RESUMEN
Currently, various functionalized nanocarrier systems are extensively studied for targeted delivery of drugs, peptides, and nucleic acids. Joining the approaches of genetic and chemical engineering may produce novel carriers for precise targeting different cellular proteins, which is important for both therapy and diagnosis of various pathologies. Here we present the novel nanocontainers based on vectorized genetically encoded Myxococcus xanthus (Mx) encapsulin, confining a fluorescent photoactivatable mCherry (PAmCherry) protein. The shells of such encapsulins were modified using chemical conjugation of human transferrin (Tf) prelabeled with a fluorescein-6 (FAM) maleimide acting as a vector. We demonstrate that the vectorized encapsulin specifically binds to transferrin receptors (TfRs) on the membranes of mesenchymal stromal/stem cells (MSCs) followed by internalization into cells. Two spectrally separated fluorescent signals from Tf-FAM and PAmCherry are clearly distinguishable and co-localized. It is shown that Tf-tagged Mx encapsulins are internalized by MSCs much more efficiently than by fibroblasts. It has been also found that unlabeled Tf effectively competes with the conjugated Mx-Tf-FAM formulations. That indicates the conjugate internalization into cells by Tf-TfR endocytosis pathway. The developed nanoplatform can be used as an alternative to conventional nanocarriers for targeted delivery of, e.g., genetic material to MSCs.
Asunto(s)
Células Madre Mesenquimatosas , Myxococcus xanthus , Transferrina , Células Madre Mesenquimatosas/metabolismo , Transferrina/metabolismo , Humanos , Myxococcus xanthus/metabolismo , Endocitosis , Receptores de Transferrina/metabolismo , Proteínas Luminiscentes/metabolismo , Proteínas Luminiscentes/genéticaRESUMEN
Cell sheet (CS) engineering using mesenchymal stromal cells (MSC) draws significant interest for regenerative medicine and this approach translates to clinical use for numerous indications. However, little is known of factors that define the timing of CS assembly from primary cultures. This aspect is important for planning CS delivery in autologous and allogeneic modes of use. We used a comparative in vitro approach with primary donors' (n = 14) adipose-derived MSCs and evaluated the impact of healthy subject's sex, MSC culture features (population doubling time and lag-phase), and extracellular matrix (ECM) composition along with factors related to connective tissue formations (α-SMA and FAP-α) on CS assembly duration. Using qualitative and quantitative analysis methods, we found that, in seeded MSCs, high contents of collagen I and collagen IV had a direct correlation with longer CS assembly duration. We found that short lag-phase cultures faster turned to a ready-to-use CS, while age, sex, fibronectin, laminin, α-SMA, and FAP-α failed to provide a significant correlation with the timing of assembly. In detachable CSs, FAP-α was negatively correlated with the duration of assembly, suggesting that its concentration rose over time and contributed to MSC activation, transitioning to α-SMA-positive myofibroblasts and ECM turnover. Preliminary data on cell density and collagen I deposition suggested that the TGF-ß1 signaling axis is of pivotal importance for ECM composition and construct maturation.
Asunto(s)
Matriz Extracelular , Células Madre Mesenquimatosas , Humanos , Células Cultivadas , Matriz Extracelular/fisiología , Colágeno Tipo I , Colágeno Tipo IV , Diferenciación CelularRESUMEN
The development of tissue fibrosis is a complex process involving the interaction of multiple cell types, which makes the search for antifibrotic agents rather challenging. So far, myofibroblasts have been considered the key cell type that mediated the development of fibrosis and thus was the main target for therapy. However, current strategies aimed at inhibiting myofibroblast function or eliminating them fail to demonstrate sufficient effectiveness in clinical practice. Therefore, today, there is an unmet need to search for more reliable cellular targets to contribute to fibrosis resolution or the inhibition of its progression. Activated stromal cells, capable of active proliferation and invasive growth into healthy tissue, appear to be such a target population due to their more accessible localization in the tissue and their high susceptibility to various regulatory signals. This subpopulation is marked by fibroblast activation protein alpha (FAPα). For a long time, FAPα was considered exclusively a marker of cancer-associated fibroblasts. However, accumulating data are emerging on the diverse functions of FAPα, which suggests that this protein is not only a marker but also plays an important role in fibrosis development and progression. This review aims to summarize the current data on the expression, regulation, and function of FAPα regarding fibrosis development and identify promising advances in the area.
