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作者：Xin Zhanga, Petra Stojkovica,c, Stefan Przyborskib, Michael Cookeb, Lyle Armstronga, Majlinda Lakoa, Miodrag Stojkovica,c作者单位：aCentre for Stem Cell Biology and Developmental Genetics, University of Newcastle, Newcastle upon Tyne, United Kingdom;bCentre for Stem Cell Biology and Regenerative Medicine, School of Biological and Biomedical Science, University of Durham, South Road, Durham, United Kingdom;cCentro de Investigaci 5 u# Q& N1 d# F
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【摘要】
5 u- S8 ]) v. Q Human embryonic stem cells (hESC) hold huge promise in modern regenerative medicine, drug discovery, and as a model for studying early human development. However, usage of embryos and derivation of hESC for research and potential medical application has resulted in polarized ethical debates since the process involves destruction of viable developing human embryos. Here we describe that not only developing embryos (morulae and blastocysts) of both good and poor quality but also arrested embryos could be used for the derivation of hESC. Analysis of arrested embryos demonstrated that these embryos express pluripotency marker genes such OCT4, NANOG, and REX1. Derived hESC lines also expressed specific pluripotency markers (TRA-1-60, TRA-1-81, SSEA4, alkaline phosphatase, OCT4, NANOG, TERT, and REX1) and differentiated under in vitro and in vivo conditions into derivates of all three germ layers. All of the new lines, including lines derived from late arrested embryos, have normal karyotypes. These results demonstrate that arrested embryos are additional valuable resources to surplus and donated developing embryos and should be used to study early human development or derive pluripotent hESC.
# {8 M/ e9 t& ^& v! t 【关键词】 Embryo Human embryonic stem cells Pluripotent stem cells Differentiation
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4 q s5 \6 ]" `( QHuman embryonic stem cells (hESC) hold huge promise in modern regenerative medicine, drug discovery, and as a model for studying early human development .- ~. d1 p( ^% ^' p7 @2 O
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Here we describe that not only developing embryos of good or poor quality but also that arrested embryos could be used for derivation of hESC. Arrested IVF embryos expressed pluripotent genes, and after removal of the zona pellucida (ZP) and exposure to more complex in vitro conditions, these embryos attached and formed primary outgrowths. Derived hESC expressed specific pluripotency markers and differentiated under in vitro and in vivo conditions into derivates of all three germ layers. This demonstrates that arrested embryos are additional valuable resources to surplus and donated developing embryos, which should be used to study early human development or derive pluripotent hESC.
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+ l1 h C) r' M P" n1 s: VMATERIALS AND METHODS Y: [. }8 b! K' J9 V
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Culture of Embryos1 D6 P6 |1 }' `- o6 v u8 C
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Surplus human embryos, produced by IVF for clinical purposes, were donated for research after patient consent was obtained at the Newcastle Fertility Centre at Life or IVF Unit, Gateshead, U.K. Embryos were cultured in G medium (Vitrolife, Kungsbacka, Sweden, http://www.vitrolife.com). Embryos of poor or good quality, which cleaved as morula on Day 4 or formed expanded blastocyst (ebl) on Day 6 were depicted as normally developing embryos and were used for derivation of new hESC lines. The chronology of normal development of human embryos up to morula and blastocyst stage under IVC conditions is presented in Figure 1. Normal developing embryos that showed low cell number, cell size irregularity, or fragmentation were morphologically classified as poor . This indicates that the early (2¨C10 cells) or late (16¨C24 cells) arrested embryos had spent a longer time in culture than the normal cleavage timing (for instance 16¨C24 cell stage on Day 5 and no further cleavage on Days 6 or 7).
