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1.
Int J Mol Sci ; 22(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203724

RESUMO

Numerous studies have shown that hedgehog inhibitors (iHHs) only partially block the growth of tumor cells, especially in vivo. Leukemia often expands in a nutrient-depleted environment (bone marrow and thymus). In order to identify putative signaling pathways implicated in the adaptive response to metabolically adverse conditions, we executed quantitative phospho-proteomics in T-cell acute lymphoblastic leukemia (T-ALL) cells subjected to nutrient-depleted conditions (serum starvation). We found important modulations of peptides phosphorylated by critical signaling pathways including casein kinase, mammalian target of rapamycin, and 5'AMP-activated kinase (AMPK). Surprisingly, in T-ALL cells, AMPK signaling was the most consistently downregulated pathway under serum-depleted conditions, and this coincided with increased GLI1 expression and sensitivity to iHHs, especially the GLI1/2 inhibitor GANT-61. Increased sensitivity to GANT-61 was also found following genetic inactivation of the catalytic subunit of AMPK (AMPKα1) or pharmacological inhibition of AMPK by Compound C. Additionally, patient-derived xenografts showing high GLI1 expression lacked activated AMPK, suggesting an important role for this signaling pathway in regulating GLI1 protein levels. Further, joint targeting of HH and AMPK signaling pathways in T-ALL cells by GANT-61 and Compound C significantly increased the therapeutic response. Our results suggest that metabolic adaptation that occurs under nutrient starvation in T-ALL cells increases responsiveness to HH pathway inhibitors through an AMPK-dependent mechanism and that joint therapeutic targeting of AMPK signaling and HH signaling could represent a valid therapeutic strategy in rapidly expanding tumors where nutrient availability becomes limiting.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Hedgehog/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Transdução de Sinais , Proteínas Quinases Ativadas por AMP/genética , Morte Celular/efeitos dos fármacos , Meios de Cultura Livres de Soro/farmacologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Células Jurkat , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Piridinas/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteína GLI1 em Dedos de Zinco/metabolismo
2.
Sci Rep ; 11(1): 13159, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162924

RESUMO

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease and strongly correlates with the growing incidence of obesity and type II diabetes. We have developed a human-on-a-chip model composed of human hepatocytes and adipose tissue chambers capable of modeling the metabolic factors that contribute to liver disease development and progression, and evaluation of the therapeutic metformin. This model uses a serum-free, recirculating medium tailored to represent different human metabolic conditions over a 14-day period. The system validated the indirect influence of adipocyte physiology on hepatocytes that modeled important aspects of NAFLD progression, including insulin resistant biomarkers, differential adipokine signaling in different media and increased TNF-α-induced steatosis observed only in the two-tissue model. This model provides a simple but unique platform to evaluate aspects of an individual factor's contribution to NAFLD development and mechanisms as well as evaluate preclinical drug efficacy and reassess human dosing regimens.


Assuntos
Adipócitos/efeitos dos fármacos , Descoberta de Drogas/instrumentação , Hepatócitos/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Dispositivos Lab-On-A-Chip , Metformina/farmacologia , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Adipócitos/metabolismo , Tecido Adiposo Branco/citologia , Comunicação Celular , Células Cultivadas , Meios de Cultura/farmacologia , Meios de Cultura Livres de Soro/farmacologia , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP3A/metabolismo , Desenho de Equipamento , Ácidos Graxos/metabolismo , Ácidos Graxos/farmacologia , Glucose/farmacologia , Hepatócitos/metabolismo , Humanos , Inflamação , Insulina/farmacologia , Fator de Necrose Tumoral alfa/farmacologia
3.
Cells ; 10(4)2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33924517

