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EXPERIMENTAL PROTOCOLS FOR EMBRYONIC STEM CELL RESEARCH |
Department of Gene Function and Development, Roslin Institute, Roslin, Midlothian, United Kingdom
Key Words. Human embryonic stem cell • Neural differentiation • Noggin • Bone morphogenetic protein • Oct4
Correspondence: Wei Cui, M.D., Ph.D., Department of Gene Function and Development, Roslin Institute, Roslin, Midlothian, EH25 9PS, U.K. Telephone: 44-0-131-527-4200; Fax: 44-0-131-440-0434; e-mail: wei.cui{at}bbsrc.ac.uk
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ABSTRACT |
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INTRODUCTION |
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Like mouse embryonic stem cells (ESCs), hESCs can be expanded to large numbers while retaining their differentiation potentials. This is particularly important for transplantation therapies, because hESCs promise an almost unlimited supply of specific cell types. However, directing hESC differentiation along specific lineage pathways remains a challenge. The clinical application of hESCs requires the generation of highly purified donor cells for specific tissues or organs. For neural differentiation of hESCs, most protocols available so far are based on the initial formation of embryoid bodies (EBs) in the presence of serum/serum replacement or coculture of hESCs with particular stromal cell lines [2–4]. In the first cases, hESCs were initially differentiated in suspension cultures to form EBs, which often contain cells of all three germ layers; clusters of neural cells are surrounded by flat non-neural cells, and even in the neural clusters there are mixed populations of neuronal (majority) and glial cells [2, 3]. Moreover, due to the complexity of multicellular aggregates, it is difficult to use this system for studying signaling pathways essential for the neural differentiation. In the latter case, the effect of stromal cells is attributed to an undefined neural-inducing activity [4].
In mouse ESCs, neuroectoderm differentiation has been achieved in an adherent monoculture system in a serum-free medium, without multicellular aggregation or coculture with other cell types [5]. This monolayer culture regimen enables us to directly visualize the process of neural conversion. In addition, because the process is in a chemically defined medium, it provides a good system in the future to investigate signaling pathways in controlling the ESC neural differentiation. Here we extend this study to hESCs, demonstrating that blocking bone morphogenetic protein (BMP) signaling with its antagonist noggin at the initial stage of the differentiation is sufficient for derivation of highly enriched and expandable populations of neural progenitors in adherent culture. Additionally, these progenitors can be further differentiated into various types of neurons in vitro but do not exhibit glial markers until long term in culture.
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MATERIALS AND METHODS |
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The growth curves were started at P5. The neural progenitors were plated at 1 x 105 per cm2 in six-well culture plates and cultured in N2B27 medium with or without 20 ng/ml bFGF. The cells were counted when splitting, and population doublings were calculated.
For tyrosine hydroxylase (TH) neuron differentiation, neural progenitors at P4 or P5 were seeded in poly-L-lysine/laminin (PLL/Lam)-coated culture dishes and induced with sonic hedgehog (300–400 ng/ml), FGF8 (100 ng/ml) [7], and ascorbic acid (160 µM) for 1–2 weeks and then followed by brain-derived neurotrophic factor (BDNF) (20 ng/ml), glial cell line–derived neurotrophic factor (GDNF) (20 ng/ml), ascorbic acid (160 µM), and laminin (0.5 µg/ml) for another 2 weeks.
Immunocytochemistry
Cells were fixed at room temperature with 4% paraformaldehyde for 10 minutes, washed with PBS, and incubated with 10% goat serum and 0.02%–0.1% Triton X-100 for 1 hour. The cells were then incubated with primary antibodies at the appropriate dilution at 4 °C overnight. Secondary antibody was applied for 30 minutes after washing with PBS. The cells were finally mounted with Mowiol (Calbiochem, San Diego, http://www.emdbiosciences.com) and then visualized and captured using Nikon TE2000-U (http://www.nikoninstruments.com) or Digital Pixel image analysis system (Brighton, U.K., http://www.digitalpixel.co.uk).
The following primary antibodies were used in this study: mouse monoclonal antibodies against nestin, polysialic acid–neural cell adhesion molecule (PSA-NCAM), and TH (1:200; all from Chemicon, Temecula, CA, http://www.chemicon.com), ß-tubulin III (1:1,000; Sigma), and 
-fetoprotein (1:500; Sigma); rabbit polyclonal antibodies against nestin and musashi (1:200), GAD65 (1:500), MAP2 and Pax6 (1:1,000; all from Chemicon), GABA and Sox1 (1:500; Sigma), GATA6 (1:50; Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), and glial fibrilliary acidic protein (GFAP) (1:500; DakoCytomation, Glos-trup, Denmark, http://www.dakocytomation.com). Secondary antibodies used were goat anti-rabbit fluorescein isothiocyanate (FITC), goat anti-mouse FITC, and goat anti-rabbit Texas red (all at 1:400; Jackson Laboratory, Bar Harbor, ME, http://www.jax.org), goat anti-mouse immunoglobulin G Alex Fluro 568, and goat anti-rabbit Alex Fluro 488 (all at 1:500–1:1,000; Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com).
