Clonal analysis of the developmental potential of 6th and 7th day visceral endoderm cells in the mouse

Development ◽  
1987 ◽  
Vol 101 (1) ◽  
pp. 143-155 ◽  
Author(s):  
D.L. Cockroft ◽  
R.L. Gardner
Keyword(s):  
Donor Cell ◽  
Host Cells ◽  
Phenotypic Change ◽  
Visceral Endoderm ◽  
Developmental Potential ◽  
Cell Clones ◽  
Donor Cells ◽  
Parietal Endoderm ◽  
Extraembryonic Membranes ◽  
Genetically Determined

Single visceral endoderm cells from the embryonic regions of 6th and 7th day conceptuses were cloned by blastocyst injection using the genetically determined presence or absence of cytoplasmic malic enzyme activity as an in situ cell marker. In the 6th day cell injection experiments wild-type donor cell clones were readily discernible in the extraembryonic membranes of more than half the midgestation null host conceptuses; a much lower cloning efficiency was encountered with 7th day cells. With one exception, the clones appeared to be confined to the extraembryonic endoderm, most occurring in the parietal endoderm only. By means of the situ marker, the morphology and arrangement of donor cells in the parietal endoderm could be compared with that of host cells, thereby demonstrating that they had undergone an appropriate phenotypic change after colonizing this tissue. Control experiments indicated that the procedures used to dissociate and select donor cells for injection were likely to have ensured that a representative sample of visceral endoderm cells had been transplanted. Hence on the 6th day of development, a high proportion of cells in the visceral endoderm seem to retain primitive endodermal characteristics, but these appear to be lost by the 7th day, when markers of visceral endoderm differentiation have first been demonstrated.

scholarly journals The extent of clonal structure in different lymphoid organs.

1992 ◽  
Vol 175 (5) ◽  
pp. 1255-1269 ◽  
Author(s):  
M H Hermans ◽  
A Wubbena ◽  
F G Kroese ◽  
S V Hunt ◽  
R Cowan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Fetal Liver ◽  
High Mobility ◽  
Donor Cell ◽  
Germinal Centers ◽  
Lymphoid Organs ◽  
Lymphoid Cells ◽  
Host Cells ◽  
Cell Clones ◽  
Donor Cells

To gain insight into the clonal organization of lymphoid organs, we studied the distribution in situ of donor-derived cells in near-physiological chimeras. We introduced RT7b fetal liver cells into nonirradiated congenic RT7a neonatal rats. The chimerism 6-20 wk after injection ranged from 0.3 to 20%. The numbers of cell clones simultaneously contributing to cell generation in a particular histological feature were deduced from the variance in donor cell distribution. In bone marrow and thymus, donor-derived lymphoid cells were found scattered among host cells, indicating a high mobility of cells. In bone marrow, donor cells were evenly distributed over the entire marrow, even at low chimerism. This indicates that leukopoiesis is maintained by the proliferation of many clones. In the thymus, the various lobules showed different quantities of donor-derived lymphoid cells. Mathematical analysis of these differences indicated that 17-18 cell division cycles occur in the cortex. In spleen, the distribution of donor-derived cells over the germinal centers indicated that 5 d after antigenic stimulation, germinal centers develop oligoclonally. The main conclusions of this work are that (a) bone marrow and thymus are highly polyclonal; (b) 17-18 divisions occur between prothymocyte and mature T cell; and (c) lymphoid cells disperse rapidly while proliferating and differentiating.

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.

Investigation of cell lineage and differentiation in the extraembryonic endoderm of the mouse embryo

Development ◽  
1982 ◽  
Vol 68 (1) ◽  
pp. 175-198
Author(s):  
R. L. Gardner
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Lineage ◽  
Early Embryo ◽  
Visceral Endoderm ◽  
Primitive Endoderm ◽  
Developmental Potential ◽  
Extraembryonic Endoderm ◽  
Parietal Endoderm ◽  
Endodermal Cells

The technique of injecting genetically labelled cells into blastocysts was used in an attempt to determine whether the parietal and visceral endoderm originate from the same or different cell populations in the early embryo. When the developmental potential of 5th day primitive ectoderm and primitive endoderm cells was compared thus, only the latter were found to colonize the extraembryonic endoderm. Furthermore, single primitive endoderm cells yielded unequivocal colonization of both the parietal and the visceral endoderm in a proportion of chimaeras. However, in the majority of primitive endodermal chimaeras, donor cells were detected in the parietal endoderm only, cases of exclusively visceral colonization being rare. Visceral endoderm cells from 6th and 7th day post-implantation embryos also exhibited a striking tendency to contribute exclusively to the parietal endoderm following blastocyst injection. The above findings lend no support to a recent proposal that parietal and visceral endoderm are derived from different populations of inner cell mass cells. Rather, they suggest that the two extraembryonic endoderm layers originate from a common pool of primitive endoderm cells whose direction of differentiation depends on their interactions with non-endodermal cells.

