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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.


Introduction
Stem cell research has vastly expanded in recent years, with the promise of revolutionizing medical therapy. An active target of this research has been Parkinson's disease (PD), a chronic, progressive, degenerative disease of the central nervous system that debilitates both motor function and speech due to the insufficient production of dopamine by pigmented cells in the substantia nigra. 1

-methyl 4-phenyl 1,2,3,6-tetrahydro pyridine (MPTP) is a selective neurotoxin of dopaminergic cells that induces PD in both animals and human beings.
Initial excitement about the therapeutic potential of embryonic stem cells for PD was damped by ethical concerns and technical difficulties, including tumour formation. Similarly, the initial enthusiasm for foetal tissue transplantation for PD was tempered in double-blind clinical trials with poor long-term results [1,2]. The role of mesenchymal stem cells (MSC) in neuro-transplantation has shown recent promise due to the ability of this subtype of stem cells to migrate to sites of damaged neural tissue following both intravenous and intracranial transplantation of bone marrow derived MSC [3][4][5] or amnionic fluid derived stem cells [6] in preclinical studies. MSC have the additional advantage of being readily manipulated for use as delivery vehicles for gene therapy in PD models [7,8].
Adult human endometrial derived stem-like cells (HEDSC) are a type of MSC that have only recently been characterized [9][10][11][12][13][14][15][16]. The endometrium displays tenacious regeneration ability due to the demands of menstruation and pregnancy, which make this tissue a promising source of dynamic stem cells suitable for use in regenerative medicine therapies. In vitro transdifferentiation of HEDSC into cartilage, bone, fat and muscle has recently been demonstrated [10,17,18]; however, neither transdifferentiation in vivo, transplantation, nor differentiation into a neurogenic cell type has previously been demonstrated. Here we demonstrate the ability of HEDSC to differentiate into dopamine-producing neurons. We also demonstrate their ability to be used for transplantation, where HEDSC engraft, migrate to the site of lesion, and are spontaneously differentiated in vivo. Furthermore, we show a therapeutic benefit, where transplantation rescues dopamine concentrations in an immunocompetent PD mouse model. To our knowledge this study is the first to demonstrate dopamine improvement using MSC in the absence of gene therapy, which could indicate a particular penchant for HEDSC use in PD transplantation.

Flow cytometry
Flow cytometry was performed to characterize the HEDSC used in this study. After two passages in culture, HEDSC displayed the following surface markers: CD31 ϩ 1.4%, ␣SMA ϩ 5.5%, CD90 ϩ 99.6%, CD45 ϩ 0.3%, which is consistent with a non-haematogenously derived endometrial stromal cell. HEDSC were strongly positive for both PDGF-R␤ ϩ 99.7% and CD146 ϩ 99.7%, which have been shown to isolate HEDSC from fresh endometrial samples.

Neurogenic in vitro differentiation of HEDSC
In vitro transdifferentiated HEDSC exhibited neurogenic morphology including long axon projections, pyramidal cell bodies and dendritic projections that appear to recapitulate synapse formation in culture (n ϭ 3) (Fig. 1). Neurogenic cell identity was demonstrated using immunostaining. Cytoplasmic expression of the neural stem cell marker nestin was observed in in vitro differentiated cells using a human nestin antibody. Dense staining in the soma and axon hillock region is evidence in some neurons, which appear to overlay the nucleus in other cells. In addition, almost all HEDSC that remained adherent after neurogenic differentiation in vitro expressed the rate-limiting enzyme involved in dopamine production tyrosine hydroxylase (TH). The presence of TH production suggests a functional phenotype, specifically dopamine synthesis. Control cells not differentiated with neurogenic media failed to demonstrate any of these indicators of neuronal identity (Fig. 1).

Electrophysiological properties of in vitro differentiated cells
In addition to morphological and immunostaining characteristics, in vitro differentiated cells expressed electrophysiological properties of neurons. A whole cell patch clamp recording method was used to measure the current characteristics of individual cells to look for evidence of barium sensitive potassium channels, which are characteristic of central neurons, including dopaminergic cells. The experiments were performed on 10 separate experi-ments derived from samples differentiated from three separate patients. In the differentiated cells, a series of voltage steps from Ϫ60 mV to Ϫ120 mV induces inward currents, which were dramatically decreased in the presence of barium (200 M), a non-specific blocker of the inwardly rectifying potassium current (Kir). The Kir current, resembling the G-protein coupled inwardly rectifying potassium current (GIRK), was only present in differentiated cells, therefore no Ba 2ϩ -sensitive inward currents were present in undifferentiated cells (Fig. 2).

