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Paracrine signaling is challenging to study in vitro, as conventional culture tools dilute soluble factors and offer little to no spatiotemporal control over signaling. Microfluidic chips offer potential to address both of these issues. However, few solutions offer both control over onset and duration of cell-cell communication, and high throughput. We have developed a microfluidic chip designed to culture cells in adjacent chambers, separated by valves to selectively allow or prevent exchange of paracrine signals. The chip features 16 fluidic inputs and 128 individually-addressable chambers arranged in 32 sets of 4 chambers. Media can be continuously perfused or delivered by diffusion, which we model under different culture conditions to ensure normal cell viability. Immunocytochemistry assays can be performed in the chip, which we modeled and fine-tuned to reduce total assay time to 1 h. Finally, we validate the use of the chip for co-culture studies by showing that HEK293Ta cells respond to signals secreted by RAW 264.7 immune cells in adjacent chambers, only when the valve between the chambers is opened.
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Técnicas Analíticas Microfluídicas , Microfluídica , Técnicas de Cocultura , Técnicas de Cultura de Células , BioensaioRESUMO
Modular construction of an autonomous and programmable multi-functional heterogeneous biochemical circuit that can identify, transform, translate, and amplify biological signals into physicochemical signals based on logic design principles can be a powerful means for the development of a variety of biotechnologies. To explore the conceptual validity, we design a CRISPR-array-mediated primer-exchange-reaction-based biochemical circuit cascade, which probes a specific biomolecular input, transform the input into a structurally accessible form for circuit wiring, translate the input information into an arbitrary sequence, and finally amplify the prescribed sequence through autonomous formation of a signaling concatemer. This upstream biochemical circuit is further wired with a downstream electrochemical interface, delivering an integrated bioanalytical platform. We program this platform to directly analyze the genome of SARS-CoV-2 in human cell lysate, demonstrating the capability and the utility of this unique integrated system.
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Técnicas Biossensoriais/métodos , Genes Virais , SARS-CoV-2/genética , COVID-19/patologia , COVID-19/virologia , Sistemas CRISPR-Cas/genética , Linhagem Celular , Técnicas Eletroquímicas , Humanos , Técnicas de Amplificação de Ácido Nucleico , RNA Guia de Cinetoplastídeos/metabolismo , SARS-CoV-2/isolamento & purificaçãoRESUMO
An accurate, rapid, and cost-effective biosensor for the quantification of disease biomarkers is vital for the development of early-diagnostic point-of-care systems. The recent discovery of the trans-cleavage property of CRISPR typeâ V effectors makes CRISPR a potential high-accuracy bio-recognition tool. Herein, a CRISPR-Cas12a (cpf1) based electrochemical biosensor (E-CRISPR) is reported, which is more cost-effective and portable than optical-transduction-based biosensors. Through optimizing the inâ vitro trans-cleavage activity of Cas12a, E-CRIPSR was used to detect viral nucleic acids, including human papillomavirusâ 16 (HPV-16) and parvovirusâ B19 (PB-19), with a picomolar sensitivity. An aptamer-based E-CRISPR cascade was further designed for the detection of transforming growth factor ß1 (TGF-ß1) protein in clinical samples. As demonstrated, E-CRISPR could enable the development of portable, accurate, and cost-effective point-of-care diagnostic systems.
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Aptâmeros de Nucleotídeos/química , Sistemas CRISPR-Cas/genética , DNA Viral/química , Papillomavirus Humano 16/genética , Ácidos Nucleicos Imobilizados/química , Parvovirus/genética , Acidaminococcus/genética , Técnicas Biossensoriais , Clivagem do DNA , Técnicas Eletroquímicas , Eletrodos , Humanos , Limite de Detecção , Células-Tronco Mesenquimais , Sensibilidade e Especificidade , Propriedades de Superfície , Fator de Crescimento Transformador beta1/análise , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Skeleton and liver are preferred organs for cancer dissemination in metastatic melanoma negatively impacting quality of life, therapeutic success and overall survival rates. At the target organ, the local microenvironment and cell-to-cell interactions between invading and resident stromal cells constitute critical components during the establishment and progression of metastasis. Mesenchymal stem cells (MSCs) possess, in addition to their cell progenitor function, a secretory capacity based on cooperativity with other cell types in injury sites including primary tumors (PT). However, their role at the target organ microenvironment during cancer dissemination is not known. We report that local MSCs, acting as pericytes, regulate the extravasation of melanoma cancer cells (MCC) specifically to murine bone marrow (BM) and liver. Intra-arterially injected wild-type MCC fail to invade those selective organs in a genetic model of perturbed pericyte coverage of the vasculature (PDGF-B(ret/ret)), similar to CD146-deficient MCC injected into wild type mice. Invading MCC interact with resident MSCs/pericytes at the perivascular space through co-expressed CD146 and Sdf-1/CXCL12-CXCR4 signaling. Implanted engineered bone structures with MSCs/pericytes deficient of either Sdf-1/CXCL12 or CD146 become resistant to invasion by circulating MCC. Collectively, the presence of MSCs/pericytes surrounding the target organ vasculature is required for efficient melanoma metastasis to BM and liver.
