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1.
J Biomed Mater Res B Appl Biomater ; 112(3): e35396, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38433653

RESUMO

Development of osteochondral tissue engineering approaches using scaffolds seeded with stem cells in association with mechanical stimulations has been recently considered as a promising technique for the repair of this tissue. In this study, an integrated and biomimetic trilayered silk fibroin (SF) scaffold containing SF nanofibers in each layer was fabricated. The osteogenesis and chondrogenesis of stem cells seeded on the fabricated scaffolds were investigated under a perfusion flow. 3-Dimethylthiazol-2,5-diphenyltetrazolium bromide assay showed that the perfusion flow significantly enhanced cell viability and proliferation. Analysis of gene expression by stem cells revealed that perfusion flow had significantly upregulated the expression of osteogenic and chondrogenic genes in the bone and cartilage layers and downregulated the hypertrophic gene expression in the intermediate layer of the scaffold. In conclusion, applying flow perfusion on the prepared integrated trilayered SF-based scaffold can support osteogenic and chondrogenic differentiation for repairing osteochondral defects.


Assuntos
Fibroínas , Animais , Coelhos , Fibroínas/farmacologia , Perfusão , Adipócitos , Bioensaio , Células-Tronco
2.
Elife ; 132024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38427029

RESUMO

A new mathematical model that can be applied to both single-cell and bulk DNA sequencing data sheds light on the processes governing population dynamics in stem cells.


Assuntos
Células-Tronco , Mutação , Análise de Sequência de DNA
3.
HLA ; 103(3): e15432, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38470345

RESUMO

HLA-C*06:372 differs from HLA-C*06:02:01:01 by a single substitution in exon 4.


Assuntos
Etnicidade , Antígenos HLA-C , Humanos , Antígenos HLA-C/genética , Alelos , Genes MHC Classe I , Células-Tronco
4.
Int J Med Sci ; 21(4): 664-673, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38464837

RESUMO

N6-Methyladenosine (m6A) has been reported to play a dynamic role in osteoporosis and bone metabolism. However, whether m6A is involved in the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) remains unclear. Here, we found that methyltransferase-like 3 (METTL3) was up-regulated synchronously with m6A during the osteogenic differentiation of hPDLSCs. Functionally, lentivirus-mediated knockdown of METTL3 in hPDLSCs impaired osteogenic potential. Mechanistic analysis further showed that METTL3 knockdown decreased m6A methylation and reduced IGF2BP1-mediated stability of runt-related transcription factor 2 (Runx2) mRNA, which in turn inhibited osteogenic differentiation. Therefore, METTL3-based m6A modification favored osteogenic differentiation of hPDLSCs through IGF2BP1-mediated Runx2 mRNA stability. Our study shed light on the critical roles of m6A on regulation of osteogenic differentiation in hPDLSCs and served novel therapeutic approaches in vital periodontitis therapy.


Assuntos
Osteogênese , Ligamento Periodontal , Humanos , Diferenciação Celular/genética , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Osteogênese/genética , Células-Tronco
5.
Hua Xi Kou Qiang Yi Xue Za Zhi ; 42(1): 37-45, 2024 Feb 01.
Artigo em Inglês, Chinês | MEDLINE | ID: mdl-38475949

