Derivation of Cloned Human Blastocysts by Histone Deacetylase Inhibitor Treatment After Somatic Cell Nuclear Transfer with β-Thalassemia Fibroblasts

Abstract

Derivation of embryonic stem cells from patient-specific cloned blastocysts by somatic cell nuclear transfer (SCNT) holds promise for both regenerative medicine and cell-based drug discovery. However, the efficiency of blastocyst formation after human SCNT is very low. The developmental competence of SCNT embryos has been previously demonstrated in several species to be enhanced by treatment with histone deacetylase inhibitors, such as trichostatin A (TSA), to increase histone acetylation. In this study, we report that treatment of SCNT embryos with 5 nM TSA for 10 h following activation incubation increased the developmental competence of human SCNT embryos constructed from β-thalassemia fibroblast cells. The efficiency of blastocyst formation from SCNT human embryos treated with TSA was approximately 2 times greater than that from untreated embryos. Cloned blastocysts were confirmed to be generated through SCNT by DNA and mitochondrial DNA fingerprinting analyses. Further, treatment of SCNT embryos with TSA improved the acetylation of histone H3 at lysine 9 in a manner similar to that observed in in vitro fertilized embryos.

Introduction

β-Thalassemia is an inherited blood disorder characterized by lowered synthesis of the hemoglobin subunit beta (hemoglobin β-chain). The molecular defects commonly observed are point mutations or small deletions influencing the transcription, splicing, or translation of the mRNA. Homozygous β-thalassemia neonates are born healthy, but the lack of β-globin results in progressive anemia as the change occurs from fetal to adult hemoglobin. The lifespan of patients with thalassemia major (also called Cooley's anemia) can only be briefly augmented with transfusion and chelation therapy treatments. Hence, β-thalassemia poses a severe health and economic burden not only for patients but also for their families. To date, no effective medical solutions have been developed to cure patients with thalassemia major.

Cell transplantation–based stem cell therapy has been proposed as the most promising approach to treat patients with genetic diseases such as β-thalassemia [1]. Somatic cell nuclear transfer (SCNT) has been successfully applied to generate autologous nuclear transfer embryonic stem NTES cells in mice and nonhuman primates (namely, the rhesus monkey) [2,3]. Moreover, a proof-of-principle study has clearly demonstrated that immune-deficient mice can be saved by bone marrow transplantation of hematopoietic stem cells differentiated from NTES cells that have undergone genetic correction [4]. However, no human NTES cells have ever been generated, although the generation of cloned blastocysts was recently reported [5 –8].

The generation of cloned human embryos by SCNT technology remains in its infancy. The efficiency of blastocyst formation after human SCNT is very low, suggesting a lack of or incomplete nuclear reprogramming with existing SCNT protocols, even though several successful human SCNT studies were recently reported [5,6,8]. Because of ethical issues and a shortage of human oocytes, discarded oocytes or embryos from in vitro fertilization (IVF) procedures were used as alternative cytoplasts. However, the NT embryos only developed to the early cleavage stage when using failed-to-fertilize human oocytes [9,10], in vitro maturation human oocytes [11], or human polyspermic zygotes [12] as alternative cytoplasts. Therefore, it is being suggested that the use of high-quality, fresh, mature oocytes from young donors is necessary for successful human SCNT [5,6,8].

