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Keywords:

  • differentiation;
  • engraftment;
  • grafting;
  • mouse

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Mouse ES (mES) cell culture and green fluorescent protein (GFP) expression
  5. In vitro differentiation assay of GFP-expressing mES cells
  6. In vitro co-culture assay
  7. mES cell preparation for transplantation
  8. Transplantation, immunosuppression and post-transplantation monitoring
  9. Immunocytochemistry
  10. Confocal laser-scanning microscopy
  11. Electrophysiology
  12. Results
  13. Establishment and characterization of GFP-positive mES cells
  14. Differentiation of mES cells to neural progenies varies among different brain regions
  15. Region-specific activities in the brain control the expansion of neural progenitors and neuronal differentiation
  16. ES cell-derived neural progenies differentiate into mature neurons
  17. ES-derived neurons gradually acquire electrophysiological properties similar to host cells
  18. Discussion
  19. Acknowledgements
  20. References

Embryonic stem (ES) cells are multipotent progenitors with unlimited developmental potential, and in vitro differentiated ES cell-derived neuronal progenitors can develop into functional neurons when transplanted in the central nervous system. As the capacity of naive primary ES cells to integrate in the adult brain and the role of host neural tissue therein are yet largely unknown, we grafted low densities of undifferentiated mouse ES (mES) cells in adult mouse brain regions associated with neurodegenerative disorders; and we demonstrate that ES cell-derived neurons undergo gradual integration in recipient tissue and acquire morphological and electrophysiological properties indistinguishable from those of host neurons. Only some brain areas permitted survival of mES-derived neural progenitors and formed instructive environments for neuronal differentiation and functional integration of naive mES cells. Hence, region-specific presence of microenvironmental cues and their pivotal involvement in controlling ES cell integration in adult brain stress the importance of recipient tissue characteristics in formulating cell replacement strategies for neurodegenerative disorders.

Abbreviations used
ALP

alkaline phosphatase

bFGF

basic fibroblast growth factor

Cy

carbocyanine

DBX

doublecortin

DMEM

Dulbecco's modified Eagle's medium

ES

embryonic stem

FCS

fetal calf serum

GFP

green fluorescent protein

LIF

leukaemia inhibitory factor

mES

mouse ES

PB

sodium phosphate buffer (0.1 M, pH 7.4)

PFA

paraformaldehyde

ir

immunoreactive

NSC

neural stem cells

TH

tyrosine hydroxylase

Tuj1

β-111-tubulin

In the central nervous system (CNS), grafts of in vitro differentiated neural stem cells (NSC) established from the embryonic, early postnatal and adult CNS, and embryonic stem (ES) cell-derived progenitors of multiple developmental stages generated in vitro survive for long periods, undergo phenotypic differentiation, and acquire neurochemical and electrophysiological properties similar to those of host neurons, with a positive impact on disease behaviour and depleted brain functions (McDonald et al. 1999; Temple 2001; Björklund et al. 2002; Kim et al. 2002). The ability of transplanted embryonic and adult NSC to generate neurons in contrast to glia; or different classes of neurons varies greatly among distinct regions of the CNS. NSC differentiate primarily into glia when transplanted into non-neurogenic brain regions such as the cerebellum, striatum and spinal cord (Gage et al. 1995; Fricker et al. 1999; Chow et al. 2000; Shihabuddin et al. 2000), and hippocampus-derived progenitors become olfactory neurons when placed in the rostral migratory stream (Suhonen et al. 1996). Transplantation of cells firmly committed to a neuronal lineage and expressing immature or mature neuronal markers have a greater propensity for neuronal differentiation in non-neurogenic brain regions, in particular the spinal cord (Shin et al. 2000). Similar results have been obtained by transplanting ES cell-derived embryoid bodies and ES cell-derived in vitro-differentiated neural cells (McDonald et al. 1999; Liu et al. 2000; Andressen et al. 2001; Zhang et al. 2001; Björklund et al. 2002; Kim et al. 2002).

Intrinsic properties of transplanted cells may contribute to differences in stem cell engraftment and differentiation in various regions of the CNS. Patterning of the body in its antero-posterior and dorso-ventral aspects imparts positional information on cells in the nervous system. Such positional information elicited by gradients of morphogens during neural induction impose a restriction in fate (Ericson et al. 1997). Both embryonic and adult NSCs established from different brain regions retain regional specification (Zappone et al. 2000; Hitoshi et al. 2002). Moreover, competence of NSCs is altered over time in many organisms; and such changes might reflect alterations in intrinsic factors controlling competence. Early, but not late, neural tube cells produce both CNS and peripheral nervous system stem cells, indicating a common progenitor at early stages (Mujtaba et al. 1998), and early stage cortical progenitors show different potential, as compared with those from later stages (Shen et al. 2002).

Whereas in vitro studies using astroglia-conditioned medium and astroglia/NSC co-culture suggest the importance of the local microenvironment for engraftment and functional integration of stem cells in the brain (Wagner et al. 1999; Song et al. 2002), transplantation of NSCs and lineage-committed ES cell-derived progenitors does not allow distinction between epigenetic and genetic factors involved in the integration of transplanted cells. Pluripotent and regionally non-specified ES cells provide a powerful tool to understand mechanisms controlling stem cell differentiation in different regions of the adult brain. It remains yet to be determined whether integration of naive, undifferentiated ES cells in neurochemically diverse regions of the adult brain follows a uniform pattern, or whether some areas are superior in promoting functional integration of ES cells transplanted at a single cell density. In this study, we have determined the influence of the recipient brain on the commitment of undifferentiated ES cells to a neural fate, their subsequent integration into the host brain, and differentiation into functional neurons.

