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Keywords:

  • EGFP;
  • Nestin;
  • Differentiation;
  • Neurones;
  • Astrocytes;
  • Oligodendrocytes

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

Expression of the enhanced green fluorescent protein (EGFP) under control of a thymidine kinase promoter/nestin second intron was specifically detected in nestin immunoreactive neural precursor cells after selection of murine embryonic stem (ES) cells in chemically defined medium. Allowing differentiation in vitro, the capacity of these cells to give rise to astroglia, oligodendroglia, and neurones was investigated. After intracerebral transplantation, long-lasting integration of precursor cells into the host tissue was observed, serving as a pool for successive neuronal and glial differentiation. EGFP expression by ES cell-derived neural precursor cells may be a valuable tool to optimize protocols for maintenance and expansion of these cells in vitro as well as in vivo after intracerebral transplantation. In addition, preparative fluorescence-activated cell sorting of EGFP-labeled neural precursor cells should be useful for standardization of a donor cell population for cell replacement therapies.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

Based on their pluripotency, embryonic stem (ES) cells are of potential interest as a possible source for cell replacement therapies. Indeed, for the nervous system ES cells have been shown to differentiate into neurones and glial cells after intracerebral transplantation [1]. Since the use of uncommitted ES cells is hampered by their possible ectopic differentiation, a selection procedure has been established for the enrichment of nestin-expressing neural precursor cells [2], which have been shown to participate in normal rat brain development [3] and differentiate into myelinating oligodendrocytes in a rat model of a human myelin disease [4]. To simplify the selection of nestin+ neural precursor cells, here we describe the use of a D3 ES cell line-derived clone with a thymidine kinase promoter/nestin second intron cis-acting enhancer-driven enhanced green fluorescent protein (EGFP) expression. By this simple on-line monitoring, selection of a pure population of appropriate precursor cells is facilitated, which allows even further purification by subsequent fluorescence-activated cell sorting (FACS) for cell replacement therapies.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

For the generation of transgenic ES cell lines, the 2 kb nestin intron II enhancer-tk promoter fragment (gift of U. Lendahl) was excised from the Nes 1852 tk/lacZ plasmid by HindIII and NotI restriction enzymes, and subcloned into pEGFP1 vector (Clontech; Palo Alto, CA; http://www.clontech.com) at the SmaI site by blunt end ligation, and designated as h-nestin-EGFP. ES cells were transfected by electroporation of the linearized h-nestin-EGFP reporter construct. Twenty-five independent neomycin-resistant clones were selected and propagated in G418 containing Dulbecco's modified Eagle's medium (DMEM) with supplements (Life Technologies; Karlsruhe, Germany; http://www.lifetech.com).

Out of 25 picked neomycin-resistant ES cell clones of the cell line D3, two clones were investigated in detail. They were cultivated feeder-independent with the supplementation of leukemia inhibitory factor (100 nM, Life Technologies) in 15% fetal calf serum (FCS) containing DMEM (Life Technologies) including supplements as described in previous studies [5]. The cells were allowed to aggregate in hanging drops to form so-called embryoid bodies (EB) as described [6]. Hanging drops with the EB were rinsed off after 2 days and subsequently cultivated in suspension (DMEM, 10% FCS) for another day. Finally, at day 3, EB were transferred to tissue culture dishes (DMEM, 10% FCS) to allow adherence within 12 hours. Selection of neural precursor cells was achieved by cultivation in an astrocyte-conditioned serum-free medium containing insulin, transferrin, selene chloride, and fibronectin in order to promote neural precursor cell selection as described [7]. Selection was performed up to 18 days.

