Differentiation Prevents Assessment of Neural Stem Cell Pluripotency after Blastocyst Injection
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《干细胞学杂志》
a Departments of Neurology,
b Biochemistry and Molecular Pharmacology,
c Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA;
d Department of Neurology and Clinical Neurosciences, Stanford University, Palo Alto, California, USA
Key Words. Neural stem cells ? Blastocyst injections ? Differentiation ? Pluripotency
Correspondence: Lawrence D. Recht, M.D., Department of Neurology, Stanford University Medical Center, 1201 Welch Road, Room P-208, Stanford, California 94305, USA. Telephone: 650-498-6320; Fax: 650-498-6262; e-mail: LRecht@stanford.edu
ABSTRACT
In the brain, the subventricular zone (SVZ) is a source of proliferating cells that replenish olfactory interneurons and glial cells . Cells from the SVZ have several characteristics that distinguish them as neural stem (NS) cells, including their capacity to self-renew indefinitely in defined media supplemented with either epidermal growth factor (EGF) or basic fibroblast growth factor (FGF) and the capacity to differentiate into neurons, astrocytes, and oligodendrocytes in vitro upon cytokine exposure .
Recent studies have suggested that under certain conditions, NS cells are not only multipotent, i.e., able to produce more than one neural cell type, but also able to differentiate into other cell types, such as blood and muscle . In fact, when NS cells are placed in embryos at the blastocyst stage (wherein differentiation between trophoblast and fetus has begun but before germ layer formation occurs) and examined later in development, pluripotency, or the ability to produce all cell types of the embryo proper, has been noted, suggesting that these cells can be reprogrammed to assume a state analogous to the embryonic stem (ES) cell . However, although cellular markers from NS cells were found in various organs of these blastocyst-injected animals, cellular reprogramming has to be reassessed in light of new data reporting potential fusion events between NS cells and ES cells . Thus, the pluripotent potential of NS cells must be further tested.
Because of our interest in NS cell pluripotency after blastocyst injection, we developed an in vitro model system to observe these cells for several days after injection. Our results indicate that these cells rapidly differentiate into glial fibrillary acidic protein (GFAP)–positive, nestin-negative cells and then regress. This behavior is strikingly different from that of ES cells but is consistent with our own experience that these cells preferentially form glia when transplanted into the central nervous system (CNS) . Furthermore, unlike other reports , we were unable to demonstrate any chimeric animals after NS cell injection into blastocysts. Because these cells consistently become astrocytes after injection, the pluripotent property of these cells under these conditions is questionable.
MATERIALS AND METHODS
Characterization of Cultured Cells
As previously noted, NS cells isolated from E16 embryos grow as nonadherent spheres when exposed to EGF in absence of any coating substrate . All cells from SC and D2NS preparations were nestinimmunoreactive, with only a few of these NS cells occasionally positive for GFAP or ?III-tubulin. Almost all ES cells were SSEA-1–positive (Fig. 1A) and nonimmunoreactive for GFAP- and ?III-tubulin (data not shown). Only a few ES cells (<8%) were nestin-immunoreactive. Additionally, GFP-ES cells expressed oct 4 mRNA (Fig. 1B). In contrast, neither SSEA-1 nor oct 4 mRNA expression was observed in SCs or D2NS cells (Fig. 1).
Figure 1. Neural stem cells lack cellular pluripotent markers. (A): Merged images of 4'6-diamidino-2-phenylindole–stained nuclei (blue) and SSEA-1 immunoreactivity (red) for GFP-ES cells (a) and GFP-NS (b). SSEA-1 staining is present at the level of the ES cell membrane and is not detected in neurospheres. Scale bar = 20 μm. (B): Reverse transcriptase-polymerase chain reaction amplification of oct 4 mRNA. An ethidium bromide–stained gel (negative image) showing oct 4 amplification (lanes 1–3) and a control amplification for gapdh (lanes 4–6). Samples were from GFP-ES cells (lanes 1 and 4), GFP single NS cells (lanes 2 and 5), and GFP-D2NS (lanes 3 and 6). The pluripotency marker, oct 4, is expressed by ES cells (lane 1) but not by NS cells (lanes 2 and 3). Abbreviations: D2NS, day 2–GFP neurosphere; ES, embryonic stem; GFP, green fluorescent protein; NS, neural stem; SSEA-1, stage-specific embryonic antigen-1.
