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Stem Cells 2003;21:248-256 www.StemCells.com
© 2003 AlphaMed Press

Manipulation of Human Pluripotent Embryonal Carcinoma Stem Cells and the Development of Neural Subtypes

Rebecca Stewart, Victoria Buyers Christie, Stefan Alexander Przyborski

School of Biological and Biomedical Sciences, University of Durham, Durham, United Kingdom

Key Words. Pluripotent stem cell • Human • Neurogenesis • Neuron • Astrocyte • Tissue culture

Correspondence: Stefan Alexander Przyborski, Ph.D., School of Biological and Biomedical Science, University of Durham, South Road, Durham DH1 3LE, United Kingdom. Telephone: 44-0-191-374-3341; Fax: 44-0-191-374-2417; e-mail: stefan.przyborski{at}durham.ac.uk


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
There are few reliable cell systems available to study the process of human neural development. Embryonal carcinoma (EC) cells are pluripotent stem cells derived from teratocarcinomas and offer a robust culture system to research cell differentiation in a manner pertinent to embryogenesis. Here, we describe the recent development of a series of culture procedures that together can be used to induce the differentiation of human EC stem cells, resulting in the formation of either pure populations of differentiated neurons, populations of differentiated astrocytes, or populations of immature neuronal cell types. Cell-type-specific markers were used to examine the induction of EC stem cell differentiation by retinoic acid. In direct response to manipulation of the culture environment, the expression of cell type markers correlated with the differentiation and appearance of distinct neural cell types, including neurons and astrocytes. These experiments demonstrate that cultured human EC stem cells provide a robust model cell system capable of reproducibly forming neural subtypes for research purposes.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Investigation of cellular differentiation is often hindered by various circumstances, including heterogeneity of starting cellular material, insufficient numbers of stem cells, and the inability to acquire specific stem cell populations at a given time during development. For obvious ethical and moral reasons, this is particularly applicable to the study of human embryogenesis and early stages of cellular development in humans. Pluripotent embryonal carcinoma (EC) stem cells, the stem cells of teratocarcinomas, provide a useful alternative to embryos for the study of mammalian cell differentiation [1]. It is now generally accepted that EC cells closely resemble embryonic stem (ES) cells and are often considered to be the malignant counterparts of ES cells [2]. Several clonal lines of mammalian EC stem cells have been described, and many of these exhibit varied abilities for differentiation [3–7]. Pioneering work by Jones-Villeneuve et al. [8] and McBurney et al. [9], first demonstrated that cultured murine EC stem cells differentiated in response to retinoic acid, forming populations of neurons and glial cells. In a subsequent paper, MacPherson and McBurney [10] described the use of mouse P19 EC cells for research in neural development and presented a range of detailed methods that are now commonly practiced for growth of neural cell types in vitro. Such techniques include the treatment of cells with retinoic acid to induce neural differentiation and the use of growth conditions that modulate cell proliferation as a means of controlling culture purity [10]. Human EC stem cells also can be used to study the development of neural cells [3–5], and sublines of the TERA2 lineage have proved particularly useful [3–6, 11–13]. In this report, we describe the development of additional strategies to manipulate human EC stem cells and induce the differentiation of specific neural subtypes. It is anticipated that these approaches will prove to be valuable tools and have multiple applications in furthering our understanding of neural development in humans.


   MATERIALS AND METHODS
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
TERA2 clone SP12 (TERA2.cl.SP12) EC cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies Ltd.; Paisley, Scotland; http://www.lifetech.com) supplemented with 10% fetal calf serum and 2 mM L-glutamine (DMEMFG) at 37°C in 5% CO2, as previously described [5]. In preparation for differentiation, cultures of confluent TERA2.cl.SP12 cells (plate 0) were briefly treated with 0.25% trypsin (Life Technologies)/2 mM EDTA in phosphate-buffered saline (PBS) for 2–3 minutes to produce a suspension of single cells. To produce neural derivatives, TERA2.cl.SP12 EC cells were induced to differentiate by exposure to 10 µM all-trans-retinoic acid (Sigma-Aldrich Company Ltd.; Poole, UK; http//www.sigmaaldrich.com) using two different procedures as follows.

