Mesenchymal "stem" cells, or facilitators for the development of regenerative macrophages? Pericytes at the interface of wound healing.

Cultured mesenchymal stromal cells are among the most used cells in clinical trials. Currently, their potential benefits include provision of mature cell types through differentiation, and secretion of various types of paracrine signaling molecules. Even though research on these cells has spanned some decades now, surprisingly, their therapeutic potential has not been fully translated into clinical practice yet, which calls for further understanding of their intrinsic nature and modes of action. In this review, after discussing pieces of evidence that suggest that some perivascular cells may exhibit mesenchymal stem cell characteristics in vivo, we examine the possibility that subpopulations of perivascular and/or adventitial cells activated after tissue injury behave as MSCs and contribute to the resolution of tissue injury by providing cues for the development of regenerative macrophages at injured sites. Under this perspective, an important contribution of cultured MSCs (or their acellular products, such as extracellular vesicles) used in cell therapies would be to instigate the development of M2-like macrophages that support the tissue repair process.

Challenges in Mesenchymal Stromal Cell-based Therapies.

Over 50 years have passed since discovering mesenchymal stromal cells (MSCs). Initially, despite gaps in the knowledge of the identity of these cells, their therapeutic aspects were recognized. Consequently, MSCs became candidates for treating a wide range of diseases. However, the therapeutic effects of MSCs are not stable in the long term, and there are inconsistent data on their clinical efficacy. Even though more than 1000 MSC-based clinical trials have been registered, and the safety of MSCbased cell therapies has been proven, data on the clinical efficacy of MSCs have not been enough to warrant FDA approval for clinical treatment and marketing purposes. The available information on MSCs still contains some controversies, perhaps owing to little progress in understanding their in vivo identity. MSCs have been used for therapeutic purposes despite poor knowledge of their in vivo origin or functions. Hence, perhaps we need to go back to the basics of MSCs and spend more time understanding the biology of these cells. An improved understanding of MSCs' location and function within tissues may improve their therapeutic efficacy and, consequently, their establishment as a cell therapy product.

Analyses of the pericyte transcriptome in ischemic skeletal muscles.

BACKGROUND: Peripheral arterial disease (PAD) affects millions of people and compromises quality of life. Critical limb ischemia (CLI), which is the most advanced stage of PAD, can cause nonhealing ulcers and strong chronic pain, and it shortens the patients' life expectancy. Cell-based angiogenic therapies are becoming a real therapeutic approach to treat CLI. Pericytes are cells that surround vascular endothelial cells to reinforce vessel integrity and regulate local blood pressure and metabolism. In the past decade, researchers also found that pericytes may function as stem or progenitor cells in the body, showing the potential to differentiate into several cell types. We investigated the gene expression profiles of pericytes during the early stages of limb ischemia, as well as the alterations in pericyte subpopulations to better understand the behavior of pericytes under ischemic conditions. METHODS: In this study, we used a hindlimb ischemia model to mimic CLI in C57/BL6 mice and explore the role of pericytes in regeneration. To this end, muscle pericytes were isolated at different time points after the induction of ischemia. The phenotypes and transcriptomic profiles of the pericytes isolated at these discrete time points were assessed using flow cytometry and RNA sequencing. RESULTS: Ischemia triggered proliferation and migration and upregulated the expression of myogenesis-related transcripts in pericytes. Furthermore, the transcriptomic analysis also revealed that pericytes induce or upregulate the expression of a number of cytokines with effects on endothelial cells, leukocyte chemoattraction, or the activation of inflammatory cells. CONCLUSIONS: Our findings provide a database that will improve our understanding of skeletal muscle pericyte biology under ischemic conditions, which may be useful for the development of novel pericyte-based cell and gene therapies.

Cancer regeneration: Polyploid cells are the key drivers of tumor progression.

