55 LIVE PLASMA MEMBRANE ANALYSIS OF EARLY APOPTOTIC CELL DEATH IN PORCINE ADULT DERMAL FIBROBLASTS PRIOR TO SOMATIC CELL CLONING

2008 ◽  
Vol 20 (1) ◽  
pp. 108
Author(s):  
M. Skrzyszowska ◽  
M. Samiec
Keyword(s):  
Plasma Membrane ◽  
Somatic Cell ◽  
Dermal Fibroblast ◽  
Chemical Activation ◽  
Annexin V ◽  
Fibroblast Cells ◽  
Apoptotic Cells ◽  
Cell Cloning ◽  
Donor Cells

The aim of our study was to determine the in vitro developmental capability of porcine nuclear-transferred (NT) embryos reconstructed with adult dermal fibroblast cells, which had been analyzed for apoptosis by live plasma membrane fluorescent labelling. Frozen/thawed fibroblasts, which had been in vitro cultured to confluency, were used for analysis. To detect the early apoptotic changes in the plasma membrane involving the externalization of phosphatidylserine molecules and the subsequent loss of lipid composition asymmetry, the fibroblasts were tagged using a conjugate of annexinV with enhanced green fluorescent protein (eGFP). In the somatic cell cloning procedure, enucleated in vitro-matured oocytes were reconstituted with non-apoptotic dermal fibroblast cell nuclei. Afterwards, NT-derived oocytes were stimulated with a combination of electrical and chemical activation. Simultaneous fusion and electrical activation were induced by application of two successive DC pulses of 1.2 kV cm–1 for 60 �s. A two-step chemical activation procedure was initiated after a 1.5–2 h delay. The cybrids were exposed to 15 µm calcium ionomycin for 5 to 7 min and then incubated in the culture medium supplemented with 10 µg mL–1 cycloheximide for 3 h. Reconstructed embryos were in vitro cultured in NCSU-23 medium for 6–7 days. Fluorescence analysis of the adult dermal fibroblast cells revealed that a relatively high proportion of donor cells exhibited proapoptotic changes in the plasma membrane. The percentage of late apoptotic cells with advanced morphological changes did not exceed 30%. Moreover, an extremely low rate (ranging from 0 to 2%) of early apoptotic cells, with a morphologically normal, i.e., smooth (non-corrugated) and intact (non-blebbing), plasmolemma but which emitted the green eGFP-derived chemiluminescence, was detected. A total of 219 enucleated oocytes were subjected to reconstruction and 185/219 (84.5%) were successfully fused with non-apoptotic nuclear donor cells. Out of 185 cultured NT embryos, 108 (58.4%) cleaved. The frequencies of cloned embryos, that reached the morula and blastocyst stages, were 84/185 (45.4%) and 26/185 (14.0%), respectively. In conclusion, annexin V-eGFP is a sensitive method able to detect the early phases of apoptosis in cultured adult dermal fibroblast cells, because it identified that very small proportion of morphologically normal cells (without shrinkage of the plasmolemma) that also emitted the annexin V-eGFP-derived biochemiluminescence. Nonetheless, the probability of their random erroneous selection for somatic cell cloning appears to be extremely low. It was also found that the preimplantation developmental potential of NT embryos originating from non-apoptotic adult dermal fibroblast cells is relatively high. This work was supported by the Scientific Net of Animal Reproduction Biotechnology.

50 FLOW CYTOMETRY-MEDIATED DETECTION OF LATE-APOPTOTIC HYPODIPLOID CELL FRACTIONS IN LIPOFECTED PORCINE ADULT DERMAL FIBROBLAST CELL LINES SELECTED FOR SOMATIC CELL NUCLEAR TRANSFER

2009 ◽  
Vol 21 (1) ◽  
pp. 125
Author(s):  
M. Samiec ◽  
M. Skrzyszowska ◽  
M. Bochenek ◽  
R. Slomski ◽  
D. Lipinski
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Somatic Cell ◽  
Dermal Fibroblasts ◽  
Fibroblast Cells ◽  
Apoptotic Cells ◽  
Facs Analysis ◽  
Donor Cells ◽  
Cell Fractions