Mouse ES (mES) cell culture and green fluorescent protein (GFP) expression

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Mouse ES (mES) cell culture and green fluorescent protein (GFP) expression
  5. In vitro differentiation assay of GFP-expressing mES cells
  6. In vitro co-culture assay
  7. mES cell preparation for transplantation
  8. Transplantation, immunosuppression and post-transplantation monitoring
  9. Immunocytochemistry
  10. Confocal laser-scanning microscopy
  11. Electrophysiology
  12. Results
  13. Establishment and characterization of GFP-positive mES cells
  14. Differentiation of mES cells to neural progenies varies among different brain regions
  15. Region-specific activities in the brain control the expansion of neural progenitors and neuronal differentiation
  16. ES cell-derived neural progenies differentiate into mature neurons
  17. ES-derived neurons gradually acquire electrophysiological properties similar to host cells
  18. Discussion
  19. Acknowledgements
  20. References

mES cells of the GSI-1 line were grown on mitotically inactivated primary mouse fibroblasts (35 Gy γ-irradiation) in Dulbecco's modified Eagle's medium (DMEM) (Gibco-BRL, Rockville, MD, USA) supplemented with (final concentrations) 15% fetal calf serum (FCS, HyClone, Logan, UT, USA), 1000 U/mL of mouse recombinant leukaemia inhibitory factor (LIF, Invitrogen, Carlsbad, CA, USA), 0.1 mm 2-mercaptoethanol (Sigma, St Louis, MO, USA), 0.1 mm non-essential amino acids, 2 mm glutamine, 100 U/mL of penicillin and 100 mg/mL streptomycin (all obtained from Gibco-BRL). mES cells were passaged using 0.25% trypsin/EDTA. mES cells were transfected by electroporation with a plasmid coding for GFP under control of the phosphoglycerate kinase promoter (QBiogen, Carlsbad, CA, USA) that ensured ubiquitous perisomatic GFP expression, irrespective of the terminal lineage of the mES progeny. Selection was performed using G418 to identify clones that had incorporated the transfected genes; and clones with high GFP expression were manually picked using fluorescence microscopy.

In vitro differentiation assay of GFP-expressing mES cells

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Mouse ES (mES) cell culture and green fluorescent protein (GFP) expression
  5. In vitro differentiation assay of GFP-expressing mES cells
  6. In vitro co-culture assay
  7. mES cell preparation for transplantation
  8. Transplantation, immunosuppression and post-transplantation monitoring
  9. Immunocytochemistry
  10. Confocal laser-scanning microscopy
  11. Electrophysiology
  12. Results
  13. Establishment and characterization of GFP-positive mES cells
  14. Differentiation of mES cells to neural progenies varies among different brain regions
  15. Region-specific activities in the brain control the expansion of neural progenitors and neuronal differentiation
  16. ES cell-derived neural progenies differentiate into mature neurons
  17. ES-derived neurons gradually acquire electrophysiological properties similar to host cells
  18. Discussion
  19. Acknowledgements
  20. References

Neurospheres were generated as previously published (Tropepe et al. 2001). In brief, mES cells (D3 and GSI-1 lines, American type culture collection; ATCC, Rockville, MD, USA) were dissociated with 0.25% trypsin and transferred to neuronal medium supplemented with N2, basic fibroblast growth factor (bFGF, 10 ng/mL) and LIF (1000 U/mL). Subsequently, neurospheres were plated on poly d-lysine/laminin and differentiated by replacement of bFGF by 1% FCS with or without retinoic acid (1 μm) where they gave rise to cells with various morphologies including neurons and glia. A mES cell clone that retained GFP expression in all classes of differentiated cells was selected for transplantation. GFP expression in neurons and glial cells was evidenced by co-localization of GFP with mouse anti-β-III-tubulin (Tuj1, 1: 2000, Promega, Madison, WI, USA) or rabbit anti-glial fibrillary acidic protein (GFAP, 1 : 800, Dako, Glostrup, Denmark), respectively. Detection of the immunosignals was performed using carbocyanine (Cy) 3-tagged species-specific secondary antibodies generated in donkey (1 : 500, Jackson ImmunoResearch, West Grove, PA, USA).

In vitro co-culture assay

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Mouse ES (mES) cell culture and green fluorescent protein (GFP) expression
  5. In vitro differentiation assay of GFP-expressing mES cells
  6. In vitro co-culture assay
  7. mES cell preparation for transplantation
  8. Transplantation, immunosuppression and post-transplantation monitoring
  9. Immunocytochemistry
  10. Confocal laser-scanning microscopy
  11. Electrophysiology
  12. Results
  13. Establishment and characterization of GFP-positive mES cells
  14. Differentiation of mES cells to neural progenies varies among different brain regions
  15. Region-specific activities in the brain control the expansion of neural progenitors and neuronal differentiation
  16. ES cell-derived neural progenies differentiate into mature neurons
  17. ES-derived neurons gradually acquire electrophysiological properties similar to host cells
  18. Discussion
  19. Acknowledgements
  20. References

Neurospheres, generated as described above, were plated on poly d-lysine/laminin in 24-well plates in the same medium and cell culture inserts containing tissue slices from 2- to 3-month-old C57BL/6 J mice (Charles River Laboratories Inc., Sulzfeld, Germany). Slices were made with a McIlwain tissue chopper (The Mickle Laboratory Engineering Co. Ltd, Guildford, UK). After 5 days, inserts were removed and the medium was supplemented with brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor (20 ng/mL each) and nerve growth factor (50 ng/mL). After three additional days, specimens were fixed with 4% paraformaldehyde (PFA) in sodium phosphate-buffered saline (0.01 m, pH 7.4). The transfer of neurospheres to laminin-coated surface leads to adherence and cell migration out of the neurospheres, resulting in a flat cell aggregate. The effect of the brain tissue on growth and morphology of the neurospheres was statistically evaluated using univariate analysis of variance (anova) with Bonferroni's post-hoc test where appropriate. A p-value of < 0.05 was considered significant. Data were expressed as means ± SEM.

mES cell preparation for transplantation

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Mouse ES (mES) cell culture and green fluorescent protein (GFP) expression
  5. In vitro differentiation assay of GFP-expressing mES cells
  6. In vitro co-culture assay
  7. mES cell preparation for transplantation
  8. Transplantation, immunosuppression and post-transplantation monitoring
  9. Immunocytochemistry
  10. Confocal laser-scanning microscopy
  11. Electrophysiology
  12. Results
  13. Establishment and characterization of GFP-positive mES cells
  14. Differentiation of mES cells to neural progenies varies among different brain regions
  15. Region-specific activities in the brain control the expansion of neural progenitors and neuronal differentiation
  16. ES cell-derived neural progenies differentiate into mature neurons
  17. ES-derived neurons gradually acquire electrophysiological properties similar to host cells
  18. Discussion
  19. Acknowledgements
  20. References

GFP-mES cells were cultured on mitotically inactivated STO fibroblasts (35 Gy γ-radiation) for at least 3 days and one to two passages prior to transplantation. FCS in the culture medium was substituted with Serum Replacement (Gibco-BRL). mES cells were then trypsinized as above, centrifuged, re-suspended in serum free DMEM and filtered to remove debris and to generate a single cell suspension. Subsequently, mES cells were washed three times in DMEM, gently centrifuged and directly used for transplantation. The density of mES cells was adjusted using a hemacytometer, and cells were diluted to ±400 and ±4000 cell/μL in DMEM. Approximately 85–90% of all transplanted cells were mES cells as determined, and their undifferentiated state was verified by staining of mES colonies for alkaline phosphatase (ALP; Ying et al. 2002).