RESUMO

Mesenchymal stem cells (MSCs) are of great interest for their use in cell-based therapies due to their multipotent differentiation and immunomodulatory capacities. In consequence of limited numbers following their isolation from the donor tissue, MSCs require extensive expansion performed in traditional 2D cell culture setups to reach adequate amounts for therapeutic use. However, prolonged culture of MSCs in vitro has been shown to decrease their differentiation potential and alter their immunomodulatory properties. For that reason, preservation of these physiological characteristics of MSCs throughout their in vitro culture is essential for improving the efficiency of therapeutic and in vitro modeling applications. With this objective in mind, many studies already investigated certain parameters for enhancing current standard MSC culture protocols with regard to the effects of specific culture media components or culture conditions. Although there is a lot of diversity in the final therapeutic uses of the cells, the primary stage of standard isolation and expansion is imperative. Therefore, we want to review on approaches for optimizing standard MSC culture protocols during this essential primary step of in vitro expansion. The reviewed studies investigate and suggest improvements focused on culture media components (amino acids, ascorbic acid, glucose level, growth factors, lipids, platelet lysate, trace elements, serum, and xenogeneic components) as well as culture conditions and processes (hypoxia, cell seeding, and dissociation during passaging), in order to preserve the MSC phenotype and functionality during the primary phase of in vitro culture.


Assuntos
Técnicas de Cultura de Células , Separação Celular/métodos , Meios de Cultura Livres de Soro/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Aminoácidos/química , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células Cultivadas , Meios de Cultura Livres de Soro/química , Humanos , Imunomodulação , Peptídeos e Proteínas de Sinalização Intercelular/química , Lipídeos/química , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/imunologia , Oligoelementos/química
4.
Sci Rep ; 11(1): 3403, 2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33564114

RESUMO

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are gaining increasing importance in the field of regenerative medicine. Although therapeutic value of MSCs is now being established through many clinical trials, issues have been raised regarding their expansion as per regulatory guidelines. Fetal bovine serum usage in cell therapy poses difficulties due to its less-defined, highly variable composition and safety issues. Hence, there is a need for transition from serum-based to serum-free media (SFM). Since SFM are cell type-specific, a precise analysis of the properties of MSCs cultured in SFM is required to determine the most suitable one. Six different commercially available low serum/SFM with two different seeding densities were evaluated to explore their ability to support the growth and expansion of BM-MSCs and assess the characteristics of BM-MSCs cultured in these media. Except for one of the SFM, all other media tested supported the growth of BM-MSCs at a low seeding density. No significant differences were observed in the expression of MSC specific markers among the various media tested. In contrary, the population doubling time, cell yield, potency, colony-forming ability, differentiation potential, and immunosuppressive properties of MSCs varied with one another. We show that SFM tested supports the growth and expansion of BM-MSCs even at low seeding density and may serve as possible replacement for animal-derived serum.


Assuntos
Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Técnicas de Cultura de Células , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Adolescente , Adulto , Meios de Cultura Livres de Soro/farmacologia , Feminino , Humanos , Masculino
5.
Nat Commun ; 12(1): 562, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33495467

RESUMO

Cell adhesion has tremendous impact on the function of culture platforms and implants. Cell-adhesive proteins and peptides have been extensively used for decades to promote cell adhesion, however, their application suffers from their easy enzymatic degradation, difficulty in large-scale preparation and expensiveness. To develop the next-generation cell-adhesive materials, we mimic the cell adhesion functions and mechanisms of RGD and KRSR peptides and design cell-adhesive cationic-hydrophobic amphiphilic ß-amino acid polymers that are stable upon proteolysis and easily prepared in large scale at low cost. The optimal polymer strongly promotes cell adhesion, using preosteoblast cell as a model, by following dual mechanisms that are independent of sequence and chirality of the statistic copolymer. Our strategy opens avenues in designing the next-generation cell-adhesive materials and may guide future studies and applications.


Assuntos
Aminoácidos/metabolismo , Oligopeptídeos/metabolismo , Polímeros/metabolismo , Sequência de Aminoácidos , Aminoácidos/química , Animais , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Linhagem Celular , Meios de Cultura Livres de Soro/farmacologia , Hidrogéis/química , Hidrogéis/metabolismo , Camundongos , Oligopeptídeos/química , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Espectroscopia Fotoeletrônica , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Polímeros/química , Propriedades de Superfície
6.
Cytotherapy ; 23(1): 88-99, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33097415

