Figure 2 - available via license: Creative Commons Attribution 4.0 International
Content may be subject to copyright.
Characterization of skin and lung MPCs. A. Representative growth curves for skin (red) and lung (green) MPCs in complete medium. One out of nine wells of representative cultures in a Real Time Cell Analyzer is shown. Cell Index represents the relative number of proliferative cells. Differences between skin and lung were statistically significant by ANOVA (*p , 0.03). B. Flow cytometry analysis of surface molecule expression on skin and lung mesenchymal precursors. One group of representative histograms out of nine independent experiments is shown. Table 1 shows the media of the mean intensity fluorescence and the variance for all nine experiments. C. RT-PCR analysis of gene expression profile in skin and lung MPCs. The panel shows one representative agarose gel analysis of amplified products out of three independent experiments for each explant. Primer sequences are shown in Table 2 and summary of the results is shown in Table 3. doi:10.1371/journal.pone.0053215.g002
Source publication
The present study reports an easy and efficient method for obtaining adult mesenchymal precursors from different adult mouse tissues.
We describe the isolation and expansion of mesenchymal precursors from skin and lung by a non-enzymatic method. Skin and lung mesenchymal precursors isolated by a modified explant technique were characterized in vitr...
Similar publications
A routine chest X-ray inspection detected a solid nodule in the right upper lobe in a 38-year-old asymptomatic female patient. It was not suspected of being malignant and had been followed up until the following year when a chest CT scan showed the slightly enlarged tumor. The patient was referred to our hospital for examination and treatment. Thor...
Recently, there has been increasing interest in stem cell transplantation therapy, to treat chronic respiratory diseases, using lung epithelial cells or alveolospheres derived from endogenous lung progenitor cells. However, optimal transplantation strategy of these cells has not been addressed. To gain insight into the optimization of stem cell tra...
Citations
... Protocols were approved by the Health Sciences Research Committee of Universidad Europea de Madrid with reference number CIPI/069/17. The isolation of s-MPs was performed using the explant technique described previously [31]. Skin explants from adult mice and adult humans were collected and dissected into 1-2 mm pieces. ...
Ultrasound is considered a safe and non-invasive tool in regenerative medicine and has been used in the clinic for more than twenty years for applications in bone healing after the approval of the Exogen device, also known as low-intensity pulsed ultrasound (LIPUS). Beyond its effects on bone health, LIPUS has also been investigated for wound healing of soft tissues, with positive results for various cell processes including cell proliferation, migration and angiogenesis. As LIPUS has the potential to treat chronic skin wounds, we sought to evaluate the effects produced by a conventional therapeutic ultrasound device at low intensities (also considered LIPUS) on the migration capacity of mouse and human skin mesenchymal precursors (s-MPs). Cells were stimulated for 3 days (20 minutes per day) using a traditional ultrasound device with the following parameters: 100 mW/cm ² with 20% duty cycle and frequency of 3 MHz. At the parameters used, ultrasound failed to affect s-MP proliferation, with no evident changes in morphology or cell groupings, and no changes at the cytoskeletal level. Further, the migration and invasion ability of s-MPs were unaffected by the ultrasound protocol, and no major changes were detected in the gene/protein expression of ROCK1, integrin β1, laminin β1, type I collagen and transforming growth factor β1. Finally, RNA-seq analysis revealed that only 10 genes were differentially expressed after ultrasound stimulation. Among them, 5 encode for small nuclear RNAs and 2 encode for proteins belonging to the nuclear pore complex. Considering the results overall, while the viability of s-MPs was not affected by ultrasound stimulation and no changes were detected in proliferation/migration, RNA-seq analysis would suggest that s-MPs do respond to ultrasound. The use of 100 mW/cm ² intensity or conventional therapeutic ultrasound devices might not be optimal for the stimulation the properties of cell populations. Future studies should investigate the potential application of ultrasound using variations of the tested parameters.
... In recent years, more and more research has been conducted using MSCs isolated from adipose tissue (16,17). In addition, it is possible to obtain MSCs from other sources, such as skin (18), lungs (19), liver (20) dental pulp (21), endometrium (22), fallopian tube (23), and anterior or posterior cruciate knee ligaments (24). ...
