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EMBRYONIC STEM CELLS |
aInstitut für Medizinische Strahlenkunde und Zellforschung, Universität Würzburg, Würzburg, Germany;
bCenter for Animal Transgenesis and Germ Cell Research, New Bolton Center, University of Pennsylvania, Kennett Square, Pennsylvania, USA
Key Words. Embryonic stem cells • Androgenetic • Differentiation • Neural progenitor cells
Correspondence: Correspondence: Albrecht M. Müller, Ph.D., Professor, Institut für Medizinische Strahlenkunde und Zellforschung, University of Würzburg, Versbacher Strasse 5, 97078 Würzburg, Germany. Telephone: 49-931-201-45848; Fax: 49-931-201-45147; e-mail: albrecht.mueller{at}mail.uni-wuerzburg.de
Received on October 18, 2007;
accepted for publication on March 18, 2008.
First published online in STEM CELLS EXPRESS March 27, 2008.
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ABSTRACT |
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 TopFootnotes Abstract IntroductionMaterials and MethodsResultsDiscussionSummaryDisclosure of Potential...AcknowledgmentsReferences |
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Disclosure of potential conflicts of interest is found at the end of this article.
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INTRODUCTION |
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TopFootnotesAbstractIntroductionMaterials and MethodsResultsDiscussionSummaryDisclosure of Potential...AcknowledgmentsReferences |
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PG ESCs isolated from monkey demonstrate the ability to differentiate into neural precursors and into cells with neuronal morphologies and characteristics of functional neurons. Additional in vivo analyses show the long-term survival of dopaminergic neurons derived from PG monkey ESCs after transplantation into immunosuppressed rats [11].
Addressing the problem of graft rejection, Kim et al. [12] demonstrated that murine PG ESCs can be selected for MHC compatibility and that injection of differentiated PG ESCs expressing matched major histocompatibility complex (MHC) antigens leads to teratoma formation in immune competent hosts. In addition, the isolation and characterization of six human PG ESC lines was described [8]. Human PG ESCs are able to differentiate into cell types from all three germ layers and they are MHC-matched with the oocyte donors [8]. In contrast to PG ESCs, AG ESCs have not been similarly examined, in part because of the abnormal phenotype of murine AG ESC chimeras [10, 13] and the lack of derivation of nonhuman primate and human AG ESCs.
Only recently has the functionality of GG and AG ESC-derived transplants been demonstrated in a mammalian species [14]. Both murine AG and GG ESC-derived fetal liver hematopoietic stem cells conveyed long-term and multilineage reconstitution of the entire hematopoietic system in transplant recipients with no associated pathologies. In contrast to defects observed in AG chimeras, AG ESC-derived cells in adult recipients did not exhibit any abnormal phenotypes, and expression levels of imprinted genes in lymphocytes of either AG or GG origin were typically low, without parent-of-origin-specific bias. It seems, therefore, that correct expression of imprinted genes is not required for normal function or that normal expression is re-established by resetting the imprinting status, at least in blood cells.
Studies on murine chimeric embryos developing following blastocyst injection of uniparental inner cell mass (ICM) cells or uniparental ESCs have provided insights into the parent-specific contribution to brain development. PG and GG cells distribute and differentiate preferentially to brain and less so to mesodermal tissues [15–17]. Analysis of fetal brain development of AG and GG chimeras developing either from aggregation of uniparental and normal fertilized (N) morulae or following injection of uniparental ICM cells into N blastocysts reveals that AG cells contribute substantially to hypothalamic structures but less to the cortex and striatum, whereas progeny of PG and GG cells seed the cortex, striatum, and hypothalamus [18]. Also, AG ESCs contribute less to brain upon blastocyst injection, although the regional distribution of AG cells in brain was not analyzed [10]. Together, these observations argue that paternal and maternal genomes have differential influence on brain development.
The apparent bias in neural differentiation of AG and GG ICM cells could also indicate that the male-derived AG ESCs would have limited neural differentiation potential and be limited or excluded for neural tissue replacement utility. Investigating the capacity of AG ESCs to undergo neural differentiation, we found that AG ESCs exhibit neural differentiation capacity in vitro and in vivo in mid-gestation chimeras, similar to ESCs derived from N embryos. AG ESC-derived cells contributed widely to embryonic day (E) 12.5 fetal brain and exhibited normal formation of neurospheres with self-renewal and neural differentiation capacity.
