First Case of Debilitating Lower Back Pain Induced by Severe Inflammation of Lumbar Joints, Healed by Intravenous Infusions of Purified Amniotic Fluid.

We report the first case of debilitating lower back pain induced by spondylitis with end plate inflammation of the lumbar spine, treated successfully by bi-weekly intravenous injections of a sterile fraction (1ml) from human purified amniotic fluid (ViX001) obtained from thoroughly screened volunteers at the time of planned c-section at the term of normal pregnancies. Our product ViX001 was generated through a proprietary process and kept in frozen one milliliter (1 ml) cryvials (protein content was ~1mg/ml) and thawed just prior to injections. Pain improvement was recorded weekly, and inflammation suppression was confirmed by monthly MRIs of the lumbar spine. While our findings need to be reproduced with a larger cohort of patients, it is instructive that ViX001 resolved pain and inflammation for a patient with severe lower back pain, the most common form of pain reported by U.S. adults.

First Case of Severe Pain and Inflammation Reduction by Application of Purified Amniotic Fluid on Active Lesions of a Patient with Pyoderma Gangrenosum.

We report the first case of severe pain and inflammation reduction by application of purified amniotic fluid on active lesions of a patient with pyoderma gangrenosum. We describe the impact of every third-day skin applications of a sterile fraction (4ml) from human purified amniotic fluid (ViX001) obtained from thoroughly screened volunteers at the time of planned c-section at the term of normal pregnancies. The product ViX001 was generated through a proprietary process and kept in frozen one or two milliliters cryovials (protein content was ~1mg/ml) and thawed just prior to applications. Pain improvement was recorded after each application, and inflammation suppression was confirmed by serial pictures of the lesions. While our findings need to be reproduced with a larger cohort of patients, preferably at an earlier stage of the disease, it is instructive that ViX001 reduced severe pain and inflammation for a patient with advanced pyoderma gangrenosum. Pyoderma gangrenosum is a dreadful skin condition consisting of noninfectious neutrophilic dermatosis that progresses to necrotic ulcers with a characteristic purple edge and extremely painful raw subdermal tissue exposure.

First Case of Accelerated Healing of a Recalcitrant Diabetic Ulcer Using Purified Amniotic Fluid.

We report the first case of recalcitrant diabetic wound treated successfully by twice-daily applications of a sterile fraction of human purified amniotic fluid (ViX001) obtained from thoroughly screened volunteers at the time of planned c-section at the term of normal pregnancies. Our product ViX001 was generated through a proprietary process and kept in frozen one milliliter (1 ml) vials (protein content was ~1mg/ml) thawed prior to applications (shelf life at 34oF of at least two weeks).

Pregnancy, a unique case of heterochronic parabiosis and peripartum cardiomyopathy.

Introduction: A loss of endogenous stem cells capable of tissue repair and regeneration drives the biological process that we recognize as "aging". Recovery of stem cell-mediated repair and regenerative functions in aged animals has been reported in murine heterochronic parabiosis experiments. Objectives: Herein we will review how pregnancy is an unusual form of heterochronic parabiosis, as the placenta prevents the exchange of most blood cells between parabionts. Instead, plasma and its content, including small extracellular vesicles, can readily cross the placental barrier. These nanosized extracellular vesicles are readily produced by the placenta, amnion, fetus and mother, and are essential for fetal organogenesis, growth and the progression of a healthy pregnancy. If defective, these extracellular vesicles can cause havoc such as in the case of peripartum cardiomyopathy. We will also review how these extracellular vesicles impact the mother substantially (including cardiac function) in the parabiosis of pregnancy. Conclusion: Extracellular vesicles generated during the course of a healthy pregnancy are essential for organogenesis and fetal growth, and also for maternal tissue repair and regeneration, and might be defective or deficient in pregnancies that result in peripartum cardiomyopathy.

Neuregulin-1, in a Conducive Milieu with Wnt/BMP/Retinoic Acid, Prolongs the Epicardial-Mediated Cardiac Regeneration Capacity of Neonatal Heart Explants.