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- k7 `% W, U0 f2 J. mFigure 1. Developmental stages and chronological time of normal early human development up to blastocyst stage. Oocyte retrieval assessed as Day 0. Early developing embryos of good quality possess equal blastomeres, absence of fragmentation, do not show delayed cleavage or do not arrest under in vitro conditions. These embryos cleave to morulae on Day 4 and to blastocysts on Day 5.9 ?! w# M4 E1 }4 d. L
2 U3 V' ~8 r3 q$ L, RDerivation of New hESC Lines' Y- W3 i0 q& N8 A1 Q5 ` H4 k
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To derive new lines, the inner cell mass (ICM) were isolated from ebl by immunosurgery .* M* p! {4 i0 o7 ?2 U i
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Growth of New hESC Lines
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; ]# l7 {7 x1 P( k( nAfter formation and mechanical dispersion of primary hESC outgrowth, hESC clumps were cultured either on inactivated mouse embryonic (MEF) or human fetal lung (HFL) feeder cells in the presence of ESC medium containing Knockout-DMEM (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), 100 µM ß-mercaptoethanol (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), 1 mM L-glutamine (Invitrogen), 100 mM nonessential amino acids, 10% serum replacement (SR, Invitrogen), 1% penicillin-streptomycin (Invitrogen) and 4 ng/ml basic fibroblast growth factor (Invitrogen) which was previously conditioned for 48 hours on hES-NCL1 line grown on MEF. Before use, conditioned medium was spun for 5 minutes at 1,000 rpm and subsequently filter-sterilized (0.22 µm) to remove any cellular material. This conditioned medium was kept at 4¡ãC until needed. Growth medium was changed every second day. Each hESC line was passaged mechanically and then transferred to freshly prepared feeders.$ `6 h5 a# N& K: F6 c$ H6 M( `( `
2 T# S' h0 C: |0 TReverse Transcription-Polymerase Chain Reaction Analysis of Embryos and hESC6 Y. \% ], |& d( _, X/ f6 l1 b
8 Y3 x& [9 y9 @+ ?4 l% NDeveloping or arrested embryos were placed into RNase-free microcentrifuge tubes containing lysis buffer and snap-frozen in liquid nitrogen before being stored at ¨C80¡ãC. Poly(A) RNA was obtained from lysed embryos by using a Dynabeads mRNA DIRECT Micro kit (Invitrogen) according to manufacturer's instructions. Complementary DNA (cDNA) was isolated from single embryos using the WT-Ovation RNA Amplification System (NuGEN Technologies, San Carlos, CA, http://www.nugenic.com) according to manufacturer's instructions. For hESC, reverse transcription-polymerase chain reaction (RT-PCR) was carried out using the cells to cDNA II kit (Ambion, Huntingdon, U.K., http://www.ambion.com) according to manufacturer's instructions. PCR and real-time RT-PCR analyses were carried out using the primers as described in supplemental Table 1, supplemental methods, and elsewhere .
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Immunocytochemical Characterization of hESC6 j* c7 O7 F9 n+ x/ j* T
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To investigate whether the new hESC lines maintain their undifferentiated and pluripotent state, we performed immunocytochemical staining of hESC surface markers as previously described .
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6 @; r! [" w( l% z; ]Karyotype Analysis and In Vitro and In Vivo Differentiation of hESC) x4 i5 R/ a+ r3 g: q: K
1 L3 R1 W# d. F5 _( n% i, ^8 XThe karyotype of 10 metaphase cells was determined by standard G-banding procedure. Differentiation of hESC under in vitro conditions and after tumor formation in severe combined immunodeficient (SCID) mice was done as previously described .5 G+ U+ }8 t* a# a9 w/ [
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/ ]$ r1 [1 y) {3 u; CFor this study, surplus and donated human embryos were used to determine whether early (2¨C10 cells) and late (16¨C24 cells) arrested embryos (schematically Fig. 2A) could be used for derivation of new hESC lines. Analysis of early or late developing and arrested embryos (Fig. 2B) demonstrated that these embryos express pluripotency genes: OCT4, NANOG, and REX1 (Fig. 2B). Of 161 donated embryos of different stages and quality (Table 1), 14 were Day 6 ebl, 15 Day 4 morulae, 119 arrested Days 3¨C5 early (4¨C10 cell stage), and 13 arrested Days 6¨C7 late (16¨C24 cell) embryos. All embryos were used for derivation of new hESC lines and plated on different feeder cells (supplemental Fig. 1). Using ebl, six (35.7%) new hESC lines were derived, one of which (hES-NCL6) was plated and cultured on HFL feeder cells (supplemental Fig. 2). Using Day 4 morulae (11 good and 4 poor quality, Fig. 3A and 3E, respectively), two (13.3%) new hESC lines (hES-NCL7 and hES-NCL8; Fig. 3D and 3H, respectively) were derived. The latter of these two cell lines was derived on HFL feeder cells. Of 119 early arrested embryos plated on feeder cells, four (3.4%) proliferated (Table 1) but without any clear signs of primary hESC-like outgrowth (supplemental Fig. 3). However, from 13 late arrested embryos (Fig. 3I), 5 (38.5%) outgrowths were observed, 2 of which showed (15.4%) a typical morphology for hESC (Fig. 3J¨CL). All hESC like outgrowths were mechanically dissected and replated either on fresh MEF or HFL feeder cells. After the replating of two outgrowths formed from late arrested embryos, one stable and fully characterized hESC line (hES-NCL9) was derived (Fig. 3L).3 x0 l2 Q* v8 x0 h( |8 w
6 b0 M& Z8 C. z+ `Figure 2. Different stages and ages of arrested human embryos. These embryos do not form blastocyst even after prolonged culture and arrest at early or late embryonic stages (A). Early and late arrested embryos could stop their cleavage with all equal (normal) blastomeres, but very frequently they have unequal or fragmented blastomeres. However, both groups of arrested and developing embryos show similar expression of pluripotency genes (B) when compared with early (a) or late (b) developing embryos. (a): ea1, two cell early arrested embryo; ea2, five cell early arrested embryo; ea3, eight cell early arrested embryos, recovered on Days 3¨C5. Two cell, 4 cell, 8 cell, 12 cell, and 16 cell are normal developing early embryos. (b): Representative micrograph of early arrested embryo (eight cell stage) stained with 4',6-diamidino-2-phenylindole (DAPI) (c). (d): Late arrested embryos (la1¨Cla3) recovered on Day 7. m1, m2, and m3 are morulae recovered on Day 4; ebl1, ebl2, and ebl3 are expanded blastocysts recovered on Day 6. (e): Representative micrograph of late arrested embryo (16 cell stage) stained with DAPI (f). Note the presence of unequal or fragmented but not stained (brown arrows) blastomeres in both, early, and late arrested embryos. Scale bar: 100 µm. Abbreviations: ea, early arrested; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; la, late arrested.
