Parthenogenetic embryonic stem cells derived from cryopreserved newborn mouse ovaries: a new approach to autologous stem cell therapy

Transcription

1 Parthenogenetic embryonic stem cells derived from cryopreserved newborn mouse ovaries: a new approach to autologous stem cell therapy Fengying Xing, Ph.D., a Zhenfu Fang, Ph.D., a Bolin Qin, M.S., a Yao Li, Ph.D., a Jian Hou, Ph.D., b and Xuejin Chen, Ph.D. a a Center for Developmental Biology, Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China; and b College of Biological Sciences, China Agricultural University, Beijing, People s Republic of China Objective: To evaluate whether cryopreserved newborn mouse ovaries can generate sufficient numbers of parthenogenetic mouse embryonic stem (pmes) cells for autologous stem cell therapy. Design: Prospective study. Setting: Reproductive clinic of Xinhua Hospital in Shanghai. Animal(s): Kunming, C57BL/6J, BALB/c, and NOD-SCID mice. Intervention(s): Cryopreserved newborn mouse ovaries were thawed, grafted into immunodeficient mice, treated with pregnant mare serum gonadotropin to promote follicular maturation, and collected oocytes activated in vitro to generate parthenogenetic embryonic stem cells. Main Outcome Measure(s): Preimplantation development and stem cell characterization. Result(s): This new protocol yielded a large number of oocytes from cryopreserved ovaries over a long period. These oocytes were used to derive pmes cell lines, which expressed embryonic stem cell specific markers and differentiated into embryoid bodies in vitro and teratomas in vivo. The pmes cell line was propagated in an undifferentiated state for more than 30 passages and maintained a diploid karyotype. Conclusion(s): The pmes cells lines established by our protocol exhibited the same degree of pluripotency as standard embryonic stem cell lines. This approach may be used for exploring autologous stem cell therapies. (Fertil Steril Ò 2009;91: Ó2009 by American Society for Reproductive Medicine.) Key Words: Newborn mouse ovaries, cryopreservation, parthenogenetic embryonic stem cells It has been suggested that somatic cell nuclear transfer techniques can produce embryonic stem (ES) cells for stem cell therapy without the risk of immune rejection. However, somatic cell nuclear transfer requires complex techniques and culture conditions and is accompanied by numerous ethical issues. In contrast, parthenogenesis involves embryos derived directly from unfertilized oocytes. Previous work demonstrated that parthenogenetic embryos always arrest in the early limb bud stage (1) and that they do not produce live offspring. Therefore, parthenogenetic ES cells used in Received October 23, 2007; revised January 8, 2008; accepted January 14, 2008; published online March 18, F.X. has nothing to disclose. Z.F. has nothing to disclose. B.Q. has nothing to disclose. Y.L. has nothing to disclose. J.H. has nothing to disclose. X.C. has nothing to disclose. Supported by grants from the National Basic Research Program of China (973 Program) (nos. 001CB509903, 001CB509904), Committee of Shanghai Agriculture (no. 5-3), and Shanghai Jiao Tong University, School of Medicine. Drs. Xing and Fang contributed equally to this work. The present address for Drs. Xing, Fang, Li, and Chen is Department of Laboratory Animal Science, School of Medicine, Shanghai Jiao Tong University, 280 South Chongqing Road, Shanghai , China. Reprint requests: Xuejin Chen, Ph.D., Center for Developmental Biology, Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University, 1665 Kongjian Road, Shanghai , People s Republic of China (FAX: ; chenxuej@yahoo.com.cn). stem cell therapy avoid ethical disputes (2). A parthenogenetic ES cell line from nonhuman primates was first established using ovulated oocytes (3). Recently, Lee et al. (2) reported that autologous ES cells can be obtained using mouse preantral follicles cultured in vitro and oocyte parthenogenesis. It has been shown that primordial ovarian follicles survive the cryopreservation process for a long time and maintain their fertility (4 