function during tooth and skeletal development. PRX1+ cells constitute an important MSC subtype that is implicated in osteogenesis. However, their potential function in tooth development and regeneration remains elusive. In the present study, we first assessed the cell fate of PRX1+ cells during molar development and periodontal ligament (PDL) formation in mice. Furthermore, single-cell RNA sequencing analysis was performed to study the distribution of PRX1+ cells in PDL cells. The behavior of PRX1+ cells during PDL reconstruction was investigated using an allogeneic transplanted tooth model. Although PRX1+ cells are spatial specific and can differentiate into almost all types of mesenchymal cells in first molars, their distribution in third molars is highly limited. The PDL formation is associated with a high number of PRX1+ cells; during transplanted teeth PDL reconstruction, PRX1+ cells from the recipient alveolar bone participate in angiogenesis as pericytes. Overall, PRX1+ cells are a key subtype of dental MSCs involved in the formation of mouse molar and PDL and participate in angiogenesis as pericytes during PDL reconstruction after tooth transplantation.


Subject(s)
Animals , Cell Differentiation , Mesenchymal Stem Cells , Mice , Molar , Osteogenesis/physiology , Periodontal Ligament
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