Reconstituted Extracellular Vesicles from Human Platelets Decrease Viral Myocarditis in Mice.

Patients with viral myocarditis are at risk of sudden death and may progress to dilated cardiomyopathy (DCM). Currently, no disease-specific therapies exist to treat viral myocarditis. Here it is examined whether reconstituted, lyophilized extracellular vesicles (EVs) from platelets from healthy men and women reduce acute or chronic myocarditis in male mice. Human-platelet-derived EVs (PEV) do not cause toxicity, damage, or inflammation in naïve mice. PEV administered during the innate immune response significantly reduces myocarditis with fewer epidermal growth factor (EGF)-like module-containing mucin-like hormone receptor-like 1 (F4/80) macrophages, T cells (cluster of differentiation molecules 4 and 8, CD4 and CD8), and mast cells, and improved cardiac function. Innate immune mediators known to increase myocarditis are decreased by innate PEV treatment including Toll-like receptor (TLR)4 and complement. PEV also significantly reduces perivascular fibrosis and remodeling including interleukin 1 beta (IL-1ß), transforming growth factor-beta 1, matrix metalloproteinase, collagen genes, and mast cell degranulation. PEV given at days 7-9 after infection reduces myocarditis and improves cardiac function. MicroRNA (miR) sequencing reveals that PEV contains miRs that decrease viral replication, TLR4 signaling, and T-cell activation. These data show that EVs from the platelets of healthy individuals can significantly reduce myocarditis and improve cardiac function.

Platelet-derived exosomes induce cell proliferation and wound healing in human endometrial cells.

Aim: To investigate the regenerative effects of a platelet-derived purified exosome product (PEP) on human endometrial cells. Materials & methods: Endometrial adenocarcinoma cells (HEC-1A), endometrial stromal cells (T HESC) and menstrual blood-derived stem cells (MenSC) were assessed for exosome absorption and subsequent changes in cell proliferation and wound healing properties over 48 h. Results: Cell proliferation increased in PEP treated T HESC (p < 0.0001) and MenSC (p < 0.001) after 6 h and in HEC-1A (p < 0.01) after 12 h. PEP improved wound healing after 6 h in HEC-1A (p < 0.01) and MenSC (p < 0.0001) and in T HESC between 24 and 36 h (p < 0.05). Conclusion: PEP was absorbed by three different endometrial cell types. PEP treatment increased cell proliferation and wound healing capacity.

The uterus has a remarkable ability to heal itself. Every month the inside lining of the uterus grows in preparation for pregnancy and sheds if no pregnancy occurs. Unfortunately, this cycle of growth, shedding and repair can be injured and lead to menstrual changes, pain or even infertility. In this study, we looked how special cell messengers ­ called exosomes ­ could help uterine cells. Exosomes are special messengers that contain substances to help the body heal and regenerate injured cells and tissues. We obtained exosomes created from human transfusion-grade platelets. We studied the exosomes' effects in three different cell types that all are important inside the uterine lining. Specifically, we studied the ability of the exosomes to help cells proliferate and migrate into a wound. In this study, exosomes were recognized by the human endometrial cells and were absorbed. Once they were inside the cells, they increased cell proliferation as well as the ability of the cells to heal a scratch wound. Furthermore, the more exosomes we presented to the cells, the more the cells were able to proliferate and move into a wound for healing. These findings lay the groundwork for future studies in animal models of uterine injury.

Exosome-Induced Vaginal Tissue Regeneration in a Porcine Mesh Exposure Model.