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Melanoma/patologia , Células-Tronco Mesenquimais/patologia , Invasividade Neoplásica/patologia , Microambiente Tumoral/fisiologia , Animais , Neoplasias Ósseas/secundário , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Hepáticas/secundário , Camundongos , Camundongos Transgênicos , Pericitos/patologiaRESUMO
Mesenchymal stem cells (MSCs) are novel therapeutics for the treatment of Crohn's disease. However, their mechanism of action is unclear, especially in disease-relevant chronic models of inflammation. Thus, we used SAMP-1/YitFc (SAMP), a chronic and spontaneous murine model of small intestinal inflammation, to study the therapeutic effects and mechanism of action of human bone marrow-derived MSCs (hMSC). hMSC dose-dependently inhibited naïve T lymphocyte proliferation via prostaglandin E2 (PGE2) secretion and reprogrammed macrophages to an anti-inflammatory phenotype. We found that the hMSCs promoted mucosal healing and immunologic response early after administration in SAMP when live hMSCs are present (until day 9) and resulted in a complete response characterized by mucosal, histological, immunologic, and radiological healing by day 28 when no live hMSCs are present. hMSCs mediate their effect via modulation of T cells and macrophages in the mesentery and mesenteric lymph nodes (mLN). Sc-RNAseq confirmed the anti-inflammatory phenotype of macrophages and identified macrophage efferocytosis of apoptotic hMSCs as a mechanism that explains their long-term efficacy. Taken together, our findings show that hMSCs result in healing and tissue regeneration in a chronic model of small intestinal inflammation and despite being short-lived, exert long-term effects via sustained anti-inflammatory programming of macrophages via efferocytosis.
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Gelatin-/chitosan-/hyaluronan-based biomaterials are used in tissue engineering as cell scaffolds. Three gamma radiation doses (1, 10 and 25 kGy) were applied to scaffolds for sterilization. Microstructural changes of the irradiated polymers were evaluated by using scanning electron microscopy (SEM) and differential scanning calorimetry (DSC). A dose of 25 kGy produced a rough microstructure with a reduction of the porosity (from 99 to 96 %) and pore size (from 160 to 123 µm). Radiation also modified the glass transition temperature between 31.2 and 42.1 °C (1 and 25 kGy respectively). Human skin cells cultivated on scaffolds irradiated with 10 and 25 kGy proliferated at 48 h and secreted transforming growth factor ß3 (TGF-ß3). Doses of 0 kGy (non-irradiated) or 1 kGy did not stimulate TGF-ß3 secretion or cell proliferation. The specific growth rate and lactate production increased proportionally to radiation dose. The use of an appropriate radiation dose improves the cell scaffold properties of biomaterials.