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MATERIALS AND METHODS |
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ESC Culture
The AG and N enhanced green fluorescent protein (eGFP)+ murine ESCs used in this study have been previously described [14]. ESCs were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM) (PAA Laboratories, Cölbe, Germany, http://www.paa.at) supplemented with 15% fetal calf serum (FCS) (HyClone, Logan, UT, http://www.hyclone.com; Thermo Fisher Scientific, Schwerte, Germany, http://www.thermofisher.com), leukemia inhibitory factor-conditioned medium [19, 20], 1% nonessential amino acids (NEAA) (PAA Laboratories), penicillin (100 U/ml)/streptomycin (100 U/ml) (PAA Laboratories), L-glutamine (2 mM; PAA Laboratories), sodium pyruvate (1 mM; PAA Laboratories), and β-mercaptoethanol (0.1 mM; Sigma-Aldrich, Schnelldorf, Germany, http://www.sigmaaldrich.com) on confluent layers of primary murine embryonic fibroblasts (MEFs). Medium was changed daily. For passage of ESCs, medium was removed, cells were washed with phosphate-buffered saline (PBS), and trypsin/EDTA (PAA Laboratories) was added. After 10 minutes at 37°C, the reaction was stopped by adding 10 ml of medium containing 15% FCS. Cell suspensions were put on gelatin-coated plates (0.1% in PBS; Sigma-Aldrich) for 45 minutes at 37°C to allow separation of MEFs and ESCs. ESCs (5 x 105) were plated on new MEF layers. Passage numbers of ESCs used for in vitro differentiation and transplantation experiments ranged from 15 to 25 (in vitro analyses) and from 15 to 36 (in vivo analyses).
ESC In Vitro Differentiation
ESCs were differentiated toward neural phenotypes as described [21–23]. Briefly, to obtain embryoid bodies (EBs), undifferentiated ESCs (AG line 1 [A1] and 2 [A2]; N line 1) were cultured in high-glucose DMEM supplemented with 10% FCS (Biochrom AG, Berlin, http://www.biochrom.de), HEPES (10 mM; PAA Laboratories), NEAA, penicillin (100 U/ml)/streptomycin (100 U/ml), L-glutamine (2 mM), sodium pyruvate (1 mM), and β-mercaptoethanol (0.1 mM) for 4 days in bacteriological Petri dishes. On day 4, free-floating EBs were transferred to tissue culture plates for adherent cells with DMEM/Ham's F-12 medium (F12) (PAA Laboratories) containing penicillin (100 U/ml)/streptomycin (100 U/ml), L-glutamine (2 mM), human transferrin (50 µg/ml), sodium selenite (30 nM), insulin (5 µg/ml), and fibronectin (2.5 µg/ml) (all from Sigma-Aldrich) to obtain attached EBs. Fresh medium was added on day 2. After 4 days of cultivation, attached EB were trypsinized (10 minutes, 37°C), and cells were transferred to poly-L-ornithine- and laminin-coated plates (both from Sigma-Aldrich) and cultured in DMEM/F12 containing penicillin (100 U/ml)/streptomycin (100 U/ml), L-glutamine (2 mM), insulin (25 µg/ml), progesterone (20 nM), putrescine (0.1 µM), sodium selenite (30 nM), human transferrin (50 µg/ml) (all from Sigma-Aldrich), basic fibroblast growth factor (bFGF) (50 ng/ml; Peprotech, Hamburg, Germany, http://www.peprotech.com), and laminin (0.7 µg/ml) for generation of pan-neural progenitor cells (pnPCs). On days 1, 2, and 3, fresh medium supplemented with bFGF (50 ng/ml) was added. After 4 days of cultivation, pnPCs were trypsinized and replated under neural differentiation conditions (Neurobasal medium with 2% B27 supplement [both from Gibco, Karlsruhe, Germany, http://www.invitrogen.com], penicillin [100 U/ml]/streptomycin [100 U/ml], and L-glutamine [2 mM], supplemented with 10% NeuroCult differentiation supplement [StemCell Technologies, St. Katharinen, Germany, http://www.stemcell.com]) for terminal differentiation into neural cell types on coverslips coated with poly-L-ornithine and laminin. Cells were cultured at 37°C in 5% CO2 for up to 13 days. Medium was changed every other day. Immunohistochemical staining for neuronal and astroglial differentiation markers was performed as described for in vitro-differentiated neurosphere cells (described below). Percentages of tubulin-β-III+ cells and glial fibrillary acidic protein (GFAP)+ cells were compared between differentiated AG and N pnPCs by two-sided Student's t test. Differences were considered statistically significant if p < .05.