SCNT is a process wherein the somatic cell gene expression can be converted to one that represents an oocyte totipotent state through an as-yet-undiscovered mechanism. Epigenetic reprogramming of the somatic cell genome has been proposed to be the key mechanism in SCNT, whereas abnormal epigenetic reprogramming contributes to cell cloning inefficiency [13,14]. Histone acetylation is an important epigenetic modification to the chromatin occurring on lysine residues of core histones. Acetylation appears to hold the greatest potential to permit unfolding chromatin to recruit different transcriptional factors and is invariably associated with cellular gene transcription activation [15 –17]. Recently, it has been reported that after cloning by SCNT, treatment with trichostatin A (TSA), an inhibitor of histone deacetylase (HDAC), resulted in a significant improvement in the blastocyst formation rate of mouse [18], bovine [19], rabbit [20], and pig [21] clones. Several studies showed that treatment of SCNT embryos with TSA improved lysine acetylation on core histones in a manner similar to that in normal mouse, bovine, rabbit, and pig fertilized embryo [19 –22]. Further, it was suggested that nuclear reorganization of centromeric/pericentromeric sequences is often abnormal in SCNT embryos and is improved by TSA treatment [23]. In addition, Li et al. found that treatment with TSA decreased the expression of DNA methylation–related genes and increased the expression of pluripotency-related genes in mouse blastocysts [24]. Similarly, Cervera et al. reported that TSA treatment of pig SCNT embryos increased the expression of pluripotency and imprinting-related genes at the blastocyst stage [25]. However, whether TSA treatment of human SCNT embryos can improve the rate of blastocyst formation has not been examined.

In the present study, we have investigated the development of cloned human embryos and the acetylation status of histone H3 at lysine 9 in cloned human embryos derived from a β-thalassemia patient's fibroblasts treated with and without TSA.

Materials and Methods

Donors

All experiments were approved by the ethical committee of The Third Affiliated Hospital of Guangzhou Medical College. All donated oocytes were obtained from 4 egg donation cycles performed at the Reproductive Medical Center of The Third Affiliated Hospital of Guangzhou Medical College, which is certified by the Ministry of Health of the People's Republic of China. We collected oocyte donations under the therapeutic cloning guidelines passed by the Ministry of Health of the People's Republic of China. No financial benefit was involved in the donation process. Oocyte donors were clearly informed of all the study details, including the oocytes' use and research destination, and they voluntarily signed detailed informed consent documents.

Ovulation induction and oocyte retrieval

Each patient underwent a basic physical examination before ovulation induction, including tests for human immunodeficiency virus, hepatitis B, hepatitis C, and contagious venereal disease. The patients had regular menstrual cycles every 29–32 days. Starting in the luteal phase of the previous cycle, on cycle day 21, eligible patients received 1.3 mg of a gonadotrophin-releasing hormone agonist (Dapherin) and then recombinant follicle-stimulating hormone (Gonalf) at a dose of 150–300 IU/day from day 3 of the menstrual cycle to promote the growth of multiple follicles. When the diameter of dominant follicles reached 18 mm, a single dose of 10,000 IU of human chorionic gonadotrophin (Profasi) was administered, and transvaginal follicular aspiration was performed 36 h later. The cumulus–oocyte complex was cultured in culture medium [Quinn's Advantage™ Fertilization Medium with 12% (v/v) TM Quinn's Advantage SPS Serum Protein Substitute; SAGE IVF, Inc.] for 2–3 h at 37°C in a humidified atmosphere of 5% CO₂ before SCNT.

Preparation of donor cells

Fibroblasts cultured from a skin biopsy was provided anonymously from a patient with homozygous β-thalassemia resulting from a 4-bp deletion (-CTTT), which caused a frameshift mutation. Skin fibroblast cells were maintained in DMEM (Invitrogen) containing 10% FBS (HyClone), 2 mM l-glutamine (Invitrogen), 50 U/mL of penicillin, and 50 mg/mL of streptomycin. For the cytogenetic analysis, donor cells were incubated in DMEM with 0.1 mg/mL of colcemid (KaryoMAX; Gibco) for 4 h. After washing with phosphate-buffered saline (PBS), the cells were trypsinized and resuspended in 0.075 M KCl for 15 min at 37°C. Then the cells were fixed 3 times with 1:3 methanol/glacial acetic acid and dropped onto glass slides. The chromosome spreads were Giemsa banded and photographed.