7). Therefore, we chose to use cryopreserved newborn mouse ovaries as a source of oocytes for establishing parthenogenetic ES cells. The procedures included cryopreservation of newborn mouse ovaries, thawing them, grafting the ovaries under the kidney capsule of immunodeficient mice, promotion of follicular maturation using pregnant mare serum gonadotropin (PMSG), and in vitro parthenogenetic activation of oocytes matured in vitro. With this new approach, large numbers of oocytes were harvested from newborn mouse ovaries and activated parthenogenetically to derive parthenogenetic mouse ES (pmes) cell lines for use in autologous cell therapy. The pmes cell lines generated by these procedures have not yet been reported. MATERIALS AND METHODS Experimental Animals Kunming, C57BL/6J, NOD-SCID, and BALB/c mice were obtained from the Shanghai Laboratory Animal Center, Chinese 1238 Fertility and Sterility â Vol. 91, No. 4, April /09/$36.00 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

2 Academy of Sciences. The institutional review board of the School of Medicine at Shanghai Jiao Tong University approved the animal care protocols used in this study. The mice were housed with food and water ad libitum and a 14- hour/10-hour light/dark cycle. Cryopreservation, Thawing, and Transplantation of Mouse Ovaries The protocol for the cryopreservation of ovaries was reported previously (8). In our studies, 120 ovaries from 60 mice were removed from (C57BL/6J BALB/c) F1 newborn mice (black skin) and immediately placed in L-15 medium (Gibco, Carlsbad, CA). After removal of the bursal membranes under a stereomicroscope, ovaries were transferred to FL-15 medium (L-15 medium supplemented with 10% [vol/vol) fetal bovine serum [FBS) and 1.5 mol/l dimethyl sulfoxide) and placed in a 0.25-mL plastic tube, both ends of which were sealed with polyvinyl chloride powder. The cryopreserved ovaries were thawed in a 37 C water bath for seconds and then flushed out and washed several times. NOD-SCID mice were used as recipients for ovarian transplantation. The mice were anesthetized, an incision was made in the abdominal wall to expose each kidney, and one thawed ovary was transplanted under the capsule of each. A total of 40 thawed ovaries were transplanted into 20 NOD-SCID mice. In Vivo Oocyte Development At 19 days after transplantation, the mice receiving ovary transplants were injected with 10 IU PMSG. At hours after injection, 33 ovaries were removed from under the kidney capsule. The enlarged follicles were punctured, and 423 oocyte cumulus cell complexes were collected and washed three times in L-15 medium, followed by incubation in mature culture medium for 17 to 18 hours at 37 C in a humidified chamber with 5% CO 2. The mature culture medium consisted of a-minimal essential medium (Gibco) containing 3 mg/ml bovine serum albumin (BSA), 1 mg/ml fetuin, 1 ng/ml epidermal growth factor, and 100 ng/ml recombinant FSH. Parthenogenetic Activation of Oocytes Three hundred ninety-four oocytes that expelled the first polar body after 17 to 18 hours of culture in vitro were chosen for parthenogenetic activation. The mature oocytes were activated parthenogenetically by exposure to 7% ethanol for 5 minutes at room temperature. The presumptive zygotes were cultured in Chatot, Ziomek, Bavister medium (9) to permit further development. After 24 hours in culture, 257 embryos developed to the two-cell stage. After approximately 96 hours of activation, 51 embryos developed into blastocysts. Five blastocysts with good morphology were selected under a stereomicroscope (Olympus, Tokyo, Japan) to facilitate the establishment of ES cells. Establishment of pmes Cell Lines The zona pellucida of the five parthenogenetic blastocysts was removed using Tyrode s solution. The inner cell mass (ICM) was isolated manually and cultured on irradiated mouse embryonic fibroblasts. The culture medium consisted of 78% Dulbecco s modified Eagle s medium (DMEM) (Gibco), 15% FBS (HyClone), 2 mmol/l b-mercaptoethanol, 1 mmol/l glutamine, 1% nonessential amino acids, and 1,000 U/mL mouse leukemia