Fig 1.
Preparation of microgrooved-topography.
(A, B): Bright field images of normal and microgrooved tissue culture flasks, respectively. (C, D): Bright field images of control and microgrooved surfaces, respectively. (E, F): SEM images of control/normal and microgrooved surfaces, respectively. (G): Quantitation of microgrooved/modified surface area (cm2) against the total cell growing surface area. Scale bars = 50 μm (C, D), 20 μm (E, F). Results shown are mean ± SEM. ***, P ≤ 0.001.
Fig 2.
Mouse bone marrow cells (BMCs) culture on normal and microgrooved.
(A, B): Bone marrow cells as original source of MSCs were isolated, counted, and then seeded at the density of 1 × 106 cells/cm2 in normal and microgrooved flasks, respectively. The images were captured using bright field microscope 5–10 minutes post-seeding. (C, D): The old medium was removed after 48 hours, and pictures of BMC culture were captured. Two types of adhered cells; small spindle-shape and round-shape cells were observed. (E): The floating cells, collected along with old media, were centrifuged and quantitated following 0.4% trypan blue staining to know percentage of attachment of BMCs and dead cells among the floating cell population. Scale bars represent = 50 μm. Results shown are mean ± SEM.
Fig 3.
Culture and expansion of mouse mesenchymal stem cells on normal and microgrooved surfaces.
(A, C, E, G): Normal surfaces showing MSCs’ morphology and density at P0, P1, P2 and P3, respectively. (B, D, F and H): Microgrooved bearing surfaces showing MMSCs density at P0, P1, P2 and P3, respectively. (I): Grouped bar chart showing quantitation of cell yield obtained from normal (MSCs) and microgrooved (MMSCs) flasks at P0, P1, P2, and P3. (J): Bar chart showing measurement of cell elongation of MSCs and MMSCs. Scale bars represent = 100 μm. Results shown are mean ± SEM. *, P ≤ 0.05, **, P≤0.01, ***, P ≤ 0.001.
Fig 4.
Study of morphological heterogeneity and morphology of P3 MSCs and MMSCs.
(A): SEM image depicting three distinct cell populations in the P3 MSCs culture: spindle-shaped, cuboidal or flattened and extremely small rapidly proliferating cells (RS cells). (B): SEM micrograph of a single P3 MSC showing typical spindle-shape morphology with oval-shape nucleus in the centre of the cell. (C): SEM image of P3 MMSCs’ culture cells showing spindle-, flattened- and round-shaped morphologies. (D): SEM micrograph of a typical spindle-shaped P3 MMSC. Scale bars = 10 μm (A, B, D), 20 μm (C).
Fig 5.
Immunophenotypic characterization of P3 MSCs and MMSCs.
(A): Immunophenotyping of the MSCs and MMSCs with respect to well-established mesenchymal-lineage markers such as CD29, CD44, Sca-1, CD45, CD34 and CD11b. The P3 MSCs and MMSCs were stained with PE-conjugated anti-mouse CD29 and FITC-conjugated anti-mouse CD44, Sca-1, CD34, CD45 and CD11b antibodies. The expression of each marker is shown along with the respective isotype control. (B): Grouped bar chart showing quantitation of cell surface markers on P3 MSCs and MMSCs along with BMCs, and data was expressed as mean ± SEM. Abbreviations: PE, phycoerythrin; FITC, fluorescein isothiocyanate.
Table 1.
Panel of antibodies used for immunophenotyping and FACS analyses.
Table 2.
Adipogenic differentiation medium.
Fig 6.
Tri-lineage differentiation of P3 MSCs and MMSCs.
(A, B): Oil red O stained adipocytes differentiated from P3 MSCs and MMSCs, respectively. (C, D): Alkaline Phosphatase staining of osteocytes derived from differentiation of MSCs and MMSCs, respectively. (E, F): Chondrocytic differentiation of MSCs and MMSCs followed by Safranin O + haematoxylin staining. Scale bars = 10 μm (A, B, E, F); 50 μm (C, D).
Table 3.
Osteogenic induction medium.
Table 4.
Chondrogenic induction medium.
Fig 7.
Expression of pluripotency-associated transcription factors in BMCs, MSCs and MMSCs.
(A): The basal expression of pluripotency-associated markers like Oct3/4, Sox-2, Nanog and Myc was measured at RNA level by RT-PCR and found to be considerably high in MSCs compared to the BMCs, and this relatively higher basal expression was even significantly higher in MMSCs. (B): Grouped bar diagram showing quantitation of pluripotency markers. Results shown are mean ± SEM of three independent experiments. *, P ≤ 0.05, **, P≤0.01, ***, P ≤ 0.001, P ≥ 0.05, n.s., not significant. Abbreviations: bp, base pair; M, 100 bp DNA ladder used as marker; Lc, loading control (452 bp); RT, reverse transcription.
Table 5.
Primers used for RT-PCR.
Fig 8.
Flow cytometric analysis of cell cycle with propidium iodide.
(A): Comparative cell cycle analysis of BMCs, MSCs and MMSCs showing percentage of cell population in various cell cycle stages (G1, S, G2/M). (B): Bar chart showing cell stage-specific quantitation among BMCs, MSCs and MMSCs populations. Abbreviations: FSCs, forward scatter; SSC, side scatter; FL2-A/W, filter 2-area/width. M1, Sub G0/G1; M2, G0/G1; M3, S-phase; M4, G2/M phase. Results shown are mean ± SEM of three independent experiments. *, P ≤ 0.05, **, P≤0.01, ***, P ≤ 0.001, P ≥ 0.05, n.s., not significant.