Transplantation, immunosuppression and post-transplantation monitoring

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Mouse ES (mES) cell culture and green fluorescent protein (GFP) expression
  5. In vitro differentiation assay of GFP-expressing mES cells
  6. In vitro co-culture assay
  7. mES cell preparation for transplantation
  8. Transplantation, immunosuppression and post-transplantation monitoring
  9. Immunocytochemistry
  10. Confocal laser-scanning microscopy
  11. Electrophysiology
  12. Results
  13. Establishment and characterization of GFP-positive mES cells
  14. Differentiation of mES cells to neural progenies varies among different brain regions
  15. Region-specific activities in the brain control the expansion of neural progenitors and neuronal differentiation
  16. ES cell-derived neural progenies differentiate into mature neurons
  17. ES-derived neurons gradually acquire electrophysiological properties similar to host cells
  18. Discussion
  19. Acknowledgements
  20. References

Young adult male C57BL/6 J mice (n = 16, 2–4 months of age; Charles River) and ROSA26 mice with ubiquitous β-galactosidase expression (n = 5; Zambrowicz et al. 1997) were used in transplantation experiments. Animals were housed individually in an air- (21 ± 2°C) and humidity (55 ± 5%)-controlled facility with a 12/12 h light/dark cycle (lights on at 08.00 h) at least 3 days prior to surgery, and kept on a normal laboratory diet and tap water ad libitum. To reduce immune reaction of the host brain and subsequent graft rejection, all animals received immunosuppression by subcutaneous injection of cyclosporin A (20 mg/kg; Sandimmun®, Novartis, Basel, Switzerland) diluted in physiological saline each day throughout the survival period, starting with a double dose 1 day before surgery (Björklund et al. 2002). Animals were anaesthetized with isoflurane [2.0% (v/v%) in 70% N2O/30% O2; 0.5 L/min flow rate] and their head position was secured in a stereotaxic frame equipped with a mouse adaptor (Model 900; Kopf Instruments, Tujunga, CA, USA). Core body temperature of the animals was kept at 37 ± 0.5°C. Single cell suspension of mES cells was infused in the medial septum (AP = +0.5 mm; L = 0.0 mm; DV = 3.4 mm), striatum (AP = +0.5 mm; L = 1.5 mm; DV = 3.0 mm), somatosensory cortex (AP =−0.9 mm; L = 3.5 mm; DV = 1.5 mm), hippocampus (AP = −2.3 mm; L = 2.4 mm; DV = 1.9 mm), or cerebellum (AP = −6.1 mm; L = 0.5 mm; DV = 2.2 mm; co-ordinates correspond to Franklin and Paxinos 1998) at a final volume of 1 μL. mES cells were grafted within 1 min with a 10-μL microsyringe (Hamilton, Bonaduz, Switzerland) that was left in place for 5 min post-infusion to allow mES cells to settle before needle removal. To minimize the number of animals, two to five brain regions were randomly grafted in each mouse. After transplantation, mice were returned to their home cages and allowed to recover under exposure to dimmed infrared light that maintained their body temperature until regaining consciousness and proper movement control. All animals survived after experimental manipulations. Body weight of the mice was controlled daily, and showed transient reduction within 48 h post operation that was followed by a gradual increase (data not shown). Experimental procedures adhered to the European Communities Council Directive (86/609/EEC) and were approved by the Swedish National Board for Laboratory Animals (N55/2002).

Immunocytochemistry

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Mouse ES (mES) cell culture and green fluorescent protein (GFP) expression
  5. In vitro differentiation assay of GFP-expressing mES cells
  6. In vitro co-culture assay
  7. mES cell preparation for transplantation
  8. Transplantation, immunosuppression and post-transplantation monitoring
  9. Immunocytochemistry
  10. Confocal laser-scanning microscopy
  11. Electrophysiology
  12. Results
  13. Establishment and characterization of GFP-positive mES cells
  14. Differentiation of mES cells to neural progenies varies among different brain regions
  15. Region-specific activities in the brain control the expansion of neural progenitors and neuronal differentiation
  16. ES cell-derived neural progenies differentiate into mature neurons
  17. ES-derived neurons gradually acquire electrophysiological properties similar to host cells
  18. Discussion
  19. Acknowledgements
  20. References

Mice were anaesthetized with isoflurane [5.0% (v/v%) in 70% N2O/30% O2; 0.5 L/min flow rate] and transcardially perfused with 100 mL of fixative composed of 4% PFA containing 0.05% glutaraldehyde in sodium phosphate buffer (PB; 0.1 m, pH 7.4), which was preceded by a short pre-rinse with ice-cold physiological saline (30 mL). Whole brains were then post-fixed in 4% PFA in PB overnight, and cryoprotected by immersion in 30% sucrose in PB for 48 h at 4°C. Series of 30-μm thick serial coronal sections were cut on a cryostat microtome. Our cutting scheme resulted in a cross-section distance of 270 μm and ensured random systematic sampling of each graft. Complete series of free-floating sections were employed for multiple immunofluorescence labelling according to standard protocols (Harkany et al. 2001; Härtig et al. 2001) with primary antibodies as follows: rabbit anti-GFP (1 : 500; Clontech Labs Inc., Palo Alto, CA, USA), rabbit anti-β-galactosidase (1 : 200; ICN Kappel/Organon, Costa Mesa, CA, USA); mouse anti-synaptophysin (1 : 50, Sigma), mouse anti-nestin (1 μg/mL; Chemicon, Temecula, CA, USA), guinea pig antidoublecortin (1 : 3000; Chemicon); rabbit anti-GFAP (1 : 400; Dako; Abraham et al. 1997); mouse anti-neuron-specific nuclear protein NeuN; (1 : 200; Chemicon; Harkany et al. 2002), goat anti-choline acetyltransferase (1 : 200; Chemicon; Harkany et al. 2001), rabbit anti-calbindin-D28k CALB; (1 : 1000; SWant, Bellenzona, Switzerland; Härtig et al. 2002), and rabbit anti-vesicular glutamate transporter 2 VGLUT2; (1 : 1000, Synaptic Systems, Göttingen, Germany; Fremeau et al. 2001; Härtig et al. 2003). Immunolabelling for GFP was developed according to the avidin-biotin method with 3,3′-diaminobenzidine tetrahydrochloride (Sigma) as chromogen, while fluorescent labelling was achieved by Cy3- and Cy5-tagged secondary antibodies (1 : 200; raised in donkey and affinity-purified with minimal cross-reactivity with mouse serum proteins; Jackson Immuno-Research). Omission of primary antibodies in control experiments resulted in the lack of any cellular labelling and switching of relevant fluorophore combinations led to identical staining patterns.

