2 THE IN VIVO DEVELOPMENTAL POTENTIAL OF PORCINE SKIN-DERIVED PROGENITORS

2012 ◽  
Vol 24 (1) ◽  
pp. 112 ◽  
Author(s):  
M. T. Zhao ◽  
X. Yang ◽  
K. Lee ◽  
J. Mao ◽  
J. M. Teson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Lineage ◽  
Pcr Amplification ◽  
Cell Types ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
Germ Layers ◽  
Developmental Potential

Skin-derived progenitors (SKP) are capable of generating both neural and mesodermal progeny in vitro: neurons, Schwann cells, adipocytes, osteocytes and chondrocytes, thus exhibiting characteristics similar to embryonic neural crest stem cells. SKP show distinct transcriptional profiles when compared with neurospheres/neural stem cells in the central nervous system (CNS) and skin-derived fibroblasts, indicating a novel type of multipotent stem cell derived from the dermis of the skin. However, it remains unclear whether SKP cells can produce ectoderm and mesoderm lineages or other germ layers in vivo, although oocyte-like structures can be induced from porcine SKP in vitro. Embryonic chimeras are a well-established tool for investigating cell lineage determination and cell potency through normal embryonic development. Thus the purpose of this study was to investigate the in vivo developmental potential of porcine SKP by chimera production. Porcine SKP cells and fibroblasts were isolated from the back skin of Day 35 to 50 GFP transgenic fetuses. Individual cells or clusters of male GFP transgenic SKP and skin-derived GFP-expressing fibroblasts were injected into pre-compact in vitro-fertilized (IVF) embryos, respectively and then transferred into corresponding surrogates 24 h post-injection. Additional injected embryos were cultured in PZM3 medium for another 2 days until the blastocyst stage and subsequently stained with Hoechst 33342. Interestingly, in some of the chimeras the injected SKP cells migrated and dispersed into different locations of the host blastocysts, whereas in others they remained as a cluster of cells within the chimeric blastocysts. In contrast, the fibroblast cells were not observed to spread around the host blastocysts. Two chimeric fetuses were recovered at the middle of gestation and a litter of viable piglets was born. Genomic DNA was extracted from various tissues of chimeric piglets and subjected to PCR amplification. Two chimeric fetuses and 2 out of 6 piglets carried the GFP transgene in SKP-derived chimeras, but GFP was not present in the fibroblast-derived chimeric fetuses (n = 6). Surprisingly, the GFP transgene was present in various tissues of two SKP-derived chimeric piglets, including lung, heart, liver, artery, kidney, brain, skin, muscle, gut, ovary, pancreas and stomach, thus representing the 3 germ layers (ectoderm, mesoderm and endoderm). In addition, SRY was detected in several tissues of the two GFP-positive female chimeric piglets, confirming the chimerism of these piglets. Therefore, it appears that porcine SKP can contribute to various cell types of the 3 germ layers and have a broader developmental potency than previously expected. Alternatively, pre-compact (4-cell and 8-cell stage) embryos may provide a unique environment for reprogramming skin-derived progenitors into a more primitive state by the process of embryonic compaction. This study was funded by NIH National Center for Research Resources (R01RR013438) and Food for the 21st Century at the University of Missouri.

scholarly journals Comparative Analyses of Single-Cell Transcriptomic Profiles between In Vitro Totipotent Blastomere-like Cells and In Vivo Early Mouse Embryonic Cells

Cells ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 3111
Author(s):  
Po-Yu Lin ◽  
Denny Yang ◽  
Chi-Hsuan Chuang ◽  
Hsuan Lin ◽  
Wei-Ju Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Embryonic Cells ◽  
Cell Stage ◽  
Developmental Potential ◽  
Functional Features ◽  
Early Mouse ◽  
Extraembryonic Tissues