RESUMO

OBJECTIVES: This study aimed to investigate the effects of sitagliptin on the proliferation, apoptosis, inflammation, and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) in lipopolysaccharide (LPS)-induced inflammatory microenvironment and its molecular mechanism. METHODS: hPDLSCs were cultured in vitro and treated with different concentrations of sitagliptin to detect cell viability and subsequently determine the experimental concentration of sitagliptin. An hPDLSCs inflammation model was established after 24 h of stimulation with 1 µg/mL LPS and divided into blank, control, low-concentration sitagliptin (0.5 µmol/L), medium-concentration sitagliptin (1 µmol/L), and high-concentration sitagliptin (2 µmol/L), high-concentrationsitagliptin+stromal cell derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) pathway inhibitor (AMD3100) (2 µmol/L+10 µg/mL) groups. A cell-counting kit-8 was used to detect the proliferation activity of hPDLSCs after 24, 48, and 72 h culture. The apoptosis of hPDLSCs cultured for 72 h was detected by flow cytometry. After inducing osteogenic differentiation for 21 days, alizarin red staining was used to detect the osteogenic differentiation ability of hPDLSCs. The alkaline phosphatase (ALP) activity in hPDLSCs was determined using a kit. The levels of inflammatory factors [tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6] in the supernatant of hPDLSCs culture were detected by enzyme-linked immunosorbent assay. The mRNA expressions of osteogenic differentiation genes [Runt-associated transcription factor 2 (RUNX2), osteocalcin (OCN), osteopontin (OPN)], SDF-1 and CXCR4 in hPDLSCs were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Western blot analysis was used to determine SDF-1 and CXCR4 protein expression in hPDLSCs. RESULTS: Compared with the blank group, the proliferative activity, number of mineralized nodules, staining intensity, ALP activity, and RUNX2, OCN, OPN mRNA, SDF-1, and CXCR4 mRNA and protein expression levels of hPDLSCs in the control group significantly decreased. The apoptosis rate and levels of TNF-α, IL-1ß, and IL-6 significantly increased (P<0.05). Compared with the control group, the proliferative activity, number of mineralized nodule, staining intensity, ALP activity, and RUNX2, OCN, OPN mRNA, SDF-1, and CXCR4 mRNA and protein expression levels of hPDLSCs in low-, medium-, and high-concentration sitagliptin groups increased. The apoptosis rate and levels of TNF-α, IL-1ß, and IL-6 decreased (P<0.05). AMD3100 partially reversed the effect of high-concentration sitagliptin on LPS-induced hPDLSCs (P<0.05). CONCLUSIONS: Sitagliptin may promote the proliferation and osteogenic differentiation of hPDLSCs in LPS-induced inflammatory microenvironment by activating the SDF-1/CXCR4 signaling pathway. Furthermore, it inhibited the apoptosis and inflammatory response of hPDLSCs.


Assuntos
Benzilaminas , Ciclamos , Lipopolissacarídeos , Ligamento Periodontal , Humanos , Ligamento Periodontal/metabolismo , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Receptores CXCR4/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Interleucina-6/metabolismo , Interleucina-6/farmacologia , Osteogênese , Transdução de Sinais , Inflamação/metabolismo , Células-Tronco , RNA Mensageiro/metabolismo , Apoptose , Proliferação de Células , Células Estromais/metabolismo , Diferenciação Celular , Células Cultivadas
6.
Front Immunol ; 15: 1368898, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38476233

RESUMO

Hepatocellular carcinoma (HCC) is a highly aggressive malignancy, with high recurrence rates and notorious resistance to conventional chemotherapy. Cancer stemness refers to the stem-cell-like phenotype of cancer cells and has been recognized to play important roles in different aspects of hepatocarcinogenesis. Small extracellular vesicles (sEVs) are small membranous particles secreted by cells that can transfer bioactive molecules, such as nucleic acids, proteins, lipids, and metabolites, to neighboring or distant cells. Recent studies have highlighted the role of sEVs in modulating different aspects of the cancer stemness properties of HCC. Furthermore, sEVs derived from diverse cellular sources, such as cancer cells, stromal cells, and immune cells, contribute to the maintenance of the cancer stemness phenotype in HCC. Through cargo transfer, specific signaling pathways are activated within the recipient cells, thus promoting the stemness properties. Additionally, sEVs can govern the secretion of growth factors from non-cancer cells to further maintain their stemness features. Clinically, plasma sEVs may hold promise as potential biomarkers for HCC diagnosis and treatment prediction. Understanding the underlying mechanisms by which sEVs promote cancer stemness in HCC is crucial, as targeting sEV-mediated communication may offer novel strategies in treatment and improve patient outcome.


Assuntos
Carcinoma Hepatocelular , Vesículas Extracelulares , Neoplasias Hepáticas , Humanos , Carcinogênese , Células-Tronco
7.
Stem Cell Reports ; 19(3): 399-413, 2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38428414

RESUMO

Degenerative bone disorders have a significant impact on global health, and regeneration of articular cartilage remains a challenge. Existing cell therapies using mesenchymal stromal cells (MSCs) have shown limited efficacy, highlighting the necessity for alternative stem cell sources. Here, we have identified and characterized MSX1+ mesenchymal progenitor cells in the developing limb bud with remarkable osteochondral-regenerative and microenvironment-adaptive capabilities. Single-cell sequencing further revealed the presence of two major cell compositions within the MSX1+ cells, where a distinct PDGFRAlow subset retained the strongest osteochondral competency and could efficiently regenerate articular cartilage in vivo. Furthermore, a strategy was developed to generate MSX1+PDGFRAlow limb mesenchyme-like (LML) cells from human pluripotent stem cells that closely resembled their mouse counterparts, which were bipotential in vitro and could directly regenerate damaged cartilage in a mouse injury model. Together, our results indicated that MSX1+PDGFRAlow LML cells might be a prominent stem cell source for human cartilage regeneration.


