Endogenously EGFP-Labeled Mouse Embryonic Stem Cells

Aging Dis. 2011 February; 2(1): 18–29.

Published online 2011 December 20.

PMCID: PMC3160738

Endogenously EGFP-Labeled Mouse Embryonic Stem Cells

Junli Zhang,⁴^# Rammohan V. Rao,²^# Patricia Spilman,² Julie Mangada,² Lin Xie,² Cathy Vitelli,² Olivia F. Gorostiza,² David T. Madden,³ Xianmin Zeng,² Kunlin Jin,² Matthew J. Hart,² Dale E. Bredesen,^2,⁴ and Veronica Galvan¹^*

¹Department of Physiology and The Sam and Ann Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, San Antonio, TX 78245-3207, USA

²The Buck Institute for Age Research, 8001 Redwood Blvd., Novato, California 94945, USA

³Touro University College of Pharmacy, Vallejo, CA 94592, USA

⁴University of California, San Francisco, San Francisco, California 94143, USA

^#These authors contributed equally to the present work

^*Correspondence should be addressed to: Dr. Veronica Galvan, Department of Physiology and The Sam and Ann Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA. Email: galvanv/at/uthscsa.edu

Received December 14, 2010; Revised December 16, 2010; Accepted December 16, 2010.

Abstract

Transplantation of embryonic stem cell (ESC)-derived precursors holds great promise for treating various disease conditions. Tracing of precursors derived from ESC after transplantation is important to determine their migration and fate. Chemical labeling, as well as transfection or viral-mediated transduction of tracer genes in ESC or in ESC-derived precursors, which are the methods that have been used in the generation of the vast majority of labeled ESCs, have serious drawbacks such as varying efficacy. To circumvent this problem we generated endogenously traceable mouse (m)ESC clones by direct derivation from blastocysts of transgenic mice expressing enhanced green fluorescent protein (EGFP) under control of the housekeeping ß-actin promoter. The only previous report of endogenously EGFP-labeled mESC derived directly from transgenic EGFP embryos is that of Ahn and colleagues (Ahn et al, 2008. Cytotherapy 10:759–769), who used embryos from a different transgenic line and used a significantly different protocol for derivation. Cells from a high-expressing EGFP-mESC clone, G11, retain high levels of EGFP expression after differentiation into derivatives of all three primary germ layers both in vitro and in vivo, and contribution to all tissues in chimeric progeny. To determine whether progenitor cells derived from G11 could be used in transplantation experiments, we differentiated them to early neuronal precursors and injected them into syngeneic mouse brains. Transplanted EGFP-expressing cells at different stages of differentiation along the neuronal lineage could be identified in brains by expression of EGFP twelve weeks after transplantation. Our results suggest that the EGFP-mESC(G11) line may constitute a useful tool in ESC-based cell and tissue replacement studies.

Keywords: Embryonic stem cell, green fluorescent protein, transplantation, mouse, drugs (NSAIDs), primary prevention

Embryonic stem cells (ESC) are pluripotent stem cells derived from the inner cell mass (ICM) of the blastocyst stage embryo [1]. ESC can give rise to cells derived from all three primary germ layers (ectoderm, endoderm and mesoderm) and can self-renew indefinitely [2]. Because of their plasticity and virtually unlimited capacity for self-renewal, ESC-based therapies have been proposed for the treatment of genetic disorders and degenerative conditions that could be addressed by cell or tissue replacement therapies [3,4]. These include juvenile diabetes [5], heart disease [6], and many injuries of the nervous system such as stroke [7], Parkinson’s disease [8–10], Alzheimer’s disease [11,12], amyotrophic lateral sclerosis [13], certain forms of blindness[14] and deafness [15] as well as spinal cord injuries [16], among others. In addition to potentially replacing lost or damaged cells, transplanted cells may also provide trophic support [12] or mobilize endogenous precursors from the injured or diseased tissue [17].

