Human blood vessel organoids reveal a critical role for CTGF in maintaining microvascular integrity.

The microvasculature plays a key role in tissue perfusion and exchange of gases and metabolites. In this study we use human blood vessel organoids (BVOs) as a model of the microvasculature. BVOs fully recapitulate key features of the human microvasculature, including the reliance of mature endothelial cells on glycolytic metabolism, as concluded from metabolic flux assays and mass spectrometry-based metabolomics using stable tracing of 13C-glucose. Pharmacological targeting of PFKFB3, an activator of glycolysis, using two chemical inhibitors results in rapid BVO restructuring, vessel regression with reduced pericyte coverage. PFKFB3 mutant BVOs also display similar structural remodelling. Proteomic analysis of the BVO secretome reveal remodelling of the extracellular matrix and differential expression of paracrine mediators such as CTGF. Treatment with recombinant CTGF recovers microvessel structure. In this work we demonstrate that BVOs rapidly undergo restructuring in response to metabolic changes and identify CTGF as a critical paracrine regulator of microvascular integrity.

PLAC8-Mediated Activation of NOX4 Signalling Restores Angiogenic Function of Endothelial Colony-Forming Cells in Experimental Hypoxia.

Ischaemic cardiovascular disease is associated with tissue hypoxia as a significant determinant of angiogenic dysfunction and adverse remodelling. While cord blood-derived endothelial colony-forming cells (CB-ECFCs) hold clear therapeutic potential due to their enhanced angiogenic and proliferative capacity, their impaired functionality within the disease microenvironment represents a major barrier to clinical translation. The aim of this study was to define the specific contribution of NOX4 NADPH oxidase, which we previously reported as a key CB-ECFC regulator, to hypoxia-induced dysfunction and its potential as a therapeutic target. CB-ECFCs exposed to experimental hypoxia demonstrated downregulation of NOX4-mediated reactive oxygen species (ROS) signalling linked with a reduced tube formation, which was partially restored by NOX4 plasmid overexpression. siRNA knockdown of placenta-specific 8 (PLAC8), identified by microarray analysis as an upstream regulator of NOX4 in hypoxic versus normoxic CB-ECFCs, enhanced tube formation, NOX4 expression and hydrogen peroxide generation, and induced several key transcription factors associated with downstream Nrf2 signalling. Taken together, these findings indicated that activation of the PLAC8-NOX4 signalling axis improved CB-ECFC angiogenic functions in experimental hypoxia, highlighting this pathway as a potential target for protecting therapeutic cells against the ischaemic cardiovascular disease microenvironment.

Retinal pigment epithelium extracellular vesicles are potent inducers of age-related macular degeneration disease phenotype in the outer retina.

Age-related macular degeneration (AMD) is a leading cause of blindness. Vision loss is caused by the retinal pigment epithelium (RPE) and photoreceptors atrophy and/or retinal and choroidal angiogenesis. Here we use AMD patient-specific RPE cells with the Complement Factor H Y402H high-risk polymorphism to perform a comprehensive analysis of extracellular vesicles (EVs), their cargo and role in disease pathology. We show that AMD RPE is characterised by enhanced polarised EV secretion. Multi-omics analyses demonstrate that AMD RPE EVs carry RNA, proteins and lipids, which mediate key AMD features including oxidative stress, cytoskeletal dysfunction, angiogenesis and drusen accumulation. Moreover, AMD RPE EVs induce amyloid fibril formation, revealing their role in drusen formation. We demonstrate that exposure of control RPE to AMD RPE apical EVs leads to the acquisition of AMD features such as stress vacuoles, cytoskeletal destabilization and abnormalities in the morphology of the nucleus. Retinal organoid treatment with apical AMD RPE EVs leads to disrupted neuroepithelium and the appearance of cytoprotective alpha B crystallin immunopositive cells, with some co-expressing retinal progenitor cell markers Pax6/Vsx2, suggesting injury-induced regenerative pathways activation. These findings indicate that AMD RPE EVs are potent inducers of AMD phenotype in the neighbouring RPE and retinal cells.

Long term high glucose exposure induces premature senescence in retinal endothelial cells.

