Stromal cell-derived inducing activity does not promote dopaminergic differentiation, but enhances differentiation and proliferation of neural stem cell-derived astrocytes

Previous evidence has shown that stromal cell-derived inducing activity (SDIA), produced by the mouse PA6 stromal cell line, promotes dopaminergic differentiation of mouse, monkey and human embryonic stem cells in vitro. To examine whether PA6 stromal cells can enhance the yield of dopaminergic differentiation from neural progenitors, we generated neurospheres from embryonic day 11.5 (E11.5) (midbrain and forebrain) and E14.5 (ventral mesencephalon and cortex) rat embryos and allowed them to differentiate in co-culture with PA6 cells or poly-l-lysine/laminin-coated dishes. We observed that SDIA did not promote dopaminergic differentiation of E11.5 and E14.5 neurospheres but more prominently, enhanced astrocyte differentiation, cell survival and astrocyte proliferation. Our results suggest that PA6 cells do not have a general capacity to promote differentiation into dopaminergic neurons from all types of stem cells, but that they may specifically induce dopaminergic differentiation of highly uncommitted stem cells such as embryonic stem cells.     

Stromal factors SDF1α, sFRP1, and VEGFD induce dopaminergic neuron differentiation of human pluripotent stem cells

Human embryonic stem cell (hESC)-derived dopaminergic (DA) neurons hold potential for treating Parkinson's disease (PD) through cell replacement therapy. Generation of DA neurons from hESCs has been achieved by coculture with the stromal cell line PA6, a source of stromal cell-derived inducing activity (SDIA). However, the factors produced by stromal cells that result in SDIA are largely undefined. We previously reported that medium conditioned by PA6 cells can generate functional DA neurons from NTera2 human embryonal carcinoma stem cells. Here we show that PA6-conditioned medium can induce DA neuronal differentiation in both NTera2 cells and the hESC I6 cell line. To identify the factor(s) responsible for SDIA, we used large-scale microarray analysis of gene expression combined with mass spectrometric analysis of PA6-conditioned medium (CM). The candidate factors, hepatocyte growth factor (HGF), stromal cell-derived factor-1 α (SDF1α), secreted frizzled-related protein 1 (sFRP1), and vascular endothelial growth factor D (VEGFD) were identified, and their concentrations in PA6 CM were established by immunoaffinity capillary electrophoresis. Upon addition of SDF1α, sFRP1, and VEGFD to the culture medium, we observed an increase in the number of cells expressing tyrosine hydroxylase (a marker for DA neurons) and βIII-tubulin (a marker for immature neurons) in both the NTera2 and I6 cell lines. These results indicate that SDF1α, sFRP1, and VEGFD are major components of SDIA and suggest the potential use of these defined factors to elicit DA differentiation of pluripotent human stem cells for therapeutic intervention in PD.     

Meningeal cells induce dopaminergic neurons from embryonic stem cells

Neural induction of midbrain dopaminergic (DA) neurons from embryonic stem (ES) cells can be achieved by culturing them on a bone marrow-derived stromal cell line, PA6, which possesses stromal cell-derived inducing activity (SDIA). The mechanism of SDIA is unknown, but clinical application of ES cell transplantation requires the use of defined factors for DA neuron induction. Here, we demonstrate that meningeal cells harvested from the developing dura can induce DA neuron differentiation from mouse and human ES cells, as assessed by midbrain DA marker expression and secretion of DA in response to potassium stimuli. Intriguingly, the inductive strength of meningeal cells depends on their developmental stage, with those harvested from embryonic day 18 embryos showing the highest activity. Among six soluble factors known to be involved in DA neuron differentiation, only Wnt-5a and transforming growth factor-β3 were expressed by both meningeal and PA6 cells, and the expression of Wnt-5a correlated with the DA neuron induction activity of these cells. Furthermore, the induction of DA neuron differentiation by PA6 cell-conditioned medium was reversed by addition of a Wnt-5a neutralizing antibody, whereas recombinant Wnt-5a promoted DA neuron induction when cells were cultured on Matrigel. These results indicate that meningeal cells can be used as feeders to induce DA neurons from ES cells, and that Wnt-5a plays an important role in DA neuron induction by SDIA.     