Tracing of transplanted precursors is important to determine their migration and fate. Fluorescent proteins are widely used markers of gene expression and protein localization. Green fluorescent protein (GFP) was first isolated from Aequorea victoria and was modified by a point mutation (S65T, [18]) to improve quantum yield, photostability and excitation at 488 nm. A 37°C folding-efficient point mutant (F64L) of GFP(S65T) [enhanced GFP (EGFP)] was then described [19] that facilitated the use of GFP in mammalian cells. Transfection or viral-mediated transduction of tracer genes such as those encoding fluorescent proteins in ESC or in precursors derived from them are the methods that have been used in the generation of the vast majority of labeled ESCs. These have important drawbacks, such as the random insertion of the tracer gene, which leads to large variations in levels of expression of the tracer in different cells and in different lineages upon differentiation, among others [20]. Transposon vectors are also used for gene transfer into ESC. Although the insertion pattern of transposons is non-random and transposon vectors can be selected that show the least preference to target genes, the insertion of these elements in regulatory non-coding regions is an important concern [21]. Transfection often results in mixed heterogeneous populations due to incomplete antibiotic selection. Moreover, transfection and viral transduction methods per se have unknown effects on ESC, which are exquisitely sensitive to stimuli or environmental perturbations. As an alternative to gene transfer methods, ESC or ESC-derived precursors can be labeled with synthetic compounds such as BrdU [22,23] or other cell-labeling dyes [24,25]. The effects of these dyes on ESC or tissue-specific precursors are also unknown. In addition, dyes tend to have short half-lives and can be progressively diluted as ESC or progenitors divide.

To avoid the pitfalls associated with gene transfer and chemical or metabolic labeling methods which have undefined effects on ESC biology, we generated unique, endogenously traceable mESC clones by direct derivation from blastocysts of transgenic mice expressing EGFP under control of the housekeeping ß-actin promoter [26]. We show that cells from EGFP-mESC clone G11 contribute to tissues in chimeric progeny and retain high levels of EGFP expression after differentiation into derivatives of all three primary germ layers both in vitro and in vivo.

To determine whether cells derived from EGFP-mESC(G11) could be used in transplantation experiments, we differentiated EGFP-mESC(G11) cells to early neuronal precursors and injected them into syngeneic mouse brains. Transplanted EGFP-expressing cells at different stages of differentiation along the neuronal lineage could be readily identified in brain 12 weeks after transplantation. Our results suggest that EGFP-mESC(G11) may constitute a useful tool in ESC-based cell and tissue replacement studies.

MATERIALS & METHODS

Mouse strains

C57BL/6J females (Jackson Laboratory, Bar Harbor, MA) were superovulated by intraperitoneal (IP) injection of 5 IU PMS (Sigma Aldrich, St. Louis, MO). 46 hours later, mice were IP injected with 5 IU HCG (Sigma). Superovulated C57BL/6J females were mated with C57BL/6-Tg(ACTB-EGFP)1Osb/J male mice (Jackson Laboratory). Blastocysts were collected at 3.5 days post-coitus (p.c.).

Derivation of GFP mES cell lines

Three-point-five day post-coitus blastocysts were transferred onto a mitomycinC-treated mouse embryonic fibroblasts (MEF) feeder layer and maintained in ES-serum replacement (SR) media [ES-SR; knock-out DMEM (Invitrogen/Gibco, Carlsbad, CA)], supplemented with 20% Knock-out serum replacement (SR) (Invitrogen/Gibco), 100U/ml Penicillin /100ug/ml Streptomycin (Invitrogen/Gibco), 2 mM L-Glutamine (Invitrogen/Gibco), 0.1 mM MEM non-essential amino acids (Invitrogen/Gibco), 100 µM β-mercaptoethanol. The cells were incubated at 37°C, 5% CO₂. Culture media was replaced every other day. Eleven inner cell masses showing strong green fluorescence under UV excitation were dissociated with 2.5% trypsin/EDTA (Invitrogen/ Gibco) and seeded onto a MEF feeder layer with FCS–ES media in which SR was replaced by fetal calf serum (FCS, Chemicon, Millipore). FCS–ES media was replaced with ESSR media on the next day. Undifferentiated ESC colonies were dissociated with 0.25% trypsin/EDTA and reseeded into new plates at day 11; counting of passage number began at this time. Five EGFP-mESC lines were established and designated G2, G5, G6, G7 and G11.

