We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.
Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51±0.8% of differentiation efficiency (n = 3; p < 0.01, Student’s t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.
1. Step 1: Mouse Embryonic Stem (mES) Cell Culture (Timing: 3 days)
1. Plating primary embryonic mouse fibroblasts (PMEF)
2. Plating mES Cells
To monitor the efficiency of mES cell differentiation into motor neurons, we used HBG3, an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb9.
2. Step 2: Neural Induction (Timing: 2 days)
To induce differentiation of mES cells into motor neurons, mES cells need to be separated from PMEF and cultivated in a suspension environment. Gelatin coated flasks are used to separate PMEF from mES cells.
3. Step 3: Motor Neuron Specification (Timing: 5 days)
4. Step 4: Preparation of Poly-DL-ornithine/laminin Coated Coverslips (Timing: 2 days)
Two days before dissociating EBs (i.e. on differentiation day 5), prepare poly-DL-ornithine/laminin-coated coverslips.
5. Step 5: Axon Elongation (Timing: 2 days)
6. Representative Results
Figure 1A shows an outline of the protocol. In step 1, mES cells are cultivated on PMEF and supplemented with LIF to prevent spontaneous differentiation. In general, undifferentiated mES colonies are round and compact, and have clearly defined edges. The colonies are not in contact with one another. Typically, mES cells should be split at a 1:4 ratio every 2~3 days. However, each individual cell line will grow at a different rate and the split ratio must be determined empirically. Arrows in Figure 1B indicate the typical appearance of mES cells after culturing on a PMEF feeder layer for 3 days. These colonies are large (50-100 μm in diameter) but still maintain round and defined edges. At this stage the colonies are ready for differentiation or subculture. An arrowhead in Figure 1B shows a small mES colony that you typically find 24 h after plating. Four days after plating, mES cells become overgrown. They show a flattened appearance and loss of defined boundaries, indicating that they are beginning to differentiate. Such cells are not optimal for differentiation into neuronal cells.
To differentiate mES cells into motor neurons, mES cells need to be cultivated in suspension conditions to allow EB formation. In step 2, ES cells are transferred onto a culture surface without a feeder layer to promote EB formation. In this step, they are incubated in Neural Differentiation medium supplemented with Noggin, bFGF, and FGF-8 for 2 days to direct cells to a neural lineage. Small EBs can be observed under the microscope at day 1 of differentiation. They should be floating in the medium. Carryover PMEF can also be found at day 1 of differentiation and must be removed. These cells adhere to bacterial dishes so are eliminated when EBs are passaged to a new bacterial dish. Thus, by day 2 of differentiation, few or no PMEF should be seen in the culture dish. In step 3, continued transfer of EBs to new dishes should ensure removal of any remaining PMEF. EBs are cultured in Motor Neuron Differentiation (MND) media supplemented with RA and SAG for 5 days to differentiate the cells into motor neurons. During culture, EBs continue to grow in size and can be seen by the naked eye at differentiation day 3~4. Figure 1C shows the microscopic appearance of EBs on differentiation day 7. Unlike the undifferentiated cells, EBs show strong fluorescence due to expression of GFP. These EBs are optimally differentiated and are ready for dissociation. Flow cytometry showed that 51% ± 0.8% of the cells from the dissociated EBs had differentiated into GFP expressing cells5. In step 5, culture of these motor neurons in the presence of GDNF, CNTF, BDNF, and NT3 results in extension of long axonal projections. Figure 1D shows the appearance of differentiated cells 2 days after plating of dissociated EBs. Note long neurites extending from the cell bodies of the plated cells. Immunofluorescent staining shows that the GFP+ cells express the pan-neuronal marker (neurofilament-medium chain) and two motor neuron specific markers, (Islet-1 and choline acetyltransferase, Figure 2).
