(자료)미국 Stemagen 사 배반포 논문

[침묵의 눈]

Development of Human Cloned Blastocysts Following Somatic Cell Nuclear Transfer with Adult Fibroblasts

1. Andrew J. French Ph.D.^1,*,
2. Catharine A. Adams²,
3. Linda S. Anderson²,
4. John R. Kitchen³,
5. Marcus R. Hughes³,
6. Samuel H. Wood^1,2

Article first published online: 17 JAN 2008

Abstract

Nuclear transfer stem cells hold considerable promise in the field of regenerative medicine and cell-based drug discovery. In this study, a total of 29 oocytes were obtained from three young (20–24 years old) reproductive egg donors who had been successful in previous cycles. These oocytes, deemed by intended parents to be in excess of their reproductive needs, were donated for research without financial compensation by both the egg donor and intended parents after receiving informed consent. All intended parents successfully achieved ongoing pregnancies with the oocytes retained for reproductive purposes. Mature oocytes, obtained within 2 hours following transvaginal aspiration, were enucleated using one of two methods, extrusion or aspiration, after 45 minutes of incubation in cytochalasin B. Rates of oocyte lysis or degeneration did not differ between the two methods. Somatic cell nuclear transfer (SCNT) embryos were constructed using two established adult male fibroblast lines of normal karyotype. High rates of pronuclear formation (66%), early cleavage (47%), and blastocyst (23%) development were observed following incubation in standard in vitro fertilization culture media. One cloned blastocyst was confirmed by DNA and mitochondrial DNA fingerprinting analyses, and DNA fingerprinting of two other cloned blastocysts indicated that they were also generated by SCNT. Blastocysts were also obtained from a limited number of parthenogenetically activated oocytes. This study demonstrates, for the first time, that SCNT can produce human blastocyst-stage embryos using nuclei obtained from differentiated adult cells and provides new information on methods that may be needed for a higher level of efficiency for human nuclear transfer.

Disclosure of potential conflicts of interest is found at the end of this article.

논문 Full Text는 아래 링크로.

 http://onlinelibrary.wiley.com/doi/10.1634/stemcells.2007-0252/full

http://onlinelibrary.wiley.com/doi/10.1634/stemcells.2007-0252/pdf

Discussion

This study demonstrates, for the first time, that SCNT can be used to generate cloned human blastocysts using differentiated adult donor nuclei remodeled and reprogrammed by human oocytes. Evidence of successful SCNT was shown with DNA fingerprinting analyses of three SCNT cloned blastocysts, where embryo genomic DNA was that of the donor fibroblast cell line and not of parthenogenetic origin or as a result of oocyte fragmentation [35, 36]. Examination of mtDNA sequence from the HVII region showed that one SCNT blastocyst (coded reference K8) was matched to egg donor 3 (coded references F1 and F2) mtDNA and was identical to the parthenogenetic embryos (coded references K1 and K6) obtained in the same experimental series.

The study describes a method allowing the derivation of human NT blastocysts using young oocytes and established adult male fibroblast lines of normal karyotype. The rate of SCNT blastocyst formation is similar to the development rates seen in standard IVF cycles (fertilization rate of 70%–80% of oocytes collected, and 40%–60% of fertilized oocytes develop into blastocysts) [37, [38]–39] and to a previous nuclear transfer study with undifferentiated hESC donor cells [15]. Total cell counts in SCNT blastocysts improved with each subsequent experimental series and appear comparable to reports for various grades of human IVF embryos [40]. Blastocysts were also derived from a limited number of parthenogenetically activated oocytes using both of the activation methods used. DNA fingerprint and mtDNA, of the HVII region, analyses of the parthenogenetic embryos (coded references K1 and K6) confirmed that they originated solely from egg donor 3 (coded references F1 and F2). The frequency of blastocyst generation is similar to that reported in the recent work of Revazova et al., where a total of 44 donated oocytes resulted in 23 blastocysts after parthenogenetic activation (52%), of which 11 (25%) had a visible inner cell mass and generated six human parthenogenetic embryonic stem cell lines [41].