Somatic cell nuclear transfer

The cumulus–oocyte complexes were cultured in vitro in culture medium [Quinn's Advantage Fertilization Medium with 12% (v/v) Quinn's Advantage SPS Serum Protein Substitute; SAGE IVF, Inc.) and then treated with 80 IU/mL hyaluronidase for 30 s before SCNT micromanipulation. Mechanical handling was performed after a brief hyaluronidase treatment. The oocytes were then cultured in an incubator for 1 h to balance the effects of in vitro manipulation. After this, 3–4 oocytes were transferred into a droplet of HTF medium [Quinn's Advantage Medium with HEPES 12% (v/v) Quinn's Advantage SPS Serum Protein Substitute) containing 5 mg/mL of cytochalasin B.

All manipulations were done on the heated stage of a Nikon microscope equipped with Hoffman modulation contrast optics. The first polar body was fixed at the 3 o'clock position using a hold needle. A 15–20-μm-inner-diameter (ID) blunt-tipped pipette was passed through the zona pellucida using a piezo device. The first polar body and adjacent cytoplasm containing the metaphase II (MII) spindle were gently squeezed and extruded with a 20-μm pipette. After enucleation, the karyoplast was stained in a separate drop with 1 μg/mL of Hoechst 33342 (Sigma) and exposed to ultraviolet (UV) light to confirm the removal of chromosomes. After culturing for 1 h in G1.5 culture medium (Vitrolife Sweden AB), the enucleated oocytes were transferred into HTF without CB. The donor cells for nuclear transfer have been cultured with 0.5% serum starvation for 2 days (Supplementary Fig. S1; Supplementary Data are available online at www.liebertonline.com/scd). Our injection needle was a 10-μm-ID blunt-tip pipette. The donor cells for nuclear transfer were chosen by the injection needle diameter. A fibroblast in interphase was aspirated into a 10-μm-ID blunt-tipped pipette, breaking the cell membrane, and then injected into an enucleated oocyte. After manipulations were completed, the reconstructed embryos were immediately transferred back into G1.5 culture medium at 37°C and incubated for 2 h before activation.

Embryo activation and TSA treatment

The preparation and treatment of TSA have been previously described [18]. Briefly, TSA was dissolved in DMSO and prepared as a 200-fold concentrated stock solution. Reconstructed embryos were exposed to 10 μM A23187 (Sigma-Aldrich) for 5 min with or without supplementation with 5 nM TSA. After extensive washing in HTF medium, the embryos were incubated at 37°C under 5% CO₂ in humidified air with 2 mM 6-DMAP (Sigma-Aldrich) for an additional 5 h with or without supplementation of 5 nM TSA. After 6-DMAP treatment, the reconstructed embryos were extensively washed in G1.5 and cultured in G1.5 with or without supplementation of 5 nM TSA for another 5 h. After 68–72 h of embryo activation, the embryos that developed to the 8-cell stage were transferred to blastocyst culture G2.5 for sequential culture to the blastocyst stage.

Parthenogenetic activation

While performing the SCNT procedure, some fresh MII oocytes were retained for parthenogenetic activation. The activation and culture conditions were the same as those for SCNT embryos (without TSA treatment).

DNA fingerprinting analyses

On day 6, 5 cloned blastocysts were used for isolation of the ICM. The isolated ICMs were then placed on mitomycin C–treated murine embryonic fibroblast (MEF) feeder layers for further culture. After 2 days, 2 ICMs were attached to the feeder layers. After 8–9 days of culture, 1 colony derived from the ICM was mechanically dispersed into 2–3 small clumps using a micropipette. The ICM clumps were then transferred to a fresh feeder layer. Unfortunately, the cells differentiated after 3 passages. The differentiated cells were used for extraction of human genomic DNA. The extracted DNA was amplified for 16 different genetic loci using the Promega PowerPlex 16 System kit (Promega). Capillary electrophoresis was carried out on an automated ABI 3100 Genetic Analyzer (Applied Biosystems). The 16 short tandem repeat (STR) loci were D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, amelogenin, vWA, D8S1179, TPOX, and FGA.

