33 PRE-AND POST-IMPLANTATION DEVELOPMENT OF EMBRYOS CLONED FROM PORCINE SKIN-DERIVED SPHERE STEM CELLS

2008 ◽  
Vol 20 (1) ◽  
pp. 97
Author(s):  
Y. H. Hao ◽  
D. Wax ◽  
Z. S. Zhong ◽  
C. N. Murphy ◽  
L. Spate ◽  
...  
Keyword(s):  
Stem Cells ◽  
Genetic Modification ◽  
Selection Process ◽  
Donor Cell ◽  
Porcine Skin ◽  
Developmental Potential ◽  
Vitro Fertilization ◽  
Donor Cells ◽  
Post Implantation

Although transgenic animals have been successfully cloned, the process is still inefficient. One of the limitations is the use of somatic donor cells that have a limited lifespan. If a genetic modification is made, the selection process must be initiated and completed rapidly or the cells will undergo senescence. Identification of a stem cell that would proliferate rapidly and not undergo senescence would prove to be very valuable. Here we report attempts at cloning by using porcine skin-derived sphere stem cells to determine if they are a suitable donor cell type. Skin-derived stem cells were isolated from fetal skin and express the neural progenitor marker NES, as well as genes that may be critical for pluripotency such as POU5F1 and STAT3. The skin-derived stem cells proliferate rapidly in vitro and retain a normal karyotype after long-term culture. In the present study, skin-derived stem cells were cultured and frozen in liquid nitrogen from passage 1 to passage 8. To investigate the developmental potential of the skin-derived stem cells, we performed nuclear transfer (NT) and compared their preimplantation developmental efficiency to that of the embryos derived from in vitro fertilization (IVF). Cumulus–oocyte complexes (COCs) were aspirated from antral follicles of ovaries from prepubertal gilts. Approximately, groups of 50-70 COCs were matured in vitro in 500 µL TCM-199 per culture well for 40–44 h at 38.5�C, in a humidified atmosphere of 5% CO2 in air. The donor cells were thawed and cultured one day before NT; skin-derived stem cells were pipetted vigorously in PBS-EDTA to isolate individual cells. For IVF, cryopreserved ejaculated spermatozoa were thawed and washed and then resuspended with fertilization medium (mTBM). The MII oocytes were co-incubated with sperm for 6 h, and then transferred to PZM3 and cultured. For NT and IVF, respectively, the percent cleavage at 48 h in PZM3 was 64.9 � 8.2% (169/208) and 62.1 � 3.1% (94/184) (P > 0.05), the percent blastocysts after 6 days was 21.5 � 5.8% (53/208) and 25.2 � 3.4% (46/184) (P > 0.05), and the number of nuclei per blastocyst was 28.5 � 1.9 (NT, maximum was 58) and 16.8 � 4.0 (IVF, maximum was 31) (P < 0.05). To determine development post-implantation, some cloned embryos were cultured in PZM3 for 15.5 h and an average of 112 cloned embryos were transferred to the oviducts of four naturally cycling gilts on Day 0–1 of standing estrus. Three of the animals were pregnant: one of them farrowed two male piglets on August 14th, with the other two due on September 8th and 9th. Future studies will involve performing NT and ET on skin-derived stem cells from a higher passage number to determine if they would be suitable for genetic modification prior to NT.

scholarly journals Protocol for controlled modeling of human epiblast and amnion development using stem cells

2019 ◽  
Author(s):  
Yi Zheng ◽  
Jianping Fu
Keyword(s):  
Stem Cells ◽  
Primordial Germ Cells ◽  
Primitive Streak ◽  
Human Embryos ◽  
Vitro Fertilization ◽  
Human Embryology ◽  
Post Implantation ◽  
Advance Knowledge ◽  
Early Human