The developmental potential within pluripotent cells in the canonical model is restricted to embryonic tissues, whereas totipotent cells can differentiate into both embryonic and extraembryonic tissues. Currently, the ability to culture in vitro totipotent cells possessing molecular and functional features like those of an early embryo in vivo has been a challenge. Recently, it was reported that treatment with a single spliceosome inhibitor, pladienolide B (plaB), can successfully reprogram mouse pluripotent stem cells into totipotent blastomere-like cells (TBLCs) in vitro. The TBLCs exhibited totipotency transcriptionally and acquired expanded developmental potential with the ability to yield various embryonic and extraembryonic tissues that may be employed as novel mouse developmental cell models. However, it is disputed whether TBLCs are ‘true’ totipotent stem cells equivalent to in vivo two-cell stage embryos. To address this question, single-cell RNA sequencing was applied to TBLCs and cells from early mouse embryonic developmental stages and the data were integrated using canonical correlation analyses. Differential expression analyses were performed between TBLCs and multi-embryonic cell stages to identify differentially expressed genes. Remarkably, a subpopulation within the TBLCs population expressed a high level of the totipotent-related genes Zscan4s and displayed transcriptomic features similar to mouse two-cell stage embryonic cells. This study underscores the subtle differences between in vitro derived TBLCs and in vivo mouse early developmental cell stages at the single-cell transcriptomic level. Our study has identified a new experimental model for stem cell biology, namely ‘cluster 3’, as a subpopulation of TBLCs that can be molecularly defined as near totipotent cells.

Abstract 4: Porcine Parthenotes as a Model to Evaluate Developmental Potential of Human Inducible Pluripotent Stem Cells

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Naoko Koyano-Nakagawa ◽  
James Dutton ◽  
Mary G Garry ◽  
Daniel J Garry
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Cellular Proliferation ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Differentiation Potential ◽  
Developmental Potential ◽  
Parthenogenetic Embryos

The use of human induced pluripotent stem cells (hiPSCs) has tremendous potential for regenerative medicine by providing an unlimited source of personalized cells. A number of protocols have been established for efficient differentiation of hiPSCs to the desired lineage in vitro, such as cardiomyocytes and blood. However, the field lacks an in vivo system to evaluate the differentiation potential and quality of hiPSCs. Developmental potential of stem cells derived from experimental animals can be readily assessed by generating blastocyst chimeras and examination of the contribution to the embryos, or by the potential of teratoma formation. However, this is not possible in the case of humans. As a potential solution for this issue, we examined whether porcine parthenotes could be used as an experimental model to test the developmental potential of the hiPSCs. Parthenotes are generated by electrical activation of the oocytes collected at the abattoir and will develop up to gestational day 53 if transferred to a pseudo-pregnant sow. The embryonic culture conditions have also been established and the zygotes can develop normally to the expanded blastocyst stage (day 7 post fertilization/activation), in vitro. We took advantage of this in vitro system and examined the ability of hiPSCs to proliferate and integrate into the parthenogenetic embryos. Parthenogenetic embryos were injected with ten undifferentiated hiPSCs at day 4 (8 cell ~ morula stage) and cultured up to 72 hours. During this period, parthenotes underwent blastocoel cavity formation and hatching. Cell tracing experiments demonstrated that hiPSCs proliferated and integrated into the parthenotes. They retained pluripotency marker expression during this period. hiPSCs and their derivatives were found both in trophoectoderm and embryo proper. We further observed that the hiPSCs underwent cellular proliferation and promoted developmental progression of the parthenote in vitro. In summary, the porcine parthenote model system is an efficient high throughput system to examine the developmental capacity of human stem cell populations.

Comparison of glucose metabolism in in vivo- and in vitro-matured tammar wallaby oocytes and its relationship to developmental potential following intracytoplasmic sperm injection

2004 ◽  
Vol 16 (6) ◽  
pp. 617 ◽  
Author(s):  
Genevieve M. Magarey ◽  
Karen E. Mate
Keyword(s):  
Glucose Metabolism ◽  
Intracytoplasmic Sperm Injection ◽  
Tammar Wallaby ◽  
Blastocyst Stage ◽  
In Vitro Fertilisation ◽  
Cell Stage ◽  
Developmental Potential ◽  
Developmental Capacity

