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EMBRYONIC STEM CELLS: CHARACTERIZATION SERIES

Derivation of Human Embryonic Stem Cells from Developing and Arrested Embryos

^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 Investigación Príncipe Felipe, Valencia, Spain

Key Words. Embryo • Human embryonic stem cells • Pluripotent stem cells • Differentiation

Correspondence: Miodrag Stojkovic, Ph.D., Centro de Investigacion Principe Felipe - Cellular Reprogramming Laboratory, C/E.P. Avda. Autopista del Saler 16-3 (junto Oceanografico) Valencia 46013, Spain. Telephone: 381-63-768-6185; e-mail: Sintocell{at}web.de

Received on June 22, 2006; accepted for publication on September 13, 2006.

First published online in STEM CELLS EXPRESS  September 21, 2006.

	ABSTRACT

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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.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
Human embryonic stem cells (hESC) hold huge promise in modern regenerative medicine, drug discovery, and as a model for studying early human development [1, 2]. However, usage of embryos and derivation of hESC for research and eventual medical application has resulted in polarized ethical debates [3] since the process involves destruction of viable developing human embryos at blastocyst [1, 2, 4] or morula stage [5]. In countries where the process of hESC derivation is allowed, these surplus embryos are donated by the couples after in vitro fertilization (IVF) treatment. These embryos are then subjected to in vitro culture (IVC) prior to being plated for derivation purposes. During this process, only small number of all IVF zygotes will develop successfully to morula and blastocyst [3, 6], and most of these embryos will succumb to embryo arrest [6–10]. For this reason, arrested embryos are also labeled as dead embryos [3], since they do not continue their cleavage even after 24 hours of observation [8]. However, not all blastomeres within the arrested embryo are abnormal nor are responsible for developmental arrest [7–10].

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.

	MATERIALS AND METHODS

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Culture of Embryos
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 [3–10]. Arrested embryos were used only when it was clear that their development had been arrested irreversibly i.e., when no blastomere from the embryo had undergone any cleavage division during the last 24–48 hours under IVC conditions [8–10]. This indicates that the early (2–10 cells) or late (16–24 cells) arrested embryos had spent a longer time in culture than the normal cleavage timing (for instance 16–24 cell stage on Day 5 and no further cleavage on Days 6 or 7).

View larger version (54K): [in this window] [in a new window]   Figure 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.

Growth of New hESC Lines
After 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 [2] 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°C until needed. Growth medium was changed every second day. Each hESC line was passaged mechanically and then transferred to freshly prepared feeders.

Reverse Transcription-Polymerase Chain Reaction Analysis of Embryos and hESC
Developing or arrested embryos were placed into RNase-free microcentrifuge tubes containing lysis buffer and snap-frozen in liquid nitrogen before being stored at –80°C. 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 [11, 12].

Immunocytochemical Characterization of hESC
To investigate whether the new hESC lines maintain their undifferentiated and pluripotent state, we performed immunocytochemical staining of hESC surface markers as previously described [2, 11].

Karyotype Analysis and In Vitro and In Vivo Differentiation of hESC
The 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 [2, 11].

	RESULTS

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For this study, surplus and donated human embryos were used to determine whether early (2–10 cells) and late (16–24 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 3–5 early (4–10 cell stage), and 13 arrested Days 6–7 late (16–24 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–L). 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).

View larger version (48K): [in this window] [in a new window]   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 3–5. 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–la3) 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.

View larger version (107K): [in this window] [in a new window]   Figure 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–D; F–H; J–L).

View larger version (80K): [in this window] [in a new window]   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 17–21). (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 17–21). (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 6–8 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–VI) and hematoxylin and eosin (II, III). Scale bars: (I, II) 500 µm; (III) 100 µm; (IV–VI) 200 µm. Abbreviation: GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

