| Abstrakt: |
Objectives: Previously we have demonstrated multi-potency of stem cells derived from human endometrium. Here we demonstrate neurogenic differentiation of human endometrial derived stem cell (HEDSC) and their therapeutic potential using a Parkinson's disease mouse model. Methods: Endometrium was obtained from reproductive aged women and standard stromal cell tissue cultures were generated. After two passages, stromal cells were characterized using flow cytometry. In vitro neurogenic differentiation into a dopaminergic pathway was then induced using a two step method. Whole cell patch clamp recording was then performed on differentiated and undifferentiated cells to examine for evidence of G-protein coupled inwardly rectifying potassium (GIRK) channels, which are characteristic of central neurons including dopaminergic cells. Differentiated cells were fixed in methanol and incubated with antibodies for Nestin or tyrosine hydroxylase (TH) followed by FITC and Alexafluore secondary antibodies, respectively. Dopamine (DA) and dihydroxy-phenylacetic acid (DOPAC) concentrations were measured in differentiated tissue cultures. A Parkinson's disease model was induced in both wild-type C57-B6 and NOD-SCID mice using methyl-phenyl-tetrahydropyridine (MPTP). Fluorescent labeled (PKH26) and unlabeled HEDSC were transplanted into the striatum, while PBS vehicle was used for control injections. Striatal DA and DOPAC concentrations were measured by HPLC. To demonstrate engraftment, HEDSC were identified in mouse brain sections using human nestin antibody. Results: HEDSC displayed the following distribution: 0.3% CD45+, 1.4% CD30+, 99.6% CD90+, 99.7% PDGFR +, and 99.7% CD146+. Cells cultured in the dopaminergic conditions displayed neurogenic morphology with axon-like projections and pyramidal cell bodies. Voltage clamping revealed the presence of barium sensitive potassium channels, which were lacking in undifferentiated cells. HEDSC induced to a dopaminergic pathway expressed nestin, a neural stem cell marker, as well as TH, a critical enzyme in dopamine synthesis pathway. DOPAC was found in the differentiated cell cultures, but not in the undifferentiated endometrial stromal cell tissue cultures. Striatal DA, DOPAC and DOPAC/DA were higher in mice transplanted with HEDSC than PBS. Engraftment of HEDSC in mouse brains was demonstrated by staining for human nestin antibody. HEDSC survival was demonstrated for at least 5 weeks after transplantation. Engraftment successfully occurred in both immunodeficient mice, as well as immunocompetent mice in this xenograph model. Conclusions: HEDSC were differentiated into dopaminergic cells. These cells display neural markers and voltage characteristics consistent with dopaminergic neurons. Dopamine metabolites are produced in neurogenic cell cultures. HEDSC successfully engraft in mouse brains, where they increase dopamine concentrations. Endometrial stem cells show therapeutic potential for use in Parkinson's disease. |