Analysis of nuclear DNA (nDNA) content of in vitro cultured somatic cells undergoing apoptosis became one of the most common methods for single-parameter flow cytometric measurement of this process. Apoptosis assessment is performed by quantification of hypodiploid cells. The cell fractions with hypodiploid (<2C) nDNA molecule number, which involve the so-called sub-G1 peak in DNA histograms are identified as late-apoptotic subpopulations. Advantage of this method is the possibility of simultaneous cell cycle measurement. The present study was conducted to investigate the preimplantation developmental outcome of porcine transgenic NT embryos reconstituted with non-apoptotic gilt ear skin-derived fibroblast cells that had been lipofected with pWAPhGH-GFPBsd gene construct. The nuclear donor cells were derived from such cell line populations whose representative random samples had been analyzed on both cell cycle and apoptosis through the non-vital nDNA fluorescent dyeing and subsequent flow cytometry (FACS). Frozen/thawed fibroblast cells, which had been cultured up to a total confluency after 2–3 passages, were used for the diagnostics. The fixed dermal fibroblasts were exposed to nDNA extraction buffer for 5 min and incubated in DNA staining solution (propidium iodide and RNAse) for 30 min. After fluorescent labeling, the cells were analyzed in the flow cytometer by reading nDNA fluorescence in the red band. Somatic cell cloned embryos, which had been created by simultaneous fusion and electrical activation, followed by delayed chemical activation of reconstructed oocytes, were cultured in NCSU-23/FBS medium for 6 to 7 days up to morula/blastocyst stages (Skrzyszowska et al. 2008 Theriogenology 70, 248–259). The FACS analysis revealed that out of all the fibroblast cells diagnosed, 94.9% were cycling and 5.1% were late-apoptotic. In turn, from among the non-apoptotic cells, an average of 92.7% were at G1/G0 stages of cell cycle, 3.1% were at S stage and 4.2% were at G2/M stages. A total of 294/348 (84.5%) enucleated oocytes were successfully fused with non-apoptotic nuclear donor cells. Out of 294 cultured NT embryos, 199 (67.7%) were cleaved. The rates of cloned embryos that reached the morula and blastocyst stages yielded 165/294 (56.1%) and 57/294 (19.4%), respectively. In conclusion, the FACS analysis for mitotic cycle of 100%-confluent lipofected adult dermal fibroblasts confirmed that the cell cycle synchronization at G1/G0 phases was highly efficient, while the frequency of late-apoptotic cells was low. It was also found that the relatively high percentages of pWAPhGH-GFPBsd transgenic blastocysts developed in vitro from NT embryos reconstructed with fibroblast cells undergoing lipofection. Furthermore, porcine cloned blastocysts exhibited approximately 100% index of reporter eGFP transgene expression, which was visually confirmed by their live-fluorescent evaluation. This work was supported by the Scientific Net of Animal Reproduction Biotechnology.

86 CREATION OF PORCINE TRANSGENIC NUCLEAR-TRANSFERRED EMBRYOS RECONSTRUCTED WITH EGFP-EXPRESSING ADULT DERMAL FIBROBLAST CELLS ANALYZED ON APOPTOSIS

2007 ◽  
Vol 19 (1) ◽  
pp. 160 ◽  
Author(s):  
M. Skrzyszowska ◽  
M. Samiec ◽  
Z. Smorag ◽  
D. Lipinski ◽  
R. Slomski
Keyword(s):  
Fluorescent Protein ◽  
Dermal Fibroblast ◽  
Annexin V ◽  
Donor Cell ◽  
Fibroblast Cells ◽  
Egfp Gene ◽  
Morphological Criteria ◽  
Donor Cells ◽  
Fluorescent Tagging

The purpose of our study was to determine the in vitro developmental competences of porcine nuclear transfer (NT) embryos reconstructed with pWAPhGH-GFPBsd transgene-nucleofected gilt ear skin-descended fibroblast cells, which had been diagnosed on apoptosis through the live-plasma membrane fluorescent tagging. Frozen–thawed fibroblast cells, which had been in vitro-cultured up to a total confluency after 2–8 passages, were used for analysis. To detect the early apoptotic changes in the fibroblast cells, single nuclear donor cell suspension was labeled with the conjugate of Annexin V and eGFP protein. The source of recipient cells were in vitro-matured oocytes. Maternal chromosomes were eliminated by a chemically assisted microsurgical technique. Fibroblast cell–ooplast couplets were simultaneously fused and activated. Reconstructed embryos were cultured in NCSU-23/BSA/FBS medium for 6–7 days. The rates of cleavage and development to morula/blastocyst stages were examined on Days 2 and 6/7, respectively. After fluorescent analysis of adult dermal fibroblast cells, it was shown that a relatively high proportion (ranging from 20 to 30%) of donor cells exhibited ultrastructural late-apoptotic or necrotic changes. In contrast, from among the morphologically normal cells, an extremely low rate (ranging from 0 to 2%) of the cells emitted the Annexin V-eGFP-derived green fluorescence, but the other ones did not emit this biochemiluminescence. This suggests that the former subpopulation of the cells was early-apoptotic, and the latter was non-apoptotic. A total of 158 enucleated oocytes were successfully fused with non-apoptotic transgenic nuclear donor cells and intended to be in vitro-cultured. Out of 158 reconstructed oocytes, 106 (67.1%) NT embryos were cleaved. The frequencies of cloned embryos that reached the morula and blastocyst stages were 48/158 (30.4%) and 21/158 (13.3%), respectively. In conclusion, the nucleofection efficiency of in vitro-cultured porcine dermal fibroblast cells as estimated by nuclear donor live-fluorescent evaluation based on the expression index of the eGFP reporter transgene was nearly 100%. Moreover, our results demonstrate that the morphological criteria commonly used for cell viability classification are a sufficient selection factor for qualitative evaluation of nuclear donor cells to somatic cell cloning. It was also found that porcine nuclear-transferred morulae and blastocysts exhibited an approximately 100% index of xenogeneic eGFP gene transcriptional activity, which revealed the live diagnostics of emission intensity for green fluorescent protein-derived biochemiluminescence. This research was supported by the State Committee for Scientific Research as a Solicited Project number PBZ-MIN-005/P04/2002/6 from year 2003 to year 2006.

