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1.
Bioact Mater ; 35: 291-305, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38370866

RESUMO

Mesenchymal stem cell (MSC) transplantation has been explored for the clinical treatment of various diseases. However, the current two-dimensional (2D) culture method lacks a natural spatial microenvironment in vitro. This limitation restricts the stable establishment and adaptive maintenance of MSC stemness. Using natural polymers with biocompatibility for constructing stereoscopic MSC microenvironments may have significant application potential. This study used chitin-based nanoscaffolds to establish a novel MSC three-dimensional (3D) culture. We compared 2D and 3D cultured human umbilical cord-derived MSCs (UCMSCs), including differentiation assays, cell markers, proliferation, and angiogenesis. When UCMSCs are in 3D culture, they can differentiate into bone, cartilage, and fat. In 3D culture condition, cell proliferation is enhanced, accompanied by an elevation in the secretion of paracrine factors, including vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), Interleukin-6 (IL-6), and Interleukin-8 (IL-8) by UCMSCs. Additionally, a 3D culture environment promotes angiogenesis and duct formation with HUVECs (Human Umbilical Vein Endothelial Cells), showing greater luminal area, total length, and branching points of tubule formation than a 2D culture. MSCs cultured in a 3D environment exhibit enhanced undifferentiated, as well as higher cell activity, making them a promising candidate for regenerative medicine and therapeutic applications.

2.
Biotechnol Lett ; 45(10): 1265-1277, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37606752

RESUMO

OBJECTIVES: Gene therapy using viral vectors and antibody-based therapies continue to expand within the pharmaceutical market. We evaluated whether Cellhesion® VP, a chitin-based material, can be used as 3D culture platform for cell lines used for the production of antibodies and viral vectors. RESULTS: The results of Cell Counting Kit-8 assay and LDH assay revealed that Cellhesion® VP had no adverse effect to Human Embryonic Kidney (HEK) 293, A549 and Chinese hamster ovary (CHO) DG44-Interferon-ß (IFN) cells. Cell growth analyses showed that Cellhesion® VP supported the 3D culture of HEK293, A549 and CHO DG44- IFN-ß cells with a spherical morphology. Importantly, subculture of these cell lines on Cellhesion® VP was easily performed without trypsinization because cells readily transferred to newly added scaffold. Our data also suggest that CHO DG44-IFNß, cultured on Cellhesion® VP secreted IFNß stably and continuously during the culture period. CONCLUSIONS: Cellhesion® VP provides a simple and streamlined expansion culture system for the production of biopharmaceuticals.


Assuntos
Produtos Biológicos , Animais , Cricetinae , Humanos , Células HEK293 , Quitina , Células CHO , Cricetulus , Técnicas de Cultura de Células
3.
Cells ; 11(6)2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-35326446

RESUMO

Mesenchymal stem cell (MSC) transplantation, in particular allogeneic transplantation, is a promising therapy for a variety of diseases. However, before performing allograft treatment it is necessary to find suitable donors, establish culture methods that maintain cell quality, and reduce cell production costs. Here, we present a new method of producing allogeneic MSCs combining human umbilical cord-derived mesenchymal stem cells (UCMSCs) and chitin-based polysaccharide fibers (Cellhesion® MS). UCMSC numbers significantly increased, and cells grew as dispersed spheres on Cellhesion® MS. Subsequent biological analyses showed that the expression levels of stemness-related and migration-related genes were significantly upregulated, including octamer-binding transcription factor 4 (OCT4), Nanog homeobox (NANOG), and C-X-C chemokine receptor type 4 (CXCR4). The secretion levels of paracrine factors such as prostaglandin E2 (PGE2), TNFα-stimulating gene (TSG)-6, fibroblast growth factor 2 (bFGF), and Angiogenin (Ang) from UCMSCs using Cellhesion® MS were significantly higher than with microcarrier and U-bottom plate culture. In addition, culture supernatant from UCMSCs with Cellhesion® MS had better angiogenic potential than that from monolayer cultured UCMSCs. Furthermore, we succeeded in a scaled-up culture of UCMSCs with Cellhesion® MS using a closed culture bag. Therefore, Cellhesion® MS is a key material for producing high-quality UCMSCs in a three-dimensional (3D) culture system.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Quitina/metabolismo , Quitina/farmacologia , Humanos , Células-Tronco Mesenquimais/metabolismo , Cordão Umbilical
4.
Sci Rep ; 11(1): 13471, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34188113

RESUMO

Extracellular vesicles (EVs) are secreted from most cells and play important roles in cell-cell communication by transporting proteins, lipids, and nucleic acids. As the involvement of EVs in diseases has become apparent, druggable regulators of EV secretion are required. However, the lack of a highly sensitive EV detection system has made the development of EV regulators difficult. We developed an ELISA system using a high-affinity phosphatidylserine-binder TIM4 to capture EVs and screened a 1567-compound library. Consequently, we identified one inhibitor and three activators of EV secretion in a variety of cells. The inhibitor, apoptosis activator 2, suppressed EV secretion via a different mechanism and had a broader cellular specificity than GW4869. Moreover, the three activators, namely cucurbitacin B, gossypol, and obatoclax, had broad cellular specificity, including HEK293T cells and human mesenchymal stem cells (hMSCs). In vitro bioactivity assays revealed that some regulators control EV secretion from glioblastoma and hMSCs, which induces angiogenesis and protects cardiomyocytes against apoptosis, respectively. In conclusion, we developed a high-throughput method to detect EVs with high sensitivity and versatility, and identified four compounds that can regulate the bioactivity of EVs.