Mitochondrial DNA analyses

DNA from peripheral blood lymphocytes of the oocyte donor, the β-thalassemia cells, and the differentiated cells from the cloned blastocysts was used to amplify mtDNA hypervariable region II (HVII). The 11 PCR primer pairs (3010, 4793, 10211, 5004, 7028, 7202, 16519, 12858, 4580, 477, and 14470, which represent the loci of SNPs) and reaction conditions were essentially as previously described [26].

Multiplex amplification of the 11 unique amplicons was carried out using the PE9700 in a total volume of 12.5 μL with 1 μL mtDNA sample. The PCR reagent concentrations were determined according to Vallone's report [26]. The PCR product was analyzed by direct nucleotide sequencing using the BigDye™ terminator Cycle Sequencing Kit and the ABI PRISM™ 3100 genetic analyzer (Applied BioSystems). Data analysis was performed using Genemapper ID 3.2 software.

Indirect immunofluorescence

After removing the zona pellucida in acidic Tyrode's medium, the reconstructed embryos were fixed with 4% paraformaldehyde in PBS for 30 min at room temperature, permeabilized with 0.5% Triton X-100 for 30 min at room temperature, blocked in 2% BSA-supplemented PBS (blocking solution) for 1 h, and incubated overnight at 4°C with primary antibodies to H3K9 acetylation (1:200). After 3 washings, the reconstructed embryos were incubated with a fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit secondary antibody for 1 h. Finally, the DNA was stained with 10 μg/mL of propidium iodide for 30 min. The embryos were mounted on slides and observed under a confocal laser scanning microscope (C1si; Nikon). Intensity of images was determined by ratio of acetylation of histone H3 at lysine 9 (AcH3K9) signal to a PI DNA signal using an image analyzer system, SigmaScan-pro V5.01 (SPSS, Inc.) [27]. The images were merged using Adobe Photoshop 9.0 software.

Statistical analysis

Embryo developmental data were analyzed by t-test using SPSS13.0. A p-value of <0.05 was regarded as significant.

Results

Karyotype of β-thalassemia donor cells

The β-thalassemia cells retained the normal morphology of fibroblasts (Fig. 1A) during propagation and exhibited a 100% normal diploid 46-XY karyotype (Fig. 1B) throughout the fifth passage, which is when they were used in SCNT experiments.

FIG. 1.

The morphology of β-thalassemia donor cells (200× magnification with an inverted microscope) (A) and the karyotype of β-thalassemia donor cells at passage 10 (B).

Assessment of developmental efficiency after TSA treatment

Four egg donors participated in our study. We obtained 27 oocytes from 2 donors (Donor 1, Donor 2), with 19 oocytes classified as MII stage and 8 oocytes classified as GV stage. Six MII-stage oocytes were used for parthenogenetic activation. Three MII-stage oocytes were used for ICSI and 10 MII-stage oocytes were used for SCNT. Nine oocytes survived after nuclear transfer. The 9 reconstructed embryos were randomly divided into 2 groups and subjected to sequential activation treatments with/without TSA. In the TSA treatment group, 3 of the 5 reconstructed embryos were activated. In the group without TSA treatment, 3 of the 4 reconstructed embryos were activated. The activated embryos were used for immunofluorescence. We obtained 40 oocytes from the other 2 donors (Donor 3, Donor 4), with 39 oocytes classified as MII stage and 1 oocyte classified as GV stage. Thirty-nine MII-stage oocytes were used for SCNT. After enucleation, 32 oocytes were successfully enucleated. The 7 oocytes were enucleated again, of which 4 oocytes were successfully enucleated and the other 3 oocytes were dead.