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( R# n! Q) O6 R/ h) zFigure 3. Developing and arrested embryos used for derivation of new human embryonic stem cell (hESC) lines. (A): Phase contrast micrograph of developing and good quality compacting Day 4 morula. Note the presence of equal blastomeres (A and schematically a1). After chemical removal of the zona pellucida, the embryo was plated on mouse feeder cells and attached after 1 day (B). After mechanical dissection, we observed primary outgrowth and hESC-like colony 15 (C) and 21 (D) days after initial plating, respectively. (E): Phase contrast micrograph of poor quality Day 4 morula. Note the presence of unequal (red arrows) and fragmented (brown arrows) blastomeres (schematically e1). The embryo was plated on human feeder cells and showed first signs of outgrowth after 10 days (F). After 16 days the outgrowth started to build hESC-like colony (G). This colony was very well formed and passaged on day 19 (H). (I): Bright-field micrograph of late arrested Day 7 embryo with equal, unequal, and fragmented blastomeres (I and schematically i1). One day after removal of zona pellucida and plating on mouse feeder cells, arrested embryos attached, and after 3 days the first signs of proliferation and primary outgrowth were noted (J). After 7 days the outgrowth proliferated further (K), and after 10 days the outgrowth showed typical morphology of hESC-like colony (L). The white arrows show the primary (B, F, J) or hESC-like outgrowths (C, G, K, D, H, L). Scale bar: 20 µm (I); 40 µm (A, E); 100 µm (B¨CD; F¨CH; J¨CL).
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Table 1. Embryos and conditions used for derivation of new hESC lines
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# u5 N O% l( R2 h& V. S: r4 pCharacterization of the new hESC lines (Table 2) including the hES-NCL9 line (Fig. 4) derived from arrested embryo demonstrated the presence of specific cell surface and intracellular (Fig. 4A and 4B) hESC markers (TRA-1-60, TRA-1-81, SSEA4, alkaline phosphatase, OCT4, NANOG, REX1, TERT). Karyotyping revealed that these new hESC lines have normal male (hES-NCL3, -4, -7, and -8), or female (hES-NCL2, -5, and -6) karyotype (Table 2). The hESC line derived from late arrested embryo (hES-NCL9) shows a normal female karyotype (Fig. 4C) and genetically differs from hES-NCL1 line (data not shown), which was used to recover conditioned medium. When allowed to differentiate spontaneously under in vitro conditions, the hES-NCL9 line produced cells of all three germ layers (Fig. 4D). Injection of hES-NCL9 cells into SCID mice resulted in consistent formation of teratomas that were primarily restricted to the site of injection. The analysis of excised tumor tissues confirmed their identity as teratomas, and histological examination revealed advanced differentiation of structures representative of all three embryonic germ layers (Fig. 4E). Real-time RT-PCR analysis (supplemental Fig. 4) revealed that the new hESC lines derived from morulae (hES-NCL7, hES-NCL8) or arrested embryo (hES-NCL9) expressed similar or higher levels of pluripotency genes when compared to already characterized and published H1 lines.5 z: i/ C7 n' y8 u- y u
2 F8 V( h" q$ p* Q( cTable 2. Characterization of new hESC lines derived from developing and arrested embryos n9 a' s$ p; O7 ?3 v" @
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Figure 4. Characterization of hES-NCL9 line derived from late arrested embryo. (A): Human embryonic stem cell (hESC) grown on mouse embryonic fibroblasts (MEF) stained with antibody recognizing the GTCM2 (a), TRA-1-60 (b), TRA-1-81 (c), SSEA-4 (d), and alkaline phosphates (e) epitopes (passages 17¨C21). (B): Reverse transcription-polymerase chain reaction (RT-PCR) analysis of undifferentiated hES-NCL9. PCR products were obtained using primers specific for OCT4, NANOG, REX1, TERT, and GAPDH (passages 17¨C21). (C): Karyotyping of hES-NCL9 cells grown on MEF show a normal female karyotype (passage 19). (D): Spontaneous differentiation of hES-NCL9 into neuronal (a), fat (b), and endoderm-like (d) cells demonstrating their differentiation ability under in vitro growth conditions. Green color represents cells stained with nestin (a) or -fetoprotein antibodies (c). Red color represents fat cells stained with oil red O staining (b). (E): Histological analysis of differentiated tissues found in teratomas formed in the testis of severe combined immunodeficient (CB17/ICR-Prkdcscid/Crl) mice following transplantation of NCL9 hESC. Teratomas were grown for a period of 6¨C8 weeks. Figures show bright-field micrographs of tissues prepared in Bouins fixative, embedded in paraffin wax, and sectioned (5 µm). (I): Low power image showing tissue heterogeneity within the tumor (b, bone; c, cartilage; g, primitive gut); (II): longitudinal profile of primitive gut (g) with accompanying submucosal muscle layer (m); (III): kidney tissue, including the glomeruli (gm) and adjacent tubules (tb); (IV): neural ganglia (ng); (V): cartilage (c); (VI): bone (b). Histological staining: Weigerts (I, IV¨CVI) and hematoxylin and eosin (II, III). Scale bars: (I, II) 500 µm; (III) 100 µm; (IV¨CVI) 200 µm. Abbreviation: GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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6 }. S, W$ [% \; zDISCUSSION