Culture method for the induction of neurospheres from mouse embryonic stem cells by coculture with PA6 stromal cells

Embryonic stem (ES) cells proliferate and maintain their pluripotency for over 1 year in vitro and may therefore provide a sufficient source for cell therapies. However, most of the previously reported methods for obtaining a source for cell therapies have not been simple. We describe here a novel method for induction of neurospheres from mouse ES cells by coculturing on PA6 cells instead of the formation of embryoid bodies. The ES cells cocultured with the PA6 stromal cell line for at least 3 days were capable of differentiating into spheres. The cells in the spheres were all green fluorescent protein (GFP) positive, showing that they were derived from GFP-expressing D3-ES cells. The spheres contained nestin-positive cells. The number of spheres increased when they were cocultured with PA6 for a longer period. Sphere formation was observed even after 10 mechanical dissociations and subculturings, showing its self-renewal ability. The cells differentiated into microtubule-associated protein-2 (MAP2)-positive neuronal cells and glial fibrillary acidic protein (GFAP)-positive glial cells. gamma-Aminobutyric acid-positive cells and tyrosine hydroxylase-positive cells were also observed in the spheres. The percentages of the MAP2- or GFAP-positive cells in the sphere changed according to the period of coculture on PA6 cells. At an early stage of coculture, more neurons were generated and, at a later period, more glial cells were generated. These results suggested that neurosphere could be generated from ES cells by coculturing with PA6, and that these cells resembled neural stem cells derived from mouse fetal brain tissue.     

Optimal conditions for in vivo induction of dopaminergic neurons from embryonic stem cells through stromal cell-derived inducing activity

A method of inducing dopamine (DA) neurons from mouse embryonic stem (ES) cells by stromal cell-derived inducing activity (SDIA) was previously reported. When transplanted, SDIA-induced DA neurons integrate into the mouse striatum and remain positive for tyrosine hydroxylase (TH) expression. In the present study, to optimize the transplantation efficiency, we treated mouse ES cells with SDIA for various numbers of days (8-14 days). SDIA-treated ES cell colonies were isolated by papain treatment and then grafted into the 6-hydroxydopamine (6-OHDA)-lesioned mouse striatum. The ratio of the number of surviving TH-positive cells to the total number of grafted cells was highest when ES cells were treated with SDIA for 12 days before transplantation. This ratio revealed that grafting cell colonies was more efficient for obtaining TH-positive cells in vivo than grafting cell suspensions. When we grafted a cell suspension of 2 x 10(5), 2 x 10(4), or 2 x 10(3) cells into the 6-OHDA-lesioned mouse striatum, we observed only a few surviving TH-positive cells. In conclusion, inducing DA neurons from mouse ES cells by SDIA for 12 days and grafting cell colonies into mouse striatum was the most effective method for the survival of TH-positive neurons in vivo.     

Hepatocyte growth factor promotes neuronal differentiation of neural stem cells derived from embryonic stem cells

We previously reported that hepatocyte growth factor (HGF) promoted proliferation of neurospheres and neuronal differentiation of neural stem cells (NSCs) derived from mouse embryonic brain. In this study, spheres from mouse embryonic stem (ES) cells were generated by floating culture following co-culture on PA6 stromal cells. In contrast to the behavior of the neurospheres derived from embryonic brain, addition of HGF to the growth medium of the floating cultures decreased the number of spheres derived from ES cells. When spheres were stained using a MAP-2 antibody, more MAP-2-positive cells were observed in spheres cultured with HGF. When HGF was added to the growth and/or differentiation medium, more MAP-2-positive cells were also obtained. These results suggest that HGF promotes neuronal differentiation of NSCs derived from ES cells.     