The efficiency of the selection procedure was continuously investigated by EGFP expression, FACS analysis and immunocytochemistry. For the FACS analysis, cells maintained in synthetic medium for 7 days were collected by trypsinisation and resuspended (5 × 105 cells/ml in phosphate buffered saline (PBS), pH 7.0, 0.1% (weight by volume) bovine serum albumin. The emitted fluorescence of EGFP was measured using FACSCalibur flow cytometer (Becton Dickinson; San José, CA; http://www.bd.com) equipped with a 488 nm argon-ion-laser (15 mW) as described [8]. D3 wild-type clones were used as a negative control. The emitted fluorescence of EGFP was measured in log scale at 530 nm (fluorescein isothiocyanate band pass filter) and analyses were performed using CellQuest software (Becton Dickinson).

After selection of neural precursor cells, the population was split. For in vitro analysis of neural precursor cell differentiation, half of the population was plated on laminin-coated dishes in a B27 (Life Technologies) supplemented Neurobasal medium (Life Technologies) and cultivated for up to 3 weeks. The other half was used for intracerebral transplantation into the right striatum of adult Han Wistar rats (300 g), using 300,000 viable neural precursor cells per animal as described [7]. For subsequent immunocytochemical investigation, differentiating neural precursor cells as well as brain tissue were fixed in PBS, pH 7.4, containing 4% paraformaldehyde (Merck; Darmstadt, Germany; http://www.merckeurolab.de). The rat brain was preserved in 18% sucrose before cryosectioning (14 μm). Cryostat sections of brains with engrafted material were evaluated 1, 2, 3, and 4 weeks after transplantation regarding EGFP fluorescence as well as immunoreactivity for markers of the neural lineage close to the injection site.

Immunocytochemistry was performed as described [7] using the following primary antibodies (anti-CD15 ATCC, Manassas, VA; http://www.atcc.org; anti-nestin, Rat 401 DSHB, Iowa City, IA; http://www.uiowa.edu/∼dshbwww; monoclonal anti-mouse-Thy-1, -MAP2, polyclonal anti-GFAP, Sigma; St. Louis, MO; http://www.sigma-aldrich.com; anti-O-4, Roche; Grenzach, Germany; http://www.rocheusa.com) or as differentiation markers for both neurones and glial cells. For fluorescence staining, Cy3-labeled goat-antirabbit and goat antimouse antibodies (Dako; Carpenteria, CA; http://www.dako.dk) were used. Nuclear counterstaining was performed by Hoechst dye. For bright field microscopy, horseradish peroxidase-labeled secondary antibodies were used, followed by visualization with diaminobenzidine as a chromogen. Dilutions of the antibodies were according to the manufacturer's instructions.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

Out of 25 independent clones, two were investigated in more detail to prove the specificity of green fluorescence in neural precursor cells following selection of these cells by a protocol similar to Okabe et al. [2]. Generally, it was found that the characteristics of the two investigated clones were absolutely comparable, especially regarding EGFP expression and neural differentiation: after 7 days of selection in the above-mentioned medium, a bright EGFP fluorescence intensity could be observed in clusters of cells with neuroepithelial morphology (Fig. 1A), and these cells were immunoreactive for Rat-401/nestin, a marker for neural precursor cells [9] (Fig. 1B), but negative for CD15, a marker for undifferentiated stem cells (not shown). Purity of the neural precursor cells was checked by Hoechst dye nuclear staining (Fig. 1C), and quantified by FACS analysis (Fig. 2A), showing about 90% gated cells in M2. Subsequent replating on poly-L-ornithin/laminin-coated dishes and incubation in B27-supplemented Neurobasal medium resulted in differentiation of precursor cells as was indicated by the decreasing number of M2 gated cells in FACS analysis, carried out after 3 and 6 days of incubation (Fig. 2B, C). Twelve days later, immunocytochemical investigations revealed a dense cell lawn consisting of MAP2+ neurones, GFAP+ astroglial cells, and O4+ oligodendroglial cells. Only occasionally EGFP-expressing cells could be discerned at that time (Fig. 1D-F). Quantification of these subpopulations was performed by counting immunoreactive cells for any of the above-mentioned antibodies correlated to the total population as determined by Hoechst dye nuclear staining (Table 1).