51 DEVELOPMENT OF EQUINE CLONED EMBRYOS DERIVED FROM IN VITRO-MATURED OOCYTES RECEIVING ADULT DERMAL FIBROBLAST CELL NUCLEI

2009 ◽  
Vol 21 (1) ◽  
pp. 125
Author(s):  
M. Skrzyszowska ◽  
M. Samiec ◽  
W. Mlodawska ◽  
J. Kochan ◽  
A. Okolski ◽  
...  
Keyword(s):  
Dermal Fibroblast ◽  
Fibroblast Cell ◽  
Vero Cells ◽  
Contact Inhibition ◽  
Fibroblast Cells ◽  
Cell Nuclei ◽  
Metaphase Chromosomes ◽  
Condensed Chromosome ◽  
Donor Cells

The purpose of our study was to determine the in vitro developmental competences of equine NT embryos reconstructed with adult dermal fibroblast cells. Frozen/thawed fibroblast cells, whose mitotic cycle had been synchronized at G1/G0 stages through a contact inhibition of their migration and proliferative activity under total confluency, were used as a source of nuclear donor cells in the somatic cell cloning procedure. In vitro-matured oocytes were used as recipient cells for fibroblast cell nuclei. The compact cumulus–oocyte complexes (cpCOCs) were collected from abattoir-derived mare ovaries and selected for in vitro maturation. The cpCOCs were cultured in TC-199 medium supplemented with 5 mU mL–1 follicle-stimulating hormone (FSH), 10% fetal bovine serum (FBS) and 75 μg mL–1 kanamycin monosulfate (kanamycin A) for 30 h at 38.2°C in a 100% water-saturated atmosphere of 5% CO2 and 95% air. Cumulus-denuded in vitro-matured oocytes were incubated in the maturation medium supplemented with 0.4 μg mL–1 demecolcine for 40 min. The treated oocytes were subsequently transferred into TC-199 medium containing 4 mg mL–1 BSA-V and 5 μg mL–1 cytochalasin B. Metaphase chromosomes, which had been allocated into the chemically-induced protrusion of the plasma membrane, were removed microsurgically. The chemically-assisted enucleation was accomplished by gently aspirating the ooplasmic cone, which contained the condensed chromosome mass, with the aid of a beveled micropipette. The single nuclear donor cells were inserted into perivitelline space of previously enucleated oocytes. Fibroblast cell-ooplast couplets were fused with two consecutive DC pulses of 2.4 kV cm–1 for 30 μs. After a 1.5-h delay, nuclear transfer-derived oocytes were chemically activated by exposure to 5 μm L–1 calcium ionomycin for 5 to 7 min, followed by their incubation in B2 medium with addition of 2 mm L–1 6-dimethylaminopurine (6-DMAP) for 4 h. Reconstructed embryos were in vitro cultured in B2 medium for 2 days. Afterwards, cleaved embryos were co-cultured with Vero cells in B2 medium supplemented with 10% FBS for 5 to 6 days up to morula/blastocyst stages. From among 88 in vitro cultured cpCOCs, 55 (62.5%) acquired meiotic nuclear and cytoplasmic maturity state after reaching the Metaphase II stage. A total of 55 enucleated oocytes underwent reconstruction and 44/55 (80.0%) were successfully fused with nuclear donor cells. Out of 44 cultured NT embryos, 21 (47.7%) were cleaved. The frequencies of cloned embryos that reached the morula and blastocyst stages were 6/44 (13.6%) and 3/44 (6.8%), respectively. In conclusion, the cell nuclei of in vitro cultured adult dermal fibroblast cells, which had undergone the contact inhibition, were able to direct the preimplantation development of equine cloned embryos to morula and blastocyst stages. This work was supported by the Scientific Net of Animal Reproduction Biotechnology.

scholarly journals Creation of cloned pig embryos using contact-inhibited or serum-starved fibroblast cells analysed intravitam for apoptosis occurrence / Uzyskiwanie klonalnych zarodków świni z wykorzystaniem komórek fibroblastycznych poddanych inhibicji kontaktowej lub deprywacji troficznej oraz analizowanych przyżyciowo w kierunku apoptozy