Abstract Due to the inaccessibility of post-implantation human embryos and the restriction on in-vitro fertilization (IVF) embryos cultured beyond 14 days, the knowledge of early post-implantation human embryogenesis remains extremely limited. Recently, we have developed a microfluidic in-vitro platform, based on human pluripotent stem cells (hPSCs), which is capable of recapitulating several key developmental landmarks of early human post-implantation embryonic development, including lumenogenesis of the epiblast (EPI), amniogenesis, and specification of primordial germ cells (PGCs) and of primitive streak (PS) cells. Given its controllability and reproducibility, the microfluidic platform provides a powerful experimental platform to advance knowledge of human embryology and reproduction. This protocol describes the preparation of the microfluidic device and its implementation for modeling human post-implantation epiblast and amnion development using hPSCs.

31 PORCINE SKIN-DERIVED STEM CELLS MAY BE A SUPERIOR SOURCE OF DONOR NUCLEI FOR EFFICIENT GENETIC MODIFICATION OF CLONED EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 116
Author(s):  
Y. H. Hao ◽  
J. W. Ross ◽  
P. Sutovsky ◽  
D. Wax ◽  
Z. S. Zhong ◽  
...  
Keyword(s):  
Stem Cells ◽  
Growth Factor ◽  
Genetic Modification ◽  
Embryonic Stem Cell Line ◽  
Embryonic Stem ◽  
High Rate ◽  
Porcine Skin ◽  
Donor Cells ◽  
Significant Difference

Somatic cell nuclear transfer (SCNT) in pigs relies primarily on the utilization of fetal-derived fibroblast cells, and the resultant clones tend to exhibit a significant level of phenotypic instability, which may be due to epigenetic reprogramming and/or genomic damage in the donor cells. In addition to the compromised phenotypic stability, production of transgenic clones through SCNT is inefficient, because the restricted lifespan of somatic donor cells in culture can be limiting when the genetic modification requires selection. In contrast, stem cells proliferate rapidly and do not undergo senescence at a high rate, so the selection process can be extended. Since there is no report of an embryonic stem cell line derived in the pig that could contribute to the germ line, we decided to investigate the utility of porcine skin-derived stem cells (SSCs). Porcine SSCs were isolated from the skin on the back of day 35 to 50 Yorkshire fetuses. The SSCs were cultured continually in SSCs medium (DMEM/F12 containing B-27, 20 ng mL–1 of epidermal growth factor, and 40 ng mL–1 of basic fibroblast growth factor) at 37.8°C, 5% CO2, 95% air. The SSCs expressed the neural progenitor marker nestin, as well as genes that are critical for pluripotency, such as Oct4 and Stat3. The SSCs proliferated actively in vitro and retained a normal karyotype after long-term culture. Electron microscopy revealed 2 distinct cell types within the spheres; elongated cells at the sphere periphery had invaginated nuclear envelopes and prominent nucleoli, and these cells displayed few, but large elongated mitochondria with transversal cristae as well as large cisternae of rough endoplasmic reticulum. In contrast, the cells in the center of the spheres were predominantly round-shaped, with a large round nucleus or cuboidal. The SSCs can be genetically modified with long-term positive selection, and 50 μg mL–1 G418 appeared to be an appropriate dose of G418 for selection of the transfected SSCs. Finally, NT embryos reconstructed with SSCs showed high rates of pre- and post-implantation development.The cell number in the blastocyst stage embryos derived from cloning with the SSC was significantly higher than those of the blastocysts derived from IVF (28.5 ± 1.9, 16.8 ± 4.0, respectively, P < 0.05), although there was no significant difference in blastocyst formation rates between these groups (21 to 25%). Three of the animals became pregnant in 4 surrogate gilts which received cloned embryos and reached to term. Two healthy male cloned piglets and 1 healthy female cloned piglet are genetically identical to the SSCs. Funding for this study was provided by the National Institutes of Health.