Procedure A   TERA2.cl.SP12 EC cells suspended in DMEMFG were seeded at 5 x 105 cells/dish into sterile 90-mm-diameter petri dishes normally used for bacterial cultures (plate A1). Suspension cultures were maintained for 1 day before the addition of retinoic acid to a final concentration of 10 µM. Retinoic-acid-induced suspension cultures were maintained for a further 2 weeks during which the retinoic-acid-induction medium was replaced every 2–3 days. Aggregations of cells were washed in DMEMFG and plated onto glass coverslips precoated with poly-D-lysine (Sigma; 10 µg/ml) and human placental laminin (10 µg/ml) (plate A2). Cells grown on coverslips were maintained in DMEMFG in the absence of retinoic acid but in the presence of mitotic inhibitors (1 µM cytosine arabinosine [for the first 7 days only]; 10 µM fluorodeoxyuridine; 10 µM uridine [all supplied by Sigma]).

Procedure B   Suspended TERA2.cl.SP12 cells were seeded at 2 x 104 cells/cm2 (equivalent of 1.5 x 106 cells per T75 tissue culture flask) in DMEMFG containing 10 µM retinoic acid, as previously reported [5] (plate B1). Induction media were changed every 3–4 days and, in some cases, treatment with retinoic acid was continued for 8 weeks. After 21 days exposure to retinoic acid, some cultures were dissociated using 0.25% trypsin/2 mM EDTA in PBS for 10 minutes at room temperature (RT). Suspensions of differentiated cells were split 1:4 and seeded into fresh tissue culture flasks containing DMEMFG without retinoic acid (plate B2). After 3–4 days, replated cells were briefly exposed to 0.1% trypsin/2 mM EDTA in PBS for 2 minutes at RT, and cells loosely attached to the surface of confluent cultures were physically dislodged by a lateral motion provided by five short, sharp blows with the palm of the hand to the side of the horizontal tissue culture flask, as previously described [13]. Cells displaced by enzymatic/mechanical disruption were immediately collected and washed in DMEMFG and seeded onto tissue culture plastic or poly-D-lysine (10 µg/ml) coated 16-mmdiameter glass coverslips at a density of ~2 x 105 cells/cm2 (plate B3). At this stage, plate B3 cultures were subsequently maintained in one of two ways: A) To enhance the purity of neuronal cultures and reduce the proliferation of non-neuronal cell types, mitotic inhibitors were included in the media as follows: 1 µM cytosine arabinosine (for the first 10 days only); 10 µM fluorodeoxyuridine; 10 µM uridine; B) to promote limited proliferation of astrocytes, the concentrations of mitotic inhibitors were reduced accordingly: 0.1 µM cytosine arabinosine (for the first 10 days only); 3 µM fluorodeoxyuridine; 5 µM uridine. After 3 weeks, almost entirely pure populations of either TERA2.cl.SP12-derived neurons or astrocytes were established on the basis of differences in cell adhesion. Astrocytes adhered well to the tissue culture surface, whereas neurons were less well attached and were easily removed by gentle enzymatic and mechanical dissociation as described above. Purified cultures of neurons (plate B4; >99% neuron purity) were maintained for up to an additional 6 weeks in DMEMFG prior to harvesting, and purified cultures of adherent astrocytes that remained attached after removal of neurons (plate B4; >95% astrocyte purity) were maintained in a similar manner.

Immunofluorescent Flow Cytometry
Cell surface antigen expression was determined on TERA2.cl.SP12 stem cells and their retinoic-acid-induced derivatives by indirect immunofluorescence using an EPICS XL cytometer (Coulter; Fullerton, CA; http://www.coulter.com) in a manner similar to that previously reported [5, 12, 14] (data not shown).

