Model of Liver Fibrosis Induction by Thioacetamide in Rats for Regenerative Therapy Studies.

Hepatic fibrosis is caused by chronic injury due to toxic, infectious, or metabolic causes, and it may progress to cirrhosis and hepatocellular carcinoma. There is currently no antifibrotic therapy authorized for human use; however, there are promising studies using cell therapies. There are also no animal models that exactly reproduce human liver fibrosis that can be used to better understand the mechanisms of its regression and identify new targets for treatment and therapeutic approaches. On the other hand, mesenchymal stem cells (MSC) have experimentally demonstrated fibrosis regression effects, but it is necessary to have an animal model of advanced liver fibrosis to evaluate the effect of these cells. The aim of this work was to establish a protocol for the induction of advanced liver fibrosis in rats using thioacetamide (TAA), which will allow us to perform trials using MSC as a possible therapy for fibrosis regression. For this purpose, we selected 24 female rats and grouped them into three experimental groups: the control group (G-I) without treatment and groups II (G-II) and III (G-III) that received TAA by intraperitoneal injection for 24 weeks. Then, 1 × 106/kg adipose mesenchymal stem cells (ASCs) were infused intravenously. Groups G-I and G-II were sacrificed 7 days after the last dose of ASC, and G-III was sacrificed 8 weeks after the last ASC infusion, all with xylazine/ketamine (40 mg/kg). The protocol used in this work established a model of advanced hepatic fibrosis as corroborated by METAVIR tests of the histological lesions; by the high levels of the markers α-SMA, CD68, and collagen type I; by functional alterations due to elevated markers of the hepatic lesions; and by alterations of the leukocytes, lymphocytes, and platelets. Finally, transplanted cells in the fibrous liver were detected. We conclude that TAA applied using the protocol introduced in this study induces a good model of advanced liver fibrosis in rats.

First report of canine morbillivirus infection of adipose tissue-derived stem cells from dogs with distemper.

Background and Aim: Ribonucleic acid viruses remain latent in different cell types, including mesenchymal stem cells; however, the distemper virus remains undetected in these cells. This study aimed to determine whether adipose stem cells (ASCs) from dogs with distemper disease are infected with the canine morbillivirus (CM). Materials and Methods: Twelve dogs with the neurological phase of the disease and who were positive for CM by reverse transcription polymerase chain reaction (RT-PCR), were studied. ASCs from adipose tissue of the lesser omentum of these infected dogs were isolated and characterized. Direct fluorescence was used to detect the viral antigen in cell cultures. Flow cytometry and RT-PCR identified detectable quantities of the virus in two cultures, while electron microscopy confirmed the CM particles within ASCs. Results: This study revealed that ASCs of the omentum of dogs with distemper disease can be infected with CM, indicating their possible involvement in this virus latency and persistence. This suggests that its detection should be considered within the quality control process of stem cells intended for regenerative medicine. Conclusion: To the best of our knowledge, this is the first study that demonstrates that omentum ASCs from dogs with distemper disease can be infected with CM and may be involved in viral latency or persistence. Our study also suggests that the detection of CM should be considered within the quality control process of stem cells intended for regenerative medicine.

Biological characteristics of a sub-population of cancer stem cells from two triple-negative breast tumour cell lines.

Triple-negative breast tumours (TNBTs) make up 15-20% of all breast tumours. There is no treatment for them, and the role that cancer stem cells (CSCs) have in carcinogenesis is still unclear, so finding markers and therapeutic targets in CSC exosomes requires these cells to exist as a homogeneous cell population. The objective of this work was to determine differences in ultrastructural morphology, proliferative capacity, and mouse-xenotransplantation characteristics of the MDA-MB-231 and MDA-MB-436 TNBT cell lines with the CD44 high /CD24 low phenotype in order to study their exosomes. The results show that the CD44 high /CD24 low MBA-MB-231 cells had a population doubling time of 41.56 h, compared to 44.79 h in the MDA-MB-436 cell line. After magnetic immunoseparation, 18.75% and 14.56% of the stem cell population of the MDA-MB-231 and MDA-MB-436 cell lines, respectively, were of the CD44 high /CD24 low phenotype, which were expanded to reach purities of 80.4% and 87.6%. The same expanded lineage in both cell lines was shown to possess the pluripotency markers Nanog and Oct4. Under a scanning electron microscope, the CD44 high /CD24 low lineage of the MBA-MD-231 cell line formed groups of more interconnected cells than this lineage of the MBA-MD-436 line. A total of 16% of the mice inoculated with the CD44 high /CD24 low lineage of either cell line presented tumours of the breast, lung, and submandibular ganglia, in whose tissues variable numbers of inoculated cells were found 30 days post-inoculation. By magnetic immunoselection, it was possible to isolate in similar quantities and characterize, expand, and xenotransplant the CD44 high /CD24 low lineage of the MDA-MB-231 and MDA-MB-436 cell lines. The former cell line has greater proliferative capacity, the two lines differ under scanning electron microscopy in how they intercommunicate, and both cell lines induce new tumours in mice and persist at least 30 days post-inoculation in the transplanted animal so their exosomes would also be different.

Cutaneous wound healing: canine allogeneic ASC therapy.