Assessment of In Vitro Chimerism after Blastocyst Injection of GFP-ES or GFP-NS Cells
Forty-eight hours after blastocyst injection, GFP-ES cells could be detected in 93% of the inner masses of hatched blastocysts (52 blastocysts with GFP cells per 56 blastocysts injected), a rate that was significantly higher than that for either GFP-SCs (62%, 40/65) or GFP-D2NS (64%, 44/69) (p < .001, 2 test, Fig. 2). At 72 and 96 hours after injection, the detection rate of GFP-ES cells decreased to 92% and 55.5%, respectively, compared with 43% and 10% for GFP-SC and 42% and 13% for GFP-D2NS. The detection rates at 72 hours (p = .009) and 96 hours (p < .001) were significantly higher for GFP-ES cells than for GFP-SC or GFP-D2NS (2 test, Fig. 2). Furthermore, by 48 hours, both the singly injected NS cells and the small neurospheres extended long cellular processes (Fig. 3A). By comparison, the ES cells maintained an undifferentiated appearance. Moreover, by 72 hours, blastocysts injected with GFP-NS cells presented only small round GFP bodies (Fig. 3B2), whereas blastocysts injected with GFP-ES cells showed a greater number of GFP cells than when initially injected (Fig. 3B1). Similar observations were made when we increased the number (up to 40 cells) of inoculated GFP-labeled or ?-galactosidase–expressing SCs into blastocysts (data not shown).
Figure 2. Time course of the presence of GFP cells after injection into blastocysts. Percentage of blastocysts containing GFP cells at 48, 72, or 96 hours after injection with GFP-ES cells, GFP-SCs, or GFP-D2NS. At all times, the percentage of blastocysts containing GFP-ES cells was significantly higher than for neural stem cell–injected blastocysts (*at 48 and 96 hours, p < .001; at 72 hours, p = .009; 2 analysis). Abbreviations: D2NS, day 2–GFP neurosphere; ES, embryonic stem; GFP, green fluorescent protein; SC, single neural stem cell.
Figure 3. Morphological differentiation of NS cells after injection into blastocysts. (A): Photomicrographs of hatched blastocysts 48 hours after injection with GFP-ES cells (a, b), single neural stem cells (c, d), and D2NS (e, f). In panels a, c, and e, nuclei were visualized with 4'6-diamidino-2-phenylindole staining. Panels b, d, and f show the corresponding GFP-expressing cells. In cultured blastocysts, GFP-ES cells maintained an undifferentiated appearance (b), whereas the neural stem cells extended long processes (d, f). Scale bar = 40 μm. (B): Photomicrographs of two hatched blastocysts injected with 10 to 15 GFP-ES cells (1) or one GFP-D2NS (2) analyzed at 48 (a, b), 72 (c, d), and 96 (e, f) hours after injection. Panels show bright field photomicrographs of the same blastocyst over time (a, c, e) and the corresponding fluorescent photomicrographs showing GFP-expressing cells (b, d, f). GFP-ES cells injected into a blastocyst remain undifferentiated 48 hours (1b) after injection and increase in number by 96 hours (1f). In contrast, GFP-D2NS cells extend long processes at 48 hours (2b) and regress by 96 hours (2f). Scale bar = 40 μm. Abbreviations: D2NS, day 2–GFP neurosphere; ES, embryonic stem; GFP, green fluorescent protein.
Immunocytochemical Characterization of GFP Cells Present in Blastocysts
Double immunocytochemistry for GFAP and bIII-tubulin, nestin, or SSEA-1 was performed on blastocysts injected with GFP cells (n = 22). GFP-ES cells in the blastocysts (n = 6) were negative for GFAP, bIII-tubulin, and nestin but were positive for SSEA-1 (Fig. 4). However, cells in the blastocysts injected with GFP-SC (n = 9 blastocysts) or GFP-D2NS (n = 7 blastocysts) were GFAP-positive and negative for nestin, ?III-tubulin, and SSEA-1 (Fig. 4). These results demonstrated an astrocytic fate for NS cells injected into blastocysts.