Adipocytes derived from PA6 cells reliably promote the differentiation of dopaminergic neurons from human embryonic stem cells

The PA6 stromal cell line comprises a heterogeneous population of cells that can induce both mouse and human embryonic stem cells to differentiate into dopaminergic neurons. This ability of PA6 cells has been termed stromal cell‐derived inducing activity (SDIA). The level of SDIA has been found to vary considerably between and within batches of PA6 cells. Not only are the molecular mechanisms that underlie SDIA unknown but also the cell type(s) within the heterogeneous PA6 cultures that underlie SDIA remain poorly defined. In this study, we reveal that adipocytes, which are present within the heterogeneous PA6 cell population, robustly release the factors mediating SDIA. Furthermore, we report that the coculture of human embryonic stem cells with PA6‐derived adipocytes reliably induces their differentiation into midbrain dopaminergic neurons. © 2014 Wiley Periodicals, Inc.     

Generation of dopamine neurons from embryonic stem cells in the presence of the neuralizing activity of bone marrow stromal cells derived from adult mice

Stromal cell lines such as PA6 and MS5 have been employed for generating dopamine (DA) neurons from embryonic stem (ES) cells. The present study was designed to test whether bone marrow stromal cells (BMSC) derived from adult mice might be available as a feeder layer to produce DA cells efficiently from ES cells. When ES cells were grown on BMSC in the presence of fibroblast growth factor 8 (FGF8) and sonic hedgehog (SHH), about 40% of TuJ1-positive neurons expressed tyrosine hydroxylase (TH). Because these cells labeled with TH were negative for dopamine-beta-hydroxylasae (DBH), the marker for noradrenergic and adrenergic neurons, the TH-positive cells were most likely DA neurons. They indeed expressed midbrain DA neuron markers such as Nurr 1, Ptx-3, and c-ret and were capable of synthesizing and releasing DA in vitro. Furthermore, DA neurons differentiated from ES cells in this differentiation protocol survived transplantation in rats with 6-hydroxydopamine lesions and reversed the lesion-induced circling behavior. The data indicate that BMSC can facilitate an efficient induction of DA neurons from ES cells and that the generated DA neurons are biologically functional both in vitro and in vivo. Insofar as BMSC have recently been employed in autologous cell therapy for ischemic heart and arteriosclerotic limb diseases, the present study raises the possibility that autologous BMSC can be applied in future cell transplantation therapy in Parkinson's disease.     

A simple method for large-scale generation of dopamine neurons from human embryonic stem cells

Dopamine (DA) neurons derived from human embryonic stem cells (hESCs) are potentially valuable in drug screening and as a possible source of donor tissue for transplantation in Parkinson's disease. However, existing culture protocols that promote the differentiation of DA neurons from hESCs are complex, involving multiple steps and having unreliable results between cultures. Here we report a simple and highly reproducible culture protocol that induces expandable DA neuron progenitors from hESCs in attached cultures. We found that the hESC-derived neuronal progenitors retain their full capacity to generate DA neurons after repeated passaging in the presence of basic fibroblast growth factor (bFGF) and medium conditioned with PA6 stromal cells. Using immunocytochemistry and RT-PCR, we found that the differentiated DA neurons exhibit a midbrain phenotype and express, e.g., Aldh1a, Ptx3, Nurr1, and Lmx1a. Using HPLC, we monitored their production of DA. We then demonstrated that the expanded progenitors are possible to cryopreserve without loosing the dopaminergic phenotype. With our protocol, we obtained large and homogeneous populations of dopaminergic progenitors and neurons. We conclude that our protocol can be used to generate human DA neurons suitable for the study of disease mechanisms, toxicology, drug screening, and intracerebral transplantation.     

