Very little is known about the determination events that commit unspecialized cells to differentiate into the more specialized cell types that appear later during embryonic development. Mouse embryonic carcinoma cells, the pluripotent stem cells of malignant teratocarcinomas, may provide a culture system with which to study these events. Lines of embryonic carcinoma cells can be isolated from teratocarcinomas and maintained in an undifferentiated state when kept in exponential growth phase in tissue culture. If left undisturbed at high density, they differentiate in vitro into a variety of cell types including epithelium, neurons, muscle, and cartilage.
The P19 mouse embryonic carcinoma cell line is an established model of cell differentiation. Developmentally, pluripotent P19 cells give rise to the formation of cell derivatives of all three germ layers and appear to differentiate by the same mechanisms as normal embryonic stem cells. When cultured in the presence of 10-6 M retinoic acid, a physiologically relevant morphogen, P19 cells differentiate into neuroectodermal cell lineage, such as neurons and glial cells neurons. However, we showed the overexpression of Shh could direct P19 cells to differentiate into neuronal cells in the absence of RA possibly through the Shh signaling pathway.
In this report, we show that P19/hShhN cells, when replated as the single cells suspension, could differentiate only into neurons in the absence of RA.
II. MATERIRALS AND METHODS
A. MATERIRALS
FITC conjugated secondary antibodies from Vector Laboratory (Burlingame, CA, U.S.A); βIII-tubulin (Tuj-1) specific antibody from Berkeley antibody company (Richmond, CA, U.S.A); neurofilament-160kDa (NF-M) specific antibody from Zymed Laboratory (South San Francisco, CA, U.S.A); 2', cyclic nucleotide 3'-phosphodiesterase (CNPase) specific antibody, Choline acetyltransferase (ChAT) and Hb9 specific antibody from Chemicon (Temecula, CA, U.S.A); DMEM and N2-supplement from GibcoBRL (Grand Island NY, U.S.A); FBS from Hyclone Inc.
(Logan, UT, U.S.A); 5E1 hybrydoma cell from DSHB (IA, U.S.A); Protein G sepharoseTM 4 Fast Flow from Amersham Bioscience (Buckinghamshire, U.K);
Mouse IgG , glial fibrillary acidic protein (GFAP) specific antibody, neuronal cell adhesion molecule (NCAM) specific antibody, All-trans-retinoic acid(RA) , Tyrosine Hydroxylase (TH) specific antibody, 2-mercaptoethanol, o-phenylenediamine dihydrochloride (OPD), Forskolin, Tris, Glycine, sodium dodecyl sulfate(SDS), polyacrylamide, bis-acrylamide, and TEMED from Sigma (St. Louis, MO, U.S.A);
Enhanced chemiluminescence (ECL) kit from Pierce (Rockford, IL, U.S.A); Westran PVDF membranes from Schleicher & Schuell (Dassel, Germany); RNAzolTM B regents from TEL-TEST Inc. (Frendwood, TX, U.S.A); Frist strand cDNA synthesis kit from Roche (Indianapolis, U.S.A); Super Taq DNA polymerase from Korea B&G
(Suwon, Korea).
All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A).
B. METHODS
1. P19 cell culture
P19 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), 100unit/ml penicillin, 100㎍/ml streptomycin in a standard humidified atmosphere at 37℃. After the cultures reached 80-90%
confluence, the cells were harvested with 0.25% trypsin and 1mM EDTA for 2-3min at 37℃, re-plated after 1:10 dilution on 100mm culture dish.
2. Purification & Titration of 5E1 antibody 2.1 Affinity chromatography
Ascitic fluid containing an anti-Shh monoclonal antibody, 5E1 was mixed with on Protein G sepharose 4 Fast Flow beads. prebound gel matrix was packed in a glass column of 1x10cm. The column was equilibrated with binding buffer (20mM sodium phosphate buffer pH 7.0). Then 10ml of binding buffer was applied to the column at a flow rate of 0.4ml/min. The 5E1 antibody was eluted with 20ml of 0.1M Glycine-HCl pH 2.8 at a flow rate of 0.8/min. Each fraction was monitored by Bradford assay and the peak fractions were neutralized with 0.05ml of 1M Tris-HCI pH 9.