Northern Blot Analysis
Poly[A+] RNA was prepared from TERA2.cl.SP12 EC cells and their retinoic-acid-induced derivatives using a commercially available RNA isolation kit (Qiagen Ltd.; Crawley, UK; http://www.qiagen.com). Northern analysis was carried out using formaldehyde denaturing gels as described by Sambrook et al. [15]. Briefly, 5 µg of poly[A+] RNA were separated and transferred onto a Gene Screen Plus nylon membrane (NEN Life Science Products, Inc.; Boston, MA; http://www.nen.com). The blot was hybridized overnight at 42°C with a 2127-bp BamHI-XbaI fragment of Pou5F1 (generously provided by Dr F. Gandolf, Institute of Anatomy, Milan, Italy) [16]. The hybridization signal was visualized by autoradiography using Kodak X-AR5 film (Eastman Kodak Company; Rochester, NY; http://www.kodak.com).

Immunocytochemistry
Cell cultures grown on glass coverslips were rinsed in PBS, fixed in ice-cold methanol (5 minutes), washed in PBS three times, and permeabilized by incubation in 0.1% saponin and 0.3% bovine serum albumin (BSA) in PBS (SBP) for 60 minutes at RT. Cells were incubated with primary mouse monoclonal antibodies diluted in SBP for 2 hours at RT as follows: anti-MAP2ab (clone AP-20, 1:250; Sigma); neurofilament 68kD (anti-NF68; clone NR4, 1:400; Sigma); glial fibrillary acidic protein (anti-GFAP; clone GA-5, 1:500; Sigma); and neuron-specific enolase (anti-NSE; clone MAB324, 1:200; Chemicon International Ltd; Harrow, UK; http://www.chemicon.com). Cells were then washed three times for 5 minutes each in SBP and subsequently incubated with fluorescein-isothiocyanate-conjugated anti-mouse IgG (Sigma; 1:400 in SBP) for 1 hour at RT. Unbound secondary antibodies were removed by five 3-minute washes in PBS. Stained coverslips were inverted and mounted on microscope slides using VectaShield (Vector Laboratories, Inc.; Burlingame, CA; http://www.vectorlabs.com) in preparation for fluorescence microscopy.

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
Poly[A+] RNA was prepared from TERA2.cl.SP12 EC cells and their retinoic-acid-induced derivatives for subsequent RT-PCR analysis as previously described [12]. Standard PCR reactions were performed in a 25-µl reaction volume using 20 pmol of specific primers corresponding to the human forms of either NSE, synaptosome-associated protein (SNAP-25), or GFAP. PCR primers were designed using Primer Select (DNAstar Inc.; Madison, WI; http://www.dnastar.com). The GenBank accession number, product size, recognition site, forward and reverse primer sequence, and annealing temperature for each primer set were as follows: NSE: M22349 [GenBank] , product 500 bp, bases 291-790, ATC GCG CCA GCC CTC ATC AGC, TTT TCC GTG TAG CCA GCC TTG TCG, 61°C; SNAP-25: NM_003081, product 324 bp, bases 620-943, AAT GAT GCC CGA GAA AAT GAA ATG, CAA TGG GGG TGG ACT GAT GTG T, 57°C; and GFAP: J04569 [GenBank] , product 424 bp, bases 959-1,382, GGG AGG CGG CCA GTT ATC AGG A, CCA GCA GAG GCG GAG CAA CTA TC, 62°C. RT-PCR was also carried out using primers specific for human ß-actin (ATC TGG CAC CAC ACC TTC TAC AAT GAG CTG CG, CGT CAT ACT CCT GCT TGC TGA TCC ACA TCT GC) to control for equivalent amounts of DNA per reaction. The specificities of all the primers were checked by sequencing the PCR products (ABI Prism Big Dye Terminator cycle sequencing; Perkin Elmer; Warrington, UK; http://www.appliedbiosystems.com) and by Southern blot analysis using standard procedures (data not shown) [15].

