Endometrial stem cell transplantation restores dopamine production in a Parkinson's disease model

Abstract

Parkinson's disease (PD) is a neurodegenerative disorder caused by the loss of dopaminergic neurons. Adult human endometrial derived stem cells (HEDSC), a readily obtainable type of mesenchymal stem-like cell, were used to generate dopaminergic cells and for transplantation. Cells expressing CD90, platelet derived growth factor (PDGF)-Rβ and CD146 but not CD45 or CD31 were differentiated in vitro into dopaminergic neurons that exhibited axon projections, pyramidal cell bodies and dendritic projections that recapitulate synapse formation; these cells also expressed the neural marker nestin and tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. Whole cell patch clamp recording identified G-protein coupled inwardly rectifying potassium current 2 channels characteristic of central neurons. A 1-methyl 4-phenyl 1,2,3,6-tetrahydro pyridine induced animal model of PD was used to demonstrate the ability of labelled HEDSC to engraft, migrate to the site of lesion, differentiate in vivo and significantly increase striatal dopamine and dopamine metabolite concentrations. HEDSC are a highly inducible source of allogenic stem cells that rescue dopamine concentrations in an immunocompetent PD mouse model.

Fig 1

In vitro neurogenic differentiation of HEDSC. HEDSC cultured in control media demonstrate typical stromal cell morphology (A), whereas cells cultured in neurogenic media demonstrated both pyramidal and dendritic cell morphology as is pictured using light microscopy (B, C). Differentiated cells visualized using: differential interference contrast (D), IF for neural stem cell marker nestin expression (E), and a merge of both (F). Differentiated cell cultures, also express TH (H), DAPI nuclei staining (G), and merge of both (I).

Fig 2

Electrophysiology using whole cell patch clamp testing. HEDSC-derived neurogenic cells display GIRK2 current characteristic of central neurons that diminishes with barium administration (right), whereas control cells do not (left).

Fig 3

HEDSC Engraft, differentiate in vivo, and migrate to the lesioned site in mice brains. (A) PCR detecting human DNA in mouse brain transplanted with HEDSC (n= 14), but not with sham transplants (n= 8) using PBS. (B) Low power view of murine brain section from sham treated [control (CTL)] and HEDSC-treated animals. An area that includes the substantia nigra (SN) is outlined. IHC using a human nestin antibody identifies cells localized to the SN in the transplanted animals. Human cells are visualized in mouse brains in the right column, and controls are shown on the left. In the top panel, all human cells are detected using a human mitochondrial antibody (hMit), which are seen here at the site of transplantation in the striatum. Spontaneous in vivo differentiation of transplanted HEDSC was observed, where they expressed nestin (hNestin). Transplanted cells adapted a neurogenic phenotype morphologically, as is visualized using red fluorescent surface labelling. Human cells were observed remote from the initial transplantation site (striatum), where they migrated to the lesioned brain area (substantia nigra) which is the area pictured in the bottom right panel (red fluorescent surface labelling).

Fig 3

HEDSC Engraft, differentiate in vivo, and migrate to the lesioned site in mice brains. (A) PCR detecting human DNA in mouse brain transplanted with HEDSC (n= 14), but not with sham transplants (n= 8) using PBS. (B) Low power view of murine brain section from sham treated [control (CTL)] and HEDSC-treated animals. An area that includes the substantia nigra (SN) is outlined. IHC using a human nestin antibody identifies cells localized to the SN in the transplanted animals. Human cells are visualized in mouse brains in the right column, and controls are shown on the left. In the top panel, all human cells are detected using a human mitochondrial antibody (hMit), which are seen here at the site of transplantation in the striatum. Spontaneous in vivo differentiation of transplanted HEDSC was observed, where they expressed nestin (hNestin). Transplanted cells adapted a neurogenic phenotype morphologically, as is visualized using red fluorescent surface labelling. Human cells were observed remote from the initial transplantation site (striatum), where they migrated to the lesioned brain area (substantia nigra) which is the area pictured in the bottom right panel (red fluorescent surface labelling).

Fig 4

HEDSC Transplantation increases dopamine concentrations in mouse striatum. Dopamine concentrations (mean ± S.E.M.) were measured in mice brains. In the first graph, animals not treated (unlesioned) with MPTP (n= 5) are shown in the left column with baseline levels. In the middle column, MPTP lesioned mice showed an expected decrease in dopamine concentrations when given sham operations with PBS (n= 8). In the right column, MPTP lesioned mice demonstrated rescued dopamine concentrations when treated with HEDSC transplantation (n= 14), P < 0.0001. A similar therapeutic effect of HEDSC is seen in the bottom graph by measuring the dopamine metabolite DOPAC concentrations in mice striatum, P= 0.008.