2.2 ELISA (Enzyme-Linked Immunosorbent Assay)
Screening for the presence of an anti-Shh mAb (5E1) in the purified 5E1 and Ascitic fluid was performed with lab-made ELISA. High binding capacity 96-well flatbottomed microtiter plates were coated with Proleukin. To each well was added 100µl of culture supernatants at 100µg/ml in 0.1% Tween-20 containing PBS (PBS-T) and then they were incubated overnight at 4℃. After washing five times with PBS-T, potential binding sites were blocked with 200µl of 5% bovine serum albumin in PBS-T and incubated for 1hr at 4℃. The purified 5E1(1:10000) and Asctic fluid (1:1000, 1:5000, 1:100 and 1:10), 100µl per well, were incubated overnight at 4℃.
After washing five times with 0.1% Tween-20 containing PBS (PBS-T), to each well was added 100µl of horse radish peroxidase (HRP)-conjugated goat anti-mouse IgG, diluted to 1:5000 in PBS-T, and then incubated for 2 hr at 37℃. After washing five times, to each well was added 100µl of OPD (o-phenylenediamine dihydrochloride) substrate and then they were incubated for 10 minutes at room temperature. The reaction was stopped by adding 50µl of 2.5M H2SO4 per well and then optical density (OD) was evaluated with an ELISA reader using either a 490nm filter.
3. Preparation of ShhN-Conditioned medium
After P19 and P19/hShhN cells had been plated in tissue culture dish at a density of 1 x 105 cells/ml in 10% fetal bovine serum (FBS)/DMEM (Gibco) for 1 day, culture medium was switched to serum-free medium supplemented with N2 supplement and to fresh 10% fetal bovine serum (FBS)/DMEM. Two days later, the
medium was collected as P19 (PCM) and P19/hShhN (SCM)-conditioned medium.
The medium filtered through a 0.2 ㎛ pore-size filter. The PCM and SCM is stored at 4℃ for use.
4. Concentration of secreted ShhN
The concentration of Shh in cultured P19/hShh and P19/hShhN conditioned media was determined using a Shh ELISA kit protocol (R&D). Using known concentrations of Shh (0-1000 pg/ml) a standard curve was calculated during each assay using the same 96-well, flat bottom, high binding, EIA/RIA. The concentration of Shh in the conditioned media fell with linear range of the standard curve and thus the Shh concentration in the media was determined from standard curve by interpolation.
5. Growth kinetics
P19, P19/hShhN cells were plated at a density of 5 x 103 cells per well of 96-well plate. Following overnight attachment, cells were maintained for 4 days with culture media, serum free media and conditioned media. Anti-Shh, 5E1 and control mIgG added into the medium to a final concentration of 10 ㎍/ml for the 4 days.
Conditioned media was collected 48 hr during P19 and P19/hShhN cells culture and filtered through a 0.2㎛ filter. Cell proliferation was measured in culture using Trypan-Blue exclusion method. Briefly, the cells were harvested with trypsin, and mixed with 0.4% Trypan-Blue solution. After 1min incubation, living cells were
counted.
6. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]assay The proliferation activity of cells was assessed using the MTT assay. This method is based on the ability of mitochondrial dehydrogenases in living cells to reduce soluble tetrazolium salts to a blue formazan product whose amount is directly proportional to the number of living cells. P19, P19/hShhN cells in suspension (500µl) were added to each well of a 4-well culture plate for a final concentration of 5x103 cells/well with 5E1 (10µg/ml) or IgG (10µg/ml) and incubated for 24h, 48h, 72h and 96h at 37°C in a CO2 incubator. At appropriate time points, 300µl of 0.5 mg/ml MTT solution were added to each well, and the cultures further incubated for 2h. Subsequently, the medium with MTT was replaced with 0.2 ml dimethyl sulphoxide. Sample (100µl) were added to each well of a 96-well culture plate. A microplate reader was used to measure absorbance at 570 nm for each well.