OBJECTIVES: The purpose of this study was to explore the utility of an injectable purified exosome product derived from human apheresis blood to (1) augment surgical closure of vaginal mesh exposures, and (2) serve as a stand-alone therapy for vaginal mesh exposure. METHODS: Sixteen polypropylene meshes (1×1-3×3 cm) were implanted in the vaginas of 7 Yorkshire-crossed pigs by urogynecologic surgeons (day 0). On day 7, group 1 underwent surgical intervention via vaginal tissue suture reclosure with (n=2 pigs, n=4 meshes) or without (n=2 pigs, n=4 meshes) exosome injection; group 2 underwent medical intervention with an exosome injection (n=3, n=8 meshes). One animal in group 2 was given oral 2'-deoxy-5-ethynyluridine to track cellular regeneration. Euthansia occurred at 5 weeks. RESULTS: Mesh exposures treated with surgical closure alone experienced reexposure of the mesh. Exosome treatment with or without surgical closure resulted in partial to full mesh exposure resolution up to 3×3 cm. Exosome-treated tissues had significantly thicker regenerated epithelial tissue (208 µm exosomes-only and 217 µm surgery+exosomes, versus 80 µm for surgery-only; P < 0.05); evaluation of 2'-deoxy-5-ethynyluridine confirmed de novo regeneration throughout the epithelium and underlying tissues. Capillary density was significantly higher in the surgery+exosomes group (P = 0.03). Surgery-only tissues had a higher inflammatory and fibrosis response as compared with exosome-treated tissues. CONCLUSIONS: In this pilot study, exosome treatment augmented healing in the setting of vaginal mesh exposure, reducing the incidence of mesh reexposure after suture closure and decreasing the area of mesh exposure through de novo tissue regeneration after exosome injection only. Further study of varied local tissue conditions and mesh configurations is warranted.

Impact of Repeat Dosing and Mesh Exposure Chronicity on Exosome-Induced Vaginal Tissue Regeneration in a Porcine Mesh Exposure Model.

OBJECTIVE: The aim of the study was to compare vaginal wound healing after exosome injection in a porcine mesh exposure model with (1) single versus multiple dose regimens and (2) acute versus subacute exposure. METHODS: Six 80-kg Yorkshire-crossed swine each had 2 polypropylene meshes implanted to create the vaginal mesh exposure model. Animals were divided into 3 groups based on number and timing of exosome injection: (1) single purified exosome product (PEP) injection (acute-single), (2) weekly PEP injections (acute-weekly, 4 total injections), and (3) delayed single injection (subacute-single). Acute and subacute injections occurred 1 and 8 weeks after mesh implantation, respectively. EdU, a thymidine analog, was given twice weekly after the first injection to track tissue regeneration. Euthanasia and tissue analysis occurred 4 weeks after the first injection. ImageJ was used to quantify epithelial thickness, cellular proliferation, and capillary density. Statistical analysis was performed using analysis of variance and post hoc Tukey test. RESULTS: Acute-single PEP injection tissues mirrored pilot study results, validating replication of protocol. Within the acute groups, weekly dosing resulted in 1.5× higher epithelial thickness (nonsignificant), 1.8× higher epithelial proliferation (P < 0.05), and 1.5× higher regenerated capillary density (P < 0.05) compared with single injection. Regarding chronicity of the exposure, the subacute group showed 1.7× higher epithelial proliferation (nonsignificant) and similar capillary density and epithelial thickness as compared with the acute group. CONCLUSIONS: Exosome redosing resulted in significantly greater epithelial proliferation with significantly higher regenerated capillary density, leading to a trend toward thicker epithelium. Subacute exposure exhibited similar regeneration to acute exposure despite a delayed injection timeline. These results contribute to a growing body of preclinical research demonstrating utility of exosomes in pelvic floor disorders.

Collagen and Fractionated Platelet-Rich Plasma Scaffold for Dermal Regeneration.