Western Blot Analysis
Protein samples were prepared from TERA2.cl.SP12 EC cells and their retinoic-acid-induced derivatives in protein isolation buffer (1% NP40, 50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1 mM MgCl2, protease inhibitors; Roche Diagnostics Ltd; Lewes, UK; http://biochem.roche.com). Protein concentration was determined using a Bradford-based assay and sample volumes were adjusted appropriately (BSA was used as a standard). Sodium dodecyl sulfate-PAGE gels were prepared according to the method of Laemmli [17]. Using the Bio-Rad minigel system (Bio-Rad Laboratories Ltd.; Hemel Hempstead, UK; http://www.bio-rad.com) loading gels (4% polyacrylamide) and separating gels (10% polyacrylamide) were poured to a thickness of 0.75 mm. Prior to sample loading, 5 µg of protein were denatured in sample loading buffer (0.5 M Tris-HCl, pH 6.8, 10% glycerol, 70 mM lauryl sulphate, 5% 2-mercaptoethanol, 15 µM bromophenol blue) for 3 minutes at 100°C. Samples were electrophoresed for 35 minutes at 200 volts (V). Resolved proteins were immediately blotted (2 hours, 100 V) onto polyvinylidene difluoride (PVDF) membranes (Amersham Pharmacia Biotech UK Ltd.; Little Chalford, UK; http://www.amershambiosciences.com) using the Bio-Rad mini-gel transfer apparatus. Confirmation of protein transfer was achieved by staining blots with 0.2% Ponceau S (w/v) in 5% acetic acid.

For immunoblotting, PVDF membranes were washed in PBS and placed in a blocking solution of 5% dried milk powder in PBS (DMPBS) for 90 minutes at RT. After blocking, the blots were incubated with the following primary mouse monoclonal (unless stated otherwise) antibodies diluted in DMPBS for 1–2 hours at RT: anti-GAP43 (clone GAP-7B10, 1:4,000; Sigma); anti-NF68 (clone NR4, 1:1,000); anti-GFAP (clone GA-5, 1:5,000); anti-ß-actin (clone AC-15, 1:5,000); anti-ß-tubulin-III (clone TU-20, 1:500; Chemicon); anti-NSE (clone MAB324, 1:2,000; Chemicon); anti-SCG10 (polyclonal rabbit antibody, 1:2,500; gift from Dr. G. Grenningloh, University of Lausanne, Switzerland). Blots were subsequently washed in DMPBS for 5 minutes times 3, followed by incubation with either mouse or rabbit IgG-horseradish peroxidase secondary antibody (Amersham; 1:1,000 in DMPBS) and three final 5-minute washes in PBS. Protein-antibody binding was detected on film (Hyperfilm ECL; Amersham) using chemiluminescence (ECL Western blotting analysis system; Amersham).

Chemicals
All chemicals and reagents were purchased from Sigma unless otherwise stated. For molecular analyses, all substances were of molecular biological grade.


   RESULTS
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To induce the formation of neural cell types, human TERA2.cl.SP12 pluripotent stem cells were exposed to 10 µM retinoic acid and were maintained either as suspended aggregation cultures (procedure A) or as confluent layers of adherent cells attached to tissue culture plastic (procedure B) (Fig. 1Go). Both procedures resulted in TERA2.cl.SP12 EC cells downregulating their expressions of pluripotent stem cell markers while also showing upregulation of cell surface antigens expressed by differentiated cells, as previously described (data not shown) [5]. Furthermore, the expression of Pou5F1, a marker of pluripotent stem cells [5, 16, 18], was suppressed after exposure to retinoic acid and the induction of cell differentiation using either of the two methods (Fig. 2Go).