Rationale: Cardiac sympathetic nerves are required for endogenous repair of the mammalian neonatal heart in vivo, but the underlying mechanism is unclear. Objective: We tested the hypothesis that a combination of cardiac developmental growth factors Wnt3a, BMP4 and Neuregulin (NRG-1), compensate for denervation and support cardiac regeneration in explanted neonatal mammalian hearts. Methods and Results: Hearts from 2-day old neonatal mice were harvested, lesioned at the apex and grown ex vivo for 21 days under defined conditions. Hearts grown in canonical cardiomyocyte culture media underwent complete coagulative necrosis, a process resembling ischemic cell death, by day 14. However, the addition of Wnt3a, BMP-4 and NRG-1, maintained cellular integrity and restored the endogenous regenerative program. None of these factors alone, or in any paired combination, were sufficient to induce regeneration in culture. rNRG-1 alone significantly reduced the accumulation of double strand DNA damage at Day 3; (-NRG-1: 60±12%; +NRG-1: 8±3%; P<0.01) and prevented coagulative necrosis at Day 14. Short-term addition of rWnt3a and rBMP-4 (day 0-3, NRG-1+) increased WT1 expression (a marker of epicardial cells) 7-fold, epicardial proliferation (78±17 cells vs. 21±9 cells; P<0.05), migration and recellularization (80±22 vs. zero cells; P<0.01; n=6) at the injury site on day 14. Conclusions: A novel explant culture system maintains three-dimensional neonatal mouse hearts and the mammalian neonatal cardiac regenerative program ex vivo. We identified that rNRG-1, plus short-term activation of Wnt- and BMP-signaling, promotes cardiac repair via epicardial cell activation, their proliferation and migration to the injury site, followed by putative cardiomyocyte recruitment. This novel technique will facilitate future studies of mammalian cardiac regeneration and may be useful in cardiac-specific drug testing.

Evaluation of exosome derivatives as bio-informational reprogramming therapy for cancer.

Exosomes are nanoparticle sized (100 ± 50 nm) extracellular vesicles (ECVs) that play important roles in cell-to-cell communication. They do this by utilizing their natural ability to shuttle signaling molecules across the cellular microenvironment and promote paracrine signaling. Currently, exosomes are being explored for their potential as therapeutic agents for various degenerative diseases including cancer. The rationale behind their therapeutic ability is that they can transfer signaling biomolecules, and subsequently induce metabolic and physiological changes in diseased cells and tissues. In addition, exosomes can be used as a drug delivery system and may be very effective at reducing toxicity and increasing bioavailability of therapeutic molecules and drugs. Although exosomes were first believed to be a waste product of the cell, current research has demonstrated that these particles can serve as modulators of the immune system, act as cancer biomarkers, cause re-differentiation of cancer cells, and induce apoptosis in diseased cells. Extensive research has been performed specifically using amniotic fluid-derived extracellular vesicles, named "cytosomes". While the use of cytosomes in clinical application is still in the early stages, researchers have shown great potential for these EVs in regenerative medicine as immune modulators, in controlling microbial infection and by inducing tissue repair through the activation of endogenous, tissue-specific stem cells. This review emphasizes the capabilities of specific subsets of extracellular vesicles that can potentially be used for cancer therapy, principally as a source of bi-informational reprogramming for malignant cells.

Use of Acellular Umbilical Cord-Derived Tissues in Corneal and Ocular Surface Diseases.

Blood derived products have become a valuable source of tissue for the treatment of ocular surface diseases that are refractory to conventional treatments. These can be obtained from autologous or allogeneic sources (patient's own blood or from healthy adult donors/umbilical cord blood, respectively). Allogeneic cord blood demonstrates practical advantages over alternatives and these advantages will be discussed herein. Umbilical cord blood (UCB) can be divided, generally speaking, into two distinct products: first, mononuclear cells, which can be used in regenerative ophthalmology, and second, the plasma/serum (an acellular fraction), which may be used in the form of eyedrops administered directly to the damaged ocular surface. The rationale for using umbilical cord serum (UCS) to treat ocular surface diseases such as severe dry eye syndrome (DES), persistent epithelial defects (PED), recurrent epithelial erosions, ocular chemical burns, graft versus host disease (GVHD), among others, is the considerably high concentration of growth factors and cytokines, mimicking the natural healing properties of human tears. Allogeneic serum also offers the opportunity for therapeutic treatment to patients who, due to poor heath, cannot provide autologous serum. The mechanism of action involves the stimulation of endogenous cellular proliferation, differentiation and maturation, which is highly efficient in promoting and enhancing corneal epithelial healing where other therapies have previously failed.

Mesenchymal Stem Cells in Cardiology.