Figure 4. NS cells differentiate into GFAP-positive cells after injection into blastocysts. GFP-SCs (A–D), GFP-D2NS (E–H), and GFP-ES cells (I–L) were injected into blastocysts that were immunostained and then analyzed by confocal microscopy. Forty-eight hours after injection, the GFP neural stem cells were no longer nestin-positive (C, G) but were GFAP-positive (B, F), as demonstrated in the merged images (D, H). On the other hand, GFP-ES cells in hatched blastocysts (I) were not GFAP-positive (J) but expressed the pluripotency marker stage-specific embryonic antigen-1 (K), as demonstrated in the merged image (L). Scale bar = 35 μm. Abbreviations: D2NS, day 2–GFP neurosphere; ES, embryonic stem; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; SC, single neural stem cell.
Assessment of In Vivo Chimerism in Neonates and Embryos after Blastocyst Injection of NS Cells
To extend our in vitro results, we also injected NS cells into blastocysts, placed the injected blastocysts into pseudo-pregnant females, and characterized the resulting pups. Blastocysts (n = 74) were injected with 10 to 15 GFP-SCs, and 42 neonates were obtained. No difference in survival was noted whether early (1–5) or late (53) passage cells were used. All neonates were screened by histology, PCR/nested PCR, or nested PCR only for the presence of GFP-expressing cells. None of the 42 pups were chimeric in the heart, liver, intestines, kidney, skin, or brain (Table 1).
Table 1. Summary of in vivo results after injections of neural stem cells in blastocysts
Given our lack of success detecting chimerism in neonates, we decided to examine embryos. Moreover, because of the possibility that the pluripotent cells represented only a small proportion of NS cells, additional injections were performed with small neurospheres selected 1 day after seeding single-cell suspensions. To ensure that we were selecting cells that would grow into mature neurospheres, we seeded one small neurosphere per well and followed its development in a separate experiment. These small neurospheres gave rise 75% of the time to mature neurospheres when singly seeded (data not shown).
Fifty-four blastocysts were injected with 1 to 12 small neurospheres (passages 1–5), and 11 embryos at day 11.5 were obtained. No chimerism was detected by histology or by real-time PCR in organs of the embryos examined. However, serial dilution of genomic DNA from GFP-expressing mice with wild-type mouse DNA showed that a quantity of 0.001 ng GFP mouse DNA (1:100,000 dilutions) resulted in a detectable amplification signal, and a quantity of 0.01 ng GFP DNA (1:10,000 dilutions) yielded a signal cleanly above background noise (Fig. 5). None of the 11 embryos showed any detectable GFP DNA signal in either heads (Fig. 5) or whole bodies (Table 1).
Figure 5. Selected neural stem cells do not form chimeric animals after injection into blastocysts reimplanted in females. Detection of GFP DNA by real-time PCR. (A): Genomic DNA from a GFP-expressing mouse was diluted with DNA from wild-type (WT; non-GFP) mouse. GFP DNA can be detected as low as 1:100,000 dilution. (B): Blastocysts were injected with GFP neurospheres, and DNA was extracted from the heads of resulting newborn pups. Real-time PCR profiles for 10 representative samples are shown. No GFP signal was detected. Abbreviations: GFP, green fluorescent protein; PCR, polymerase chain reaction.
To check for the possibility that a more developed neurosphere might better incorporate into blastocysts, we chose to inject D2NS when the spheres were more developed but still small in diameter. Blastocysts (n = 44) were injected with one neurosphere (passages 1–5), and 26 embryos between E 13.5 and E 15.5 were obtained. No chimerism was detected in any organs of the 26 embryos by real-time PCR (Table 1). Overall, we used multiple methods to examine 82 embryos or neonates and found no evidences for chimerism.