inhibitory factor. Four to five days after the initial culture, cells growing out of the ICM were manually disrupted into small patches that were transferred to dishes containing irradiated fresh mouse embryonic fibroblasts. Parthenogenetic mouse ES cells were passaged using 0.05% trypsin/ethylenediaminetetraacetic acid and plated at cells/ml. Karyotyping Parthenogenetic mouse ES cells were incubated with nocodazole (100 ng/ml) for 2 hours at 37 C in a humidified atmosphere containing 5% CO 2. Cells were trypsinized and collected by centrifugation at 1,000 rpm for 5 minutes (Eppendorf centrifuge, 5810 R), followed by resuspension in 5 ml of hypotonic solution (0.075 mol/l KCl), incubation at 37 C for 15 minutes, the addition of 1 ml of fixative (1:3 acetic acid:methanol), and incubation at room temperature for 5 minutes. Cells were collected and resuspended in 5 ml of fixative; the previous steps were then repeated twice. After the final step, cells were kept in the fixative, shaken gently to yield a cell suspension, and dropped onto prechilled glass slides. Chromosome spreads were stained using 1% Giemsa solution. At least 20 photographs of metaphase spreads were counted. Reverse Transcriptase Polymerase Chain Reaction Analysis Total RNA was extracted from pmes cells using TRIzol reagent (Invitrogen, Carlsbad, CA). Genomic DNA was degraded by treatment with DNAse I at 37 C for 20 minutes. First strand synthesis was performed using M-MLV reverse transcriptase (Promega, Madison, WI) using random primed synthesis according to the manufacturer s instructions. Polymerase chain reaction (PCR) reactions used the following parameters: a denaturation step (94 C, 4 minutes), 35 amplification cycles consisting of a denaturation step (94 C, 30 seconds), a primer annealing step (30 seconds), and an extension step (72 C, 40 seconds), and a final extension step (72 C, 5 minutes). Annealing temperatures were 55 C for Oct-4 and 60 C for Nanog. As a negative control for genomic DNA contamination, total RNA was subjected to reverse transcriptase- PCR (RT-PCR) in the absence of the reverse transcriptase enzyme, followed by PCR reactions using the PCR primer sets. Beta-actin was used as an internal standard. The primer sequences of Oct-4 and Nanog are shown in Table 1. Fertility and Sterility â 1239

3 TABLE 1 Primers used in RT-PCR experiments. Primer Oct-4 Sense:5 0 -TGAGAACCTTCAGGAGATATGCAA-3 0 Antisense:5 0 -CTCAATGCTAGTTCGCTTTCTCTTC-3 0 Nanog Sense:5 0 -CAGAAAAACCAGTGGTTGAAGACTAG-3 0 Antisense:5 0 -GCAATGGATGCTGGGATACTC-3 0 NF Sense: 5 0 -GCAGACATTGCCTCCTACC-3 0 Antisense: 5 0 -TCACTCCTTCCGTCACCC-3 0 Nestin Sense: 5 0 -GTCTGATGGGTTTGCTGA-3 0 Antisense: 5 0 -AATCGCTTGACCTTCCTC-3 0 BMP4 Sense: 5 0 -ACTGCCGTCGCCATTCAC-3 0 Antisense: 5 0 -CACCACCTTGTCATACTCATCC-3 0 Myoglobin Sense: 5 0 -CCGCCAAGTACAAGGAGC-3 0 Antisense: 5 0 -CATCTCGCCCAAAGGTCA-3 0 AFP Sense: 5 0 -ATTCCTCCCAGTGCGTGAC-3 0 Antisense: 5 0 -GCAGTCTTCTTGCGTGCC-3 0 Albumin Sense: 5 0 -AGCCCGAAAGAAACGAAT-3 0 Antisense: 5 0 -GACGGACAGATGAGACCAAT-3 0 Immunocytochemistry Parthenogenetic mouse ES cell colonies were washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 15 minutes. Colonies were washed twice in TBST buffer (20 mmol/l Tris-HCl, ph 7.4, 0.15 mol/l NaCl, 0.05% Tween-20), treated with 0.1% Triton X-100/PBS for 10 minutes, washed twice in TBST buffer, and incubated in blocking solution (3% BSA/PBS) for 1 hour at room temperature. The primary antibodies used in these experiments were anti-ssea-1 (1:40), anti-ssea-3 (1:40), anti-ssea-4 (1:40) (Chemicon, Temecula, CA), and Oct-4 (1:100) (Santa Cruz Biotechnology, Santa Cruz, CA). All antibodies were diluted in 3% BSA/PBS, and samples were incubated overnight at 4 C. The following day, colonies were washed twice with TBST and incubated with secondary antibodies at room temperature for 30 minutes. Secondary antibodies (1:100) corresponding to the primary antibodies were fluorescein isothiocyanate conjugate (FITC)- conjugated goat anti-mouse IgG plus IgM, FITC-conjugated goat anti-rat IgG plus IgM, FITC-conjugated donkey anti-mouse