Cardiovascular disease (CVD) accounts for more deaths globally than any other single disease. There are on average 1.5 million episodes of myocardial infarction (heart attack) each year in the United States alone with roughly one-third resulting in death. There is therefore a major need for developing new and effective strategies to promote cardiac repair. Intramyocardial transplantation of mesenchymal stem cells (MSCs) has emerged as a leading contender in the pursuit of clinical intervention and therapy. MSCs are potent mediators of cardiac repair and are therefore an attractive tool in the development of preclinical and clinical trials. MSCs are capable of secreting a large array of soluble factors, which have had demonstrated effects on pathogenic cardiac remolding, fibrosis, immune activation, and cardiac stem cell proliferation within the damaged heart. MSCs are also capable of differentiation into cardiomyocytes, endothelial cells, and vascular smooth muscle cells, although the relative contribution of trilineage differentiation and paracrine effectors on cardiac repair remains the subject of active investigation.

cKit+ cardiac progenitors of neural crest origin.

The degree to which cKit-expressing progenitors generate cardiomyocytes in the heart is controversial. Genetic fate-mapping studies suggest minimal contribution; however, whether or not minimal contribution reflects minimal cardiomyogenic capacity is unclear because the embryonic origin and role in cardiogenesis of these progenitors remain elusive. Using high-resolution genetic fate-mapping approaches with cKit(CreERT2/+) and Wnt1::Flpe mouse lines, we show that cKit delineates cardiac neural crest progenitors (CNC(kit)). CNC(kit) possess full cardiomyogenic capacity and contribute to all CNC derivatives, including cardiac conduction system cells. Furthermore, by modeling cardiogenesis in cKit(CreERT2)-induced pluripotent stem cells, we show that, paradoxically, the cardiogenic fate of CNC(kit) is regulated by bone morphogenetic protein antagonism, a signaling pathway activated transiently during establishment of the cardiac crescent, and extinguished from the heart before CNC invasion. Together, these findings elucidate the origin of cKit(+) cardiac progenitors and suggest that a nonpermissive cardiac milieu, rather than minimal cardiomyogenic capacity, controls the degree of CNC(kit) contribution to myocardium.

Sympathetic Reinnervation Is Required for Mammalian Cardiac Regeneration.

RATIONALE: Although mammalian cardiac regeneration can occur in the neonatal period, the factors involved in this process remain to be established. Because tissue and limb regeneration require concurrent reinnervation by the peripheral nervous system, we hypothesized that cardiac regeneration also requires reinnervation. OBJECTIVE: To test the hypothesis that reinnervation is required for innate neonatal cardiac regeneration. METHODS AND RESULTS: We crossed a Wnt1-Cre transgenic mouse with a double-tandem Tomato reporter strain to identify neural crest-derived cell lineages including the peripheral autonomic nerves in the heart. This approach facilitated the precise visualization of subepicardial autonomic nerves in the ventricles using whole mount epifluorescence microscopy. After resection of the left ventricular apex in 2-day-old neonatal mice, sympathetic nerve structures, which envelop the heart under normal conditions, exhibited robust regrowth into the regenerating myocardium. Chemical sympathectomy inhibited sympathetic regrowth and subsequent cardiac regeneration after apical resection significantly (scar size as cross-sectional percentage of viable left ventricular myocardium, n=9; 0.87%±1.4% versus n=6; 14.05±4.4%; P<0.01). CONCLUSIONS: These findings demonstrate that the profound regenerative capacity of the neonatal mammalian heart requires sympathetic innervation. As such, these data offer significant insights into an underlying basis for inadequate adult regeneration after myocardial infarction, a situation where nerve growth is hindered by age-related influences and scar tissue.

Impaired endothelial progenitor cell mobilization and dysfunctional bone marrow stroma in diabetes mellitus.

BACKGROUND: Circulating Endothelial Progenitor Cell (EPC) levels are reduced in diabetes mellitus. This may be a consequence of impaired mobilization of EPC from the bone marrow. We hypothesized that under diabetic conditions, mobilization of EPC from the bone marrow to the circulation is impaired -at least partly- due to dysfunction of the bone marrow stromal compartment. METHODS: Diabetes was induced in mice by streptozotocin injection. Circulating Sca-1(+)Flk-1(+) EPC were characterized and quantified by flow cytometry at baseline and after mobilization with G-CSF/SCF injections. In vivo hemangiogenic recovery was tested by 5-FU challenge. Interaction within the bone marrow environment between CD34(+) hematopoietic progenitor cells (HPC) and supporting stroma was assessed by co-cultures. To study progenitor cell-endothelial cell interaction under normoglycemic and hyperglycemic conditions, a co-culture model using E4Orf1-transfected human endothelial cells was employed. RESULTS: In diabetic mice, bone marrow EPC levels were unaffected. However, circulating EPC levels in blood were lower at baseline and mobilization was attenuated. Diabetic mice failed to recover and repopulate from 5-FU injection. In vitro, primary cultured bone marrow stroma from diabetic mice was impaired in its capacity to support human CFU-forming HPC. Finally, hyperglycemia hampered the HPC supportive function of endothelial cells in vitro. CONCLUSION: EPC mobilization is impaired under experimental diabetic conditions and our data suggest that diabetes induces alterations in the progenitor cell supportive capacity of the bone marrow stroma, which could be partially responsible for the attenuated EPC mobilization and reduced EPC levels observed in diabetic patients.