Formation of embryoid bodies (EB)

EGFP-mESC(G11) cells were plated onto Petri dishes (Falcon) at a density of 5 x 10⁴ cells cm⁻² in knock-out DMEM/F12 media (Invitrogen/Gibco, Carlsbad, CA), supplemented with 17.6% Knock-out serum replacement (SR) (Invitrogen/Gibco), 100U/ml Penicillin /100ug/ml Streptomycin (Invitrogen/Gibco), 2 mM L-Glutamine (Invitrogen/Gibco), 0.1 mM MEM non-essential amino acids (Invitrogen/Gibco), 100 µM β-mercaptoethanol and 1,000 units/ml leukemia inhibitory factor (Chemicon, Millipore, Billerica, MA). The cells were incubated at 37°C, 5% CO₂. Culture media was replaced every other day.

Generation of chimeras

Chimeras were generated by microinjection of disaggregated EGFP-mESC(G11) cells into C57BL/6J or FVB blastocysts following a standard method [29]. EGFP expression in chimeras ranged between 5–95%. Chimeras with a high percentage of chimerism died within 3 weeks of birth.

Generation of neuronal progenitors

ESC were plated onto 6-well tissue culture plates (NUNC) without feeders at a density of 1x10⁵ cells cm⁻² in knock-out DMEM/F12 media (Invitrogen, Carlsbad, CA) supplemented with 17.6% SR (Invitrogen), 100U/ml Penicillin, 100μg/ml Streptomycin (Invitrogen), 2 mM L-Glutamine (Invitrogen), 0.1 mM MEM non-essential amino acids (Invitrogen), and 100 µM β-mercaptoethanol to allow for the formation of ESC aggregates. Cultures were maintained at 37°C/5% CO₂ and media was replaced the next day. To change media, aggregates were first aspirated out of the plate and transferred to a 15 ml Falcon tube and allowed to settle for 5 minutes. Aggregates were briefly centrifuged for 2 min at 200xg, collected, resuspended in fresh media and transferred to a fresh 6-well plate. Media was changed every day. After 4 days EB were plated on a tissue culture plate pre-coated with 20 μg/ml fibronectin. Twenty-four hours later the media was replaced with knock-out DMEM-F12 media supplemented with HEPES (Invitrogen), N2 (Invitrogen), 20 ng/ml FGF-2 (Millipore), 2 mM L-Glutamine (Invitrogen), 0.01 mM MEM non-essential amino acids (Invitrogen), 1 ug/ml Laminin, and 15 ug/ml fibronectin. Media was changed daily for the first two days and every other day thereafter.

PCR

Eleven days after plating ES cells on 0.1% gelatin coated plates, DNA was extracted from each ES cell line. The sex of cultures was determined by PCR to amplify a Y chromosome-specific sequence within the Zfy locus using the following primers: 5′CCTATTGC ATGGACAGCAGCTTATG3′ and 5′GCATAGACA TGTCTTAACATCTGTCC3′.

Karyotyping ESC

To determine the chromosome complement of EGFP-mESC(G11) cells karyotype was performed at Cell Line Genetics (Cell Line Genetics, Madison, WI).

Alkaline phosphatase (AP) staining

ESCs were plated on a MEF feeder layer, and media was replaced every other day. At day 4, the media was removed and ESCs were fixed with 4% paraformaldehyde (PFA) for 1–2 minutes. ESC were washed 3 times with PBS and incubated with FAST BCIP/NBT buffered substrate solution (Sigma) in the dark at room temperature for 25 minutes.

Immunocytochemistry

Immunocytochemistry and staining procedures of EB were performed as described previously [30]. Eight-day EB were embedded in OCT blocks and sectioned on a cryostat. Sections were then stained with antibodies specific for AFP, TUBB3, MAP2, and Brachyury as described [30]. Briefly, cells were fixed with 2% paraformaldehyde for half an hour, blocked in blocking buffer (5% donkey serum, 1% BSA, 0.1% Triton X-100) for 1 hour followed by incubation with the primary antibody at 4°C overnight in 5% donkey serum, 1% BSA, 0.1% Triton X-100. Secondary antibodies coupled to Alexa Fluor 555 (Molecular Probes, Invitrogen) were used for single labeling. Slides were mounted with ProLong Gold with DAPI for nuclei identification. Images were captured on a Nikon fluorescence microscope.