RNA Isolation and Reverse Transcription–Polymerase Chain Reaction
Total RNA was extracted using QIAGEN RNeasy kit and DNase I treatment (Qiagen, Inc., Valencia, CA, http://www.qiagen.com). First-strand cDNA was synthesized with 2 µg total RNA in 20–25 µl as described previously [8]. The cDNAs were then diluted to 225 µl with distilled H2O. Diluted cDNA 3–10 µl was used for polymerase chain reaction (PCR) in 50-µl reactions. PCR conditions were optimized and linear amplification range was determined for each pair of primers by varying annealing temperature and cycle numbers. Primer sequences and their PCR conditions are provided upon request. The expression levels were analyzed using multianalysis software and standardized by ß-actin housekeeping gene expression.
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RESULTS |
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On both matrigel and PLL/Lam, hESC plating efficiency was improved. After 4 days of culture in N2B27 medium, flat cells started to emerge, which were very similar in appearance to BMP2-treated hESCs reported earlier by Pera et al. [9]. This flat cell population expanded rapidly (Figs. 1A, 1B, 1D, 1E
), pushing other cell types into clumps, eventually forcing them to detach into the medium. This was most apparent in H9 cells and then H1 and its clonal derivatives and less evident in H7 cells. These flat cells showed positive staining with GATA6 antibody (Fig. 1G) and sometimes formed fluid-filled cysts (Fig. 1B, arrow) that expressed -fetoprotein (AFP) (Fig. 1H). GATA6 has been implicated in mammalian yolk sac endoderm differentiation and function [10, 11]. Forced expression of GATA6 in mouse ESCs triggers differentiation to visceral endoderm [12]. Therefore, the results suggest that these cells are extraembryonic endoderm [9].
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), whereas follistatin did not show evident neuroectoderm formation, although it partially inhibited extraembryonic endoderm formation. The hESCs in follistatin-treated cultures exhibited mainly fibroblast-like morphology (Fig. 1I).
Analysis of gene expression in H1, T5 (a clonal Oct4-GFP cell line derived from H1 [17]), and H7 cell lines in the noggin-treated as well as nontreated culture conditions revealed that at day 6 of differentiation (Fig. 2A), noggin-treated cultures showed apparent downregulation of GATA6, Id1, and Id3 transcripts in all tested cell lines. Additionally, the downregulation of BMP4 was observed in most tested lines. Id proteins have been identified as targets of BMP signaling, and BMP2 induces expression of Id1 and Id3 [18]. Both Id1 and Id3 repress the promoter activation induced by neurogenic bHLH factors and inhibit neurogenesis [18]. These results indicate that noggin inhibited BMP signal pathways, and this may have been the mechanism that blocked ESC differentiation to extraembryonic endoderm. However, the levels of BMP2 expression exhibited upregulation in noggin-treated cultures. This could be due to the feedback regulation of downstream genes. There was no obvious expression of AFP at day 6 of differentiation in both conditions. By day 13, however, AFP expression was significantly upregulated in non–noggin-treated cultures (Fig. 2B), whereas in noggin-treated cultures, no or very low levels of AFP expression were detected.
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Oct4-GFP expression was readily visible in most cells throughout the first week of hESC differentiation (Fig. 3Aa). After approximately 10 days of differentiation, downregulation of GFP expression was initiated. The areas where the differentiated cells became smaller and the nuclei were less visible lost GFP expression, whereas the areas where the cells retained morphology of hESCs were still positive for GFP (Fig. 3Ab). After approximately 1 month of differentiation, cells exhibited typical neural progenitor morphology, and almost no GFP was visible (Fig. 3Ac). However, if the hESCs were cultured in N2B27 medium without noggin, approximately 50% of cells lost Oct4-GFP expression during the first week of differentiation and those cells exhibited extraembryonic endoderm-like morphology (Fig. 3B). The observations of GFP expression were compatible with the reverse transcription (RT)-PCR results (Fig. 4A) in which Oct4 expression was retained longer during neural differentiation than other hESC markers, such as Nanog and human telomerase RT catalytic unit (hTERT). This is consistent with previous published reports [19, 4], suggesting that transiently sustained Oct4 levels may be required to bypass the initial differentiation to extraembryonic endoderm-like cells.