Assuntos
Cartilagem Articular , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Humanos , Camundongos , Animais , Células-Tronco , Terapia Baseada em Transplante de Células e Tecidos , Mesoderma , Transplante de Células-Tronco Mesenquimais/métodos , Diferenciação Celular , Fator de Transcrição MSX1/genética
8.
Int J Mol Sci ; 25(5)2024 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-38473976

RESUMO

Extracellular vesicles (EVs), a diverse group of cell-derived exocytosed particles, are pivotal in mediating intercellular communication due to their ability to selectively transfer biomolecules to specific cell types. EVs, composed of proteins, nucleic acids, and lipids, are taken up by cells to affect a variety of signaling cascades. Research in the field has primarily focused on stem cell-derived EVs, with a particular focus on mesenchymal stem cells, for their potential therapeutic benefits. Recently, tissue-specific EVs or cell type-specific extracellular vesicles (CTS-EVs), have garnered attention for their unique biogenesis and molecular composition because they enable highly targeted cell-specific communication. Various studies have outlined the roles that CTS-EVs play in the signaling for physiological function and the maintenance of homeostasis, including immune modulation, tissue regeneration, and organ development. These properties are also exploited for disease propagation, such as in cancer, neurological disorders, infectious diseases, autoimmune conditions, and more. The insights gained from analyzing CTS-EVs in different biological roles not only enhance our understanding of intercellular signaling and disease pathogenesis but also open new avenues for innovative diagnostic biomarkers and therapeutic targets for a wide spectrum of medical conditions. This review comprehensively outlines the current understanding of CTS-EV origins, function within normal physiology, and implications in diseased states.


Assuntos
Vesículas Extracelulares , Células-Tronco Mesenquimais , Neoplasias , Humanos , Vesículas Extracelulares/metabolismo , Neoplasias/metabolismo , Células-Tronco/metabolismo , Células-Tronco Mesenquimais/metabolismo , Comunicação Celular/fisiologia
9.
Cells ; 13(5)2024 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-38474399

RESUMO

Numerous reports have demonstrated the breakdown of the blood-CNS barrier (B-CNS-B) in amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease. Re-establishing barrier integrity in the CNS is critical to prevent further motor neuron degeneration from harmful components in systemic circulation. Potential therapeutic strategies for repairing the B-CNS-B may be achieved by the replacement of damaged endothelial cells (ECs) via stem cell administration or enhancement of endogenous EC survival through the delivery of bioactive particles secreted by stem cells. These cellular and noncellular approaches are thoroughly discussed in the present review. Specific attention is given to certain stem cell types for EC replacement. Also, various nanoparticles secreted by stem cells as well as other biomolecules are elucidated as promising agents for endogenous EC repair. Although the noted in vitro and in vivo studies show the feasibility of the proposed therapeutic approaches to the repair of the B-CNS-B in ALS, further investigation is needed prior to clinical transition.


Assuntos
Esclerose Amiotrófica Lateral , Doenças Neurodegenerativas , Humanos , Esclerose Amiotrófica Lateral/metabolismo , Células Endoteliais/metabolismo , Doenças Neurodegenerativas/metabolismo , Neurônios Motores/metabolismo , Células-Tronco/metabolismo
10.
Nat Commun ; 15(1): 2258, 2024 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-38480714

RESUMO

Complex biological processes, such as cellular differentiation, require intricate rewiring of intra-cellular signalling networks. Previous characterisations revealed a raised network entropy underlies less differentiated and malignant cell states. A connection between entropy and Ricci curvature led to applications of discrete curvatures to biological networks. However, predicting dynamic biological network rewiring remains an open problem. Here we apply Ricci curvature and Ricci flow to biological network rewiring. By investigating the relationship between network entropy and Forman-Ricci curvature, theoretically and empirically on single-cell RNA-sequencing data, we demonstrate that the two measures do not always positively correlate, as previously suggested, and provide complementary rather than interchangeable information. We next employ Ricci flow to derive network rewiring trajectories from stem cells to differentiated cells, accurately predicting true intermediate time points in gene expression time courses. In summary, we present a differential geometry toolkit for understanding dynamic network rewiring during cellular differentiation and cancer.