Teratoma formation

EGFP-mESC(G11) were plated on gelatin-coated plates and allowed to grow in ES-SR media for 48 hours. Cells were then trypsinized and resuspended in growth media at 1.4x10⁵ cells/μl. Cells were injected with a Hamilton syringe in 1μl 2.6 mm anterior to bregma, 1.8 mm lateral to midline, and 2.0 mm beneath the dura into the hippocampal area. Four weeks later, mice were perfused with PBS and brains were dissected and flash frozen. Twenty-μm sections were mounted in DAPI-mounting media (Vector Laboratories) and imaged under UV excitation.

Transplantation of NSC

NSC derived from EGFP-mESC(G11) at passage 4 were dissociated with trypsin and resuspended at a concentration of 2.5 x 10⁴ cells per μl in 3 μl or at 5 x 10⁴ cells per μl in 3 μl of cell preparation medium. Cells were injected with a Hamilton syringe into the hippocampal area in two stages: (a) 3μl of cell suspension (−2.6 mm anterior to bregma, 1.8 mm lateral to midline, and 2.6 mm beneath the dura); and followed by (b) 2μl of cell suspension (−2.6 mm anterior to bregma, 1.8 mm lateral to midline, and 2.0 mm beneath the dura). Experimental protocols were in accordance with the National Institutes of Health Guidelines for Use of Live Animals and were approved by the Animal Care and Use Committee at the Buck Institute. All possible efforts were made to minimize the number of animals used and their suffering.

Immunohistochemistry

Twelve weeks after injection, brains injected with NSC were perfused with 4% PFA, washed in phosphate-buffered saline (PBS) and cryoprotected first in 10%, followed by 20% and later in 30% sucrose/1X PBS in subsequent overnight incubations, snap frozen on dry ice and cryosectioned to 20μm sections. Sections were then mounted on charged glass slides and stored at −80°C. As EGFP fluorescence can be lost with fixation, and autofluorescence (particularly from lipofuscin) competes with the EGFP signal, antibody-based labeling was performed. Sections were hydrated in PBS and underwent antigen retrieval using the microwave method at 95°C for 10 minutes in 10mM sodium citrate at pH6. Tissue was allowed to cool to room temperature and was permeabilized with PBS containing 0.05% Triton X100 for 30 minutes at room temperature. Non-specific binding was blocked with 5% normal horse serum (NHS) in PBST (PBS with 0.1% Tween 20) for 1 hour at room temperature. For colorimetric IHC, a rabbit polyclonal anti-GFP antibody was used at 1:200 in PBST with NHS overnight at 4°C. After washing with PBST, sections were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit antibody (Vector, 1:500 in PBST) with NHS for 2 hours at room temperature, washed again and developed for 10 minutes with the Vector DAB kit. Slides were mounted with glycerol and images were taken on a Nikon FM microscope using a 40X objective and Nikon Act 1 software.

Sections of brains receiving NSC transplants were observed under a fluorescent microscope and images were taken following UV excitation. For double labeling experiments, we followed the protocol described above with the following modifications: after permeabilization, endogenous mouse IgG was blocked with 5% mouse-on-mouse reagent in PBST (“M.O.M.”, Vector Laboratories, Burlingame, CA) for 2 hours at room temperature. Primary antibodies were “Living Colors” anti-GFP mouse monoclonal (Clontech, Mountain View, CA) and rabbit polyclonal anti-nestin (#5968, Abcam, Cambridge, MA). The two primary antibodies were incubated overnight at 4°C, then the sections were washed and incubated with biotinylated anti-rabbit antibody, (1:500 in PBST, Vector Laboratories) in NHS for 1 hour at room temperature, then washed again and incubated with a mix of strepavidin conjugated AlexaFluor488 at 1:500 and anti-mouse AlexaFluor555 at 1:500 in PBST with NHS for 1 hour at room temperature. The final washes included two with PBST and one with PBS alone. The slides were mounted using Vectashield with DAPI (Vector Laboratories), viewed on a Nikon FM microscope using the FITC, TRITC and DAPI filters and imaged using Nikon Act 1 software. Images were taken at 20X. Confocal images were taken using a Zeiss 510 LSM microscope at 100X.