RESUMO

BACKGROUND AIMS: Mesenchymal stem/stromal cells (MSCs) are of interest for the treatment of graft-versus-host disease, autoimmune diseases, osteoarthritis and neurological and cardiovascular diseases. Increasing numbers of clinical trials emphasize the need for standardized manufacturing of these cells. However, many challenges related to diverse isolation and expansion protocols and differences in cell tissue sources exist. As a result, the cell products used in numerous trials vary greatly in characteristics and potency. METHODS: The authors have established a standardized culture platform using xeno- and serum-free commercial media for expansion of MSCs derived from umbilical cord (UC), bone marrow and adipose-derived (AD) and examined their functional characteristics. RESULTS: MSCs from the tested sources stably expanded in vitro and retained their biomarker expression and normal karyotype at early and later passages and after cryopreservation. MSCs were capable of colony formation and successfully differentiated into osteogenic, adipogenic and chondrogenic lineages. Pilot expansion of UC-MSCs and AD-MSCs to clinical scale revealed that the cells met the required quality standard for therapeutic applications. CONCLUSIONS: The authors' data suggest that xeno- and serum-free culture conditions are suitable for large-scale expansion and enable comparative study of MSCs of different origins. This is of importance for therapeutic purposes, especially because of the numerous variations in pre-clinical and clinical protocols for MSC-based products.


Assuntos
Técnicas de Cultura de Células/instrumentação , Meios de Cultura Livres de Soro/farmacologia , Células-Tronco Mesenquimais/fisiologia , Adipogenia , Tecido Adiposo , Adulto , Medula Óssea , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Condrogênese , Meios de Cultura Livres de Soro/metabolismo , Humanos , Osteogênese , Cordão Umbilical
7.
J Mol Neurosci ; 71(3): 565-582, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32789724

RESUMO

Parkinson's disease (PD) is a chronic neurodegenerative condition characterized by motor symptoms such as bradykinesia, resting tremor, and rigidity. PD diagnosis is based on medical history, review of signs, symptoms, neurological and physical examinations. Unfortunately, by the time the disease is diagnosed, dopamine (DA) neuronal loss is often extended, thereby resulting in ineffective therapies. Recent evidence suggests that neuroinflammation may be pivotal during PD onset and progression. However, suitable cellular models and biomarkers to detect early signs of neuroinflammation are still missing. In this study, we developed a well-differentiated DAergic neuronal cell line where we triggered a neuroinflammatory response to assess the temporal expression of the tissue- and urokinase plasminogen activators (tPA and uPA) and their endogenous inhibitor (PAI-1) along with that of pro-inflammatory mediators and the neuronal marker nNOS. Human neuroblastoma cells SH-SY5Y were differentiated into DAergic neuronal-like cells using a combination of 12-O-tetradecanoylphorbol-13-acetate (TPA) and serum depletion. Terminally-differentiated neurons were then exposed to lipopolysaccharide (LPS) for short (up to 24 h) or long term (up to 10 days) to mimic acute or chronic inflammation. Results demonstrated that uPA protein expression was stably upregulated during chronic inflammation, whereas the expression of nNOS protein better reflected the cellular response to acute inflammation. Additional studies revealed that the temporal induction of uPA was associated with increased AKT phosphorylation, but did not seem to involve cAMP-responsive element-binding protein (CREB) activation, nor the mitogen-activated protein kinase (MAPK) pathway. In conclusion, our in vitro data suggests that nNOS and uPA may serve as viable candidate biomarkers of acute and chronic neuroinflammation.


Assuntos
Técnicas de Reprogramação Celular/métodos , Neurônios Dopaminérgicos/citologia , Doença de Parkinson/metabolismo , Linhagem Celular Tumoral , Meios de Cultura Livres de Soro/farmacologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Humanos , Lipopolissacarídeos/farmacologia , Neurogênese , Óxido Nítrico Sintase Tipo I/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Acetato de Tetradecanoilforbol/farmacologia
8.
Methods Mol Biol ; 2174: 19-29, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32813242

RESUMO

Glioblastomas (GBM) are the most frequent and aggressive brain tumors due to their recurrence and resistance to current therapies. These characteristics are associated with the presence of glioma stem cells (GSCs), mainly identified by the detection of the membrane antigens CD133 and CD15. The main source of GSCs has been biopsies of tumors. However, alternatives are sought from cell lines because more homogeneous populations can be obtained with high yields. This chapter describes a method for the enrichment and characterization of GSCs from cell lines derived from human GBM by selective culture with serum-free neural stem cell medium and growth factors. The technique offers alternatives for the enrichment and characterization of GSCs, that could contribute to a better understanding of the biology of GBMs.