Stromal cell‐secreted factors promote the survival of embryonic stem cell‐derived early neural stem/progenitor cells via the activation of MAPK and PI3K‐Akt pathways

Neural stem/progenitor cells (NS/PCs) have been studied extensively with the hope of using them clinically to repair the damaged central nervous system. However, little is known about the signals that regulate the proliferation, survival, and differentiation of NS/PCs in early development. To clarify the underlying mechanisms, we took advantage of an in vitro ES cell differentiation system from which we can obtain neurospheres containing NS/PCs with characteristics of the early caudal neural tube, by treating embryoid bodies (EBs) with a low concentration of retinoic acid (RA). We found that conditioned medium from the PA6 stromal cell line (PA6CM) increased the efficiency of neurosphere formation by suppressing apoptosis and promoting the survival of the NS/PCs. PA6CM also induced the phosphorylation of Erk1/2 and Akt1 in cells derived from the EBs. Furthermore, inhibitors of the MAPK and PI3K‐Akt signaling pathways, U0126 and LY294002, attenuated the effects of PA6CM, significantly increasing the number of apoptotic cells and decreasing the number of viable cells among the ES cell‐derived NS/PCs. Thus, PA6CM appears to contain soluble factors that promote the survival of ES cell‐derived early NS/PCs through the activation of the MAPK and PI3K‐Akt pathways. © 2009 Wiley‐Liss, Inc.     

Overexpression of a truncated TrkB isoform increases the proliferation of neural progenitors

The truncated isoform of TrkB, TrkB.T1, has been shown to be expressed in the neurogenic region of rodent brain. TrkB.T1 lacks tyrosine kinase activity and it may modify the action of the full-length TrkB. We show here that the full-length TrkB and TrkB.T1 are expressed at the same relative expression levels in mouse neural progenitor cell cultures. The number of neurosphere-forming progenitors was reduced and apoptosis increased in neurospheres generated from mice overexpressing TrkB.T1 when compared with wild-type neurospheres. The proliferation of the transgenic neural progenitors was increased, as indicated by the larger average diameter of spheres (140% of control), the increased cell growth in an MTT assay (137% of control) and the faster rate of 3H-thymidine incorporation (128% of control) in the transgenic cell cultures than in controls. The proliferation of neural progenitors was also increased after lentivirus-mediated TrkB.T1 overexpression. A significant increase in 3H-thymidine incorporation (119% of control) and the average diameter of spheres (112% of control) in the TrkB.T1-transduced neurospheres compared with neurospheres transduced with the control vectors confirmed the role of TrkB.T1 in proliferation of neural progenitor. When induced to differentiate, progenitors overexpressing TrkB.T1 generated two to three times more neurons than did wild-type cells. The increase in the number of neurons correlated with an increase in the number of apoptotic cells (two-fold) at these time points. The data indicate that changes in the relative expression levels of different TrkB isoforms influence the replicative capacity of neural progenitors.     

The generation of olfactory epithelial neurospheres in vitro predicts engraftment capacity following transplantation in vivo

The stem and progenitor cells of the olfactory epithelium maintain the tissue throughout life and effectuate epithelial reconstitution after injury. We have utilized free-floating olfactory neurosphere cultures to study factors influencing proliferation, differentiation, and transplantation potency of sphere-grown cells as a first step toward using them for therapeutic purposes. Olfactory neurospheres form best and expand most when grown from neonatal epithelium, although methyl bromide-injured or normal adult material is weakly spherogenic. The spheres contain the full range of epithelial cell types as marked by cytokeratins, neuron-specific antigens, E-cadherin, Sox2, and Sox9. Globose basal cells are also prominent constituents. Medium conditioned by growth of phorbol ester-stimulated, immortalized lamina propria-derived cells (LP_Imm) significantly increases the percentage of Neurog1eGFP(+) progenitors and immature neurons in spheres. Sphere-forming capacity resides within selected populations; FACS-purified, Neurog1eGFP(+) cells were poorly spherogenic, while preparations from ΔSox2eGFP transgenic mice that are enriched for Sox2(+) basal cells formed spheres very efficiently. Finally, we compared the potency following transplantation of cells grown in spheres vs. cells derived from adherent cultures. The sphere-derived cells engrafted and produced colonies with multiple cell types that incorporated into and resembled host epithelium; cells from adherent cultures did not. Furthermore, cells from spheres grown in conditioned media from the phorbol ester-activated LP_Imm line gave rise to significantly more neurons after transplantation as compared with control. The current findings demonstrate that sphere formation serves as a biomarker for engraftment capacity and multipotency of olfactory progenitors, which are requirements for their eventual translational use.     