Confocal laser-scanning microscopy

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Mouse ES (mES) cell culture and green fluorescent protein (GFP) expression
  5. In vitro differentiation assay of GFP-expressing mES cells
  6. In vitro co-culture assay
  7. mES cell preparation for transplantation
  8. Transplantation, immunosuppression and post-transplantation monitoring
  9. Immunocytochemistry
  10. Confocal laser-scanning microscopy
  11. Electrophysiology
  12. Results
  13. Establishment and characterization of GFP-positive mES cells
  14. Differentiation of mES cells to neural progenies varies among different brain regions
  15. Region-specific activities in the brain control the expansion of neural progenitors and neuronal differentiation
  16. ES cell-derived neural progenies differentiate into mature neurons
  17. ES-derived neurons gradually acquire electrophysiological properties similar to host cells
  18. Discussion
  19. Acknowledgements
  20. References

Specimens were analyzed using a confocal laser-scanning microscope (Model 510, Zeiss, Jena, Germany), where images were acquired separately in green, red and infrared emission channels using 488, 543 and 633 nm excitation lasers, respectively; and appropriate excitation filters (narrow band-pass filters) for maximal channel separation. Qualitative analysis of our specimens was performed by systematic evaluation of parallel series of sections spanning the grafts in each brain region. mES cell-derived tumours were defined as clusters of transplanted cells found demarcated in the host parenchyma. In ROSA26 mice, the simultaneous presence of GFP and β-galactosidase signals was determined in each section containing mES cell grafts and all cells with GFP expression were included in the analysis irrespective of their lineages. Optical section thickness was kept to a minimum (< 1.0 μm at 63 × primary magnification), and cells were viewed both as single xy-images and merged composites along the z-axis. Cells were recognized as double or triple labelled when no spatial separation of the immunosignals was observed in any optical section of ± 1 μm and in each rotated orthogonal view.

Electrophysiology

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Mouse ES (mES) cell culture and green fluorescent protein (GFP) expression
  5. In vitro differentiation assay of GFP-expressing mES cells
  6. In vitro co-culture assay
  7. mES cell preparation for transplantation
  8. Transplantation, immunosuppression and post-transplantation monitoring
  9. Immunocytochemistry
  10. Confocal laser-scanning microscopy
  11. Electrophysiology
  12. Results
  13. Establishment and characterization of GFP-positive mES cells
  14. Differentiation of mES cells to neural progenies varies among different brain regions
  15. Region-specific activities in the brain control the expansion of neural progenitors and neuronal differentiation
  16. ES cell-derived neural progenies differentiate into mature neurons
  17. ES-derived neurons gradually acquire electrophysiological properties similar to host cells
  18. Discussion
  19. Acknowledgements
  20. References

Sagittal brain slices (300 μm) spanning the mES cell grafts in somatosensory cortex and hippocampus of ROSA26 mice (n = 2) were prepared 12 days post-transplantation. Transplanted cells were identified using combined infra-red-differential interference contrast and fluorescent microscopy with appropriate filter sets (Olympus, Melville, NY, USA). All experiments were performed in oxygenated extracellular solution at 32°C. The extracellular solution contained (in mM): 125 NaCl, 2.5 KCl, 25 glucose, 25 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, and 1 MgCl2, while the pipette solution contained (in mM): 100 K-gluconate, 20 KCl, 4 ATP-Mg, 10 Na-phosphocreatine, 0.3 GTP, and 10 HEPES (pH 7.3, 310 mOsm/L). Electrical signals in GFP-positive cells (n = 14) were recorded in whole-cell voltage-clamp configuration as previously described in detail (Holmgren and Zilberter 2001). During recording, cells were intracellularly filled with 2.0 mg/mL neurobiotin in order to enable subsequent visualization of the cellular morphology. Brain slices were subsequently fixed in 4% PFA in PB overnight, and neurobiotin was visualized by Cy3-conjugated streptavidin (2.5 μg/mL in PB for 6 h; Jackson ImmunoResearch), and the cells were inspected by confocal laser-scanning microscopy as described above. Co-localization of GFP (in green) and neurobiotin (in red) signals was defined as being simultaneously present in ± 0.8-μm thick optical slices.

Establishment and characterization of GFP-positive mES cells

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Mouse ES (mES) cell culture and green fluorescent protein (GFP) expression
  5. In vitro differentiation assay of GFP-expressing mES cells
  6. In vitro co-culture assay
  7. mES cell preparation for transplantation
  8. Transplantation, immunosuppression and post-transplantation monitoring
  9. Immunocytochemistry
  10. Confocal laser-scanning microscopy
  11. Electrophysiology
  12. Results
  13. Establishment and characterization of GFP-positive mES cells
  14. Differentiation of mES cells to neural progenies varies among different brain regions
  15. Region-specific activities in the brain control the expansion of neural progenitors and neuronal differentiation
  16. ES cell-derived neural progenies differentiate into mature neurons
  17. ES-derived neurons gradually acquire electrophysiological properties similar to host cells
  18. Discussion
  19. Acknowledgements
  20. References

Mouse GSI-1 ES cells constitutively expressing GFP were established by electroporation of the cells with an expression construct and subsequent selection of G418 resistant clones. Individual undifferentiated, ALP-positive clones (Fig. 1a) were allowed to differentiate and to form embryoid bodies. Neurospheres and neural progenitors were generated from embryoid bodies and placed in differentiation medium where they gave rise to all major types of neural progeny including differentiation to cells with an apparent neuronal morphology (data not shown). A stage specific embryonic antigen 1 and ALP-positive GFP-mES clone was selected that retained GFP expression in neurons, as indicated by Tuj1 immunoreactivity (Fig. 1b), as well as GFAP-immunoreactive (ir) astroglial cells (Fig. 1c) after in vitro differentiation. However, we observed that the intensity of GFP fluorescence was overall reduced in differentiated cells, as compared with the undifferentiated mES cell clone, and we occasionally identified Tuj1-positive cells that were GFP-negative (Fig. 1b, arrowhead). The GFP-expressing mouse GSI-1 mES cell line retained GFP expression also after grafting in the adult mouse brain (Figs 1d–g).

