BackgroundEarly-stage diabetic retinopathy (DR) is characterized by neurovascular defects. In this study, we hypothesized that human adipose-derived stem cells (ASCs) positive for the pericyte marker CD140b, or their secreted paracrine factors, therapeutically rescue early-stage DR features in an Ins2Akita mouse model.MethodsIns2Akita mice at 24 weeks of age received intravitreal injections of CD140b-positive ASCs (1000 cells/1 μL) or 20× conditioned media from cytokine-primed ASCs (ASC-CM, 1 μL). Age-matched wildtype mice that received saline served as controls. Visual function experiments and histological analyses were performed 3 weeks post intravitreal injection. Biochemical and molecular analyses assessed the ASC-CM composition and its biological effects.ResultsThree weeks post-injection, Ins2Akita mice that received ASCs had ameliorated decreased b-wave amplitudes and vascular leakage but failed to improve visual acuity, whereas Ins2Akita mice that received ASC-CM demonstrated amelioration of all aforementioned visual deficits. The ASC-CM group demonstrated partial amelioration of retinal GFAP immunoreactivity and DR-related gene expression but the ASC group did not. While Ins2Akita mice that received ASCs exhibited occasional (1 in 8) hemorrhagic retinas, mice that received ASC-CM had no adverse complications. In vitro, ASC-CM protected against TNFα-induced retinal endothelial permeability as measured by transendothelial electrical resistance. Biochemical and molecular analyses demonstrated several anti-inflammatory proteins including TSG-6 being highly expressed in cytokine-primed ASC-CM.ConclusionsASCs or their secreted factors mitigate retinal complications of diabetes in the Ins2Akita model. Further investigation is warranted to determine whether ASCs or their secreted factors are safe and effective therapeutic modalities long-term as current locally delivered therapies fail to effectively mitigate the progression of early-stage DR. Nonetheless, our study sheds new light on the therapeutic mechanisms of adult stem cells, with implications for assessing relative risks/benefits of experimental regenerative therapies for vision loss.Electronic supplementary materialThe online version of this article (10.1186/s13287-018-1059-y) contains supplementary material, which is available to authorized users.

Diabetic retinopathy (DR), a complication of diabetes, is one of the leading causes of blindness in working-age adults. The pathology of the disease prevents the endogenous stem cells from participating in the natural repair of the diseased retina. Current treatments, specifically stem cell therapeutics, have shown variable efficacy in preclinical models due to the multi-faceted nature of the disease. Among the various adult stem cells, mesenchymal stem cells, especially those derived from adipose tissue and bone marrow, have been explored as a possible treatment for DR. This review summarizes the current literature around the various adult stem cell treatments for the disease and outlines the benefits and limitations of the therapeutics that are being explored in the field. The paracrine nature of adipose stem cells, in particular, has been highlighted as a potential solution to the lack of a homing and conducive environment that poses a challenge to the implantation of exogenous stem cells in the target tissue. Various methods of mesenchymal stem cell priming to adapt to a hostile retinal microenvironment have been discussed. Current clinical trials and potential safety concerns have been examined, and the future directions of stem cell therapeutics in DR have also been contemplated.

PurposeOur goal was to generate and characterize a new mouse model in which only angiogenesis- and glaucoma-relevant tissues would be naturally fluorescent. The Matrix Gla (MGP) gene is highly expressed in vascular smooth muscle cells (VSMC) and trabecular meshwork (TM). We sought to direct our Mgp-Cre.KI mouse recombinase to VSMC/TM cells to produce their longitudinal fluorescent profiles.MethodsHomozygous Mgp-Cre.KI mice were crossed with Ai9 homozygous reporter mice harboring a loxP-flanked STOP cassette preventing transcription of a DsRed fluorescent protein (tdTomato). The F1 double-heterozygous (Mgp-tdTomato) was examined by direct fluorescence, whole mount, histology, and fundus photography. Custom-made filters had 554/23 emission and 609/54 exciter nanometer wavelengths. Proof of concept of the model's usefulness was conducted by inducing guided imaging laser burns. Evaluation of a vessel's leakage and proliferation was followed by noninvasive angiography.ResultsThe Mgp-tdTomato mouse was viable, fertile, with normal IOP and ERG. Its phenotype exhibited red paws and snout (cartilage expression), which precluded genotyping. A fluorescent red ring was seen at the limbus and confirmed to be TM expression by histology. The entire retinal vasculature was red fluorescent (VSMC) and directly visualized by fundus photography. Laser burns on the Mgp-tdTomato allowed separation of leakiness and neovascularization evaluation parameters.ConclusionsThe availability of a transgenic mouse naturally fluorescent in glaucoma-relevant tissues and retinal vasculature brings the unique opportunity to study a wide spectrum of single and combined glaucomatous conditions in vivo. Moreover, the Mgp-tdTomato mouse provides a new tool to study mechanisms and therapeutics of retinal angiogenesis longitudinally.