Bisulfite Sequencing
Genomic DNA isolated from ESCs and pnPCs using the DNeasy Tissue Kit (Qiagen, Hilden, Germany, http://www1.qiagen.com) was modified using the EZ DNA Methylation-Gold kit (Zymo Research, Orange, CA, http://www.zymoresearch.com) according to the manufacturer's instructions. Modified DNA was amplified by nested and seminested polymerase chain reaction (PCR) as described [24]. The amplified DNA fragments were subcloned into pJet1/blunt (Fermentas, Glen Burnie, MD, http://www.fermentas.com) for sequencing. Analysis of sequences and diagram generation was performed using BiQ Analyzer (http://biq-analyzer.bioinf.mpi-sb.mpg.de)[25].
Quantitative Reverse Transcription-PCR
RNAs were isolated from AG and N pnPCs by using peqGOLD RNAPure (peqLab Biotechnologie, Göttingen, Germany, http://www.peqlab.de). Before generation of cDNA, RNA preparations were treated with DNase I (Ambion, Austin, TX, http://www.ambion.com; Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com). RNA was reverse-transcribed into cDNA using Moloney murine leukemia virus reverse transcriptase (Invitrogen, Karlsruhe, Germany, http://www.invitrogen.com). Reverse transcription (RT)-PCRs were performed and quantified using a Rotor-Gene 3000 (Corbett Life Science, Wasserburg, Germany, www.corbettlifescience.com) and ABsolute QPCR SYBR Green Mix (ABgene, Hamburg, Germany, http://www.abgene.com). Differences in gene expression were calculated with the 2–
CT method. The housekeeping gene β-actin was used for normalization. The 2–CT values of N pnPCs were set to 1 for tested genes to determine expression differences between AG and N pnPCs. The primer sequences were as follows: β-actin: forward (f), 5'-GATATCGCTGCGCTGGTCGTC-3'/reverse (r), 5'-ACGCAGCTCATTGTAGAAGGTGTGG-3'; Igf2: f, 5'-CTAAGACTTGGATCCCAGAACC-3'/r, 5'-GTTCTTCTCCTTGGGTTCTTTC-3'; Impact: f, 5'-ACGTTTCCCCATTTTACAAG-3'/r, 5'-CTCTACATATGATTTTCTCTAC-3'; Igf2r: f, 5'-TAGTTGCAGCTCTTTGCACG-3'/r, 5'-ACAGCTCAAACCTGAAGCG-3'; Ube3a: f, 5'-CACATATGATGAAGCTACGA-3'/r, 5'-CACACTCCCTTCATATTCC-3' [14]; Zim1: f, 5'-GAGAAGCCGTACTGCTGTCA-3'/r, 5'-CTTGCACCGGTACCTGGAGT-3' [26]; H19: f, 5'-CATGTCTGGGCCTTTGAA-3'/r, 5'-TTGGCTCCAGGATGATGT-3' [27].
Generation of Chimeric Embryos by Blastocyst Injection
To obtain blastocysts for the injection of ESCs, 6–8-week-old female NMRI mice were superovulated by intraperitoneal injection of 10 units of pregnant mare's serum followed 48 hours later by an injection of 10 units of human chorionic gonadotropin (both from Intervet, Unterschleißheim, Germany, http://www.intervet.de), caged with stud males, and checked for vaginal plugs the next morning. The day of finding the plug was designated 0.5 days post coitum (dpc). At 3.5 dpc, pregnant mice were sacrificed, and ovaries and oviducts were removed and transferred into M16 medium (Sigma-Aldrich). Blastocysts were flushed from the oviducts and kept for 1–2 hours in M2 medium (Sigma-Aldrich) prior to the injection of ESCs. To induce pseudopregnancy in recipient foster animals, 6–8-week-old female NMRI mice were mated with vasectomized NMRI males of proven sterility. ESCs were trypsinized, and MEFs were removed by incubation on gelatin-coated plates for 45 minutes. ESC single-cell suspensions were prepared in M2 medium prior to blastocyst injection. Ten to 15 ESCs were injected per blastocyst. Afterward, blastocysts were transferred into pseudopregnant foster animals. At 12.5 and 14.5 dpc, pregnant mice were sacrificed, and embryos were isolated from the uteri. Pictures of embryos were taken, embryo sizes were measured, fetal livers were isolated and triturated, and liver single-cell suspensions were analyzed by flow cytometry (FACSCalibur; BD Biosciences, Heidelberg, Germany, http://www.bdbiosciences.com) for grade of chimerism by measuring the percentage of eGFP+ cells. Gates were defined by using fetal liver cells from wild-type and eGFP transgenic embryos [21]. Embryos were considered chimeric if the eGFP+ cell frequency was 
1%. Heads of chimeric embryos were either fixed for cryosections and immunohistochemistry or used to establish neurosphere cultures.
