Figure 1.
Growth characteristics of pSSCs in vitro.
(A) Proliferation of pSSCs: Given is the CPD (cumulative population doubling) of pSSCs as function of days in culture. Data are expressed as means ± SEM from three independent pSSC strains. (B) Average CFU efficiency of one representative pSSCs strain after 22 CPD. Mean of three experiments ± SEM. (C) Image of cell colonies (stained with trypan-blue) formed after 11 days seeded with 10 cells per cm2 growth surface. Scale bar: 1 cm. (D, E) Morphological appearance of pSSCs. Scale bars: (D) 200 µm, (E) 100 µm.
Figure 2.
Expression of stem cell related genes and translated proteins.
(A) RT-PCR analysis of stem cell related genes. Line 1–3: results of three independent cell strains, line 4: negative control (-RT) from one representative cell strain. (B) Flow cytometric analysis of cell surface markers CD9, CD29, CD44, CD90, CD105, and nestin. Data are expressed as means ± SEM from three independent pSSCs strains after 22 CPD. (C) Immunocytochemistry revealed a subpopulation of pSSCs expressing nestin. (D) Double staining indicates the coexpression of nestin and fibronectin. Scale bars: 50 µm.
Figure 3.
Neuro-muscular differentiation potential of pSSCs.
(A) After 30 days of induced neuronal differentiation one cell strain developed a structured cell network of linked bodies. (B–D) Cells show morphologies similar of neuronal cells (white arrows) and smooth muscle cells (black arrows). Scale bars: (A, B) 500 µm, (C) 50 µm, (D) 100 µm. (C) Represents the marked region in Fig. 3B. (E–G) Immunocytochemistry revealed the expression β-III-tubulin and GFAP in the centre and some surrounding cells of the bodies, whereas α-SMA is exclusively expressed in the outer regions of the bodies and some surrounding cells. Scale bars: 50 µm. (H, I) β-III-tubulin is expressed in a subpopulation of cells that are morphological consistent with neurons. (J, K) Other subpopulations of differentiated pSSCs expressed the neuronal markers GFAP and neurofilament M. (L, M) Subpopulations with a morphology consistent of smooth muscle cells expressed the smooth muscle marker α-SMA. Scale bars: 50 µm.
Figure 4.
Differentiation-related regulation of proteins.
Expression of the neuronal and muscular lineage markers nestin, beta-III-tubulin, GFAP, and α-SMA before differentiation (white bars) and after differentiation (grey bars) revealed by flow cytometry. Data are expressed as means ± SEM from three independent cell strains.
Figure 5.
Induced adipogenic differentiation of pSSCs.
(A) The adipogenic lineage differentiation is shown by bright field pictures of positive Oil Red O stained lipid vacuoles within the cytoplasm of the pSSCs after 30 days of induced differentiation. (B) Oil red O staining of pSSCs cultured in control medium. Scale bars: 50 µm. (C) Leptin expression shown by RT-PCR in adipogenic differentiated and undifferentiated pSSC cultures. Hypoxanthin-guanin phosphoribosyltransferase (HPRT) was used as an internal control for each PCR.