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- Materials and Methods
- LITERATURE CITED
- Supporting Information
Pericyte perivascular cells, believed to originate mesenchymal stem cells (MSC), are characterized by their capability to differentiate into various phenotypes and participate in tissue reconstruction of different organs, including the brain. We show that these cells can be induced to differentiation into neural-like phenotypes. For these studies, pericytes were obtained from aorta ex-plants of Sprague–Dawley rats and differentiated into neural cells following induction with trans retinoic acid (RA) in serum-free defined media or differentiation media containing nerve growth and brain-derived neuronal factor, B27, N2, and IBMX. When induced to differentiation with RA, cells express the pluripotency marker protein stage-specific embryonic antigen-1, neural-specific proteins β3-tubulin, neurofilament-200, and glial fibrillary acidic protein, suggesting that pericytes undergo differentiation, similar to that of neuroectodermal cells. Differentiated cells respond with intracellular calcium transients to membrane depolarization by KCl indicating the presence of voltage-gated ion channels and express functional N-methyl-D-aspartate receptors, characteristic for functional neurons. The study of neural differentiation of pericytes contributes to the understanding of induction of neuroectodermal differentiation as well as providing a new possible stem-cell source for cell regeneration therapy in the brain. © 2011 International Society for Advancement of Cytometry
The evidence for a role of pericytes in neural tissue repair was obtained from animal studies with experimentally induced brain ischemia, demonstrating that pericytes and pericyte-like cells, termed adventitial cells, originate neurons in the subgranular zone (SGZ) and glial cells in the dentate gyrus of hippocampus in monkeys (1). Moreover, the expression of stromal-derived factor-1 and angiopoietin-1 in the vascular niche close to the infarct areas is related with the guidance and survival of tissue-specific intrinsic progenitors in the subventricular zone (SVZ) of mice after focal cortical stroke (2). Pericytes surrounding brain blood vessels constitutively express nestin, a marker of neural progenitor cells (3), and, therefore, might participate in neuronal regeneration (4). The existence of mesenchymal stem cells (MSC) in perivascular niches supports the hypothesis that vessels in different tissues participate in originating such stem cells (SCs) (5–8). These findings do not agree with the idea of distinct developmental origins of all MSC, such as from neural crest (9, 10) and other mesodermal derivatives (11). The ability of these cells to differentiate into several cell types in vitro probably reflects the efficacy of the perivascular niche in the maintenance of their stemness. Evidence collected so far indicates that the pericyte in the perivascular compartment functions as a SC in the postnatal organism (7).
Neurogenesis in the brain occurs in close proximity to blood vessels and may be associated with angiogenesis (8). A web of growth and morphogenetic factors, including sonic hedgehog, bone morphogenetic proteins, Ephs/ephrins, Notch and fibroblast growth factor, nitric oxide, and erythropoietin present in adult neural SC niches (SVZ and SGZ of the hippocampal zone), participate in regulation of angiogenesis (12–15). In addition, blood vessels are conduits for the delivery of paracrine factors, such as hormones (sexual hormones, glucocorticoids, and prolactin) and cytokines, from distant sources. These “long-distance” cues may act directly on neural stem and progenitor cells and endothelial cells to regulate angiogenesis and neurogenesis (16, 17). The leptomeninges (pia mater), closely associated with blood vessels and also with microvascular pericytes/perivascular cells throughout the central nervous system (CNS), reveal neural stem/progenitor cell activity in response to ischemia and can generate neurons (18).
The therapeutic potential of SCs has already been recognized. Pericytes were able to regenerate skeletal muscle and promote functional recovery of the diseased heart and kidney (19). Actually, the pericyte is considered to be a pluripotent SC and, therefore, should be able to differentiate into tissues originated from the three germ layers (20). Recent data have shed light on the role of the CNS capillary pericytes as regulatory cells with SC capability (18, 21, 22). Brain pericytes originate from pluripotent neural crest cells like the neurons, supporting the hypothesis of one common cell lineage origin (23). CNS pericytes may be a source for purified viable SC with the potential of directed neurogenesis and could be important for future therapeutic strategies (21, 22). Roles for neural pericytes in brain remodeling after injury are unquestionable; however, the question whether outer CNS pericytes have the same SC properties needs yet to be resolved. Therefore, we now show in the present work that non-CNS perivascular pericytes obtained from rat aorta ex-plants and induced to neural differentiation express stage-specific embryonic antigen (SSEA-1), suggesting that they pass through a pluripotent stage and then differentiate into neural phenotypes in the presence of appropriate stimuli.