Assuntos
Neoplasias Encefálicas/patologia , Regulação Neoplásica da Expressão Gênica/genética , Glioblastoma/patologia , Células-Tronco Neoplásicas/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Antígeno AC133/análise , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Meios de Cultura Livres de Soro/química , Meios de Cultura Livres de Soro/farmacologia , Citometria de Fluxo , Glioblastoma/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Antígenos CD15/análise , Células-Tronco Neoplásicas/fisiologia , Células-Tronco Neurais/citologia
9.
Front Immunol ; 11: 614972, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33363548

RESUMO

Human induced Pluripotent Stem Cell (hiPSC) models are a valuable new tool for research into neurodegenerative diseases. Neuroinflammation is now recognized as a key process in neurodegenerative disease and aging, and microglia are central players in this. A plethora of hiPSC-derived microglial models have been published recently to explore neuroinflammation, ranging from monoculture through to xenotransplantation. However, combining physiological relevance, reproducibility, and scalability into one model is still a challenge. We examine key features of the in vitro microglial environment, especially media composition, extracellular matrix, and co-culture, to identify areas for improvement in current hiPSC-microglia models.


Assuntos
Técnicas de Cultura de Células , Microambiente Celular , Células-Tronco Pluripotentes Induzidas/citologia , Microglia/citologia , Modelos Biológicos , Animais , Células Cultivadas , Técnicas de Cocultura , Meios de Cultura/farmacologia , Meios de Cultura Livres de Soro/farmacologia , Xenoenxertos , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/transplante , Inflamação/imunologia , Camundongos , Microglia/efeitos dos fármacos , Doenças Neurodegenerativas/imunologia , Doenças Neurodegenerativas/patologia
10.
Int J Mol Sci ; 21(20)2020 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-33086620

RESUMO

Perinatal stem cells have been regarded as an attractive and available cell source for medical research and clinical trials in recent years. Multiple stem cell types have been identified in the human placenta. Recent advances in knowledge on placental stem cells have revealed that human amniotic epithelial stem cells (hAESCs) have obvious advantages and can be used as a novel potential cell source for cellular therapy and clinical application. hAESCs are known to possess stem-cell-like plasticity, immune-privilege, and paracrine properties. In addition, non-tumorigenicity and a lack of ethical concerns are two major advantages compared with embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). All of the characteristics mentioned above and other additional advantages, including easy accessibility and a non-invasive application procedure, make hAESCs a potential ideal cell type for use in both research and regenerative medicine in the near future. This review article summarizes current knowledge on the characteristics, therapeutic potential, clinical advances and future challenges of hAESCs in detail.


Assuntos
Âmnio/citologia , Células Epiteliais/citologia , Células-Tronco/citologia , Plasticidade Celular/efeitos dos fármacos , Meios de Cultura Livres de Soro/farmacologia , Humanos , Transplante de Células-Tronco
11.
J Biosci ; 452020.
Artigo em Inglês | MEDLINE | ID: mdl-32975235

RESUMO

Low back pain due to degeneration of intervertebral disc (IVD) is a major health problem resulting in significant disability as well as adding to the economic burden. Discectomy is a very common procedure done worldwide to relieve this pain. At present all the surgically removed disc tissue is mostly discarded. However, there are reports that state that progenitor cells in the IVD can be grown ex vivo and have the potential to be used for IVD repair and regeneration. We report here that viable cells can be harvested from surgically removed, herniated disc tissue and can be potentially used in cell based therapy. Further, we have successfully replaced xenogenic supplements such as foetal bovine serum with either autologous serum or human platelet lysate for culturing IVD cells from patient's surgically removed disc tissue, without loss of any cell characteristics, including cell surface markers, growth factor secretion in the conditioned medium and osteogenic and chondrogenic differentiation potential in vitro. The present work will not only contribute to overcoming some of the major barriers in carrying out human clinical trials, but also provide a cheap, alternate source of proteins and growth factors for growing IVD cells ex vivo for therapy.


