Differential Developmental Ability of Embryos Cloned from Tissue-Specific Stem C



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江边孤钓

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发表于 2009-3-5 00:54 |显示全部帖子

作者：Kimiko Inoue, Shinichi Noda, Narumi Ogonuki, Hiromi Miki, Shinichi Inoue, Kazufumi Katayama, Kazuyuki Mekada, Hiroyuki Miyoshi, Atsuo Ogura ( h7 Y! Z. k* v' y
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      【摘要】5 ^6 w: r7 {& j. w& w+ G
   Although cloning animals by somatic cell nuclear transfer is generally inefficient, the use of certain nuclear donor cell types may significantly improve or deteriorate outcomes. We evaluated whether two multipotent stem cell lines produced in vitro¡ªneural stem cells (NSCs) and mesenchymal stem cells (MSCs)¡ªcould serve as nuclear donors for nuclear transfer cloning. Most (76%) NSC-derived embryos survived the two-cell¨Cto¨Cfour-cell transition, the stage when the major zygotic gene activation occurs. Consistent with this observation, the expression patterns of zygotically active genes were better in NSC-derived embryos than in fibroblast clone embryos, which arrested at the two-cell stage more frequently. Embryo transfer experiments demonstrated that at least some of these NSC embryos had the ability to develop to term fetuses (1.6%, 3/189). In contrast, embryos reconstructed using MSCs showed a low rate of in vitro development and never underwent implantation in vivo. Chromosomal analysis of the donor MSCs revealed very frequent aneuploidy, which probably impaired the potential for development of their derived clones. This is the first demonstration that tissue-specific multipotent stem cells produced in vitro can serve as donors of nuclei for cloning mice; however, these cells may be prone to chromosomal aberrations, leading to high embryonic death rates. We found previously that hematopoietic stem cells (HSCs) are very inefficient donor cells because of their failure to activate the genes essential for embryonic development. Taken together, our data led us to conclude that tissue-specific stem cells in mice, namely NSCs, MSCs, and HSCs, exhibited marked variations in the ability to produce cloned offspring and that this ability varies according to both the epigenetic and genetic status of the original genomes.

Disclosure of potential conflicts of interest is found at the end of this article. 0 A% Q3 E: ~7 A' @
      【关键词】 Cloning Stem cell Genotype Chromosome Gene activation
   INTRODUCTION

Cloning animals by somatic cell nuclear transfer depends on many factors, most of which remain unknown. Cloning studies in different animal species have shown that the donor cell type is one of the most important factors determining the success of cloning . Because low Hdac1 expression level is an inherent characteristic of HSCs and is assumed to be related to their stem cell characters, the poor development of HSC-derived cloned embryos may be unique and not common to other stem cell clones. We were interested in investigating the developmental ability of embryos cloned from other stem cell types.& ]1 I  X2 z6 z5 u/ `! R
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For reliable nuclear transfer experiments, the donor cells for cloning must be identified precisely by their morphology or should be prepared as a suspension with nearly 100% purity. At present, the mouse stem cells that fulfill this requirement are neural stem cells (NSCs) and mesenchymal stem cells (MSCs), both of which can be established by selective culture in vitro and are fully capable of differentiating in vitro. In this study, we used NSCs and MSCs as nuclear donors for cloning experiments and examined the developmental potential of the resultant embryos in vitro and in vivo. We also performed gene expression analysis of the cloned embryos and karyotyped the donor cells to clarify the results.
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MATERIALS AND METHODS7 I4 v5 X: o" {

Preparation of Donor Cells
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We used male (C57BL/6 x 129/Sv-ter) F1 strain mice (called B6 x 129F1 for brevity) to prepare the donor cells. NSCs were obtained from the brains of fetuses at 12.5 days postcoitum as described previously . In brief, cells were dispersed by repeated pipetting in phosphate-buffered saline (PBS; pH 7.6), and were cultured in Dulbecco's modified Eagle's medium/Ham's F12 medium (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) containing 0.6% glucose, 100 µg/ml bovine transferrin (Invitrogen), 25 µg/ml bovine insulin (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), 10 µg/ml putrescine (Sigma), 30 nM sodium selenite (Sigma), 20 nM progesterone (Sigma), 20 ng/ml human epidermal growth factor (EGF; Sigma), and 20 ng/ml human fibroblast growth factor (FGF; Peprotech, Rocky Hill, NJ, http://www.peprotech.com). Cells were cultured for 1 month by changing the medium every week until neurospheres formed. They were further cultured for more than 1 month until other contaminating cells were depleted from neurospheres.