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Materiais Biocompatíveis/química , Pele/citologia , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Varredura Diferencial de Calorimetria , Proliferação de Células/efeitos da radiação , Quitosana/química , Relação Dose-Resposta à Radiação , Raios gama , Gelatina/química , Humanos , Ácido Hialurônico/química , Lactatos/metabolismo , Microscopia Eletrônica de Varredura , Porosidade , Temperatura , Fator de Crescimento Transformador beta3/metabolismoRESUMO
Objective: Mesenchymal stem cells (MSCs) are novel therapeutics for treatment of Crohn's disease. However, their mechanism of action is unclear, especially in disease-relevant chronic models of inflammation. Thus, we used SAMP-1/YitFc, a chronic and spontaneous murine model of small intestinal inflammation, to study the therapeutic effect and mechanism of human bone marrow-derived MSCs (hMSC). Design: hMSC immunosuppressive potential was evaluated through in vitro mixed lymphocyte reaction, ELISA, macrophage co-culture, and RT-qPCR. Therapeutic efficacy and mechanism in SAMP were studied by stereomicroscopy, histopathology, MRI radiomics, flow cytometry, RT-qPCR, small animal imaging, and single-cell RNA sequencing (Sc-RNAseq). Results: hMSC dose-dependently inhibited naïve T lymphocyte proliferation in MLR via PGE 2 secretion and reprogrammed macrophages to an anti-inflammatory phenotype. hMSC promoted mucosal healing and immunologic response early after administration in SAMP model of chronic small intestinal inflammation when live hMSCs are present (until day 9) and resulted in complete response characterized by mucosal, histological, immunologic, and radiological healing by day 28 when no live hMSCs are present. hMSC mediate their effect via modulation of T cells and macrophages in the mesentery and mesenteric lymph nodes (mLN). Sc-RNAseq confirmed the anti-inflammatory phenotype of macrophages and identified macrophage efferocytosis of apoptotic hMSCs as a mechanism of action that explains their long-term efficacy. Conclusion: hMSCs result in healing and tissue regeneration in a chronic model of small intestinal inflammation. Despite being short-lived, exert long-term effects via macrophage reprogramming to an anti-inflammatory phenotype. Data Transparency Statement: Single-cell RNA transcriptome datasets are deposited in an online open access repository 'Figshare' (DOI: https://doi.org/10.6084/m9.figshare.21453936.v1 ).
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The production of a clinically useful engineered cartilage is an outstanding and unmet clinical need. High-throughput RNA sequencing provides a means of characterizing the molecular phenotype of populations of cells and can be leveraged to better understand differences among source cells, derivative engineered tissues, and target phenotypes. In this study, small RNA sequencing is utilized to comprehensively characterize the microRNA transcriptomes (miRNomes) of native human neonatal articular cartilage and human bone marrow-derived mesenchymal stem cells (hBM-MSCs) differentiating into cartilage organoids, contrasting the microRNA regulation of engineered cartilage with that of a promising target phenotype. Five dominant microRNAs are upregulated during cartilage organoid differentiation and disproportionately regulate transcription factors: miR-148a-3p, miR-140-3p, miR-27b-3p, miR-140-5p, and miR-181a-5p. Two microRNAs that dominate the miRNomes of hBM-MSCs, miR-21-5p and miR-143-3p, persist throughout the differentiation process and may limit the ability of these cells to differentiate into an engineered cartilage resembling target native articular cartilage. By using predictive bioinformatics tools and antagomir inhibition, these persistent microRNAs are shown to destabilize the mRNA of genes with known or potential roles in cartilage biology including FGF18, TGFBR2, TET1, STOX2, ARAP2, N4BP2L1, LHX9, NFIA, and RPS6KA5. These results shed light on the extent to which only a few microRNAs contribute to the complex regulatory environment of hBM-MSCs for engineered tissues. Impact statement MicroRNAs are emerging as important controlling elements in the differentiation of human bone marrow-derived mesenchymal stem cells (hBM-MSCs). By using a robust bioinformatic approach and further validation in vitro, here we provide a comprehensive characterization of the microRNA transcriptomes (miRNomes) of a commonly studied and clinically promising source of multipotent cells (hBM-MSCs), a gold standard model of in vitro chondrogenesis (hBM-MSC-derived cartilage organoids), and an attractive in vivo target phenotype for clinically useful engineered cartilage (neonatal articular cartilage). These analyses highlighted a specific set of microRNAs involved in the chondrogenic program that could be manipulated to acquire a more robust articular cartilage-like phenotype. This characterization provides researchers in the cartilage tissue engineering field a useful atlas with which to contextualize microRNA involvement in complex differentiation pathways.
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Cartilagem Articular , Células-Tronco Mesenquimais , MicroRNAs , Diferenciação Celular/genética , Condrogênese/genética , MicroRNAs/genética , MicroRNAs/metabolismoRESUMO
Chondrocytes are the only cell type in cartilage. The dense cartilage extracellular matrix surrounding the chondrocytes makes isolating these cells a complex and lengthy task that subjects the cells to harsh conditions. Protocols to isolate expand and maintain these cells have been improved over the years, providing ways to obtain viable cells for tissue engineering and clinical applications. Here we describe a method to obtain populations of chondrocytes that are able to expand and maintain a native-like phenotype.