Assuntos
Condrócitos/citologia , Misturas Complexas/farmacologia , Deslocamento do Disco Intervertebral/patologia , Disco Intervertebral/citologia , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , Antígenos CD/biossíntese , Antígenos CD/genética , Biomarcadores/metabolismo , Plaquetas/química , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Misturas Complexas/química , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/metabolismo , Meios de Cultura Livres de Soro/farmacologia , Expressão Gênica , Antígenos HLA-DR/biossíntese , Antígenos HLA-DR/genética , Humanos , Integrina alfa6/biossíntese , Integrina alfa6/genética , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/genética , Disco Intervertebral/metabolismo , Deslocamento do Disco Intervertebral/metabolismo , Deslocamento do Disco Intervertebral/cirurgia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Cultura Primária de Células/métodos
12.
Biochem Biophys Res Commun ; 532(2): 195-199, 2020 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-32859376

RESUMO

GOLPH3, an oncoprotein, plays crucial roles in tumor etiology. Compelling evidences have demonstrated that GOLPH3 contributes to regulate tumor cell growth, migration and invasion under normal nutrient condition. However, the oncogenic activity of GOLPH3 under serum starvation remains largely unknown. In this study, we reported that GOLPH3 depletion led to marked reduction in adhesion of glioma U251 cells, particularly under serum deprivation. We found that silencing of GOLPH3 expression reduced the protein amount of ITGB1 only in serum-free medium. Further insights into the mechanism between GOLPH3 and ITGB1, we applied proteasome or lysosome inhibitor to block the degradation of ITGB1, and identified GOLPH3 silencing can prompt ITGB1 lysosomal degradation under serum starvation. Finally, we found the reductions in glioma cell adhesion and ITGB1 protein amount could be rescued by ITGB1 overexpression. Taken together, these results show that GOLPH3 contributes to the adhesion of glioma cells by regulating the lysosomal degradation of ITGB1 under serum starvation.


Assuntos
Glioma/patologia , Integrina beta1/metabolismo , Proteínas de Membrana/genética , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Linhagem Celular Tumoral , Meios de Cultura Livres de Soro/farmacologia , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Humanos , Lisossomos/metabolismo , Proteínas de Membrana/metabolismo
13.
Cytotherapy ; 22(9): 511-518, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32631696

RESUMO

Cytokine-Induced (CIK) cells represent an attractive approach for cell-based immunotherapy, as they show several advantages compared with other strategies. Here we describe an original serum-free protocol for CIK cell expansion that employs G-Rex devices and compare the resulting growth, viability, phenotypic profile and cytotoxic activity with conventional culture in tissue flasks. CIK cells were obtained from buffy coats, seeded in parallel in G-Rex and tissue flasks, and stimulated with clinical-grade IFN-γ, anti-CD3 antibody and IL-2. G-Rex led to large numbers of CIK cells, with a minimal need for technical interventions, thus reducing the time and costs of culture manipulation. CIK cells generated in G-Rex showed a less differentiated phenotype, with a significantly higher expression of naive-associated markers such as CD62L, CD45RA and CCR7, which correlates with a remarkable expansion potential in culture and could lead to longer persistence and a more sustained anti-tumor response in vivo. The described procedure can be easily translated to large-scale production under Good Manufacturing Practice. Overall, this protocol has strong advantages over existing procedures, as it allows easier, time-saving and cost-effective production of CIK effector cells, fostering their clinical application.


Assuntos
Técnicas de Cultura de Células/instrumentação , Meios de Cultura Livres de Soro/farmacologia , Células Matadoras Induzidas por Citocinas/citologia , Gases/química , Morte Celular/efeitos dos fármacos , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Células Matadoras Induzidas por Citocinas/imunologia , Citotoxicidade Imunológica/efeitos dos fármacos , Humanos , Memória Imunológica/efeitos dos fármacos , Permeabilidade , Fenótipo
14.
Adv Biosyst ; 4(8): e2000008, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32700474

RESUMO

Human mesenchymal stromal cells (hMSCs) have enormous potential for the treatment of various inflammatory and degenerative diseases. Their manufacturing for cell-based therapies requires extensive ex vivo expansion and optimal growth conditions. To support cell adhesion, spreading, and growth in serum-free culture conditions, the applied plasticware needs to be functionalized with essential biochemical cues. By employing a recently developed screening tool, a chemically defined functional matrix composed of dextran sulfate and a bone-related extracellular matrix peptide is identified, which supports long-term culture of bone marrow-derived hMSCs in serum-free culture conditions. Cells grown under these conditions display rapid proliferation and high viability while maintaining their differentiation and immunomodulatory capacity, characteristic cell morphology, expression of hMSC-specific surface antigens as well as important markers of stemness and differentiation potential. The chemically defined, serum-free culture environment enables reliable and reproducible expansion of hMSCs important for cell based-therapies, drug screening, and disease modeling.


