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8 i1 ?8 j. i, v! K ) j C4 m" `' a! T1 Z# e: w 【关键词】骨髓基质干细胞6 x; }3 w' |- N3 |" ]
Production of blastocysts by transfer of nuclei from nonquiescent adult rabbits bone mesenchymal stem cells and adipose stromal stem cellsYAN Quan-Jian LI Liu-Jin YANG Gui-Zhong ZHANG Yu-Mei WANG Yue-Feng WANG He SHAO Guo-XingKeywords:nuclear transfer;adult rabbits;bone mesenchy-mal stem cells;adipose stromal stem cells;oocytes donation;blastocysts Abstract:AIM To study the isolation and in vitro culture of rabbit's bone mesenchymal stem cells（BMSCs）and adipose stromal stem cells（ASSCs）and the use of both BMSCs and ASSCs cells in nuclear transfer（NT）to produce totipotent blastocysts.METHODS Both cell lines were derived from New Zealand rabbits and used as donor cells.The cell lines were cultured as a low cell density suspension in the Dulbecco's modified Eagle's medium（DMEM）and150mL?L-1 fetal calf serum（FCS）for7d before use in nuclear transfer.Direct intracystplasm microinjection techniques were used to reconstruct one-cell embryos.Sire differences were noted in the ability of both BMSCs and ASSCs to form blastocysts.RESULTS A total of986nuclear transfer clones were made by fusing both BMSCs and ASSCs cells nuclei into enucleated oocytes.After culture of the clones for7d in vitro，88（16.9%）and73（15.7%）of those fused with BMSCs and ASSCs cells nuclei became blastocysts（P>0.05，Yates corrected）.CONCLUSION BMSCs and ASSCs can be used as the NT donor cells to form blastocysts.There is no significant difference in blastocyst development rate（BDR）between them. 第四军医大学: 1 西京医院泌尿外科， 2 实验动物中心，陕西西安710033 关键词: 核移植;成年兔;骨髓基质干细胞;脂肪基质干细胞;卵母细胞移植;囊胚摘 要:目的研究非休眠期成年兔骨髓基质干细胞和脂肪基质干细胞作为供体，以体外成熟去核兔卵母细胞作为受体进行核移植构建重组胚胎的可行性及囊胚形成率. 方法首先利用新西兰兔骨髓和脂肪为来源分别培养骨髓基质干细胞和脂肪基质干细胞，然后用他们作为核供体，采用卵细胞胞质内直接注射法构建重组胚胎，7d后比较囊胚形成率. 结果核移植操作后卵细胞存活率达80.0%，骨髓基质干细胞和脂肪基质干细胞作为供体重组胚胎囊胚形成率分别为88/522（16.9%）和73/464（15.7%），（P>0.05，Yates corrected）. 结论处于非休眠期的成年兔骨髓基质干细胞和脂肪基质干细胞移植到去核卵母细胞后能够进行早期胚胎发育，两者囊胚形成率无明显差别. 3 O9 n/ i6 N+ ~: I
INTRODUCTION Nuclear transfer（NT）technology has been used to derive live clones in several species including sheep［1］ ，cattle［2］ ，goats［3］ ，pigs［4，5］ and mice［6］ .With one notable exception，poor survival of NT embryos has so far been independent of the donor tissue;a significantly higher fraction of blastocysts cloned from embryo stem（ES）cell nuclei than from any somatic cell type survive to adulthood［7，8］ .This result is consistent with the idea that the nucleus from an undifferentiated embryonic cell might be more amenable to or require less reprogramming than the nucleus from a differentiated somatic cell.BMSCs and ASSCs are undifferentiated stem cells.In this research we examined whether BMSCs and ASSCs were suitable to reconstruct embryos. MATERIALS AND METHODS Preparation of donor BMSCs and ASSCs Materials were purchased from Sigma unless otherwise sta-ted.All tissue culture reagents were purchased from Life Technologies.Fetal bovine serum（FBS）were purchased from Hyclone.For isolation of rab-bits BMSCs［9，10］ ，tibias and femurs were dissected from8-to12-week-old New Zealand female rab-bits.The ends of the bones were cut，and the mar-row was extruded with5mL of Dulbecco's modified Eagle's medium（DMEM，Gibco）by using a needle and syringe（or the marrow aspirates were pumped at the extent of1.0～2.5cm of anterointernal bone surface below the space of knee joint on the upper part of tibia puncture area）.Between100and200×106 whole marrow cells were plated on175cm2 tis-sue culture flask in DMEM/100mL L-1 FBS.After24h，the nonadherent cells were removed by replac-ing the medium.The medium was replaced every2～3d as the cells were grown to confluency.The cells were lifted by incubation with2.5g L-1 