400 KARYOTYPIC ANALYSIS AND EPIGENETIC MODIFICATIONS OF CULTURED PORCINE ADIPOSE TISSUE-DERIVED STEM CELLS

2010 ◽  
Vol 22 (1) ◽  
pp. 356
Author(s):  
K. J. Williams ◽  
K. R. Bondioli ◽  
R. A. Godke
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Stem Cells ◽  
Chromatin Remodeling ◽  
Primary Cultures ◽  
Donor Cell ◽  
Global Methylation ◽  
Histone 3 ◽  
Donor Cells

The introduction of genetic modifications in donor cells for NT requires a significant number of population doublings (PD), and the deleterious effects, which may be attributed to aneuploidy or changes in DNA methylation and histone acetylation, are difficult at this time to circumvent. We hypothesize that the identification of a donor cell that is genetically stable for a long period of time in vitro such as somatic stem cells or those cells that demonstrate stem-like characteristics may be reprogrammed more completely, thus providing the key to increasing the efficiency of NT. Regulators of development in undifferentiated cells are suggested to be silenced by the presence of a bivalent domain modification pattern in which a large region of repressive histone 3 lysine 27 trimethylation (H3K27me3) contains smaller regions of activating histone 3 lysine 4 trimethylation (H3K4me3).The dual marks work to silence developmental genes in embryonic stem cells while simultaneously keeping them receptive to activation. The objectives of the current study were to determine the chromosomal stability of porcine adipose tissue-derived adult stem cells (pASC) through in vitro culture, to analyze pASC alongside fetal porcine fibroblasts (FPF) for gene expression profiles of chromatin remodeling proteins and global methylation and acetylation patterns, and to determine the presence of a co-enrichment of H3K27me3 and H3K4me3 within the promoter regions of developmentally important transcription factors. Metaphase spreads were prepared, and the presence of H3K27me3 and H3K4me3 was investigated in each of 3 individual pASC primary cultures for each analysis; whereas, gene expression and global methylation and acetylation were analyzed in each of 4 individual pASC and FPF primary cultures. Of 714 metaphases analyzed, 509 (71.3%) were aneuploid and only 205 (28.7%) were normal diploid porcine cells. For each cell population, we found a remarkable percentage of aneuploidies (43.7, 48.9, and 47.3, with a 46.6 ± 1.5 average) present immediately after the cultures were established. Chi-square analysis indicated that the percent of aneuploid cells during PD 1-10 was significantly less than that for PD 11-20 and PD 21-30. Also, porcine ASC demonstrated a consistently lower level of DNA methylation and histone acetylation through passages 2 through 7; whereas, the patterns for FPF varied. The expression levels of chromatin remodeling transcripts remained lower in pASC throughout culture when compared with FPF. Finally, porcine ASC possess a co-enrichment of H3K27me3 and H3K4me3 on the promoter region of the developmentally important transcription factor OCT-4. In vitro-cultured porcine ASC used as donor cells for NT should be chosen from early PD because of increased levels of aneuploidy at later PD. With a more complete characterization of porcine ASC, a donor cell population that can be more efficiently reprogrammed following fusion with the oocyte might be identified.

Analysis of Heterogeneous Mitochondria Distribution in Somatic Cell Nuclear Transfer Porcine Embryos

2008 ◽  
Vol 14 (5) ◽  
pp. 418-432 ◽  
Author(s):  
Zhisheng Zhong ◽  
Yanhong Hao ◽  
Rongfeng Li ◽  
Lee Spate ◽  
David Wax ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Mouse Fibroblast ◽  
Mitochondrial Morphology ◽  
Fibroblast Cells ◽  
Developmental Potential ◽  
Donor Cells

