Ilknur Özen

The Adult Human Brain Harbors Multipotent Perivascular Mesenchymal Stem Cells

Blood vessels and adjacent cells form perivascular stem cell niches in adult tissues. In this perivascular niche, a stem cell with mesenchymal characteristics was recently identified in some adult somatic tissues. These cells are pericytes that line the microvasculature, express mesenchymal markers and differentiate into mesodermal lineages but might even have the capacity to generate tissue-specific cell types. Here, we isolated, purified and characterized a previously unrecognized progenitor population from two different regions in the adult human brain, the ventricular wall and the neocortex. We show that these cells co-express markers for mesenchymal stem cells and pericytes in vivo and in vitro, but do not express glial, neuronal progenitor, hematopoietic, endothelial or microglial markers in their native state. Furthermore, we demonstrate at a clonal level that these progenitors have true multilineage potential towards both, the mesodermal and neuroectodermal phenotype. They can be epigenetically induced in vitro into adipocytes, chondroblasts and osteoblasts but also into glial cells and immature neurons. This progenitor population exhibits long-term proliferation, karyotype stability and retention of phenotype and multipotency following extensive propagation. Thus, we provide evidence that the vascular niche in the adult human brain harbors a novel progenitor with multilineage capacity that appears to represent mesenchymal stem cells and is different from any previously described human neural stem cell. Future studies will elucidate whether these cells may play a role for disease or may represent a reservoir that can be exploited in efforts to repair the diseased human brain.

Brain pericytes acquire a microglial phenotype after stroke.

Abstract Pericytes are located on the abluminal side of endothelial cells lining the microvasculature in all organs. They have been identified as multipotent progenitor cells in several tissues of the body including the human brain. New evidence suggests that pericytes contribute to tissue repair, but their role in the injured brain is largely unknown. Here, we investigate the role of pericytes in ischemic stroke. Using a pericyte-reporter mouse model, we provide unique evidence that regulator of G-protein signaling 5 expressing cells are activated pericytes that leave the blood vessel wall, proliferate and give rise to microglial cells after ischemic brain injury. Consistently, we show that activated pericytes express microglial markers in human stroke brain tissue. We demonstrate that human brain-derived pericytes adopt a microglial phenotype and upregulate mRNA specific for activated microglial cells under hypoxic conditions in vitro. Our study indicates that the vasculature is a novel source of inflammatory cells with a microglial phenotype in brain ischemia and hence identifies pericytes as an important new target for the development of future stroke therapies.

Perivascular mesenchymal stem cells in the adult human brain: a future target for neuroregeneration?

Perivascular adult stem cells have been isolated from several tissues, including the adult human brain. They have unique signatures resembling both pericytes and mesenchymal stem cells. Understanding the nature of these cells in their specific vascular niches is important to determine their clinical potential as a new adult stem cell source. Indeed, they have promising features in vitro in terms of multipotency, immunomodulation and secretion of growth factors and cytokines. However, their in vivo function is less known as yet. Recent emerging data show a crucial role of perivascular mesenchymal stem cells in tissue homeostasis and repair. Furthermore, these cells may play an important role in adult stem cell niche regulation and in neurodegeneration. Here we review the recent literature on perivascular mesenchymal stem cells, discuss their different in vitro functions and highlight especially the specific properties of brain-derived perivascular mesenchymal stem cells. We summarize current evidence that suggests an important in vivo function of these cells in terms of their regenerative potential that may indicate a new target cell for endogenous tissue regeneration and repair.

Endogenous brain pericytes are widely activated and contribute to mouse glioma microvasculature.