trypsin and1mM EDTA，passed three or four times，and stored frozen.To prevent spontaneous differentiation，cells were maintained at subconflu-ent levels. For isolation of rabbits ASSCs［11，12］ ，Chinchilla adi-pose tissue was obtained from epiploon excision pro-cedures under846-extender injection anesthesia.In this procedure，the raw lipoaspirate（～100mL）was processed according to established methodolo-gies to obtain ASSCs.To isolate the ASSCs，lipoaspirates were washed extensively with equal volumes of phosphate-buffered saline（PBS），then digested at37℃for30min with0.75g L-1 collage-nase.Enzyme activity was neutralized with DMEM，containing150mL L-1 FBS and centrifuged at1200g for10min to obtain a high-density ASSCs pellet.The pellet was resuspended in160mM NH4 Cl and incubated at room temperature for10min to lyse contaminating red blood cells.The ASSCs was col- lected by centrifugation，as described above，fil-tered through a100-mm nylon mesh to remove cel-lular debris and incubated overnight at37℃/50mL L-1 CO2 in control medium（DMEM，100mL L-1 FBS，5g L-1 gentamicin solution）.Following incu-bation，the plates were washed extensively with PBS to remove residual nonadherent red blood cells.ASSCs obtained from excised adipose tissue were maintained at37℃/50mL L-1 CO2 in noninductive control medium.Cells did not require specific FBS sera lots for expansion and differentiation. Donor cells and recipient oocyte preparation and nu┐clear transfer BMSCs and ASSCs were used as donor cells before reaching confluency（cells at pas-sage5only）.Immediately before nuclear transfer，donor cells were trypsinized，washed by centrifuga-tion，and resuspended in PBS supplemented with5mL L-1 FBS.Recipient oocyte collection，matura-tion，and enucleation were as described［13，14］ at about24h after maturation culture.Cells with an approximate diameter of10～15μm were trans-ferred directly to the recipient cytoplast［15］ .Fusion was then confirmed by microscopic examination.All fused embryos were further activated by culturing with35μm cycloheximide in CR1aa medium［16］ for additional5h. In vitro culture of cloned embryos The nuclear transferred embryos were cultured in CR1aa medi-um for48h at38.5℃in a humidified atmosphere of50mL L-1 CO2 .Cleavage rates were recorded，and cleaved embryos were cultured further in CR1aa medium supplemented with50mL L-1 FBS with cumulus-cell coculture for5d.On d7，blastocyst development was recorded. $ n3 `5 |; Z/ ^, O4 n# WStatistics analyses Single table analyses were car-ried out with the Epi Info6.04d，（USD，Inc.2075A West Park Place，Stone Mountain，GA30087U.S.A.）.Differences between groups were consid-ered significant when P Tab1 Results of nuclear transfer 略0 R% Y- \) d) X9 ~ r
RESULTS Results of nuclear transfer Results of nuclear transfer are showed in Tab1. Morphology of donor cells and reconstructed em┐bryos Morphology of donor cells and reconstructed embryos are shown in Fig1，2. N. o( B7 B; k$ |% D8 j3 M& p I) H Fig1 Morphology of BMSCs 略Fig2 略 DISCUSSION Many attempts have been made to clarify cell cycle effects of donor nuclei on the developmental ability of reconstructed embryos［17-23］ .It is thought that donor nuclei at G1 or G1 /S phase are at a suitable stage for embryo cloning;therefore，attempts to control the cell cycle of embryonic nuclei have used chemicals with low toxicity such as aphidicolin，as well as DNA polymerase I inhibitor.In rabbits，ex-periments on nuclear transfer of G1 -phase and late S-phase nuclei synchronized with colcemid and aphidicolin showed that G1 -phase nuclei have a greater potential to contribute development of the reconstituted embryos［24］ .Cheong et al［25］ have re-ported that transferring nuclei at G1 phase from two-，four-，and eight-cell mouse embryos into