Figure 1. Differentiation of mES cells into motor neurons (picture modified from Wu et al.5). A) Scheme of the differentiation protocol from mES cells to motor neurons. B) Undifferentiated mES cells form round colonies on the top of a fibroblast feeder layer. They have weak GFP expression. C) mES cells were separated from feeder layers and cultured on low-attachment dishes to form EBs. During the first two days of the differentiation process, cells were exposed to 50 ng/ml Noggin, 20 ng/ml bFGF, and 20 ng/ml FGF-8. Subsequently, they were induced to differentiate with 1 μM retinoic acid and 1 μM sonic hedgehog agonist SAG for 5 days. Differentiated mES cells expressed strong GFP fluorescence. D) After 7-day differentiation, EBs were dissociated and plated on poly-DL-ornithine/laminin coated plates. The differentiated cells extended long neural processes after 2 days in culture. Scale bar = 200 μm. MN = motor neuron. B, C and D are bright field images and B’, C’ and D’ are corresponding fluorescence images.
Figure 2. Characterization of mES cell-derived motor neurons. Immunofluorescence staining for neurofilament medium chain (NF-M, red), ChAT (red), and Islet-1 (red) was coincident with GFP (green) expression. DAPI (blue) was used to identify the nuclei. Scale bar = 50 μm.
The quality of mES cells is the most critical parameter for efficient generation of motor neurons. mES cells must be cultivated on PMEF to prevent spontaneous differentiation. Addition of LIF helps maintain the mES cells in an undifferentiated state. As mES cells divide every 12-15 h, the culture medium becomes depleted rapidly and must be replaced daily.
Efficient activation of the Shh pathway is a critical parameter needed to induce motor neuron specification. Shh protein (R&D System), and Shh signaling agonists such as Hh agonist HhAg1.3 (Curis, Inc.), purmorphamine (Calbiochem), and SAG (EMD Chemicals) have all been used to promote the formation of motor neurons1-3,10,11. We found SAG to be a more effective molecule than purmorphamine in differentiating cells towards a motor neuronal phenotype5. 1- 2.5 μM purmorphamine with 1 μM RA never gave a differentiation efficiency of more than 20%. However in our hands a combination of 1 μM of RA and 1 μM of SAG gave a differentiation efficiency of approximately 25% in procedures without a neural induction step.
To further enhance differentiation efficiency, we add a 2-day neural induction step prior to the motor neuron specification step. This approach takes advantage of the fact that both Noggin and FGF signaling are crucial for the early stage of neural induction when embryonic cells first enter the neural lineage6-9. In addition to neural induction, FGF signaling maintains cultures as neural precursor cells. Overexpression of Fgf8 in the developing midbrain leads to a dramatic expansion of the neural precursor population in the ventricular zone12. A combination of FGFs and Noggin acts synergistically in inducing neural tissue formation by promoting a posterior neural identity9. Thus the 2-day neural induction step is designed to direct the mES cells towards the neural lineage prior to initiation of the process of motor neuron specification.
The protocol described here is a modification and enhancement of the original established protocol described previously1. Although the timeline of generating motor neurons is the same, we have made a number of modifications to streamline the procedure and improve the yield of motor neurons. By adding a neural induction step to the differentiation protocol, we are able to increase differentiation efficiency from 25% to 50%. Our protocol provides an efficient approach to enrich motor neurons derived from mES cells. Motor neurons cannot be obtained from SMA patients and the poor health of primary motor neurons from SMA mice precludes isolation of cells of sufficient quantity, quality and purity to perform quantitative analyses of proteins affected in this disease. Using this mES cell protocol, we were able to obtain a motor neuron cell population of sufficient quantity and purity for a proteomic study to identify molecular pathways affected in spinal muscular atrophy5.
The authors have nothing to disclose.
This manuscript is dedicated to the memory of Dr. Wenlan Wang who passed away on May 26, 2011. We thank Dr Douglas A. Kerr for generously providing the HBG3 mES cells used in this study. This work was funded by Nemours, a grant (2 RR016472-10) under the INBRE program of the National Center for Research Resources (NCRR), and a COBRE grant award from the NCRR (5 P20 RR020173-05) to support the Center for Pediatric Research at the Alfred I. duPont Hospital for Children, Wilmington, Delaware, USA.