The 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 [17, 18]. Using non-invasive lineage tracing it was demonstrated that one of the two cell stage blastomeres of cleaving mouse embryos tends to contribute to embryonic tissues, while the other contributes predominantly to extraembryonic part of the blastocyst [17, 19]. Previously it was demonstrated that the asymmetrical distribution of Cdx2 gene product in mouse oocytes and embryos defines the lineage of trophectoderm [20]. The blastomeres that happen to be on the outside of early mouse embryos will form the trophoblast, whereas the cells that happen to be inside will generate the embryo [17–21]. It was suggested that in human embryos this process happens during compaction of the early embryo [6, 10]. These studies and our study support the view that blastomeres of early embryos have dissimilar fates and proliferative potential and that reorganization of blastomeres during embryo development plays crucial role in further cell lineage and normal development. However, quite frequently, human embryos arrest due to the different developmental abnormalities including presence of unequal blastomere or blastomere fragmentation [6–10]. The proportion of arrested embryos and degree of equal/unequal blastomeres or fragmentation during early cleavage is universally used as an indicator of embryo quality and to define IVC conditions, which lead to reduced blastocyst formation [3, 8–10]. Nevertheless, the level of whole embryo markers of normal embryonic genome activation in arrested human embryos is indistinguishable from that of developing embryos [22]. In addition, poor grade human embryos harbor blastomeres with normal karyotype [23] and a normal or a high-level of pluripotency marker such as OCT4 [24], and when isolated from arrested embryos nearly 40% of blastomeres demonstrate the ability to divide [7]. hESC derived from arrested embryos possess a normal karyotype, which points to the suggestion that arrested embryos, although possessing some blastomeres with chromosomal or other abnormalities might preserve a self-normalization mechanism(s) [23], which protects the ICM [6, 8, 10] and the subsequently derived hESC from these abnormalities. In view of this, our failure to derive hESC from early arrested embryos (in total 119) can be attributed to the suboptimal in vitro conditions, incorrect transition from maternal to embryonic genome, smaller number of normal blastomeres with ICM predisposition necessary for attachment, certain blastomere interaction, polarization, reorganization, and further proliferation. The very similar phenomenon of interaction and proliferation has been noted after the unsuccessful attempts to derive murine or hESC from single blastomeres [25, 26]. This is also supported by several recent studies, which show that hESC are more viable as aggregates rather than single cells [27, 28]. Here we demonstrated that arrested embryos have viable blastomeres with the potential to proliferate and that there are no biological or technical obstacles to derive hESCs using these arrested embryos. However, future studies are necessary to increase derivation efficiency, identify molecular mechanisms, autocrine and paracrine factors, which support attachment, outgrowth, and proliferation, and development of early and late arrested embryos using feeder-free and animal-free conditions [29, 30].

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 [3, 25, 26, 31]. It was demonstrated previously [26] 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.

Our 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 [25, 26], second, to set up conditions for derivation of clonal lines from the same embryo (and to find out how clonal they are), third, to study and compare normal and abnormal early human development, fourth, to study genetic/epigenetic profiles of embryos and derived hESC, and finally, to derive new hESC lines of clinical grade for cell replacement therapies or drug discoveries. In addition, arrested embryos derived after nuclear transfer (NT) could be a crucial source for successful derivation of patient-specific stem cells, since derivation of human NT blastocysts is not an efficient procedure [32].

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 [31, 33]. 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.

	DISCLOSURES

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The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
The 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.

	REFERENCES

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 

1. Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell line from human blastocysts. Science 1998;282:1145–1147.[Abstract/Free Full Text]

2. Stojkovic M, Lako M, Stojkovic P et al. Derivation of human embryonic stem cells from Day 8 blastocysts recovered after three-step in vitro culture. STEM CELLS 2004;22:790–797.[Abstract/Free Full Text]

3. Landry DW, Zucker HA. Embryonic death and the creation of human embryonic stem cells. J Clin Invest 2004;114:1184–1186.[CrossRef][Medline]

4. Mitalipova M, Calhoun J, Shin S et al. Human embryonic stem cell lines derived from discarded embryos. STEM CELLS 2003;21:521–526.[Abstract/Free Full Text]

5. Strelchenko N, Verlinsky O, Kucharenko V et al. Morula-derived human embryonic stem cells. RBM Online 2004;9:623–629.

6. Jurisicova A, Acton BM. Deadly decisions: the role of genes regulating programmed cell death in human preimplantation embryo development. Reproduction 2004;128:281–291.[Abstract/Free Full Text]

7. Geber S, Sampaio M. Blastomere development after embryo biopsy: a new model to predict embryo development and to select for transfer. Hum Reprod 1999;14:782–786.[Abstract/Free Full Text]

8. Martinez F, Rienzi L, Iacobelli M et al. Caspase activity in preimplantation human embryos is not associated with apoptosis. Hum Reprod 2002;17:1584–1590.[Abstract/Free Full Text]

9. Munné S, Grifo J, Cohen J et al. Chromosome abnormalities in human arrested preimplantation embryos: a multiple-probe FISH study. Am J Hum Genet 1994;55:150–159.[Medline]