Mesenchymal stem cells (MSCs) are non-hematopoietic, multipotent stem cells derived from mesoderm, which can be easily isolated from many sources such as bone marrow, umbilical cord or adipose tissue. MSCs provide support for hematopoietic stem cells and have an ability to differentiate into multiple cell lines. Moreover, they have proangiogenic, protective and immunomodulatory properties. MSCs have the capacity to modulate both innate and adaptive immune responses, which accompany many diseases, by inhibiting pro-inflammatory reactions and stimulating anti-inflammatory activity. Recent findings revealed that the positive effect of MSCs is at least partly associated with the production of extracellular vesicles (EVs). EVs are small membrane structures, containing proteins, lipids and nuclei acids, which take part in intra-cellular communication. Many studies indicate that EVs contain protective and pro-regenerative properties and can modulate an immune response that is activated in various diseases such as CNS diseases, myocardial infarction, liver injury, lung diseases, ulcerative colitis or kidney injury. Thus, EVs have similar functions as their cells of origin and since they do not carry the risk of cell transplantation, such as tumor formation or small vessel blockage, they can be considered a potential therapeutic tool for cell-free therapy.
... At the end of the study period (20 weeks), ASCs were isolated from the PAT of the three groups of mice using the "explant" technique (Bernal, Fernandez, Perez, San Martin, & Galvez, 2012;Galvez, San Martin & Rodriguez, 2009). Briefly, small pieces of fresh PAT were isolated from mice, aged-matched to 6.5 months, and were placed on Matrigel™-coated plates (BD Biosciences, Franklin Lakes, NJ) in complete Dulbecco's modified Eagles's medium (DMEM; ...
Pericardial adipose tissue (PAT), a visceral fat depot enveloping the heart, is an active endocrine organ and a source of free fatty acids and inflammatory cytokines. As in other fat adult tissues, PAT contains a population of adipose stem cells; however, whether these cells and/or their environment play a role in physiopathology is unknown. We analyzed several stem cell‐related properties of pericardial adipose stem cells (PSCs) isolated from obese and ex‐obese mice. We also performed RNA‐sequencing to profile the transcriptional landscape of PSCs isolated from the different diet regimens. Finally, we tested whether these alterations impacted on the properties of cardiac mesoangioblasts isolated from the same mice. We found functional differences between PSCs depending on their source: specifically, PSCs from obese PSC (oPSC) and ex‐obese PSC (dPSC) mice showed alterations in apoptosis and migratory capacity when compared with lean, control PSCs, with increased apoptosis in oPSCs and blunted migratory capacity in oPSCs and dPSCs. This was accompanied by different gene expression profiles across the cell types, where we identified some genes altered in obese conditions, such as BMP endothelial cell precursor‐derived regulator (BMPER), an important regulator of BMP‐related signaling pathways for endothelial cell function. The importance of BMPER in PSCs was confirmed by loss‐ and gain‐of‐function studies. Finally, we found an altered production of BMPER and some important chemokines in cardiac mesoangioblasts in obese conditions. Our findings point to BMPER as a potential new regulator of PSC function and suggest that its dysregulation could be associated with obesity and may impact on cardiac cells.
... Cardiomyocytes were isolated from neonatal mice and cultured as described previously [22]. Murine ASC and CP cells were isolated using the "explant" technique [23,24]. Briefly, fresh small pieces of subcutaneous or visceral adipose tissue, or cardiac tissue from ventricles, were isolated from control and obese mice, aged-matched to 4 months, and were placed on 1% gelatin-coated plates in complete Dulbecco's modified Eagles's medium (DMEM; Sigma, St. Louis MO) containing 10% fetal bovine serum (FBS; Sigma), 100 U/ml penicillin/streptomycin, 2 mM Lglutamine, and 10 mM Hepes (all from Lonza, Basel, Switzerland). ...
Background:
The role of adipose tissue in the pathophysiology of cardiovascular disease remains a major subject of research. The objective of the present study was to dissect the molecular mechanisms that regulate the survival and differentiation of cardiac cells in an obese environment.
Material and methods:
We isolated murine/human cardiac cells from adult hearts of control and obese mice/subjects and analyzed the communication between cardiac cells and adipocytes in vitro, as well as the effects on their main functions such as survival and differentiation.