AbstractWe previously reported that translocation of mitochondria from the oocyte cortex to the perinuclear area indicates positive developmental potential that was reduced in porcine somatic cell nuclear transfer (SCNT) embryos compared to in vitro–fertilized (IVF) embryos (Katayama, M., Zhong, Z.-S., Lai, L., Sutovsky, P., Prather, R.S. & Schatten, H. (2006). Dev Biol299, 206–220.). The present study is focused on distribution of donor cell mitochondria in intraspecies (pig oocytes; pig fetal fibroblast cells) and interspecies (pig oocytes; mouse fibroblast cells) reconstructed embryos by using either pig fibroblasts with mitochondria-stained MitoTracker CMXRos or YFP-mitochondria 3T3 cells (pPhi-Yellow-mito) as donor cells. Transmission electron microscopy was employed for ultrastructural analysis of pig oocyte and donor cell mitochondria. Our results revealed donor cell mitochondrial clusters around the donor nucleus that gradually dispersed into the ooplasm at 3 h after SCNT. Donor-derived mitochondria distributed into daughter blastomeres equally (82.8%) or unequally (17.2%) at first cleavage. Mitochondrial morphology was clearly different between donor cells and oocytes in which various complex shapes and configurations were seen. These data indicate that (1) unequal donor cell mitochondria distribution is observed in 17.2% of embryos, which may negatively influence development; and (2) complex mitochondrial morphologies are observed in IVF and SCNT embryos, which may influence mitochondrial translocation and affect development.

Characterization of mesenchymal stem cells and their application in experimental embryology

2013 ◽  
Vol 16 (3) ◽  
pp. 593-599 ◽  
Author(s):  
J. Opiela ◽  
M. Samiec
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Somatic Cell ◽  
Fertilization In Vitro ◽  
Cell Cloning ◽  
Mammalian Embryo ◽  
Vitro Fertilization ◽  
Experimental Embryology ◽  
Genomic Engineering

Abstract The efficiency of somatic cell cloning (somatic cell nuclear transfer; SCNT) as well as in vitro fertilization/in vitro embryo production (IVF/IVP) in mammals stay at relatively same level for over a decade. Despite plenty of different approaches none satisfactory break-through took place. In this article, we briefly summarize the implementation of mesenchymal stem cells (MSCs) for experimental embryology. The advantages of using MSCs as nuclear donors in somatic cell cloning and in vitro embryo culture are described. The description of results obtained with these cells in mammalian embryo genomic engineering is presented.

scholarly journals Dynamic integration of enteric neural stem cells in ex vivo organotypic colon cultures

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Georgina Navoly ◽  
Conor J. McCann
Keyword(s):  
Stem Cells ◽  
Neural Stem Cells ◽  
Organotypic Culture ◽  
Ex Vivo ◽  
Donor Cell ◽  
Colonic Tissue ◽  
Cell Therapies ◽  
Enteric Ganglia ◽  
Donor Cells

AbstractEnteric neural stem cells (ENSC) have been identified as a possible treatment for enteric neuropathies. After in vivo transplantation, ENSC and their derivatives have been shown to engraft within colonic tissue, migrate and populate endogenous ganglia, and functionally integrate with the enteric nervous system. However, the mechanisms underlying the integration of donor ENSC, in recipient tissues, remain unclear. Therefore, we aimed to examine ENSC integration using an adapted ex vivo organotypic culture system. Donor ENSC were obtained from Wnt1cre/+;R26RYFP/YFP mice allowing specific labelling, selection and fate-mapping of cells. YFP+ neurospheres were transplanted to C57BL6/J (6–8-week-old) colonic tissue and maintained in organotypic culture for up to 21 days. We analysed and quantified donor cell integration within recipient tissues at 7, 14 and 21 days, along with assessing the structural and molecular consequences of ENSC integration. We found that organotypically cultured tissues were well preserved up to 21-days in ex vivo culture, which allowed for assessment of donor cell integration after transplantation. Donor ENSC-derived cells integrated across the colonic wall in a dynamic fashion, across a three-week period. Following transplantation, donor cells displayed two integrative patterns; longitudinal migration and medial invasion which allowed donor cells to populate colonic tissue. Moreover, significant remodelling of the intestinal ECM and musculature occurred upon transplantation, to facilitate donor cell integration within endogenous enteric ganglia. These results provide critical evidence on the timescale and mechanisms, which regulate donor ENSC integration, within recipient gut tissue, which are important considerations in the future clinical translation of stem cell therapies for enteric disease.