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  		Figure 1. Phase contrast micrographs showing TERA2.cl.SP12 EC cells and their retinoic-acid-induced derivatives. A) TERA2.cl.SP12 human pluripotent stem cells (plate 0); B) Cellular differentiation by induction (procedure A): suspended aggregations of differentiated TERA2.cl.SP12 cells after 2 weeks exposure to 10 µM retinoic acid (plate A1); C) Cellular differentiation by induction (procedure B): differentiated derivatives adhere to tissue culture plastic after exposure to 10 µM retinoic acid for 35 days (plate B1). Scales bars: (A) 30 µm; (B) 600 µm; (C) 750 µm.

 


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  		Figure 2. Northern analysis of the pluripotent stem cell marker, Pou5F1, in TERA2.cl.SP12 stem cells and their differentiated derivatives. Samples of RNA were isolated from cultures of stem cells (ECs); cells were exposed to 14 days retinoic acid using either procedure A (a: 2w RA) or procedure B (b: 2w RA) and populations of purified neurons and astrocytes. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control.

 
Suspensions of human TERA2.cl.SP12 stem cells formed disorganized floating aggregations when grown in bacteriological dishes for the first 24 hours. After maintaining the culture in the presence of 10 µM retinoic acid for 14 days, the cells had aggregated into tight spherical structures that sometimes fused with one another (Fig. 1BGo). Aggregations of retinoic-acid-induced cells adhered to poly-D-lysine and laminin-coated glass and showed significant evidence of neurite formation (Fig. 3AGo), suggesting the presence of neuroprogenitor cells. Confirmation of the neural nature of these structures was demonstrated by the vast majority of cells showing positive staining for the neuronal markers NF 68 and NSE (Figs. 3BGo and 3CGo, respectively).



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  		Figure 3. Differentiation of neurons from aggregations of TERA2.cl.SP12 cells grown in suspension and exposed to retinoic acid for 2 weeks. A) Cell aggregations sprout large numbers of branching neurites when attached to poly-D-lysine/laminin-coated surfaces for 7 days (plate A2). (B,C) Immunocytochemical localization of NF68 and NSE respectively in attached cell aggregations showing neurite formation. Scale bars: (A) 600 µm; (B, C) 150 µm.

 
Human TERA2.cl.SP12 EC stem cells that were seeded onto tissue culture plastic grew as adherent monolayers, responded rapidly to 10 µM retinoic acid, and produced a range of cell types including morphologically identifiable neurons (Figs. 1CGo and 4Go). The characteristic structure of individual neurons could be identified among populations of non-neuronal cells after 14 days differentiation (Fig. 4AGo); however, it was not until weeks 3 and 4 that obvious aggregations of neuronal perikarya and their linking bundles of neurites became visible on the surfaces of the culture monolayers (Fig. 1CGo). Dissociation, splitting, and reseeding of differentiated cultures (plate 2B) appeared to release large numbers of neural cells that formed small aggregations on the surfaces of monolayers of large flat cells (Fig. 4BGo). Separation and maintenance of these loosely attached cells resulted in neuronal cultures of >95% purity (Fig. 4CGo, plate 3B). The purity of neuronal cultures was further enhanced (>99%) by the suppression of cell proliferation and by an additional round of cell separation (Fig. 4DGo, plate 4B). A reduction in the concentration of mitotic inhibitors allowed limited cell proliferation and the formation of colonies of GFAP+ cells that were indicative of astrocytes (Fig. 5Go, plate 4B). Separation of less-adherent contaminating neurons increased the purity of astrocyte cultures to >95%.



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  		Figure 4. Differentiation of neurons from TERA2.cl.SP12 human pluripotent stem cells. A) Expression of NF68 after 21 days exposure to 10 µM retinoic acid (plate B1). B) Phase bright aggregation of neuronal perikarya-expressing neurites over a background monolayer of non-neuronal cells (plate B2). C) Enriched populations of TERA2.cl.SP12-derived neurons grown on poly-D-lysine in high concentrations of mitotic inhibitors (plate B3) for 14 days. D) Immunocytochemical localization of the neural cytoskeletal marker microtubule-associated protein 2 in pure (>99%) populations of TERA2.cl.SP12-derived neurons (plate B4). Scale bars: (A, D) 100 µm; (B, C) 50 µm.