DISCUSSION
We want to thank Dr. Anthony van den Pol for the gift of the GFP transgenic mice and Drs. Todd Savarese and William Schwartz for their helpful comments on the writing of this manuscript. This work was supported in part by NS43879 from the National Institute of Neurological Disorders and Stroke (L.R.).
REFERENCES
Peretto P, Merighi A, Fasolo A et al. The subependymal layer in rodents: a site of structural plasticity and cell migration in the adult mammalian brain. Brain Res Bull 1999;49:221– 243.
Brazel CY, Romanko MJ, Rothstein RP et al. Roles of the mammalian subventricular zone in brain development. Prog Neurobiol 2003;69:49–69.
Reynolds BA, Tetzlaff W, Weiss S. A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes. J Neurosci 1992;12:4565–4574.
Reynolds BA, Weiss S. Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell. Dev Biol 1996; 175:1–13.
Bjornson CRR, Rietze RL, Reynolds BA et al. Turning brain into blood: a hematopoietic fate adopted by adult neural stem cells in vivo. Science 1999;283:534–537.
Galli R, Borello U, Gritti A et al. Skeletal myogenic potential of human and mouse neural stem cells. Nature Neurosci 2000;3:986–991.
Clarke DL, Johansson CB, Wilbertz J et al. Generalized potential of adult neural stem cells. Science 2000;288: 1660–1663.
Kirschof N, Harder F, Petrovic S et al. Developmental potential of hematopoietic and neural stem cells: unique or all the same. Cell Tissues Organs 2002;171:77–89.
Ying Q-L, Nichols J, Evans EP et al. Changing potency by spontaneous fusion. Nature 2002;416:545–548.
Pells S, Di Domenico AI, Gallagher EJ et al. Multipotentiality of neuronal cells after spontaneous fusion with embryonic stem cells and nuclear reprogramming in vitro. Cloning Stem Cells 2002;4:331–338.
Mitome M, Low HP, van den Pol AN et al. Towards the reconstruction of central nervous system white matter using neural precursor cells. Brain 2001;124:2147–2161.
van den Pol AN, Ghosh PK. Selective neuronal expression of green fluorescent protein with cytomegalovirus promoter reveals entire neuronal arbor in transgenic mice. J Neurosci 1998;18:10640–10651.
Zambrowicz BP, Imamoto A, Fiering S et al. Disruption of overlapping transcripts in the ROSA bgeo 26 gene trap strain leads to widespread expression of ?-galactosidase in mouse embryos and hematopoietic cells. Proc Natl Acad Sci U S A 1997;94:3789–3794.
Hulspas R, Tiarks C, Reilly J et al. In vitro cell density dependent clonal growth of EGF responsive murine neural precursor cells under serum-free conditions. Exp Neurol 1997;148:147–156.
Lachyankar MB, Condon PJ, Quesenberry PJ et al. Embryonic precursor cells that express Trk receptors: induction of different cell fates by NGF, BDNF, NT-3, and CNTF. Exp Neurol 1997;144:350–360.
Geiger H, Sick S, Bonifer C et al. Globin gene expression is reprogrammed in chimeras generated by injecting adult hematopoietic stem cells into mouse blastocysts. Cell 1998; 93:1055–1065.
Liang L, Bickenbach JR. Somatic epidermal stem cells can produce multiple cell lineages during development. STEM CELLS 2002;20:21–31.
Morshead CM, Benveniste P, Iscove NN et al. Hematopoietic competence is a rare property of neural stem cells that may depend on genetic and epigenetic alterations. Nature Med 2002;8:268–273.
Alvarez-Buylla A, Garcia-Verdugo JM, Tramontin AD. A unified hypothesis on the lineage of neural stem cells. Nature Rev 2001;2:287–293.
Goldman S. Glia as neural progenitor cells. Trends Neurosci 2003;26:590–596.
Niwa H. Molecular mechanism to maintain stem cell renewal of ES cells. Cell Struct Funct 2001;26:137–148.