scholarly journals Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation

Reproduction ◽  
2007 ◽  
Vol 133 (1) ◽  
pp. 219-230 ◽  
Author(s):  
Feikun Yang ◽  
Ru Hao ◽  
Barbara Kessler ◽  
Gottfried Brem ◽  
Eckhard Wolf ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Epigenetic Mark ◽  
Cell Type ◽  
Developmental Potential ◽  
Acetylation Status ◽  
Donor Cells ◽  
Donor Cell Type

The epigenetic status of a donor nucleus has an important effect on the developmental potential of embryos produced by somatic cell nuclear transfer (SCNT). In this study, we transferred cultured rabbit cumulus cells (RCC) and fetal fibroblasts (RFF) from genetically marked rabbits (Alicia/Basilea) into metaphase II oocytes and analyzed the levels of histone H3-lysine 9-lysine 14 acetylation (acH3K9/14) in donor cells and cloned embryos. We also assessed the correlation between the histone acetylation status of donor cells and cloned embryos and their developmental potential. To test whether alteration of the histone acetylation status affects development of cloned embryos, we treated donor cells with sodium butyrate (NaBu), a histone deacetylase inhibitor. Further, we tried to improve cloning efficiency by chimeric complementation of cloned embryos with blastomeres fromin vivofertilized or parthenogenetic embryos. The levels of acH3K9/14 were higher in RCCs than in RFFs (P<0.05). Although the type of donor cells did not affect development to blastocyst, after transfer into recipients, RCC cloned embryos induced a higher initial pregnancy rate as compared to RFF cloned embryos (40 vs 20%). However, almost all pregnancies with either type of cloned embryos were lost by the middle of gestation and only one fully developed, live RCC-derived rabbit was obtained. Treatment of RFFs with NaBu significantly increased the level of acH3K9/14 and the proportion of nuclear transfer embryos developing to blastocyst (49 vs 33% with non-treated RFF,P<0.05). The distribution of acH3K9/14 in either group of cloned embryos did not resemble that inin vivofertilized embryos suggesting that reprogramming of this epigenetic mark is aberrant in cloned rabbit embryos and cannot be corrected by treatment of donor cells with NaBu. Aggregation of embryos cloned from NaBu-treated RFFs with blastomeres fromin vivoderived embryos improved development to blastocyst, but no cloned offspring were obtained. Two live cloned rabbits were produced from this donor cell type only after aggregation of cloned embryos with a parthenogenetic blastomere. Our study demonstrates that the levels of histone acetylation in donor cells and cloned embryos correlate with their developmental potential and may be a useful epigenetic mark to predict efficiency of SCNT in rabbits.

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.

In Vitro Fertilization and the Embryonic Revolution

2009 ◽  
pp. 609-622
Author(s):  
D. Gareth Jones
Keyword(s):  
Stem Cells ◽  
In Vitro Fertilization ◽  
Human Embryo ◽  
Human Life ◽  
Genetic Diagnosis ◽  
Embryonic Stem ◽  
Reproductive Technologies ◽  
Vitro Fertilization ◽  
Human Embryology

The advent of in vitro fertilization (IVF) marked a watershed in the scientific understanding of the human embryo. This, in turn, led to a renaissance of human embryology, accompanied by the ability to manipulate the human embryo in the laboratory. This ability has resulted in yet further developments: refinements of IVF itself, preimplantation genetic diagnosis, the derivation and extraction of embryonic stem cells, and even various forms of cloning. There are immense social and scientific pressures to utilize the artificial reproductive technologies in ways that have little or no connection with overcoming infertility. As the original clinical goals of IVF have undergone transformation ethical concerns have escalated, so much so that they are condemned by some as illustrations of ‘playing God’, while any babies born via some of these procedures are labelled as ‘designer babies’. Both terms reflect the fear and repugnance felt by some at the interference with the earliest stages of human life by the artificial reproductive technologies. It is at these points that bioethical analyses have an important contribution to make.

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