Although marsupial oocytes undergo nuclear maturation in vitro, there is, at present, no indication of their developmental potential, largely owing to the lack of in vitro fertilisation and related technologies for marsupials. Glucose metabolism has proven a useful indicator of oocyte cytoplasmic maturation and developmental potential in several eutherian species. Therefore, the aims of the present study were to compare: (1) the rates of glycolysis and glucose oxidation in immature, in vitro-matured and in vivo-matured tammar wallaby oocytes; and (2) the metabolic rate of individual oocytes with their ability to form pronuclei after intracytoplasmic sperm injection. The rates of glycolysis measured in immature (2.18 pmol oocyte–1 h–1), in vitro-matured (0.93 pmol oocyte–1 h–1) and in vivo-matured tammar wallaby oocytes (0.54 pmol oocyte–1 h–1) were within a similar range to values obtained in eutherian species. However, unlike the trend observed in eutherian oocytes, the glycolytic rate was significantly higher in immature oocytes compared with either in vivo- or in vitro-matured oocytes (P < 0.001) and significantly higher in in vitro-matured oocytes compared with in vivo-matured oocytes (P < 0.001). No relationship was identified between glucose metabolism and the developmental capacity of oocytes after intracytoplasmic sperm injection when assessed after 17–19 h. Oocytes that became fertilised (two pronuclei) or activated (one or more pronucleus) were not distinguished from others by their metabolic rates. Longer culture after intracytoplasmic sperm injection (e.g. blastocyst stage) may show oocyte glucose metabolism to be predictive of developmental potential; however, culture to the single-cell stage did not reveal any significant differences in normally developing embryos.

The neural crest population responding to endothelin-3 in vitro includes multipotent cells

1997 ◽  
Vol 110 (14) ◽  
pp. 1673-1682 ◽  
Author(s):  
J.G. Stone ◽  
L.I. Spirling ◽  
M.K. Richardson
Keyword(s):  
Neural Crest ◽  
Schwann Cells ◽  
Cell Types ◽  
Developmental Potential ◽  
Colony Assay ◽  
Cellular Targets ◽  
Multipotent Cells ◽  
Endothelin 3

The peptide endothelin 3 (EDN3) is essential for normal neural crest development in vivo, and is a potent mitogen for quail truncal crest cells in vitro. It is not known which subpopulations of crest cells are targets for this response, although it has been suggested that EDN3 is selective for melanoblasts. In the absence of cell markers for different precursor types in the quail crest, we have characterised EDN3-responsive cell types using in vitro colony assay and clonal analysis. Colonies were analysed for the presence of Schwann cells, melanocytes, adrenergic cells or sensory-like cells. We provide for the first time a description of the temporal pattern of lineage segregation in neural crest cultures. In the absence of exogenous EDN3, crest cells proliferate and then differentiate. Colony assay indicates that in these differentiated cultures few undifferentiated precursors remain and there is a low replating efficiency. By contrast, in the presence of 100 ng/ml EDN3 differentiation is inhibited and most of the cells maintain the ability to give rise to mixed colonies and clones containing neural crest derivatives. A high replating efficiency is maintained. In secondary culture there was a progressive decline in the number of cell types per colony in control medium. This loss of developmental potential was not seen when exogenous EDN3 was present. Cell type analysis suggests two novel cellular targets for EDN3 under these conditions. Contrary to expectations, one is a multipotent precursor whose descendants include melanocytes, adrenergic cells and sensory-like cells; the other can give rise to melanocytes and Schwann cells. Our data do not support previous claims that the action of EDN3 in neural crest culture is selective for cells in the melanocyte lineage.

scholarly journals From blastocyst to gastrula: gene regulatory networks of embryonic stem cells and early mouse embryogenesis

2014 ◽  
Vol 369 (1657) ◽  
pp. 20130542 ◽  
Author(s):  
David-Emlyn Parfitt ◽  
Michael M. Shen
Keyword(s):  
Stem Cells ◽  
Gene Networks ◽  
Regulatory Networks ◽  
Embryonic Stem ◽  
Cell Lineage ◽  
Network Models ◽  
Cell Types ◽  
Mammalian Development ◽  
A Genome

