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
] 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
] 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 (), 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 (). Alkaline phosphatase (AP) activity is a specific marker of the undifferentiated state [31
]. As shown in , 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 (). This was confirmed by PCR amplification of the Zfy
gene sequence [32
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 (). 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
] (). 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 (). 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) (). 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 , 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 () 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 (). 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 ().
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 ( a–b). Only one strongly EGFP-expressing cell was found in the anterior pretectal nucleus, having migrated into the parenchyma (). Clusters of cells expressing both nestin and EGFP () 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 (). 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 ().
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 (). Occasionally, EGFP-expressing cells expressed ß3T (). The cytoplasm of EGFP/DCX-expressing cells was consistently smaller than that of EGFP/ß3T-expressing cells (). 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 () 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.