Assuntos
Materiais Biomiméticos/farmacologia , Meios de Cultura Livres de Soro/farmacologia , Sulfato de Dextrana/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Peptídeos/farmacologia , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Adesão Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Colágeno/farmacologia , Meios de Cultura Livres de Soro/química , Matriz Extracelular/química , Fibronectinas/farmacologia , Expressão Gênica , Humanos , Laminina/farmacologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Vitronectina/farmacologia
15.
Int J Mol Sci ; 21(7)2020 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-32224849

RESUMO

Dental pulp stem cells (DPSCs) have excellent proliferative properties, mineralization potential and can be easily obtained from third molar teeth. Recently, many studies have focused on isolation and differentiation of DPSCs. In our study, we focused on biological properties of non-differentiated DPSCs in comparison with osteogenic differentiated cells from DPSCs. We analyzed morphology as well as mineralization potential using three varied osteogenic differentiation media. After fifteen days of differentiation, calcium deposit production was observed in all three osteogenic differentiation media. However, only one osteogenic medium, without animal serum supplement, showed rapid and strong calcification-OsteoMAX-XF™ Differentiation Medium. Therefore, we examined specific surface markers, and gene and protein expression of cells differentiated in this osteogenic medium, and compared them to non-differentiated DPSCs. We proved a decrease in expression of CD9 and CD90 mesenchymal stem cell surface markers, as well as downregulation in the expression of pluripotency genes (NANOG and OCT-4) and increased levels of expression in osteogenic genes (ALP, BSP, OCN and RUNX2). Moreover, osteogenic proteins, such as BSP and OCN, were only produced in differentiated cells. Our findings confirm that carefully selected differentiation conditions for stem cells are essential for their translation into future clinical applications.


Assuntos
Diferenciação Celular , Técnicas de Reprogramação Celular/métodos , Polpa Dentária/citologia , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , Adulto , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Cálcio/metabolismo , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Meios de Cultura Livres de Soro/química , Meios de Cultura Livres de Soro/farmacologia , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Osteoblastos/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo
16.
Cytotherapy ; 22(6): 322-328, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32278551

RESUMO

BACKGROUND: Optimal expansion of therapeutic natural killer (NK) cell products has required media supplementation with human or fetal bovine serum, which raises safety and regulatory concerns for clinical manufacturing. Serum-free media (SFM) have been optimized for T-cell expansion, but few SFM systems have been developed for NK cells. Here, we compare six commercial clinical-grade SFM with our standard fetal bovine serum-containing medium for their ability to support NK cell expansion and function. METHODS: Human peripheral blood NK cells were expanded in selected media by recursive weekly stimulation with K562-based feeder cells expressing membrane-bound interleukin-21 and CD137L. Expansion was the primary readout, and the best-performing SFM was then compared with standard medium for cytotoxicity, phenotype, degranulation and cytokine secretion. Multiple lots were compared for consistency, and media was analyzed throughout for nutrient consumption and metabolic byproducts. RESULTS: TexMACS, OpTmizer, SCGM, ABS-001 and StemXVivo demonstrated equal or inferior NK cell expansion kinetics compared with standard medium, but expansion was markedly superior with AIM V + 5% Immune Cell Serum Replacement (ICSR; mean 5448 vs. 2621-fold expansion in 14 days). Surprisingly, NK cells expanded in AIM V + ICSR also showed increased cytotoxicity, tumor necrosis factor α secretion and DNAM-1, NKG2D, NKp30, FasL, granzyme B and perforin expression. Lot-to-lot variability was minimal. Glucose and glutamine consumption were inversely related to lactate and ammonia production. DISCUSSION: The AIM V + ICSR SFM system supports excellent ex vivo expansion of clinical-grade NK cells with the phenotype and function needed for adoptive immunotherapy.