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Cartilagem Articular/citologia , Técnicas de Cultura de Células , Separação Celular , Condrócitos/citologia , Separação Celular/métodos , Células Cultivadas , Humanos , Esferoides CelularesRESUMO
Chronic nontuberculous mycobacterial infections with Mycobacterium avium and Mycobacterium intracellulare complicate bronchiectasis, chronic obstructive airway disease, and the health of aging individuals. These insidious intracellular pathogens cause considerable morbidity and eventual mortality in individuals colonized with these bacteria. Current treatment regimens with antibiotic macrolides are both toxic and often inefficient at providing infection resolution. In this article, we demonstrate that human marrow-derived mesenchymal stem cells are antimicrobial and anti-inflammatory in vitro and in the context of an in vivo sustained infection of either M. avium and/or M. intracellulare.
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Anti-Infecciosos , Células-Tronco Mesenquimais , Infecção por Mycobacterium avium-intracellulare , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Humanos , Infecção por Mycobacterium avium-intracellulare/complicações , Infecção por Mycobacterium avium-intracellulare/tratamento farmacológico , Infecção por Mycobacterium avium-intracellulare/microbiologia , Micobactérias não TuberculosasRESUMO
OBJECTIVE: Cartilage is avascular and numerous studies have identified the presence of single anti- and pro-angiogenic factors in cartilage. To better understand the maintenance hyaline cartilage, we assessed the angiogenic potential of complete cartilage releasate with functional assays in vitro and in vivo. DESIGN: We evaluated the gene expression profile of angiogenesis-related factors in healthy adult human articular cartilage with a transcriptome-wide analysis generated by next-generation RNAseq. The effect on angiogenesis of the releasate of cartilage tissue was assessed with a chick chorioallantoic membrane (CAM) assay as well as human umbilical vein endothelial cell (HUVEC) migration and proliferation assays using conditioned media generated from tissue-engineered cartilage derived from human articular and nasal septum chondrocytes as well as explants from bovine articular cartilage and human nasal septum. Experiments were done with triplicate samples of cartilage from 3 different donors. RESULTS: RNAseq data of 3 healthy human articular cartilage donors revealed that the majority of known angiogenesis-related factors expressed in healthy adult articular cartilage are pro-angiogenic. The releasate from generated cartilage as well as from tissue explants, demonstrated at least a 3.1-fold increase in HUVEC proliferation and migration indicating a pro-angiogenic effect of cartilage. Finally, the CAM assay demonstrated that cartilage explants can indeed attract vessels; however, their ingrowth was not observed. CONCLUSION: Using multiple approaches, we show that cartilage releasate has an inherent pro-angiogenic capacity. It remains vessel free due to anti-invasive properties associated with the tissue itself.
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Cartilagem Articular , Membrana Corioalantoide , Adulto , Animais , Cartilagem Articular/metabolismo , Bovinos , Condrócitos/metabolismo , Membrana Corioalantoide/irrigação sanguínea , Membrana Corioalantoide/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Neovascularização Patológica/metabolismoRESUMO
Fibrin has been used extensively in cell encapsulation because it has important biological properties. Keratinocyte encapsulation in fibrin is a widely used technique in skin tissue engineering. The production of growth factors (EGF, TGF-beta1 and PDGF-BB) was evaluated when keratinocytes are encapsulated in fibrin. Secretions of TGF-beta1 and PDGF-BB increased more than five times compared to monolayer cultures. Encapsulated cells secreted about 80% active form of TGF-beta1 (monolayer cells only secreted inactive form). An enhanced secretion of TGF-beta1 and PDGF-BB was found in encapsulated cells, showing that fibrin capsules are favourable for the production of these growth factors.