Purpose: Features of cellular senescence have been described in diabetic retinal vasculature. The aim of this study was to investigate how the high glucose microenvironment impacts on the senescence program of retinal endothelial cells. Methods: Human retinal microvascular endothelial cells were cultured under control and high glucose conditions of 5 mM and 25 mM D-glucose, respectively. Isomeric l-glucose was used as the osmotic control. Cells were counted using CASY technology until they reached their Hayflick limit. Senescence-associated ß-Galactosidase was used to identify senescent cells. Endothelial cell functionality was evaluated by the clonogenic, 3D tube formation, and barrier formation assays. Cell metabolism was characterized using the Seahorse Bioanalyzer. Gene expression analysis was performed by bulk RNA sequencing. Retinal tissues from db/db and db/+ mice were evaluated for the presence of senescent cells. Publicly available scRNA-sequencing data for retinas from Akimba and control mice was used for gene set enrichment analysis. Results: Long term exposure to 25 mM D-Glucose accelerated the establishment of cellular senescence in human retinal endothelial cells when compared to 5 mM D-glucose and osmotic controls. This was shown from 4 weeks, by a significant slower growth, higher percentages of cells positive for senescence-associated ß-galactosidase, an increase in cell size, and lower expression of pRb and HMGB2. These senescence features were associated with decreased clonogenic capacity, diminished tubulogenicity, and impaired barrier function. Long term high glucose-cultured cells exhibited diminished glycolysis, with lower protein expression of GLUT1, GLUT3, and PFKFB3. Transcriptomic analysis, after 4 weeks of culture, identified downregulation of ALDOC, PFKL, and TPI1, in cells cultured with 25 mM D-glucose when compared to controls. The retina from db/db mice showed a significant increase in acellular capillaries associated with a significant decrease in vascular density in the intermediate and deep retinal plexuses, when compared to db/+ mice. Senescent endothelial cells within the db/db retinal vasculature were identified by senescence-associated ß-galactosidase staining. Analysis of single cell transcriptomics data for the Akimba mouse retina highlighted an enrichment of senescence and senescence-associated secretory phenotype gene signatures when compared to control mice. Conclusion: A diabetic-like microenvironment of 25 mM D-glucose was sufficient to accelerate the establishment of cellular senescence in human retinal microvascular endothelial cells.

Advances in cell therapies using stem cells/progenitors as a novel approach for neurovascular repair of the diabetic retina.

BACKGROUND: Diabetic retinopathy, a major complication of diabetes mellitus, is a leading cause of sigh-loss in working age adults. Progressive loss of integrity of the retinal neurovascular unit is a central element in the disease pathogenesis. Retinal ischemia and inflammatory processes drive interrelated pathologies such as blood retinal barrier disruption, fluid accumulation, gliosis, neuronal loss and/or aberrant neovascularisation. Current treatment options are somewhat limited to late-stages of the disease where there is already significant damage to the retinal architecture arising from degenerative, edematous and proliferative pathology. New preventive and interventional treatments to target early vasodegenerative and neurodegenerative stages of the disease are needed to ensure avoidance of sight-loss. MAIN BODY: Historically, diabetic retinopathy has been considered a primarily microvascular disease of the retina and clinically it is classified based on the presence and severity of vascular lesions. It is now known that neurodegeneration plays a significant role during the pathogenesis. Loss of neurons has been documented at early stages in pre-clinical models as well as in individuals with diabetes and, in some, even prior to the onset of clinically overt diabetic retinopathy. Recent studies suggest that some patients have a primarily neurodegenerative phenotype. Retinal pigment epithelial cells and the choroid are also affected during the disease pathogenesis and these tissues may also need to be addressed by new regenerative treatments. Most stem cell research for diabetic retinopathy to date has focused on addressing vasculopathy. Pre-clinical and clinical studies aiming to restore damaged vasculature using vasoactive progenitors including mesenchymal stromal/stem cells, adipose stem cells, CD34+ cells, endothelial colony forming cells and induced pluripotent stem cell derived endothelial cells are discussed in this review. Stem cells that could replace dying neurons such as retinal progenitor cells, pluripotent stem cell derived photoreceptors and ganglion cells as well as Müller stem cells are also discussed. Finally, challenges of stem cell therapies relevant to diabetic retinopathy are considered. CONCLUSION: Stem cell therapies hold great potential to replace dying cells during early and even late stages of diabetic retinopathy. However, due to the presence of different phenotypes, selecting the most suitable stem cell product for individual patients will be crucial for successful treatment.