To investigate the developmental potential of human oocytes after artificial activation, 6 oocytes were subjected to parthenogenetic activation without an SCNT attempt, and the developmental efficiency was observed and recorded every 24 h. All 6 oocytes were activated after 6-DMAP treatment. Of these, all embryos developed to the 2-cell stage, and 2 successfully progressed to blastocysts (Fig. 2PA1, PA2). Blastocyst quality was defined according to the criteria presented by Gardner et al. [28]. The 2 parthenogenetic blastocysts were scored as grade A.

FIG. 2.

Parthenogenetic blastocyst and β-thalassemia cloned blastocysts (200× magnification with an inverted microscope). (PA1, PA2) Parthenogenetic blastocyst. (A) A cloned blastocyst without trichostatin A (TSA) treatment. (B–E) Cloned blastocysts generated with TSA treatment.

To investigate whether TSA treatment of human SCNT embryos could improve the rate of blastocyst formation, 36 reconstructed embryos were divided into 2 groups at random. Of these reconstructed embryos, 22 were activated with TSA treatment and 14 were activated without TSA. In the TSA treatment group, 18 of the 22 reconstructed embryos were activated, and 11 of the 18 cleaved to the 2-cell stage. In the group without TSA treatment, 12 of the 14 reconstructed embryos were activated, and 7 of the 12 cleaved to the 2-cell stage. Six embryos developed to the 8-cell stage and 4 progressed to the blastocyst stage in the TSA treatment group (Fig. 2B–E, scored as grade B, B, A, and B, respectively). Two embryos developed to the 8-cell stage, and 1 progressed to the blastocyst stage in the group without TSA treatment (Fig. 2A, scored as grade C). Treating the reconstructed embryos with TSA resulted in a 2-fold (22.2%, 4 of 18 vs. 8.3%, 1 of 12) increase in the blastocyst formation rate compared with that of untreated embryos (Table 1).

Table 1.

The Developmental Rates of Parthenogenetic Activation and Somatic Cell Nuclear Transfer Embryos Treated with or without Trichostatin A

	No. of reconstructed	No. of activated (%)	No. of 2-cell embryos (% activated)	No. of 8-cell embryos (% activated)	No. of blastocysts (% activated)
Parthenogenetic activation	6	6 (100)	6 (100)	5 (83.3)	2 (33.3)^a
Somatic cell nuclear transfer embryos	14	12 (85.7)	7 (58.3)	2 (16.7)	1 (8.3)^a
Trichostatin A–somatic cell nuclear transfer embryos	22	18 (81.8)	11 (61.1)	6 (33.3)	4 (22.2)^a

Denote that values do not differ significantly within that column (P>0.05).

Confirmation of cloned blastocysts by STR analysis

The peripheral blood lymphocytes of the oocyte donor, the differentiated cells from the cloned blastocysts (Fig. 3C), and the β-thalassemia cells were used for DNA fingerprinting to compare microsatellite markers of both samples. The 16 STR loci were scattered in different regions of 12 different chromosomes, and the results revealed that both donor cells and NT blastocysts had the same genetic origin (Fig. 4).

FIG. 3.

The cloned ICM attached and differentiated on feeder cells (100× magnification with an inverted microscope). (A) The cloned ICM attached after 2 days. (B) The ICM after 9 days in culture. (C) The ICM differentiated after passage 3.

FIG. 4.

DNA fingerprinting analysis using the differentiated cells from the cloned blastocyst, the β-thalassemia cells, and the peripheral blood lymphocytes of the oocyte donor.

SNP identification of the mtDNA

The differentiated cells from the cloned blastocysts, the β-thalassemia cells, and the peripheral blood lymphocytes of the oocyte donor were used for mtDNA SNP identification. The assay was signed to detect 11 SNP sites located in the mitochondrial genome. The differentiated cells from the cloned blastocysts showed a T at position 10211 and matched that of the oocyte donor (T at position 10211). The β-thalassemia cells showed a C at position 10211 (Fig. 5). These results show that mtDNA in the differentiated cells from the cloned blastocysts contained the SNP site of oocyte donor somatic cells, and we could conclude that mtDNA in the differentiated cells from the cloned blastocyst was derived from its recipient oocyte.