Endothelial cells are essential for the self-renewal and repopulation of Notch-dependent hematopoietic stem cells.

Bone marrow endothelial cells (ECs) are essential for reconstitution of hematopoiesis, but their role in self-renewal of long-term hematopoietic stem cells (LT-HSCs) is unknown. We have developed angiogenic models to demonstrate that EC-derived angiocrine growth factors support in vitro self-renewal and in vivo repopulation of authentic LT-HSCs. In serum/cytokine-free cocultures, ECs, through direct cellular contact, stimulated incremental expansion of repopulating CD34(-)Flt3(-)cKit(+)Lineage(-)Sca1(+) LT-HSCs, which retained their self-renewal ability, as determined by single-cell and serial transplantation assays. Angiocrine expression of Notch ligands by ECs promoted proliferation and prevented exhaustion of LT-HSCs derived from wild-type, but not Notch1/Notch2-deficient, mice. In transgenic notch-reporter (TNR.Gfp) mice, regenerating TNR.Gfp(+) LT-HSCs were detected in cellular contact with sinusoidal ECs. Interference with angiocrine, but not perfusion, function of SECs impaired repopulation of TNR.Gfp(+) LT-HSCs. ECs establish an instructive vascular niche for clinical-scale expansion of LT-HSCs and a cellular platform to identify stem cell-active trophogens.

Generation of a functional and durable vascular niche by the adenoviral E4ORF1 gene.

Vascular cells contribute to organogenesis and tumorigenesis by producing unknown factors. Primary endothelial cells (PECs) provide an instructive platform for identifying factors that support stem cell and tumor homeostasis. However, long-term maintenance of PECs requires stimulation with cytokines and serum, resulting in loss of their angiogenic properties. To circumvent this hurdle, we have discovered that the adenoviral E4ORF1 gene product maintains long-term survival and facilitates organ-specific purification of PECs, while preserving their vascular repertoire for months, in serum/cytokine-free cultures. Lentiviral introduction of E4ORF1 into human PECs (E4ORF1(+) ECs) increased the long-term survival of these cells in serum/cytokine-free conditions, while preserving their in vivo angiogenic potential for tubulogenesis and sprouting. Although E4ORF1, in the absence of mitogenic signals, does not induce proliferation of ECs, stimulation with VEGF-A and/or FGF-2 induced expansion of E4ORF1(+) ECs in a contact-inhibited manner. Indeed, VEGF-A-induced phospho MAPK activation of E4ORF1(+) ECs is comparable with that of naive PECs, suggesting that the VEGF receptors remain functional upon E4ORF1 introduction. E4ORF1(+) ECs inoculated in implanted Matrigel plugs formed functional, patent, humanized microvessels that connected to the murine circulation. E4ORF1(+) ECs also incorporated into neo-vessels of human tumor xenotransplants and supported serum/cytokine-free expansion of leukemic and embryonal carcinoma cells. E4ORF1 augments survival of PECs in part by maintaining FGF-2/FGF-R1 signaling and through tonic Ser-473 phosphorylation of Akt, thereby activating the mTOR and NF-kappaB pathways. Therefore, E4ORF1(+) ECs establish an Akt-dependent durable vascular niche not only for expanding stem and tumor cells but also for interrogating the roles of vascular cells in regulating organ-specific vascularization and tumor neo-angiogenesis.

T-bet regulates metastasis rate in a murine model of primary prostate cancer.

The local progression of primary tumors is extrinsically controlled by type 1 immune responses, particularly via the cytokine IFN-gamma, whose secretion is highly dependent on helper T cells. The T-box transcription factor T-bet (Tbx21) plays a critical role in the development of type 1 helper T cells and is essential for the production of IFN-gamma. Here, the T-bet pathway in the autochthonous transgenic adenocarcinoma mouse prostate model is demonstrated to have only a modest effect on the characteristics of primary prostate cancers but rather exerts a significant suppressor function in the development of metastatic disease.