Current concepts on endothelial stem cells definition, location, and markers.

Ischemic vascular disease is a major cause of mortality and morbidity worldwide, and regeneration of blood vessels in perfusion-deficient tissues is a worthwhile therapeutic goal. The idea of delivering endothelial stem/progenitor cells to repair damaged vasculature, reperfuse hypoxic tissue, prevent cell death, and consequently diminish tissue inflammation and fibrosis has a strong scientific basis and clinical value. Various labs have proposed endothelial stem/progenitor cell candidates. This has created confusion, as there are profound differences between these cell definitions based on isolation methodology, characterization, and reparative biology. Here, a stricter definition based on stem cell biology principles is proposed. Although preclinical studies have often been promising, results from clinical trials have been highly contradictory and served to highlight multiple challenges associated with disappointing therapeutic benefit. This article reviews recent accomplishments in the field and discusses current difficulties when developing endothelial stem cell therapies. Emerging evidence that disputes the classic view of the bone marrow as the source for these cells and supports the vascular wall as the niche for these tissue-resident endothelial stem cells is considered. In addition, novel markers to identify endothelial stem cells, including CD157, EPCR, and CD31low VEGFR2low IL33+ Sox9+ , are described.

miR-130a activates the VEGFR2/STAT3/HIF1α axis to potentiate the vasoregenerative capacity of endothelial colony-forming cells in hypoxia.

Hypoxia modulates reparative angiogenesis, which is a tightly regulated pathophysiological process. MicroRNAs (miRNAs) are important regulators of gene expression in hypoxia and angiogenesis. However, we do not yet have a clear understanding of how hypoxia-induced miRNAs fine-tune vasoreparative processes. Here, we identify miR-130a as a mediator of the hypoxic response in human primary endothelial colony-forming cells (ECFCs), a well-characterized subtype of endothelial progenitors. Under hypoxic conditions of 1% O2, miR-130a gain-of-function enhances ECFC pro-angiogenic capacity in vitro and potentiates their vasoreparative properties in vivo. Mechanistically, miR-130a orchestrates upregulation of VEGFR2, activation of STAT3, and accumulation of HIF1α via translational inhibition of Ddx6. These findings unveil a new role for miR-130a in hypoxia, whereby it activates the VEGFR2/STAT3/HIF1α axis to enhance the vasoregenerative capacity of ECFCs.

The Long Pentraxin PTX3 as a New Biomarker and Pharmacological Target in Age-Related Macular Degeneration and Diabetic Retinopathy.

Age related macular degeneration (AMD) and diabetic retinopathy (DR) are multifactorial, neurodegenerative and inflammatory diseases of the eye primarily involving cellular and molecular components of the outer and inner blood-retina barriers (BRB), respectively. Largely contributed by genetic factors, particularly polymorphisms in complement genes, AMD is a paradigm of retinal immune dysregulation. DR, a major complication of diabetes mellitus, typically presents with increased vascular permeability and occlusion of the retinal vasculature that leads, in the proliferative form of the disease, to neovascularization, a pathogenic trait shared with advanced AMD. In spite of distinct etiology and clinical manifestations, both pathologies share common drivers, such as chronic inflammation, either of immune (in AMD) or metabolic (in DR) origin, which initiates and propagates degeneration of the neural retina, yet the underlying mechanisms are still unclear. As a soluble pattern recognition molecule with complement regulatory functions and a marker of vascular damage, long pentraxin 3 (PTX3) is emerging as a novel player in ocular homeostasis and a potential pharmacological target in neurodegenerative disorders of the retina. Physiologically present in the human eye and induced in inflammatory conditions, this protein is strategically positioned at the BRB interface, where it acts as a "molecular trap" for complement, and modulates inflammation both in homeostatic and pathological conditions. Here, we discuss current viewpoints on PTX3 and retinal diseases, with a focus on AMD and DR, the roles therein proposed for this pentraxin, and their implications for the development of new therapeutic strategies.

Tibetan PHD2, an allele with loss-of-function properties.

