Nestin-GFP transgenic mice neural stem cell culture

B. METHODS

1. Nestin-GFP transgenic mice neural stem cell culture

The E12.5 transgenic mouse embryos, which have transgene containing eGFP with nestin promoter and the nestin second intron (Fig. 3A) expressed bright GFP in whole central nervous system. The GFP expression means that nestin gene is expressed by regulation of nestin second intron. Also, it means that the neural stem cells expressing nestin protein exist all around CNS.

To obtain neural stem cells from nestin-GFP transgenic mice, the forebrain of the embryonic day 12.5 embryo was dissected and treated with Accutase to acquire single cell. The primary neural stem cells were cultured in DMEM:F12(1:1) with N2 supplement and supplied EGF and bFGF 20ng/ml. The media also contained 2μg/ml heparin sulfate to stabilize bFGF in the media (Caldwell et al, 2004). After 6-7days from primary culture, I could observe 100-200μm in diameter sphere shaped cell aggregation (neurosphere), and about 70% of the cells in the spheres were expressing GFP (Fig. 3B).

Neurosphere were sliced with 5μm thick and stained with various stem cell markers (Fig. 3C). Cells in neurospheres were expressing nestin and other stem cell markers such as musashi, sox2. The proliferating cell marker Ki67 showed in small population of sphere (~1%), but it can not be said that the neurosphere does not proliferate. The Ki67 antibody detects proliferating cell just before cells were fixed.

Also, it can be possible the size of neurosphere inhibit to proliferation of neurosphere

more. I also stained with differentiated cell marker Tuj1 (neuron) and GFAP (astrocyte), but I could not detect any Tuj1 or GFAP positive cell in the neurosphere.

With these results, I could conclude that the GFP(+) and GFP(-) cells in neurosphere are proliferating and not differentiated neural stem (progenitor) cells in our culture condition.

Fig. 3. GFP+ cells express neural precursor cell markers in neurospheres derived from nestin-GFP transgenic mice. (A) Schematic diagram on nestin reporter gene in the transgenic mouse. (B) primery neurosphere population has GFP(+) and (-) cells in neurosphere (C) Various neural stem cell / progenitor markers (nestin, Ki67, musashi and sox2) were coexpressed with GFP, but differentiated cell markers (GFAP and Tuj1) were not.

2. Regulation of nestin gene by the nestin enhancer in the second intron

Nestin gene is composed with four exons and three introns. The second intron of the gene is known that regulate gene expression in central nervous system. Also, the nestin second intron has evolutionary conserved sequence in posterior 3’ half of the sequence - more than 70% of sequence homology among mouse, rat and human: for 1.8kbp rat nestin second intron, it is located sequence from 1162bp to 1798bp (Fig.

1). For convenience, I named this 637base pair sequence as “Nestin Enhancer”

(N2E).

To test nestin gene regulation by nestin sequence and nestin enhancer, I performed luciferase assay in P19 cell line. The reporter gene used in this experiment was pNE1.7luc and pNE0.7Luc+2, which contains whole nestin second intron or two copies of nestin enhancer in pGL3 promoter vector. As transfected amount of bHLH transcription factor neurogenin1 increased, the luciferase activity was increased in both reporter gene (pNE1.7Luc and pNE0.7Luc+2) in dose dependent matter (Fig. 4).

However, the nestin enhancer contained reporter gene has more sensitive activity on ngn1. It can be possible the activity is higher in pNE0.7Luc+2 caused by the more binding ngn1 on enhancer due to double copies of nestin enhancer on reporter gene, but the anterior part of the intron might acts as the repressor of nestin expression in neural stem cells as a report previous (Zhang et al., 2005). At least, I could conclude that the nestin enhancer is regulating nestin expression by proneural gene neurogenin.

2^ndIntron

Fig. 4. ngn1 activated the nestin enhancer in P19 cells. (A) Nestin 2nd intron and enhancer containing luciferase reporter constructs (B) Reporter gene activity of pNE0.7luc+2 was highly increased by ngn1 in a dose dependent manner.