FIG. 5.

Mitochondrial DNA analysis using peripheral blood lymphocytes of the oocyte donor, the β-thalassemia cells, and the differentiated cells from the cloned blastocysts. (A) Mitochondrial DNA of oocyte donor somatic cells at position 10211 was T; (B) mitochondrial DNA of the differentiated cells from the cloned blastocysts at 10211 was T; (C) mitochondrial DNA of β-thalassemia cells at 10211 was C.

Distribution patterns of acetylation on lysine residue 9 of histone H3 in ICSI embryos, cloned embryos without TSA treatment, and TSA-treated cloned embryos

The AcH3K9 signal was detected in both male and female pronuclei in ICSI embryos (Fig. 6A). In SCNT embryos, the signal was observed at 10 h after activation (Fig. 6B), and TSA-treated SCNT embryos displayed a relatively strong staining (Fig. 6C). Following activation, restoration of H3K9 acetylation in the pseudopronuclei made them similar to those in normal ICSI embryos. Quantification of AcH3K9/DNA signal intensity in TSA-treated cloned embryos is similar to those in normal ICSI embryos (the ICSI, NT, and TSA-NT embryos' AcH3K9/DNA signal ratio is 0.85, 0.62, and 0.91 respectively; n=3) The intensity of H3K9 acetylation in cloned embryos was much weaker than that in normal embryos, whereas the intensities of H3K9 acetylation were similar in the pseudopronuclei of cloned embryos treated with TSA and normal embryos.

FIG. 6.

Acetylation of histone H3 at lysine 9 in a 1-cell embryo during the pronuclear stage at 10 h postactivation in fertilized (A, A1, A2), TSA-cloned (B, B1, B2), and cloned (C, C1, C2) embryos. The embryos were immunostained with anti–acetylation of histone H3 at lysine 9, and the DNA was stained with propidium iodide.

Discussion

In the present study, we investigated the effect of TSA on the in vitro developmental potential and histone acetylation of SCNT embryos using β-thalassemia fibroblast cells. We are reporting for the first time that SCNT can be used to generate patient-specific cloned blastocysts using differentiated adult donor nuclei from human oocytes. Through DNA fingerprinting analyses and mtDNA SNP identification, we demonstrated that the genomic DNA of one of the SCNT-cloned blastocysts was that of the β-thalassemia fibroblast cell line and was not of parthenogenetic origin or a result of oocyte fragmentation.

The efficiency of generation of SCNT blastocysts using differentiated somatic cell nuclei from human oocytes is extremely low, which is one of the major obstacles to successful application of patient-specific NTSC in regenerative medicine. The developmental ability of cloned embryos is related to the nuclear transfer technique used to create them. It has been suggested that the developmental potential of monkey SCNT embryos is limited by microtubule motor and centrosomal protein deletions during meiotic spindle removal, which is followed by defective mitotic spindle formation from the transferred nucleus [29]. Zhou et al. also reported that a 1-step micromanipulation technique was superior in the routine production of developmentally competent SCNT embryos in monkeys [30]. In this study, several modifications to the nuclear transfer process were investigated. The piezo-assisted method used to create a “blind” enucleation technique, thus avoiding the need for “squish” enucleation, was efficient, at least in our experiments, because it enucleated more than 80% of the oocytes. The use of Hoechst 33342/UV was avoided during the oocyte spindle removal step because the karyoplast was stained with Hoechst 33342 in a separate drop to confirm spindle removal. Recently, Byrne et al. reported that nonhuman primate SCNT removing the least cytoplasm possible during enucleation and avoiding the use of Hoechst (by using the oocyte spindle imaging system; CRI, Inc.) were crucial to successfully derive 2 NTSC lines [2]. In our experience, 1 oocyte “blind” enucleation can be finished within 1 min. However, 1 oocyte enucleation using the oocyte spindle imaging system required more than 3 min [8]. The oocyte operation time in vitro was decreased using our method. However, in our study, approximately 40% of the NT embryos did not cleave. This may be because an increased volume of cytoplasm was removed to successfully enucleate the oocytes, and cytoplasm removal is detrimental to embryo development. Thus, further optimization of the human NT technique is required.

