Direct conversion of mouse fibroblasts to GABAergic neurons with combined medium without the introduction of transcription factors or miRNAs

<p>Degeneration or loss of GABAergic neurons frequently may lead to many neuropsychiatric disorders such as epilepsy and autism spectrum disorders. So far no clinically effective therapies can slow and halt the progression of these diseases. Cell-replacement therapy is a promising strategy for treatment of these neuropsychiatric diseases. Although increasing evidence showed that mammalian somatic cells can be directly converted into functional neurons using specific transcription factors or miRNAs via virus delivery, the application of these induced neurons is potentially problematic, due to integration of vectors into the host genome, which results in the disruption or dysfunction of nearby genes. Here, we show that mouse fibroblasts could be efficiently reprogrammed into GABAergic neurons in a combined medium composed of conditioned medium from neurotrophin-3 modified Olfactory Ensheathing Cells (NT3-OECs) plus SB431542, GDNF and RA. Following 3 weeks of induction, these cells derived from fibroblasts acquired the morphological and phenotypical GABAerigic neuronal properties, as demonstrated by the expression of neuronal markers including Tuj1, NeuN, Neurofilament-L, GABA, GABA receptors and GABA transporter 1. More importantly, these converted cells acquired neuronal functional properties such as synapse formation and increasing intracellular free calcium influx when treated with BayK, a specific activator of L-type calcium channel. Therefore, our findings demonstrate for the first time that fibroblasts can be directly converted into GABAergic neurons without ectopic expression of specific transcription factors or miRNA. This study may provide a promising cell source for the application of cell replacement therapy in neuropsychiatric disorders.</p

Human UC-MSCs combined with rUGSSs can generate prostate glands.

<p>Mice were sacrificed 2 months after co-transplantation surgery, and the kidneys from the cell implanted nude mice were collected. (A) Graft initiated with hUC-MSCs alone and (B) rUGSSs alone were used as negative control, respectively. (C) Graft derived with hUC-MSCs and rUGSSs. (D–F) Histological analyses of the sections of the graft stained for haematoxylin and eosin (H&E). (D) Note that while hUC-MSCs alone and (E) rUGSSs single cell type transplantation fail to regenerate prostate glandular structures. (F) co-transplantation of hUC-MSCs and rUGSSs gives rise to prostate glandular structures. Scale bar 50 µm.</p

Regulation and Methylation of Tumor Suppressor MiR-124 by Androgen Receptor in Prostate Cancer Cells

<div><p>Prostate cancer (PCa) is the most frequently diagnosed cancer for men in the developed world. Androgen receptor signaling pathway plays an important role in prostate cancer progression. Recent studies show that microRNA miR-124 exerts a tumor suppressive function in prostate cancer. However, the relationship between AR and miR-124 is unclear. In the present study, we found a negative feedback loop between AR and miR-124 expression. On one hand, miR-124 was a positively regulated target gene of the AR, on the other hand, overexpression of miR-124 inhibited the expression of AR. In addition, we found that miR-124-2 and miR-124-3 promoters were hypermethylated in AR-negative PCa cells. Furthermore, overexpression of miR-124 inhibited proliferation rates and invasiveness capacity of PCa cells <i>in vitro</i>, and suppressed xenograft tumor growth <i>in vivo</i>. Taken together, our results support a negative feedback loop between AR and miR-124 expression. Methylation of miR-124-2 and miR-124-3 may serve as a biomarker for AR-negative PCa cells, and overexpression of miR-124 might be of potential therapeutic value for the treatment of PCa.</p></div

Regenerated prostates resemble the phenotypes of normal prostates.

<p>(A, C, E, G) Immunofluorescence analysis of the expression of CK8, p63, CK5 and androgen receptor (AR) in regenerated prostate tissue. (F) shows triple-staining for the basal cell marker CK5 (green), luminal marker CK8 (red) and DAPI counter staining (blue). (B, D, F, H) The tissue sections were counterstained with 4, 6-diamidino-2-phenylindole (DAPI; blue). Scale bar 50 µm.</p

Detection of human cells in the regeneration of prostate.

