http://www.liebertonline.com/doi/abs/10.1089/scd.2009.0340
Cell therapy remains an experimental treatment for neurological disorders. A major obstacle in pursuing the clinical application of this therapy is finding the optimal cell type that will allow benefit to a large patient population with minimal complications. A cell type that is a complete match of the transplant recipient appears as an optimal scenario. Here, we report that menstrual blood may be an important source of autologous stem cells. Immunocytochemical assays of cultured menstrual blood reveal that they express embryonic-like stem cell phenotypic markers (Oct4, SSEA, Nanog), and when grown in appropriate conditioned media, express neuronal phenotypic markers (Nestin, MAP2). In order to test the therapeutic potential of these cells, we used the in vitro stroke model of oxygen glucose deprivation (OGD) and found that OGD-exposed primary rat neurons that were co-cultured with menstrual blood-derived stem cells or exposed to the media collected from cultured menstrual blood exhibited significantly reduced cell death. Trophic factors, such as VEGF, BDNF, and NT-3, were up-regulated in the media of OGD-exposed cultured menstrual blood-derived stem cells. Transplantation of menstrual blood-derived stem cells, either intracerebrally or intravenously and without immunosuppression, after experimentally induced ischemic stroke in adult rats also significantly reduced behavioral and histological impairments compared to vehicle-infused rats. Menstrual blood-derived cells exemplify a source of “individually tailored” donor cells that completely match the transplant recipient, at least in women. The present neurostructural and behavioral benefits afforded by transplanted menstrual blood-derived cells support their use as a stem cell source for cell therapy in stroke.
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Aliquots derived from the 2 samples of cells, passaged either 6 or 9, and grown for additional 3 passages in culture, were found to behave similarly, with cell yields, number of adherent cells and those expressing neuronal phenotypes, as well as graft survival and functional effects showing near complete resemblance of the 2 cell populations. Thus, the data from these 2 cell populations were collapsed into a single treatment condition. A flow chart of experimental procedures is provided (Fig. 1).
Immunocytochemical assays of cultured menstrual blood reveal that they express embryonic-like stem cell phenotypic markers (Oct4, SSEA, Nanog) (Fig. 2). Greater than 90% of the cells were positive for these pluripotent markers. They maintained these stemness properties at least up to 9 passages plus the additional 3 culture passages (ie, longest time point the cells were cultured in this study). In addition, their growth rate or proliferative capacity did not change over time. Of note, human ES cells showed the typical specific nuclear staining, but we detected a few cells positive for Oct4 show cytoplasmic labeling, which appears to be the pattern of staining displayed by majority of menstrual blood-derived stem cells. While we do not have a solid explanation for such cytoplasmic labeling, this differential pattern of Oct4 labeling may distinguish ES cells from menstrual blood-derived stem cells. Furthermore, menstrual blood-derived stem cells (75%) were CXCR4-positive, a stem cell chemotaxis marker, also expressed by human ES cells. The cells were plated on a coated 10-cm dish in DMEM/F12 supplemented with ITS, and the medium was changed twice a week throughout the study.
 View larger version(37K) | FIG. 2. Cultured menstrual blood cells display embryonic stem cell-like features. Panels A and B are positive control images taken from human embryonic stem cells expressing the phenotypic markers Oct4 and CXCR4. Immunocytochemical assays of cultured menstrual blood reveal that these cells (75%) were CXCR4-positive, a stem cell chemotaxis marker (Panel C). Furthermore, they express embryonic-like stem cell phenotypic markers Oct4, SSEA, and Nanog as shown in panels D–F, respectively. Greater than 90% of the cells were positive for these pluripotent markers. They maintained these stemness properties at least up to passage 9 plus the additional 3 passages in culture (ie, longest time point the cells were cultured in this study). In addition, their growth rate or proliferative capacity did not change over time. The cells were plated on a coated 10-cm dish in DMEM/F12 supplemented with ITS and medium was changed twice a week throughout the study. |
After passage 6 or 9, the cells were transferred to coated dishes in neural induction medium (DMEM/F12 supplemented with N2 and FGF-2) for a week, and retinoic acid was added to the medium over the next 3 weeks. Cells were Nestin-positive, indicative of an early neural lineage commitment, and readily differentiated into intermediate neuronal (30% MAP2-positive, but the mature neuronal marker, NeuN, labeling not detected) and astrocytic phenotype (40% GFAP-positive) upon withdrawal of FGF-2 (Fig. 3). Thus, when grown in appropriate conditioned media, cultured menstrual blood stem cells express neural phenotypic markers (Nestin, MAP2).
 View larger version(23K) | FIG. 3. Cultured menstrual blood cells can be steered toward neural lineage. Morphological changes in cultured menstrual blood cells immediately following thawing (A), after a few hours (B), and prolonged exposure (C) in neural induction medium (DMEM/F12 supplemented with N2 and FGF-2). After passage 6 or 9, the cells were transferred to coated dishes in neural induction medium for a week, and retinoic acid was added to the medium over the next 3 weeks. Cells were Nestin-positive, indicative of an early neural lineage commitment, and readily differentiated into intermediate neuronal (30% MAP2-positive, but the mature neuronal marker, NeuN, labeling not detected) and astrocytic phenotype (40% GFAP-positive) upon withdrawal of FGF-2. Thus, when grown in appropriate conditioned media, cultured menstrual blood stem cells express neural phenotypic markers (Nestin, MAP2). |
In order to test the therapeutic potential of these cells, we used the in vitro OGD stroke model and found that OGD-exposed primary rat neurons that were co-cultured with menstrual blood-derived stem cells or exposed to the media collected from cultured menstrual blood-derived stem cells exhibited significantly protected against ischemic cell death (Fig. 4). ANOVA revealed significant treatment effects in both Trypan blue exclusion method (F 2,6 = 58.78, P < 0.0001) and MTT assay (F 2,6 = 45.60, P < 0.001) for detecting cell death and cell survival, respectively. Post hoc tests revealed that menstrual blood-derived stem cells (Trypan blue exclusion method and MTT assay, P < 0.0001 vs. controls) or the media collected from cultured menstrual blood-derived stem cells (Trypan blue exclusion method, P < 0.0001 vs. controls; MTT assay, P < 0.001 vs. controls) significantly reduced cell death and improved cell survival of OGD-exposed primary neurons. There were no significant differences in the protective effects afforded by co-culturing with menstrual blood-derived stem cells and exposure to the media collected from cultured menstrual blood-derived stem cells (P > 0.1).
 View larger version(11K) | FIG. 4. Co-cultured menstrual blood-derived stem cells protects against in vitro stroke insult. Cell viability tests using Trypan blue exclusion method and MTT assay revealed that oxygen glucose deprivation (OGD)-exposed primary rat neurons that were co-cultured with menstrual blood-derived stem cells or exposed to the media collected from cultured menstrual blood-derived stem cells exhibited significantly protected against ischemic cell death. There were no significant differences in the protective effects afforded by co-culturing with menstrual blood-derived stem cells and exposure to the media collected from cultured menstrual blood-derived stem cells. Error bars represent standard deviations. Data were generated from 2 triplicates of 2 different menstrual blood-derived stem cell samples. Asterisk corresponds to statistically significant difference between conditioned media or menstrual blood-derived stem cells and control. |
As an approach to reveal a mechanism of action underlying the therapeutic benefits of cultured menstrual blood-derived stem cells, we assayed for growth factors implicated as neuroprotective in stroke models. ELISA data showed elevated levels of trophic factors, such as VEGF, BDNF, and NT-3, in the media of OGD-exposed cultured menstrual blood-derived stem cells (Table 1).
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