RESULTS

Transplantation of embryonic stem cell (ESC)-derived precursors holds great promise for treating many disease conditions. Tracing of transplanted precursors derived from ESC is important to determine their migration and fate. Transfection or transduction of tracer genes such as those encoding fluorescent proteins in ESC or in precursors derived from them have important drawbacks, such as the random insertion of the tracer gene, among others[20]. Synthetic compounds such as BrdU or other cell-labeling dyes[22–25] have unknown effects on ESC or tissue-specific precursors, tend to have short half-lives, and can be progressively diluted as ESC or progenitors divide. To circumvent the pitfalls associated with gene transfer or cell labeling procedures in ESC or in ESC-derived precursors, we used the protocol described by Bryja et al [27,28] to derive ESC lines endogenously expressing EGFP from a cDNA transgene driven by the ß-actin promoter [26]. Mouse (m)ESC lines were derived from blastocysts obtained from crosses of EGFP-transgenic mice (Tg(ACTB-EGFP)1Osb/0m [26]) in the C57BL/6J background. All tissues of EGFP transgenic mice, including the germline and with the exception of hair and erythrocytes, show strong green fluorescence under UV excitation [26]. Embryonic day 3.5 blastocysts from crosses of EGFP transgenic mice with C57BL/6J females were obtained as described in Methods. Blastocysts were allowed to hatch and expand for six days in ES medium, which selectively favors the growth of ESCs at the expense of other cell types. Of a total of 11 EGFP-expressing inner cell masses dissociated from blastocysts, 5 ESC lines were established and designated as G2, G5, G6, G7 and G11. All ESC lines strongly expressed EGFP and markers of pluripotency, and showed the expected morphology and growth characteristics of ESC colonies (data not shown).

The only previous report of endogenously EGFP-labeled mESC derived directly from transgenic EGFP embryos is that of Ahn et al.[35], who used embryos from a different transgenic line (TgN(act-EGFP)OsbC15-001-FJ001) that were homozygous or heterozygous for the EGFP transgene. EGFP-mESC(G11) cells were derived from a different transgenic line (C57BL/6-Tg(CAG-EGFP)1Osb/J), are heterozygous for the EGFP transgene, and were derived using a significantly different protocol that involves only defined media.

We selected a clone (G11) that showed excellent morphology and high levels of EGFP expression for further characterization. Colonies of the EGFP-expressing mouse ESC clone G11 [EGFP-mESC(G11)] line showed typical ESC morphology (Figure 1 a–c), rapid growth, and remained undifferentiated in the presence of leukemia inhibitory factor (LIF) when grown on mitomycin C-treated mouse embryonic fibroblasts (MEF) for at least 14 passages. At passage 9 and thereafter, EGFP-mESC(G11) cells continued to express high levels of EGFP (Fig. 1b–c). Alkaline phosphatase (AP) activity is a specific marker of the undifferentiated state [31]. As shown in Fig. 1d, EGFP-mESC(G11) colonies maintained high AP activity after at least 14 passages in culture.

Figure 1.

Generation of the EGFP-mESC(G11) clone. a, EGFP expression in an EGFP-mESC ICM. b, EGFP expression in EGFP-mESC(G11) colonies at passage 2 after 20 days in culture. c, EGFP expression in EGFP-mESC(G11) colonies after 9 passages in culture. d, Alkaline (more ...)

The karyotype of EGFP-mESC(G11) cells was determined at passage 3 by G-banding and chromosome analysis. EGFP-mESC(G11) cells showed normal diploid karyotype and male chromosome complement (Fig. 1e). This was confirmed by PCR amplification of the Zfy gene sequence [32](Fig. 1f).