7. Differentiation of P19 cells
7.1 Conventional neuronal differentiation
P19 cells were allowed to aggregate in bacterial Petri dishes at a seeding density of 1 x 105 cells/ml in the presence of 0.5 µM all-trans-retinoic acid in 10%
fetal bovine serum (FBS)/DMEM. After 4 days of aggregation, cells were dissociated into single cells by 0.05 % trypsin-EDTA , and were replated in tissue culture dish at a density of 1 x 105 cells/ml in a N2 serum-free medium [DMEM/F12 supplemented
with 5 ㎍/ml insulin, 50 ㎍/ml human transferring, 20 mM progesterone, 60 µM putresine, and 30 nM sodium selenite] supplemented with or without final concentration 10 µM forskolin. The cells were then allowed to adhere and cultured for 4 days with replacement of the medium every 48h.
7.2 Current neuronal differentiation
P19 cells were plated in tissue culture dish at a density of 2 x 105 cells/ml in 10% fetal bovine serum (FBS)/DMEM. To induce neuronal differentiation, the media were removed, and the cells were washed with HBSS and was replaced with N2 serum-free medium with or without final concentration 10 µM forskolin. The cells maintained for 4 days. The same protocol was used for P19/hShhN cells.
- Conventional method
- Current method
Neuronal cells P19 cells Embryoid body
10% FBS N2
4-8d
ShhN, N2
P19 cells Neuronal cells
4d RA +2d/-2d
Fig. 4. The method for neural induction of P19 cells.
8. Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)
Total RNA was isolated using a RNAzolTM B and cDNA was synthesized using First-strand cDNA synthesis kit and 1㎍ of total RNA following the manufacture’s instructions. The PCR reactions were carried out according to standard protocols. Primer sequences (forward and reverse) and product sizes (base pairs) were as follows (Table 1). The PCR products were analyzed on a 1.5% agarose gel.
Table 1. Primer sequence for RT-PCR
Forward Reverse Size (bp)
9. Western blotting
Whole cell lysates were prepared as follows; Cells were washed with cold phosphate buffered saline (PBS) twice and drained. Cells were resuspended in RIPA buffer [150mM Sodium chloride, 1% NP-40, 0.1% SDS, 50mM Tris-HCl (pH8.0), 0.5% sodium deoxycholate] and kept on ice for 5 min. After centrifugation at 12000 rpm for 20min, the supernatant was collected and protein content was assayed by Bradford method. Each 60 ㎍ proteins were separated on 8% or 12% SDS polyacrylamide gel electrophoresis and transferred to PVDF membranes. Membranes were incubated in blocking solution [5% nonfat dry milk in 10mM Tris-HCl (ph7.4), 100mM NaCl, and 0.1% Tween 20] for 1 hour at room temperature and with mouse anti- ShhN (1:100), mouse anti-βIII -tubulin (Tuj-1, 1:1,000), mouse anti-neuronal cell adhesion molecule (NCAM, 1:1,000), mouse anti-neurofilament-160kD (NF-M, 1:1,000), mouse anti-glial fibrillary acidic protein (GFAP, 1:1,000), and mouse anti-2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase, 1:1,000) antibody in TTBS [10mM Tris-HCl (ph7.4), 100mM NaCl, and 0.1% Tween 20] solution for overnight at 4°C. The secondary antibody was horseradish-peroxidase (HRP)-conjugated to goat anti-mouse IgG (1:5,000) or anti-goat IgG (1:5000) and the proteins were visualized using an enhanced chemiluminescence (ECL) kit following the manufacture’s recommendation.
10. Immunocytochemistry
Differentiated P19 and P19/hShhN cells were fixed with 2~4%
paraformaldehyde solution or 100% methanol for 10 min at 4℃. The fixed was washed with PBS solution, three times and incubated with 10% normal horse serum and goat serum, 0.1% BSA and 0.03% Triton X-100 in PBS (PBS-T) for 1hour. After sufficient washing, the cells were incubated with primary antibodies overnight at 4℃.
The following primary antibodies were used at the concentrations given: Rat 401 (1:200), GFP (1:200), Ki-67 (1:200), Tuj1 (1:200), NFM (1:200), TH (1:200), Isl-1 (1:200), ChAT (1:200) and Hb9 (1:200). Following several washing with 0.03%
Triton X-100 containing PBS-T solution, cells were incubated in FITC conjugated anti-mouse IgG (1:500), anti-rabbit IgG (1:500), Alexa 488 anti-mouse IgG (1:500), or Alexa 594 anti-rabbit IgG (1:500) secondary antibody for 1 hour in dark chamber.
