Sean S. Liour

Differentiation of radial glia-like cells from embryonic stem cells.

Radial glial cells play important roles in neural development. They provide support and guidance for neuronal migration and give rise to neurons and glia. In vitro, neurons, astrocytes, and oligodendrocytes can be generated from neural and embryonic stem cells, but the generation of radial glial cells from these stem cells has not yet been reported. Since the differentiation of radial glial cells is indispensable during brain development, we hypothesize that stem cells also generate radial glial cells during in vitro neural differentiation. To test this hypothesis, we utilized five different clones of mouse embryonic (ES) and embryonal carcinoma (EC) stem cell lines to investigate the differentiation of radial glial cells during in vitro neural differentiation. Here, we demonstrate that radial glia‐like cells can be generated from ES/EC cell lines. These ES/EC cell‐derived radial glia‐like cells are similar in morphology to radial glial cells in vivo, i.e., they are bipolar with an unbranched long process and a short process. They also express several cytoskeletal markers, such as nestin, RC2, and/or GFAP, that are characteristics of radial glial cells in vivo. The processes of these in vitro generated radial glia‐like cells are organized into parallel arrays that resemble the radial glial scaffolds in neocortical development. Since radial glia‐like cells were observed in all five clones of ES/EC cells tested, we suggest that the differentiation of radial glial cells may be a common pathway during in vitro neural differentiation of ES cells. This novel in vitro model system should facilitate the investigation of regulation of radial glial cell differentiation and its biological function. GLIA 42:109–117, 2003.

Expression of gangliosides in neuronal development of P19 embryonal carcinoma stem cells

Gangliosides are constituents of the cell membrane and are known to have important functions in neuronal differentiation. We employed an embryonal carcinoma stem cell line P19 as an in vitro model to investigate the expression of gangliosides during neuronal development. After treatment with retinoic acid, these cells differentiate synchronously into neuron‐like cells by a series of well‐defined events of development. We examined several aspects of ganglioside metabolism, including the changes of ganglioside pattern, the activities and gene expression of several enzymes at different stages of differentiation, and the distribution of gangliosides in differentiating neurons. Undifferentiated P19 cells express mainly GM3 and GD3. After P19 cells were committed to differentiation, the synthesis of complex gangliosides was elevated more than 20‐fold, coinciding with the stage of neurite outgrowth. During the maturation of differentiated cells, the expression of c‐series gangliosides was downregulated concomitantly with upregulation of the expression of a‐ and b‐series gangliosides. We also examined the distribution of gangliosides in differentiating neurons by confocal and transmission electron microscopy after cholera toxin B subunit and sialidase treatment. Confocal microscopic studies showed that gangliosides were distributed on the growth cones and exhibited a punctate localization on neurites and soma. Electron microscopic studies indicated that they also are enriched on the plasma membranes of neurites and the filopodia as well as on the lamellipodia of growth cones during the early stage of neurite outgrowth. Our data demonstrate that the expression of gangliosides in P19 cells during RA‐induced neuronal differentiation resembles that of the in vivo development of the vertebrate brain, and hence validates it as an in vitro model for investigating the function of gangliosides in neuronal development. J. Neurosci. Res. 62:363–373, 2000.

Further characterization of embryonic stem cell-derived radial glial cells

Previously, we showed that radial glia‐like (RG) cells differentiated from embryonic stem (ES) cells after retinoic acid induction (Liour and Yu, 2003 : Glia 42:109–117). In the present study, we demonstrate that the production of RG cells from ES cells is independent of the neural differentiation protocol used. These ES cell‐derived RG (ES‐RG) cells are similar in morphology to RG cells in vivo and express several characteristic RG cell markers. The processes of these ES‐RG cells are organized into radial arrays similar to the RG scaffold in developing CNS. Expression of Pax6, along with other circumstantial data, suggests that at least some of these ES‐RG cells are neural progenitors. The progression of neurogenesis into gliogenesis during the in vitro neural differentiation of ES cells recapitulates the in vivo developmental process. The identification of two cell surface markers, SSEA‐1 and GM1, on both the native embryonic RG cells and ES‐RG cells, may facilitate purification of radial glial cells for future studies and cell therapy. Overall, our study suggests that differentiation of radial glial cells is a common pathway during the neural differentiation of ES cells.