Mitsui K, Tokuzawa Y, Itoh H et al. The homeoprotein nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 2003;113:631–642.
Rossant J. Stem cells from the mammalian blastocyst. STEM CELLS 2001;19:477–482.
Rao MS, Mattson MP. Stem cells and aging: expanding the possibilities. Mech Aging Dev 2001;122:713–734.
Craig CG, Tropepe V, Morshead CM et al. In vivo growth factor expansion of endogenous subependymal neural precursor cell populations in the adult mouse brain. J Neurosci 1996;16:2649–2658.
Gritti A, Frolichsthal-Schoeller P, Galli R et al. Epidermal and fibroblast growth factors behave as mitogenic regulators for a single multipotent stem cell-like population from the subventricular region of the adult mouse forebrain. J Neurosci 1999;19:3287–3297.
Rao MS. Multipotent and restricted precursors in the central nervous system. Anat Rec 1999;257:137–148.
Ciccolini F. Identification of two distinct types of multipotent neural precursors that appear sequentially during CNS development. Mol Cell Neurosci 2001;17:895–907.
Tada M, Takahama Y, Abe K et al. Nuclear reprogramming of somatic cells by in vitro hybridization with ES cells. Curr Biol 2001;11:1553–1558.
Terada N, Hamazaki T, Oka M et al. Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature 2002;416:542–545.
Winkler C, Fricker RA, Gates MA et al. Incorporation and glial differentiation of mouse EGF-responsive neural progenitor cells after transplantation into the embryonic rat brain. Mol Cell Neurosci 1998;11:99–116.(Béatrice Grécoa, Hoi Pang)
b Biochemistry and Molecular Pharmacology,
c Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA;
d Department of Neurology and Clinical Neurosciences, Stanford University, Palo Alto, California, USA
Key Words. Neural stem cells ? Blastocyst injections ? Differentiation ? Pluripotency
Correspondence: Lawrence D. Recht, M.D., Department of Neurology, Stanford University Medical Center, 1201 Welch Road, Room P-208, Stanford, California 94305, USA. Telephone: 650-498-6320; Fax: 650-498-6262; e-mail: LRecht@stanford.edu
ABSTRACT
In the brain, the subventricular zone (SVZ) is a source of proliferating cells that replenish olfactory interneurons and glial cells . Cells from the SVZ have several characteristics that distinguish them as neural stem (NS) cells, including their capacity to self-renew indefinitely in defined media supplemented with either epidermal growth factor (EGF) or basic fibroblast growth factor (FGF) and the capacity to differentiate into neurons, astrocytes, and oligodendrocytes in vitro upon cytokine exposure .
Recent studies have suggested that under certain conditions, NS cells are not only multipotent, i.e., able to produce more than one neural cell type, but also able to differentiate into other cell types, such as blood and muscle . In fact, when NS cells are placed in embryos at the blastocyst stage (wherein differentiation between trophoblast and fetus has begun but before germ layer formation occurs) and examined later in development, pluripotency, or the ability to produce all cell types of the embryo proper, has been noted, suggesting that these cells can be reprogrammed to assume a state analogous to the embryonic stem (ES) cell . However, although cellular markers from NS cells were found in various organs of these blastocyst-injected animals, cellular reprogramming has to be reassessed in light of new data reporting potential fusion events between NS cells and ES cells . Thus, the pluripotent potential of NS cells must be further tested.
Because of our interest in NS cell pluripotency after blastocyst injection, we developed an in vitro model system to observe these cells for several days after injection. Our results indicate that these cells rapidly differentiate into glial fibrillary acidic protein (GFAP)–positive, nestin-negative cells and then regress. This behavior is strikingly different from that of ES cells but is consistent with our own experience that these cells preferentially form glia when transplanted into the central nervous system (CNS) . Furthermore, unlike other reports , we were unable to demonstrate any chimeric animals after NS cell injection into blastocysts. Because these cells consistently become astrocytes after injection, the pluripotent property of these cells under these conditions is questionable.