In spite of significant advancements of therapies for initial eradication of cancers, tumor relapse remains a major challenge. It is for a long time known that polyploid malignant cells are a main source of resistance against chemotherapy and irradiation. However, therapeutic approaches targeting these cells have not been appropriately pursued which could partly be due to the shortage of knowledge on the molecular biology of cell polyploidy. On the other hand, there is a rising trend to appreciate polyploid/ multinucleated cells as key players in tissue regeneration. In this review, we suggest an analogy between the functions of polyploid cells in normal and malignant tissues and discuss the idea that cell polyploidy is an evolutionary conserved source of tissue regeneration also exploited by cancers as a survival factor. In addition, polyploid cells are highlighted as a promising therapeutic target to overcome drug resistance and relapse.

Are Liver Pericytes Just Precursors of Myofibroblasts in Hepatic Diseases? Insights from the Crosstalk between Perivascular and Inflammatory Cells in Liver Injury and Repair.

Cirrhosis, a late form of liver disease, is characterized by extensive scarring due to exacerbated secretion of extracellular matrix proteins by myofibroblasts that develop during this process. These myofibroblasts arise mainly from hepatic stellate cells (HSCs), liver-specific pericytes that become activated at the onset of liver injury. Consequently, HSCs tend to be viewed mainly as myofibroblast precursors in a fibrotic process driven by inflammation. Here, the molecular interactions between liver pericytes and inflammatory cells such as macrophages and neutrophils at the first moments after injury and during the healing process are brought into focus. Data on HSCs and pericytes from other tissues indicate that these cells are able to sense pathogen- and damage-associated molecular patterns and have an important proinflammatory role in the initial stages of liver injury. On the other hand, further data suggest that as the healing process evolves, activated HSCs play a role in skewing the initial proinflammatory (M1) macrophage polarization by contributing to the emergence of alternatively activated, pro-regenerative (M2-like) macrophages. Finally, data suggesting that some HSCs activated during liver injury could behave as hepatic progenitor or stem cells will be discussed.

Induction of Expression of CD271 and CD34 in Mesenchymal Stromal Cells Cultured as Spheroids.

Cultured mesenchymal stromal cells (MSCs) are cells that can be used for tissue engineering or cell therapies owing to their multipotency and ability to secrete immunomodulatory and trophic molecules. Several studies suggest that MSCs can become pericytes when cocultured with endothelial cells (ECs) but failed to use pericyte markers not already expressed by MSCs. We hypothesized ECs could instruct MSCs to express the molecules CD271 or CD34, which are expressed by pericytes in situ but not by MSCs. CD271 is a marker of especial interest because it is associated with multipotency, a characteristic that wanes in MSCs as they are culture expanded. Consequently, surface expression of CD271 and CD34 was detected in roughly half of the MSCs cocultured with ECs as spheroids in the presence of insulin-like growth factor 1 (IGF-1). Conversely, expression of CD271 and CD34 was detected in a similar proportion of MSCs cultured under these conditions without ECs, and expression of these markers was low or absent when no IGF-1 was added. These findings indicate that specific culture conditions including IGF-1 can endow cultured MSCs with expression of CD271 and CD34, which may enhance the multipotency of these cells when they are used for therapeutic purposes.

How Plastic Are Pericytes?

Pericytes are defined by both their anatomical location and molecular markers. Numerous publications have reported their role as stem cells, contributing to the formation of tissues other than blood vessels. However, using cell-lineage tracing in a new transgenic mouse model, a recent study shows that in the context of aging and some pathologies, Tbx18+ pericytes do not function as stem cells in vivo. This study challenges the current view that pericytes can differentiate into other cells and reopen questions about their plasticity. This emerging knowledge is important not only for our understanding of development but may also inform treatments for diseases.

Neurotrauma: The Crosstalk between Neurotrophins and Inflammation in the Acutely Injured Brain.