image

Figure 1. Differentiation of ALP-positive mES cells with ubiquitous GFP expression and their integration in the brain. (a)  Cells from colonies of undifferentiated, ALP-positive mES cells expressing GFP. (b, c) Neural progenitors from GFP expressing mES cells were in vitro differentiated and stained for Tuj1 (b)  or GFAP (c).  Note a Tuj1-positive GFP-expressing neuron (arrow). Arrowhead indicates a Tuj1-positive GFP-negative neuron. Arrow in (c) denotes a GFAP-positive GFP-expressing cells. (d, e) GFP immunoreactivity of transplanted mES cell-derived progenies in the adult mouse brain. mES cells gave rise to cells with neuron-like (d)  and glia-like (e , arrow) morphology. Arrowhead in (e) points to likely undifferentiated progenitors. (f, g and g′) Immunofluorescence detection of GFP in mES cell-derived cells in the hippocampus of ROSA26 mice with ubiquitous β-galactosidase (β-Gal) expression. (f)  Transplanted mES cells retained their cellular identity in adult brain as was evidenced by the lack of spontaneous fusion of GFP-labelled mES cells with β-galactosidase (β-Gal)-positive host cells in ROSA26 mice 14 days post-transplantation. GFP-positive mES cells (arrows) appeared single-labelled in the dentate gyrus of the hippocampus. (g, g′) High-power images demonstrate the integration of a GFP-positive mES cell-derived cell with neuronal morphology in the granular layer of the dentate gyrus. Scale bars: (a) 50 μm (b, c, e) 30 μm (d, g) 15 μm (f) 70 μm.

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We then performed transplantation of ± 400 or ± 4000 mES cells in the medial septum, striatum, somatosensory cortex, hippocampus and cerebellum of adult male mice. After transplantation, residual cells were stained for ALP to confirm the transplantation of undifferentiated mES cells (Fig. 1a). Whereas ±400 mES cells integrated in the adult mouse brain and showed a propensity to form teratomas only in the somatosensory cortex, transplants of ±4000 mES cells led to abundant tumour formation (Björklund et al. 2002) except of the medial septum where engraftment of mES cells appeared very limited (Table 1). Single cell density mES cell grafts in all brain regions led to the development of cells with neuron- and glia-like morphologies, as detected by both immunocytochemistry for GFP (Figs 1d and e) and direct immunofluorescence detection of GFP (Figs 1f and g). To eliminate the possibility that the presence of GFP-positive cells in recipient tissue was a consequence of spontaneous fusion of mES cells with host neurons and glia (Terada et al. 2002; Ying et al. 2002), we performed mES cell transplantation (±400 cells) in the medial septum, cerebral cortex and hippocampus of ROSA26 mice with ubiquitous β-galactosidase expression and screened the grafts for cells with GFP/β-galactosidase co-expression after a survival period of 14 days. None of the GFP-positive cells we examined in the different brain areas co-expressed β-galactosidase [medial septum: 44/0 cell, somatosensory cortex: 105/0 cell, hippocampus: 109/0 cell (GFP-positive/β-galactosidase-positive); Figs 1f and g] indicating that GFP-positive cells originated from mES cells.

Table 1.  Marked differences of frequency of tumour formation among brain regions after transplantation of ±400 or ±4000 mES cells in various regions of the adult mouse brain ( n  = 3–7 per group)
 400 mES cells (%)4000 mES cells (%)
5 days14 days5 days14 days
  1. Complete series of sections spanning each individual graft were histologically analyzed for tumours. Tumours were defined as dense clusters of transplanted cells found demarcated in the host parenchyma.

Medial septum0000
Striatum00100100
Cortex3343100100
Hippocampus014100100
Cerebellum00100100

Differentiation of mES cells to neural progenies varies among different brain regions

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Mouse ES (mES) cell culture and green fluorescent protein (GFP) expression
  5. In vitro differentiation assay of GFP-expressing mES cells
  6. In vitro co-culture assay
  7. mES cell preparation for transplantation
  8. Transplantation, immunosuppression and post-transplantation monitoring
  9. Immunocytochemistry
  10. Confocal laser-scanning microscopy
  11. Electrophysiology
  12. Results
  13. Establishment and characterization of GFP-positive mES cells
  14. Differentiation of mES cells to neural progenies varies among different brain regions
  15. Region-specific activities in the brain control the expansion of neural progenitors and neuronal differentiation
  16. ES cell-derived neural progenies differentiate into mature neurons
  17. ES-derived neurons gradually acquire electrophysiological properties similar to host cells
  18. Discussion
  19. Acknowledgements
  20. References

To explore the potential of different brain regions to provide permissive environments for mES cells to generate neural progenitors, we investigated whether mES cells (±400 cell/graft) can give rise to nestin-ir neural progenitors. Five days post-transplantation, nestin-ir/GFP-positive cells were observed in all brain regions with varying densities (Figs 2a, b and e); with highest frequency of nestin-ir cells being present in the hippocampus and cerebellum and lowest in the medial septum and striatum. Many GFP-positive cells in the medial septum and striatum appeared atrophic that was apparent both 5 and 14 days after transplantation (Figs 2b and 4b and c). In addition, grafts of GFP-positive cells were surrounded by nestin-ir activated astroglia of mES and host origin (Fig. 2f) with apparently higher abundance of activated astroglia in the hippocampus and cerebellum (Fig. 2g). Doublecortin (DBX), a marker for migrating neural precursors (Gage 2000), labelled GFP-positive cells in major commissural pathways 5 days after transplantation (Figs 2a, c and d). In contrast, a loss of nestin and DBX immunoreactivity in GFP-positive cells was observed after a 14-day survival period. These data suggest that mES cells can give rise to neural progenitors in several regions of the adult brain.

image

Figure 2. GFP-positive mES cells were transplanted into the medial septum, striatum, somatosensory cortex, hippocampus and cerebellum of adult mice and analyzed 5 days later. (a)  Grey bars indicate sites of transplantations; ( red circle) nestin-immunoreactive (ir) neural precursor; (blue circle): doublecortin (DBX)-ir migrating cell; (grey circle) GFP-positive cell; (—•—) GFP-positive cell with processes. (b)  Nestin-positive mES cells in medial septum. (c)  Nestin-negative globular GFP-positive cell in corpus callosum. Inset shows DBX-ir GFP-positive cell in corpus callosum. (d)  DBX-ir GFP-positive cells in the CA1 subfield of hippocampus. Arrowheads point to cells in the core of the transplant while arrows indicate distant cells. (e)  Nestin-ir neural precursor of GFP-positive mES cell origin (arrow) intermingled with host cells in the hippocampus. (f)  Grafts were surrounded by nestin-ir reactive astroglia of host origin, e.g. in cerebellum. (g)  Survival and differentiation of mES cells in adult mouse brain 5 days after transplantation. Qualitative observations were based on analysis of serial sections stained for multiple differentiation and phenotypic markers (see above). Symbols: (–) negative; (±) very limited; (+) rarely observed; (++) present in tissue; (+++) regularly observed; (++++) frequently observed. The entire cortical mantle together with the corpus callosum forms the area designated as cortex. Scale bars: (b, c inset) 10 μm (c) 15 μm (d, e) 20 μm (f) 60 μm.