Assuntos
Meios de Cultura Livres de Soro/farmacologia , Células Alimentadoras/efeitos dos fármacos , Células Matadoras Naturais/efeitos dos fármacos , Antígenos de Diferenciação de Linfócitos T/metabolismo , Técnicas de Cultura de Células/métodos , Proliferação de Células/efeitos dos fármacos , Meios de Cultura/química , Meios de Cultura/farmacologia , Meios de Cultura Livres de Soro/química , Citotoxicidade Imunológica , Proteína Ligante Fas/metabolismo , Humanos , Células K562 , Células Matadoras Naturais/citologia , Células Matadoras Naturais/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Receptor 3 Desencadeador da Citotoxicidade Natural/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
17.
PLoS One ; 15(4): e0231664, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32302338

RESUMO

Natural killer (NK) cells are innate lymphocytes with functions that include target cell killing, inflammation and regulation. NK cells integrate incoming activating and inhibitory signals through an array of germline-encoded receptors to gauge the health of neighbouring cells. The reactive potential of NK cells is influenced by microRNA (miRNA), small non-coding sequences that interfere with mRNA expression. miRNAs are highly conserved between species, and a single miRNA can have hundreds to thousands of targets and influence entire cellular programs. Two miRNA species, miR-155-5p and miR-146a-5p are known to be important in controlling NK cell function, but research to best understand the impacts of miRNA species within NK cells has been bottlenecked by a lack of techniques for altering miRNA concentrations efficiently and without off-target effects. Here, we describe a non-viral and straightforward approach for increasing or decreasing expression of miRNA in primary human NK cells. We achieve >90% transfection efficiency without off-target impacts on NK cell viability, education, phenotype or function. This opens the opportunity to study and manipulate NK cell miRNA profiles and their impacts on NK cellular programs which may influence outcomes of cancer, inflammation and autoimmunity.


Assuntos
Engenharia Celular/métodos , Células Matadoras Naturais/metabolismo , MicroRNAs/genética , Transfecção/métodos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Meios de Cultura Livres de Soro/farmacologia , Voluntários Saudáveis , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/transplante , MicroRNAs/agonistas , MicroRNAs/antagonistas & inibidores , Cultura Primária de Células
18.
Sci Rep ; 10(1): 3775, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111895

RESUMO

Brown adipocytes coordinate systemic energy metabolism associated with the pathogenesis of obesity and related metabolic diseases including type 2 diabetes. We have previously reported chemical compound-induced brown adipocytes (ciBAs) converted from human dermal fibroblasts without using transgenes. In this study, to reveal a precise molecular mechanism underlying the direct conversion and human adipocyte browning, we developed serum-free brown adipogenic medium (SFBAM) with an optimized chemical cocktail consisting of Rosiglitazone, Forskolin, and BMP7. During the direct conversion, treatment with BMP7 enhanced Ucp1 expression rather than the conversion efficiency in the absence of BMP signalling inhibitors. Moreover, treatment with a TGF-ß signalling pathway inhibitor was no longer required in the serum-free medium, likely because the TGF-ß pathway was already suppressed. SFBAM and the chemical cocktail efficiently converted human dermal fibroblasts into ciBAs within four weeks. The ciBAs exhibited increased mitochondrial levels, elevated oxygen consumption rate, and a response to ß-adrenergic receptor agonists. Thus the ciBAs converted by the serum-free medium and the chemical cocktail provide a novel model of human brown (beige) adipocytes applicable for basic research, drug screening, and clinical applications.


Assuntos
Adipócitos Marrons/metabolismo , Diferenciação Celular , Derme/metabolismo , Fibroblastos/metabolismo , Transdução de Sinais , Adipócitos Marrons/citologia , Proteína Morfogenética Óssea 7/química , Proteína Morfogenética Óssea 7/farmacologia , Colforsina/química , Colforsina/farmacologia , Meios de Cultura Livres de Soro/química , Meios de Cultura Livres de Soro/farmacologia , Derme/citologia , Fibroblastos/citologia , Humanos , Rosiglitazona/química , Rosiglitazona/farmacologia
19.
Food Funct ; 11(3): 2477-2488, 2020 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-32134068