 


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  		Figure 5. Differentiation of astrocytes from TERA2.cl.SP12 human pluripotent stem cells. Phase contrast (A, C) and corresponding GFAP immunofluorescence (B, D) micrographs of retinoic- acid-induced differentiated TERA2.cl. SP12 cells maintained in low concentrations of mitotic inhibitors (plate B3) for 14 days. The arrow indicates a population of neurons that is negative for GFAP. Scale bars: (A, B) 200 µm; (C, D) 75 µm.

 
Undifferentiated TERA2.cl.SP12 EC cells expressed very low levels of NSE; however, the stem cells did not show evidence of transcription for markers associated with the more mature stages of neural differentiation, such as the SNAP-25, or the astrocyte marker, GFAP (Fig. 6Go). Transcription of a variety of neural genes, including NSE, SNAP-25, and GFAP, were all markedly upregulated as cultures of adherent cells responded to 10 µM retinoic acid. Similarly, the translation of neural transcripts, including the growth-associated proteins SCG10 and GAP43 and the pan-neuronal markers ß-tubulin-III and NF68, also displayed regulated expression correlating with the appearance of the mature morphologically identifiable neuron phenotype (Fig. 7Go). Note that the transcriptional profiles for both NSE and GFAP compared favorably with their corresponding protein expression patterns. Molecular analysis also showed that the expressions of neural markers in cultures of purified neural subtypes (grown according to procedure B) were generally cell-type specific. The absolute specificities of these markers and the potential for small numbers of contaminating cell types may explain why such differential cell-type expression was not entirely exclusive.



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  		Figure 6. Transcriptional regulation of mRNA in TERA2.cl.SP12 EC cells and their retinoic acid (RA)-induced derivatives determined by RT-PCR analysis. Cultures were exposed to RA for up to 8 weeks (plate B1) before RNA collection. Populations of neurons and astrocytes were maintained in the appropriate media and sampled after 21 days (plate B3). Remaining non-neuronal cells (see Fig. 4BGo) were harvested immediately after first cell shake-off. ß-actin was used as a loading control.

 


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  		Figure 7. Regulation of protein expression during the differentiation of TERA2.cl.SP12 stem cells by Western analysis. Cultures were exposed to retinoic acid for up to 8 weeks (plate B1) before protein collection. Populations of neurons and astrocytes were maintained in the appropriate media and sampled after 21 days (plate B3). ß-actin was used as loading control.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present investigation describes a series of relatively simple procedures that enable the production of large, almost entirely pure populations of neural subtypes from human pluripotent stem cells. The ability to reproduce such differentiation and the capacity to scale up this process to produce larger quantities of differentiated cell types offer distinct advantages over existing systems. The application of these techniques to the EC stem cells provides a robust model to study the process of human neurogenesis and the specification of certain neural subtypes, and allows the production of neural tissues for further research, drug screening, toxicological testing, and potential therapeutic development.

Several earlier investigations have described the use of stem cells to study neurogenesis in mammals. Pluripotent ES cells derived from inner cell masses of preimplantation embryos have proved to be powerful tools for the study of the mechanisms of mammalian development [19, 20]. Neural cell types have been produced from cultured murine [21, 22] and human [23] ES cells. Indeed, there are some similarities in the way in which Bain et al. [21] induced murine ES cells to form neuronal derivatives with the aggregation method (procedure A) described in the present study. However, handling human ES cells appears more technically challenging, which is partly due to their requirement for growth on feeder cells to maintain their stem cell status [2]. In comparison, the maintenance of human EC cells and their differentiation into neural derivatives is currently less demanding and is more amenable to scaling up and increasing the yield of differentiated cell types. While pluripotent EC tumor cell lines are relatively straightforward to culture, enabling rapid cell population expansion, it is worth noting that the malignant characteristics of certain clonal lines may impinge on their capacities to differentiate, and therefore, their abilities to produce a full range of neural subtypes could be restricted.