Immunohistochemistry of E12.5 Brain Cryosections
For cryosections, whole E12.5 embryo heads were fixed for 12 hours in PBS/4% paraformaldehyde (Applichem, Darmstadt, Germany, http://www.applichem.de). Following fixation, tissues were dehydrated in PBS/16% glucose (Applichem), embedded in Tissue-Tek OCT Compound (Sakura Finetek, Heppenheim, Germany, http://www.sakura.com), and frozen at –80°C. Ten-micrometer sagittal sections were stained with primary chicken antibody against eGFP (Abcam, Cambridge, U.K., http://www.abcam.com) and with secondary goat anti-chicken IgY Cy2-conjugated antibody (Abcam). For neuronal cell staining, primary mouse antibody against tubulin-β-III (clone TUJ-1; R&D Systems Inc., Minneapolis, http://www.rndsystems.com) or primary mouse antibody against neuronal nuclei (NeuN) (Chemicon, Schwalbach, Germany, http://www.chemicon.com) and secondary mouse IgG Cy3-conjugated antibody (Chemicon) were used. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). For analysis of colocalization of green and red signals, 10 µm z-stacks with 1-µm picture distance were taken using a BioZero microscope (magnification, x600) (Keyence, Neu-Isenburg, Germany, http://www.keyence.de), yielding xy pictures and orthogonal xz and yz projections of z-stacks. For intranuclear staining, tissue slices were incubated for 20 minutes at 96°C in 10 mM citrate buffer (pH 6.0) for antigen retrieval. To visualize proliferating cells, 5-µm transversal sections were stained with primary rabbit antibody against proliferating cell nuclear antigen (PCNA) (BD Biosciences) and secondary goat anti-rabbit IgG Cy3-conjugated antibody (Chemicon). Apoptotic cells were detected using primary mouse antibody against cleaved-caspase-3 (Cell Signaling Technology, NEB, Frankfurt, Germany, http://www.cellsignal.com) and secondary goat anti-mouse IgG Cy3-conjugated antibody (Chemicon). Nuclei were counterstained with DAPI (Sigma-Aldrich).
Quantification of eGFP+ ESC-Derived Cell Contribution in E12.5 Chimeric Brains
To quantify the contribution of eGFP+ ESC-derived cells, the percentage of eGFP+ cells was determined for distinct brain regions by counting the numbers of DAPI-stained nuclei and the numbers of eGFP+ cells in three representative 100 x 100-µm squares per brain region. Regions analyzed were striatum, hypothalamus, cortex, and brain stem. Percentages of eGFP+ cells in striatum, hypothalamus, and cortex were calculated relative to brain stem. Means of eGFP+ cell percentages relative to brain stem for striatum, hypothalamus, and cortex from AG and N chimeric brains were considered significantly different if p < .05 (two-sided Student's t test).