Traumatic brain injury (TBI) is a major cause of morbidity and mortality among young individuals worldwide. Understanding the pathophysiology of neurotrauma is crucial for the development of more effective therapeutic strategies. After the trauma occurs, immediate neurologic damage is produced by the traumatic forces; this primary injury triggers a secondary wave of biochemical cascades together with metabolic and cellular changes, called secondary neural injury. In the scenario of the acutely injured brain, the ongoing secondary injury results in ischemia and edema culminating in an uncontrollable increase in intracranial pressure. These areas of secondary injury progression, or areas of "traumatic penumbra", represent crucial targets for therapeutic interventions. Neurotrophins are a class of signaling molecules that promote survival and/or maintenance of neurons. They also stimulate axonal growth, synaptic plasticity, and neurotransmitter synthesis and release. Therefore, this review focuses on the role of neurotrophins in the acute post-injury response. Here, we discuss possible endogenous neuroprotective mechanisms of neurotrophins in the prevailing environment surrounding the injured areas, and highlight the crosstalk between neurotrophins and inflammation with focus on neurovascular unit cells, particularly pericytes. The perspective is that neurotrophins may represent promising targets for research on neuroprotective and neurorestorative processes in the short-term following TBI.

The gene expression profile of non-cultured, highly purified human adipose tissue pericytes: Transcriptomic evidence that pericytes are stem cells in human adipose tissue.

Pericytes (PCs) are a subset of perivascular cells that can give rise to mesenchymal stromal cells (MSCs) when culture-expanded, and are postulated to give rise to MSC-like cells during tissue repair in vivo. PCs have been suggested to behave as stem cells (SCs) in situ in animal models, although evidence for this role in humans is lacking. Here, we analyzed the transcriptomes of highly purified, non-cultured adipose tissue (AT)-derived PCs (ATPCs) to detect gene expression changes that occur as they acquire MSC characteristics in vitro, and evaluated the hypothesis that human ATPCs exhibit a gene expression profile compatible with an AT SC phenotype. The results showed ATPCs are non-proliferative and express genes characteristic not only of PCs, but also of AT stem/progenitor cells. Additional analyses defined a gene expression signature for ATPCs, and revealed putative novel ATPC markers. Almost all AT stem/progenitor cell genes differentially expressed by ATPCs were not expressed by ATMSCs or culture-expanded ATPCs. Genes expressed by ATMSCs but not by ATPCs were also identified. These findings strengthen the hypothesis that PCs are SCs in vascularized tissues, highlight gene expression changes they undergo as they assume an MSC phenotype, and provide new insights into PC biology.

Identification of suitable reference genes for quantitative gene expression analysis in rat adipose stromal cells induced to trilineage differentiation.

This study was designed to (i) identify stable reference genes for the analysis of gene expression during in vitro differentiation of rat adipose stromal cells (rASCs), (ii) recommend stable genes for individual treatment conditions, and (iii) validate these genes by comparison with normalization results from stable and unstable reference genes. On the basis of a literature review, eight genes were selected: Actb, B2m, Hprt1, Ppia, Rplp0, Rpl13a, Rpl5, and Ywhaz. Genes were ranked according to their stability under different culture conditions as assessed using GenNorm, NormFinder, and RefFinder algorithms. Although the employed algorithms returned different rankings, the most frequently top-ranked genes were: B2m and/or Ppia for all 28day treatments (ALL28); Ppia and Hprt1 (adipogenic differentiation; A28), B2m (chondrogenic differentiation; C28), Rpl5 (controls maintained in complete culture medium; CCM), Rplp0 (osteogenic differentiation for 3days; O3), Rpl13a and Actb (osteogenic differentiation for 7days; O7), Rplp0 and Ppia (osteogenic differentiation for 14days; O14), Hprt1 and Ppia (osteogenic differentiation for 28days; O28), as well as Actb (all osteogenesis time points combined; ALLOSTEO). The obtained results indicate that the performance of reference genes depends on the differentiation protocol and on the analysis time, thus providing valuable information for the design of RT-PCR experiments.