Multipotent neural stem cells present in the mammalian embryonic brain have been isolated and grown in culture using a variety of approaches [24–28]. Many of these procedures involve the expansion of neuroprogenitor cells in vitro using growth factors (e.g., basic fibroblast growth factor and epidermal growth factor), followed by changes to the culture environment (e.g., exposure to additional growth factors and varying culture surface substrates) to induce the formation of neural derivatives. Neural development from fetal tissues is often variable [26], especially when using cell samples derived from humans. This is partly because it is difficult to control the exact timing of when material is harvested, hence the developmental potential of the explanted cells may vary. In addition, excised samples of embryonic neuroepithelium contain a mixture of cell types at various stages of differentiation, and such heterogeneity may also influence the behavior of cells explanted in culture. In comparison, cultures of human TERA2.cl.SP12 stem cells are derived from a single clone and are, to the best of our knowledge, homogeneous and reproducibly form neural derivatives in a similar manner when using different batches of TERA2.cl.SP12 stem cells. Furthermore, we describe methods that employ simple cell handling techniques and require the use of few inexpensive media supplements in an attempt to minimize parameters and reduce variability.

The induction and differentiation of neural derivatives from EC stem cells has been recorded previously [3-13, 29]. In each case and as described here, retinoic acid was used as the primary agent to induce cellular differentiation. However, the manner in which EC cells were cultured during the exposure to retinoic acid and the subsequent stages of handling cell differentiation differ between various research groups. Whether this impacts directly on the specification of cell fate and differentiation is unknown, since different clonal lineages of human and mouse EC cells have been used, which themselves are likely to have differing developmental potentials. We have previously shown that TERA2.cl.SP12 stem cells differ from their TERA2 relatives and appear to have a greater ability for differentiation [5]. For the first time, we describe here methods that can be applied to the same human EC cell lineage that result in either the formation of pure populations of differentiated neurons, populations of astrocytes, or spherical aggregations of undifferentiated neuronal cell types.

The regulated pattern of gene and protein expression reported in this study indicates that the differentiation and commitment of human EC stem cells follow a similar pathway to that observed by neural ectodermal precursors during vertebrate neurogenesis in vivo. While there is much information about the terminally differentiated phenotype of human EC-stem-cell-derived neurons [30–34], there are few data describing the process of their differentiation. Our current data concerning the formation of neurons by retinoic-acid-induced TERA2.cl.SP12 stem cells agree favorably with our early work examining neurogenesis in the human EC line NTERA-2 [12]. However, we also demonstrate, in the current paper, the progression of astrocyte differentiation and describe means by which two different neural subtypes can be produced from a common population of EC stem cells, thus providing a useful model of cell fate specification.

In conclusion, human EC stem cells provide a robust cell system for the production of neurons and astrocytes in vitro. In response to retinoic acid, TERA2.cl.SP12 stem cells regulate neural genes and proteins in a conserved manner and may, therefore, provide a useful model for the study of human neural development in vitro.


   ACKNOWLEDGMENT
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The authors would like to thank Dr. Gandolfi, Institute of Anatomy, Milan, Italy, for providing the Pou5F1 cDNA clone; Dr. G. Grenningloh, University of Lausanne, Switzerland, for providing the SCG10 polyclonal antibody; and Professor Andrews, University of Sheffield, UK, for providing the monoclonal antibodies used in cytometric analyses. This work was supported in part by awards to S.A.P. by The Peel Medical Research Trust, London, UK (Registered Charity: 214683); The Royal Society, London, UK (Registered Charity: 207043); and The Wellcome Trust (Registered Charity: 210183).


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received August 19, 2002; accepted for publication October 21, 2002.



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