Generation of embryonic stem cells and transgenic mice expressing green fluorescence protein in midbrain dopaminergic neurons

We have generated embryonic stem (ES) cells and transgenic mice with green fluorescent protein (GFP) inserted into the Pitx3 locus via homologous recombination. In the central nervous system, Pitx3-directed GFP was visualized in dopaminergic (DA) neurons in the substantia nigra and ventral tegmental area. Live primary DA neurons can be isolated by fluorescence-activated cell sorting from these transgenic mouse embryos. In culture, Pitx3-GFP is coexpressed in a proportion of ES-derived DA neurons. Furthermore, ES cell-derived Pitx3-GFP expressing DA neurons responded to neurotrophic factors and were sensitive to DA-specific neurotoxin N-4-methyl-1, 2, 3, 6-tetrahydropyridine. We anticipate that the Pitx3-GFP ES cells could be used as a powerful model system for functional identification of molecules governing mDA neuron differentiation and for preclinical research including pharmaceutical drug screening and transplantation. The Pitx3 knock-in mice, on the other hand, could be used for purifying primary neurons for molecular studies associated with the midbrain-specific DA phenotype at a level not previously feasible. These mice would also provide a useful tool to study DA fate determination from embryo- or adult-derived neural stem cells.     

Primate embryonic stem cell-derived neuronal progenitors transplanted into ischemic brain.

Transplantation of stem cells has the possibility of restoring neural functions after stroke damage. Therefore, we transplanted neuronal progenitors generated from monkey embryonic stem (ES) cells into the ischemic mouse brain to test this possibility. Monkey ES cells were caused to differentiate into neuronal progenitors by the stromal cell-derived inducing activity method. Focal cerebral ischemia was induced by occluding the middle cerebral artery by the intraluminal filament technique. The donor cells were transplanted into the ischemic lateral striatum at 24 h after the start of reperfusion. The cells transplanted into the ischemic brain became located widely around the ischemic area, and, moreover, the transplanted cells differentiated into various types of neurons and glial cells. Furthermore, at 28 days after the transplantation, over 10 times more cells in the graft were labeled with Fluorogold (FG) by stereotactic focal injection of FG into the anterior thalamus and substantia nigra on the grafted side when compared with the number at 14 days. From these results we confirmed the survival and differentiation of, as well as network formation by, monkey ES-cell-derived neuronal progenitors transplanted into the ischemic mouse brain.     

Neurosphere generation from dental pulp of adult rat incisor

Dental pulp is a potential source of cells that can be used in cell replacement therapy for various nervous system disorders. Here we report that adult rat dental pulp cells have the ability to form neurospheres when cultured in serum-free culture medium on super-hydrophilic plates. The cells within small spheres continued to grow, and the dental pulp-derived cells generated large spheres. Sphere formation was dependent on exogenously supplied basic-fibroblast growth factor, but not on epidermal growth factor, and the formation and growth of dental pulp-derived spheres were negatively regulated by transforming growth factor-β. Plating cells that were dissociated from spheres on an adhesive substrate resulted in differentiation into Tuj1- and MAP2-positive neuronal cells. Analysis of the three-dimensional structure of dental pulp-derived spheres shows that they contained nestin-positive progenitors, Tuj1-positive neuronal cells and S100-positive glial cells. We found that spheres contained CD81 (TAPA1) and nestin double-positive cells, and identified a small population of CD81 and nestin double-positive cells in the odontoblast layer of the dental pulp. Flow cytometric analysis showed that CD81-positive cells were enriched in the spheres compared with the dental pulp tissue. Bromodeoxyuridine (BrdU) staining showed that nestin- and BrdU-positive cells were located only in the apical portion of the dental pulp, and the apical portion produced a large number of large-sized spheres. These data suggest that the CD81 and nestin double-positive cells localized in the odontoblast layer of the apical portion of the dental pulp may have the ability to grow and form neurospheres.     