To date, many regulatory genes and signalling events coordinating mammalian development from blastocyst to gastrulation stages have been identified by mutational analyses and reverse-genetic approaches, typically on a gene-by-gene basis. More recent studies have applied bioinformatic approaches to generate regulatory network models of gene interactions on a genome-wide scale. Such models have provided insights into the gene networks regulating pluripotency in embryonic and epiblast stem cells, as well as cell-lineage determination in vivo . Here, we review how regulatory networks constructed for different stem cell types relate to corresponding networks in vivo and provide insights into understanding the molecular regulation of the blastocyst–gastrula transition.

scholarly journals Guiding T lymphopoiesis from pluripotent stem cells by defined transcription factors

2019 ◽  
Author(s):  
Rongqun Guo ◽  
Fangxiao Hu ◽  
Qitong Weng ◽  
Cui Lv ◽  
Hongling Wu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Time Window ◽  
Immune Surveillance ◽  
Cell Stage ◽  
Immunodeficient Mice ◽  
Cell Transcriptome ◽  
T Lymphopoiesis

ABSTRACTAchievement of immunocompetent and therapeutic T lymphopoiesis from pluripotent stem cells is a central aim in T cell regenerative medicine. To date, preferentially regenerating T lymphopoiesis in vivo from pluripotent stem cells (PSC) remains a practical challenge. Here we documented that synergistic and transient expression of Runx1 and Hoxa9 restricted in the time window of endothelial to hematopoietic transition and hematopoietic maturation stages induced in vitro from PSC (iR9-PSC) preferentially generated engraftable hematopoietic progenitors capable of homing to thymus and developing into mature T (iT) cells in primary and secondary immunodeficient recipients. Single-cell transcriptome and functional analyses illustrated the cellular trajectory of T lineage induction from PSC, unveiling the T-lineage specification determined at as early as hemogenic endothelial cell stage and identifying the bona fide pre-thymic progenitors. The iT cells distributed normally in central and peripheral lymphoid organs and exhibited abundant TCRαβ repertoire. The regenerative T lymphopoiesis rescued the immune-surveillance ability in immunodeficient mice. Furthermore, gene-edited iR9-PSC produced tumor-specific-T cells in vivo that effectively eradicated tumor cells. This study provides insight into universal generation of functional and therapeutic T lymphopoiesis from the unlimited and editable PSC source.

scholarly journals Modulating the Substrate Stiffness to Manipulate Differentiation of Resident Liver Stem Cells and to Improve the Differentiation State of Hepatocytes

2016 ◽  
Vol 2016 ◽  
pp. 1-12 ◽  
Author(s):  
Angela Maria Cozzolino ◽  
Valeria Noce ◽  
Cecilia Battistelli ◽  
Alessandra Marchetti ◽  
Germana Grassi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Culture Method ◽  
Cell Types ◽  
Culture Conditions ◽  
Precursor Cells ◽  
Substrate Stiffness ◽  
Growth And Survival ◽  
Liver Stem Cells

In many cell types, several cellular processes, such as differentiation of stem/precursor cells, maintenance of differentiated phenotype, motility, adhesion, growth, and survival, strictly depend on the stiffness of extracellular matrix that,in vivo, characterizes their correspondent organ and tissue. In the liver, the stromal rigidity is essential to obtain the correct organ physiology whereas any alteration causes liver cell dysfunctions. The rigidity of the substrate is an element no longer negligible for the cultivation of several cell types, so that many data so far obtained, where cells have been cultured on plastic, could be revised. Regarding liver cells, standard culture conditions lead to the dedifferentiation of primary hepatocytes, transdifferentiation of stellate cells into myofibroblasts, and loss of fenestration of sinusoidal endothelium. Furthermore, standard cultivation of liver stem/precursor cells impedes an efficient execution of the epithelial/hepatocyte differentiation program, leading to the expansion of a cell population expressing only partially liver functions and products. Overcoming these limitations is mandatory for any approach of liver tissue engineering. Here we propose cell lines asin vitromodels of liver stem cells and hepatocytes and an innovative culture method that takes into account the substrate stiffness to obtain, respectively, a rapid and efficient differentiation process and the maintenance of the fully differentiated phenotype.