MSCs were obtained from bone marrow cells according to the method of Sun et al.  with slight modifications. Approximately 7.6 x 107 bone marrow cells were collected from four-week-old male mice and cultured in -minimal essential medium (Invitrogen) containing 10% fetal bovine serum. The medium was changed every 3 days. After four passages, nonhematopoietic cells were collected using a fluorescence-activated cell sorter Vantage SE (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com) as a cell population that was negative for an anti-CD45.2 antibody (eBioscience, San Diego, http://www.ebioscience.com/). Single cells were seeded onto wells of a 96-well plate, and putative MSCs were allowed to proliferate clonally. The cells were used for nuclear transfer shortly after cell line establishment (5 o- j. e5 n6 X
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The ability of the NSCs and MSCs to differentiate was tested in vitro before they were used for nuclear transfer experiments. For NSC differentiation, neurospheres were allowed to adhere to poly(L-ornithine) (Sigma)-coated plates (Lab-Tec chamber slides; Nunc, Roskilde, Denmark, http://www.nuncbrand.com) in EGF/FGF-free medium containing 2% bovine calf serum for 4 days . The NSCs proliferated, extended their neurites, and differentiated into neurons. Differentiated cell types were identified by staining using specific antibodies. NSC-derived differentiated cells were fixed in 4% paraformaldehyde in PBS at 25¡ãC for 30 minutes and washed thoroughly with PBS. The cells were permeabilized in 0.3% Triton X-100 in PBS for 5 minutes, washed in PBS, and treated with 10% normal goat serum in PBS for 1 hour. The primary antibodies used were as follows (dilutions in parentheses): rabbit anti-mouse MAP-2 polyclonal antibody (1:500¨C1:1,000; Chemicon, Temecula, CA, http://www.chemicon.com); mouse anti-GFAP monoclonal IgG1 (1:500; Chemicon); and mouse anti-O4 monoclonal IgM (1:73; Chemicon). After washing in PBS, the cells were treated with secondary antibodies as follows: Alexa Fluor 488-anti-rabbit IgG (1:400; Invitrogen); Alexa Fluor 594-anti-mouse IgG1 (1:400; Invitrogen); and Alexa Fluor 350-anti-mouse IgM (1:400; Invitrogen). After washing in PBS, the cells were observed under a fluorescence microscope.! o' [) [0 E2 w6 L/ w8 Q
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MSCs were induced to differentiate in vitro using methods reported previously (osteoblasts, adipocytes ). To identify the specific cell types, the differentiated cells were stained with a reaction mixture for alkaline phosphatase for osteoblasts (Nichirei Biosciences Inc, Tokyo, Japan, http://www.nichirei.co.jp/bio/english/index.html), Oil red O for adipocytes (Sigma), and Alcian blue (Sigma) for chondrocytes.

Fibroblasts as sources of control nuclei were obtained from the tail tips of adult (2¨C3 months old) male mice by confluent culture as described previously .' x' j, x8 y9 b" {! G% P9 q! d
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Oocyte Collection# P% H1 _7 F4 D+ W