scholarly journals Effects of Donor Cell Types on the Development of Bovine Embryos Using Cytoplasm Injection Cloning Technology

2021 ◽  
Vol 22 (11) ◽  
pp. 5841
Author(s):  
Lianguang Xu ◽  
Seok-Hwan Song ◽  
Muhammad Idrees ◽  
Ayman Mesalam ◽  
Myeong-Don Joo ◽  
...  
Keyword(s):  
Dna Methyltransferase ◽  
Cell Mass ◽  
Donor Cell ◽  
Cell Types ◽  
Histone Deacetylation ◽  
Bovine Embryos ◽  
Developmental Potential ◽  
Donor Cells ◽  
Cloning Technology

Cytoplasm injection cloning technology (CICT) is an efficient technique for evaluating the developmental potential of cloned embryos. In this study, we investigated the effects of donor cell type on the developmental potential and quality of cloned bovine embryos. Adult fibroblasts (AFs) and embryonic cells (ECs) were used as donor cells to clone bovine embryos using CICT. We initially used AF cells to develop cloned embryos and then cultured the cloned day-8 blastocysts for 10 days to obtain ECs as donor cells for second embryo cloning. We found that the bovine blastocysts cloned using AF cells had significantly reduced developmental rates, embryo quality, and ratios of inner cell mass (ICM) to the total number of cells compared to those using ECs as donor cells. Furthermore, there were significant differences in the DNA methyltransferase-, histone deacetylation-, apoptosis-, and development-related genes at the blastocyst stage in embryos cloned from AFs compared to those in embryos cloned from ECs. Our results suggest that using ECs as donor cells for nuclear transfer enhances the quantity and quality of cloned embryos. However, further investigation is required in terms of determining pregnancy rates and developing cloned embryos from different donor cell types.

Microchimerism Detection by HLA-Specific Real-Time Polymerase Chain Reaction Analysis in Recipients of Allogeneic Epstein Barr Virus Specific Cytotoxic T Lymphocytes.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2648-2648
Author(s):  
Kenneth G. Lucas ◽  
Qi Sun ◽  
Karen Chorney ◽  
Timothy Erickson ◽  
J. Lee Nelson
Keyword(s):  
Hodgkin's Disease ◽  
Hodgkin’S Disease ◽  
Donor Cell ◽  
Polymerase Chain Reaction Analysis ◽  
Host Cells ◽  
Str Analysis ◽  
Donor Cells ◽  
Post Infusion ◽  
The Subject ◽  
Polymerase Chain

Abstract Adoptive immunotherapy with allogeneic EBV specific cytotoxic T lymphocytes (CTL) has been used as a treatment strategy for EBV induced lymphoproliferative disease in stem cell transplant and organ transplant recipients, and is currently being investigated for patients with EBV positive Hodgkin’s disease. Currently available molecular methods for detecting chimerism, such as single tandem repeat (STR) analysis, have a sensitivity range that does not permit the detection of allogeneic effector cells when doses of 106 to 107 cells/kg are infused. We have demonstrated that the infusion of EBV CTL from allogeneic donors can be associated with clinical responses in patients with therapy refractory Hodgkin’s disease, however donor cells could not be detected by STR analysis. We employed HLA-specific real-time polymerase chain reaction analysis (Q-PCR) to test for donor microchimerism in two groups of subjects with relapsed, therapy refractory, EBV positive Hodgkin’s disease. Non-shared, donor-specific HLA sequences (HLA B, DRB1, DRB3, DRB4, DRB5, DQA1, and DQB1) were targeted to detect the DNA equivalent of 1 donor cell in a background of 500,000 host cells. The first cohort consisted of three subjects, each receiving three allogeneic EBV CTL infusions consisting of 5 x 106 cells/kg/dose, with no chemotherapy prior to the infusions. A second cohort received a single infusion of 1.5 x 107 EBV CTL/kg, which was preceded by fludarabine 30 mg/m2/day for three days (in order to attempt to achieve donor CTL chimerism). Informative HLA-specific Q-PCR assays were available for one subject not receiving chemotherapy and for the three subjects receiving fludarabine prior to a single CTL infusion. Results are expressed as genome equivalent number of donor cells per million host cells (gEq/mil). The subject who did not receive fludarabine had no detectable donor DNA prior to the CTL infusion and had 105 gEq/mil 2 days following the first infusion, 230 gEq/mil one day following the second infusion, and only 12 gEq/mil one day after the third infusion. This data suggests that the subject had been sensitized following the first CTL infusion and possibly rejected subsequent infusions. All three subjects who received fludarabine prior to a single EBV CTL infusion had increases in donor DNA post-infusion. One of these subjects had 13,061 gEq/mil one day post-infusion, which decreased to slowly to 124 gEq/mil by 21 days post-infusion, and was at 193 gEq at 3.5 months post-infusion. The other two CTL recipients who received immunosuppressive chemotherapy prior to CTL had increases in donor DNA post-infusion but not of the same maximum amplitude (5026 and 740 gEq/mil at one week post-infusion for each of these subjects). Since subsequent CTL infusions were not given to the second cohort, it is unclear whether the decrease in donor cells over time in this group represented cell rejection or the natural kinetics of allogeneic CTL infused in this setting. These studies demonstrate that quantification of donor cell microchimerism is feasible using Q-PCR for mismatched HLA alleles for subjects receiving allogeneic CTL.