0 1 2 3 4

Ngn1

-

-pNE0.7luc+2 pNE1.7luc

Relative luciferase activity (F. I.)

pNE0.7luc+2

pNE1.7luc

_TATA

A

B

1kb 0.7kb _TATA

Luciferase

1kb 0.7kb

Luciferase

0.7kb 0.7kb _TATA

Luciferase

0.7kb 0.7kb _TATA

Luciferase

3. E-boxes on Nestin Enhancer

” to regulate gene expressi

gene by ngn1. However, the first and m

4. Regulation of nestin expression by E-box on Nestin Enhancer

y the second E-box or not, I constructed

activate nestin gene expression by second E-box on nestin enhancer.

bHLH transcription factors binds to DNA sequence called “E-box (CANNTG) on. The second intron of nestin gene has 14 putative E-boxes, and 6 of the E-box are located on the nestin enhancer (Fig. 1). Especially, the first and second E-box of the nestin enhancer are evolutionary conserved among mouse, rat and human. So, we constructed luciferase reporter gene with those two E-box or mutant E-E-box to exam the binding affinity of bHLH transcription factor, ngn1.

The luciferase activity was increased in the second E-box containing reporter utated second E-box didn’t show activity increase by ngn1 (Fig. 5). With this data, we predicted that ngn1 mediated nestin gene expression is regulated by the second E-box on nestin enhancer.

To exam whether bHLH transcription factor activates nestin enhancer b βgnlacZ expression vector containing nestin enhancer or second E-box mutated nestin enhancer. I transfected the reporter gene with ngn1 in P19 cell line and perform β-gal assay and X-gal stain (Fig. 6). By increasing the copies of nestin enhancers, the transactivity of β-gal was increased by ngn1. However, the second E-box mutated nestin enhancer didn’t show any increase by ngn1. With this result, we could conclude that the bHLH transcription factor may

Fig 5. ngn1 specifically activated the E-box mediated reporter gene activity in P19 cells.

(A), (B) E-box sequences and their mutant forms-containing luciferase reporter constructs (C) E2-mediated reporter gene activity was increased by ngn1 in a dose dependent manner.

5. bHLH transcription factors the effects on nestin gene expression

Development of central nervous system is known to be regulated by many signaling molecules including bHLH transcription factors. We were targeting on

bHLH transcription factor on nestin enhanc

bHLH transcription factor on nestin expression, we examined the effects of many er. To exam the effect of various bHLH transcription factors on nestin gene expression, nestin enhancer containing pNE0.7luc+2 reporter vector and bHLH were transfected on P19 cell line and evaluated luciferase activity (Fig. 7). The early proneural genes- ngn1, ngn2 and mash1- during development of CNS induced luciferase activity, but relatively late proneural genes –math1, math3 and neuroD- didn’t induce luciferase activity on pNE0.7luc+2. Because nestin and proneural genes expression are overlapped in some period during CNS development, this presents the proneural bHLH transcription factors may be enhancing nestin expression by regulating nestin enhancer.

Fig 6. Transactivation of nestin enhancer by ngn1. (A) construction of βgnlacZ-N2E(+n) and N2EM(+n). (B) β-gal assay of reporter gene that contains wild type nestin enhancer and 2^nd E-box mutated enhancer. (C) X-gal staining of βgnlacZ-N2E(+n) in P19 cell line

Fig 7. Specific induction of the 2nd intron by early proneural bHLH proteins. (A) A reporter gene containing the 3’ half of the 2nd intron. (B) Early proneural bHLH proteins prominently promoted luciferase activity whereas the effects of late proneural bHLH proteins such as math1, math3, and neuroD were minor.

6. Nestin Ehnahcer (N2E) / Nestin Enhancr mutant (N2Em) - LacZ transgenic mice

To examine the effect of nestin enhancer and second E-box of nestin enhancer in vivo, we construct transgenic mice with βgnlacZ-N2E(+4) and βgnlacZ-N2Em(+4).