Glioblastoma multiforme (GBM) is the most common brain tumor in adults. It presents an extremely challenging clinical problem, and treatment very frequently fails due to the infiltrative growth, facilitated by extensive angiogenesis and neovascularization. Pericytes constitute an important part of the GBM microvasculature. The contribution of endogenous brain pericytes to the tumor vasculature in GBM is, however, unclear. In this study, we determine the site of activation and the extent of contribution of endogenous brain pericytes to the GBM vasculature. GL261 mouse glioma was orthotopically implanted in mice expressing green fluorescent protein (GFP) under the pericyte marker regulator of G protein signaling 5 (RGS5). Host pericytes were not only activated within the glioma, but also in cortical areas overlying the tumor, the ipsilateral subventricular zone and within the hemisphere contralateral to the tumor. The host-derived activated pericytes that infiltrated the glioma were mainly localized to the tumor vessel wall. Infiltrating GFP positive pericytes co-expressed the pericyte markers platelet-derived growth factor receptor-β (PDGFR-β) and neuron-glial antigen 2. Interestingly, more than half of all PDGFR-β positive pericytes within the tumor were contributed by the host brain. We did not find any evidence that RGS5 positive pericytes adopt another phenotype within glioma in this paradigm. We conclude that endogenous pericytes become activated in widespread areas of the brain in response to an orthotopic mouse glioma. Host pericytes are recruited into the tumor and constitute a major part of the tumor pericyte population.

Platelet-derived growth factor-BB has neurorestorative effects and modulates the pericyte response in a partial 6-hydroxydopamine lesion mouse model of Parkinson's disease

Parkinsons disease (PD) is a neurodegenerative disease where the degeneration of the nigrostriatal pathway leads to specific motor deficits. There is an unmet medical need for regenerative treatments that stop or reverse disease progression. Several growth factors have been investigated in clinical trials to restore the dopaminergic nigrostriatal pathway damaged in PD. Platelet-derived growth factor-BB (PDGF-BB), a molecule that recruits pericytes to stabilize microvessels, was recently investigated in a phase-1 clinical trial, showing a dose-dependent increase in dopamine transporter binding in the putamen of PD patients. Interestingly, evidence is accumulating that PD is paralleled by microvascular changes, however, whether PDGF-BB modifies pericytes in PD is not known. Using a pericyte reporter mouse strain, we investigate the functional and restorative effect of PDGF-BB in a partial 6-hydroxydopamine medial forebrain bundle lesion mouse model of PD, and whether this restorative effect is accompanied by changes in pericyte features. We demonstrate that a 2-week treatment with PDGF-BB leads to behavioural recovery using several behavioural tests, and partially restores the nigrostriatal pathway. Interestingly, we find that pericytes are activated in the striatum of PD lesioned mice and that these changes are reversed by PDGF-BB treatment. The modulation of brain pericytes may contribute to the PDGF-BB-induced neurorestorative effects, PDGF-BB allowing for vascular stabilization in PD. Pericytes might be a new cell target of interest for future regenerative therapies.

Ischemia/reperfusion in rat: Antioxidative effects of enoant on EEG, oxidative stress and inflammation

Primary objective: The present study was undertaken to evaluate whether enoant, which is rich in polyphenols, has any effect on electroencephalogram (EEG), oxidative stress and inflammation in ischemia/reperfusion (I/R) injury. Methods: Ischemia was induced by 2-hour occlusion of bilateral common carotid artery. Animals orally received enoant. Group 1 was the ischemic control group. Group 2 was treated with enoant of 1.25 g kg−1 per day for 15 days after I/R. Group 3 received the same concentration of enoant as in group 2 for 15 days before and after I/R. Group 4 was the sham operation group. EEG activities were recorded and the levels of TNF-α, IL-1β and IL-6, TBARS and GSH were measured in the whole brain homogenate. Results: There were significant changes in EEG activity in groups treated with enoant either before or after ischemia when compared with their basal EEG values. TNF-α, IL-6 and IL-1β levels were significantly increased after I/R. GSH levels in group 3 treated with enoant in both pre- and post-ischemic periods were significantly increased and TBARS concentration was decreased compared with the ischemic group. Conclusion: The findings support that both pre-ischemic and post-ischemic administrations of enoant might produce neuroprotective action against cerebral ischemia.