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image

Figure 4. Neuronal differentiation of mES cells in adult brain 14 days after transplantation. (a)  Survival and distribution of mES cell-derived neurons 14 days post-transplantation. Grey bars denote the site of transplantation; (—•—) GFP-positive cell with processes; (burgundy) NeuN-ir GFP-positive cell; (green) GFP + cell receiving vesicular glutamate transporter 2 (VGLUT2)-ir innervation; (orange) calbindin D-28k (CALB)-ir GFP-positive cell; (blue) choline-acetyltransferase (ChAT)-ir GFP-positive cell. (b–g) GFP-positive cells surrounded by neurons (red) and astroglia (color-coded in blue) of host origin in medial septum (b, c), neocortex (d, e), hippocampus (f) and cerebellum (g).  Note the lack of integration into host parenchyma, atrophic appearance (arrowhead) and a predominant absence of NeuN, GFAP, CALB-ir in the medial septum and striatum (a–c). Note neurons of apparent mature morphologies in cerebral cortex (d, e) hippocampus (f) and cerebellum (g) that expressed CALB or NeuN (arrowheads indicate the absence, while arrows denote the presence of immunoreactivity). (f ′) Projection image of a GFP-positive cell in hippocampus receiving VGLUT2-ir and synaptophysin-ir innervation. (h)  Survival and differentiation of mES cells in adult mouse brain 14 days after transplantation. Qualitative observations were based on analysis of serial sections stained for multiple differentiation and mature phenotypic markers (see above). Symbols: (–) negative; (+) rarely observed; (++) present in tissue; (+++) regularly observed; (++++) frequently observed. The neocortex together with the corpus callosum forms the area designated as cortex. Scale bars : 25 μm (b) , 12 μm (c–g).

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Region-specific activities in the brain control the expansion of neural progenitors and neuronal differentiation

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Mouse ES (mES) cell culture and green fluorescent protein (GFP) expression
  5. In vitro differentiation assay of GFP-expressing mES cells
  6. In vitro co-culture assay
  7. mES cell preparation for transplantation
  8. Transplantation, immunosuppression and post-transplantation monitoring
  9. Immunocytochemistry
  10. Confocal laser-scanning microscopy
  11. Electrophysiology
  12. Results
  13. Establishment and characterization of GFP-positive mES cells
  14. Differentiation of mES cells to neural progenies varies among different brain regions
  15. Region-specific activities in the brain control the expansion of neural progenitors and neuronal differentiation
  16. ES cell-derived neural progenies differentiate into mature neurons
  17. ES-derived neurons gradually acquire electrophysiological properties similar to host cells
  18. Discussion
  19. Acknowledgements
  20. References

We established a cell culture assay to compare activities between different brain regions on proliferation and neuronal differentiation of ES-derived neural progenitor cells grown as neurospheres. In this assay, proliferation of neural progenitors under differentiation conditions (i.e. presence of laminin and absence of bFGF) was examined in the presence or absence of conditioned medium from adult hippocampus, cerebellum and striatum, presented from slices of these brain regions placed in cell culture inserts. All cultures were examined daily for pyknotic cells. Overt cell death was not observed in any of the conditions. During 5 days in culture, 306 ± 45 cells arose from timed neurospheres under control conditions. Conditioned medium from all brain regions significantly reduced proliferation (F = 17.655, p < 0.001) with the non-neurogenic brain region striatum carrying the strongest inhibiting condition (80 ± 13 cells) and the neurogenic region hippocampus being the most permissive environment (214 ± 15 cells; Fig. 3a). We next examined regional effects on neuronal differentiation of the neural progenitor cells by culturing the cells for 8 days under differentiation conditions with or without conditioned medium during the first 5 days (F = 5.410, p = 0.014). While conditioned medium from the cerebellum and striatum had no effect on the number of Tuj1-positive cells, as compared with control cultures, a nearly 4-fold increase in the number of Tuj1-positive cells was seen in cultures with conditioned medium from the hippocampus (Fig. 3b). Thus, distinct properties among brain regions allowing differentiation and maintenance of nestin-positive progenitors in vivo correlate with soluble activities released from brain slices to stimulate the expansion of ES-derived neural progenitors and promote neuronal differentiation.

image

Figure 3. The effects of distinct region-specific activities in the brain on proliferation and neuronal differentiation of neural progenitor cells in vitro . (a)  The impact of conditioned medium from adult brain regions on cell proliferation. The number of cells was counted in cultures of timed mES cell-derived neurospheres grown under conditions promoting differentiation (i.e. without bFGF and with poly d -lysine/laminin) without (control) or with conditioned medium from tissue slices of hippocampus (HC), cerebellum (CB) or striatum (Str) of adult mice. Note the significantly reduced cell numbers in cultures with striatum-conditioned medium and that the neurogenic hippocampal region shows the smallest decrease of cell numbers. (b)  The impact of conditioned medium from adult brain regions on neurogenesis measured by counting Tuj1-positive neurons. Note a nearly 4-fold increase in neurogenesis from neural progenitor cells in the presence of hippocampus conditioned medium. Statistical analysis was performed using univariate anova with Bonferroni's post-hoc test. Data were expressed as means ± SEM.