Assuntos
Apoptose/efeitos dos fármacos , Vesículas Extracelulares/metabolismo , Células-Tronco Mesenquimais/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Avaliação Pré-Clínica de Medicamentos , Células HCT116 , Células HEK293 , Humanos , Células Jurkat , Células K562 , Camundongos , Células NIH 3T3 , Células THP-1
5.
Biomaterials ; 271: 120742, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33706111

RESUMO

Mesenchymal stem cell (MSC) transplantation is a promising therapy for regenerative medicine. However, MSCs grown under two-dimensional (2D) culture conditions differ significantly in cell shape from those in the body, with downregulated stemness genes and secretion of paracrine factors. Here, we evaluated the effect of 3D culture using Cellhesion VP, a water-insoluble material composed of chitin-based polysaccharide fibers, on the characteristics of human Wharton's jelly-derived MSCs (hMSCs). Cellhesion VP significantly increased cell proliferation after retrieval. Transcriptome analyses suggested that genes involved in cell stemness, migration ability, and extracellular vesicle (EV) production were enhanced by 3D culture. Subsequent biochemical analyses showed that the expression levels of stemness genes including OCT4, NANOG, and SSEA4 were upregulated and migration capacity was elevated in 3D-cultured hMSCs. In addition, EV production was significantly elevated in 3D cells, which contained a distinct protein profile from 2D cells. Gene and drug connectivity analyses revealed that the 2D and 3D EVs had similar functions as immunomodulators; however, 3D EVs had completely distinct therapeutic profiles for various infectious and metabolic diseases based on activation of disease-associated signaling pathways. Therefore, EVs from Cellhesion VP-primed hMSCs offer a new treatment for immune and metabolic diseases.


Assuntos
Vesículas Extracelulares , Células-Tronco Mesenquimais , Geleia de Wharton , Humanos , Fatores Imunológicos , Medicina Regenerativa
6.
Pharm Res ; 28(10): 2467-76, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21562928

RESUMO

PURPOSE: Peroxisome proliferator-activated receptor α (PPARα) is an important transcriptional factor that regulates genes encoding endo/xenobiotic enzymes and lipid metabolizing enzymes. In this study, we investigated whether microRNAs (miRNAs) are involved in the regulation of PPARα in human liver. METHODS: Precursor or antisense oligonucleotide for miR-21 or miR-27b was transfected into HuH7 cells; expression of PPARα and acyl-CoA synthetase M2B was determined by Western blot and real-time RT-PCR. Luciferase assay was performed to identify the functional miRNA recognition element (MRE). Expression levels of PPARα, miR-21, and miR-27b in a panel of 24 human livers were determined. RESULTS: The overexpression and inhibition of miR-21 or miR-27b in HuH7 cells significantly decreased and increased the PPARα protein level, respectively, but not PPARα mRNA level. The miRNA-dependent regulation of PPARα affected the expression of its downstream gene. Luciferase assay identified a functional MRE for miR-21 in the 3'-untranslated region of PPARα. In human livers, the PPARα protein levels were not correlated with PPARα mRNA, but inversely correlated with the miR-21 levels, suggesting a substantial impact of miR-21, although the contribution of miR-27b could not be ruled out. CONCLUSIONS: We found that PPARα in human liver is regulated by miRNAs.


Assuntos
Regulação da Expressão Gênica , Fígado/fisiologia , MicroRNAs/genética , PPAR alfa/genética , Regiões 3' não Traduzidas , Linhagem Celular Transformada , Linhagem Celular Tumoral , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Fígado/metabolismo , MicroRNAs/metabolismo , PPAR alfa/metabolismo , RNA Mensageiro/genética , Transfecção
7.
J Biol Chem ; 285(7): 4415-22, 2010 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-20018894

RESUMO

Hepatocyte nuclear factor (HNF) 4alpha is a key transcription factor regulating endo/xenobiotic-metabolizing enzymes and transporters. We investigated whether microRNAs are involved in the regulation of human HNF4alpha. Potential recognition elements for miR-24 (MRE24) were identified in the coding region and the 3'-untranslated region (3'-UTR), and those for miR-34a (MRE34a) were identified in the 3'-UTR in HNF4alpha mRNA. The HNF4alpha protein level in HepG2 cells was markedly decreased by the overexpression of miR-24 and miR-34a. The HNF4alpha mRNA level was significantly decreased by the overexpression of miR-24 but not by miR-34a. In luciferase analyses in HEK293 cells, the reporter activity of plasmid containing the 3'-UTR of HNF4alpha was significantly decreased by miR-34a. The reporter activity of plasmid containing the HNF4alpha coding region downstream of the luciferase gene was significantly decreased by miR-24. These results suggest that the MRE24 in the coding region and MRE34a in the 3'-UTR are functional in the negative regulation by mRNA degradation and translational repression, respectively. The down-regulation of HNF4alpha by these microRNAs resulted in the decrease of various target genes such as cytochrome P450 7A1 and 8B1 as well as morphological changes and the decrease of the S phase population in HepG2 cells. We also clarified that the expressions of miR-24 and miR-34a were regulated by protein kinase C/mitogen-activated protein kinase and reactive oxygen species pathways, respectively. In conclusion, we found that human HNF4alpha was down-regulated by miR-24 and miR-34a, the expression of which are regulated by cellular stress, affecting the metabolism and cellular biology.


Assuntos
Ciclo Celular/fisiologia , Fator 4 Nuclear de Hepatócito/metabolismo , MicroRNAs/fisiologia , Regiões 3' não Traduzidas/genética , Western Blotting , Ciclo Celular/genética , Eletroforese em Gel de Poliacrilamida , Células Hep G2 , Fator 4 Nuclear de Hepatócito/genética , Humanos , MicroRNAs/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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