The microinjection toward fertilized eggs was performed in Sookmyoung women’s

university. Integration of transgene was checked with PCR, and then the transgene integration was confirmed with X-gal stain of E11.5 embryos. The LacZ gene was highly expressed in entire CNS including dosal root ganglion, trigeminal nerve in the N2E-lacZ embryo (Fig. 8A, D and G). However, the lacZ expression of N2Em-lacZ is various in each embryo (Fig. 8B, C, E, H, F and I). Some of embryo showed that is normal expression pattern on surface, and others showed ectopic expression. When we section the embryos, the expression patterns of LacZ were quite different between wildtype and E-box mutated nestin enhancer (Fig. 8J and K). The LacZ expression showed radial formation from ventricle to marginal layer in wildtype nestin enhancer, but the LacZ expression was obserbed only in marginal layer in second E-box mutaed transgenic mice. Because nestin gene expression begins at E7.75 and neurogenin1 expression started at E 8.5 in development mouse embryo, the lacZ on marginal layer can be explained as the nestin gene expression is regulated by factors rather than ngn1 with nestin enhancer before E8.5 and enhancing the expression by ngn1 after E8.5.

X-gal stained embryos were sectioned and stained with stem cell marker anti-nestin and anti-sox2. Also, they were stained with bHLH transcription factor neurogenin1. As a result, the dark blue x-gal stained cells were also stained with stem cell markers, anti-nestin and sox2. Furthermore, the dark blue lacZ expressing cells also expressed neurogenin1 (Fig. 9).

Fig. 8. Transgenic mice expressing LacZ driven by nestin enhancer with wild type and mutated E-box (A~C) X-gal staining of whole embryos at E11.5 (D~F) Dorsal view of LacZ expression in the brain (G~I) Dorsal view of LacZ expression in the spinal cord (J~L) Transverse sections of the telencephalic vesicles. Note high levels of LacZ expression in the ventricular zone of the wild type transgenic embryos. (M) Summary of LacZ expression in the transgenic mice

Fig. 9. Colocalization of LacZ and neural stem cell markers in E11.5 transgenic mouse brain carrying the wild type E-box. (A), (D), (G) Telencephalone, (B),(E), (H) midbrain (C), (F), (I) hindbrain

7. ngn1 expression on GFP(+) cell of Nestin-GFP transgenic mouse NSC

We observed the LacZ expression that expressed by regulation of nestin enhancer was colocalized with ngn1 in N2E-LacZ transgenic mice. To observe colocalization of neurogenin and GFP in nestin-GFP transgenic mice, we cultured neural stem cells from transgenic mice to form neurosphere. The neurosphere were sectioned and

stained with anti-GFP and anti-ngn1 and observed with confocal laser scanning microscope. The cells in neurosphere expressed nestin. Noticeable observation is that the GFP positive cells are all expressing neurogenin1 in the neurosphere (Fig. 10).

Hoechst

Nestin

GFP Hoechst

ngn1

GFP

Fig. 10. Coexpression of nestin, GFP and ngn1 in neurospheres derived from nestin-GFP transgenic mice. Expression of nestin-GFP and nestin (A) and nestin-GFP and ngn1 (B) in the neurosphere

8. Characterization of GFP(+) cells using microarray analysis

The GFP expression of nestin-GFP transgenic mice is regulated by nestin second intron. However, not all cells in nestin-GFP transgenic mice neurosphere expressing GFP, even though they are expressing nestin protein (Fig. 3 and 10). The GFP positive cells in nestin-GFP transgenic mice neurosphere were dissociated and counted on slide glass with cover glass using the fluorescence microscope. As a

result, the GFP positive populations were decreased by each passage (Fig. 11), even though the total cells generated were increased in each passage. This might caused by the proliferation rate of GFP positive cells was much smaller than the GFP negative cells. Also, it is possible that GFP positive cells were differentiated to progenitors that still expressing nestin gene but not regulated by nestin enhancer.

Fig. 11. Percentage of positive cells at different passages. The percentage of GFP-positive cells decreased in each passage. PC : primary culture, p1-3 : passage 1 to 3.