Adipose-derived stem cells (ASCs) are multipotent mesenchymal progenitor cells that have functional and phenotypic overlap with pericytes lining microvessels in adipose tissue. The role of CD140b [platelet-derived growth factor receptor-β (PDGFR-β)], a constitutive marker expressed by ASCs, in the angiogenic behavior of human retinal endothelial cells (HREs) is not known. CD140b was knocked down in ASCs using targeted siRNA and lipofectamine transfection protocol. Both CD140b+ and CD140b− ASCs were tested for their proliferation (WST-1 reagent), adhesion (laminin-1 coated plates), and migration (wound-scratch assay). Angiogenic effect of CD140b+ and CD140b− ASCs on HREs was examined by co-culturing ASCs:HREs in 12:1 ratio for 6 days followed by visualization of vascular network by Isolectin B4 staining. The RayBio® Membrane-Based Antibody Array was used to assess differences in human cytokines released by CD140b+ or CD140b− ASCs. Knockdown of CD140b in ASCs resulted in a significant 50% decrease in proliferation rate, 25% decrease in adhesion ability to Laminin-1, and 50% decrease in migration rate, as compared to CD140b+ ASCs. Direct contact of ASCs expressing CD140b+ with HREs resulted in robust vascular network formation that was significantly reduced with using CD140b− ASCs. Of the 80 proteins tested, 45 proteins remained unchanged (>0.5-<1.5 fold), 6 proteins including IL-10 downregulated (<0.5 fold) and 29 proteins including IL-16 & TNF-β were upregulated (>1.5 fold) in CD140b− ASCs compared to CD140b+ ASCs. Our data demonstrate a substantial role for CD140b in the intrinsic abilities of ASCs and their angiogenic influence on HREs. Future studies are needed to fully explore the signaling of CD140b in ASCs in vivo for retinal regeneration.

Endothelial progenitor cells (EPCs) promote revascularization and tissue repair mainly by paracrine actions. In the present study, we investigated whether EPC-secreted factors in the form of conditioned medium (EPC-CM) can protect cultured brain microvascular endothelial cells against an ischemic insult. Furthermore, we addressed the type of factors that are involved in the EPC-CM-mediated functions. For that purpose, rat brain-derived endothelial cells (rBCEC4 cell line) were exposed to EPC-CM pretreated with proteolytic digestion, heat inactivation, and lipid extraction. Moreover, the involvement of VEGF and IL-8, as canonical angiogenic factors, was investigated by means of neutralizing antibodies. We demonstrated that EPC-CM significantly protected the rBCEC4 cells against an ischemic insult mimicked by induced oxygen-glucose deprivation followed by reoxygenation. The cytoprotective effect was displayed by higher viable cell numbers and reduced caspase 3/7 activity. Heat inactivation, proteolytic digestion, and lipid extraction resulted in a significantly reduced EPC-CM-dependent increase in rBCEC4 viability, tube formation, and survival following the ischemic challenge. Notably, VEGF and IL-8 neutralization did not affect the actions of EPC-CM on rBCEC4 under both standard and ischemic conditions. In summary, our findings show that paracrine factors released by EPCs activate an angiogenic and cytoprotective response on brain microvascular cells and that the activity of EPC-CM relies on the concerted action of nonproteinaceous and proteinaceous factors but do not directly involve VEGF and IL-8.