IgG, and FITC-conjugated donkey anti-goat IgG (Jackson, West Grove, PA), respectively. The mouse ES cell line D3 was used as a positive control, and a negative control group was added with isotype antibody replacing the primary antibody. Fluorescent signals were visualized with a confocal microscope (Olympus). To detect alkaline phosphatase activity, pmes cell colonies were fixed in 4% paraformaldehyde, rinsed in Tris buffer (100 mmol/ L Tris-HCl, 100 mmol/l NaCl, 50 mmol/l MgCl 2,pH9.5),and reacted with NBT/BCIP in the dark. The reaction was stopped when the color had developed sufficiently. In Vitro Differentiation of pmes Cells Parthenogenetic mouse ES cells were harvested after 0.05% trypsin digestion and resuspended in a nonadherent Petri dish containing culture medium (78% DMEM, 20% FBS, 2 mmol/l L-glutamine, 1% nonessential amino acids, and 0.1 mmol/l b-mercaptoethanol); after 4 6 days in culture, the pmes cells formed embryoid bodies. The culture medium was changed every other day. To induce further differentiation, embryoid bodies were transferred to gelatin-coated culture dishes. After approximately 3 weeks in culture, a variety of morphologically distinct cells grew from the embryoid bodies. Total RNA extracted from these cells was analyzed by RT-PCR to determine whether genes associated with each of the three germ layers were expressed: neurofilament (NF), nestin, bone morphologic protein 4(BMP4), myoglobin, alpha-fetoprotein (AFP), and albumin. The RT-PCR primer sets are shown in Table 1. In Vivo Differentiation of pmes Cells Approximately morphologically undifferentiated pmes cells were injected into the thigh muscle of 4-weekold NOD-SCID mice. Tumor masses that formed at the injection site were dissected, fixed in 4% paraformaldehyde, processed for paraffin sectioning, and stained with hematoxylin and eosin for histologic examination. RESULTS Development of Transplanted Ovaries and Maturation of Oocytes Newborn mouse ovaries grafted under the kidney capsule of immunodeficient mice increased in size and formed large follicles visible on the surface of the ovary (Fig. 1Aa). Of 40 grafted ovaries, 33 (82.5%) were retrieved. We collected 423 oocytes from the 33 ovaries, retrieving approximately 13 oocytes from each one. Of 423 mature oocytes cultured 1240 Xing et al. pmes cells derived of newborn ovaries Vol. 91, No. 4, April 2009

4 FIGURE 1 Development of oocytes, cellular morphology, and the karyotype of a pmes cell line derived from cryopreserved ovaries transplanted under the kidney capsule of immunodeficient mice. (A) Development of oocytes derived from cryopreserved ovaries transplanted under the kidney capsule of immunodeficient mice. (a) Histologic section of an ovary transplanted under the kidney capsule showed many antral follicles, hematoxylin and eosin staining. Bar ¼ 1 mm. (b, c) Micrographs of oocytes matured in vitro and blastocysts activated parthenogenetically. Bar ¼ 50 mm. (B) Cellular morphology and the karyotype of a pmes cell line. (a) Micrograph of a colony of pmes cells. Bar ¼ 50 mm. (b) Karyotype of a pmes cell line (diploid: 40 chromosomes), Giemsa staining. Bar ¼ 10 mm. in vitro for 17 to 18 hours, 173 (40.9%) underwent germinal vesicle breakdown, and 154 (89.0%) progressed to the meiosis II stage (Fig. 1Ab). were propagated for more than 30 passages. The morphology resembled normal ES cells. The molecular characteristics and multi-differentiation capacity were analyzed in one of these cells lines at passages Establishment of pmes Cell Lines After parthenogenetic activation, 257 of 394 embryos developed to the two-cell stage, and the cleavage rate was 65.2% after 24 hours in culture. Fifty-one embryos developed to blastocysts after another 3 days in culture (Fig. 1Ac). The rate of blastocyst development of cleavage embryos reached 19.8%. Five ICM cells from parthenogenetic blastocysts were transferred to a mouse feeder cell layer. After 4 to 5 days in culture, these cells underwent marked expansion and formed colonies. Analysis of cellular morphology showed a high nuclear cytoplasmic ratio with