BACKGROUND: Wound healing is a complex biological process comprised of a series of sequential events aiming to repair injured tissue. Adult mesenchymal stem cells (MSCs) have been used in cellular therapy in preclinical animal studies; a promising source of MSCs is adipose tissue (AT). In this paper, we evaluated the clinical value and safety of the application of cultured allogenic MSCs from AT for acute and chronic skin wound healing in a canine model. METHODS: Twenty-four dogs of different breeds between 1 and 10 years of age with acute and chronic wounds were studied. Morphology of the wounded skin was monitored for changes over time via serial photographs and histopathological studies. RESULTS: The percentage of the wounds that exhibited contraction and re-epithelialization were significantly different between wounds treated with adipose mesenchymal stem cells (ASCs) and control wounds; this effect was observed in both acute and chronic conditions. At 90 days, re-epithelization of acute and chronic wounds reached more than 97%. Histopathological study revealed a reduction in inflammatory infiltrate and the presence of multiple hair follicles on day 7 after treatment with ASCs, promoting epidermal and dermal regeneration. To guarantee the safety of our treatment, we determined the serum levels of cytokine markers in our patients. ASC treatment upregulated granulocyte-macrophage colony stimulating factor (GM-CSF) at the gene level, which may contribute to the recruitment of cells that participate in skin repair to the site of injury. CONCLUSIONS: The development of an allogenic ASC therapy to improve wound healing in a canine model could have a clinical impact in human treatment.

Regenerative potential of allogeneic adipose tissue-derived mesenchymal cells in canine cutaneous wounds.

BACKGROUND: Mesenchymal stem cells (MSCs) have generated a great amount of interest over the past decade as a novel therapeutic treatment for a variety of diseases. Emerging studies have indicated that MSCs could enhance the repair of injured skin in canine cutaneous wounds. CASE PRESENTATION: A healthy 2 years old Bodeguero Andaluz dog was presented with multiple skin bite wounds. Antibiotic and anti-inflammatory therapy was administered for 8 days. On day three, 107 allogeneic adipose-derived mesenchymal stem cells (ASCs) were intradermally injected approximately equidistant to the ASCs treated wounds. Control wounds underwent conventional treatment with a topical antibacterial ointment until wound healing and closure. Wounds, skin morphology and healing progress were monitored via serial photographs and histopathology of biopsies obtained at day seven after ASC treatment. Histopathology revealed absence of inflammatory infiltrates and presence of multiple hair follicles in contrast to the non-ASCs treated control wounds indicating that ASC treatment promoted epidermal and dermal regeneration. ASCs were identified by flow cytometry and RT-PCR. The immunomodulatory role of ASCs was evidenced by coculturing peripheral blood mononuclear cells with allogeneic ASCs. Phytohemagglutinin was administered to stimulate lymphocyte proliferation. Cells were harvested and stained with an anticanine CD3-FITC antibody. The ASCs inhibited proliferation of T lymphocytes, which was quantified by reduction of carboxyfluorescein succinimidyl ester intensity using flow cytometry. CONCLUSIONS: Compared with conventional treatment, wounds treated with ASCs showed a higher regenerative capacity with earlier and faster closure in this dog.

Multidose intramuscular allogeneic adipose stem cells decrease the severity of canine atopic dermatitis: A pilot study.

AIM: The aim of this pilot study was to evaluate the therapeutic and safety performance of an intramuscular treatment protocol of multidose of allogeneic adipose stem cells (ASCs) isolated, characterized, and expanded ex vivo from a healthy canine donor. MATERIALS AND METHODS: Twelve dogs diagnosed with canine atopic dermatitis (CAD) were intramuscularly treated with 0.5×106 of cryopreserved ASCs from a healthy immunized young canine Ehrlichia canis free donor weekly for 6 weeks. Treatment efficacy was evaluated by the pruritus index and the CAD Lesion Index (CADLI) test. Safety and adverse effects were determined by injection site reaction, weight, blood chemistry, liver function, and whole blood count. RESULTS: Canine ASCs obtained from a donor met the minimum qualities required for this type of cells and showed viability of 90% after thawing. The efficacy of the CADLI score and the pruritus index in 12 dogs with atopic dermatitis was statistically significant efficacy. No adverse reactions were observed at the intramuscular application site, or in relation to animal weight, blood cell populations, or liver and renal function. CONCLUSION: These results suggest that intramuscular administration of cryopreserved ASCs to dogs with atopic dermatitis is a promising cellular therapeutic product for the relief of the symptoms of this disease; however, the duration of the effects obtained with this dose and with other doses should be evaluated, as well as possible immune reactions. As far as we know, this is the first report of the use of multiple intramuscular doses cryopreserved ASCs to treat atopic dermatitis.

Stem cell factor supports migration in canine mesenchymal stem cells.

Adult Mesenchymal Stem Cells (MSC) are cells that can be defined as multipotent cells able to differentiate into diverse lineages, under appropriate conditions. These cells have been widely used in regenerative medicine, both in preclinical and clinical settings. Initially discovered in bone marrow, MSC can now be isolated from a wide spectrum of adult and foetal tissues. Studies to evaluate the therapeutic potential of these cells are based on their ability to arrive to damaged tissues. In this paper we have done a comparative study analyzing proliferation, surface markers and OCT4, SOX9, RUNX2, PPARG genes expression in MSC cells from Bone marrow (BMMSC) and Adipose tissue (ASC). We also analyzed the role of Stem Cell Factor (SCF) on MSC proliferation and on ASCs metalloproteinases MMP-2, MMP-9 secretion. Healthy dogs were used as BMMSC donors, and ASC were collected from omentum during elective ovariohysterectomy surgery. Both cell types were cultured in IMDM medium with or without SCF, 10% Dog Serum (DS), and incubated at 38 °C with 5% CO2. Growth of BMMSCs and ASCs was exponential until 25-30 days. Flow citometry of MSCs revealed positive results for CD90 and negative for CD34, CD45 and MCH-II. Genes were evaluated by RT-PCR and metalloproteinases by zymografy. Our findings indicate morphological and immunological similarities as well as expression of genes from both origins on analyzed cells. Furthermore, SCF did not affect proliferation of MSCs, however it up-regulated MMP-2 and MMP-9 secretion in ASCs. These results suggest that metalloproteinases are possibly essential molecules pivoting migration.