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ES cell-derived neural progenies differentiate into mature neurons

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Mouse ES (mES) cell culture and green fluorescent protein (GFP) expression
  5. In vitro differentiation assay of GFP-expressing mES cells
  6. In vitro co-culture assay
  7. mES cell preparation for transplantation
  8. Transplantation, immunosuppression and post-transplantation monitoring
  9. Immunocytochemistry
  10. Confocal laser-scanning microscopy
  11. Electrophysiology
  12. Results
  13. Establishment and characterization of GFP-positive mES cells
  14. Differentiation of mES cells to neural progenies varies among different brain regions
  15. Region-specific activities in the brain control the expansion of neural progenitors and neuronal differentiation
  16. ES cell-derived neural progenies differentiate into mature neurons
  17. ES-derived neurons gradually acquire electrophysiological properties similar to host cells
  18. Discussion
  19. Acknowledgements
  20. References

We next investigated whether mES cell-derived neural progenitors seen at 5 days differentiate into neurons in the adult mouse brain, and used NeuN, CALB and VGLUT2 as markers of terminally differentiated neurons and of their innervation by host nerve cells. While GFP-positive cells were immunonegative for any of the mature neuronal markers 5 days post-transplantation, mES cell-derived neurons with neuroanatomical hallmarks similar to neighbouring host cells were identified after a 14-day post-transplantation period (Fig. 4a). Limited survival of mES cell grafts was detected in the medial septum (Figs 4a–c) and striatum (Fig. 4a), and remaining cells appeared atrophic, accompanied by a lack of GFP-positive cell integration in host parenchyma (Figs 4b and c). In contrast, GFP-positive cells with neuronal morphology were present in cerebral cortex, hippocampus and cerebellum (Figs 4a and d–g), expressed the adult neuronal markers NeuN (Figs 4f and g), CALB (Fig. 4d) and received synaptophysin and VGLUT2-positive innervation of host origin (Fig. 4f′). Figure 4(h) summarizes semiquantitative measurements of neuronal precursors, migration and maturation into neurons at 14 days post-transplantation.

ES-derived neurons gradually acquire electrophysiological properties similar to host cells

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Mouse ES (mES) cell culture and green fluorescent protein (GFP) expression
  5. In vitro differentiation assay of GFP-expressing mES cells
  6. In vitro co-culture assay
  7. mES cell preparation for transplantation
  8. Transplantation, immunosuppression and post-transplantation monitoring
  9. Immunocytochemistry
  10. Confocal laser-scanning microscopy
  11. Electrophysiology
  12. Results
  13. Establishment and characterization of GFP-positive mES cells
  14. Differentiation of mES cells to neural progenies varies among different brain regions
  15. Region-specific activities in the brain control the expansion of neural progenitors and neuronal differentiation
  16. ES cell-derived neural progenies differentiate into mature neurons
  17. ES-derived neurons gradually acquire electrophysiological properties similar to host cells
  18. Discussion
  19. Acknowledgements
  20. References

In order to verify functional integration of mES cell-derived neurons in host neuronal circuitries, we performed whole-cell recordings in cerebral cortex and hippocampus with simultaneous intracellular labelling of GFP-positive cells to seek morphological correlates of electrical activity. Two subsets of transplanted cells emerged, where the majority of GFP-positive cells resembled primordial neurons in an early stage of differentiation (Strubing et al. 1995; Finley et al. 1996) with small perikarya of 8–10 μm in diameter and aspiny dendrites. In the cerebral cortex, GFP-positive neurons were predominantly found scattered in layers 2/3, while mES cells grafted in hippocampus migrated in e.g. principal cell layers of the CA1-CA3 subfields and attained morphological features of pyramidal cells (Fig. 5a). These immature GFP-positive neurons exhibited resting membrane potentials in a range of −30.6 to −45.0 mV, and did not generate spikes when injected with depolarizing currents (Fig. 5a and d). In two of 14 cells, however, mES cells transplanted in the hippocampus differentiated into functional neurons within 14 days post-transplantation and exhibited morphological hallmarks indistinguishable from neighbouring host cells, e.g. extensive dendritic arbors packed with dendritic spines (Figs 5b and c). Whole-cell recordings revealed electrophysiological properties of mES cell-derived neurons identical to host nerve cells with a similar resting potential (−65 and −68 mV) and depolarization-induced trains of action potential firing (Fig. 5e).

image

Figure 5. ES cell-derived neurons in the hippocampus become electrically active. Functional integration of mES cell-derived neurons was determined by whole-cell recordings combined with intracellular labelling. (a, b) Neurobiotin (NB)-filled GFP-positive cells in the CA1 subfield of the hippocampus. (c)  High magnification of the cell in (b)  displaying extensive dendritic branches packed with dendritic spines. (d, e) The neuron in (a)  had a resting potential of −45 mV and was electrically inactive (d) , while the neuron in (b) had a resting potential of −65 mV and generated trains of action potentials (e).  Scale bars : 35 μm (a, b), 12 μm (c).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Mouse ES (mES) cell culture and green fluorescent protein (GFP) expression
  5. In vitro differentiation assay of GFP-expressing mES cells
  6. In vitro co-culture assay
  7. mES cell preparation for transplantation
  8. Transplantation, immunosuppression and post-transplantation monitoring
  9. Immunocytochemistry
  10. Confocal laser-scanning microscopy
  11. Electrophysiology
  12. Results
  13. Establishment and characterization of GFP-positive mES cells
  14. Differentiation of mES cells to neural progenies varies among different brain regions
  15. Region-specific activities in the brain control the expansion of neural progenitors and neuronal differentiation
  16. ES cell-derived neural progenies differentiate into mature neurons
  17. ES-derived neurons gradually acquire electrophysiological properties similar to host cells
  18. Discussion
  19. Acknowledgements
  20. References

We conclude that naive, undifferentiated mES cells can execute a programme of lineage specification that leads to the generation of functionally integrated neurons in adult brain. Previous transplantation experiments (Deacon et al. 1998; Björklund et al. 2002) using moderate to high numbers of dissociated cells from mES cell-derived embryoid bodies revealed frequent tumour formation in CNS that was attributed to discordant host-to-ES cell communication. Our data evidence that naive mES cells, when transplanted as a single cell suspension, are capable of adequately responding to microenvironmental cues of host tissues and undergo terminal differentiation in brain parenchyma. In contrast, predominant ES-to-ES cell signalling triggers mitotic activity and tumour formation.

Teratomas are tumours composed of well-differentiated somatic tissues of the three germ layers which have limited capacity for growth. Teratomas can be generated by transplanting ES cells into extra-uterine sites (Stevens 1970; Skreb et al. 1971). Among ectodermal cell derivatives formed in teratomas are neural tissue. By transplanting very low cell numbers and using a transplantation strategy with optimal ES cell to host interaction, tumour growth was largely prevented. This allowed us to determine whether distinct brain regions differently promote neuronal differentiation of ES cells. The striatum and medial septum, two non-neurogenic areas in the brain, exhibited a very poor ability to direct ES differentiation into nestin-positive neural progenitor cells, and those that were detected appeared atrophic. In contrast, numerous nestin and DBX-positive neural progenitor cells were seen in the cerebral cortex, hippocampus and cerebellum. Thus, the latter brain regions contain cues that either instruct neuronal differentiation and/or promote their survival. Consistent with our in vivo results, secreted molecules from the striatum were ineffective in the maintenance and growth of neurospheres in vitro under differentiating conditions. These results suggest that the hippocampus, cerebral cortex and cerebellum provide unique niches with properties different from the striatum and medial septum.

