Figure 1.
S100 expression in differentiated mesenchymal stem cells.
(A) RT-PCR analysis of S100 from undifferentiated MSC (uMSC) and differentiated MSC (dMSC) harvested from young and old donors at early passage. The amplicons (size shown in base pairs; bp) were resolved on a 2% agarose gel and stained with Gel Red dye. (B) Cell cultures were stained for S100 protein (green) and the nuclei counter-stained with DAPI (blue).
Figure 2.
Mesenchymal stem cells enhance neurite outgrowth of co-cultured DRG neurons.
(A) Immunocytochemical staining for βIII tubulin (Alexa Fluor 488) visualized neurite outgrowth from DRG following 24 h co-culture with medium only controls (α-MEM and diff α-MEM) and undifferentiated MSC (uMSC) and differentiated MSC (dMSC) from young and old donors (T3) seeded in tissue culture inserts above the neurons. (B) Quantitative analysis of total neurite outgrowth in the co-cultures. ***P<0.001 significantly different to respective media only controls. n.s not significantly different (α-MEM and diff α-MEM versus DRG alone and dMSC versus uMSC old donors).
Figure 3.
Neurotrophic factor expression in mesenchymal stem cells.
(A) RT-PCR analysis of neurotrophic factor transcripts NGF, GDNF, NT-3, BDNF, VEGF from undifferentiated MSC (uMSC) and differentiated MSC (dMSC) harvested from young and old donors. Amplicon size is shown in base pairs (bp). (B and C) ELISA was used to determine BDNF and VEGF protein levels in cell culture supernatants. ***P<0.001 differentiated MSC significantly higher compared with undifferentiated MSC. (D) Neurite outgrowth of DRG neurons after 24 h in the absence (DRG alone) or presence of young donor dMSC alone or with BDNF and VEGF blocking antibodies (ab) or when treated with conditioned medium (cond med) prepared from dMSC. *P<0.05; **P<0.01 significantly different from DRG alone; ns, no significant difference between groups.
Figure 4.
Mesenchymal stem cells enhance neurite outgrowth from the human SH-SY5Y neuronal cell line.
(A) Immunocytochemical staining for βIII tubulin (Alexa Fluor 488) visualized neurite outgrowth (highlighted with arrows) from SH-SY5Y cells following 24 h co-culture with medium only controls (α-MEM and diff α-MEM) and undifferentiated MSC (uMSC) and differentiated MSC (dMSC) from young and old donors seeded in tissue culture inserts above the neurons. (B) Quantitative analysis of total neurite outgrowth in the co-cultures. ***P<0.001 significantly different to respective media only controls; *P<0.05 significantly different young dMSC versus old dMSC; ns not significantly different to respective media only controls.
Figure 5.
Change in undifferentiated stem cell morphology and proliferation after time in culture.
(A) Undifferentiated MSC showed spindle shaped morphology at T2 but cells from old donors show a bigger more flattered shape at T12. (B) uMSC were cultured for nine weeks and passaged and counted on a weekly basis. Cumulative population doublings were calculated and plotted against time.
Figure 6.
Late passage mesenchymal stem cells enhance neurite outgrowth of co-cultured DRG neurons.
(A) Immunocytochemical staining for βIII tubulin (Alexa Fluor 488) visualized neurite outgrowth from DRG following 24 h co-culture with medium only controls (α-MEM and diff α-MEM) and undifferentiated MSC (uMSC) and differentiated MSC (dMSC) from young and old donors (T12) seeded in tissue culture inserts above the neurons. (B) Quantitative analysis of total neurite outgrowth in the co-cultures. ***P<0.001 significantly different to respective media only controls. n.s not significantly different (α-MEM and diff α-MEM versus DRG alone and dMSC versus uMSC old donors).