Tibetans have adapted to the chronic hypoxia of high altitude and display a distinctive suite of physiologic adaptations, including augmented hypoxic ventilatory response and resistance to pulmonary hypertension. Genome-wide studies have consistently identified compelling genetic signatures of natural selection in two genes of the Hypoxia Inducible Factor pathway, PHD2 and HIF2A The product of the former induces the degradation of the product of the latter. Key issues regarding Tibetan PHD2 are whether it is a gain-of-function or loss-of-function allele, and how it might contribute to high-altitude adaptation. Tibetan PHD2 possesses two amino acid changes, D4E and C127S. We previously showed that in vitro, Tibetan PHD2 is defective in its interaction with p23, a cochaperone of the HSP90 pathway, and we proposed that Tibetan PHD2 is a loss-of-function allele. Here, we report that additional PHD2 mutations at or near Asp-4 or Cys-127 impair interaction with p23 in vitro. We find that mice with the Tibetan Phd2 allele display augmented hypoxic ventilatory response, supporting this loss-of-function proposal. This is phenocopied by mice with a mutation in p23 that abrogates the PHD2:p23 interaction. Hif2a haploinsufficiency, but not the Tibetan Phd2 allele, ameliorates hypoxia-induced increases in right ventricular systolic pressure. The Tibetan Phd2 allele is not associated with hemoglobin levels in mice. We propose that Tibetans possess genetic alterations that both activate and inhibit selective outputs of the HIF pathway to facilitate successful adaptation to the chronic hypoxia of high altitude.

NOX4 is a major regulator of cord blood-derived endothelial colony-forming cells which promotes post-ischaemic revascularization.

AIMS: Cord blood-derived endothelial colony-forming cells (CB-ECFCs) are a defined progenitor population with established roles in vascular homeostasis and angiogenesis, which possess low immunogenicity and high potential for allogeneic therapy and are highly sensitive to regulation by reactive oxygen species (ROS). The aim of this study was to define the precise role of the major ROS-producing enzyme, NOX4 NADPH oxidase, in CB-ECFC vasoreparative function. METHODS AND RESULTS: In vitro CB-ECFC migration (scratch-wound assay) and tubulogenesis (tube length, branch number) was enhanced by phorbol 12-myristate 13-acetate (PMA)-induced superoxide in a NOX-dependent manner. CB-ECFCs highly-expressed NOX4, which was further induced by PMA, whilst NOX4 siRNA and plasmid overexpression reduced and potentiated in vitro function, respectively. Increased ROS generation in NOX4-overexpressing CB-ECFCs (DCF fluorescence, flow cytometry) was specifically reduced by superoxide dismutase, highlighting induction of ROS-specific signalling. Laser Doppler imaging of mouse ischaemic hindlimbs at 7 days indicated that NOX4-knockdown CB-ECFCs inhibited blood flow recovery, which was enhanced by NOX4-overexpressing CB-ECFCs. Tissue analysis at 14 days revealed consistent alterations in vascular density (lectin expression) and eNOS protein despite clearance of injected CB-ECFCs, suggesting NOX4-mediated modulation of host tissue. Indeed, proteome array analysis indicated that NOX4-knockdown CB-ECFCs largely suppressed tissue angiogenesis, whilst NOX4-overexpressing CB-ECFCs up-regulated a number of pro-angiogenic factors specifically-linked with eNOS signalling, in parallel with equivalent modulation of NOX-dependent ROS generation, suggesting that CB-ECFC NOX4 signalling may promote host vascular repair. CONCLUSION: Taken together, these findings indicate a key role for NOX4 in CB-ECFCs, thereby highlighting its potential as a target for enhancing their reparative function through therapeutic priming to support creation of a pro-reparative microenvironment and effective post-ischaemic revascularization.

Vascular Regeneration for Ischemic Retinopathies: Hope from Cell Therapies.

Retinal vascular diseases, such as diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, ocular ischemic syndrome and ischemic optic neuropathy, are leading causes of vision impairment and blindness. Whilst drug, laser or surgery-based treatments for the late stage complications of many of these diseases are available, interventions that target the early vasodegenerative stages are lacking. Progressive vasculopathy and ensuing ischemia is an underpinning pathology in many of these diseases, leading to hypoperfusion, hypoxia, and ultimately pathological neovascularization and/or edema in the retina and other ocular tissues, such as the optic nerve and iris. Therefore, repairing the retinal vasculature may prevent progression of ischemic retinopathies into late stage vascular complications. Various cell types have been explored for their vascular repair potential. Endothelial progenitor cells, mesenchymal stem cells and induced pluripotent stem cells are studied for their potential to integrate with the damaged retinal vasculature and limit ischemic injury. Clinical trials for some of these cell types have confirmed safety and feasibility in the treatment of ischemic diseases, including some retinopathies. Another promising avenue is mobilization of endogenous endothelial progenitors, whereby reparative cells are moved from their niche to circulating blood to target and home into ischemic tissues. Several aspects and properties of these cell types have yet to be elucidated. Nevertheless, we foresee that cell therapy, whether through delivery of exogenous or enhancement of endogenous reparative cells, will become a valuable and beneficial treatment for ischemic retinopathies.