The neurosphere expressing nestin but not GFP shows that the expression of nestin gene is regulated by other elements rather than the nestin second intron in CNS. This means the cells in neurosphere might have various staged cells. To investigate the difference between GFP positive and GFP negative cells in nestin-GFP transgenic mice NSC, we performed microarray after sorting nestin-GFP positive and negative cells with FACS. Because the GFP positive cell population was gradually

decrease by the passage, I used only primary sphere to sort using FACS analyzer to maximize the GFP positive cell yield (Fig. 12). The intermediate region of GFP expression were not collected to minimize the cross contamination. Also, I assumed that weak GFP expression means that the GFP transcription was already turned off and remaining GFP detected. When the GFP positive cells after sorting were re-analyzed with FACS, about 98.5% of cells were detected as strong GFP expressing cells (data not shown). It means the FACS analyzer separate GFP positive and negative cells not perfectly. This might caused by the GFP positive and negative cells in same drop are detected as GFP positive by the FACS. Even though the separation was not perfect, it was enough to see the difference of gene expression between GFP positive and negative.

Sorted GFP positive and negative cells were collected in 5ml round bottom tube and the total RNA was prepared with RNA sol. Bee™. Then, the total RNA was cleaned up using Qiagen RNeasy mini kit. Because the purity of input RNA is important for successful analysis, the total RNA were cleaned up using RNeasy mini kit and the quality of RNA in each step were checked with gel image (Fig. 13). It showed that the quality of input RNA and final fragmented cRNA were good to perform microarray. Also, I could conclude the array were performed well with the report file: the value of background & noise, housekeeping control probe set and spike control were meet with the acceptable range.

Fig. 12. Sorting out GFP positive and negative cells from nestin-GFP transgenic mice neurosphere using FACS. GFP negative cells were in R2 and GFP positive cells were in R3. The intermediate regions were not collected.

Gene expression profile from microarray showed that 93 annotated genes were decreased and 104 genes were increased relatively in GFP positive cells. When the genes with normalized expression level increased or decreased by two-fold or more are selected, 30(decreased) and 31(increased) were remained. Then, the genes with

“absent” expression in both samples were eliminated, and finally 16 genes in GFP positive (Table 2) and 8 genes in GFP negative (Table 3) were shown the increased expression. For neurogenin1 (neurod3), the expression level were ranked as “absent”

in the gene profile, but the expression level were shown to be slightly increased in GFP positive cells (Table 4).

Fig. 13. Total RNA and cRNA preparation for microarray. the gel image for quality control in each step were shown. The purified total RNA (left), purifed cRNA (middle), and fragmented cRNA(right)

Table 2. The gene expression increased in GFP(+) cells of nestin-GFP transgenic mouse neurosphere. The gene expression increased more than two folds in GFP positive cells were described with brief gene function

Table 3. The gene expression increased in GFP (-) cells of nestin-GFP transgenic mouse neurosphere. The gene expression increased more than two folds in GFP negative cells were described with brief gene function

Table 4. neurogenin 1 expression level in GFP (+) and (-) cells of nestin-GFP transgenic mice neurosphere. 1438441_at and 1450836_at are the probe ID of Affimetrix Gene chip representing neurogenin1 (neuroD3). Both in GFP(+) and (-) gene expression were ranked as

“Absent”, but the expression were slightly decreased in GFP(-) cells comparing to input.

IV. DISCUSSION

Intermediate filament is characterized that expressed during specific period during development. The class VI intermediate filament, nestin protein is expressed in neural stem (precursor) cells in CNS, and it is replaced by other cell type specific intermediate filament such as GFAP, NF4 during differentiation of NSC (Dahlstrand et al,. 1995). The nestin expression is known that one of the characteristic for the neural stem cells. If the live neural stem cells can be isolated efficiently, the stem cells can be easily used in cell therapy for neurodegenerative diseases. Therefore, many researchers have been tried to identify the neural stem cells using physical properties or non-fixed stain (i.e. surface marker) (Murayama et al,. 2002).

In this study, the neural stem cells were isolated and cultured from E12.5 nestin-GFP transgenic mice embryo, and the cultured neural stem cells were formed neurosphere. The cells in neurosphere were expressed neural stem cell markers such as nestin, sox 2 and musashi, but not differentiated cell markers (Tuj1 and GFAP).