enu-cleated oocytes resulted in the birth of live off-spring.However，Kwon OY and Kono T repeated the experiments precisely but failed to confirm their results.The reason for this is not clear，but it is ex-tremely difficult to obtain embryonic nuclei at G1 -phase because it is too transitory.The early cell cycles of mouse embryos are easily synchronized at metaphase by culturing the embryos with nocoda-zole，which inhibits tubulin polymerization.Expo-sure of embryos to the concentration of nocodazole necessary to cause cell cycle arrest does not affect the ability of the manipulated embryos to complete development to term［26］ .Thus，large numbers of identical embryos can be produced from the donor nuclei. Development to the blastocyst stage was identical（16.9%to15.7%）when donor cell nuclei from the BMSCs and ASSCs were transferred into enucleated parthenogenetic one-cell embryos.This shows that the ability of cytoplasm to support development has no difference between the BMSCs and ASSCs nuclei reconstructed embryos.The exact reason is un-known.One possibility is that new transcripts from the BMSCs and ASSCs genome have less difference in the development of the reconstituted embryos，because it has been reported that zygotic genome ac-tivation［27］ and exogenous gene expression［28］ occur in the late one-cell mouse embryos.Therefore，when BMSCs and ASSCs were used as the donor nuclei，they got the same blastocyst development rate.Alternatively，nucleocytoplasmic interac-tion［29］ in hybrid embryos may be responsible for this phenomenon. Attention has focused on the cell cycle stage of the donor nucleus.For instance，the success in somatic cell cloning in sheep was attributed by the authors to their unique approach to cell cycle staging，wherein they starved nuclear donor cells and forced them into a quiescent or G0stage.In contrast，in the cow，nonquiescent fetal fibroblasts have been employed successfully［30，31］ ，suggesting that staging or forcing the cell out of an active cycle into a quies-cent state is not always required.More recently，noncultured cumulus cells in the G /G1 phase of the cell cycle were used in cloning mice［32］ . Our results show clearly that metaphase-stage nu-clei from BMSCs and ASSCs are reprogrammed by the present nuclear transfer system，and the recon-structed embryos have the capacity to complete em-bryonic development to term.Nevertheless，how and when donor metaphase nuclei are reprogrammed and acquire totipotency is not known.Perhaps the reprogramming occurs mainly during decondensa-tion of nuclei in reconstructed oocytes after parthenogenetic activation.The present manipula-tive techniques suggest that larger scale cloning by nuclear transfer is possible in rabbits and other species. REFERENCES: ［1］Wilmut I，Schnieke AE，McWhir J，Kind AJ，Campbell KH.Viable offspring derived from fetal and adult mammalian cells ［J］.Nature，1997;385（6619）:810-813. $ ^& x3 m& B7 E* Q9 d) S［2］Kato Y，Tani T，Sotomaru Y，Kurokawa K，Kato JY，Doguchi H，Yasue H，Tsunoda Y.Eight calves cloned from somatic cells of a single adult ［J］.Science，1998;282（2095）:2095-2098." G% }0 l2 S9 G) N
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［32］Wakayama T，Perry AC，Zuccotti M，Johnson KR，Yanagi-machi R.Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei ［J］. . Z2 y, l# \# \4 a, t9 V Biography:YAN Quan-Jian（male，born on Much4，1971in Zhouk-ou city，Henan province，China），a M.D.in the Department of Urology，Xijing Hospital，the Fourth Military Medical University，Supervisor:Prof.SHAO Guo-Xing and Prof.LI Liu-Jin.Tel 862913709180100 Email.godriwg@263.net 1 Department of Urology，Xijing Hospital， 2 Laboratory Animal Center，Fourth Military Medical Uni-versity，Xi'an710033，China） Editor XU Fu-Ming作者: 剑啸寒 时间: 2015-6-15 08:01