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). Stage 1 is hESC culture (Fig. 5Ba). hESCs were cultured in matrigel-coated six-well culture plates with MEF-CM supplemented with bFGF as previously described [6, 17]. When cells were almost confluent, they were disassociated with EDTA, seeded at a 1:5 ratio into new plates (~3 x 104 per cm2), coated with either matrigel or PLL/lam, and cultured in N2B27 medium supplemented with mouse recombinant noggin at 100 ng/ml. These cells were defined as passage 1 (P1). During the initial week of induction stage, the cells formed more compacted colonies, devoid of surrounding spontaneously differentiated cells (Fig. 5Bb, stage 2, neural induction) compared with hESCs grown in MEF-CM culture. Gradually the cells in some areas started to look darker and smaller with less clearly visible nuclei, a sign of early neuroectoderm formation. These cells were split with collagenase at a 1:3 ratio (5 to 7 x 104 per cm2) at days 8 through 14 and continuously cultured in the same medium. Collagenase was used rather than EDTA at this stage because the seeding efficiency with EDTA was much lower even though there were no significant morphological changes observed. The neural tube–like structure became clearly visible at P2 (approximately day 20), and some early rosette neural progenitors also started to appear (Figs. 5Bc, 5Bd, stage 3, formation of neuroectoderm). These early neural progenitors still prefer to be propagated in small clumps to give a high yield of neural lineages. We found that if we disassociated them into single cells at this stage, some of these cells became other cell types rather than neural progenitors. This could be due to three possibilities. One is that some cells were still undifferentiated at this stage so they could respond to the change in the microenvironment to become other cell types. Alternatively, the early neuroectoderm cells retained the capacity to generate other cell types in response to environment, or it could be a simple in vitro selection for non-neural cells.
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) and showed robust staining with nestin antibody (Fig. 6A), indicating their neural progenitor properties. From P3, the hESC-derived neural progenitor cells could be disassociated and seeded efficiently with TrypLE express at approximately 1 x 105 per cm2. Most cells exhibited a homogenous appearance of neural precursor/early neurons, as shown in Figure 5Bf, and showed positive staining for neural precursor markers, nestin, musashi, and PSA-NCAM (Figs. 6B–6D). By counting the cells positive for these markers over the number of cells positive for DAPI, 97.7% ± 0.55%, 96.3% ± 2.87%, and 92.3% ± 0.96% were positive for nestin, mushashi, and PSA-NCAM, respectively. We have repeated the experiments several times on H1, H7, and T5 cell lines, and the results are similar. The data were from three independent differentiation experiments of T5 cells. At P3, cells at the edge of the rosette also stained positive for MAP2 and ß-tublin III, and by P4, even more cells were positive for MAP2 and ß-tublin III (Figs. 6E, 6F). However, no GFAP-positive cells were observed (Fig. 6K), although they started to emerge after more than 80 days of differentiation in culture (Fig. 6L).
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). At P3 without bFGF, cells in the center of the rosette retained strong proliferative potential, as shown by BrdU staining, and the surrounding cells positive for MAP2 antibody showed less BrdU incorporation (Fig.6J). Some of the hESC-derived cells have been cultured for longer than 4 months and still proliferate vigorously with the addition of bFGF. We also found that although addition of bFGF from the beginning of differentiation enhanced the cell proliferation and slightly delayed the time of differentiation, it did not affect the overall neural differentiation outcome.
Characterization of hESC-Derived Neural Progenitors and Neurons
Expression analysis of the hESCs and their derived neural progenitors at various stages of differentiation confirmed morphological observations and immunostaining results. The semi-quantitative RT-PCR showed a time-dependent reduction in expression of ESC markers, including Oct4, hTERT, and nanog (Figs. 4A, 4B), and the reduction of Oct4 was slower than hTERT and nanog. Furthermore, the reduction in expression of ESC markers was associated with increased expression of neural progenitor markers, such as Sox1, nestin, Pax6, and the neuronal marker MAP2 (Figs. 4A, 4C). The glial marker GFAP, however, was not detected, which corresponded to the immunostaining results. The mesodermal and endodermal markers AFP, GATA6, and brachyury showed almost undetectable expression without consistent increase or decrease during differentiation (Fig. 4A). Pax6 antibody staining was observed by the second week of differentiation and became more homogenous along the progress of differentiation (Fig. 4D, upper panel). No clear Sox1 antibody staining was observed until after 4 weeks of differentiation (Fig. 4D, lower panel). The sequence of Pax6 and Sox1 expression is similar to the neural differentiation with multicellular aggregates [20]. Musashi showed a similar timeline as Sox1 (Fig. 6C). Together, these results suggest that hESCs cultured with this serum-free adherent protocol mainly differentiated into the neural lineage, particularly neuronal cells.