Table Table 1.. Cell quantification for the evaluation of neuronal differentiation of selected neural precursor cells (NPC) after selection (day 0) and on days 2, 5, and 14 after replating in B27/Neurobasal medium (days post-plating [dpp]), given in %. (n.d. = not detected). Time-dependent differentiation of neural precursor cells in vitro.
 NPC (Nestin+, EGFP)Neurones (MAP2+)Astrocytes (GFAP+)Oligodendrocytes (O4+)
  1. a

    The data are the mean ± standard error of 1,000 cells counted in five independent experiments.

 0 dpp95.1 ± 1.4n.d.n.d.n.d.
 3 dpp68.8 ± 2.15.6 ± 1.65.3 ± 2.40
5 dpp21.8 ± 1.741.2 ± 2.735.1 ± 3.42.1 ± 4.6
14 dpp2.4 ± 0.435.7 ± 1.955.3 ± 1.77.7 ± 3.4
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Figure Figure 2.. FACS analysis of clone 1 EGFP fluorescent neural precursor cells selected for 7 days (A) showing purity of 90% precursor cells gated in M2.Three days after replating precursor cells in B27/Neurobasal medium, a second peak occurs lacking significant fluorescence intensities, representing an increasing population of differentiating neurones and glial cells (B). As shown for clone 2, further differentiation for another 3 days resulted in increasing cell population of below the M2 level, (C), approximating the control level of respective D3 wild-type cells (D).

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thumbnail image

Figure Figure 1.. Selection of EGFP+neural precursor cells reveals cluster of fluorescent cells 7 days after selection (A) identified as a near pure population of Rat401/nestin neuroepithelial precursor cells (B) by Hoechst dye nuclear counterstaining (C).Fourteen days after replating in B27/Neurobasal medium, cultivation results in a near pure population of neural cells, as revealed by colabeling with MAP2+neurones (black), and GFAP+astroglia (red) (D, E) respective O4+oligodendroglia (red), and EGFP fluorescent precursor cells, in combination with nuclear counterstain (F). EGFP expression is restricted to only a few cells with radial glial cell morphology (arrowheads). Intracerebral grafting experiments with neural precursor cells (G-K) allow easy identification of EGFP-expressing cells in the recipient tissue, shown for 1 week after transplantation (G), and this EGFP-expressing population (H) is immunoreactive for nestin (I). Four weeks after transplantation, Thy-1+mouse neuronal cells are detectable in the vicinity of EGFP fluorescent cells (J). Additionally some EGFP-expressing cells are identified as GFAP+astroglia (yellow, arrowheads in K). Scale bar: 20 μm in A-C, 50 μm in D-K.

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Transplantation experiments revealed localization of EGFP-expressing cells within and close to the injection site 7 days after intrastriatal grafting (Fig. 1G). These EGFP-expressing cells (Fig. 1H) exhibit a neuroepithelial morphology and are immunoreactive for nestin (Fig. 1I). Even 4 weeks after transplantation, clusters of neural precursor cells can still be found close to the injection site where they subsequently differentiated into neurones as identified by the mouse-specific marker Thy-1+ (Fig. 1J). Differentiation into glial cells was shown by occasionally detected EGFP fluorescent cells also expressing GFAP+ (Fig. 1K). We never observed EGFP-expressing cells migrating towards or even settling down at the subventricular zone of the lateral ventricle, where intrinsic neural precursor cells reside.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

Data from our in vitro studies point out that the D3-derived clones with the specific EGFP expression under the control of the human nestin second intron regulatory sequence showed high fluorescence signals with a strong correlation to undifferentiated neural precursor cells. These in vitro observations are in line with those described in vivo for transgenic mice using either the same regulatory sequence [10] or the homologous rat regulatory intron [11].