Transcriptomic comparisons between cultured human adipose tissue-derived pericytes and mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs), sometimes called mesenchymal stem cells, are cultured cells able to give rise to mature mesenchymal cells such as adipocytes, osteoblasts, and chondrocytes, and to secrete a wide range of trophic and immunomodulatory molecules. Evidence indicates that pericytes, cells that surround and maintain physical connections with endothelial cells in blood vessels, can give rise to MSCs (da Silva Meirelles et al., 2008 [1]; Caplan and Correa, 2011 [2]). We have compared the transcriptomes of highly purified, human adipose tissue pericytes subjected to culture-expansion in pericyte medium or MSC medium, with that of human adipose tissue MSCs isolated with traditional methods to test the hypothesis that their transcriptomes are similar (da Silva Meirelles et al., 2015 [3]). Here, we provide further information and analyses of microarray data from three pericyte populations cultured in pericyte medium, three pericyte populations cultured in MSC medium, and three adipose tissue MSC populations deposited in the Gene Expression Omnibus under accession number GSE67747.

Mesenchymal stem cells and their relationship to pericytes.

Our body contains cells that can be propagated in vitro and give rise to cells with mature mesenchymal phenotypes. These cells are interesting not only because of their differentiation capability, which could be used for tissue engineering, but also because they secrete molecules which have trophic, chemoattractant, and immunomodulatory properties. Along decades of study, these cells have been referred to as fibroblastic cells, stromal cells, or mesenchymal stem cells. There is evidence that pericytes, cells that wrap endothelial cells in blood vessels, behave as stem cells in the tissues, and give rise to these progenitor cells when removed from the body and expanded in culture - a process that may reflect changes that occur in vivo under injury conditions. Here, we discuss the evidence that favors this thesis, and discuss culture methods, clinical and preclinical applications of mesenchymal stem cells under this perspective.

Cultured Human Adipose Tissue Pericytes and Mesenchymal Stromal Cells Display a Very Similar Gene Expression Profile.

Mesenchymal stromal cells (MSCs) are cultured cells that can give rise to mature mesenchymal cells under appropriate conditions and secrete a number of biologically relevant molecules that may play an important role in regenerative medicine. Evidence indicates that pericytes (PCs) correspond to mesenchymal stem cells in vivo and can give rise to MSCs when cultured, but a comparison between the gene expression profiles of cultured PCs (cPCs) and MSCs is lacking. We have devised a novel methodology to isolate PCs from human adipose tissue and compared cPCs to MSCs obtained through traditional methods. Freshly isolated PCs expressed CD34, CD140b, and CD271 on their surface, but not CD146. Both MSCs and cPCs were able to differentiate along mesenchymal pathways in vitro, displayed an essentially identical surface immunophenotype, and exhibited the ability to suppress CD3(+) lymphocyte proliferation in vitro. Microarray expression data of cPCs and MSCs formed a single cluster among other cell types. Further analyses showed that the gene expression profiles of cPCs and MSCs are extremely similar, although MSCs differentially expressed endothelial cell (EC)-specific transcripts. These results confirm, using the power of transcriptomic analysis, that PCs give rise to MSCs and suggest that low levels of ECs may persist in MSC cultures established using traditional protocols.

Mesenchymal Stem Cells Improve Heart Rate Variability and Baroreflex Sensitivity in Rats with Chronic Heart Failure.