Results:
We found that the presence of visceral or subcutaneous adipocytes in the environment of cardiomyocytes or cardiac precursors provoked apoptosis or blocked differentiation, respectively, and these effects were mediated by secreted adipokines. Remarkably, cardiac precursors changed their fate and differentiated into mature adipocytes, contributing to the overall increase in adipose cell content. Inhibiting the adipokines TNF-α, visfatin, or HMGB1 could block the deleterious effects of adipokines on cardiac cells.
Conclusions:
Our findings demonstrate that mouse and human visceral adipose tissue contributes negatively to the homeostasis and regeneration of the heart. Moreover, our results suggest that blocking the action of certain adipokines might enhance cardiac differentiation and survival.
... In a previous study, a slightly modified method for the isolation of pre-committed adult mesenchymal precursors from various tissue sources that are more easily accessible was established (Bernal et al., 2012). By addition of ECM rich in factors that attract the mesenchymal precursors resulting in increasing the quantity and shorten the timing for obtaining these cell populations. ...
Dental pulp stem cells have emerged as a preferred source of mesenchymal stem cells, because of its easy availability and high stem cell content. Dental pulp is a specific fibrous tissue that contains heterogeneous populations of odontoblasts, fibroblasts, pericytes, progenitors, stem cells, leukocytes and neuronal cells. In this study, we propose sustained explant culture as a simple, economical and efficient process to isolate dental pulp stem cells from human Dental pulp Tissue. Historically explant cultures were used to get fibroblast cells from embryonic chick heart using plasma clot cultures. The subculture was performed by lifting mother explant (original explant) and grafting it in a new plasma clot. We modified this age old technique to suit the modern times. Here we demonstrate for the first time that the mother explant (E0) of human dental pulp tissue could be sub‐cultured consecutively seven times (E7) without displacement. This technique is highly reproducible and permits growth and proliferation of dental pulp stem cells yielding an enriched homogeneous mesenchymal stem cells population in the first passage itself as revealed by surface marker expression. These dental pulp stem cells exhibit differentiation into adipogenic, chondrogenic and osteogenic lineage revealing their mesenchymal stem cell nature. We propose that dental pulp stem cells isolated by sustained explant culture are phenotypically and functionally comparable to those obtained by enzymatic method. It is a simple, inexpensive and gentle method, which may be preferred over the conventional techniques for obtaining stem cells from other tissue sources as well especially in cases of limited starting material.
... Mesenchymal stem cells (MSCs) seem to be good candidates for cell therapies since they have self-renewal capacity and present great potential to differentiate into multiple cell types. Multiple adult tissues contain this population of mesenchymal stem/ progenitor cells [1][2][3], and they have been well characterized as progenitors residing in specific locations or niches into the adult tissues. Adult stem cells reside in stem cell niches that are necessary to maintain tissue homeostasis and repair by controlling stem cell behavior. ...
... It has been reported the presence of MSCs in almost every adult tissue allowing the possibility to obtain diverse stem cells from different sources [4]. Here, we provide a method to isolate MSCs from diverse adult tissue niches by the explant technique [1,3]. The question of which stem cell source will be optimal for Beatriz de Lucas and Laura M. Pérez contributed equally to this work. ...
It has been described that adult tissues contain mesenchymal stem cell populations. The specific areas where stem cells reside are known as niches. Crosstalk between cells and their niche is essential to maintain the correct functionality of stem cell. MSCs present a set of abilities such as migration, invasion, and angiogenic potentials, which make them ideal candidates for cell-based therapies. In order to test the regenerative capacity of these cells, we have described a methodology for the collection and for the evaluation of these mesenchymal precursors from different niches.
... MSCs can be found in nearly all tissues and are generally located in perivascular niches [14]. The major MSC reservoirs include bone marrow [7], peripheral blood [15], adipose tissue [16,17], lung [18,19] and neonatal tissues [20]. MSCs have three main characteristics that make them ideal for the creation of off-the-shelf therapies. ...
... Lung progenitor cells have been identified as small populations of lung resident cells that display stem cell features [35]. Several studies have shown that lung progenitors share some common characteristics with MSCs [19]. The existence of pancreatic progenitor cells, which possess abilities of self-renewal and multipotency, is controversial. ...
