Cardiopulmonary and histological characterization of an acute rat lung injury model demonstrating safety of mesenchymal stromal cell infusion.

BACKGROUND AIMS: In the field of cellular therapy, potential cell entrapment in the lungs following intravenous administration in a compromised or injured pulmonary system is an important concern that requires further investigation. We developed a rat model of inflammatory and fibrotic lung disease to mimic the human clinical condition of obliterative bronchiolitis (OB) and evaluate the safety of intravenous infusion of mesenchymal stromal cells (MSCs). This model was used to obtain appropriate safety information and functional characterization to support the translation of an ex vivo-generated cellular product into human clinical trials. To overcome spontaneous recovery and size limitations associated with current animal models, we used a novel multiple dose bleomycin strategy to induce lasting lung injury in rats. METHODS: Intratracheal instillation of bleomycin was administered to rats on multiple days. MSCs were intravenously infused 7 days apart. Detailed pulmonary function tests including forced expiratory volume, total lung capacity, and invasive hemodynamic measurements were conducted to define the representative disease model and monitor cardiopulmonary hemodynamic consequences of the cell infusion. Post-euthanasia assessments included a thorough evaluation of lung morphology and histopathology. RESULTS: The double dose bleomycin instillation regimen resulted in severe and irreversible lung injury and fibrosis. Cardiopulmonary physiological monitoring reveled that no adverse events could be attributed to the cell infusion process. DISCUSSION: Although our study did not show the infusion of MSCs to result in an improvement in lung function or rescue of damaged tissue this study does confirm the safety of MSC infusion into damaged lungs.

Mesenchymoangioblast-derived mesenchymal stromal cells inhibit cell damage, tissue damage and improve peripheral blood flow following hindlimb ischemic injury in mice.

BACKGROUND AIMS: Existing treatments have limited success in modifying the course of peripheral artery disease, which may eventually lead to limb-threatening ulcers and amputation. Cellular therapies have the potential to provide a new treatment option for this condition, but isolation of cells by conventional means has limitations with respect to reproducibility and scalability. METHODS: Induced pluripotent stem cells (iPSCs) were differentiated into precursor cells known as mesenchymoangioblasts (MCAs) and subsequently into mesenchymal stromal cells (MSCs). Hindlimb ischemia in mice was created by ligating both the iliac and femoral arteries of one hindlimb. Immediately after surgery, each animal received intramuscular injections of 5 × 10(6) cells or media in the ischemic limb. Toe necrosis was assessed visually, and hindlimb blood flow was measured by laser Doppler using a set region of interest (ROI) and by tracing the entire foot. Myofiber heterogeneity, nuclear centralization, fatty degeneration, fibrosis and capillary angiogenesis in the gastrocnemius muscle were assessed histologically. RESULTS: Blood flow in the MCA-derived MSC-treated animals was higher at each day (P <0.006), and these mice recovered faster than control animals (3.6 vs. 2.5 for set ROI; 7.5 vs. 4.1 foot tracing; slope; P <0.001). There was significantly less myofiber heterogeneity, nuclear centralization, fatty degeneration and fibrosis in MCA-derived MSC-treated animals, indicating less tissue damage. DISCUSSION: MCA-derived MSCs improved limb blood flow, reduced necrosis and maintained muscle mass and gross muscle appearance. We conclude that MCA-derived MSCs have a significant and protective effect against ischemic insults.

Intravenous Followed by X-ray Fused with MRI-Guided Transendocardial Mesenchymal Stem Cell Injection Improves Contractility Reserve in a Swine Model of Myocardial Infarction.

The aim of this study is to determine the effects of early intravenous (IV) infusion later followed by transendocardial (TE) injection of allogeneic mesenchymal stem cells (MSCs) following myocardial infarction (MI). Twenty-four swine underwent balloon occlusion reperfusion MI and were randomized into 4 groups: IV MSC (or placebo) infusion (post-MI day 2) and TE MSC (or placebo) injection targeting the infarct border with 2D X-ray fluoroscopy fused to 3D magnetic resonance (XFM) co-registration (post-MI day 14). Continuous ECG recording, MRI, and invasive pressure-volume analyses were performed. IV MSC plus TE MSC treated group was superior to other groups for contractility reserve (p = 0.02) and freedom from VT (p = 0.03) but had more lymphocytic foci localized to the peri-infarct region (p = 0.002). No differences were observed in post-MI remodeling parameters. IV followed by XFM targeted TE MSC therapy improves contractility reserve and suppresses VT but does not affect post-MI remodeling and may cause an immune response.

Pituitary cystadenoma, enterolipidosis, and cutaneous mycosis in an Everglades ratsnake (Elaphe obsoleta rossalleni).

An 11-yr-old captive-born male Everglades ratsnake (Elaphe obsoleta rosalleni) presented with dysecdysis, hyperkeratosis, and inappetance. Two skin biopsies demonstrated a diffuse hyperkeratosis with both a bacterial and fungal epidermitis. Fusarium oxysporum was cultured from both biopsies and considered an opportunistic infection rather than a primary pathogen. Medical management was unsuccessful, and the snake was euthanized. Histologic findings included a pituitary cystadenoma arising from the pars intermedia, severe intestinal lipidosis, generalized epidermal hyperkeratosis, and lesions consistent with sepsis. It is hypothesized that endocrine derangements from the pituitary tumor may have caused the skin and intestinal lesions.

Dynamic regulation of the human dopachrome tautomerase promoter by MITF, ER-alpha and chromatin remodelers during proliferation and senescence of human melanocytes.

Senescent cells are known to display altered gene expression of differentiation-associated genes. We have previously demonstrated that the melanocyte transcriptional regulator microphthalmia-associated protein (MITF) is down-regulated in senescent melanocytes. Since virtually nothing is known regarding the differentiated function of senescent melanocytes, we analyzed the transcriptional regulation of Dopachrome tautomerase (DCT), a member of the tyrosinase gene family, in proliferating and in senescent human melanocytes. Computational analysis of the region containing the M-box that includes the MITF CATGTG binding motif demonstrated that this sequence overlaps with the estrogen receptor alpha (ER-alpha), USF-1, TFE-3, Isl-1 and AP-1 binding elements. Electrophoresis gel-shift analysis using an oligonucleotide containing MITF and ERE elements identified MITF and ER-alpha complexes in proliferating melanocytes, whereas only ER-alpha complexes were detected in senescent cells. Importantly, a promoter-reporter analysis demonstrated that the coactivator p300/CBP switched MITF from a repressor to an activator of DCT transcription. p300/CBP was also required by ER-alpha and MITF to induce high, synergistic activation of the DCT promoter. We have also found that transcription of the DCT gene is differentially regulated by major melanocyte mitogens. In contrast to the activating effect of cAMP inducers, 12-O-tetradecanoylphorbolacetate (TPA) was a potent repressor of DCT transcription, suggesting that this gene can be differentially regulated by multiple environmental signals and promoter context. In support of this conclusion, trichostatin A, a histone deacetylase inhibitor, counteracted the TPA-mediated repression, and restored high levels of DCT protein in cultured melanocytes. We conclude that senescent melanocytes display dramatic changes in the expression of differentiation-related proteins; such changes may in turn result in altered melanocyte function and survival to environmental stresses.