Most neurons generated with this differentiation protocol by passage 5 (~60 days in differentiation) are immunopositive for 
-aminobutyric acid (GABA) (Fig. 6G) and GAD65, an enzyme required for GABA synthesis (Fig. 6H), with very few TH-positive neurons. No glutamatergic neurons were detected, as indicated by negative staining with GluRI and GLAST antibodies. However, exposing the cells to SHH, FGF8, and ascorbic acid for 1–2 weeks at passages 4 through 5 of differentiation (~40–50 days of differentiation) followed by treatment with BDNF, GDNF, ascorbic acid, and laminin for another 1–2 weeks resulted in a significant increase in the numbers of TH-positive neurons (Fig. 6I). These results indicate that the hESC-derived neural progenitors with this protocol have the potential for terminal differentiation into other neuronal types other than GABA neurons if optimal culture conditions for a particular neuronal type are provided. In the current protocol, neither GFAP-positive nor gal C–positive and O4-positive cells were detected by passage 5. However, GFAP-positive cells were detectable after long-term culturing (>80 days).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mouse ESCs have been successfully converted to neuroectodermal precursors in adherent monoculture [5]. However, due to the differences between mouse ESCs and hESCs, we failed to differentiate hESCs efficiently into neural lineage using the same protocol. First, mouse ESCs can grow in culture after disassociation into single cells and seeding at low density, whereas hESCs are prone to spontaneous differentiation into flat or fibroblast-like cells if plated at low density [6, 9], indicating that hESCs require cell-cell contacts to control their fate. Second, there is no problem to culture mouse ESCs in gelatin-coated culture plastics, whereas hESCs are unable to efficiently attach in these dishes. This may reflect the differences of cell-surface molecules, including adhesion molecules, between these two ESCs. For example, mouse ESCs express SSEA1 cell-surface marker, but hESCs express SSEA3 and SSEA4. Therefore, instead of gelatin, matrigel and poly-lysine/laminin were used. Similar to mouse ESCs, maybe even more prevalent, hESCs produced large numbers of flattened cells when plated on matrigel or laminin. These flat cells were positive for GATA6 and AFP, indicating that they are extraembryonic endoderm [21, 22]. Addition of BMP antagonist noggin but not follistatin into the culture medium of hESCs effectively blocked the extraembryonic endoderm formation and promoted neural development. Such an effect of noggin has been observed in Xenopus laevis [23].
The neural progenitors generated in this study were mainly neuronal precursors. There were no detectable GFAP- and O4-positive cells until late passage in long-term cultures. These results differ from the neural progenitors produced by multicellular aggregates in the presence of serum. In the latter culture system, GFAP-positive glial cells are visible in less than 1 month of differentiation [2, 24]. This could be due to the application of BMP antagonist because BMP has been indicated as an important factor for glial cell formation [25]. The addition of noggin into the medium might have blocked the BMP signaling and inhibited the glial cell differentiation; withdrawing noggin at passage 3 or 4 may permit glial development in the long-term culture. It is also possible that cells in multicellular aggregates are at various developmental stages due to their complex microenvironments; some cells are more developmentally advanced than others, so they may develop into glial cells when most of the cells are still neuronal cells.
The adherent culture protocol facilitates visualization of the process of neural conversion. This process seemed to recapitulate early steps of the nervous system development in vivo in that undifferentiated ESCs become more compact and then neural tube–like structures are formed. This could provide an experimental tool to study factors affecting human neural tube formation under controlled conditions. We have used this system to investigate Oct4 expression during neural differentiation and found that Oct4 expression is temporally retained before downregulation to become neural progenitor type of cells. The cells that lost Oct4 expression rapidly did not commit to neural fate, instead turning into the flattened extraembryonic cells. Our observations support previous findings that forced rapid downregulation of Oct4 in ESCs pushes them into extraembryonic cells [26, 27]. Our results and others [4, 19] suggest that transiently sustained levels of Oct4 expression may be required for in vitro differentiation of human ESCs into neural lineages because rapid downregulation of Oct4 expression in ESCs might have promoted the formation of primitive endoderm.
Understanding how to direct hESCs toward a specific lineage pathway and generate appropriate cell types robustly is very important not only for the study of developmental biology but also for potentially using these cells to treat human diseases. The results reported here provide a simple and relatively defined system for differentiation of hESCs to neural lineages, particularly neurons. This system will be useful to study factors for the determination of neural and other lineages.
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ACKNOWLEDGMENTS |
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DISCLOSURES
The authors indicate no potential conflicts of interest.
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REFERENCES |
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