After sufficient washing with PBS-T, cells were mounted on acras or cover slips by using Vectashield (mounting medium for fluorescence, Vector) with DAPI or Hoechst (Molecular probe) and photographed using a fluorescent microscope.
III. RESULTS
1. Expression of hShhN in P19 cells
To determine whether Shh could play a role in neuronal differentiation, the expression of components of the Shh signaling pathway in P19 cells was analyzed in western blot. Shh was expressed in P19 cells (Figure 5 A). The N-terminal fragment receives two lipid modifications and, after being secreted, can tether to the membrane of producing cells or diffuse, establishing concentration gradients (Porter et al. 1996; Pepinsky et al. 1998; Lewis et al. 2001; Zeng et al. 2001). Medium conditioned by P19/hShhN cells contained Shh-N (Figure 5 A). The components of the Shh receptor complex, Ptc and Smo, as well as the three Gli transcription factors involved downstream of Shh signaling, have been demonstrated by RT-PCR to be expressed on P19 cells (Figure 5 B).
A.
P19 P19/hShhN
C M C M
Shh
B.
Smo
Ptc
Gli
GAPDH P19
P19/hShhN
Fig. 5. Generation of ShhN-Producing P19 Cell Lines. (A) Cells, P19 and P19/hShhN, were harvested and media collected, separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to PVDF membrane, and probed with antibodies direct against the anti-ShhN. ShhN protein as a 20 kDa was detected both cell extracts and medium in P19/hShhN cell. (B) Expression level of Smo, Ptc and Gli mRNA was measured by RT-PCR.
2. Preparation of 5E1, a neutralizing antibody against Shh
5E1 hybridoma cells were obtained from DSHB (IA, U.S.A). The cells were grown in the presence of HAT (Hypoxanthine-Aminopterin Thymidine). The cells were injected into the peritoneal cavity of BalbC female (6~8 week) to obtain ascitic fluid. The titer of 5E1 ascitic fluid was determined by ELISA using P19/hShhN conditioned medium as an antigen. 5E1 reacted with secreted ShhN in ELISA at dilutions up to >1:1000 (ascitic fluid) (Fig 6 A). IgG fractions were obtained by affinity purification on protein G–sepharose (Fig 6 B). Purified 5E1 were measured the concentration of ShhN by coomassie blue staining and showed neutralizing activity with ShhN in growth kinetics. P19/hShhN were inhibited proliferation at 10~30 ㎍/ml 5E1 (Fig 7).
3. The Concentration of secreted Shh
Shh detected in media conditioned by P19/hShhN using an ELISA. As this concentration of Shh is near the low limit of detection we used the same ELISA protocol to measure Shh in growth media containing 10% FBS, conditioned media form P19/hShhN and differentiation media containing N2 supplement in DMEM:F12 from P19/hShh. The concentration of secreted Shh has 0.7ng/ml in differentiation media and 0.85 ng/ml in conditioned media.
A. B.
Fig. 6. Preparation of 5E1, a neutralizing antibody against Shh. 5E1 reacted with secreted ShhN in ELISA at dilutions up to 1:1000~5:000 (ascitic fluid) (A). The titer of 5E1 was determined by ELISA using P19/hShhN conditioned medium as an antigen and its purified by affinity chromatography (B). Purified 5E1 were measured the concentration of ShhN by coomassie blue staining (C).
Mouse IgG5E1 dilution of 5E1 monoclonal antibody (mAb) or IgG and the total living cell number were measured by Trypan blue dye exclusion method.
4. Shh induces proliferation of P19 cells
To test whether Shh regulates P19 cell growth and differentiation, we cultured cells for 96hr in the presence of 5E1, as Shh neutralizing antibody, or control IgG and analyzed the cultures by cell counting using Trypan-blue dye exclusion method.