282 IMPROVED QUALITY OF PORCINE BLASTOCYSTS BY AGGREGATION OF EMBRYOS AT THE 4 - 8-CELL STAGE

2006 ◽  
Vol 18 (2) ◽  
pp. 248
Author(s):  
S.-G. Lee ◽  
C.-H. Park ◽  
D.-H. Choi ◽  
H.-Y. Son ◽  
C.-K. Lee
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Transgenic Animals ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
Vitro Fertilization

Use of blastocysts produced in vitro would be an efficient way to generate embryonic stem (ES) cells for the production of transgenic animals and the study of developmental gene regulation. In pigs, the morphology and cell number of in vitro-produced blastocysts are inferior to these parameters in their in vivo counterparts. Therefore, establishment of ES cells from blastocysts produced in vitro might be hindered by poor embryo quality. The objective of this study was to increase the cell number of blastocysts derived by aggregating 4–8-cell stage porcine embryos produced in vitro. Cumulus–oocyte complexes were collected from prepubertal gilt ovaries, and matured in vitro. Embryos at the 4–8-cell stage were produced by culturing embryos for two days after in vitro fertilization (IVF). After removal of the zona pellucida with acid Tyrode’s solution, one (1X), two (2X), and three (3X) 4–8-cell stage embryos were aggregated by co-culturing them in aggregation plates followed by culturing to the blastocyst stage. After 7 days, the developmental ability and the number of cells in aggregated embryos were determined by staining with Hoechst 33342 and propidium iodide. The percentage of blastocysts was higher in both 2X and 3X aggregated embryos compared to that of 1X and that of intact controls (Table 1). The cell number of blastocysts also increased in aggregated embryos compared to that of non-aggregated (1X) embryos and controls. This result suggests that aggregation might improve the quality of in vitro-fertilized porcine blastocysts by increasing cell numbers, thus becoming a useful resource for isolation and establishment of porcine ES cells. Further studies are required to investigate the quality of the aggregated embryos in terms of increasing the pluripotent cell population by staining for Oct-4 and to apply improved aggregation methods in nuclear-transferred (NT) porcine embryos. Table 1. Development, cell number, and ICM ratio of aggregated porcine embryos

58 HISTONE DEACETYLASES INHIBITOR, SCRIPTAID, IS BENEFICIAL TO THE REPROGRAMMING OF SOMATIC NUCLEI FOLLOWING NUCLEAR TRANSFER

2009 ◽  
Vol 21 (1) ◽  
pp. 129
Author(s):  
J. G. Zhao ◽  
J. W. Ross ◽  
Y. H. Hao ◽  
D. M. Wax ◽  
L. D. Spate ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Histone Deacetylases ◽  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Untreated Group ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Reconstructed Embryos