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Fibrina/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Queratinócitos/metabolismo , Técnicas de Cultura de Células , Células Cultivadas , HumanosRESUMO
Mesenchymal stem cells (MSCs) are at the forefront as therapeutic tools for an extensive number of tissue engineering and regenerative medicine applications. MSC differentiation properties have been extensively studied in vitro by this laboratory and many others. The generation and validation of in vivo potency assays would be a valuable tool for the study of cellular properties relevant for in vivo applications. We have developed a unique system, we call the Habitat assay, in which porous ceramic cube carrier loaded with human bone marrow (BM)-MSCs (hMSCs) is subcutaneously implanted into immune-compromised mice. These cells have the capacity to create bone tissue and reconstitute the hematopoietic microenvironment within the "Habitat." These donor-derived hMSCs form bone structures by 3-4 weeks and associate as perivascular MSCs. In this study, we have extensively analyzed data generated with the habitat (ceramic cube in vivo assay) using cells derived from 117 hMSC-donors (iliac aspiration); this analysis provides a validation of the platform as a way to study the in vivo effect of several variables involved in the generation of the bony Habitat. These studies show that passage number and the age of the hMSC donor influence the sequence of in vivo bone formation within the Habitat. These variables have been shown to have an effect on in vitro properties of MSCs; in this study, for the first time, we show these effects to be important on an in vivo setting.
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Células-Tronco Mesenquimais , Osteogênese , Alicerces Teciduais , Animais , Células da Medula Óssea , Diferenciação Celular , Cerâmica , Xenoenxertos , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Engenharia TecidualRESUMO
The engineering of a native-like articular cartilage (AC) is a long-standing objective that could serve the clinical needs of millions of patients suffering from osteoarthritis and cartilage injury. An incomplete understanding of the developmental stages of AC has contributed to limited success in this endeavor. Using next generation RNA sequencing, we have transcriptionally characterized two critical stages of AC development in humans-that is, immature neonatal and mature adult, as well as tissue-engineered cartilage derived from culture expanded human mesenchymal stem cells. We identified key transcription factors (TFs) and long noncoding RNAs (lncRNAs) as candidate drivers of the distinct phenotypes of these tissues. AGTR2, SCGB3A1, TFCP2L1, RORC, and TBX4 stand out as key TFs, whose expression may be capable of reprogramming engineered cartilage into a more expandable and neonatal-like cartilage primed for maturation into biomechanically competent cartilage. We also identified that the transcriptional profiles of many annotated but poorly studied lncRNAs were dramatically different between these cartilages, indicating that lncRNAs may also be playing significant roles in cartilage biology. Key neonatal-specific lncRNAs identified include AC092818.1, AC099560.1, and KC877982. Collectively, our results suggest that tissue-engineered cartilage can be optimized for future clinical applications by the specific expression of TFs and lncRNAs.
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Cartilagem/patologia , Células-Tronco Mesenquimais/citologia , RNA Longo não Codificante/metabolismo , Engenharia Tecidual/métodos , Fatores de Transcrição/metabolismo , Transcriptoma , Adulto , Cartilagem Articular/citologia , Diferenciação Celular , Condrócitos/citologia , Condrogênese , Feminino , Humanos , Lactente , Masculino , Osteoartrite/metabolismo , Fenótipo , RNA/metabolismo , Transcrição Gênica , Adulto JovemRESUMO
With rising demand for cartilage tissue repair and replacement, the differentiation of mesenchymal stem cells (BMSCs) into cartilage tissue forming cells provides a promising solution. Often, the BMSC-derived cartilage does not remain stable and continues maturing to bone through the process of endochondral ossification in vivo. Similar to the growth plate, invasion of blood vessels is an early hallmark of endochondral ossification and a necessary step for completion of ossification. This invasion originates from preexisting vessels that expand via angiogenesis, induced by secreted factors produced by the cartilage graft. In this study, we aimed to identify factors secreted by chondrogenically differentiated bone marrow-derived human BMSCs to modulate angiogenesis. The secretome of chondrogenic pellets at day 21 of the differentiation program was collected and tested for angiogenic capacity using in vitro endothelial migration and proliferation assays as well as the chick chorioallantoic membrane (CAM) assay. Taken together, these assays confirmed the pro-angiogenic potential of the secretome. Putative secreted angiogenic factors present in this medium were identified by comparative global transcriptome analysis between murine growth plate cartilage, human chondrogenic BMSC pellets and human neonatal articular cartilage. We then verified by PCR eight candidate angiogenesis modulating factors secreted by differentiated BMSCs. Among those, Serpin E1 and Indian Hedgehog (IHH) had a higher level of expression in BMSC-derived cartilage compared to articular chondrocyte derived cartilage. To understand the role of these factors in the pro-angiogenic secretome, we used neutralizing antibodies to functionally block them in the conditioned medium. Here, we observed a 1.4-fold increase of endothelial cell proliferation when blocking IHH and 1.5-fold by Serpin E1 blocking compared to unblocked control conditioned medium. Furthermore, endothelial migration was increased 1.9-fold by Serpin E1 blocking and 2.7-fold by IHH blocking. This suggests that the pro-angiogenic potential of chondrogenically differentiated BMSC secretome could be further augmented through inhibition of specific factors such as IHH and Serpin E1 identified as anti-angiogenic factors.