Female B6D2F1 strain mice, 7¨C10 weeks old, were superovulated with 7.5 IU of pregnant mare serum gonadotropin and 7.5 IU of human chorionic gonadotropin (hCG) at 48-hour intervals and killed 16 hours after hCG injection. Mature meiosis stage II (MII) oocytes were collected from their oviducts. Cumulus cells were released in potassium-modified simplex-optimized medium (KSOM)  containing 0.1% hyaluronidase and washed several times with fresh medium. Oocytes were cultured in KSOM at 37.5¡ãC in an atmosphere of 5.5% CO2 in air until enucleation.' {/ W# |  x. Q: E5 R
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Nuclear Transfer/ N! ~+ d3 p6 z0 P
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Nuclear transfer was carried out as described previously . MII oocytes were placed in HEPES-buffered KSOM including 7.5 µg/ml cytochalasin B (Calbiochem, San Diego, http://www.emdbiosciences.com), and nuclei were removed with a small amount of cytoplasm. Enucleated oocytes were cultured in KSOM in an incubator (as above) for 30¨C60 minutes to allow the cell membrane to recover. NSCs and MSCs were enucleated using glass micropipettes and the nuclei of donor cells were injected into the ooplasm using a Piezo-driven micromanipulator (PrimeTech, Tsuchiura, Japan)./ R& ~* F$ n2 ~' y3 r7 x  k
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Adult fibroblasts were prepared from tail tips as reported . After nuclear transfer, reconstructed oocytes were cultured with KSOM for 1¨C2 hours and transferred into Ca2 -free KSOM, including 3 mM SrCl2 and 5 µg/ml cytochalasin B. One hour later, activated oocytes were transferred into KSOM containing only 5 µg of cytochalasin B and cultured further for 5 hours. After washing, the oocytes were cultured in fresh KSOM at 37.5¡ãC in an atmosphere of 5.5% CO2 for 48 hours., Q3 N$ C9 L; Y, g, [6 u5 s: t7 }
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Embryo Transfer/ s3 f2 H6 {- B7 Q, m: l3 e1 y) X

Reconstructed embryos that reached the 4¨C8-cell stage after 48 hours of culture in KSOM were transferred into the oviducts of pseudopregnant ICR strain female mice mated with vasectomized male mice the day before. On day 20, the recipient female mice were examined for the presence of fetuses, and live pups were nursed by lactating ICR female mice.

Chromosomal Analysis1 s% G; y5 o+ L* q3 \/ X' e* P

NSC and MSC cell lines established as described above were subjected to chromosomal analysis. NSCs and MSCs in culture dishes were treated with 25 ng/ml colcemide for 30 minutes, and the round cells (composed mostly of cells in metaphase) were collected, spread onto clean glass slides, and allowed to dry in air. Q-banding staining was performed by a combined quinacrine-33258 Hoechst method . Metaphase images were observed under a fluorescent microscope (Axio Photo 2; Carl Zeiss, Jena, Germany, http://www.zeiss.com) and karyotype analysis was performed using an Ikaros karyotyping system (Carl Zeiss).
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Gene Expression Analysis5 Q* I. [, a% i+ L$ B
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We selected six zygotic genes, Dppa2, Dppa3 (Stella or PGC7), Dppa4, ERV-L, Hdac1, and eIF-1A, based on previous studies on global or specific gene expression  were treated with acid Tyrode's solution to remove the zona pellucida, and cDNA was extracted using Cell-to-cDNA II kits (Ambion, Austin, TX, http://www.ambion.com). PCR products amplified with the primers in Table 1 were diluted serially and used as external standards for quantitative real-time PCR. Measurements of gene expression levels were carried out using an ABI7900HT Sequence Detection system (Applied Biosystems, Foster City, CA, http://www.appledbiosystems.com) with QuantiTect Syber Green or QuantiTect Probe PCR kits (QIAGEN, Hilden, Germany, http://www.qiagen.com).

Table 1. Primer sequence for gene expression analysis) r5 V  v  E8 C  ]. E% p, r
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Statistical Analysis  x2 X% M" {2 N: i1 B, V" J) Q
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Development rates of embryos in vitro and in vivo were compared between groups using Fisher's exact probability test. The relative transcription levels of embryos or donor cells determined by quantitative real-time PCR were analyzed by one-way analysis of variance followed by a post hoc procedure using Scheff¨¦'s F test for multiple comparisons between groups where appropriate. All animals were maintained and used for experiments in accordance with the guidelines of the RIKEN Institute, Japan.( E& u% w$ G9 T9 E0 V8 N8 w