Therefore, the neurosphere in our culture method maintained the undifferentiated condition. Even though, the Ki67 positive cells in neurosphere were around 1%, the total cell number always increased after a week culture from passage. Ki67 is known as a nuclear antigene associated with cell proliferation and is present throughout the active cell cycle (G1,S, G2 and M phases) but absent in resting cells (Lalor et al., 1987). The percentage of Ki67 positive cells in the neurospheres in figure 3 may be

relatively small because the Ki67 antibody only detected the proliferating cells when the neurospheres were fixed. Also, it is possible that the cells in neurosphere proliferate faster when the size of neurosphere is smaller and getting slower after the size is bigger (over 50μm). Because smaller sized neurospheres were more easily stuck in plastic wears during preparing neurosphere sample, I could collect neurosphere at least 50μm in diameter.

I constructed various reporter genes that regulate reporter gene expression by nestin enhancer of nestin second intron, and evaluated that the effect of bHLH transcription factor ngn1 on nestin enhancer activation. Even though the nestin expression begins before ngn1 expression during neurodevelopment, the nestin enhancer was activated by ngn1 (also activated by early proneural gene ngn2 and mash1) in reporter gene assay. It could be possible that proneural bHLH groups are enhancing the nestin expression in specific period when the bHLH transcription factors binds to nestin enhancer. By evaluate reporter gene assays using E-boxes and E-box mutant of nestin enhancer and E-box mutation on nestin enhancer, I could conclude that the second E-box of nestin enhancer is crucial for activation of nestin enhancer by bHLH transcription factors.

The transgenic mice in this study have great advantage to investigate nestin gene regulation. Because the N2E-LacZ transgenic mice contained nestin enhancer, β-globin promoter and LacZ gene in the transgene, the lacZ gene expression is only regulated by only nestin enhancer; we could observe the effect of nestin enhancer rather nestin promoter or fore part of nestin second intron. Also, multicopies of

nestin enhancer in the transgene help to enhance sensitively the lacZ gene expression by the regulation of nestin enhancer (Fig. 6). This may overcome the limitation of sensitivity to β-galactosidase activity detection in the comparison with GFP (Chiocchetti et al., 1997). Therefore, the comparison with nestin-GFP transgenic mice may give a cue to explain the regulation of nestin gene. Also, if the nestin-GFP and N2E-lacZ transgenic mice are hybridized by crossing each other, it will help to visualize the difference of nestin gene regulation by nestin 2^nd intron and nestin enhancer.

When the E12.5 nestin-GFP transgenic mice forebrain were primary cultured, sphere shaped cell aggregation, called “neurosphere” were formed. The neurosphere forming is known as one of the characteristic of neural stem cells (Gage, 2000; Bottai et al., 2003). I could observe that not all nestin expressing cells in neurosphere were expressing GFP; nestin was expressed even in GFP negative cells. It could be explained that the nestin second intron is crucial for expression of nestin gene (Dahlstrand et al., 1995; Lendahl et al., 1997) but many other factors and pathways (ie. Notch signaling) also can regulate the nestin expression in neural stem cells (Mellodew et al., 2004). The enhancer in eukaryotic cells is important to regulating gene expression, but the promoter of one gene has a role in actual transcription of the gene. I could find a report that the ubiquitous transcription factor that related transcription of house keeping gene, SP1 and SP3 are important to nestin gene expression (Cheng et al., 2004).

The GFP expression in nestin-GFP transgenic mice neurosphere was decreased when they were cultured in growth medium in vitro. It could be possible that the GFP expressing cells are differentiated further linage and turned off the GFP, but the nestin expression and sphere formation ability were not vanished in our neurosphere.

The stem cells in CNS are limited in number and suppressed the proliferation in adult (Mazurova et al., 2006), and the GFP expression cells has slow proliferation rate (Ma et al., 2006). With those report, I could conclude that the GFP positive cells in neurosphere are real stem cells and the negative cells are progenitors. To investigate the difference between GFP positive and negative cells, I performed gene array with RNA of cells isolated by GFP. With the array profile, we can check the different

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