The timetable of efficient neural precursor cell enrichment within 7 days of selection is concordant with that observed in the D3 wild-type clone (own observations) as well as other ES cell lines [2], suggesting that EGFP expression does not affect differentiation of ES cells into the neural lineage. Moreover, the two clones investigated in this study did not show any significant differences. Likewise, cardiac-specific EGFP expression in ES cell-derived cardiomyocytes as well as in transgenic animals altered neither electrophysiological properties nor normal development [8, 12].

Immunostaining of MAP2+ neurones, GFAP+ astrocytes as well as O4+ oligodendrocytes after cultivation for 14 days in B27-supplemented Neurobasal medium showed the physiologic capacity of the selected neural precursor cells to enter both the neuronal and glial lineage. These cells along with those exhibiting green fluorescence made up 95% of the total cell number. Purity and their restricted differentiation capacity towards the neural lineage were indirectly validated by absence of immunoreactivity, e.g., alpha actin for striated muscle or PECAM for endothelial cells under conditions suitable for differentiation of these cells [7].

Actually, these results indicate that the combination of a specific selection protocol for neural precursor cells together with the specific EGFP expression in these cells may offer a good opportunity to gain a pure cell population for cell replacement therapies in neurodegenerative diseases. This is even more valuable as the physiological behavior and the in vivo differentiation capacity of neural stem cells seems not to be altered by EGFP expression as shown in our previous studies [8, 12].

In contrast to earlier grafting studies using non-selected ES cells as transplants, which resulted in spheroid-like aggregates with differentiated neurones inside [1], transplantation of selected neural precursor cells shows a better integration into the host tissue [7]. Additionally, with the here described nestin-EGFP clones, we are able to show for the first time that there is a good integration capacity of the precursor cells themselves in localizations outside the subventricular zone, where precursor cells normally reside in the adult brain [13]. Thus, observation of these cells for up to 4 weeks reveals that they are ideal material for cell replacement therapies in progressive neurodegenerative diseases with a gradual migration and differentiation into the surrounding tissue.

Transplantation of genetically modified cells incorporating the expression cassette for the EGFP gene, of course might raise the question of a T-cell immune response against EGFP as it was shown by Stripecke et al. [14]. However, as in our previous studies using EGFP-expressing neural precursor cells [7], a rejection problem due to an immune reaction against EGFP can most likely be neglected in this system, probably as the brain is regarded as an immunologically protected compartment.

Implementing a clinical use of the here described selected neural precursor cells, the proliferative capacity of neural precursor cells after transplantation might be considered as another safety issue. However, investigation of the injection site after transplantation did not give any hints for an uncontrolled proliferation or even tumor formation after grafting of precursor cells. Furthermore, in order to definitely exclude any risk for an enhanced proliferation of these cells, a “suicide gene” such as HSV-1 thymidine kinase could be additionally used, as it is also suggested for gene therapeutical treatment of hyperproliferative disorders [15].

Compared to the Tα1 tubulin EGFP construct, which has been used to isolate neuronal progenitors from embryonic forebrain [16], the nestin EGFP construct used in this study offers a broader selection spectrum allowing the parallel differentiation of neurones and glia. In fact, after transplantation, differentiated glial cells will act as an additional environmental factor producing a support tissue and scaffolding for the neuronal cells differentiating in the host tissue. Furthermore, specific EGFP expression in true neural precursor cells allows their easy identification and quantification, as well as their functional characterization facilitating studies unraveling mechanisms involved in either neuronal or glial cell lineage commitment.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

EGFP labeling under the control of the second intron of the human nestin gene guarantees easy cell selection of neural precursor cells. Combined with the exclusion of ectopic differentiation which might lead to tumorigeneity, the good integration capacity of these cells with continuous differentiation into neurones and glia makes them a suitable population for establishing cell replacement therapies.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

The authors wish to thank Mrs. J. Siodlaczek for excellent technical help. This work was supported by the Hochhaus Stiftung (CA 3640-15).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References