Assuntos
Neoplasias , Transdução de Sinais , Humanos , Diferenciação Celular , Neoplasias/genética , Neoplasias/metabolismo , Células-Tronco/metabolismo
11.
Methods Mol Biol ; 2783: 13-24, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38478223

RESUMO

Adipose tissue is an abundant and accessible source of stem cells with multipotent properties suitable for tissue engineering and regenerative medical applications. Adipose-derived stromal/stem cells (ASCs) have been widely used in tissue engineering and cell therapy. In addition, the clinical application of ASCs in the treatment of inflammation and injury has been proven a success. Here, we describe methods from our own laboratory and the literature for the isolation and expansion of Adipose-derived stromal/stem cells (ASCs). We present a large-scale procedure suitable for processing >100 mL volumes of lipoaspirate tissue specimens by collagenase digestion, a related procedure suitable for processing adipose tissue aspirates without digestion, and a procedure suitable for intact human adipose tissue, such as buccal fat pads in the maxillofacial region.


Assuntos
Adipócitos , Tecido Adiposo , Humanos , Células Estromais , Células-Tronco , Engenharia Tecidual/métodos , Diferenciação Celular , Células Cultivadas
12.
Methods Mol Biol ; 2783: 93-107, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38478227

RESUMO

Murine models of obesity or reduced adiposity are a valuable resource for understanding the role of adipocyte dysfunction in metabolic disorders. Adipose tissue stromal vascular cells or primary adipocytes derived from murine adipose tissue and grown in culture are essential tools for studying the mechanisms underlying adipocyte development and function. Herein, we describe methods for the isolation, expansion, and long-term storage of murine adipose-derived stromal/stem cells, along with protocols for inducing adipogenesis to white or beige adipocytes in this cell population and osteogenic differentiation. Isolation of the adipose stromal vascular fraction cells for flow cytometric analysis is also described.


Assuntos
Adipogenia , Adiposidade , Camundongos , Humanos , Animais , Citometria de Fluxo/métodos , Osteogênese , Adipócitos , Tecido Adiposo , Diferenciação Celular , Obesidade/metabolismo , Células-Tronco
13.
Methods Mol Biol ; 2783: 109-114, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38478228

RESUMO

Adipose tissue provides a valuable cell source for tissue engineering, regenerative medicine, and adipose tissue biology studies. The most widely used adipose-derived stromal/stem cells (ASCs) isolation protocol involves enzymatic digestion with collagenase. However, the yield of the method often proves to be poor if not impossible for collection of sufficient stromal vascular fraction (SVF) for expansion when the sample size is small, for instance when only newborn mice are available for cell culture. Here, we describe an efficient protocol for the isolation and expansion of ASCs using explant culture as an alternative. Briefly, adipose tissue was minced after removing excess liquid. Then, the minced tissue was placed in culture dishes or flasks. The cells will migrate out of tissue and adhere to the culture surface after one or more days.


Assuntos
Adipócitos , Tecido Adiposo , Camundongos , Animais , Células Estromais , Engenharia Tecidual/métodos , Obesidade , Células-Tronco , Diferenciação Celular
14.
Methods Mol Biol ; 2783: 195-207, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38478234

RESUMO

Adipose-derived stromal/stem cells (ASCs) and decellularized adipose tissue (DAT) are adipose tissue products obtained from individuals undergoing fat removal procedures like liposuction, lipectomy, or breast reduction. DAT hydrogel is prepared by removing the cells from the adipose tissue and digesting it to form a liquid material that forms a gel at physiological temperature. ASCs seeded on DAT have displayed osteogenic potential in vitro and in animal models of bone defects. Herein, we describe the methods for preparing DAT hydrogel, ASC seeding in DAT hydrogel, osteogenic differentiation of ASCs, creation of critical-sized femur defect model in mice, its treatment with ASC-DAT hydrogel, and analyses.


Assuntos
Hidrogéis , Osteogênese , Animais , Camundongos , Osteogênese/fisiologia , Tecido Adiposo , Adipócitos , Diferenciação Celular/fisiologia , Células-Tronco
15.
Methods Mol Biol ; 2783: 235-262, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38478237

RESUMO

Advances in technology and automation over the past several decades have made it feasible to perform high-throughput compound screening with cell spheroids, a valuable approach for drug discovery. It is entirely feasible to generate multiple 384-well plates containing adipose spheroids from cryopreserved, single-donor, adipose stem cells, thus incorporating genetic diversity into the discovery stages of research. In this protocol, we describe our method for isolating primary human adipose stem cells and synthesizing cell spheroids comprised of mature adipocytes and stromal cells. Also included are representative outcome measurements useful for characterizing adipocyte metabolism and health. Wherever possible, we describe technologies that can be used to automate characterization and increase throughput.