BACKGROUND: Current options for in vivo regeneration of dermal tissue remain limited. The purpose of this study was to engineer a unique scaffold capable of recruiting dermal stem cells from adjacent tissue, thus circumventing the need to seed the scaffolds with stem cells before implantation, leading to skin regeneration. METHODS: A hydrogel scaffold was created through combination of type I collagen along with fractionated platelet-rich plasma. This was compared to a control hydrogel consisting of type I collagen and fetal bovine serum. Hydrogels were cultured with fresh human skin tissue and incubated with supplemental media. Gels were digested weekly for cellular content as examined by flow cytometry at the 4- and 8-week time points. The fractionated platelet-rich plasma and collagen gels were then implanted onto full-thickness skin defects on the backs of rats and compared to wounds healing by secondary intention. Wound area was evaluated for epithelialization and neovascularization. RESULTS: Platelet-rich plasma fractionation increased platelet-derived growth factors. In contrast to collagen scaffolds, fractionated platelet-rich plasma-supplemented scaffolds recruited more dermal-derived stem cells from fresh skin tissue compared with collagen hydrogels at the 4- and 8-week time points. Furthermore, fractionated platelet-rich plasma-supplemented hydrogels accelerated wound healing, angiogenesis, and hair and sweat gland formation, ultimately regenerating a dermis-like tissue. CONCLUSIONS: Generation of hydrogels with fractionated platelet-rich plasma was able to improve cellular recruitment and growth and differentiation of dermal-derived stem cells, leading to hair growth and sweat gland formation. This provides a novel approach to regenerate skin for treating large defects.

An Athymic Rat Model for Mandibular Osteoradionecrosis Allowing for Direct Translation of Regenerative Treatments.

OBJECTIVE: We aim to create a model of mandibular osteoradionecrosis in athymic rats. Athymic rats provide an immunosuppressed environment whereby human stem cells and biomaterials can be used to investigate regenerative solutions for osteoradionecrosis, bridging the gap between in vivo testing and clinical application. STUDY DESIGN: Prospective animal study. SETTING: Academic otolaryngology department laboratory. SUBJECTS AND METHODS: After Institutional Animal Care and Use Committee approval, 10 athymic nude rats were divided into 2 groups. The experimental group (n = 6) underwent irradiation (20 Gy), while the control group (n = 4) underwent sham irradiation catheter placement only. All 10 rats underwent extraction of the second mandibular molar 7 days later. The rats were sacrificed 28 days after dental extraction, and their mandibles were harvested. The mandibles were examined with histologic analysis and bone volume analysis based on 3-dimensional micro-computed tomography. RESULTS: All 10 rats survived the experiment period. Radiographic and histologic analysis revealed decreased bone formation in the experimental group compared with the control group. Jaw region volume ratio was 0.83 for the experimental group versus 0.97 in the control group (P = .003). The region-of-interest volume ratio was 0.75 in the experimental group and 0.97 in the control group (P = .005). Histologically, there were increased osteoclasts (P = .02) and decreased osteoblasts (P = .001) as well as increased fibrosis in the experimental group versus the control group. CONCLUSION: Mandibular osteoradionecrosis can be effectively and reproducibly produced in an athymic rat model. This will allow further research to study regenerative medicine in an athymic rat model.

Adipose-derived Mesenchymal Stem Cells Are Phenotypically Superior for Regeneration in the Setting of Osteonecrosis of the Femoral Head.

