Figure 1.
Naive hOMSC express constitutively neuronal and dopaminergic markers.
Immunofluorescence of neuronal (A–C) and dopaminergic (D–F) markers in naïve hOMSC, scale bars 100 µM.
Figure 2.
hOMSC developed neuronal-like morphology and gene expression after DA differentiation protocol.
Bright field microscopy of hOMSC cultured in serum free medium for 17 days and of hOMSC-DA (A–B), scale bar 200 µM. Real time PCR analysis of differentiated hOMSC-DA from four different donors (C), showing a reduction in the expression of Nanog, Oct4 and Sox2 and a concomitant increased expression of the neuronal markers β-III tubulin and NCAM1 compared to the naïve hOMSC which served as baseline (relative value = 1). Differentiated hOMSC also showed increased expression of genes related to dopaminergic differentiation and mature dopaminergic neurons. Data is expressed as Mean ± SEM. Significance levels: *p<0.05, **p<0.01, ***p<0.001.
Figure 3.
Increased expression and nuclear translocation of dopaminergic transcription factors was observed in hOMSC-DA.
Immunofluorescent detection of Pitx3, Lmx1A and Nurr1 evaluated in naïve and differentiated hOMSC, midbrain primary culture, hOMSC-derived osteoblast-like cells and mouse embryonic fibroblasts (A). Arrows point to the nuclear localization of these transcription factors in the hOMSC-differentiated and midbrain DA cells. Scale bar = 50 µM. Quantification of cells expressing the evaluated transcription factors in the whole cell (nuclei+cytoplasm) or only in the nuclei before and after DA-differentiation (B).
Figure 4.
Induced hOMSC show a mature dopaminergic-like phenotype in vitro.
Immunofluorescence analysis and cell positive counts of neuronal and DA markers in naïve, DA-hOMSC and fibroblasts before and after differentiation, scale bar = 50 µM (A). Complete evaluation of naïve, differentiated hOMSC and fibroblasts marker expression can be seen in supplemental figure S2. Evaluation of Pitx3 and Tyrosine hydroxylase (TH) expression levels by western blot and band densitography, showing increased protein expression after differentiation (B). Medium dopamine content evaluation in naïve or hOMSC-DA in presence or absence of the non-specific membrane depolarizer agent KCl, showing increased levels of dopamine after membrane depolarization of differentiated hOMSC (C). Data is presented as Mean±SEM. Significance levels: *p<0.05, **p<0.01, ***p<0.001.
Figure 5.
Induced hOMSC-DA show sustained therapeutic effect in a PD rat model after transplantation.
Amphetamine-induced rotations (A), rotorod (B) and cylinder (C) test evaluation of 6-OHDA affected rats, following intra-striatal transplantation of saline, naive and hOMSC-DA. Data is presented as Mean±SEM. Significance levels: *p<0.05, **p<0.01, ***p<0.001.
Figure 6.
Transplanted cells engrafted the injured hemisphere and increased dopamine levels.
Ten weeks post-transplantation TH immunostaining (A) show decreased levels of TH in injured striatum and substantia nigra compared to healthy ones. TH striatal expression, analyzed by TH-DAB immunochemistry was detected in hOMSC-DA transplanted hemispheres, while saline or naïve hOMSC transplanted hemispheres showed significant reduced levels (black arrows indicate TH expressing cells). Injured hemispheres transplantated with hOMSC contain PKH26 (red) labeled cells. In hOMSC-DA transplanted hemispheres analyzed by immunofluorescence, TH expression (green) and PKH26 were found in the same cell clusters. Scale bar = 50 µm. (B) Whole hemisphere dopamine and serotonin HPLC quantification from healthy, and 6-OHDA rats treated with saline, naive and hOMSC-DA. Data is presented as Mean±SEM. Significance levels: *p<0.05, **p<0.01, ***p<0.001.