<p>The field of prostate epithelial regeneration can be seen within the graft transplanted with hUC-MSCs and rUGSSs. (A, B) Immunofluorescent staining for CK8 in the new graft. (C, D) Detection of human cells in the new graft (green). (E, F) Note that the human nuclear antigen+ cells (green) in the graft can be co-stained with CK8. (G, H) The grafts express the prostate specific antigen (PSA). Scale bar 50 µm.</p

DKK1 inhibits proliferation and migration of prostate cancer cells.

<p>(A) Tritiated thymidine incorporation in PC3 cultures in the absence or presence of Wnt3a or DKK1. While high concentration of Wnt3a (10nM) enhanced PC3 cell proliferation, DKK1 inhibited PC3 proliferation in a dose-dependent manner. (B) Regulation of PC3 cell migration by Wnt signaling. Data were collected from 6–8 cultures per group and are expressed as mean+SEM (t-test). Note that Wnt3a increased the number of migrated cells, whereas DKK1 inhibited cell migration.</p

Immunophenotype analysis of P0-P8 hUC-MSCs by FACS.

<p>Cells were from P0-P8 hUC-MSCs and stained with CD29, CD105, CD34, CD45, and CD31 antibodies. The upper half of the figure is the flow histogram of single antibody staining. The shaded area shows the profile of the negative control. The lower half of the figure is CD29-FITC and CD105-PE expression of P0-P8 hUC-MSCs. (A) hUC-MSCs exhibit positive surface expression of integrin marker (CD29), (B) MSC marker (CD105), (C-E) but are negative for hematopoietic lineage markers (CD34, CD45) nor the platelet/endothelial cell adhesion molecule (CD31). The CD29+CD105+ hUC-MSC population accounts for 85%∼99.9% of all cells.</p

Methylation Status of MiR-124-1, MiR-124-2 and MiR-124-3 CpG Islands.

<p>Schematic summary of CpG sites in the miR-124-1, miR-124-2 and miR-124-3 promoter regions. (A) Methylation analysis was done in 10 clones from each cell line. Each row of circles represents a single clone, and each circle represents a single DNA methylated or demethylated site. (B) The methylation percentages of 10 clones from each of the cell lines are summarized in the bar graph. Data are shown as the means ± SEM. Asterisks indicate P<0.05, double asterisks indicate P<0.001.</p

Wnt signaling prevents prostatic epithelial cell differentiation. (A,B,C) p63 immunocytochemistry (red) of the P2 rat ventral prostate organ cultures maintained for 7 days in the absence (A) or presence of 50 nM of Wnt3a (B) or 400 nM of DKK1 (C).

<p>The tissue sections were counterstained with DAPI (blue). While p63 positive cells (purple) represent basal cells where progenitor cells reside, the blue cells (arrows) that are negative to p63 in the epithelium are differentiated luminal cells. (D) Quantification of p63 positive cells over total epithelial cells. Data were collected from randomly selected 22–27 ductal units from sections of the organ cultures per group and are expressed as mean + SEM (t-test). Note that while Wnt3a led to a significant increase in the number of basal cells, DKK1 resulted in a reduction in basal cells. Bar, 50 µm for A-C.</p

Wnt3a and DKK1 regulate prostatic epithelial branching morphogenesis.

<p>Whole mount ventral prostates were prepared from P2 rats and maintained for 7 days in serum-free medium in the absence (A) or presence of 50 nM of Wnt3a (B) or 400 nM of DKK1 (C). Similar patterns were consistently seen in 3 repeat experiments of 5–6 prostate organs per group in each individual experiment. Note that addition of either Wnt3a or DKK1 to the culture resulted a change in epithelial branching morphogenesis. Quantification of the cultures by measuring the diameter of the cultured prostates (D) and the ductal tips (E) using Axiovision software and the branching points (F) were done by analyzing 4 randomly selected cultures per group. Data are expressed as mean + SEM (t-test, compared to control cultures). Bar, 400 µm.</p