RESUMO

The most significant cost driver for efficient bio-production of edible animal proteins is the cell culture media, where growth factors account for up to 96% of the total cost. The culture media must be serum-free, affordable, contain only food-grade ingredients, be efficient to promote cell growth and available in massive quantities. The commercially available serum substitutes are expensive and not necessarily food-grade. Identifying inexpensive food-safe alternatives to serum is crucial. By-products from food production are available in massive quantities, contain potential factors that can promote growth and are promising ingredients for serum replacement. The main goal of this study was to explore if food-grade by-product materials can be used as growth promoting agents in skeletal muscle cell culture to develop a tailor-made serum free media. Different by-products, including chicken carcass, cod backbone, eggshell membrane, egg white powder and pork plasma were enzymatically or chemically hydrolyzed. The hydrolysates in addition to lyophilized pork plasma and yeast extract were further characterized by size-exclusion chromatography, elemental combustion analysis and degree of hydrolysis. The materials were used as supplement to or replacement of commercial serum and further evaluated for their effect on metabolic activity, cell proliferation and cell cytotoxicity in muscle cells cultured in vitro. Our results indicate that none of the materials were cytotoxic to the skeletal muscle cells. Hydrolysates rich in peptides with approximately 2-15 amino acids in length were shown to improve cell growth and metabolic activity. Of all the materials tested pork plasma hydrolysates and yeast extract were the most promising. Pork plasma hydrolysates increased metabolic activity by 110% and cell proliferation with 48% when cultured in serum-free conditions for 3 days compared with control cells cultured with full serum conditions. Most interestingly, this response was dependent on both material and choice of enzyme used. We suggest that these materials have the potential to replace serum during cultivation and as such be included in a tailor-made serum-free media.


Assuntos
Técnicas de Cultura de Células/métodos , Meios de Cultura Livres de Soro/farmacologia , Indústria Alimentícia , Músculo Esquelético/citologia , Animais , Proliferação de Células/efeitos dos fármacos , Galinhas , Hidrólise , Suínos
20.
Biomedica ; 40(1): 72-88, 2020 03 01.
Artigo em Inglês, Espanhol | MEDLINE | ID: mdl-32220165

RESUMO

INTRODUCTION: The olfactory neuro-epithelium has an intrinsic capability of renewal during lifetime provided by the existence of globose and horizontal olfactory precursor cells. Additionally, mesenchymal stromal olfactory cells also support the homeostasis of the olfactory mucosa cell population. Under in vitro culture conditions with Dulbecco modified eagle/F12 medium supplemented with 10% fetal bovine serum, tissue biopsies from upper turbinate have generated an adherent population of cells expressing mainly mesenchymal stromal phenotypic markers. A closer examination of these cells has also found co-expression of olfactory precursors and ensheathing cell phenotypic markers. These results were suggestive of a unique property of olfactory mesenchymal stromal cells as potentially olfactory progenitor cells. OBJECTIVE: To study whether the expression of these proteins in mesenchymal stromal cells is modulated upon neuronal differentiation. MATERIALS AND METHODS: We observed the phenotype of olfactory stromal cells under DMEM/F12 plus 10% fetal bovine serum in comparison to cells from spheres induced by serum-free medium plus growth factors inducers of neural progenitors. RESULTS: The expression of mesenchymal stromal (CD29+, CD73+, CD90+, CD45-), horizontal basal (ICAM-1/CD54+, p63+, p75NGFr+), and ensheathing progenitor cell (nestin+, GFAP+) proteins was determined in the cultured population by flow cytometry. The determination of Oct 3/4, Sox-2, and Mash-1 transcription factors, as well as the neurotrophins BDNF, NT3, and NT4 by RT-PCR in cells, was indicative of functional heterogeneity of the olfactory mucosa tissue sample. CONCLUSIONS: Mesenchymal and olfactory precursor proteins were downregulated by serum-free medium and promoted differentiation of mesenchymal stromal cells into neurons and astroglial cells.


Assuntos
Células-Tronco Mesenquimais/metabolismo , Mucosa Nasal/citologia , Mucosa Olfatória/citologia , Biossíntese de Proteínas , Adipogenia , Antígenos de Diferenciação/análise , Técnicas de Cultura de Células , Diferenciação Celular , Células Cultivadas , Condrogênese , Meios de Cultura/farmacologia , Meios de Cultura Livres de Soro/farmacologia , Proteína Glial Fibrilar Ácida/biossíntese , Proteína Glial Fibrilar Ácida/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Mucosa Nasal/metabolismo , Fatores de Crescimento Neural/biossíntese , Fatores de Crescimento Neural/genética , Nestina/biossíntese , Nestina/genética , Neuroglia/metabolismo , Neurônios/metabolismo , Mucosa Olfatória/metabolismo , Osteogênese , Proteínas Recombinantes/farmacologia , Esferoides Celulares , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Conchas Nasais
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