To determine whether EGFP-mESC(G11) could differentiate into cell types from all germ layers, we grew them in suspension culture for 8 days as described in Methods to trigger cell aggregation. Differentiation is initiated in pluripotent cells upon aggregation and results in the formation of embryoid bodies (EB). This process recapitulates, to a limited extent, the events of embryogenesis, giving rise to cells of the three germ layers. EGFP-mESC(G11) readily formed EB that expressed EGFP (Figure 2a). After 8 days of culture, EB derived from EGFP-mESC(G11) cells were comprised of a large variety of differentiated cell types arising from all three primary germ layers, as shown by expression of microtubule-associated protein 2 (MAP2) or tubulin beta-3 chain (TUBB3, ß-3-tubulin, ectoderm), Brachyury (Bry, mesoderm), and alpha fetoprotein (AFP, endoderm)[30] (Figure 2a). We allowed 4 day-old EB to attach to fibronectin-coated plates and grow on the same media for an additional 4 days. Cells in EB spread on the plates and became elongated upon attachment. EB-derived cells retained high expression of MAP2 and Bry and became strongly immunoreactive for nestin, a type VI intermediate filament protein that is expressed in neuronal precursors both during development and in the adult brain (Figure 2b). Thus, EGFP-mESC(G11) could readily form EB and differentiate into cell types from all germ layers in vitro.

Figure 2.

EGFP-mESC(G11) are pluripotent. a, EB generated from EGFP-mESC(G11) strongly expressed EGFP and gave rise to cells derived from the three germ layers (MAP2 and TUBB3, ectoderm; Bry, mesoderm; AFP, endoderm). b, Cells derived from EB continue to be immunoreactive (more ...)

The ability of stem cells to form noncancerous tumors called teratomas is one of their defining traits. To determine whether EGFP-mESC(G11) could give rise to cell types from all germ layers in vivo we injected EGFP-mESC(G11) cells in brains of syngeneic adult mice in order to produce differentiating teratomas after a single passage. The brain is an immunologically privileged site, and we performed syngeneic transplants, thus did not observe immune rejection, as expected. The animals were sacrificed 4 weeks later. EGFP-mESC(G11) cells formed fluid-filled cysts containing solid teratomas that contained morphologically differentiated cells and tissues derived from all three germ layers, such as glandular epithelium (endoderm), cartilage, smooth muscle and striated muscle (mesoderm), as well as neural epithelium and stratified squamous epithelium (ectoderm) (Figure 2c). Thus, EGFP-mESC(G11) could differentiate into cell types from all germ layers in vivo.

To determine whether EGFP-mESC(G11) cells could contribute to chimeric progeny of syngeneic and non-syngeneic mice, we injected EGFP-mESC(G11) cells into C57BL/6J or FVB blastocysts and screened progeny for chimeric expression of the EGFP transgene. As shown in Fig. 2d, EGFP-expressing chimeric mice of either sex were obtained at the expected frequency (12 from each C57BL/6J and FVB injections out of a total of 16 and 13 pups respectively). Skin EGFP expression of chimeras, detected under UV illumination, ranged from 5–95%. Thus, EGFP-mESC(G11) cells can contribute to tissues in chimeric progeny and retain high levels of EGFP expression after differentiation in vivo. Chimeras showing a high percentage of EGFP expression were smaller than other chimeras and died within 3 weeks after birth. Although progeny were obtained from moderate (~20–40%) chimeras, the transgene did not segregate at the expected frequency (20–40%) regardless of genetic background (C57BL/6J or FVB). The lack of viable EGFP-expressing progeny from crosses of chimeric C57BL/6J or FVB mice independent of degree of chimerism suggests that cells from the EGFP-mESC(G11) line were excluded from the germline, or alternatively, that high levels of EGFP overexpression may interfere with normal gametogenesis. This is consistent with the observation that homozygous transgenic EGFP-expressing mice are not viable, and that the EGFP transgene does not segregate at the expected frequency (1:1) in heterozygous crosses. Thus, EGFP-mESC(G11) may not be suitable for gene targeting procedures.

To determine whether EGFP-mESC(G11) could be differentiated into neuronal precursors suitable for transplantation experiments, we cultured EGFP-mESC(G11) in conditions that favor their differentiation into early neuronal precursors using a modified version of the protocol of Lee et al [9] as described in Methods. EGFP-mESC(G11) cells differentiated into homogeneous cultures of EGFP-expressing cells (Fig. 3a) that extended neuronal-like processes and showed morphological characteristics of early neuronal progenitors (NP) such as pyramidal cell body shape and limited cytoplasmic volume in the soma (Fig. 3b). Immunostaining of differentiated EGFP-mESC(G11) cultures showed that >90% of cells expressed markers of neural stem cells or early neuronal precursors such as SRY(sex-determining region Y)-box 2 (Sox2), and Nestin (Fig. 3c).

Figure 3.