Human neurospheres derived from the fetal central nervous system are regionally and temporally specified but are not committed

Proliferating single cells were isolated from various CNS regions (telencephalon, diencephalon, midbrain, cerebellum, pons and medulla, and spinal cord) of human fetal cadavers at 13 weeks of gestation and grown as neurospheres in long-term cultures. We investigated whether neural stem cells (NSCs) or progenitors within spheres have specific regional or temporal characteristics with regard to growth, differentiation, and region-specific gene expression, and whether these molecular specifications are reversible. Regardless of regional origin, all of the neurospheres were found to contain cells of different subtypes, which suggests that multipotent NSCs, progenitors or radial glial cells co-exist with restricted neuronal or glial progenitors within the neurospheres. Neurospheres from the forebrain grew faster and gave rise to significantly more neurons than did those from either the midbrain or hindbrain, and regional differences in neuronal differentiation appeared to be sustained during long-term passage of neurospheres in culture. There was also a trend towards a reduction in neuronal emergence from the respective neurospheres over time in culture, although the percentages of neurons generated from cerebellum-derived neurospheres increased dramatically. These results suggest that differences in neuronal differentiation for the various neurospheres are spatially and temporally determined. In addition, the properties of glial fibrillary acidic protein (GFAP)-, glutamate-, and gamma-aminobutyric acid (GABA)-expressing cells derived from neurospheres of the respective CNS regions appear to be regionally and temporally different. Isolated human neurospheres from different CNS compartments expressed distinctive molecular markers of regional identity and maintained these patterns of region-specific gene expression during long-term passage in vitro. To determine the potential of human neurospheres for regional fate plasticity, single spheres from the respective regions were co-cultured with embryonic day 16.5 (E16.5 d) mouse brain slices. Specific cues from the developing mouse brain tissues induced the human neurospheres to express different marker genes of regional identity and to suppress the expression of their original marker genes. Thus, even the early regional identities of human neurospheres may not be irreversible and may be altered by local inductive cues. These findings have important implications for understanding the characteristics of growth, differentiation, and molecular specification of human neurospheres derived from the developing CNS, as well as the therapeutic potential for neural repair.     

Neuronal differentiation of stem cells isolated from adult muscle

Lineage uncommitted pluripotent stem cells reside in the connective tissue of skeletal muscle. The present study was carried out with pluripotent stem cells (PPSCs) isolated from 6-month old rat muscle. Before differentiation, these cells were vimentin+, CD90+, CD45-, and varied in their expression of CD34. The PPSCs were expanded as non-adherent aggregates under similar conditions to those used to generate neurospheres from embryonic or neural stem cells. The PPSC-derived neurospheres were positive for nestin, an early marker present in neuronal precursors, and expressed the two alternative mRNA forms of the neuroectodermal marker Pax-6, as well as mRNA for Oct-4, a gene related to the pluripotentiality of stem cells. To confirm their neural potential, PPSC-derived neurospheres were plated on coated coverslips under varying conditions: Neurobasal medium with N2 or B27, and either NT3 or BDNF. After 4-6 days the cells expressed neuronal (Tuj1+, NF68), astrocytic (GFAP) and oligodendrocytic (MOSP+, MBP+) markers, both by immunocytochemistry and RT-PCR. In addition, PPSCs were cultured as monolayers under adherent conditions, exposed to growth factors and defined differentiating conditions for 5 hr, and subsequently kept for 2 days in a maturation medium. At this point they gave rise to a mixed population of early neural progenitors (Nestin+ or NG2+), immature and mature neurons (Tuj1+ and NF145+) and myelin producing oligodendrocytes (CNPase + and MOSP+). Our study shows that PPSCs present in adult muscle can overcome germ lineage restrictions and express the molecular characteristics of brain cells. Therefore, PPSCs isolated from adult muscle could provide a novel source for autologous cell replacement in neurodegenerative and demyelinating diseases.