RESULTS; e" C2 D8 w7 u3 A

Determination of Pluripotency of the Donor NSCs and MSCs
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Before the cloning experiments, we characterized the donor NSC and MSC lines for their ability to differentiate in vitro. Under appropriate culture conditions, NSCs differentiated into neurons, astrocytes, and oligodendrocytes, and MSCs differentiated into adipocytes, osteoblasts, and chondrocytes (Fig. 1). Thus, the stem cell lineages used here had differentiation potentials similar to those reported elsewhere .
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Figure 1. In vitro differentiation of neural stem cells (NSCs) and mesenchymal stem cells (MSCs) used as nuclear donors in this study. (A): Undifferentiated NSC neurospheres. (B): NSC-derived neurons (anti-MAP2 staining). (C): NSC-derived astrocytes (anti-glial fibrillary acidic protein staining). (D): NSC-derived oligodendrocytes (anti-O4 staining). (E): Undifferentiated MSC cells. (F): MSC-derived adipocytes (oil-red O staining). (G): MSC-derived osteoblasts (alkaline phosphatase staining). (H): MSC-derived chondrocytes (Alcian blue staining). Scale bar, 100 µm.( u- x3 G% T1 r2 S9 d- z7 N0 H2 ]

Development of NSC and MSC Cloned Embryos( n- |" ~3 H) W. ^9 p

Cloned embryos reconstructed with NSC or MSC nuclei were cultured in vitro for 48 hours until they should have reached the four-cell stage. As shown in Table 2, more than half of the reconstructed embryos developed to the two-cell stage, whereas the remaining embryos did not divide. Because these one-cell-arrested embryos had formed pseudopronuclei successfully from the donor nuclei, the likely cause of their arrest was cell cycle asynchrony between the donor cell nucleus and the recipient ooplasm, as reported for cloning with ES cells and immature Sertoli cells .

Table 2. In vitro and in vivo development of embryos cloned from NSCs, MSCs, and adult fibroblasts' R: E) n* d0 U3 O

Among the two-cell embryos, those derived from NSCs showed a higher rate of growth to the four-cell stage (75.9%) than those from MSCs (45.9%) or fibroblasts (41.7%) (p # M( o! M) {: Y, _4 i

Figure 2. Cloned mouse pups born after nuclear transfer using neural stem cells (NSCs) as donors. Shortly after Caesarian section at full term, two pups recovered their movement and respiration.

Chromosomal Analysis of Donor Cells+ }! E7 a& r: ~4 _; v: I
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We examined the chromosome constitutions of the MSC line used in this study. Fifty-two (67%) of the 78 metaphase chromosome spreads examined had 41 chromosomes because of monosomy 4, trisomy 6, and two Y chromosomes (Fig. 3A). The remaining 26 spreads showed the normal number of chromosomes (2n = 40 in the mouse), but they also had aneuploidy of monosomy four and trisomy six (Fig. 3B). Heteromorphisms were often observed on chromosome 16.

Figure 3. Cytogenetic analysis of neural stem cell (NSC) and mesenchymal stem cell (MSC) lines. (A, B): The two chromosome types found in the MSC line used for nuclear transfer. Some MSCs had 41 chromosomes with monosomy 4, trisomy 6, and two Y chromosomes (arrowheads in A). Others had the normal number of chromosomes (2n = 40), but they also had the same monosomy 4 and trisomy 6 (arrowheads in B). Heteromorphism was observed on chromosome 16 in both types (arrows in A and B). The chromosomes of MSCs were especially prone to morphological and numerical abnormalities. (C): The distribution of the cells classified according to the chromosome numbers in different MSC lines. All MSC lines comprise cells with abnormal chromosome numbers. (D): The distribution of the cells classified according to the chromosome numbers in two NSC lines. In contrast to the MSC lines, NSC lines comprise predominantly cells with the normal ploidy (2n = 40).

We investigated five other MSC lines and found highly frequent abnormal ploidy in all (Fig. 3C). Their chromosomal patterns were more severely affected compared with that of the donor MSC line (Fig. 3A, 3B), probably because of their longer culture in vitro (additional 1 to 2 months in culture). In contrast, the NSC line had relatively normal ploidy levels (Fig. 3D).