Involvement of gangliosides in proliferation of immortalized neural progenitor cells

The CNS consists of neuronal and glial cells generated from common neural progenitor cells during development. Cellular events for neural progenitor cells, such as proliferation and differentiation, are regulated by multiple intrinsic and extrinsic cell signals. Although much is known on the importance of the proteinous factors in regulating the fate of neural progenitor cells, the involvement of other molecules such as gangliosides, sialic acid‐containing glycosphingolipids, remains to be clarified. To elucidate the biological functions of gangliosides in neural progenitor cells, we transfected an immortalized neural progenitor cell line, C17.2, which does not express GD3 ganglioside, with a fusion protein of GD3‐synthase (ST‐II) and enhanced green fluorescent protein (ST‐II‐EGFP). Analysis of the ST‐II transfectants revealed the ectopic expression of b‐ and c‐series gangliosides. In the ST‐II transfectants, proliferation induced by epidermal growth factor (EGF) was severely retarded. EGF‐induced proliferation of C17.2 cells was dependent on the Ras‐mitogen‐activated protein kinase (Ras‐MAPK) pathway, and the EGF‐induced activation of this pathway was significantly repressed in the transfectants. Thus, ST‐II overexpression retarded proliferation of C17.2 cells via repression of the Ras‐MAPK pathway. The result supports the concept that gangliosides may play an important role in regulating the proliferation of neural progenitor cells.

Glial‐guided neuronal migration in P19 embryonal carcinoma stem cell aggregates

During development of the nervous system, neuronal precursors that originated in proliferative regions migrate along radial glial fibers to reach their final destination. P19 embryonal carcinoma (EC) stem cells exposed to retinoic acid (RA) differentiate into neurons, glia, and fibroblast‐like cells. In this work, we induced P19 aggregates for 4 days with RA and plated them onto tissue culture dishes coated with poly‐L‐lysine. Several cells migrated out of and/or extended processes from the aggregates after 24 hr. Some cell processes were morphologically similar to radial glial fibers and stained for glial fibrillar acidic protein (GFAP) and nestin. Large numbers of migrating cells showed characteristics similar to those of bipolar migrating neurons and expressed the neuronal marker microtubule‐associated protein 2. Furthermore, scanning electron microscopy analysis revealed an intimate association between the radial fibers and the migrating cells. Therefore, the migration of neuron‐like cells on radial glia fibers in differentiated P19 aggregates resembled some of the migration models used thus far to study gliophilic neuronal migration. In addition, HPTLC analysis in this system showed the expression of 9‐O‐acetyl GD3, a ganglioside that has been associated with neuronal migration. Antibody perturbation assays showed that immunoblockage of 9‐O‐acetyl GD3 arrested neuronal migration in a reversible manner. In summary, we have characterized a new cell culture model for investigation of glial‐guided neuronal migration and have shown that 9‐O‐acetyl GD3 ganglioside has an important role in this phenomenon.

Differential Effects of Three Inhibitors of Glycosphingolipid Biosynthesis on Neuronal Differentiation of Embryonal Carcinoma Stem Cells

Gangliosides have been implicated in having important roles in neural development. It has been shown that disruption of ganglioside biosynthesis inhibits neurite outgrowth. However, many contradictory results have been reported. The inconsistency of these reports may result from the differential use of neuronal cell lines and inhibitors for ganglioside biosynthesis. In order to clarify the inconsistency in these studies, we utilized an in vitro neuronal differentiation model using an embryonic caricinoma (EC) stem cell line to elucidate the relationship between ganglioside expression and neural development. These cells were exposed to three different inhibitors of glucosylceramide synthase, the first enzyme committed for the biosynthesis of most of the brain gangliosides. All three inhibitors, d-threo-1-phenyl-2-decanoylamino-3-morphlino-1-propanol (D-PDMP), d-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (D-PPPP), and N-butydeoxynojirimycin (NB-DNJ) can inhibit greater than 90% of ganglioside biosynthesis at certain concentrations, respectively. D-PDMP significantly slowed down cellular proliferation in undifferentiated P19 EC cells, inhibited neurite outgrowth, and eventually caused cell death in differentiated cells. However, no retardation in cell growth, neuronal differentiation, and neurite outgrowth was observed in cultures treated with D-PPPP or NB-DNJ despite the depletion of gangliosides. These results indicate that the effect of D-PDMP on cellular proliferation, neurite outgrowth, and survival of differentiated cells is independent of the inhibition of ganglioside biosynthesis.

Spatiotemporal expression of GM1 in murine medial pallial neural progenitor cells.

The expression of gangliosides is developmentally regulated in the central nervous system. The expression of GM1 in the neural progenitor cells of the telencephalonic ventricular zone (VZ) has been reported in several studies. However, information on the spatial and temporal regulation of GM1 expression in the VZ is still lacking. In this study, we characterized the expression of GM1 in the developing mouse telencephalon. At E13, GM1 is expressed in neuronal cells as well as in the VZ. The initial expression of GM1 in the VZ is restricted to regions close to the medial pallium. Fluorescence‐activated cell sorting (FACS) analysis and characterization of E14 GM1‐positive cells showed that they contain progenitor cells that proliferate in response to epidermal growth factor (EGF) and/or basic fibroblast growth factor (bFGF) stimulation. The results obtained from quantitative gene expression analysis of region‐specific genes (Emx1, Lhx2, Ngn1, Ngn2, Pax6, Dlx2, Gsh2, Mash1, and Nkx2.1), using real‐time polymerase chain reaction indicate that FACS of GM1‐expressing cells in the fetal forebrain enriches for the medial pallial neural progenitor cells. J. Comp. Neurol. 491:330–338, 2005.