DNA fingerprinting analysis of the adult donor fibroblasts, egg donor cumulus cells, and all blastocysts observed following nuclear transfer was conducted to determine whether the embryonic pathways in the donor nucleus had been reinitiated. Of the five putative SCNT blastocysts generated, successful DNA fingerprints from three SCNT blastocysts were consistent with those of the somatic cell donor used, with no evidence of contamination from the egg donors, indicating that embryonic development was being controlled by the donor cell genome. The DNA fingerprint comparisons were not identical, which was not unexpected given the reported incidence of ADO and unvisualized alleles in single-cell DNA fingerprinting systems [42, 43].

Further amplification of the samples for forward and reverse HVII region mtDNA sequencing showed three alterations in sequencing in one cloned blastocyst, T or C at 33 bp, G or A at 34 bp, and 8C or 9C at 270–271 bp, and indicates that mtDNA in the cloned blastocyst K8 was from egg donor 3 (coded references F1 and F2) and not cell donor AF2 (coded references C5 and C6). It should be noted that the stutter observed after the poly(C) stretch is almost certainly due exclusively to the repetitive nature of the sequence; there are no heterozygotes in the K cell series at the T or C and the G or A loci, with only the T allele and the G allele from egg donor 3 (coded references F1 and F2) clearly visualized. This would indicate that if any mitochondria from fibroblast donor AF2 (coded references C5 and C6) were transferred along with the nuclear material, they were vastly outnumbered by those of egg donor 3 (coded references F1 and F2).

Sequence data in both directions are appropriate until the string of C homopolymer is reached in the forward direction or G homopolymer in the reverse direction. The presence of repetitive sequence, especially in the case of homopolymer and dinucleotide repeats (less so for higher order repeats), causes slippage by DNA polymerase and causes stutter in sequence downstream of that sequence. The presence of poly(C) and poly(G) is especially problematic, and significant sequence degradation can be seen even with fewer than 10 repeats (much more so than poly(A) or poly(T), which can tolerate ∼13–15 repeats). In the case of the sequences seen here, their overall quality gives the expected result, with significant degradation of sequence quality once the critical repeat number is exceeded for the repetitive sequence in question.

Several modifications to the nuclear transfer process were investigated. Using a different method of enucleation (either extrusion or aspiration of the metaphase plate) resulted in no discernible differences in terms of embryo lysis or degeneration or in the developmental competence of SCNT embryos. The longer incubation of oocytes in a cell-permeable mycotoxin, cytochalasin B (45 minutes compared with 15–20 minutes), prevents the formation of contractile microfilaments and disruption of actin filaments and polymerization. Incubation in cytochalasin B using the time intervals reported in previous studies using failed-to-fertilize and MI oocytes resulted in increased oocyte lysis. Increasing the exposure of oocytes to cytochalasin B (45 minutes) decreased oocyte lysis and supported the findings of a previous report [16]. It is not known whether the use of recombinant human hyaluronidase, instead of the commonly used bovine source, to remove the cumulus matrix also contributed to the efficiency of this procedure, but additional studies with this new product are certainly warranted.

Eleven successful enucleation attempts did not remove the first polar body because of untoward location that would not have allowed us to achieve our goal of minimizing the amount of cytoplasm removed during the process. Increasing the amount of cytoplasm removed during the enucleation process has an impact on the development potential of nuclear transfer embryos [44, 45]. Anecdotally, removal of the first polar body with either technique appeared to increase the amount of cytoplasm removed.

Although mice have been generated from the first polar body following NT, that method of transfer required intracytoplasmic injection of the polar body, primarily as a result of apoptotic events associated with the breakdown and degeneration of the first polar body in the mouse [46]. The presence of the first polar body is of little concern because, in practice, it is difficult to initiate successful electrical fusion of the polar body with the enucleated oocyte and because the donor cell is easily distinguished from the polar body during the microscopic examinations required as part of the electrical fusion. The presence of two pronuclei during the assessment of remodeling, which would have indicated fusion of the first polar body, was not detected. In addition, DNA fingerprinting analysis did not confirm‎ the presence of maternal DNA in the SCNT blastocysts.

In this study, parthenogenetic activation using either CI in combination with protein synthesis (6-DMAP) or protein kinase inhibitors (CHX/CYTD) artificially activated fresh oocytes that subsequently underwent pronuclear formation and cleavage division in a manner consistent with previous reports [24, 47, [48]–49]. Prevention of second polar body extrusion by the inhibition of either protein synthesis pathways (6-DMAP) or protein kinase pathways (CHX/CYTD) can alter the developmental competence of SCNT embryos [50]. However, in our assessment, there appeared to be no difference between the two activation treatments in terms of pronuclei and blastocyst formation, although the limited number of activated human oocytes used makes it difficult to draw conclusions. Of note, parthenogenetically activated blastocysts observed were of lower embryonic grades due to a slower development rate and lower total cell number.