Asunto(s)
Fibroblastos , Serina Endopeptidasas , Humanos , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Fibroblastos/metabolismo , Gelatinasas/metabolismo , Fibrosis , Células del Estroma/metabolismoRESUMEN
The aim of the study was to investigate the relationship between established clinical systemic biomarkers of ageing and the development of age-associated diseases and senescent cell biomarkers at tissue and cellular levels. Thirty-eight patients (mean age 70 ± 4.9 years) who were assessed for traditional risk factors for cardiovascular diseases were included. From all patients we obtained biomaterials (peripheral blood, skin, subcutaneous fatty tissue) and isolated different cell types (peripheral blood mononuclear cells (PBMC), fibroblasts (FB) and mesenchymal stem/stromal cells (MSC)). Isolated cells were analyzed using several senescent cells biomarkers such as telomere length and telomerase activity, proliferation rate, cell cycle inhibitor expression (p16 and p21), b-galactosidase activity, gH2AX expression. CD34+ cell content in peripheral blood was determined by flow cytometry. Systemic senescent cell-associated factors (insulin-like growth factor 1 (IGF-1), fibroblast growth factor 21 (FGF-21), osteoprogerin, ferritin, soluble vascular cell adhesion molecule (VCAM-1), intercellular adhesion molecule 1 (ICAM-1)) in peripheral blood as well as senescence-associated secretory phenotype (SASP) components in MSC and FB secretome were evaluated by ELISA. Skin and adipose tissue biopsy samples were analyzed histologically to assess senescent cell markers. A strong significant association of tissue p16 expression with age (r = 0.600, p < 0.001), pulse wave velocity (PWV) (r = 0.394, p = 0.015), vascular cell adhesion molecule (VCAM-1) content (r = 0.312, p = 0.006) in the systemic blood stream and p16 mRNA level in the blood mononuclear cells (MNCs) (r = 0.380, p = 0.046) were confirmed by correlation analysis. Statistically significant correlations were found with indicators of FBs and MSCs proliferation in culture and acquisition of SASP by the cells. Thus, p16 expression in tissues correlated significantly with interleukin-6 (IL-6) (r = 0.485, p < 0.05) and monocyte chemoattractant protein type 1 (MCP-1) (r = 0.372, p < 0.05) secretion by isolated cells. The results of regression analysis confirmed that, regardless of age, the expression of p16 was associated with the proliferation of isolated cells and IL-6 within SASP. Based on these findings, two models have been proposed to predict the level of p16 expression in tissues from the levels of other markers of senescent cell accumulation determined by non-invasive methods and available in clinical practice.
Asunto(s)
Senescencia Celular , Molécula 1 de Adhesión Celular Vascular , Senescencia Celular/genética , Leucocitos Mononucleares/metabolismo , Interleucina-6 , Análisis de la Onda del Pulso , Biomarcadores/metabolismo , Células CultivadasRESUMEN
We report a comparative study of multipotent mesenchymal stromal cells (MSC) delivered by injection, MSC-based cell sheets (CS) or MSC secretome to induce healing of cutaneous pressure ulcer in C57Bl/6 mice. We found that transplantation of CS from adipose-derived MSC resulted in reduction of fibrosis and recovery of skin structure with its appendages (hair and cutaneous glands). Despite short retention of CS on ulcer surface (3-7 days) it induced profound changes in granulation tissue (GT) structure, increasing its thickness and altering vascularization pattern with reduced blood vessel density and increased maturation of blood vessels. Comparable effects on GT vascularization were induced by MSC secretome, yet this treatment has failed to induce repair of skin with its appendages we observed in the CS group. Study of secretome components produced by MSC in monolayer or sheets revealed that CS produce more factors involved in pericyte chemotaxis and blood vessel maturation (PDGF-BB, HGF, G-CSF) but not sprouting inducer (VEGF165). Analysis of transcriptome using RNA sequencing and Gene Ontology mapping found in CS upregulation of proteins responsible for collagen binding and GT maturation as well as fatty acid metabolism enzymes known to be negative regulators of blood vessel sprouting. At the same time, downregulated transcripts were enriched by factors activating capillary growth, suggesting that in MSC sheets paracrine activity may shift towards matrix remodeling and maturation of vasculature, but not activation of blood vessel sprouting. We proposed a putative paracrine trigger mechanism potentially rendering an impact on GT vascularization and remodeling. Our results suggest that within sheets, MSC may change their functional state and spectrum of soluble factors that influence tissue repair and induce more effective skin healing inclining towards regeneration and reduced scarring.
Asunto(s)
Fibrosis/genética , Trasplante de Células Madre Mesenquimatosas , Úlcera por Presión/terapia , Cicatrización de Heridas/genética , Tejido Adiposo/trasplante , Animales , Cicatriz/genética , Cicatriz/patología , Fibrosis/patología , Fibrosis/terapia , Tejido de Granulación/metabolismo , Tejido de Granulación/patología , Humanos , Células Madre Mesenquimatosas/metabolismo , Ratones , Úlcera por Presión/genética , Úlcera por Presión/patología , Piel/metabolismo , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Regeneration is a fundamental process attributed to the functions of adult stem cells. In the last decades, delivery of suspended adult stem cells is widely adopted in regenerative medicine as a leading means of cell therapy. However, adult stem cells cannot complete the task of human body regeneration effectively by themselves as far as they need a receptive microenvironment (the niche) to engraft and perform properly. Understanding the mechanisms underlying mammalian regeneration leads us to an assumption that improved outcomes of cell therapy require a specific microenvironment that is generated in damaged areas prior to stem cell delivery. To a certain extent, it may be achieved by the delivery of mesenchymal stromal cells (MSCs), not in dispersed form, but rather in self-organized cell sheets (CS) â» tissue-like structures comprised of viable cells and microenvironment components: extracellular matrix and soluble factors deposited in the matrix. In this review, we highlight the potential role of MSCs as regeneration organizers and speculate that this function emerges in CS. This concept shifts our understanding of the therapeutic mechanism underlying a widely known CS-based delivery method for regenerative medicine.