Fig 2: NTF-SCs differentiation and characterization (A–D). A Flow cytometry histograms of three passaged ADMSCs for (CD105, CD90) were positive and negative for (CD45), indicating the ability to express of ADMSCs surface markers by isolated cells. B Bright-field imaging of ADMSCs, pre-induction ADMSCs (third passage) and NTF-SCs cultures showed the expanded ADMSCs have a fibroblast-like phenotype and the NTF-SCs with an astrocyte-like appearance. C Immunohistochemistry imaging for GFAP astrocyte cells marker and DAPI staining for the nucleus of the cells demonstrated that 80.3% of the differentiated cells expressed the GFAP marker. D In Western blot analysis for NTF secretion in NTF-SCs-CM, the blots were developed with NGF and BDNF antibodies, and an average of 35 KDa and 15 KDa of NGF and BDNF were detected, respectively. One-way ANOVA and Tukey post hoc analysis. *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001. NTF-SCs, Neurotrophic-factor-secreting astrocyte-like cells. ADMSCs, Adipose-derived mesenchymal stem cells. NTF, Neurotrophic factor, CM, Culture medium

Fig 3: Experimental procedure schema and multipotent mesenchymal stem cell (MSC) and alveolar type II cell (ATII) purity. (A) Animal experimental design: rats of 200–225 g body weight at the beginning of the experiment were randomized in six experimental groups as indicated. At 0, 2, and 9 h an intratracheal instillation was administered as indicated; the body weights were recorded every 24 h; and all the animals were sacrificed 72 h after starting the experiment. (B) Alveolar type II (ATII) cells stained with alkaline phosphatase (100×). In dark pink, we can identify the positive cells, therefore the real ATII. The purity for the ATII cells was 86 ± 3%. (C) Surfactant C staining for alveolar type II cells. Surfactant C (in green, fluorescein isothiocyanate (FITC)) and nuclear staining (in blue, Hoechst 33342) to confirm the ATII cell purity. (D) Mesenchymal stem cells stained by CD44 (in red, Texas Red), CD90 (in green, FITC), CD105 (in green, FITC), and CD34 (in green, FITC). Nuclei can be observed in blue by Hoechst 33342 staining. Magnification used is 100×. MSC should express CD44, CD90, and CD105 and should be negative for CD34. (E) MSC were differentiated into different lineages. Panel E shows the differentiation to osteocytes stained with Alizarin Red, to chondrocytes stained with Alzian Blue, and to adipocytes stained in oil-red -O staining (200× magnification). The purity of MSC was 78 ± 5%.

Fig 4: Characterization of cPDLSCs. (A) Colony formation and morphology of cPDLSCs. Magnification, ×4. (B) Flow cytometric analysis of cell surface markers CD45, CD73, CD90, CD105 and STRO-1. Blue presents the blank control and red is the surface marker. (C) Alizarin Red S staining of mineralized nodule formation after 28 days of osteogenesis induction. Magnification, ×20. (D) Oil Red O staining of lipid droplet after 28 days of adipogenesis induction. Magnification, ×10. (E) Immunofluorescence imaging of βIII-tubulin after 28 days of neurogenesis induction (scale bar, 500 µm). cPDLSCs, canine periodontal ligament stem cells.

Fig 5: Derivation and culture of canine CDCs. (A) Schematic diagram showing the derivation of canine CDC a of Beagle dog. (B) Outgrowth cells from plated myocardial tissues. (C) Cardiosphere formation in suspension culture. (D) CDCs dissociated from cardiospheres. (E) Expressions of CD105, CD90, ckit by flow cytometry in canine CDCs. Scale bars = 50 μm in all images.