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.

46 RECLONING USING TRANSGENIC FETAL FIBROBLASTS AS NUCLEI DONORS INCREASES DEVELOPMENTAL POTENTIAL OF RECONSTRUCTED EMBRYOS IN CATTLE

2010 ◽  
Vol 22 (1) ◽  
pp. 180
Author(s):  
F. F. Bressan ◽  
M. S. Miranda ◽  
F. Perecin ◽  
T. H. C. De Bem ◽  
M. Bajgelman ◽  
...  
Keyword(s):  
Genetically Modified ◽  
Cell Lineage ◽  
Donor Cell ◽  
Pregnancy Rates ◽  
Control Group ◽  
Specific Cell ◽  
Nuclear Reprogramming ◽  
Developmental Potential ◽  
Donor Cells ◽  
Fetal Fibroblasts

Genetically modified animals have numerous applications ranging from basic research to agriculture production. Cloning by nuclear transfer (NT) has made possible the production of transgenic animals using previously genetically modified cell lineages. Gene expression studies and adequate selection of the nuclei donor cell for NT guarantees the presence of the gene construction in the offspring and the absence of deleterious mutations caused by the random insertion of transgenes in functional areas of the genome. Embryonic development after NT requires a change in the transcriptome of the donor cell from a somatic to an embryonic pattern, causing cloning efficiencies to be low because of incomplete or defective nuclear reprogramming. Therefore, the establishment of methodologies able to increase cloning success is highly desirable. The experiment was designed to test if recloning of transgenic fetal fibroblasts increases cloned blastocyst production and the pregnancy rates of transgenic cloned embryos produced by NT. This study compared the developmental potential of cloned embryos reconstructed with fetal fibroblasts genetically modified by lentivirus random integration (control group) expressing the green fluorescent protein gene (eGFP), with a transgenic fetal fibroblast cell line established from a 30-day transgenic pregnancy (recloning group). Fusion, cleavage (72 h post-activation, hpa), blastocyst production (168 hpa), and 30-day pregnancy rates were analyzed. A total of 1213 embryos were reconstructed; 884 (10 replicates) with random transgene insertion fibroblasts and 329 (4 replicates) with cells derived from the cloned fetus. Results were analyzed by chi-square test at 5% significance. No difference was observed (P > 0.05) between control and recloned groups regarding fusion rate (n = 550, 62.22% and n = 189, 57.25%; respectively) or cleavage rate (n = 383, 69.45% and n = 132, 69.84%, respectively). The recloned group, however, showed a higher blastocyst development rate (P < 0.01) compared with the control group (n = 51, 26.98%, and n = 79, 14.36%, respectively) and a higher 30-day pregnancy rate (n = 6, 15.38% and n = 3, 5.56%, respectively). In conclusion, recloning of transgenic fibroblasts from a successfully established pregnancy augments the efficiency in the production of embryos and pregnancy establishment compared with the control group. We speculate that a second round of NT enhances the nuclear reprogramming of donor cells, and moreover, the use of a transgenic cell lineage already proven to be successfully reprogrammed may indicate that transgene integration is not deleterious in that specific cell lineage, resulting in a good source of donor cells to be used in order to produce a homogeneous bioreactor herd. Financial support: FAPESP, Brazil.

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.

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.

Mannose-binding lectin genetics: from A to Z

2008 ◽  
Vol 36 (6) ◽  
pp. 1461-1466 ◽  
Author(s):  
Peter Garred
Keyword(s):  
Epidemiological Studies ◽  
Mannose Binding Lectin ◽  
Host Cells ◽  
Lectin Pathway ◽  
Mannose Binding ◽  
The Stability ◽  
Genetically Determined ◽  
Micro Organisms ◽  
Binding Lectin

MBL (mannose-binding lectin) is primarily a liver-derived collagen-like serum protein. It binds sugar structures on micro-organisms and on dying host cells and is one of the four known mediators that initiate activation of the complement system via the lectin pathway. Common variant alleles situated both in promoter and structural regions of the human MBL gene (MBL2) influence the stability and the serum concentration of the protein. Epidemiological studies have suggested that genetically determined variations in MBL serum concentrations influence the susceptibility to and the course of different types of infectious, autoimmune, neoplastic, metabolic and cardiovascular diseases, but this is still a subject under discussion. The fact that these genetic variations are very frequent, indicates a dual role of MBL. This overview summarizes the current molecular understanding of human MBL2 genetics.

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