Assuntos
Adipócitos , Tecido Adiposo , Humanos , Tecido Adiposo/metabolismo , Adipócitos/metabolismo , Esferoides Celulares , Células Estromais , Obesidade/metabolismo , Células-Tronco/metabolismo , Diferenciação Celular
16.
Methods Mol Biol ; 2783: 177-193, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38478233

RESUMO

With the increase in decellularization of different tissue sources, an understanding of the viscoelastic properties of these soft materials is important for determining practical applications. The purpose of this chapter is to better define a series of experiments to profile important rheological properties for adipose-based hydrogels. While there are numerous mechanical characterizations that are done experimentally, the protocol outlined in this chapter provides a step-wise approach to determine the gelation characteristics and native hydrogel network properties. A more complete understanding of adipose-derived hydrogel mechanical properties would provide vital information for downstream applicability in fields such as disease modeling or soft tissue regeneration.


Assuntos
Adipócitos , Hidrogéis , Células Estromais , Cicatrização , Células-Tronco
17.
Methods Mol Biol ; 2783: 263-268, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38478238

RESUMO

Compared to two-dimensional monolayer culture, cells cultured in three-dimensional (3D) platforms provide a more biochemically and physiologically relevant environment to study cell-cell and cell-extracellular matrix interactions in vitro. Using the liquid overlay technique, a scaffold-free method to generate 3D spheroids from human adipose-derived stem cells is described.


Assuntos
Esferoides Celulares , Células-Tronco , Humanos , Tecido Adiposo , Adipócitos , Matriz Extracelular , Células Cultivadas
18.
Methods Mol Biol ; 2783: 209-220, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38478235

RESUMO

Amniotic membrane, being part of the placenta, is discarded as medical waste after childbirth. It can be decellularized to convert it into an acellular material while retaining the extracellular matrix. Such amniotic membrane grafts support stem cell adhesion, growth, and proliferation. These properties make it a useful candidate to be used as a bio-scaffold in regenerative medicine. This chapter describes a method for the decellularization of the amniotic membrane. Furthermore, the method for seeding adipose-derived stem cells on the decellularized amniotic membrane is described.


Assuntos
Âmnio , Tecidos Suporte , Adipócitos , Matriz Extracelular/metabolismo , Células-Tronco , Engenharia Tecidual/métodos
19.
Methods Mol Biol ; 2783: 309-322, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38478243

RESUMO

We have developed a hollow fiber bioreactor-based production system for manufacturing large quantities of extracellular vesicles (EVs) containing exosomes from adult human adipose-derived stromal/stem cells (ASCs). By manipulating the cellular bioreactor environment, we have found that we can alter ASC EV production, secretion, and surface protein composition. The aims of this chapter are to describe the methodology for culturing and tuning of adipose ASCs in a bioreactor, along with the collection and isolation of the EVs containing exosomes demonstrating increased HSP70 content.


Assuntos
Exossomos , Vesículas Extracelulares , Adulto , Humanos , Exossomos/metabolismo , Células Estromais , Vesículas Extracelulares/metabolismo , Adipócitos , Obesidade/metabolismo , Células-Tronco , Tecido Adiposo
20.
Methods Mol Biol ; 2783: 349-365, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38478246

RESUMO

It is critical that human adipose-derived stromal/stem cell (hASC) tissue engineering therapies possess appropriate mechanical properties in order to restore the function of the load-bearing tissues of the musculoskeletal system. In an effort to elucidate hASC response to mechanical stimulation and develop mechanically robust tissue-engineered constructs, recent research has utilized a variety of mechanical loading paradigms, including cyclic tensile strain, cyclic hydrostatic pressure, and mechanical unloading in simulated microgravity. This chapter will describe the methods for applying these mechanical stimuli to hASC to direct differentiation for functional tissue engineering of the musculoskeletal system.


Assuntos
Sistema Musculoesquelético , Ausência de Peso , Humanos , Engenharia Tecidual/métodos , Pressão Hidrostática , Diferenciação Celular , Células-Tronco , Células Cultivadas
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