Gene Expression Patterns in NSC Embryos# R% z5 s6 ~1 Y" y% a

Because zygotic genes are programmed to activate at specific stages during preimplantation development, their expression pattern is a good indicator of the success of genomic reprogramming by nuclear transfer. We analyzed the expression levels of six genes by real-time quantitative PCR using two-cell NSC-derived cloned embryos, control fibroblast-derived cloned embryos, control IVF embryos, and MII oocytes. As shown in Figure 4, the gene expression patterns of NSC-derived embryos were similar to those of IVF embryos in all genes examined except Hdac1, which was more actively expressed in the NSC-derived embryos than in IVF embryos (p / Q2 A+ K; x6 K7 N2 v8 a" X
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Figure 4. Quantification by real-time reverse transcriptase-polymerase chain reaction of mRNA expression of various zygotic-activated genes in single oocytes and embryos. Genotype (B6 x 129F1)-matched two-cell IVF embryos, two-cell Fi embryos, and two-cell NSC-derived clone embryos were analyzed. MII oocytes were derived from B6D2F1 females, as in the nuclear transfer experiments. Each dot represents a single embryo. Values are expressed relative to those in the IVF group (value = 1). Values with different letters differ significantly (p

DISCUSSION
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We aimed to evaluate whether tissue-specific stem cells, NSCs and MSCs, could be used as nuclear donor cells for cloning mice. Because we undertook detailed analysis for the effects of the donor cell type and genotype on cloning efficiency , the standard oocyte donors for mouse cloning experiments. In this study, therefore, we used B6 x 129F1 male mice as the source of NSCs and MSCs, and we now add the former to the clonable cell type list in mice. Table 3 shows the efficiencies of cloning mice using seven cell types with the male B6 x 129F1 genotype. These data show apparent cell type-specific differences in cloning efficiency ranging from 0% to 9.4% per embryo transferred. In view of the rates of four-cell embryos (per two cells) and offspring (per transfer), NSCs seem to be moderately efficient sources of nuclei for transfer.* c4 q% Q- x2 v% Y- @* ^) H# i

Table 3. Efficiencies of cloning male mice from different cell types with the B6 x 129F1 genotype. q" w" u7 y; j$ ^1 q- Z

After nuclear transfer into the ooplasm, the donor somatic cell genome should be reprogrammed to a state equivalent to that of a fertilized embryo for further development. During the first and second cell cycles in cloned mouse embryos, this reprogramming is manifested in the structural remodeling of the donor nucleus into pseudopronuclei and the initiation of embryo-specific transcription, termed zygotic gene activation (ZGA) . These findings show clear associations between the expression levels of certain genes and subsequent embryonic development. Taken together, we expect that the reprogrammability of the different donor cells can be assessed as early as at the two-cell stage by analyzing the expression of appropriate genes as indicators.! n# }4 q. E8 T9 G) t# F! c6 z9 o
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In general, genetic factors as well as epigenetic factors may considerably affect the development of cloned embryos. The implantation failure found here for MSC-derived embryos is strongly suggestive of chromosomal abnormalities, as documented by Bosch et al. .( f; n# ], \6 U# a3 O' v0 {
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We conclude that tissue-specific stem cells in mice, namely NSCs, MSCs, and HSCs, can show marked variations in their ability to produce cloned offspring, according to both the epigenetic and genetic status of their original genomes.+ s7 W8 {" `) u0 w

NOTE ADDED IN PROOF

Very recently, cloning mice from neonatal neural stem cells has been reported by Mizutani et al. .) y3 h" g9 }, T, C$ i+ s6 d0 Y; X

DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

The authors indicate no potential conflicts of interest.8 {  G1 A6 ^1 e/ j" [# F
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ACKNOWLEDGMENTS! @9 K' E# t, y6 Z

This research was supported by grants from MEXT, MHLW, CREST, and the Human Science Foundation in Japan. K.K. is currently affiliated with The Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan.: m6 W4 o& E9 [9 K' k6 b9 F
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