It is well known that faulty epigenetic reprogramming of somatic nuclei is likely to be a major cause of the low efficiency observed in the production of all mammals through SCNT. Recent studies demonstrated that a 5 nM TSA treatment could dramatically increase the quantity and quality of blastocysts in mouse cloned embryos [18]. Further, a 50 nM TSA treatment led to increased blastocyst rates in porcine NT [31]. Because of the limited fresh human oocytes, we did not test the toxicity of TSA on human embryos. We chose 5 nM as our TSA treatment concentration and 10 h following activation incubation, conditions that have been demonstrated to significantly improve the efficiency of mouse cloning [18]. However, whether this concentration is the most appropriate one for human embryos needs to be investigated further. Consistent with other studies, the efficiency of blastocyst formation for SCNT human embryos treated with TSA was approximately 2 times greater than that of untreated embryos. Previous reports have indicated that increased acetylation of specific lysine residues might be important for embryo development, especially zygotic gene activation [32,33]. We found that TSA increased the signal intensity of H3K9 acetylation in the pseudopronuclei of cloned embryos, which may account for the improved development of cloned embryos treated with TSA. Recently, it has been demonstrated that a new HDAC inhibitor, scriptaid, which has low toxicity, enhances transcriptional activity and protein expression. When scriptaid is used in mouse [34] and pig [35] nuclear transfer, it improves the cloning efficiency to a greater degree than TSA. We also found that a new HDAC inhibitor, m-carboxycinnamic acid bishydroxamide (CBHA), could increase the somatic cell reprogramming efficiency and NTES cell derivation efficiency in the mouse model. CBHA appears even more efficient than TSA [36]. Whether these new HDAC inhibitors would be more efficient than TSA in human somatic cell reprogramming needs to be investigated further.

Recently, we have proven that differentiated β-thalassemia fibroblast cells could be reprogrammed to a pluripotent state by forced expression of 4 transcription factors mediated by viral transduction [37,38]. The induced pluripotent stem (iPS) cells possess a similar differentiation potential to those of embryonic stem cells (ESCs), and hematopoietic cells have been differentiated from these iPS cells. However, iPS cells suffer serious limitations because the virus-mediated delivery of reprogramming factors introduces unacceptable risks of permanent transgene integration into the genome. The resulting genomic alteration and possible reactivation of viral transgenes pose serious clinical concerns. Continuous overexpression of exogenous factors is also problematic secondary to the incomplete silencing of transgenes during differentiation. Moreover, several studies have reported distinct differences between iPSCs and ESCs [39 –43]. More studies need to be performed to further improve the quality of iPS cells. Reprogramming by oocyte-specific factors after SCNT avoids these pitfalls because it employs endogenous epigenetic programs. Currently, we are pursuing the generation of patient-specific ESC lines from SCNT embryos using donor oocytes. The recent achievements that ESC lines can be generated from individual blastomeres of human [44] and cloned mouse embryos [45], human-arrested embryos [46] and primate somatic cell blastocysts [2], and human recombinant leukemia inhibitory factor and human basic fibroblast growth factor significantly increase the number and quality of human blastocysts formed, as well as the efficiency of human ESC derivation from poor-quality embryos [47] may hasten the attainment of this goal.

Footnotes

Acknowledgments

This work was supported by grants from Guangzhou City Science and Technology Administration (2006Z1-E0021 and 2008A1-E4011-3), National Natural Science Foundation of China (30871378), and Guangdong Province Science and Technology Administration (2009A030200010 and 2008B090500258).

Author Disclosure Statement

No competing financial interests exist.

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