MATERIALS AND METHODS
Characterization of Cultured Cells
As previously noted, NS cells isolated from E16 embryos grow as nonadherent spheres when exposed to EGF in absence of any coating substrate . All cells from SC and D2NS preparations were nestinimmunoreactive, with only a few of these NS cells occasionally positive for GFAP or ?III-tubulin. Almost all ES cells were SSEA-1–positive (Fig. 1A) and nonimmunoreactive for GFAP- and ?III-tubulin (data not shown). Only a few ES cells (<8%) were nestin-immunoreactive. Additionally, GFP-ES cells expressed oct 4 mRNA (Fig. 1B). In contrast, neither SSEA-1 nor oct 4 mRNA expression was observed in SCs or D2NS cells (Fig. 1).
Figure 1. Neural stem cells lack cellular pluripotent markers. (A): Merged images of 4'6-diamidino-2-phenylindole–stained nuclei (blue) and SSEA-1 immunoreactivity (red) for GFP-ES cells (a) and GFP-NS (b). SSEA-1 staining is present at the level of the ES cell membrane and is not detected in neurospheres. Scale bar = 20 μm. (B): Reverse transcriptase-polymerase chain reaction amplification of oct 4 mRNA. An ethidium bromide–stained gel (negative image) showing oct 4 amplification (lanes 1–3) and a control amplification for gapdh (lanes 4–6). Samples were from GFP-ES cells (lanes 1 and 4), GFP single NS cells (lanes 2 and 5), and GFP-D2NS (lanes 3 and 6). The pluripotency marker, oct 4, is expressed by ES cells (lane 1) but not by NS cells (lanes 2 and 3). Abbreviations: D2NS, day 2–GFP neurosphere; ES, embryonic stem; GFP, green fluorescent protein; NS, neural stem; SSEA-1, stage-specific embryonic antigen-1.
Assessment of In Vitro Chimerism after Blastocyst Injection of GFP-ES or GFP-NS Cells
Forty-eight hours after blastocyst injection, GFP-ES cells could be detected in 93% of the inner masses of hatched blastocysts (52 blastocysts with GFP cells per 56 blastocysts injected), a rate that was significantly higher than that for either GFP-SCs (62%, 40/65) or GFP-D2NS (64%, 44/69) (p < .001, 2 test, Fig. 2). At 72 and 96 hours after injection, the detection rate of GFP-ES cells decreased to 92% and 55.5%, respectively, compared with 43% and 10% for GFP-SC and 42% and 13% for GFP-D2NS. The detection rates at 72 hours (p = .009) and 96 hours (p < .001) were significantly higher for GFP-ES cells than for GFP-SC or GFP-D2NS (2 test, Fig. 2). Furthermore, by 48 hours, both the singly injected NS cells and the small neurospheres extended long cellular processes (Fig. 3A). By comparison, the ES cells maintained an undifferentiated appearance. Moreover, by 72 hours, blastocysts injected with GFP-NS cells presented only small round GFP bodies (Fig. 3B2), whereas blastocysts injected with GFP-ES cells showed a greater number of GFP cells than when initially injected (Fig. 3B1). Similar observations were made when we increased the number (up to 40 cells) of inoculated GFP-labeled or ?-galactosidase–expressing SCs into blastocysts (data not shown).
Figure 2. Time course of the presence of GFP cells after injection into blastocysts. Percentage of blastocysts containing GFP cells at 48, 72, or 96 hours after injection with GFP-ES cells, GFP-SCs, or GFP-D2NS. At all times, the percentage of blastocysts containing GFP-ES cells was significantly higher than for neural stem cell–injected blastocysts (*at 48 and 96 hours, p < .001; at 72 hours, p = .009; 2 analysis). Abbreviations: D2NS, day 2–GFP neurosphere; ES, embryonic stem; GFP, green fluorescent protein; SC, single neural stem cell.