Previous studies have reported that CNS stem cells form virtually only glia in the striatum and spinal cord (Gage et al. 1995; Fricker et al. 1999; Chow et al. 2000; Shihabuddin et al. 2000). Thus, when a reduced number of neurons are detected in some regions of the CNS, it could be the consequence of a predominant differentiation into glial cells. Due to difficulties of robustly and quantitatively identifying GFP-positive glia, we were not able to address the proportion of glial cells generated from engrafted mES cells. However, nestin-positive neural progenitor cells have the capacity to form both neurons and glia. In addition, it was recently suggested that some nestin-positive radial glia at mid-gestation might be stem cells (Alvarez-Buylla et al. 2001). Because the striatum and medial septum showed very few nestin-positive cells and even fewer DBX-positive cells established from transplanted mES cells, we conclude that our results represent differences between these niches that are not only caused by differences in acquiring neuronal versus glial fate.

Cytochemical profiling of transplanted ES- and CNS-derived neural progenitor cells is commonly used to determine whether these cells are progenitors, neurons, or glial cells, and might provide indices for integration into the host parenchyma. Functional integration of cells can also be implored by behavioural recovery associated with the transplanted cells in disease models. It is important to understand how far transplanted cells can differentiate and mature, whether they integrate into the host, and are functional and electrically active. By viral labelling of dividing cells in the hippocampus, newly formed neurons were demonstrated to have action potentials and functional synaptic inputs, similar to host granule cells (van Praag et al. 2002). Adult rat CNS stem cells differentiated in co-culture with astrocytes also give rise to electrically active neurons in vitro (Song et al. 2002), and CNS stem cells established from the neocortex and transplanted in fetal hippocampus can also differentiate into neurons that are electrically active and functionally connected (Auerbach et al. 2000). In a recent report, tyrosine hydroxylase (TH)-positive neurons derived from ES cells were shown to integrate and develop properties of functional dopaminergic neurons in the adult brain (Kim et al. 2002). In this former study, ES cell-derived in vitro differentiated TH-positive neurons were transplanted into the adult striatum. Electrophysiological recordings of cells at the host-graft interphase were performed one to several months after transplantation. ES cell-derived TH-positive neurons exhibited electrophysiological properties similar to those of mesencephalic dopaminergic neurons. Here, we explored by morphology, immunohistochemistry and electrophysiology the functional potential of naive mES cells transplanted into different regions of the adult brain. These mES cells transiently expressed neural progenitor markers, displayed polarized morphology and integrated into the host parenchyma. Many transplanted mES cells acquired markers also expressed by mature neurons. However, often these cells had a resting potential reminiscent of immature neurons and were electrically inactive. A few, however, received VGLUT2- and synaptophysin-positive innervation of host origin and were electrically active with a resting potential resembling that of host neurons. Taking into account that these results were obtained only 14 days post-transplantation, it is conceivable that a number of silent neurons could still become active given more time to mature. These results demonstrate for the first time that naive ES cells can differentiate and mature into electrically active neurons upon transplantation in the adult brain.

The formation of functional neurons from naive ES cells only in some brain regions demonstrates that instructive host-to-ES cell communication, in a manner similar to adult neurogenesis, is a prerequisite for neuronal differentiation, subsequent maturation, and functional integration of ES cell-derived neurons. It is likely that local environmental cues could be modified by environmental or pharmacological intervention. For instance, marked changes of neurogenesis are observed in some regions by antidepressants, adrenal steroid hormones, stress leading to behavioural despair, physical exercise and enriched environment (Kempermann et al. 1997; Malberg and Duman 2003; Ormerod et al. 2003; Santarelli et al. 2003). An alteration of the local environment could also be expected during pathological conditions of the brain, in which regenerative cell replacement is explored as a possible treatment strategy. Given the impact of the local environmental factors on grafted cells, identification of such factors could facilitate strategies to overcome non-permissible environments and allow a better access of the brain for cell-based therapy. Thus, the impinging host interaction with grafted cells suggest that (i) determination of area-specific composition of key microenvironmental cues in adult brain must become an integral part of successful transplantation strategies and (ii) the lack or dysbalance of environmental factors might also be considered to form a basis of neurodegenerative disorders by inhibiting adult neurogenesis in affected brain areas.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Mouse ES (mES) cell culture and green fluorescent protein (GFP) expression
  5. In vitro differentiation assay of GFP-expressing mES cells
  6. In vitro co-culture assay
  7. mES cell preparation for transplantation
  8. Transplantation, immunosuppression and post-transplantation monitoring
  9. Immunocytochemistry
  10. Confocal laser-scanning microscopy
  11. Electrophysiology
  12. Results
  13. Establishment and characterization of GFP-positive mES cells
  14. Differentiation of mES cells to neural progenies varies among different brain regions
  15. Region-specific activities in the brain control the expansion of neural progenitors and neuronal differentiation
  16. ES cell-derived neural progenies differentiate into mature neurons
  17. ES-derived neurons gradually acquire electrophysiological properties similar to host cells
  18. Discussion
  19. Acknowledgements
  20. References

The authors are grateful to J. Frisén and K. Meletis for ROSA26 mice; O. K. Penz and A. Ahlsén for skilled assistance during histochemical procedures; and L. Amaloo for secretarial assistance. This work was supported by the Swedish Medical Research Council, the Michael J. Fox Foundation, EU Biotechnology programme (QLG3-CT-2000–01343), the Swedish Foundation for Strategic Research (CEDB grant to PE), the Hungarian National Science Fund (OTKA, #F035254; to TH), and Loo and Hans Ostermans Foundation for Medical Research (to TH and MA). TH and MA. are recipients of postdoctoral fellowships of the Karolinska Institutet.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Mouse ES (mES) cell culture and green fluorescent protein (GFP) expression
  5. In vitro differentiation assay of GFP-expressing mES cells
  6. In vitro co-culture assay
  7. mES cell preparation for transplantation
  8. Transplantation, immunosuppression and post-transplantation monitoring
  9. Immunocytochemistry
  10. Confocal laser-scanning microscopy
  11. Electrophysiology
  12. Results
  13. Establishment and characterization of GFP-positive mES cells
  14. Differentiation of mES cells to neural progenies varies among different brain regions
  15. Region-specific activities in the brain control the expansion of neural progenitors and neuronal differentiation
  16. ES cell-derived neural progenies differentiate into mature neurons
  17. ES-derived neurons gradually acquire electrophysiological properties similar to host cells
  18. Discussion
  19. Acknowledgements
  20. References
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