These assays were performed in the presence or absence of specific Shh inhibitor, the Shh neutralizing monoclonal antibody 5E1. Purified 5E1 (10µ g/ml) was added to the medium and the medium was replaced every 2 days. Cultures grown in P19 cells demonstrated low proliferation rate. However, a 2-fold, significant increase in cell proliferation was observed in P19/hShhN. P19/hShhN cells were almost completely inhibited by 5E1 monoclonal antibody, but unaffected by mIgG (Figure 8 A). The hShhN effect was blocked by 5E1. To test whether secreted Shh regulates proliferation of P19 cells, we cultured for 4 days in P19 cells conditioned medium and P19/hShhN cells conditioned medium. Conditioned medium from P19/hShhN increased the proliferation of P19 cells, which was blocked by 5E1 (Fig 8 B). Unlike P19 cells, the proliferating potential of P19/hshhN cells were maintained under the differentiation condition of P19 (Fig 8 C).
To confirm the effect of Shh on P19 cell proliferation, proliferation was studied by using 5E1 monoclonal antibody. Neutralizing activity of 5E1 monoclonal antibody was studied by incubating the antibody with 5E1 or IgG. The MTT assay was then performed after 92 hours incubation. As shown in Figure 9, P19/hShhN cells increased proliferation and inhibited by 5E1 (10 ㎍/ml) by compared to the control groups (Fig 9).
A. B.
Fig. 8. Shh induces proliferation of P19 cells. P19 andP19/hShhN cells were plated at a density of 3 x 103 cells per well of 48-well plate and cultured with DMEM supplemented 10% FBS (A), 0.5% FBS (B) and N2 (C) for 4 days. Cells incubated with 10µg/ml of 5E1 monoclonal antibody (mAb) or IgG and the total living cell number were measured by Trypan blue dye exclusion method. Data are expressed as a mean of duplicate per time point.
0 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. P19, P19/hShhN cells in suspension (500µl) were added to each well of a 4-well culture plate for a final concentration of 5x103 cells/well with 5E1 (10µg/ml) or IgG (10µg/ml) and incubated for 24h, 48h, 72h and 96h at 37°C in a CO2 incubator. At appropriate time points, 300µl of 0.5 mg/ml MTT solution were added to each well, and the cultures further incubated for 2h. Subsequently, the medium with MTT was replaced with 0.2 ml dimethyl sulphoxide. Sample (100µl) were added to each well of a 96-well culture plate. A microplate reader was used to measure absorbance at 570 nm for each well.
5. Shh induces neuronal differentiation of P19 cells
In vitro neuronal differentiation of P19 cells and P19/hShhN cells were grown without or with 10µM forskolin in the presence of N2 supplement for 4 days.
P19/hShhN cells formed neurite-like processes and showed a neuron-like morphology on 4 days (Figure 10 A). RT-PCR analyzed for differentiation of P19 and P19/hShhN cells into neural stem/precursors (Nestin), neurons (NF-M) (Fig 10 B). Expression of a neuron-specific protein, NF-M and β-tubulin III were increased in P19/hShhN, which were further increased by addition of 10µM forskolin.
Expression of proteins specific for oligodendrocyte (CNPase) and astrocytes (GFAP) were not obvious (Figure 10 C). Furthermore, immunocytochemistry revealed that cells were positive for neuron-specific markers Tuj1, suggesting that they could be induced into neurons (Figure 10 D).
A.
C.
Fig. 10. Neuronal differentiation of P19, P19/hShhN cells. Morphological changes of P19 cells by Shh or Foskolin (10µM) were examined under a phase contrast microscope (Magnification : x200) (A). Expression of Nestin, as neural stem cell marker, and NF-M, as neuronal marker, mRNA was measured by RT-PCR (B).
Neuronal differentiation was measured by western blot analysis with anti-Tuj1 and NF-M specific antibody (C) and Immunocytochemistry with anti-Tuj1 specific antibody (D).