Transplantation of HEDSC in Parkinson's disease mouse model
HEDSC were successfully transplanted into both immunodeficient and immunocompetent MPTP lesioned mice, where engraftment was demonstrated up to 5 weeks following transplantation using multiple techniques. First, human genomic DNA was detected within transplanted mouse brains using PCR (Fig. 3A). Next, engrafted cells were visualized within the mouse brain using four different techniques. A human mitochondrial antibody, which does not cross react with the mouse antigen, was used to detect human cells in mice brains. Human cells were found around the transplantation site in the striatum; however, they were also found to have migrated to the substantia nigra (Fig. 3B). In contrast, when transplantations were performed with differentiated HEDSC, localization to the substantia nigra was not observed.
The transplanted human cells were shown to exhibited neural stem cell markers by staining with the human nestin antibody (Fig. 3B). Mice that were transplanted with HEDSC were also found to express human TH by RT-PCR, whereas sham transplanted animals did not (data not shown). As cells were observed to successfully engraft in both immunodeficient as well as immunocompetent mice, wild-type mice were used for subsequent experiments.
Engraftment and migration was confirmed using two different types of fluorescently labelled HEDSC for transplantation: PKH26 was used for surface labelling and whereas green fluorescent protein (GFP) transfection was used for cytoplasmic labelling. First, PKH26 red labelled HEDSC were identified at the site of transplantation in the striatum; however, they also demonstrated the ability to migrate to the site of lesion, localizing in the substantia nigra. Furthermore, these migrating cells were able to differentiate from an endometrial phenotype into a neurogenic phenotype in vivo (Fig. 3B). GFP transfected HEDSC were also able to be visualized within the mouse brains, but this method was limited by the low transfection efficiency of approximately 10% of HEDSC in culture prior to use for transplantation.
Intracranial transplantation with HEDSC resulted in a significant

Discussion
Here we demonstrate the ability of HEDSC to differentiate into dopamine-producing neurons, where in vitro cultures demonstrate characteristic neuron morphology, express markers of neural cell phenotype and enzymatic function, and display electrophysiological properties specific to dopamine-producing neurons. Furthermore, we demonstrate the ability of HEDSC to be used for transplantation for the first time, even in immunocompetent animals. This was shown by detecting human DNA in mouse brains after HEDSC transplantation, visualizing human HEDSC in mouse brains using antibodies specific to human cells, identifying HEDSC labelled with red fluorescent dye and identifying GFP fluorescing human HEDSC. These cells survive in the location they are transplanted, but also spontaneously migrate to areas of damage and spontaneously differentiate in vivo. HEDSC exert a therapeutic benefit by rescuing dopamine concentrations in this PD animal model.
HEDSC represent an important source of stem cells that can be obtained from a routine office procedure. Further, they can be used as an autologous or allogenic stem cell source, thereby obviating concerns regarding rejection in human beings. The lack of rejection of human stem cells in this murine model could be due to several possibilities. First it is possible this is a result of immune-privilege provided by the blood-brain barrier. Lack of rejection after neuro-transplantation has been demonstrated in several studies [3,4,[19][20][21][22][23][24][25][26][27]. However, evidence suggests that MSC have immuno-privileged properties themselves compared to other types of stem cells [26,[28][29][30], lending additional support to the potential of stem cells from the endometrium. In fact, spontaneous microchimerism of maternal tissue from foetal cells is well established in the setting of pregnancy [31,32] and following blood transfusions. In addition, MSC can home to and engraft mouse bone marrow [33]. It is also possibly that MSC, or HEDSC in particular, display a particular penchant for neural regeneration such as PD treatment due to an underlying disposition of these cells towards neural cells [34]. An additional advantage of MSC is  their low potential for teratoma formation. It is also possible that PD disease is particularly amenable to exogenous stem cells transplantation due to the lack of gliosis, which impedes MSC influx in pathological disease pathologies like stroke.
To date, stem cells derived from the endometrium that demonstrate the ability to transdifferentiate have been isolated in two ways: (1) by performing flow cytometry to select for cells that are both PDGF-R␤ ϩ and CD146 ϩ [9,18] and (2) by passaging routine stromal cell endometrial cultures [10,11,17]. It is interesting that merely passaging cells in a routine manner selects cells that are strongly positive for the same markers shown to prospectively isolate HEDSC, and serves to mutually validate both reports. However, the differences in cells collected in these two manners remains to be more fully characterized. As our understanding of stem cell biology grows, we are re-evaluating their role in tissue repair. It now appears that stem or progenitor cells are present in most tissues [35]. An expanding body of evidence suggests that stem cells play a role in processes that were previously unrecognized; for instance, it was once thought that neurons in the brain did not undergo regeneration after completing development, when in fact neurons are involved in adult tissue remodelling [36]. It is possible that stem cells may actually be involved in a dynamic state of disease and repair relevant to many chronic disease processes. In fact, it is possible that chronic disease may actually be an indicator of the lifetime burden of regeneration and stem cell depletion. This theory can be illustrated by the cases of early onset PD in high regenerative burden states such as boxing, post-concussion syndrome or drug abuse. Stem