Figure 3. Morphological differentiation of NS cells after injection into blastocysts. (A): Photomicrographs of hatched blastocysts 48 hours after injection with GFP-ES cells (a, b), single neural stem cells (c, d), and D2NS (e, f). In panels a, c, and e, nuclei were visualized with 4'6-diamidino-2-phenylindole staining. Panels b, d, and f show the corresponding GFP-expressing cells. In cultured blastocysts, GFP-ES cells maintained an undifferentiated appearance (b), whereas the neural stem cells extended long processes (d, f). Scale bar = 40 μm. (B): Photomicrographs of two hatched blastocysts injected with 10 to 15 GFP-ES cells (1) or one GFP-D2NS (2) analyzed at 48 (a, b), 72 (c, d), and 96 (e, f) hours after injection. Panels show bright field photomicrographs of the same blastocyst over time (a, c, e) and the corresponding fluorescent photomicrographs showing GFP-expressing cells (b, d, f). GFP-ES cells injected into a blastocyst remain undifferentiated 48 hours (1b) after injection and increase in number by 96 hours (1f). In contrast, GFP-D2NS cells extend long processes at 48 hours (2b) and regress by 96 hours (2f). Scale bar = 40 μm. Abbreviations: D2NS, day 2–GFP neurosphere; ES, embryonic stem; GFP, green fluorescent protein.
Immunocytochemical Characterization of GFP Cells Present in Blastocysts
Double immunocytochemistry for GFAP and bIII-tubulin, nestin, or SSEA-1 was performed on blastocysts injected with GFP cells (n = 22). GFP-ES cells in the blastocysts (n = 6) were negative for GFAP, bIII-tubulin, and nestin but were positive for SSEA-1 (Fig. 4). However, cells in the blastocysts injected with GFP-SC (n = 9 blastocysts) or GFP-D2NS (n = 7 blastocysts) were GFAP-positive and negative for nestin, ?III-tubulin, and SSEA-1 (Fig. 4). These results demonstrated an astrocytic fate for NS cells injected into blastocysts.
Figure 4. NS cells differentiate into GFAP-positive cells after injection into blastocysts. GFP-SCs (A–D), GFP-D2NS (E–H), and GFP-ES cells (I–L) were injected into blastocysts that were immunostained and then analyzed by confocal microscopy. Forty-eight hours after injection, the GFP neural stem cells were no longer nestin-positive (C, G) but were GFAP-positive (B, F), as demonstrated in the merged images (D, H). On the other hand, GFP-ES cells in hatched blastocysts (I) were not GFAP-positive (J) but expressed the pluripotency marker stage-specific embryonic antigen-1 (K), as demonstrated in the merged image (L). Scale bar = 35 μm. Abbreviations: D2NS, day 2–GFP neurosphere; ES, embryonic stem; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; SC, single neural stem cell.
Assessment of In Vivo Chimerism in Neonates and Embryos after Blastocyst Injection of NS Cells
To extend our in vitro results, we also injected NS cells into blastocysts, placed the injected blastocysts into pseudo-pregnant females, and characterized the resulting pups. Blastocysts (n = 74) were injected with 10 to 15 GFP-SCs, and 42 neonates were obtained. No difference in survival was noted whether early (1–5) or late (53) passage cells were used. All neonates were screened by histology, PCR/nested PCR, or nested PCR only for the presence of GFP-expressing cells. None of the 42 pups were chimeric in the heart, liver, intestines, kidney, skin, or brain (Table 1).
Table 1. Summary of in vivo results after injections of neural stem cells in blastocysts
Given our lack of success detecting chimerism in neonates, we decided to examine embryos. Moreover, because of the possibility that the pluripotent cells represented only a small proportion of NS cells, additional injections were performed with small neurospheres selected 1 day after seeding single-cell suspensions. To ensure that we were selecting cells that would grow into mature neurospheres, we seeded one small neurosphere per well and followed its development in a separate experiment. These small neurospheres gave rise 75% of the time to mature neurospheres when singly seeded (data not shown).