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' t' \$ n) _) [2 Y$ GMany hESC lines have been isolated to date, and several of these lines have already been derived from low-grade embryos .
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/ J+ n2 c5 P8 x4 [& iThe efficiency of hESC derivation was highest when we used Day 6 ebl. However, the arrested embryos also had the potential to proliferate and form primary outgrowth and hESC-like colonies once freed from ZP and subjected to more complex IVC conditions. This suggests that they have viable blastomeres with a predisposition to form ICM cells and the potential to proliferate. In mice, the first fate decision of a blastomere, whether it will become ICM or trophectoderm appears to be specified by its position during the first cleavage .
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Derivation of hESC from embryos with arrested cleavage corroborates with several proposals and alternative ways to derive new hESC without destruction of viable human embryos that hESC lines could be derived from blastomeres obtained by a single cell biopsy. This inefficient procedure requires improvements and skilful micromanipulation of early human embryos, which sometimes has adverse effects. In addition, the growth conditions are complicated since the biopsied blastomere needs to be cocultured with a previously derived hESC line. This also introduces the possibility of contamination of the new hESC line by cells of the coculture.
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/ ^* G6 |; V7 VOur work demonstrates that whole arrested embryos with clear signs of fragmentation and the presence of unequal blastomeres do not resume cell division and cannot be stated as live; however they possess some viable blastomeres. These can be induced to restart dividing or resume their developmental potential by transferring them to the more complex milieu conducive to ICM and hESC demands. The usage of arrested embryos offers an attractive option to further refine strategies for research and derivation of hESC as follows: first, using vital staining to identify and isolate viable blastomeres .
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- `1 Z/ N+ B$ [7 }In countries with a non-flexible policy, arrested embryos provide a more ethical source for research, and hESC derivation from these embryos correlates with the significant scientific effort that tries to resolve some of the political issues surrounding research using human embryos . Our opinion is that all surplus and consented arrested and developing embryos whether of poor or good quality should be used for research or derivation of hESC and not discarded. The latter is for sure less ethical since these embryos provide an appealing source and instrument to understand early human development and eventually cure human diseases.) ~. a3 p$ z* `* a
! c8 h& Z4 x, \DISCLOSURES
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The authors indicate no potential conflicts of interest.* g- H8 y X4 X( b7 Q2 p
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ACKNOWLEDGMENTS
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; N( s+ C& b4 g: t9 J ZThe authors are very thankful to the patients and clinical embryologists of Newcastle Fertility Centre at Life and IVF Unit Gateshead for donation and supply of embryos, J. Evans for karyotyping, and Louise Allcroft (Geneblitz, U.K.) for DNA profiling. This study was supported by One NorthEast Regional Development Agency and MRC Grant number G0301182. Author contribution: X.Z. and P.S. contributed equally to this work. X.Z. and P.S. derived, cultured, and characterized the lines. L.A. and M.L. helped with gene expression and characterization analysis and S.P. and M.C. with histological analysis of teratomas. M.S. designed the study, helped with derivation, growth and characterization of new hESC lines and wrote the manuscript. All authors discussed the results and commented on the manuscript. P.S. and M.S. are currently affiliated with Sintocell, Norvezanska 16, Leskovac, Serbia.6 z# l9 j$ k; U; |& `2 P- A( i
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