Neurosphere Cell Isolation and Culture
To establish neurosphere cultures, E12.5 fetal brains were isolated from chimeric embryos. Whole brains were triturated, and single-cell suspensions were obtained by pipetting cells through a cell strainer (70 µm; BD Biosciences). Cell suspensions were cultured under neurosphere growth conditions (Neurobasal medium with 2% B27 supplement, penicillin [100 U/ml]/streptomycin [100 U/ml], and L-glutamine [2 mM], supplemented with human growth factors epidermal growth factor [20 ng/ml] and basic fibroblast growth factor [20 ng/ml] [both from Peprotech]) [28]. After 2 days of culture, free-floating neurospheres started to grow. At the day (d) of isolation (d0), d5, and d10, the grade of eGFP+ ESC-derived contribution was assessed by flow cytometry. For flow cytometric analysis at d5 and d10, single-cell suspensions of neurospheres were prepared by incubating neurospheres with AccuMax (PAA Laboratories) for 7 minutes at 37°C and pipetting cells through cell strainers. To isolate AG brain cells, single-cell suspensions were prepared from chimeric brains, and cells were expanded for two passages under neurosphere growth conditions, as primary brain cells were found to be sensitive to fluorescence-activated cell sorting (FACS). Single-cell suspensions were prepared from neurospheres, and cells were subjected to FACS according eGFP fluorescence. The purity of eGFP+ and eGFP– populations after sorting was >98%. Sorted eGFP+ and eGFP– neurosphere cells were expanded for two to four passages under neurosphere growth conditions to obtain sufficient cell numbers for analysis.
Neurosphere Cell Self-Renewal
To determine the neurosphere-initiating cell (NIC) number, single-cell suspensions from neurosphere cultures were produced by AccuMax treatment. Cells were counted, and viability was assessed by trypan blue exclusion. Five thousand single neurosphere cells were seeded per well, and cells were cultured under neurosphere growth conditions. After 5 days, newly formed neurospheres were counted. Percentages of NICs were calculated and considered significantly different if p < .05 (two-sided Student's t test).
Neurosphere Cell In Vitro Differentiation
For neural differentiation, single-cell suspensions from neurospheres were cultured under differentiation conditions. Single-cell suspensions from neurospheres were generated by AccuMax treatment. Thereafter, cells were seeded onto poly-L-ornithine/laminin-coated coverslips at 5 x 105 cells per coverslip and cultured for up to 13 days under neural differentiation conditions. Subsequent to differentiation, cells were stained using the following antibodies: for neurons, primary mouse antibody against tubulin-β-III and secondary goat anti-mouse IgG Cy3-conjugated antibody; for astroglia, primary rabbit antibody against GFAP (Dako, Hamburg, Germany, http://www.dako.com) and secondary goat anti-rabbit IgG Cy3-conjugated antibody (Chemicon); and for oligodendrocytes, primary mouse antibody against O4 (R&D Systems) and secondary goat anti-mouse IgG Cy3-conjugated antibody (Chemicon). Nuclei were counterstained with DAPI. Percentages of neurons, astroglia, and oligodendroglia were assessed by differential counting total cell numbers (DAPI signals) and numbers of tubulin-β-III+, GFAP+, and O4+ cells. Mean values for AG and N neurosphere cells, as well as values for AG and blastocyst-derived cells, were analyzed by two-sided Student's t test. Values were considered statistically different if p < .05.
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RESULTS |
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TopFootnotesAbstractIntroductionMaterials and MethodsResultsDiscussionSummaryDisclosure of Potential...AcknowledgmentsReferences |
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As expected for AG cells, the frequency of dead or absorbed embryos increased from E12.5 to E14.5 for AG ESC-injected blastocysts. Following injection of AG ESCs, we detected that at E12.5 18 of 60 (30%) and at E14.5 15 of 40 (38%) embryos were dead or absorbed, whereas following N ESC injection at E12.5 6 of 36 (17%) and at E14.5 7 of 25 (28%) embryos were dead or absorbed. In addition, we assessed the size and appearance of embryos. The consequences of abnormal genomic imprinting associated with the AG cells in E14.5 chimeric embryos included overall larger size, as well as characteristic limb, head, and trunk growth distortion and evidence of organomegaly [10, 13] (supplemental online Fig. 1A, 1B).
Contribution of AG cells to fetuses was determined by flow cytometric analysis for eGFP expression in fetal livers, since previously it was shown that AG ESCs contribute to this organ [14]. Six independent injections of AG ESCs (four injections with line A1; two injections with line A2) yielded, in total, 42 live E12.5 embryos, of which 22 (52%) were chimeric. Heads of 8 of 22 AG chimeras were fixed and used for immunohistochemical analyses. From the brains of the remaining embryos, single-cell suspensions were prepared for further analysis of proliferation and differentiation properties (described below). In addition, of 30 living E12.5 embryos that developed out of four independent injections of N ESCs, 24 (80%) showed eGFP+ cells in the fetal liver. Fifteen of 24 E12.5 chimeric heads were processed for immunohistochemistry, and brain cell cultures were established from 9 of 24 embryos. Furthermore, 25 E14.5 embryos originating from eight independent injections of AG ESCs were analyzed, and 8 of 25 (32%) embryos showed eGFP+ cells in liver (data not shown). As controls, 18 embryos were generated by blastocyst injection of N ESCs, producing 9 (50%) chimeras. All E12.5 or E14.5 embryos with eGFP+ cells in liver also had eGFP+ cells present in the brain. Because of the elevated death rate of AG chimeras [10, 13], we did not analyze advanced developmental stages and focused on E12.5 chimeras.