10. Hardy K, Spanos S, Becker D et al. From cell death to embryo arrest: Mathematical models of human preimplantation embryo development. Proc Natl Acad Sci U S A 2001;98:1655–1660.[Abstract/Free Full Text]

11. Stojkovic P, Lako M, Stewart R et al. An autogeneic feeder cell system that efficiently supports growth of undifferentiated human embryonic stem cells. STEM CELLS 2005;23:306–314.[Abstract/Free Full Text]

12. Hyslop L, Stojkovic M, Armstrong L et al. Downregulation of NANOG induces differentiation of human embryonic stem cells to extraembryonic lineages. STEM CELLS 2005;23:1035–1043.[Abstract/Free Full Text]

13. Sus-Toby E, Gerecht-Nir S, Amit M et al. Derivation of a diploid human embryonic stem cell line from a mononuclear zygote. Hum Reprod 2004;19:670–675.[Abstract/Free Full Text]

14. Cowan CA, Klimanskaya I, McMahon J et al. Derivation of embryonic stem-cell lines from human blastocysts. N Engl J Med 2004;350:1353–1356.[Free Full Text]

15. Chen H, Qian K, Hu J et al. The derivation of two additional human embryonic stem cell lines from day 3 embryos with low morphological scores. Hum Reprod 2005;20:2201–2206.[Abstract/Free Full Text]

16. Hovatta O, Mikkola M, Gertow K et al. A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum Reprod 2003;18:1404–1409.[Abstract/Free Full Text]

17. Piotrowska K, Wianny F, Pedersen RA et al. Blastomeres arising from the first cleavage division have distinguishable fates in normal mouse development. Development 2001;128:3739–3748.[Abstract/Free Full Text]

18. Gardner RL. The initial phase of embryonic patterning in mammals. Int Rev Cytol 2001;203:233–290.[Medline]

19. Fujimori T, Kurotaki Y, Miyazaki J et al. Analysis of cell lineage in two- and four-cell mouse embryos. Development 2003;130:5113–5122.[Abstract/Free Full Text]

20. Deb K, Sivaguru M, Yong HY et al. Cdx2 gene expression and trophectoderm lineage specification in mouse embryos. Science 2006;311:992–996.[Abstract/Free Full Text]

21. Sutherland AE, Speed TP, Calarco PG. Inner cell allocation in the mouse morula: the role of oriented division during fourth cleavage. Dev Biol 1990;137:13–25.[CrossRef][Medline]

22. Dobson AT, Raja R, Abeyta MJ et al. The unique transcriptome through day 3 of human preimplantation development. Hum Mol Genet 2004;13:1461–1470.[Abstract/Free Full Text]

23. Munné S, Velilla E, Colls P et al. Self-correction of chromosomally abnormal embryos in culture and implications for stem cell production. Fertil Steril 2005;84:1328–1334.[CrossRef][Medline]

24. Hansis C, Tang YX, Grifo JA et al. Analysis of Oct-4 expression and ploidy in individual human blastomeres. Mol Hum Reprod 2001;7:155–161.[Abstract/Free Full Text]

25. Chung Y, Klimanskaya I, Becker S et al. Embryonic and extraembryonic stem cell lines derived from single mouse blastomeres. Nature 2006;439:216–219.[CrossRef][Medline]

26. Klimanskaya I, Chung Y, Becker S et al. Human embryonic stem cell lines derived from single blastomeres. Nature 2006; in press.

27. Taylor RA, Cowin PA, Cunha GR et al. Formation of human prostate tissue from embryonic stem cells. Nat Methods 2006;3:179–181.[CrossRef][Medline]

28. Trounson A. The production and directed differentiation of human embryonic stem cells. End Rev 2006;27:208–219.[Abstract/Free Full Text]

29. Klimanskaya I, Chung Y, Meisner L et al. Human embryonic stem cells derived without feeder cells. Lancet 2005;365:1636–1641.[CrossRef][Medline]

30. Ludwig TE, Levenstein ME, Jones JM et al. Derivation of human embryonic stem cells in defined conditions. Nat Biotechnol 2006;24:185–187.[CrossRef][Medline]

31. The President's Council on Bioethics. White Paper: Alternative Sources of Pluripotent Stem Cells. http://bioethics.gov/reports/white_paper/text.html.

32. Stojkovic M, Stojkovic P, Leary C et al. Derivation of a human blastocyst after heterologous nuclear transfer to donated oocytes. RBM Online 2005;11:226–231.

33. Weissman IL. Medicine: Politic stem cells. Nature 2005;439:145–147.[CrossRef]

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