BACKGROUND: Bone marrow-derived mesenchymal stem cells (bmMSCs) have been used as a cellular therapeutic option for treatment of osteonecrosis of the femoral head. However, use of bmMSCs as a treatment adjuvant for orthopaedic disorders in general has achieved limited success. Adipose-derived MSCs (aMSCs) may be a more-efficient regenerative cell source given their greater quantity and protection from physiologic stress. QUESTIONS/PURPOSES: We asked the following questions in a paired analysis of MSCs from patients with osteonecrosis: (1) Is there a difference in proliferation potential between aMSCs and bmMSCs? (2) Is there a difference in osteogenic differentiation potential between aMSCs and bmMSCs? (3) Are genetic pathways differentially expressed between aMSCs and bmMSCs that may govern functional phenotypic discrepancies? METHODS: Periarticular samples of adipose tissue and bone marrow from the femoral canal were obtained from 15 patients undergoing hip replacement for late-stage (Steinberg Stages III-VI) osteonecrosis. MSCs were isolated from both tissue sources and taken through a standardized 20-day cell division protocol to establish cumulative cell count. They also were grown in osteogenic differentiation media for 14 days with subsequent measurement of alkaline phosphatase in units of optical density. RNA was isolated from aMSCs and bmMSCs in five patients to assess differentially expressed genetic pathways using the Affymetrix GeneChip Human Transcriptome Array 2.0 platform. RESULTS: Proliferation capacity was increased by fourfold in aMSCs compared with bmMSCs after 20 days in culture. The mean difference in cumulative cell count was 3.99 × 10(8) cells (SD = 1.67 × 10(8) cells; 95% CI, 3.07 × 10(8)-4.92 × 10(8) cells; p < 0.001). Bone differentiation efficiency as measured by optical density was increased by 2.25-fold in aMSCs compared with bmMSCs. The mean difference in optical density was 1.27 (SD = 0.34; 95% CI, 1.08-1.46; p < 0.001). RNA transcriptome analysis showed 284 genes that met statistical (p < 0.05) and biological (fold change > 1.5) significance cutoffs for differential expression between cell populations. Subsequent network topology of differentially expressed genes showed alterations in pathways critical for musculoskeletal tissue development in addition to many nonspecific findings. CONCLUSIONS: aMSCs outperform bmMSCs in growth rate and bone differentiation potential in the setting of osteonecrosis, suggesting they may provide a more-potent regenerative therapeutic strategy in this population. Differential expression of genes and cellular pathways highlighted in this study may provide therapeutic targets for cellular optimization or acellular treatment strategies. CLINICAL RELEVANCE: aMSCs may provide a more robust cellular therapeutic than bmMSCs for treatment of osteonecrosis. Ideally, a well-designed prospective study will be able to evaluate the efficacy of these cellular therapies side-by-side in patients with bilateral early stage disease.

Optimized delivery system achieves enhanced endomyocardial stem cell retention.

BACKGROUND: Regenerative cell-based therapies are associated with limited myocardial retention of delivered stem cells. The objective of this study is to develop an endocardial delivery system for enhanced cell retention. METHODS AND RESULTS: Stem cell retention was simulated in silico using 1- and 3-dimensional models of tissue distortion and compliance associated with delivery. Needle designs, predicted to be optimal, were accordingly engineered using nitinol, a nickel and titanium alloy displaying shape memory and superelasticity. Biocompatibility was tested with human mesenchymal stem cells. Experimental validation was performed with species-matched cells directly delivered into Langendorff-perfused porcine hearts or administered percutaneously into the endocardium of infarcted pigs. Cell retention was quantified by flow cytometry and real-time quantitative polymerase chain reaction methodology. Models, computing optimal distribution of distortion calibrated to favor tissue compliance, predicted that a 75°-curved needle featuring small-to-large graded side holes would ensure the highest cell retention profile. In isolated hearts, the nitinol curved needle catheter (C-Cath) design ensured 3-fold superior stem cell retention compared with a standard needle. In the setting of chronic infarction, percutaneous delivery of stem cells with C-Cath yielded a 37.7±7.1% versus 10.0±2.8% retention achieved with a traditional needle without effect on biocompatibility or safety. CONCLUSIONS: Modeling-guided development of a nitinol-based curved needle delivery system with incremental side holes achieved enhanced myocardial stem cell retention.

Human CD55 expression blocks hyperacute rejection and restricts complement activation in Gal knockout cardiac xenografts.