Generation of neuronal progenitors from EGFP-mESC(G11). a, Bright field (left) and epifluorescent (right) images of cultures of neuronal progenitors derived from EGFP-mESC(G11) 9 days after EB attachment. b, Higher magnification (400X) images of NP cultures. (more ...)

To determine whether NP derived from EGFP-mESC(G11) (EGFP-NP) could survive and differentiate in vivo, we deposited 1.5 x 10⁵ and 1 x 10⁵ dissociated EGFP-NP cells in the corpus callosum and into the polymorph layer of the dentate gyrus of the hippocampus, respectively, of 12 week-old syngeneic C57BL/6J mice using a two-step stereotaxic injection protocol. Twelve weeks after transplantation, brains were dissected and processed for immunohistochemical determinations as described in Methods. Since autofluorescent lipofuscin deposits can develop in response to injury such as that associated with intracranial injection, we perfused animals with 4% paraformaldehyde to quench lipofuscin autofluorescence and used specific anti-GFP antibodies to detect EGFP-expressing cells. Twelve weeks after grafting, EGFP-expressing cells were abundant along the needle track (Fig. 4 a–b). Only one strongly EGFP-expressing cell was found in the anterior pretectal nucleus, having migrated into the parenchyma (Fig. 4a). Clusters of cells expressing both nestin and EGFP (Figure 4b) were found immediately adjacent to the injection track. Some cells in these clusters expressed EGFP but not nestin, suggesting that these cells may have further differentiated along the neuronal lineage and thus down-regulated the expression of nestin, a marker of early neuronal progenitors (Fig. 4b). The specificity of the anti-nestin antibodies was confirmed by staining of endogenous neuronal progenitors in the subventricular zone (SVZ), a prominent spontaneously neurogenic area in the adult rodent brain (Fig. 4c).

Figure 4.

Transplantation of NP derived from EGFP-mESC(G11). a, Location of the injection track (red arrow) and colorimetric expression of EGFP (40X). b, Nestin- and EGFP-expressing cells lining (upper panel) and projecting from (lower panel) the track space. (more ...)

Doublecortin (DCX) is a microtubule-associated protein expressed almost exclusively in immature neurons. Down-regulation of DCX begins at the same time that immature neurons turn into mature neurons and begin to express neuron-specific markers including ß-III-tubulin (ß3T) and microtubule-associated protein 2 (MAP2). To determine whether transplanted EGFP-NP continued differentiation along the neuronal lineage, we stained brain sections of mice injected with EGFP-NP with antibodies specific for DCX and ß3T. Cells expressing both EGFP and DCX were frequent along the injection tract (Fig. 4e–f). Occasionally, EGFP-expressing cells expressed ß3T (Fig. 4e). The cytoplasm of EGFP/DCX-expressing cells was consistently smaller than that of EGFP/ß3T-expressing cells (Fig. 4e–f). These observations are consistent with prior studies showing that DCX-expressing neuronal progenitors in neurospheres increase their cytoplasmic volume concomitantly with an increase in expression of ß3T, and that this event precedes neurite extension [33]. Interestingly, DCX-expressing NP cells that did not express EGFP (Fig. 4f) were also present along the needle track and in the corpus callosum (not shown) suggesting that endogenous NP may migrate to the site of injection as described for other brain injuries [34], possibly from SVZ. Albeit at very low frequency, NP derived from EGFP-mESC gave rise to mature neuron-like cells, showing ß3T-positive neurites that migrated a small distance (approximately one cell layer) away from the injection track (Fig. 5g).

Survival of transplanted precursors in adult brain was relatively low, and the proportion of transplanted cells that further differentiated into mature neuron-like cells was even lower. Moreover, we observed that only a few precursors had migrated away from the injection site 12 weeks after transplantation. These results are not unexpected since (a) it has been shown that most cells transplanted in the parenchyma remain associated in a ‘core’ structure [23], (b) we used relatively low numbers of progenitors for transplantation, and (c) we did not administer growth factors concomitant with transplantation[23]. In spite of these limitations, our results show that EGFP-mESC(G11) can be differentiated in vitro along the neuronal lineage, and that EGFP-NP can continue differentiation in vivo and retain high EGFP expression after delivery into tissues of adult syngeneic mice. Thus, progenitors derived from EGFP-mESC(G11) can be used in transplantation experiments.