Additional modifications to the activation method, as suggested by a number of recent studies, may improve efficacy. For example, cytosolic extracts from sperm have been shown to cause Ca²⁺ oscillations in a range of different mammalian oocytes, including humans [51]. In a further refinement, Saunders et al. [52] isolated the specific isoform of phospholipase C (PLC) [53] thought to be responsible for generating Ca²⁺ release and inducing inositol trisphosphate production. Microinjection of cRNA encoding human PLC_ζ induced a response in aged human oocytes that closely mimicked the repetitive calcium Ca²⁺ oscillations stimulus provided by the sperm during human fertilization and induced parthenogenesis and development to the blastocyst stage [54].

The protocols produced high rates of pronuclear formation (66%), early cleavage (47%), and blastocyst development (23%) following incubation in standard IVF culture media. As would be expected, a range of blastocyst grades were observed following both parthenogenetic activation and SCNT; however, all showed the presence of a defined inner cell mass. The observation of morphologically normal SCNT blastocysts is the prelude to obtaining embryonic stem cell lines, which will now be the focus of this research. This study examined the reinitiation of the embryonic genome in cloned blastocysts using the methods of DNA fingerprinting and mtDNA sequencing. However, future studies to support the total cell number findings will examine the quality of the SCNT blastocyst and examine chromosome number, ICM/trophectoderm ratio, and expression‎ of critical embryonic genes at the time of blastocyst formation (Oct4, Cdx2) [55, 56]. The process of preparing the samples for DNA fingerprinting, however, precluded the application of these methods.

Other factors that may have contributed to the success of SCNT procedures include the quality of the oocytes obtained from young egg donors. The best donor candidates are likely to be younger women, without a history of infertility, who respond well to ovarian stimulation. A major impediment to conducting studies of this type is the difficulty of obtaining suitable donor oocytes, something that is influenced by both ethical and legal considerations. Models for donation include a range of potential options, including altruistic oocyte donation, monetary inducement, or compensation in the form of reduced treatment fees. After obtaining the appropriate informed consent from the egg donor and intended parents, this study involved using oocytes that were excess to reproductive needs and that were donated without compensation. All three intended parents were successful in achieving ongoing pregnancies with the oocytes they retained for reproductive purposes. The average in vitro development rates of assisted reproductive technologies oocytes following either in vitro fertilization and/or intracytoplasmic sperm injection was 76% for normal fertilization (two pronuclei) and 92% for cleavage to good-quality embryos (grades I and II) determined on days 2–4 (data not shown). Since a significant percentage of couples undergoing fertility treatments appear willing to participate in this type of research [28, 29], we believe that the method described to obtain donated oocytes is a viable and ethically acceptable strategy that can enable optimization of the nuclear transfer stem cell method to produce embryonic stem cells [10]. Although it allows the acquisition of only limited numbers of oocytes and thus small groups of embryos from an individual egg donor, it clearly is a more efficacious process than those in previous studies, which obtained oocytes predominantly from patients of increasing maternal ages undergoing a variety of IVF treatments [15, 24].

We are currently pursuing the generation of embryonic stem cell lines from SCNT embryos generated using these protocols. The recent findings with mouse [57] and human [58] embryos showing the generation of ESC lines from individual blastomeres in human from arrested embryos [59] and primate embryonic stem cells following somatic cell nuclear transfer [60] may hasten the attainment of this goal.

In conclusion, it has been demonstrated that heterologous SCNT blastocysts can be successfully and reliably generated from two male adult fibroblast lines using oocytes from young oocyte donors obtained shortly after an oocyte retrieval‎ that followed a standard oocyte donation cycle. Following SCNT, the somatic cell nuclei underwent remodeling, as evidenced by pronuclear formation. Of the five putative cloned blastocysts produced, only one cloned blastocyst was confirmed by DNA and mtDNA fingerprinting analyses; however, DNA fingerprinting of two other cloned blastocysts indicated that they were generated by SCNT, although we were unable to conclusively support these finding with mitochondrial DNA analyses.