Pericytes secrete pro-regenerative molecules in response to platelet-derived growth factor-BB

Brain pericytes not only maintain the anatomical, biochemical and immune blood–brain barrier, but display features of mesenchymal stem cells (MSCs) in vitro. MSCs have pro-regenerative properties attributed to their secretome. However, whether also brain pericytes possess such pro-regenerative capacities is largely unknown. Here we characterize the secretome and microvesicle (MV) release of human brain pericytes mediated by platelet-derived growth factor-BB (PDGF-BB)/PDGF receptor beta (PDGFRβ) signalling. Upon PDGF-BB, pericytes release not only a plethora of growth factors and a panel of cytokines, but also MVs containing BDNF, FGFb, βNGF, VEGF and PLGF, a response that is specific for PDGFRβ signalling and activation of the ERK 1/2 pathway. In contrast, lipopolysaccharide (LPS), an activator of the innate immune system, stimulates the secretion of much higher amounts of mainly inflammatory cytokines and activates the NFκB pathway. Pericytes change their morphology and undergo opposite changes in surface marker expression, respectively. Our findings provide evidence that the secretome of human brain pericytes varies greatly depending on the exogenous stimulus. The differential secretory functions of pericytes may play an important role in either regulating neuroinflammation or contributing to neurorestoration and identify a possible new target cell for neuroregeneration.

The pericyte secretome : Potential impact on regeneration

Personalized and regenerative medicine is an emerging therapeutic strategy that is based on cell biology and biomedical engineering used to develop biological substitutes to maintain normal function or restore damaged tissues and organs. The secretory capacities of different cell types are now explored as such possible therapeutic regenerative agents in a variety of diseases. A secretome can comprise chemokines, cytokines, growth factors, but also extracellular matrix components, microvesicles and exosomes as well as genetic material and may differ depending on the tissue and the stimulus applied to the cell. With regard to clinical applications, the secretome of mesenchymal stem cells (MSC) is currently the most widely explored. However, other cell types such as pericytes may have similar properties as MSC and the potential therapeutic possibilities of these cells are only just beginning to emerge. In this review, we will summarize the currently available data describing the secretome of pericytes and its potential implications for tissue regeneration, whereby we especially focus on brain pericytes as potential new target cell for neuroregeneration and brain repair.

STAT3 precedes HIF1α transcriptional responses to oxygen and oxygen and glucose deprivation in human brain pericytes

Brain pericytes are important to maintain vascular integrity of the neurovascular unit under both physiological and ischemic conditions. Ischemic stroke is known to induce an inflammatory and hypoxic response due to the lack of oxygen and glucose in the brain tissue. How this early response to ischemia is molecularly regulated in pericytes is largely unknown and may be of importance for future therapeutic targets. Here we evaluate the transcriptional responses in in vitro cultured human brain pericytes after oxygen and/or glucose deprivation. Hypoxia has been widely known to stabilise the transcription factor hypoxia inducible factor 1-alpha (HIF1α) and mediate the induction of hypoxic transcriptional programs after ischemia. However, we find that the transcription factors Jun Proto-Oncogene (c-JUN), Nuclear Factor Of Kappa Light Polypeptide Gene Enhancer In B-Cells (NFκB) and signal transducer and activator of transcription 3 (STAT3) bind genes regulated after 2hours (hs) of omitted glucose and oxygen before HIF1α. Potent HIF1α responses require 6hs of hypoxia to substantiate transcriptional regulation comparable to either c-JUN or STAT3. Phosphorylated STAT3 protein is at its highest after 5 min of oxygen and glucose (OGD) deprivation, whereas maximum HIF1α stabilisation requires 120 min. We show that STAT3 regulates angiogenic and metabolic pathways before HIF1α, suggesting that HIF1α is not the initiating trans-acting factor in the response of pericytes to ischemia.