Expression of gangliosides in an immortalized neural progenitor/stem cell line.

Glycosphingolipids (GSLs) are known to play important roles in cellular growth and differentiation in the nervous system. The change in expression of gangliosides is correlated with crucial developmental events and is evolutionarily conserved among many vertebrate species. The emergence of neural progenitors represents a crucial step in neural development, but little is known about the exact composition and subcellular localization of gangliosides in neural progenitor cells. The C17.2 cell line was derived after v‐myc transformation of neural progenitor cells isolated from neonatal mouse cerebellar cortex. The developmental potential of C17.2 cells is similar to that of endogenous neural progenitor/stem cells in that they are multipotential and capable of differentiating into all neural cell types. We characterized the GSL composition of C17.2 cells and found the presence of only a‐series gangliosides. Subcellular localization studies revealed that GM1 and GD1a are localized mainly on the plasma membrane and partly in the cytoplasm, both as punctate clusters. Reverse transcription‐polymerase chain reaction revealed the absence of ST‐II transcripts in C17 cells, which most likely accounts for the lack of expression of b‐ and c‐series complex gangliosides in this cell line. These data suggest that the divergence in ganglioside expression in C17.2 cells is regulated at the transcriptional level.

Cryobanking of Embryoid Bodies to Facilitate Basic Research and Cell-Based Therapies

Pluripotent stem cells offer unique opportunities for curing debilitating diseases. However, further comprehensive research is needed to better understand cell signaling during the differentiation of pluripotent cells into different cell lineages and accordingly to develop clinically applicable protocols. One of the limiting steps for differentiation studies is proper culture and expansion of pluripotent stem cells, which is labor intensive, expensive, and requires a great deal of expertise. This limiting step can be overcome by successful banking and distribution of embryoid bodies (EBs), which are aggregates of pluripotent stem cells and typically the starting point of differentiation protocols. The objective of this study was to investigate the feasibility of EB banking by studying survival and functionality of cryopreserved EBs. To this end, EBs were formed by culturing mouse 129 embryonic stem (ES) cells in the absence of leukemia inhibitory factor (LIF) in hanging drops and then subjected to different cryopreservation protocols. In a series of experiments, we first tested the postthaw survival of EBs as a function of dimethylsulfoxide (DMSO) and extracellular trehalose concentrations and cooling rates. Next, we studied the functionality of cryopreserved EBs by assessing their postthaw attachment, growth, and differentiation into various cell types. Higher (≥5%) DMSO concentrations alone or in combination with trehalose (0.1 M and 0.2 M) yielded good postthaw survival rates of >80%, whereas cooling of EBs at 1°C/min in the presence of 5% DMSO +0.1 M trehalose gave the best attachment and growth rates, with differentiation into cell lineages of three germ layers. Taken together, our results suggest that EBs are tolerant to cryopreservation-associated stresses and retain their differentiation potential after freezing and thawing. Furthermore, our experiments with dissociated EB cells and nondissociated EBs suggest that the extracellular matrix may play a beneficial role in the cryotolerance of EBs. Overall, our data support the feasibility of EB banking, which would facilitate advancement of cell-based therapies.

Inhibition of neuronal migration by JONES antibody is independent of 9-O-acetyl GD3 in GD3-synthase knockout mice.

It has been shown previously that the migration of granule neurons in neonatal cerebellum can be inhibited by a monoclonal antibody (Mab) JONES. Because the inhibition is presumed to be mediated through binding of the JONES antibody to 9‐O‐acetyl GD3, we used GD3‐synthase knockout (GD3S−/−) mice that do not express 9‐O‐acetyl GD3 and also have no detectable defect in brain development, to examine the mechanism of the inhibitory effect. We found no difference between the migration of granule neurons in the neonatal cerebellar explant culture in GD3S−/− mice and in wild‐type mice. Addition of the Mab JONES, but not Mab R24 or A2B5, in the culture medium blocked the neuronal migration in the explant culture of the wild‐type mice. The inhibitory effect of Mab JONES was also observed, however, in the explant culture of GD3S−/− mice. Immuno‐HPTLC analysis showed at least two JONES‐positive glycolipids bands in the lipid extract of GD3S+/+ mice, and none was detected in that of GD3S−/− mice. Western blot analysis of the cerebellum homogenate of wild‐type and GD3S−/− mice identified at least 3 JONES‐positive protein bands, one of which is β1‐integrin. Because the JONES antibody also blocked neuronal migration in the cerebellar explant culture of GD3S−/− mice that do not express 9‐O‐acetyl‐GD3, it suggested an alternative mechanism for the inhibitory effect of the antibody, at least in the GD3S knockout mice, and the inhibitory effect of the JONES antibody on neuronal migration could be mediated through its binding to β1‐integrin.