No evidence of Gremlin1-mediated activation of VEGFR2 signaling in endothelial cells.

Canonical Gremlin1 (GREM1) signaling involves binding to and sequestering bone morphogenetic proteins (BMPs) in the extracellular matrix, preventing the activation of cognate BMP receptor. Exquisite temporospatial control of the GREM1-BMP interaction is required during development, and perturbation of this balance leads to abnormal limb formation and defective kidney development. In addition to inhibition of BMP signaling, several other noncanonical signaling modalities of GREM1 have been postulated. Some literature reports have suggested that GREM1 can bind to and activate vascular endothelial growth factor receptor-2 (VEGFR2) in endothelial cells, human kidney epithelial cells, and others. These reports suggest that the GREM1 → VEGFR2 signaling can drive angiogenesis both in vitro and in vivo We report here that, despite exhaustive attempts, we did not observe GREM1 activation of VEGFR2 in any of the cell lines reported by the above-mentioned studies. Incubation of endothelial colony-forming cells (ECFCs) or human umbilical vein endothelial cells (HUVECs) with recombinant VEGF triggered a robust increase in VEGFR2 tyrosine phosphorylation. In contrast, no VEGFR2 phosphorylation was detected when cells were incubated with recombinant GREM1 over a range of time points and concentrations. We also show that GREM1 does not interfere with VEGF-mediated VEGFR2 activation, suggesting that GREM1 does not bind with any great affinity to VEGFR2. Measurements of ECFC barrier integrity revealed that VEGF induces barrier function disruption, but recombinant human GREM1 had no effect in this assay. We believe that these results provide an important clarification of the potential interaction between GREM1 and VEGFR2 in mammalian cells.

Therapies for Type 1 Diabetes: Current Scenario and Future Perspectives.

Type 1 diabetes (T1D) is caused by autoimmune destruction of insulin-producing ß cells located in the endocrine pancreas in areas known as islets of Langerhans. The current standard-of-care for T1D is exogenous insulin replacement therapy. Recent developments in this field include the hybrid closed-loop system for regulated insulin delivery and long-acting insulins. Clinical studies on prediction and prevention of diabetes-associated complications have demonstrated the importance of early treatment and glucose control for reducing the risk of developing diabetic complications. Transplantation of primary islets offers an effective approach for treating patients with T1D. However, this strategy is hampered by challenges such as the limited availability of islets, extensive death of islet cells, and poor vascular engraftment of islets post-transplantation. Accordingly, there are considerable efforts currently underway for enhancing islet transplantation efficiency by harnessing the beneficial actions of stem cells. This review will provide an overview of currently available therapeutic options for T1D, and discuss the growing evidence that supports the use of stem cell approaches to enhance therapeutic outcomes.

Selection of a Real-Time PCR Housekeeping Gene Panel in Human Endothelial Colony Forming Cells for Cellular Senescence Studies.

Endothelial Colony Forming Cells (ECFCs) represent a subset of endothelial progenitors with well-documented vasoreparative capacity. However, cellular senescence, which occurs due to aging, diabetes, smoking, or tissue inflammation, renders these cells dysfunctional. Therefore, there is growing interest in studying expression of senescence markers in ECFCs. RT-qPCR is the most commonly used technique to quantify gene expression and the proper choice of reference genes used for data normalization is critical for accurate quantification. It has been reported that the expression of commonly used housekeeping genes is often unstable in senescence. To identify the most suitable reference genes for ECFC senescence studies, we analyzed a microarray dataset, which compared the gene expression between proliferating and senescent ECFCs. In addition to replicative senescence, the data included X-ray-induced and Etoposide-induced senescence. We used the geNorm algorithm to identify the most stable genes across all studied conditions. Gene Ontology analysis found that the most stable genes belonged to the KEGG category of Genetic Information Processing. The optimal combination of housekeeping genes for ECFC senescence was found to include four ribosomal protein genes; RPL13, RPL31, RPL37, and RPL30. The RT-qPCR validation confirmed that normalization with our novel panel was more sensitive in identifying senescence markers compared to commonly used genes such as ACTB, UBC, and GAPDH.