Somatic cell nuclear transfer (SCNT) is a promising technology with potential applications in both agriculture and regenerative medicine. The reprogramming of differentiated somatic nuclei into totipotent embryonic state following NT is not efficient and the mechanism is currently unknown. However, accumulating evidence suggests that faulty epigenetic reprogramming is likely to be the major cause of low success rates observed in all mammals produced through SCNT. It has been demonstrated that increased histone acetylation in reconstructed embryos by applying histone deacetylases inhibitor (HDACi) such as trychostatin A (TSA) significantly enhanced the developmental competence in several species in vitro and in vivo. However TSA has been known to be teratogenic. Compared with TSA, Scriptaid is a low toxic but more efficient HDACi (Su GH et al. 2000 Cancer Res. 60, 3137–3142). The objectives of this study were: 1) to investigate and optimize the application Scriptaid to the NT using Landrace fetal fibroblast cells (FFCs) as donor; 2) investigate the effect of increased histone acetylation on the developmental competence of reconstructed embryos from NIH mini inbred FFCs in vitro and in vivo. The reconstructed embryos were treated with Scriptaid at different concentrations (0 nm, 250 nm, 500 nm and 1000 nm) after activation for 14 to 16 h. IVF embryos without treatment were produced as an additional control. Developmental rates to the 2-cell and blastocyst stage were determined. Developmental potential was determined by transferring Day 1 NT zygotes to the oviducts of surrogates on the day of, or one day after, the onset of estrus. Experiments were repeated at least 3 times and data were analyzed with chi-square tests using SAS 6.12 program (SAS institute, Inc., Cary, NC, USA). The percentage blastocyst of cloned embryos using Landrace FFCs as donors treated with 500 nm Scriptaid was the highest and was significantly higher than untreated group (25% v. 11%, P < 0.05). Percent cleaved was not different among four treatment groups. We used 500 nm Scriptaid for 14 to 16 h after activation for all subsequent experiments. Developmental rate to the blastocyst stage was significantly increased in cloned embryos derived from NIH mini inbred FFCs after treating with Scriptaid (21% v. 9%, P < 0.05), while the blastocyst rate in IVF group was 30%. Embryo transfer (ET) results showed that 5/6 (Transferred embryos No. were 190, 109, 154, 174, 152, and 190, respectively) surrogates (83%) became pregnant resulting in 2 healthy piglets from 2 litters (recipients received 190 and 154 embryos, respectively) in the Scriptaid treatment group, while no pregnancies were obtained in the untreated group from 5 ET (Embryos transferred No. are 140, 163, 161, 151 and 151, respectively). These results suggest that 500 nm Scriptaid treatment following activation increase both the in vitro and in vivo development of porcine SCNT embryos from NIH mini inbred FFCs and the hyperacetylation might actually improve reprogramming of the somatic nuclei after NT. Funding from the National Institutes of Health National Center for Research Resources RR018877.

180 IDENTIFICATION AND QUANTIFICATION OF DIFFERENTIALLY REPRESENTED TRANSCRIPTS IN PREIMPLANTATION BOVINE EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 169 ◽  
Author(s):  
C. E. McHughes ◽  
G. K. Springer ◽  
L. D. Spate ◽  
R. Li ◽  
R. J. Woods ◽  
...  
Keyword(s):  
Relative Abundance ◽  
Nuclear Transfer ◽  
Developmental Stages ◽  
Reproductive Technologies ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Production Techniques

Identification of transcripts that are present at key development stages of preimplantation embryos is critical for a better understanding of early embryogenesis. To that end, this project had two goals. The first was to characterize the relative abundance of multiple transcripts during several developmental stages, including metaphase II-stage oocytes (MPII), and 2-cell-stage (2-cell), precompact morula (PCM), and in vitro-produced blastocyst-stage (IVTBL) embryos. The second was to characterize differences in the relative abundance of transcripts present in in vivo- (IVVBL), in vitro-, and nuclear transfer-produced (NTBL) blastocysts. It was our hypothesis that the identification of differentially represented transcripts from these stages would reveal not only developmentally important genes, but also genes that might be aberrantly expressed due to embryo production techniques. Individual clusters from a large bovine EST project (http://genome.rnet.missouri.edu/Bovine/), which focused on female reproductive tissues and embryos, were compared using Fisher's exact test weighted by number of transcripts per tissue by gene (SAS PROC FREQ; SAS Institute, Inc., Cary, NC, USA). Of the 3144 transcripts that were present during embryogenesis, 125 were found to be differentially represented (P < 0.01) in at least one pairwise comparison (Table 1). Some transcripts found to increase in representation from the MPII to the 2-cell stage include protein kinases, PRKACA and CKS1, as well as the metabolism-related gene, PTTG1. These same transcripts were also found to decrease in representation from the 2-cell to the PCM stage. RPL15 (translation) and FTH1 (immune function) were both more highly represented in the PCM than in the 2-cell stage. From PCM to IVTBL, we saw an increase in RPS11, another translation-related transcript. When comparing blastocyst-stage embryos from different production techniques, several transcripts involved in energy production (e.g., COX7B and COX8A) were found to be more highly represented in the NTBL than in the IVTBL. COX8A was also more highly represented in the IVVBL than in the IVTBL. By investigating these differentially represented transcripts, we will be able to better understand the developmental implications of embryo manipulation. We may also be able to better develop reproductive technologies that lead to in vitro- and nuclear transfer-derived embryos which more closely follow a normal program of development. Table 1. Differentially represented transcripts between developmental stages

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