Name of the reagent | Company | Catalogue number | Final concentration |
PMEF | Millipore | PMEF-H | N.A. |
PMEF medium | |||
DMEM | Invitrogen | 11965-118 | N.A. |
FBS | Invitrogen | 16140-071 | 10% |
L-glutamine | Invitrogen | 25030-081 | 1% |
Penicillin/streptomycin | Invitrogen | 15240-063 | 1% |
mES medium | |||
DMEM | StemCell Technologies | 36250 | N.A. |
ES Cell-Qualified FBS | Invitrogen | 16141-079 | 15% |
GlutaMax-I | Invitrogen | 35050-061 | 1% |
Non-essential amino acids | Invitrogen | 11140-050 | 1% |
Nucleosides | Millipore | ES-008-D | 1% |
β-mercaptoethanol | Millipore | ES-007-E | 0.1 mM |
Penicillin/streptomycin | Millipore | TMS-AB2-C | 1% |
LIF | Millipore | LIF2010 | 10 ng/ml |
Neural Differentiation medium | |||
DMEM | StemCell Technologies | 36250 | N.A. |
ES Cult FBS | StemCell Technologies | 06905 | 15% |
Non-essential amino acids | Invitrogen | 11140-050 | 1% |
Mono-thio glycerol | Sigma-Aldrich | M-6145 | 1mM |
Noggin | Invitrogen | PHC1506 | 50 ng/ml |
FGF-8 | Invitrogen | PHG0274 | 20 ng/ml |
bFGF | Invitrogen | PHG0024 | 20 ng/ml |
MND medium (differentiation) | |||
ES-Cult Basal Medium-A | StemCell Technologies | 5801 | N.A. |
Knockout serum replacement | Invitrogen | 10828-028 | 10% |
N-2 supplement | Invitrogen | 17502-048 | 1% |
ITS Supplement-B | StemCell Technologies | 07155 | 1% |
Ascorbic acid | StemCell Technologies | 07157 | 1% |
Penicillin/streptomycin | Millipore | TMS-AB2-C | 1% |
GlutaMax-I | Invitrogen | 35050-061 | 1% |
D-glucose | Sigma-Aldrich | G-8270 | 0.15% in d2H2O |
Fibronectin | StemCell Technologies | 07159 | 5 μg/ml |
Heparin | Sigma-Aldrich | H3149 | 20 μg/ml in d2H2O |
β-mercaptoethanol | Millipore | ES-007-E | 0.1 mM |
Retinoic Acid | Sigma-Aldrich | R-2625 | 1 μM |
SAG | EMD Chemicals | 566660 | 1 μM |
MND medium (Motor Neuron culture) | |||
ES-Cult Basal Medium-A | StemCell Technologies | 5801 | N.A. |
Knockout serum replacement | Invitrogen | 10828-028 | 10% |
N-2 supplement | Invitrogen | 17502-048 | 1% |
ITS Supplement-B | StemCell Technologies | 07155 | 1% |
Ascorbic acid | StemCell Technologies | 07157 | 1% |
Penicillin/streptomycin | Millipore | TMS-AB2-C | 1% |
GlutaMax-I | Invitrogen | 35050-061 | 1% |
D-glucose | Sigma-Aldrich | G-8270 | 0.15% in d2H2O |
Fibronectin | StemCell Technologies | 07159 | 5 μg/ml |
Heparin | Sigma-Aldrich | H3149 | 20 μg/ml in d2H2O |
β-mercaptoethanol | Millipore | ES-007-E | 0.1 mM |
BDNF | R&D Systems | 248-BD-005/CF | 10 ng/ml |
CNTF | R&D Systems | 257-NY-010/CF | 10 ng/ml |
GDNF | R&D Systems | 212-GD-010/CF | 10 ng/ml |
NT-3 | R&D Systems | 267-N3-005/CF | 10 ng/ml |
N.A. = Non-applicable.
Table 1. PMEF and Media for mES cell culture or differentiation.
Name of the reagent | Company | Catalogue number | Final Concentration |
0.1% Gelatin | StemCell Technologies | 07903 | N.A. |
Poly-DL-ornithine | Sigma-Aldrich | P0421 | 0.1 mg/ml in d2H2O |
Mouse laminin | Millipore | CC095 | 2 μg/ml in PBS |
0.25% Trypsin/EDTA | StemCell Technologies | 07901 | N.A. |
Accumax | Millipore | SCR006 | N.A. |
N.A. = Non-applicable.
Table 2. Reagents for coating and dissociation.