Yuyo Yasumura

Mesenchymal stromal cells (MSCs) are a promising cell source for the treatment of dogs with refractory chronic inflammatory enteropathy (CIE). Although sev

Mesenchymal stem cells (MSCs) are a promising cell source for stem cell therapy of intractable diseases in veterinary medicine, but donor-dependent cellular heterogeneity is an issue that influences therapeutic efficacy. Thus, we previously established immortalized cells that maintain the fundamental properties of primary cells, but functional evaluation had not been performed. Therefore, we evaluated the immunomodulatory capacity of the immortalized canine adipose-derived MSCs (cADSCs) in vitro and in vivo to investigate whether they maintain primary cell functions. C57BL/6J mice were treated with dextran sulfate sodium (DSS) to induce colitis, injected intraperitoneally with immortalized or primary cADSCs on day 2 of DSS treatment, and observed for 10 days. Administration of immortalized cADSCs improved body weight loss and the disease activity index (DAI) in DSS-induced colitic mice by shifting peritoneal macrophage polarity from the M1 to M2 phenotype, suppressing T helper (Th) 1/Th17 cell responses and inducing regulatory T (Treg) cells. They also inhibited the proliferation of mouse and canine T cells in vitro. These immunomodulatory effects were comparable with primary cells. These results highlight the feasibility of our immortalized cADSCs as a cell source for stem cell therapy with stable therapeutic efficacy because they maintain the immunomodulatory capacity of primary cells.

Mesenchymal stem cells are expected to be a cell source for stem cell therapy of various diseases in veterinary medicine. However, donor-dependent cell heterogenicity has been a cause of inconsistent therapeutic efficiency. Therefore, we established immortalized cells from canine adipose tissue-derived mesenchymal stem cells (ADSCs) to minimize cellular heterogeneity by reducing the number of donors, evaluated their properties, and compared them to the primary cells with RNA-sequencing. Immortalized canine ADSCs were established by transduction with combinations of the R24C mutation of human cyclin-dependent kinase 4 (CDKR24C), canine cyclin D1, and canine TERT. The ADSCs transduced with CDK4R24C, cyclin D1, and TERT (ADSC-K4DT) or with CDK4R24C and cyclin D1 (ADSC-K4D) showed a dramatic increase in proliferation (population doubling level >100) without cellular senescence compared to the primary ADSCs. The cell surface markers, except for CD90 of the ADSC-K4DT and ADSC-K4D cells, were similar to those of the primary ADSCs. The ADSC-K4DT and ADSC-K4D cells maintained their trilineage differentiation capacity and chromosome condition, and did not have a tumorigenic development. The ability to inhibit lymphocyte proliferation by the ADSC-K4D cells was enhanced compared with the primary ADSCs and ADSC-K4DT cells. The pathway analysis based on RNA-sequencing revealed changes in the pathways mainly related to the cell cycle and telomerase. The ADSC-K4DT and ADSC-K4D cells had decreased CD90 expression, but there were no obvious defects associated with the decreased CD90 expression in this study. Our results suggest that ADSC-K4DT and ADSC-K4D cells are a potential novel cell source for mesenchymal stem cell therapy.

Mesenchymal stem cells (MSC) are currently being investigated for their therapeutic applications in a wide range of diseases. Although many studies examined peripheral venous administration of MSC, few have investigated the detailed intravenous administration procedures of MSC from their preparation until they enter the body. The current study therefore aimed to explore the most efficient infusion procedure for MSC delivery by preparing and infusing them under various conditions. Canine adipose-derived mesenchymal stem cells (cADSC) were infused using different infusion apparatuses, suspension solutions, allogenic serum supplementation, infusion time and rates, and cell densities, respectively. Live and dead cell counts were then assessed by manual measurements and flow cytometry. Efficiency of live- and dead-cell infusion and cell viability were calculated from the measured cell counts and compared under each condition. Efficiency of live-cell infusion differed significantly according to the infusion apparatus, infusion rate, and combination of cell density and serum supplementation. Cell viability after infusion differed significantly between the infusion apparatuses. The optimal infusion procedure resulting in the highest cell delivery and viability involved suspending cADSC in normal saline supplemented with 5% allogenic serum at a density of 5 × 10⁵ cells/mL, and infusing them using an automatic infusion device for 15 min. This procedure is therefore recommended as the standard procedure for the intravenous administration of ADSC in terms of cell-delivery efficiency.

An 11-year-old intact female Papillion weighing 2.1 kg was referred to our institution with the main complaint of shallow, rapid breathing. At the first visit (day 0), although clinical signs improved due to the use of medication from the primary hospital, transthoracic radiography and echocardiography revealed left heart enlargement and left ventricular dysfunction. A clinical diagnosis of dilated cardiomyopathy (DCM) was made and oral administration of pimobendan, temocapril, and taurine was initiated. However, on day 10, the respiratory status worsened and furosemide was prescribed. On day 54, no significant improvement in heart size was observed. Additionally, the diet that this patient received met the recommendation for diet-related DCM by the U.S. Food and Drug Administration, and the patient’s diet was changed from a grain-free diet to a grain-containing diet. On day 1191, the patient’s respiratory status was stable and no clinical signs were observed. Transthoracic radiography and echocardiography revealed an improvement in left heart size. Additionally, improvements in the left and right ventricular myocardial strains were observed after changing the diet. We suggest that it may be necessary to suspect a dietary association with dilated cardiomyopathy, and a good prognosis might be expected by dietary modification, even in small-breed dogs.

Pulmonary hypertension (PH) is a fatal condition that affects many dogs. In humans, PH is often treated with beraprost sodium (BPS). However, the effectiveness of BPS for canine PH has not been established. This study aimed to evaluate the clinical and cardiovascular response of BPS in canine patients with PH of various causes. Sixteen dogs with PH (post-capillary PH, n = 8; pre-capillary PH, n = 8) were included. BPS was continuously administered twice daily at 15 µg/kg. All dogs underwent echocardiography, including speckle-tracking analysis and blood pressure measurement, before and after BPS administration. Continuous BPS administration (range: 13.2–22.0 µg/kg) significantly decreased the pulmonary and systemic vascular impedance and increased left and right ventricular myocardial strain. In dogs with post-capillary PH, BPS administration caused no significant worsening of the left atrial pressure indicators. No side effects of BPS were observed in any dog. BPS also improved cardiac function and pulmonary circulation through pulmonary vasodilation, suggesting that BPS may be an additional treatment option for canine PH of various causes. Particularly, BPS increased left ventricular function and systemic circulation without worsening the left heart loading condition in dogs with post-capillary PH.

Mesenchymal stem cells (MSCs) have excellent anti-inflammatory and immunomodulatory capabilities and therapeutic effects in some viral diseases. The therapeutic impact of MSCs mainly relies on the paracrine effects of various secreted substances. Feline calicivirus (FCV) and feline herpesvirus type 1 (FHV1) are common and highly prevalent pathogens causing upper respiratory diseases, and FCV is associated with gingivostomatitis in cats. Recently, feline MSC treatment has been reported to improve the clinical symptoms of feline chronic gingivostomatitis, but the antiviral effects of feline MSCs on FCV and FHV1 are not known. In this study, we evaluated the antiviral efficacy of using feline MSC secretome as a conditioned medium on FCV and FHV1 viral replication in Crandell–Reese feline kidney (CRFK) cells, and RNA sequencing was used to analyze how the CRFK cells were altered by the MSC secretomes. The feline MSC secretome did not inhibit FCV or FHV1 viral entry into the CRFK cells but had antiviral effects on the replication of both FCV and FHV1 in a dose-dependent manner.