[침묵의 눈]

ㅎㅎㅎㅎ, 눈치 채셨구만요. ㅋㅋ, 눈이야 뜨고 하겠지요. ㅎㅎ. 눈감고 감각으로 할때, 제 1극체가 난자 핵과 가까이 없는 경우가 너무 많으니, 처녀 방지하려고 자꾸만 많이 짜내게 됨다. ㅋㅋ

[침묵의 눈]

원숭이 복제 줄기세포 성공시킨 기법에서도 난자의 핵을 편광 작용을 이용한 전자 현미경으로 난자 핵 위치를 보면서 탈핵시키고, 이번 Stemagen 방법에서도 염색하여 난자 핵 위치를 보면서 탈핵시킵니다. 이렇게 하면, 탈핵시에 난자의 세포질 소실이 최소화되는 장점과 처녀생식을 방지시킬 수 있는 장점이 있지요. ㅎㅎ
쥐어짜기에 새튼도 홀딱 반해 버렸으니...

물론 세포질 소실을 초소화 시키는 것만으로 모든 문제점이 해결된다고는 보지 못할 지도 모르지만, 최소한 과다 소실 문제점만은 해결되어야 할 것으로 보입니다.

[침묵의 눈]

알아요, 염색하지 않는 이유. 그리고 불을 밝히면 난자 배 발달에 안 좋다는 이유가 있다는 것을.... 그러나 지금 나타난 다른 사람들의 연구 결과로는 세포질 소실 최소화가 더 중요한 요인인 것 같고, 처녀 방지 차원에서도 더 유효한 듯이 보입니다.

[봄이다]

냅~^^... 전 아무말도 안 한 겁니다.ㅋㅋ

[융프라우]

수암에서도...핵염색을 생각지 못한 것은 아니었을 거라 생각합니다. ..처움부터 배반포의 질을 목표했을거란 생각...

[침묵의 눈]

Stemagen논문이 중국 논문보다 더 빠르지 않아요? 2008년 1월이니...

[봄이다]

제 기억으론 중국논문이 후가 맞는거 같습니다. 이 논문은 별로 기억에 없습니다. 제가 한국에 있을 때, 떡진님이 어느날 갑자기 문자가 날라와서, 숨 넘어가는 어투로, 중국에서 배반포 논문 나왓다고 하는데 사실이냐? 그 제보로 논문 다운받아 읽어보았던 기억이 납니다. 참 부지런한 황빠들이였지요. 논문마저도 입에 떠넣어 주는 자상하고 의욕에 넘치던 황빠들... 제가 진도 못 따라가네요. 월요일에 중국논문 찾아서 올릴게요. 그 당시 특허출원내용까지 제가 확인했었던 기억이 납니다.

[침묵의 눈]

Stemagen논문이 2008년 1월이면, 아마 특허는 2007 년도 출원되었을 것으로 보이네요. 이미 공개되었겠네...

[봄이다]

배반포 특허 함 검색해 보세용^^. 전 낼은 또 교회 가고... 무쟈게 바ㅡ쁩니당. 님께서 특허 찾아올리시면 함께 공부하세용. ㅋ

[융프라우]

수암이 안하는 스터디를 수암밖에서 해주고 있네요...죽순임...침묵님....춘박님...덕진님...랄락님....밀밭님.....감사합니다....자랑스럽습니다...

[dangoonjason]

이 정도의 실험 결과 들이면, 그 걸 토대로 탈핵 시나리오 시물레이션을 통한 최적 조건 산출은 안됍니까? 굳이 임상적으로만, physical하게 trial & error를 반복하지 말고요...

하이고, 전혀 이해도 못하는 놈이 헛소리 한번 해 봅니다.

[봄이다]

탈핵 시나리오 시물레이션을 통한 현 단계의 최적조건이, 현재 거론되고 있는 본 배반포 논문에 사용되었다고 보셔도 무방합니다. 죽순님과 침묵님께서 저 위에 조목조목 설명하신 대로. 고로, 6년전 기술과 조건은, 흑백티비 시대라고 제가 비유한 겁니다. 지금은 칼라티비, 아이폰, 아이패드 시대...

[신지은]

연구적이고 학문적인...저는 어려워 추천만 누릅니다^^*