Multipotent perivascular mesenchymal stem cells in the human brain

K questions in adult stem cell biology revolve around origin, tissue home and physiological role of adult stem cell populations. Mesenchymal stem cells (MSC) have remained elusive with regard to their in vivo physiology. Recent observations suggest that almost all adult tissues contain mesenchymal-like progenitors in a perivascular niche. Those cells can differentiate into mesodermal cell types and may even be endowed with tissue specific differentiation capacities. We have isolated and characterized a previously unrecognized progenitor cell population in the adult human brain. This cell population exhibits characteristics of mesenchymal stem cells (CD105, CD90, CD73, CD29) but in its native state does not express hematopoetic (CD34, CD45), endothelial (CD31), microglial (CD14, CD11b), glial and neuronal progenitor markers (GFAP, O4). We demonstrate at a clonal level, that the progenitors have true multilineage potential not only towards a mesodermal but also neuroectodermal phenotype and can differentiate into neurons. Thus, the vasculature in the adult human brain contains progenitor cells with multilineage capacity that may represent a reservoir that can be exploited in attempts to repair the damaged or diseased brain.T cell-environment communication is a dynamic procedure. Therefore, when biomaterials are developed for regulating cell behaviour, the functionality of biomaterials needs to be regulated in a dynamic manner to satisfy dynamic cell requirements. To this end, we have developed a new generation of adaptive hydrogels that can mimic the functions of extracellular matrices (ECMs). ECMs have complicated, dynamic functions. The artificial ECMs play three critical roles when interacting with cells. First, the hydrogel networks are used as the fundamental structural support to provide cells with a biocompatible environment to survive. Second, multiple growth factors can be sequentially released at desired time points. Third, the interactions between cell receptors and aptamers tethered to the hydrogel networks can be switched on and off. Therefore, the artificial ECMs can dynamically provide cells with biophysical and biochemical cues. In this presentation, we will discuss the synthesis of aptamerfunctionalized hydrogels, the sequential release of multiple growth factors from artificial ECMs, and the dynamic regulation of cell-aptamer interactions in the artificial ECMs. It is believed that this research will open a new avenue of developing adaptive biomaterials for tissue regeneration.I optimal conditions, damaged bone heals by a precisely regulated multi-stage process that takes a few weeks. Although the physiological mechanisms that govern the repair of different types of bone are somewhat variable, they all exhibit a highly anabolic repair phase of finite duration and capacity. If the extent of trauma is too great, or if healing potential is diminished by bone disease, the bone simply does not heal. This is a significant health concern given a current fracture non-union rate of 10%, a spinal fusion failure rate of up to 40%, at least 10 million osteoporosis sufferers in the United States, and about half of all cancer sufferers face destructive metastases to bone. Here, we discuss the lessons our group have learned from the presumptive progenitors of osteoblasts, the bone marrow mesenchymal stem cell (MSC). This work has led to the development of novel cytotherapeutic preparations and biologically complex matrices for unprecedented levels of stem cell retention and bone repair in experimental models. The study of malignant bone disease is also discussed, and how our work on MSCs has led to the characterization of bone degradation mechanisms caused by malignancy. Novel methods for the reversal of bone damage during malignancy are introduced.C therapy with small RNA agents against multiple viral targets is critical to efficient inhibition of viral production. We have previously validated this approach by expressing multiple anti-HIV RNAs from independent Pol III promoters within a single gene therapy construct, however, the high transcription rate of Pol III promoters often leads to toxicity as a consequence of saturating the endogenous RISC machinery. An alternative approach takes advantage of an endogenous polycistronic miRNA cluster driven by a Pol II promoter which has been engineered as a multiplexing platform. We tested combinations of different classes of therapeutic anti-HIV RNAs expressed within the context of an intronic MCM7 platform replacing the endogenous miRNAs with siRNAs targeting HIV-1 tat and rev messages, a nucleolar-localizing RNA ribozyme that targets the conserved U5 region of HIV-1 transcripts for degradation, or nucleolar TAR and RBE RNA decoys. We demonstrate the versatility of the MCM7 platform for expression and efficient processing of all RNA elements. Three of the combinatorial constructs tested potently suppressed viral replication during a one-month HIV challenge, with greater than five-logs of inhibition compared to unprotected HIV-1 infected CEM T-cells. One of the most effective constructs contains an anti-HIV siRNA combined with a nucleolar-localizing U5 ribozyme and nucleolar-localizing TAR RNA decoy. To the best of our knowledge, this is the first successful demonstration of functional combinations of different types of small inhibitory RNAs expressed from a single intron transcriptional unit.