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Mesenchymal stem cells (MSCs) are promising cells to treat cartilage defects due to their chondrogenic differentiation potential. However, an inflammatory environment during differentiation, such as the presence of the cytokine TNFα, inhibits chondrogenesis and limits the clinical use of MSCs. On the other hand, it has been reported that exposure to TNFα during in vitro expansion can increase proliferation, migration, and the osteogenic capacity of MSCs and therefore can be beneficial for tissue regeneration. This indicates that the role of TNFα on MSCs may be dependent on the differentiation stage. To improve the chondrogenic capacity of MSCs in the presence of an inflamed environment, we aimed to determine the effect of TNFα on the chondrogenic differentiation capacity of MSCs. Here, we report that TNFα exposure during MSC expansion increased the chondrogenic differentiation capacity regardless of the presence of TNFα during chondrogenesis and that this effect of TNFα during expansion was reversed upon TNFα withdrawal. Interestingly, pre-treatment with another pro-inflammatory cytokine, IL-1ß, did not increase the chondrogenic capacity of MSCs indicating that the pro-chondrogenic effect is specific for TNFα. Finally, we show that TNFα pre-treatment increased the levels of SOX11 and active ß-catenin suggesting that these intracellular effectors may be useful targets to improve MSC-based cartilage repair. Overall, these results suggest that TNFα pre-treatment, by modulating SOX11 levels and WNT/ß-catenin signaling, could be used as a strategy to improve MSC-based cartilage repair.
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The chondrogenic differentiation of mesenchymal stem cells (MSCs) is mediated by transcription factors and small noncoding RNAs such as microRNAs (miRNAs). Each miRNA is initially transcribed as a long transcript, which matures to produce -5p and -3p strands. It is widely believed that the mature and functional miRNA from any given pre-miRNA, usually the -5p strand, is functional, while the opposing -3p strand is degraded. However, recent cartilage literature started to show functional -3p strands for a few miRNAs. This study aimed at examining both -5p and -3p strands of two key miRNAs miR-140 and miR-145, known to be involved in the chondrogenic differentiation of MSCs. The level (copy number) of both -5p and -3p strands of miR-145 and miR-140 along the time line of MSC chondrogenic differentiation was determined by polymerase chain reaction. The gene expression profiles of several genes related to MSC chondrogenesis were compared with these miRNA profiles along the same timeline. While miR-145-3p is declining in step with miR-145-5p in pellet cultures during the process, the -3p strand is only 1-2% of the total miR-145 products. In contrast, the mature -3p and -5p products of miR-140 are found to increase with near-equal molar expression throughout chondrogenic differentiation. Numerous genes are expressed by cartilage progenitor cells during development. One such target gene, Sox9, is a regulatory target of the dominant miR-145-5p, consistent with the data. Further experimental validations are warranted to confirm that ACAN, FOXO1, and RUNX3 as direct targets of miR-145-5p in the context of MSC chondrogenesis. Similarly, TRSP1 and ACAN are worth further validation as direct targets of miR-145-3p. For miR-140, SOX4 shall be further validated as a direct target of miR-140-5p, while KLF4, PTHLH, and WNT5A can be validated as direct targets of miR-140-3p.