The stem cell field has grown very rapidly during the last decade, offering the promise of innovative therapies to treat disease. Different stem cell populations have been isolated from various human adult tissues, mainly from bone marrow and adipose tissue, but many other body tissues harbor a stem cell population. Adult tissue stem cells are invariably found in discrete microenvironments termed niches, where they play key roles in tissue homeostasis by enabling lifelong optimization of organ form and function. Some diseases are known to strike at the stem cell population, through alterations in their specific microenvironments, making them non-viable. Furthermore, it has been shown that a transformed stem cell population could prompt the development of certain cancers. This review focuses on the potential negative aspects of a range of diseases on the activity of stem cells and how their potential use in cell therapies may be affected.
... Nevertheless, cells are not only affected by their immediate environment. The arrival of soluble factors such as cytokines [64,65] or growth factors [66,67], presumably produced in more remote areas, is sufficient to activate receptors and initiate migration signalling. Stem cell migration is also affected by the arrival of hormones of different origins [68]. ...
Cell migration is an essential process throughout the life of vertebrates, beginning during embryonic development and continuing throughout adulthood. Stem cells have an inherent ability to migrate, that is as important as their capacity for self-renewal and differentiation, enabling them to maintain tissue homoeostasis and mediate repair and regeneration. Adult stem cells reside in specific tissue niches, where they remain in a quiescent state until called upon and activated by tissue environmental signals. Cell migration is a highly regulated process that involves the integration of intrinsic signals from the niche and extrinsic factors. Studies using three-dimensional in vitro models have revealed the astonishing plasticity of cells in terms of the migration modes employed in response to changes in the microenvironment. These same properties can, however, be subverted during the development of some pathologies such as cancer. In this review, we describe the response of adult stem cells to migratory stimuli and the mechanisms by which they sense and transduce intracellular signals involved in migratory processes. Understanding the molecular events underlying migration may help develop therapeutic strategies for regenerative medicine and to treat diseases with a cell migration component.
... Adipose stem cells were obtained from non-obese or obese mice and humans as described previously [21,28]. Murine tissues were collected from adult C57BL6 mice as the non-obese individuals (non-obesity-derived ASCs) and from 4-month-old mice with diet-induced obesity as the obese individuals (obesity-derived ASCs). ...
Obesity affects the functional capability of adipose-derived stem cells (ASCs) and their effective use in regenerative medicine through mechanisms still poorly understood. Here we employed a multiplatform (LC/MS, CE/MS, GC/MS) metabolomics untargeted approach to investigate the metabolic alteration underlying the inequalities observed in obese-derived ASCs. The metabolic fingerprint (metabolites within the cells) and footprint (metabolites secreted in the culture medium) from humans or mice, obese and non-obese derived ASCs, were characterized by providing valuable information. Metabolites associated to glycolysis, TCA, pentose phosphate pathway and polyol pathway were increased in the footprint of obese-derived human ASCs indicating alterations in the carbohydrate metabolism; whereas from the murine model, deep differences in lipid and amino acid catabolism were highlighted. Therefore, new insights on the ASCs metabolome were provided that enhance our understanding of the processes underlying the ASCs stemness capacity and its relationship with obesity, in different cell models.
... MPs were isolated from adult mouse tissue explants and characterized as it was described before [25]. Briefly, mice were sacrificed by CO 2 chamber. ...
Mesenchymal precursors (MPs) present some advantageous features, such as differentiation and migration, which make them promising candidates for cell therapy. A better understanding of MP migration characteristics would aid the development of cell delivery protocols. Traditionally, cell migration is thought to occur only through the formation of lamellipodia. More recently, contractility-driven bleb formation has emerged as an alternative mechanism of motility. Here we report that MPs derived from different tissues present spontaneously dynamic cytoplasmic projections in sub-confluent culture, which appear as a combination of lamellipodia with blebs in the leading edge. Upon initial seeding, however, only bleb structures could be observed. Immunofluorescence revealed the presence of pERM, RhoA and F-actin during the blebbing process. Results from migration assays in the presence of blebbistatin, a myosin II inhibitor, showed that bleb formation correlated with migratory capacity, suggesting a functional role for blebs in migration. Bleb formation might be a useful mechanism to improve cell migration in cellular therapy protocols.