DISCUSSION

Here we report the derivation of an endogenously EGFP-labeled pluripotent mouse ESC line, EGFP-mESC(G11). Our results show that EGFP-mESC(G11) cells are totipotent and maintain high levels of EGFP expression after differentiation into cells derived from all three germ layers in vitro and in vivo. EGFP-mESC(G11) cells contributed to chimeric progeny when injected into syngeneic and non-syngeneic blastocysts. Animals with a high degree of chimerism, however, showed delayed development and died within 3 weeks of birth. Although heterozygous EGFP transgenic mice develop normally, they do not segregate at the expected mendelian frequency for the transgenic allele (1:1), and homozygous animals die within a few days of birth. It is thus possible that very high levels of expression of EGFP are detrimental when present in every cell of the organism except hair and erythrocytes [26]. The high expression of EGFP at the individual cell level in stem cell colonies or in cell cultures of differentiated precursors, however, did not result in any detectable decrease in viability (Figures 1 and and2)2) nor in the down-regulation of expression of the EGFP transgene, suggesting that organismal effects are an emergent property of high levels of EGFP expression when present in a large proportion of tissues. Thus, no deleterious effects of high EGFP expression should be expected in transplantation studies using cells derived from EGFP-mESC(G11).

We further show that EGFP-mESC(G11) can be used in transplantation experiments, circumventing the pitfalls associated with gene transfer or cell labeling procedures in ESC. The most closely related previous study is that of Ahn et al.[35], who used embryos arising from crosses of animals carrying the same EGFP transgene but belonging to a different transgenic line (TgN(act-EGFP)OsbC15-001-FJ001), to study the incorporation of ESC in allotransplantation experiments in cochleae of experimentally deafened mice. Their cells were derived from double heterozygous crosses. Since no indication was provided regarding the dosage of the EGFP transgene, whether ESC derived by Ahn and colleagues were homozygous or heterozygous for the EGFP transgene is not known. Ahn and colleagues found that the EGFP-expressing mESC expressed markers of the undifferentiated state, formed morphologically defined EB, and were incorporated in their undifferentiated state in several locations throughout the cochleae of deafened mice. Our study differs in that we (a) generated EGFP-mESC(G11) from a different transgenic line (C57BL/6-Tg(CAG-EGFP)1Osb/J); (b) derived the mESC from heterozygous crosses of EGFP transgenic animals with C57BL/6J breeders, thus EGFP-mESC(G11) cells are heterozygous for the EGFP transgene; (c) used a different protocol for derivation of ESC that did not include incubation in FBS, avoiding batch-dependent effects; used primary MEF instead of a transformed cell line (STO cells), and did not use retinoic acid; (d) determined karyotype; (e) determined totipotency of ESC both in vitro and in vivo by the generation of EB and chimeric mice; and (f) determined the ability of EGFP-mESC(G11) to be differentiated in vitro along a given lineage, in our case neuronal; (g) determined the feasibility of tracing differentiated cells derived from EGFP-mESC(G11) cells in transplantation experiments.

We sought to generate a mESC line that would yield progenitors readily identifiable in transplantation or co-culture experiments, avoiding the need for prior labeling manipulations, because the elimination of potential confounds in studies of differentiation and transplantation of ESC-derived progenitors is an important goal. We showed that genetically labeled EGFP-mESC(G11) are totipotent, can give rise to cells derived from all three germ layers in vitro and in vivo, and can be differentiated along a defined lineage (in the present studies to neuronal progenitors) in vitro and give rise to mature neuron-like cells in vivo. Our data thus provide proof-of-principle for the use of EGFP-mESC(G11) cells in differentiation and transplantation studies. EGFP-mESC(G11) cells may therefore constitute a useful experimental tool for the study of ESC biology and for the investigation of the potential for cell replacement and tissue repair approaches in a variety of disease conditions.

Acknowledgments

This work was supported by the NIH Nathan Shock Center Core B and C: P30 AG025708 and by California Institute of Regenerative Medicine, CIRM-TG2-01155 to DEB.

Footnotes

AUTHOR DISCLOSURE STATEMENT

No competing financial interests exist.

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