Flow cytometric analysis revealed that AG ESC derivatives contributed substantially to the brains of fetal chimeras at E12.5 (supplemental online Fig. 2). We detected variation of ESC contribution between chimeras but no indication of exclusion of AG compared with N cells to fetal brain other than by stochastic variation [29]. For AG chimeric embryos, AG cell contribution ranged from 10% to 76% in the brain. The chimerism in N chimeric embryos varied from 1% to 38% in the brain.
To analyze the regional distribution of AG or N ESC-derived eGFP+ cells in E12.5 chimeric fetal brains, sagittal sections of AG and N chimeric brains were assessed for eGFP and tubulin-β-III signals. As shown in Figure 3A and 3B, AG and N ESC-derived cells displayed similarly widespread distribution in all brain regions. Sections of the striatum, hypothalamus, cortex, and brain stem region showed strong tubulin-β-III+ AG or N ESC-derived contribution (Fig. 3Aa– 3Ae, 3Ba– 3B3, insets). Staining of sagittal brain sections of AG and N chimeras for eGFP and neuronal nuclei (NeuN) also showed widespread NeuN+ AG or N ESC-derived cells contributing to these brain regions (supplemental online Fig. 3A, 3B).
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Cell Proliferation and Apoptosis in AG Chimeric Fetal Brains
To evaluate the proliferative and apoptotic properties of AG cells in chimeric brains, transverse brain sections were assessed for PCNA and cleaved-caspase-3, a crucial component of the apoptosis pathway [30, 31] (Fig. 4A, 4B). As expected at this early stage of embryonic development, brain tissues are highly proliferative, with almost all cells positive for PCNA, including eGFP+ AG ESC-derived cells (Fig. 4Ad, inset). We detected no colocalization of eGFP+ AG ESC-derived signals with cleaved-caspase-3 staining (Fig. 4Bd, inset) and, overall, a very low number of apoptotic cells.
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DISCUSSION |
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TopFootnotesAbstractIntroductionMaterials and MethodsResultsDiscussionSummaryDisclosure of Potential...AcknowledgmentsReferences |
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As shown for biparental ESCs, AG ESCs gave rise to pnPCs and neural and glial cells when subjected to appropriate culture conditions [22, 23, 32]. The analysis of methylation patterns of the imprinting control regions of Snrpn and Igf2r genes indicated that epigenetic marks did not change during neural in vitro differentiation. Gene expression analysis of a selected set of imprinted genes expressed in brain tissue [33] showed that AG pnPCs maintain parent-of-origin-specific gene expression for the majority of genes analyzed, with the exception of the maternally expressed Ube3a gene, which is substantially upregulated in AG-derived pnPCs. It is unclear whether expression of Ube3a in neural derivatives of AG ESCs is a consequence of changes in the imprinting control mechanisms for this gene during the in vitro differentiation process of AG ESCs or whether neural commitment is undergone by a subpopulation of AG ESCs with lack of silencing of this gene. Ube3a exhibits maternal-specific expression in some neural lineages [34] but is expressed biallelically in glia cells; differential imprinting is established during neurogenesis [35]. It remains to be determined to what extent these imprints are established in in vitro derivatives of AG ESCs.
The brain seeding capacity of AG ICM cells in E10–E12 embryos was previously described [17]. However, as whole brain extracts were analyzed for donor glucose-6-phosphate isomerase-1 isoform (Gpi1) contribution, this did not include analysis of regional distribution. In a second study, AG cells arising either from aggregation of AG and N morulae or from injection of AG ICM cells into N blastocysts were found to substantially contribute to the developing E13 and E17 hypothalamus but not to the cortex, and AG cell contribution was associated with smaller brain size [18]. The limited contribution of AG ICM cells to brain establishes an expectation that AG cells would show restricted neural differentiation potential compared with N cells. Despite the apparent phenotype and lethality in AG chimeras, our analyses of AG contribution to brain development at E12.5 show a widespread and balanced distribution of AG ESC-derived cells to forebrain regions, including striatum, hypothalamus, and cortex.