BACKGROUND: Transgenic expression of human complement regulatory proteins reduces the frequency of hyperacute rejection (HAR) in Gal-positive cardiac xenotransplantation. In this study, we examined the impact of human CD55 (hCD55) expression on a Gal knockout (GTKO) background using pig-to-primate heterotopic cardiac xenotransplantation. METHODS: Cardiac xenotransplantation was performed with GTKO (group 1; n=6) and GTKO.hCD55 (group 2; n=5) donor pigs using similar immunosuppression. Cardiac biopsies were obtained 30 min after organ reperfusion. Rejection was characterized by histology and immunohistology. Intragraft gene expression, serum non-Gal antibody, and antibody recovered from rejected hearts were analyzed. RESULTS: HAR of a GTKO heart was observed. Remaining grafts developed delayed xenograft rejection. Median survival was 21 and 28 days for groups 1 and 2, respectively. Vascular antibody deposition was uniformly detected 30 min after organ reperfusion and at explant. A higher frequency of vascular C5b deposition was seen in GTKO organs at explant. Serum non-Gal antibody, antibody recovered from the graft, and intragraft gene expression were similar between the groups. CONCLUSION: HAR of GTKO hearts without hCD55 may occur. Expression of hCD55 seemed to restrict local complement activation but did not improve graft survival. Chronic vascular antibody deposition with evidence of protracted endothelial cell activation was seen. These observations suggest that non-Gal antibody-induced chronic endothelial cell activation coupled to possible hemostatic incompatibilities may be the primary stimulus for delayed xenograft rejection of GTKO hearts. To avoid possible HAR, future clinical studies should use donors expressing human complement regulatory proteins in the GTKO background.

Identification of new carbohydrate and membrane protein antigens in cardiac xenotransplantation.

BACKGROUND: α1,3-Galactosyltransferase gene knockout (GTKO) pigs reduced the significance of antibody to galactose alpha 1,3-galactose (Gal) antigens but did not eliminate delayed xenograft rejection (DXR). We hypothesize that DXR of GTKO organs results from an antibody response to a limited number of non-Gal endothelial cell (EC) membrane antigens. In this study, we screened a retrovirus expression library to identify EC membrane antigens detected after cardiac xenotransplantation. METHODS: Expression libraries were made from GT:CD46 and GTKO porcine aortic ECs. Viral stocks were used to infect human embryonic kidney cells (HEK) that were selected by flow cytometry for IgG binding from sensitized cardiac heterotopic xenograft recipients. After three to seven rounds of selection, individual clones were assessed for non-Gal IgG binding. The porcine complementary DNA was recovered by polymerase chain reaction amplification, sequenced, and identified by homology comparisons. RESULTS: A total of 199 and 317 clones were analyzed from GT:CD46 and GTKO porcine aortic EC complementary DNA libraries, respectively. Sequence analysis identified porcine CD9, CD46, CD59, and the EC protein C receptor. We also identified porcine annexin A2 and a glycosyltransferase with homology to the human ß1,4 N-acetylgalactosaminyl transferase 2 gene. CONCLUSION: The identified proteins include key EC functions and suggest that non-Gal antibody responses may compromise EC functions and thereby contribute to DXR. Recovery of the porcine ß1,4 N-acetylgalactosaminyl transferase 2 suggests that an antibody response to a SD-like carbohydrate may represent a new carbohydrate moiety involved in xenotransplantation. The identification of these porcine gene products may lead to further donor modification to enhance resistance to DXR and further reduce the level of xenograft antigenicity.

Progenitor cell therapy in a porcine acute myocardial infarction model induces cardiac hypertrophy, mediated by paracrine secretion of cardiotrophic factors including TGFbeta1.