prominent nucleoli, and the colonies had clear edges (Fig. 1Ba). Three pmes cell lines derived from five parthenogenetic blastocysts were established. The cloning rate was 60%. The pmes cell lines Karyotypic Analysis and Gene Expression of pmes Cells The results of karyotypic analyses showed that 31% of the pmes cells were normal diploid (Fig. 1Bb). In addition, RT-PCR showed that they expressed Oct-4 and Nanog (Fig. 2A). As shown in Fig. 2B, the pmes cells expressed Oct-4 and SSEA-1, but not SSEA-3 or SSEA-4. In addition, the cell line had a high level of alkaline phosphatase activity (Fig. 2C). Embryoid Bodies and Teratomas Derived from pmes Cell Lines Embryoid bodies derived from the pmes cell line were round, and the surface was smooth (Fig. 3A). Cells growing out from Fertility and Sterility â 1241

5 FIGURE 2 Characterization of pmes cells. (A) Expression of Oct-4 and Nanog transcripts in pmes cells. Oct-4 and Nanog transcripts were expressed in pmes cells. As a control for genomic DNA contamination, the RT-PCR reaction was carried out in the absence of the reverse transcriptase enzyme [pmes(-)], followed by PCR using the appropriate primer sets. Beta-actin RT-PCR reactions served as a loading control. (B) Immunofluorescence staining to determine the expression of Oct-4, SSEA-1, SSEA-3, and SSEA-4 in pmes cells. Oct-4 and SSEA-1 were expressed, but SSEA-3 and SSEA-4 were not. Isotype antibody replaced the primary antibody in negative control samples, although all other steps were the same. Bar ¼ 50 mm. (C) Photomicrographs showing alkaline phosphatase activity in pmes cells. Bar ¼ 100 mm. the embryoid bodies were highly heterogeneous in morphology. Reverse transcriptase polymerase chain reaction analyses using primer sets specific for NF or nestin (ectoderm), BMP4 or myoglobin (mesoderm), and AFP or albumin (endoderm) showed that these cells expressed marker genes representative of all three germ layers (Fig. 3B). One of three pmes cell lines was injected into NOD- SCID mice. The three mice tested all developed teratomas weeks after injection. Pathologic examination showed that they contained multi-differentiated tissues, including keratinized stratified squamous epithelium, neural epithelium (ectoderm), bone (mesoderm), and pseudostratified ciliated columnar epithelium (endoderm) (Fig. 3C). DISCUSSION This study demonstrated that newborn mouse ovaries cryopreserved and grafted into NOD/SCID mice after PMSG injection produced large numbers of mature oocytes. These mature oocytes were activated parthenogenetically in vitro to develop into blastocysts. Three pmes cell lines were successfully established from the ICM of parthenogenetic blastocysts. The success rate was 60%. The pmes cells had the same morphologic characteristics as mouse ES cells and were propagated in an undifferentiated state for more than 30 generations. Cells growing out of the embryoid bodies of pmes cells expressed transcripts corresponding to all three germ layers: NF, nestin (ectoderm), BMP4, myoglobin (mesoderm), AFP, and albumin (endoderm). After being injected 1242 Xing et al. pmes cells derived of newborn ovaries Vol. 91, No. 4, April 2009

6 FIGURE 3 Differentiation of pmes cells in vivo and in vitro. (A) Micrographs showing embryoid body formation by cultured pmes cells. Bar ¼ 100 mm. (B) Reverse transcriptase-polymerase chain reaction showing that differentiated cells were derived from pmes cells; the differentiated cells expressed genes corresponding to each of the three embryonic germ layers (lane 1): NF and nestin (ectoderm); BMP4 and myoglobin (mesoderm); AFP and albumin (endoderm). Lane 2 is a negative control, and b-actin was used as a loading control. (C) Histologic analysis of teratomas derived from pmes cells injected into the thigh muscle of immunocompromised mice. Teratomas at the injection site contained multi-differentiated tissues. (a, b) Ectoderm (keratinized stratified squamous epithelium, neural epithelium), (c) mesoderm (cartilage), and (d) endoderm (pseudostratified ciliated columnar epithelium), hematoxylin and eosin staining. Bar ¼ 100 mm. into immunodeficient mice, the pmes cells formed teratomas that contained cell populations with characteristics of all three germ layers. Therefore, our protocol provided a large number of mature oocytes and produced pmes cell lines. This sets up a new means of exploring autologous stem cell therapy. Previous studies demonstrated that primary mouse oocytes can be frozen without inducing injury (10); however, they mature slowly in the preantral follicles and show a low rate of development after in vitro activation (2). Liu et al. (7) reported that growth of preantral follicles under the kidney capsule requires 14 days, followed by 12 days of in vitro culture in the presence of hcg, when germinal vesicle breakdown occurs. To expedite this process, newborn mouse ovaries were cryopreserved and grafted into immunodeficient mice after thawing. The mice were treated with PMSG at day 19 after grafting. At hours after injection, large numbers of mature oocytes appeared in the grafted ovaries. The mature oocytes removed from under the kidney capsule developed into antral follicles, thereby reducing the time required for culture in vitro. Although the maturation rate Fertility and Sterility in vitro did not increase, the cleavage rate of parthenogenetically activated oocytes was 65.2%, and the development rate to blastocysts was 19.8%. Both of these rates were higher than those reported in the literature (21% and 18%, respectively [2]). Chimera tests have been used to determine the degree of totipotency of ES cell lines (11). We carried out chimera experiments using passage mouse ES cells established with the same protocol as the pmes cells, and chimeric mice with mixed colors were produced (data not shown). However, chimera experiments with pmes cells did not generate live chimeric mice. Perhaps further experiments are needed to show the ability of pmes to make chimeras. But fertilization experiments in vitro with the mature mouse oocytes prepared using the procedures described above did successfully produce live offspring (data not shown). Therefore, the new protocol established in our laboratory provides a robust approach for obtaining mature mouse oocytes and parthenogenetic ES cells and may contribute to the development of future autologous stem cell therapies. 1243

7 REFERENCES 1. Lerou P, Yabuuchi A, Lengerke C, Ng K, West J, Kirby A, et al. Histocompatible embryonic stem cells by parthenogenesis. Science 2007;315: Lee ST, Choi MH, Lee EJ, Gong SP, Jang M, Park SH, et al. Establishment of autologous embryonic stem cells derived from preantral follicle culture and oocyte parthenogenesis. Fertil Steril. In press. 3. Cibelli JB, Grant KA, Chapman KB, Cunniff K, Worst T, Green HL, et al. Parthenogenetic stem cells in nonhuman primates. Science 2002;295: Hovatta O, Silye R, Krausz T, Abir R, Margara R, Trew G, et al. Cryopreservation of human ovarian tissue by using dimethylsulphoxide and propanediol-sucrose as cryoprotectants. Hum Reprod 1996;11: Newton H, Aubard Y, Rutherford A, Sharma V, Gosden RG. Low temperature storage and grafting of human ovarian tissue. Hum Reprod 1996;11: Gook DA, Edgar DH, Stern C. Effect of cooling rate and dehydration regimen on the histological appearance of human ovarian cortex following cryopreservation in 1,2-propanediol. Hum Reprod 1999;14: Liu J, Van der Elst J, Van den Broecke R, Dhont M. Live offspring by in vitro fertilization of oocytes from cryopreserved primordial mouse follicles after sequential in vivo transplantation and in vitro maturation. Biol Reprod 2001;64: Gosden RG. Low temperature storage and grafting of human ovarian tissue. Mol Cell Endocrinol 2000;163: Chatot CL, Ziomek CA, Bavister BD, Lewis JL, Torres I. An improve culture medium supports development of random bred 1-cell mouse embryos in vitro. J Reprod Fertil 1989;86: Carroll J, Gosden RG. Transplantation of frozen-thawed mouse primordial follicles. Hum Reprod 1993;8: Rossant J. Stem cells from the mammalian blastocyst. Stem Cell 2001;19: Xing et al. pmes cells derived of newborn ovaries Vol. 91, No. 4, April 2009