Fifty-four blastocysts were injected with 1 to 12 small neurospheres (passages 1–5), and 11 embryos at day 11.5 were obtained. No chimerism was detected by histology or by real-time PCR in organs of the embryos examined. However, serial dilution of genomic DNA from GFP-expressing mice with wild-type mouse DNA showed that a quantity of 0.001 ng GFP mouse DNA (1:100,000 dilutions) resulted in a detectable amplification signal, and a quantity of 0.01 ng GFP DNA (1:10,000 dilutions) yielded a signal cleanly above background noise (Fig. 5). None of the 11 embryos showed any detectable GFP DNA signal in either heads (Fig. 5) or whole bodies (Table 1).
Figure 5. Selected neural stem cells do not form chimeric animals after injection into blastocysts reimplanted in females. Detection of GFP DNA by real-time PCR. (A): Genomic DNA from a GFP-expressing mouse was diluted with DNA from wild-type (WT; non-GFP) mouse. GFP DNA can be detected as low as 1:100,000 dilution. (B): Blastocysts were injected with GFP neurospheres, and DNA was extracted from the heads of resulting newborn pups. Real-time PCR profiles for 10 representative samples are shown. No GFP signal was detected. Abbreviations: GFP, green fluorescent protein; PCR, polymerase chain reaction.
To check for the possibility that a more developed neurosphere might better incorporate into blastocysts, we chose to inject D2NS when the spheres were more developed but still small in diameter. Blastocysts (n = 44) were injected with one neurosphere (passages 1–5), and 26 embryos between E 13.5 and E 15.5 were obtained. No chimerism was detected in any organs of the 26 embryos by real-time PCR (Table 1). Overall, we used multiple methods to examine 82 embryos or neonates and found no evidences for chimerism.
DISCUSSION
We want to thank Dr. Anthony van den Pol for the gift of the GFP transgenic mice and Drs. Todd Savarese and William Schwartz for their helpful comments on the writing of this manuscript. This work was supported in part by NS43879 from the National Institute of Neurological Disorders and Stroke (L.R.).
REFERENCES
Peretto P, Merighi A, Fasolo A et al. The subependymal layer in rodents: a site of structural plasticity and cell migration in the adult mammalian brain. Brain Res Bull 1999;49:221– 243.
Brazel CY, Romanko MJ, Rothstein RP et al. Roles of the mammalian subventricular zone in brain development. Prog Neurobiol 2003;69:49–69.
Reynolds BA, Tetzlaff W, Weiss S. A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes. J Neurosci 1992;12:4565–4574.
Reynolds BA, Weiss S. Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell. Dev Biol 1996; 175:1–13.
Bjornson CRR, Rietze RL, Reynolds BA et al. Turning brain into blood: a hematopoietic fate adopted by adult neural stem cells in vivo. Science 1999;283:534–537.
Galli R, Borello U, Gritti A et al. Skeletal myogenic potential of human and mouse neural stem cells. Nature Neurosci 2000;3:986–991.
Clarke DL, Johansson CB, Wilbertz J et al. Generalized potential of adult neural stem cells. Science 2000;288: 1660–1663.
Kirschof N, Harder F, Petrovic S et al. Developmental potential of hematopoietic and neural stem cells: unique or all the same. Cell Tissues Organs 2002;171:77–89.
Ying Q-L, Nichols J, Evans EP et al. Changing potency by spontaneous fusion. Nature 2002;416:545–548.
Pells S, Di Domenico AI, Gallagher EJ et al. Multipotentiality of neuronal cells after spontaneous fusion with embryonic stem cells and nuclear reprogramming in vitro. Cloning Stem Cells 2002;4:331–338.
Mitome M, Low HP, van den Pol AN et al. Towards the reconstruction of central nervous system white matter using neural precursor cells. Brain 2001;124:2147–2161.
van den Pol AN, Ghosh PK. Selective neuronal expression of green fluorescent protein with cytomegalovirus promoter reveals entire neuronal arbor in transgenic mice. J Neurosci 1998;18:10640–10651.
Zambrowicz BP, Imamoto A, Fiering S et al. Disruption of overlapping transcripts in the ROSA bgeo 26 gene trap strain leads to widespread expression of ?-galactosidase in mouse embryos and hematopoietic cells. Proc Natl Acad Sci U S A 1997;94:3789–3794.
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