Administration of endothelial progenitor cells (EPC) is a promising therapy for post-infarction cardiac repair. However, the mechanisms that underlie apparent beneficial effects on myocardial remodeling are unclear. In a porcine model of acute myocardial infarction, we investigated the therapeutic effects of a mixed population of culture modified peripheral blood mononuclear cells (termed hereafter porcine EPC). Porcine EPC were isolated using methods identical to those previously adopted for harvest of EPC in human cell therapy studies. In addition the therapeutic effects of paracrine factors secreted by these cells was evaluated in vitro and in vivo. Intracoronary injection of autologous porcine EPC was associated with increased infarct territory mass and improved regional ventricular systolic function at 2 months compared to control. Treatment with conditioned media derived from autologous EPC was associated with similar improved effects on infarct territory mass and function. Histologic analysis of the infarct territory revealed significantly increased cardiomyocyte size in EPC and conditioned media treated groups, when compared to controls. A paracrine EPC effect was also verified in a pure myocardial preparation in which cardiomyocytes devoid of fibroblast, neuronal and vascular elements directly responded by increasing cell mass when exposed to the same conditioned media. Analysis of conditioned media revealed elevated levels of TGFbeta1 (human 267.3+/-11.8 pg/ml, porcine 57.1+/-6.1 pg/ml), a recognized mediator of hypertrophic signaling in the heart. Neutralizing antibodies to TGFbeta1 attenuated the pro-hypertrophic effect of conditioned media, and use of recombinant TGFbeta1 added to fresh media replicated the pro-hypertrophic effects of conditioned media in vitro. These data demonstrate the potential of paracrine factors secreted from endothelial progenitor cells to induce cardiomyocyte hypertrophy contributing to increased infarct territory LV mass, with favorable medium term effects on regional function following myocardial infarction.

Smooth muscle cells in human coronary atherosclerosis can originate from cells administered at marrow transplantation.

Atherosclerosis is the major cause of adult mortality in the developed world, and a significant contributor to atherosclerotic plaque progression involves smooth muscle cell recruitment to the intima of the vessel wall. Controversy currently exists on the exact origin of these recruited cells. Here we use sex-mismatched bone marrow transplant subjects to show that smooth muscle cells throughout the atherosclerotic vessel wall can derive from donor bone marrow. We demonstrate extensive recruitment of these cells in diseased compared with undiseased segments and exclude cell-cell fusion events as a cause for this enrichment. These data have broad implications for our understanding of the cellular components of human atherosclerotic plaque and provide a potentially novel target for future diagnostic and therapeutic strategies.

Smooth muscle progenitor cells in human blood.

BACKGROUND: Recent animal data suggest that vascular smooth muscle cells within the neointima of the vessel wall may originate from bone marrow, providing indirect evidence for circulating smooth muscle progenitor cells (SPCs). Evidence for circulating SPCs in human subjects does not exist, and the mechanism whereby such putative SPCs may home to sites of plaque formation is presently not understood but is likely to involve expression of specific surface adhesion molecules, such as integrins. In this study, we aimed to culture smooth muscle outgrowth cells (SOCs) from SPCs in human peripheral blood and characterize surface integrin expression on these cells. METHODS AND RESULTS: Human mononuclear cells isolated from buffy coat were seeded on collagen type 1 matrix and outgrowth cells selected in endothelial growth medium (EGM-2) or EGM-2 and platelet-derived growth factor BB. Selection in platelet-derived growth factor BB-enriched medium caused rapid outgrowth and expansion of SOC to >40 population doublings in a 4-month period. These SOCs were positive for smooth muscle cell-specific alpha actin (alphaSMA), myosin heavy chain, and calponin on immunofluorescence and Western blotting and were also positive for CD34, Flt1, and Flk1 receptor but negative for Tie-2 receptor expression, suggesting a potential bone marrow angioblastic origin. In contrast, endothelial outgrowth cells (EOCs) grown in EGM-2 alone and the initial MNC population were negative for these smooth muscle-specific markers. Integrin alpha5beta1 expression by FACS and Western blotting was significantly increased in SOCs compared with EOCs, and this was confirmed by 8-fold greater adhesion of SOC to fibronectin (P<0.001), an effect that could be decreased using an alpha5beta1 antibody. Finally, SOC showed a significantly greater in vitro proliferative potential compared with EOCs of similar passage (P<0.001). CONCLUSIONS: This study demonstrates for the first time outgrowth of smooth muscle cells with a specific growth, adhesion, and integrin profile from putative SPC in human blood. These data have implications for our understanding of adult vascular smooth muscle cell differentiation, proliferation, and homing.