6. Shh promotes neuronal progenitor cell/ motor neurons survival or proliferation
Nestin is expressed in the myotome the neural progenitor cells of the developing embryo. While the regulatory region for expression in myotome resides in the first intron of the Nestin gene, that for expression in neural progenitor cells exists in the second intron. To ascertain if Shh was important for neuronal progenitor cell survival we added Shh conditioned medium, as a secreted Shh, to cultures of P19/pNPEeGFP cell lines (Figure 11 A). By 4 days in culture, <4% of Nestin-positive cells were present in the control conditioned medium, whereas 25-30%
survive in Shh conditioned medium. GFP-positive cells were increased by secreted Shh (Figure 11 B). The expression of Nestin was further confirmed by RT-PCR. Shh increased the GFP and Nestin expression driven by the Nestin promoter/enhancer (Figure 11 C).
A.
Fig. 11. eGFP-labeled neuronal precursors in P19/pNPEeGFP. Construct for generation of P19/pNPEeGFP (A). P19/pNPEeGFP cells were cultured in Shh conditioned medium or control conditioned medium and counted GFP-positive cells (B). RT-PCR analyzed expression of Nestin (C).
7. Shh induces motor neuron differentiation in P19 cells.
The induction of MN progenitors depends on Shh activity (Briscoe and Ericson, 2001). To examine whether the spinal progenitors present in P19/hShhN can differentiate into MN progenitor, we monitored the expression of homeobox domain and bHLH transcription factors. In vitro neuronal differentiation of P19 cells was induced by Shh and forskolin. The P19/ShhN cells showed the morphology of the cells changed gradually. P19/hShhN cells formed neurite-like processes and showed a neuron-like morphology on 4 days. To examine whether the spinal progenitors present in P19/hShhN cells can differentiate into MN progenitors, we monitored the expression of HD and bHLH transcription factors that delineate sets of neural progenitor cells. Spinal MN progenitors are found in P19/hShhN cells and expressed Nkx6.1, Olig2 and Ngn2. To determine whether MNs are generated from progenitor cells present in P19/hShhN cells, we analyzed the expression of Hb9 and ChAT using RT-PCR and Immunocytochemistry. P19/ShhN cells increased expression of Hb9 and ChAT, suggesting that they could be induced into motor neurons (Fig 12).
Immunocytochemistry revealed that the differentiated P19/hShhN cells increased positive for motor neuron specific markers including Hb9 and ChAT (Fig 13 and 14).
0 1 2 3 4 1 2 3 4
0 µM FSK 10 µM FSK
Nkx6.1 Olig2 Ngn2 NeuroD
GAPDH ChAT Hb9
P19
Nkx6.1 Olig2 Ngn2 NeuroD
GAPDH ChAT Hb9
P19/hShhN
Fig. 12. RT-PCR of the expression of HD, bHLH, and motor neuron specific markers in P19 and P19/hShhN. Neuronal induction of P19 cells was performed as described in materials and methods. P19 and P19/hShhN were treated with 10µM forskolin for 4 days. The efficiency of MN differentiation were measured by expression level of motor neuron marker using RT-PCT. (
-
/+ indicates that forskolin treatment)Hb9Tuj1Hb9Tuj1Hb9Hoechst
P19 P19/hShhN
Tuj1
10µM Fsk 0µM Fsk
10µM Fsk 0µM Fsk
Fig. 13. Shh expression induces motor neuron differentiation of P19 cells.
Neuronal induction of P19 cells was performed as described in materials and methods. P19 and P19/hShhN were treated with 10µM forskolin for 4 days. Cells were visualized by Hoechst (blue) and immuno-stained with neuron specific antibody, Tuj1 (green) and motor neuron specific antibody, Hb9 (red). (Magnification : x200)
P19 P19/hShhN
Tuj1
10µM Fsk 0µM Fsk
10µM Fsk 0µM Fsk
ChATTuj1ChATTuj1ChATHoechst
Fig. 14. Shh expression induces motor neuron differentiation of P19 cells.
Neuronal induction of P19 cells was performed as described in materials and methods. P19 and P19/hShhN were treated with 10µM forskolin for 4 days. Cells were visualized by Hoechst (blue) and immuno-stained with neuron specific antibody, Tuj1 (green) and motor neuron specific antibody, ChAT (red). (Magnification : x200)
8. Shh induces dopaminergic neuron and motor neuron differentiation in P19
8. Shh induces dopaminergic neuron and motor neuron differentiation in P19