Sample collection
Human endometrial tissue was collected by curettage from nine reproductive aged women undergoing surgery for benign gynaecological conditions. Standard endometrial stromal cell cultures were generated in a routine fashion, which produced an unfractionated stromal cell population. (

Neurogenic in vitro differentiation
After the second passage, cells were treated with a two-step dopaminergic differentiation protocol adapted from Blondheim et al. [34]. HEDSC were first treated with differentiation medium I for

In vitro electrophysiology
Whole cell patch clamp recording was then performed on in vitro differentiated and undifferentiated endometrial cells from three different patients to examine for evidence of GIRK2 channels, which are characteristic of central neurons including dopaminergic cells. Briefly, cells were maintained in a recording chamber with artificial cerebrospinal fluid (bubbled with 5% CO2 and 95% O2) containing (in mM): NaCl 124, KCl

Visualization of human cells in mice brains
Mice were killed at intervals up to 5 weeks following HEDSC transplantation. Some mice were overdosed with dimethyl ether and transcardially perfused with fixative (4% paraformaldehyde, 0.1% glutaraldehyde and 15% picric acid in PBS) for immunofluorescent experiments. To examine for evidence of human cell engraftment, brains were removed from mice and were post-fixed overnight in fixative without glutaraldehyde. Serial 50 m coronal sections were cut through mouse brains using a vibratome and were collected in microtitre plates.
To detect all human cells present in the mouse brains, a generic antibody against human cells that does not cross react with mouse tissue was used: human 60 kD mitochondrial antibody (Millipore, Bedford, MA, USA). Brain sections were then counterstained with the fluorescent secondary antibody donkey antimouse IgG 488 Alexafluore (1:200; Molecular Probes) for 1 hr. Engraftment in mouse brains was further demonstrated by identifying immunofluorescent PKH26 labelled HEDSC (n ϭ 6) and GFP transfected HEDSC (n ϭ 3) in situ. To detect transplanted human cells that expressed neural stem cell markers, sections of mouse brain were also stained with a human nestin antibody using the same protocol described above for in vitro immunostaining.

Dopamine concentrations in transplanted mice
The functional ability of HEDSC to rescue dopamine production in a PD mouse model was evaluated by measuring DA and DOPAC from the striatum of HEDSC transplanted (n ϭ 14) and control (n ϭ 8) immunocompetent C57-Black 6 mice. Five unlesioned mice were injected with intraperitoneal saline to be used as an MPTP model control. Control mice were treated with MPTP, but underwent a sham intracranial transplant with PBS. Test mice were treated with MPTP and underwent a therapeutic transplant with HEDSC. Five weeks after transplantation, mice were killed. Striata from each mouse were rapidly dissected on a chilled glass plate and frozen at Ϫ80ЊC. Samples from each mouse were processed and concentrations of dopamine and metabolites were measured using HPLC as previously described [37]. Results were expressed as nanograms per milligram of protein (mean Ϯ S.E.M.). N-values represent number of mice samples that were examined.

Statistical analysis
Mean dopamine and DOPAC concentrations between groups were analysed using One way ANOVA and result with a P-value Ͻ0.05 were considered significant using SPSS statistical software (Chicago, IL, USA).

Ethics statement
Endometrial tissue was collected under an approved Human Investigations Committee protocol at Yale University. Informed written consent was obtained prior to surgery and to collect research samples. Mice were used under an approved Institutional Animal Care and Use Committee protocol at Yale University.