The vasoreparative potential of endothelial colony-forming cells in the ischemic retina is enhanced by cibinetide, a non-hematopoietic erythropoietin mimetic.

PURPOSE: Retinal ischemia remains a common sight threatening end-point in blinding diseases such as diabetic retinopathy and retinopathy of prematurity. Endothelial colony forming cells (ECFCs) represent a subpopulation of endothelial progenitors with therapeutic utility for promoting reparative angiogenesis in the ischaemic retina. The current study has investigated the potential of enhancing this cell therapy approach by the dampening of the pro-inflammatory milieu typical of ischemic retina. Based on recent findings that ARA290 (cibinetide), a peptide based on the Helix-B domain of erythropoietin (EPO), is anti-inflammatory and tissue-protective, the effect of this peptide on ECFC-mediated vascular regeneration was studied in the ischemic retina. METHODS: The effects of ARA290 on pro-survival signaling and function were assessed in ECFC cultures in vitro. Efficacy of ECFC transplantation therapy to promote retinal vascular repair in the presence and absence of ARA290 was studied in the oxygen induced retinopathy (OIR) model of retinal ischemia. The inflammatory cytokine profile and microglial activation were studied as readouts of inflammation. RESULTS: ARA290 activated pro-survival signaling and enhanced cell viability in response to H2O2-mediated oxidative stress in ECFCs in vitro. Preconditioning of ECFCs with EPO or ARA290 prior to delivery to the ischemic retina did not enhance vasoreparative function. ARA290 delivered systemically to OIR mice reduced pro-inflammatory expression of IL-1ß and TNF-α in the mouse retina. Following intravitreal transplantation, ECFCs incorporated into the damaged retinal vasculature and significantly reduced avascular area. The vasoreparative function of ECFCs was enhanced in the presence of ARA290 but not EPO. DISCUSSION: Regulation of the pro-inflammatory milieu of the ischemic retina can be enhanced by ARA290 and may be a useful adjunct to ECFC-based cell therapy for ischemic retinopathies.

The Vasoreparative Potential of Endothelial Colony Forming Cells: A Journey Through Pre-clinical Studies.

For over a decade various cell populations have been investigated for their vasoreparative potential. Cells with the capacity to promote blood vessel regeneration are commonly known as endothelial progenitor cells (EPCs); although such a definition is currently considered too simple for the complexity of cell populations involved in the reparative angiogenic process. A subset of EPCs called endothelial colony forming cells (ECFCs) have emerged as a suitable candidate for cytotherapy, primarily due to their clonogenic progenitor characteristics, unequivocal endothelial phenotype, and inherent ability to promote vasculogenesis. ECFCs can be readily isolated from human peripheral and cord blood, expanded ex vivo and used to revascularize ischemic tissues. These cells have demonstrated efficacy in several in vivo preclinical models such as the ischemic heart, retina, brain, limb, lung and kidney. This review will summarize the current pre-clinical evidence for ECFC cytotherapy and discuss their potential for clinical application.

Characterization of a murine mixed neuron-glia model and cellular responses to regulatory T cell-derived factors.