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Diferenciação Celular , Condrogênese , Regulação da Expressão Gênica , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/metabolismo , Perfilação da Expressão Gênica , Humanos , Fator 4 Semelhante a Kruppel , Células-Tronco Mesenquimais/citologia , MicroRNAs/genéticaRESUMO
Chondrogenic cell differentiation constitutes a multistep program that is spatially and temporally modulated by combinations of bioactive factors that drives the establishment of specific cellular phenotypes. This sequence of events results in the fabrication of a distinctive structural and functional extracellular matrix which determines the quality of the cartilaginous tissue and, thus, its potential in vivo implantability as a tissue-engineered implant. Current assessments of engineered cartilage rely on destructive methodologies typically applied at the end of the fabrication period that make it difficult to predict failures early in the process. The high inherent variability of engineered tissues raises questions regarding reproducibility and the validity of using such end-stage representative samples to characterize an entire batch of engineered tissues. Therefore, the development of dynamic, multimodal, nondestructive, and noninvasive technology toolsets to monitor cell differentiation (and secondarily tissue phenotypes) in real time is of paramount importance. In this study, we report the creation of cell-based probes to directly interrogate cell differentiation events during in vitro chondrogenesis and in vivo osteogenesis. For that, native promoters of well-established chondrogenic (Sex Determining Region Y-Box 9 [Sox9] and Aggrecan [AGG]) and osteogenic (Osteocalcin [OC]) differentiation biomarkers were used to create independent probes incorporating a traceable signal (Luciferase) and transduced into human bone marrow-derived mesenchymal stem cells. The probes were used to monitor the progression throughout in vitro chondrogenic differentiation program in aggregate (pellet) cultures and in vivo osteogenic differentiation in heterotopic ossicles. These tissue differentiation constructs were positively tested in conditions known to modulate the differentiation program at various phases that confirmed their sensitivity and reproducibility. This technology toolset allows a nondestructive and noninvasive, imaging-based longitudinal reconstruction of the in vitro chondrogenic differentiation program, while providing an analytical assessment of phenotypic changes of engineered cartilage in real time.
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Células da Medula Óssea/citologia , Diferenciação Celular/fisiologia , Condrogênese/fisiologia , Diagnóstico por Imagem/métodos , Fibroblastos/citologia , Células-Tronco Mesenquimais/citologia , Osteogênese/fisiologia , Células da Medula Óssea/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Condrogênese/genética , Fibroblastos/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Osteogênese/genéticaRESUMO
Regenerative medicine and tissue engineering studies are actively developing novel means to repair adult articular cartilage defects using biological approaches. One such approach is the harnessing of adult human therapeutic cells such as those referred to as mesenchymal stem cells. Upon exposure to chondrogenic signals, these cells differentiate and initiate the production of a complex and voluminous cartilaginous matrix that is crucial to both the structure and function of cartilage. Furthermore, this complexity requires the time-sensitive activation of a large number of genes to produce the components of this matrix. The current study analyzed the kinetics of matrix production in an aggregate culture model where adult human mesenchymal stem cells were induced to differentiate as chondrocytes. The results indicate the existence of a biphasic mode of differentiation and maturation during which matrix genes and molecules are differentially activated and secreted. These results have important implications for developing novel approaches for the creation of tissue engineered articular cartilage. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1757-1766, 2018.
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Condrócitos/citologia , Matriz Extracelular/metabolismo , Células-Tronco Mesenquimais/citologia , Agregação Celular , Diferenciação Celular , Colágeno/biossíntese , Humanos , Proteoglicanas/biossínteseRESUMO
Tissue engineering requires large numbers of cells with enhanced differentiation properties. Thus, the effect of expansion conditions must be explored. Human and rat marrow-derived mesenchymal stem cells (hMSCs and rMSCs, respectively) were comparatively culture expanded through seven passages in the presence of either fibroblast growth factor-2 (FGF-2) or platelet-derived growth factor BB (PDGF-BB). Proliferation of both hMSCs and rMSCs was enhanced by FGF-2 and PDGF-BB. Population doubling times for hMSCs were 2.4 days for control and 1.75 and 2.0 days for FGF-2 and PDGF-BB, respectively, and 3.25, 3.06, and 2.95 days for rMSCs. Supplementation with FGF-2 during cell expansion resulted in significantly greater in vivo bone formation for hMSCs. Use of PDGF-BB resulted in greater bone formation than that observed for control conditions, but the differences were only significant for P1. For rMSCs, significant increases in bone formation were noted in either FGF-2 or PDGF-BB expanded cells implanted at P4 or P7, but not for P1. Under in vitro osteogenic stimulation, calcium content was elevated and bone matrix deposition was enhanced for P1 and P7 rMSCs expanded with FGF-2. Although culture conditions, including FBS, were held constant, these observations suggest that medium must be optimized separately for each species of MSCs.