In addition, analyses of AG-derived compared with N-derived brain cells revealed no differences in NIC and neural differentiation frequencies. Although the ability of ICM-derived AG cells in fetal chimeras to form neurospheres is unknown, our findings indicate that AG ESCs and ICM cells differ in their developmental potential in vivo. One reason for this apparent discrepancy may be that during the establishment of ESCs from ICM cells, regulators of differentiation are modified or the epigenome undergoes changes [36–38].
We observed differences in the neuronal lineage differentiation frequencies between ESC-derived and in vitro-expanded fetal brain-derived cells. This difference in neuronal differentiation capacity is probably caused by the in vitro expansion of neurosphere cells over four to six passages. It was previously reported that ESC-derived neural progenitor cells maintain their neuronal potential during in vitro expansion, whereas E12.5 fetal brain-derived neural progenitor/stem cells do not [39]. In mixed whole brain cell cultures of AG and N chimeric embryos, we detected, on average, a minor growth disadvantage for both AG and N neurosphere cells in comparison with the blastocyst-derived cells. As both AG and N cells exhibit this feature, it is likely that this is caused by the different mouse strain backgrounds of ESCs and blastocysts or is due to eGFP transgene expression [40].
Uniparental cells of AG ESC origin, like N ESCs, can generate adult-transplantable hematopoietic stem cells that can repopulate the hematopoietic system of adult transplant recipients [14]. Likewise, in vitro differentiation of AG ESCs leads to neuronal and glial cell frequencies similar to those of N ESCs [21]. Furthermore, proliferation and differentiation properties of fetal brain-derived AG and N neurosphere cells did not differ. This indicates that, outside the normal developmental paradigm, the differentiation potential of uniparental ESCs may be much less restricted than that of uniparental cells in chimeras.
What are potential strategies for the generation of patient-specific stem cells, and what are the advantages of uniparental ESCs? Pluripotent cells have been isolated from several tissues, such as bone marrow (multipotent adult progenitor cells [MAPCs] [41]) and adult testes (spermatogonial stem cells [42, 43]). Although MAPCs were reported from human sources, human spermatogonial stem cells have so far not been described. Both cell types are pluripotent in vitro and in vivo; however, such cells are rare, and their full potential is unknown. A promising approach for producing autologous pluripotent cells is the reprogramming of somatic cells to ESC-like cells by the expression of defined factors in vitro (induced pluripotent stem [iPS] cells), which has been demonstrated in mouse [44] and also in human [45, 46]. Although iPS cell derivatives have been successfully used for hematopoietic tissue replacement in the mouse [47], the developmental and therapeutic potential of human iPS cells is uncertain, in particular as some human iPS cell lines exhibit limitations in their neural differentiation potential [46].
Uniparental ESCs may represent alternative sources for patient-specific pluripotent stem cells and are derived from gametic rather than somatic genomes. Both in mouse and in human, PG ESC lines with the full MHC complement of the oocyte donor have been derived [8, 12]. Generated without using fertilized eggs and without destroying fertilized human embryos, human PG ESCs have thus far shown a differentiation capability similar to that of stem cells derived from human embryos and are considered pluripotent. Although the generation of uniparental ESCs may circumvent the destruction of viable embryos, it still requires the manipulation and destruction of donated oocytes. Although the number of imprinted genes is small, many of them are expressed in the brain, affecting neurodevelopment, and thereby influence brain function and behavior [33]. Derivation of human AG ESCs remains to be demonstrated; however, the formation of hydatidiform moles provides some evidence for the early developmental potential of human AG conceptuses [48, 49].
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SUMMARY |
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TopFootnotesAbstractIntroductionMaterials and MethodsResultsDiscussionSummaryDisclosure of Potential...AcknowledgmentsReferences |
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST |
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TopFootnotesAbstractIntroductionMaterials and MethodsResultsDiscussionSummaryDisclosure of Potential...AcknowledgmentsReferences |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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-eGFP; ESC-derived cells), red (
