Table 1.
Primer sequences used for qRT-PCT.
The sequences were obtained using Primer Express (Applied Biosystems) and validated through BLAST and BLAT.
Fig 1.
Isolation, characterization and differentiation of murine epidermal stem cells.
The epidermal stem cells were simultaneously isolated with two other cell populations and selected for their ability to adhere to collagen IV. (A) Micrograph displaying holoclonic colonies at the 4th through the 10th passage of subculturing. mRNA relative levels for specific markers of these stem cells were evaluated by qRT-PCR: (B) Δn p63; (C) α6 integrin; (D) β1 integrin. The normalization factor was calculated with the GeNorm algorithm (HPRT, GAPDH and HMBS were used as endogenous control). (E) Cells after reaching the fourth passage were induced to differentiate by increasing levels of calcium/serum. The efficiency of this process was assessed by evaluating the mRNA levels (F) of a differentiation marker, namely filaggrin. The results are presented as the mean ± SD of values obtained in three independent experiments performed in triplicates. Statistical analyzes were performed using ANOVA analysis of variance followed by Tukey test. All groups were measured versus the control (TS = total skin, P1, P2, P4, P10 = passages from 1 to 10, C = control not induced to differentiate, E = Epidermal differentiation), ns = not significant; * p ≤ 0.05; ** p≤0.001.
Fig 2.
Isolation and characterization of skin stem cell precursors (SKP).
The SKPs were selected by culture in serum-free suspension, generating cell aggregates, as may be observed in the micrograph shown in (A). To assess the success of selection, we analyzed the levels of mRNA and protein of negative (B) Vimentin and positive (C) Nestin and (D) Fibronectin markers through qRT-PCR and Western blotting. The same markers were observed with fluorescence microscopy to show the distribution/localization of stem cells in the mass of aggregates (E) Bar = 50μm. The results are presented as the mean ± SD of values obtained in three independent experiments performed in triplicates. Statistical analyzes were performed using ANOVA analysis of variance followed by Tukey test to post-Kramer. All groups were assessed versus control (in the case of stem cell markers relative to the total dermis, TS = total skin), ns = not significant; * p ≤ 0.05; ** p≤0.001.
Fig 3.
Differentiation of murine skin stem cell precursors (SKP).
The differentiation of SKPs was induced through the dissociation of cell aggregates into single cells followed by serum exposure and, as may be seen in the micrographs after 7 days (A), 14 days (B) and 21 days (C), these cells presented several elongated extensions. The mRNA levels of astrocyte markers (D) GFAP; oligodendrocytes (E) CNPase; and neurons (F) βIII tubulin were measured by qRT-PCR and validated at the protein level (G) by immunofluorescence microscopy, Bar = 50μm. The results are presented as the mean ± SD of values obtained in three independent experiments performed in triplicates. Statistical analyzes were performed using ANOVA analysis of variance followed by Tukey test to post-Kramer. All groups were measured versus the undifferentiated control in the shortest time of differentiation, ns = not significant; * p ≤ 0.05; ** p≤0.001.
Fig 4.
Isolation and characterization of mesenchymal stromal stem cells from murine dermis.
The mesenchymal stem cells from murine dermis were selected for their ability to form colonies at low densities (CFU), as shown in the micrograph (A). These colonies expand and form a confluent layer at P0, as shown in (A). These cells are then passaged whenever they reached 80% confluence and expanded until passage 10 (B). Cells at P4 were used to detect the presence of positive and negative markers of MSCs by flow cytometry, namely: CD34, CD31, CD45, CD44, CD90, CD29 and CD105 (C). All groups marked with conjugated antibody (blue line) were compared to their respective controls (gray filled line); at least 50.000 events were collected for analysis. IgG isotype controls conjugated with Alexa 488 and APC were used as negative controls. Results are representative of those obtained in three independent experiments performed in triplicates.
Fig 5.
Mesenchymal stem cells were stimulated to undergo adipogenesis, osteogenesis and chondrogenesis.
Cells were exposed to differentiation medium at P4 and samples were collected at 7, 14 and 21 days. Important morphological changes may be seen in micrographs under an inverted microscope after 21 days (A), Bar = 50μm. The same is reflected in increased production of lipid droplets (adipogenesis), calcified matrix (osteogenesis) and glycosaminoglycans (chondrogenesis) as shown by immune-histochemistry (B), the control group is in the inserts (21 days after induction). The relative expression levels of mRNA for specific markers of these processes were evaluated in (C) Leptin; (D) Alkaline Phosphatase; (E) Collagen II. The results are presented as the mean ± SD of values obtained in three independent experiments performed in triplicate. Statistical analyzes were performed using ANOVA analysis of variance followed by Tukey test. All groups were measured versus the undifferentiated control in the shortest differentiation period of time, ns = not significant; * p ≤ 0.05; ** p≤0.001.