- Top of page
- Materials and Methods
- LITERATURE CITED
- Supporting Information
Recent research efforts have largely focused on the detection, phenotypic expression characterization, and in vitro replication and differentiation of SCs from human adult tissue. The determination of the origin and identity of SCs together with their niches in adult tissue also provides important information on their participation in endogenous tissue regeneration and their possible applications as pluri- or multipotent cells in cellular regeneration therapy (6, 7, 27–33). Evidence accumulated in the last few years show that the adult macro- and microvessels contain multi- or pluripotent SCs, including MSCs, and/or pericytes, as well as hemopoietic SCs, and lineage committed progenitors, such as vascular walls endothelial progenitor and Sca-1+ smooth muscle progenitor cells (6, 7, 23, 29–31, 33–36). Pericyte SCs have been isolated from different vascular tissues including abdominal adipose, bone marrow, dental pulp, and umbilical cord (6, 7, 23, 29–31, 33–35). Actual discussions of the role of pericytes as SCs propose that pericytes are a source for MSC (29). It has yet not been resolved how MSCs/pericytes contribute to the formation, maturation, and homeostasis of all vascularized tissues (5, 7). Because blood vessels and with them pericytes are part of all tissues and organs, there would be many therapeutic applications if these cells could be induced to differentiate into defined phenotypes (37). In view of that, we have studied the capability of these cells to differentiate into neural phenotypes.
During embryonic development, neural crest cells surround aortic vessels providing all components including pericytes and musculature-connective tissue except the endothelial cells (23). Brain microvascular pericytes show a pluripotential SC activity, express neural markers as nestin, GFAP, NF1, and oligodendrocyte O4 antigen when induced to differentiation (22), supporting the idea that pericytes are pluripotent cells in the blood–brain barrier. The neural crest cell origin of pericytes, in addition to a high similarity with MSCs and other sources of SCs, support the idea that SCs have an embryonic neuroectodermal/epiblast common origin that will give rise to cells of the three germ layers: ectoderm, mesoderm, and endoderm (1, 23, 38). This assumption would also explain why undifferentiated SCs express neural genes (39). It is unquestionable that CNS pericytes have a role in the brain remodeling after injury; however, whether non-CNS pericytes possess SC properties, such as pluri- or multipotency, is yet being discussed. Therefore, we have developed an in vitro protocol for differentiation of pericytes into neural phenotypes.
Pericyte isolation and culture did not depend on FACS selection or other complex equipment for cell separation. With the described protocol, 75% of cells expanded from the aorta explants express Thy-1/CD90 (Supporting Information) and pericyte markers (αSMA, PDGFRs). Our protocol reveals advantages over previously described methods for the differentiation of CNS microvascular pericytes (21, 22). Aortic pericytes required 8 days of differentiation for the acquisition of neural morphology, excitability, and expression of functional NMDA-glutamate receptors. Besides neural stem and progenitor cells that are intensively studied for the potential in regeneration therapy of neurodegenerative diseases (38), pericyte SCs might be an alternative SC source based on the easiness of isolation of these cells. In the near future, in vivo studies will reveal the possible therapeutic potential of these cells for the treatment of neurodegenerative diseases. Experimental support has been already obtained for the common regulation of angiogenesis and neurogenesis during developmental processes and regeneration following ischemic stroke (40, 41), being in agreement with our observation of pericytic cells with angiogenic potential being involved in neuronal regeneration.
The medium for the induction of neural differentiation contained BDNF and RA, factors known to induce neural differentiation of embryonic cells (42), of neural stem cells (43), bone-marrow MSCs, and adipose-derived mesenchymal cells with pericyte-specific markers, producing functional response profiles to stimulation by neurotransmitters (33, 34, 44, 45). In agreement with the characteristics of excitable neuronal cells, differentiated pericytes responded with [Ca2+]i transients to membrane depolarization by KCl indicating the presence of voltage-operated ion channels. Moreover, differentiated cells expressed functional NMDA-glutamate receptors such as in neural-differentiated adipose-derived SC where the presence of NMDA receptors served as an indicator and marker of the efficiency of neuronal differentiation (46). However, the presence of RA alone is not enough for maintaining pericytes in their neural-differentiated stage, as evaluated by their morphology, expression of neural marker proteins and excitability. However, the presence of the used morphogenetic factors (NGF, BNDF, B27, and N2) and IBMX maintained neural phenotypes of differentiated pericytes, but no excitability could be observed in the presence of RA alone. Consequently, RA and the cocktail of morphogenetic factors contributed to differentiation induction in independent manners by activation of different cell signaling pathways. RA activates nuclear receptors, while the used growth factors and IBMX mediate membrane receptor dependent down-stream signaling pathways.
In summary, pericyte cells are pluripotent SCs that can be induced to neural differentiation. Expression of SSEA-1 suggests that cells pass through a pluripotent stage, followed by expression of β3-tubulin, GFAP and NF200 such as observed during differentiation of neural crest cells. Differentiated cells express a protein characteristic for functional NMDA-glutamate receptors and voltage-gated ion channels. Following studies will elucidate whether neural-differentiated pericytes can be integrated in neuronal networks and participate in synaptic transmission. Besides being important for understanding mechanisms of neuronal differentiation, pericytes may provide a novel source of SCs for cell regeneration therapy in the CNS.