One of the unmet clinical needs in demyelinating diseases such as Multiple Sclerosis (MS) is to provide therapies that actively enhance the process of myelin regeneration (remyelination) in the central nervous system (CNS). Oligodendrocytes, the myelinating cells of the CNS, play a central role in remyelination and originate from oligodendrocyte progenitor cells (OPCs). We recently showed that depletion of regulatory T cells (Treg) impairs remyelination in vivo, and that Treg-secreted factors directly enhance oligodendrocyte differentiation. Here we aim to further characterize the dynamics of Treg-enhanced oligodendrocyte differentiation as well as elucidate the cellular components of a murine mixed neuron-glia model. Murine mixed neuron-glia cultures were generated from P2-7 C57BL/6 mice and characterized for percentage of neuronal and glial cell populations prior to treatment at 7 days in vitro (div) as well as after treatment with Treg-conditioned media at multiple timepoints up to 12 div. Mixed neuron-glia cultures consisted of approximately 30% oligodendroglial lineage cells, 20% neurons and 10% microglia. Furthermore, a full layer of astrocytes, that could not be quantified, was present. Treatment with Treg-conditioned media enhanced the proportion of MBP+ oligodendrocytes and decreased the proportion of PDGFRα+ OPCs, but did not affect OPC proliferation or survival. Treg-enhanced oligodendrocyte differentiation was not caused by Treg polarizing factors, was dependent on the number of activation cycles Treg underwent and was robustly achieved by using 5% conditioned media. These studies provide in-depth characterization of a murine mixed neuron-glia model as well as further insights into the dynamics of Treg-enhanced oligodendrocyte differentiation.

The Vasoreparative Function of Myeloid Angiogenic Cells Is Impaired in Diabetes Through the Induction of IL1ß.

Myeloid angiogenic cells (MACs) promote revascularization through the paracrine release of angiogenic factors and have been harnessed as therapeutic cells for many ischemic diseases. However, their proangiogenic properties have been suggested to be diminished in diabetes. This study investigates how the diabetic milieu affects the immunophenotype and function of MACs. Both MACs isolated from diabetic conditions and healthy cells exposed to a diabetic environment were used to determine the potential of MACs as a cell therapy for diabetic-related ischemia. MACs were isolated from human peripheral blood and characterized alongside proinflammatory macrophages M (LPS + IFNγ) and proangiogenic macrophages M (IL4). Functional changes in MACs in response to high-d-glucose were assessed using an in vitro 3D-tubulogenesis assay. Phenotypic changes were determined by gene and protein expression analysis. Additionally, MACs from type 1 diabetic (T1D) patients and corresponding controls were isolated and characterized. Our evidence demonstrates MACs identity as a distinct macrophage subtype that shares M2 proangiogenic characteristics, but can be distinguished by CD163hi expression. High-d-glucose treatment significantly reduced MACs proangiogenic capacity, which was associated with a significant increase in IL1ß mRNA and protein expression. Inhibition of IL1ß abrogated the antiangiogenic effect induced by high-d-glucose. IL1ß was also significantly upregulated in MACs isolated from T1D patients with microvascular complications compared to T1D patients without microvascular complications or nondiabetic volunteers. This study demonstrates that Type 1 diabetes and diabetic-like conditions impair the proangiogenic and regenerative capacity of MACs, and this response is mediated by IL-1ß. Stem Cells 2018;36:834-843.

Preclinical Evaluation and Optimization of a Cell Therapy Using Human Cord Blood-Derived Endothelial Colony-Forming Cells for Ischemic Retinopathies.

Cell therapy using endothelial progenitors holds promise for vascular repair in ischemic retinopathies. Using a well-defined subpopulation of human cord blood-derived endothelial progenitors known as endothelial colony-forming cells (ECFCs), we have evaluated essential requirements for further development of this cell therapy targeting the ischemic retina, including dose response, delivery route, and toxicity. First, to evaluate therapeutic efficacy relating to cell dose, ECFCs were injected into the vitreous of mice with oxygen-induced retinopathy. Using angiography and histology, we found that intravitreal delivery of low dose (1 × 103 ) ECFCs was as effective as higher cell doses (1 × 104 , 1 × 105 ) in promoting vascular repair. Second, injection into the common carotid artery was tested as an alternative, systemic delivery route. Intracarotid ECFC delivery conferred therapeutic benefit which was comparable to intravitreal delivery using the same ECFC dose (1 × 105 ), although there were fewer human cells observed in the retinal vasculature following systemic delivery. Third, cell immunogenicity was evaluated by injecting ECFCs into the vitreous of healthy adult mice. Assessment of murine ocular tissues identified injected cells in the vitreous, while demonstrating integrity of the host retina. In addition, ECFCs did not invade into the retina, but remained in the vitreous, where they eventually underwent cell death within 3 days of delivery without evoking an inflammatory response. Human specific Alu sequences were not found in healthy mouse retinas after 3 days of ECFC delivery. These findings provide supportive preclinical evidence for the development of ECFCs as an efficacious cell product for ischemic retinopathies. Stem Cells Translational Medicine 2018;7:59-67.