Heart failure induced by myocardial infarct (MI) attenuates the heart rate variability (HRV) and baroreflex sensitivity, which are important risk factors for life-threatening cardiovascular events. Therapies with mesenchymal stem cells (MSCs) have shown promising results after MI. However, the effects of MSCs on hemodynamic (heart rate and arterial pressure) variability and baroreflex sensitivity in chronic heart failure (CHF) following MI have not been evaluated thus far. Male Wistar rats received MSCs or saline solution intravenously 1 week after ligation of the left coronary artery. Control (noninfarcted) rats were also evaluated. MI size was assessed using single-photon emission computed tomography (SPECT). The left ventricular ejection fraction (LVEF) was evaluated using radionuclide ventriculography. Four weeks after MSC injection, the animals were anesthetized and instrumented for chronic ECG recording and catheters were implanted in the femoral artery to record arterial pressure. Arterial pressure and HRVs were determined in time and frequency domain (spectral analysis) while HRV was also examined using nonlinear methods: DFA (detrended fluctuation analysis) and sample entropy. The initial MI size was the same among all infarcted rats but was reduced by MSCs. CHF rats exhibited increased myocardial interstitial collagen and sample entropy combined with the attenuation of the following cardiocirculatory parameters: DFA indices, LVEF, baroreflex sensitivity, and HRV. Nevertheless, MSCs hampered all these alterations, except the LVEF reduction. Therefore, 4 weeks after MSC therapy was applied to CHF rats, MI size and myocardial interstitial fibrosis decreased, while baroreflex sensitivity and HRV improved.

Phenotypic analysis and differentiation of murine mesenchymal stem cells.

Mesenchymal stem cells (MSCs) hold great promise as therapeutic tools to treat different types of disease and the use of preclinical animal models is mandatory for the development of novel MSC-based therapies. The mouse is one of the most important species used for preclinical experiments and, by extension, so is the isolation and characterization of murine MSCs. This chapter presents methods for the phenotypic analysis of cultured murine MSCs.

MSC frequency correlates with blood vessel density in equine adipose tissue.

Mesenchymal stem cells (MSCs) are multipotent cells that have the capacity to develop into different mature mesenchymal cell types. They were originally isolated from bone marrow, but MSC-like cells have also been isolated from other tissues. The common feature of all of these tissues is that they all house blood vessels. It is, thus, possible that MSCs are associated with perivascular locations. The objective of this work was to test the hypothesis that MSCs are associated with blood vessels by verifying if MSC frequency positively correlates with blood vessel density. To this end, samples from highly and poorly vascularized adipose tissue sites of two equine donors were collected and processed for histology and cell isolation. MSC frequency in these samples was estimated by means of CFU-F assays, which were performed under MSC conditions. Culture-adherent cells from equine adipose tissue and bone marrow were culture expanded, tested for differentiation into mesenchymal cell types in vitro, and implanted in vivo in porous ceramic vehicles to assess their osteogenic capacity, using human MSCs and brain pericytes as controls. The differentiation assays showed a difference between adipose tissue-derived cells as compared to equine bone marrow MSCs. While differences in CFU-F frequencies between both donors were evident, the CFU-F numbers correlated directly with blood vessel densities (r(2) = 0.86). We consider these preliminary data as further evidence linking MSCs to blood vessels.

In search of the in vivo identity of mesenchymal stem cells.

In spite of the advances in the knowledge of adult stem cells (ASCs) during the past few years, their natural activities in vivo are still poorly understood. Mesenchymal stem cells (MSCs), one of the most promising types of ASCs for cell-based therapies, are defined mainly by functional assays using cultured cells. Defining MSCs in vitro adds complexity to their study because the artificial conditions may introduce experimental artifacts. Inserting these results in the context of the organism is difficult because the exact location and functions of MSCs in vivo remain elusive; the identification of the MSC niche is necessary to validate results obtained in vitro and to further the knowledge of the physiological functions of this ASC. Here we show an analysis of the evidence suggesting a perivascular location for MSCs, correlating these cells with pericytes, and present a model in which the perivascular zone is the MSC niche in vivo, where local cues coordinate the transition to progenitor and mature cell phenotypes. This model proposes that MSCs stabilize blood vessels and contribute to tissue and immune system homeostasis under physiological conditions and assume a more active role in the repair of focal tissue injury. The establishment of the perivascular compartment as the MSC niche provides a basis for the rational design of additional in vivo therapeutic approaches. This view connects the MSC to the immune and vascular systems, emphasizing its role as a physiological integrator and its importance in tissue repair/regeneration.