2013 ◽  
Vol 13 (2) ◽  
pp. 275-293 ◽  
Author(s):  
Marcin Samiec ◽  
Maria Skrzyszowska ◽  
Jolanta Opiela
Keyword(s):  
Dermal Fibroblast ◽  
Somatic Cells ◽  
Fibroblast Cell ◽  
Serum Starvation ◽  
Fibroblast Cells ◽  
Blastocyst Formation ◽  
Cell Nuclei ◽  
Donor Cells ◽  
Cloned Pig

Abstract Somatic cell cloning efficiency is determined by many factors. One of the most important factors is the structure-functional quality of nuclear donor cells. Morphologic criteria that have been used to date for qualitative evaluation of somatic cells may be insufficient for practical application in the cloning. Biochemical and biophysical changes that are one of the earliest symptoms in the transduction of apoptotic signal may be not reflected in the morphologic changes of somatic cells. For this reason, adult cutaneous or foetal fibroblast cells that, in our experiments, provided the source of genomic DNA for the cloning procedure had been previously analysed for biochemical and biophysical proapoptotic alterations with the use of live-DNA (YO-PRO-1) and plasma membrane (Annexin V-eGFP) fluorescent markers. In Groups IA and IB, the generation of nucleartransferred (NT) embryos using non-apoptotic/non-necrotic contact-inhibited or serum-starved adult cutaneous fibroblast cells yielded the morula and blastocyst formation rates of 125/231 (54.1%) and 68/231 (29.4%) or 99/237 (41.8%) and 43/237 (18.1%), respectively. In Groups IIA and IIB, the frequencies of embryos reconstituted with non-apoptotic/non-necrotic contact-inhibited or serum-starved foetal fibroblast cell nuclei that reached the morula and blastocyst stages were 171/245 (69.8%) and 97/245 (39.6%) or 132/227 (58.1%) and 63/227 (27.8%), respectively. In conclusion, contact inhibition of migration and proliferative activity among the subpopulations of adult dermal fibroblast cells and foetal fibroblast cells resulted in considerably higher morula and blastocyst formation rates of in vitro cultured cloned pig embryos compared to serum starvation of either type of fibroblast cell line. Moreover, irrespective of the methods applied to artificially synchronize the mitotic cycle of nuclear donor cells at the G0/G1 phases, developmental abilities to reach the morula/blastocyst stages were significantly higher for porcine NT embryos that had been reconstructed with non-apoptotic/non-necrotic foetal fibroblast cells than those for NT embryos that had been reconstructed with non-apoptotic/non-necrotic adult dermal fibroblast cells. To our knowledge, the generation of cloned pig embryos using abattoir-derived oocytes receiving cell nuclei descended from contact-inhibited or serum-deprived somatic cells undergoing comprehensive vital diagnostics for the absence of biochemical and biophysical proapoptotic alterations within their plasmalemmas has not been reported so far.

scholarly journals Assessment of in Vitro Developmental Capacity of Porcine Nuclear-Transferred Embryos Reconstituted with Cumulus Oophorus Cells Undergoing Vital Diagnostics for Apoptosis Detection / Ocena zdolności rozwojowych in vitro klonalnych zarodków świni rekonstytuowanych z jąder komórek wzgórka jajonośnego poddawanych przyżyciowej diagnostyce w kierunku wykrywania apoptozy

2013 ◽  
Vol 13 (3) ◽  
pp. 513-529 ◽  
Author(s):  
Marcin Samiec ◽  
Maria Skrzyszowska
Keyword(s):  
Plasma Membranes ◽  
Dermal Fibroblast ◽  
Annexin V ◽  
Cumulus Cells ◽  
Fibroblast Cells ◽  
Developmental Potential ◽  
Cumulus Oophorus ◽  
Group I ◽  
Cloned Pig

Abstract The objective of the current investigation was to extensively compare the in vitro developmental capabilities between cloned pig embryos reconstructed with the cell nuclei of either cumulus oophorus cells or adult dermal fibroblast cells that were both evaluated as non-apoptotic on the basis of YO-PRO-1- and Annexin V-eGFP-mediated vital analysis for programmed cell suicide. In Group I, the competences of nuclear-transferred (NT) embryos that were derived from non-apoptotic/ non-necrotic (i.e., YO-PRO-1- and Annexin V-eGFP-negative) cumulus cells to complete their development to the morula and blastocyst stages were maintained at the proportions of 155/364 (42.6%) and 54/364 (14.8%), respectively. In Group II, NT embryos that were reconstituted with non-apoptotic and/or non-necrotic adult cutaneous fibroblast cells developed to the morula and blastocyst stages at the rates of 207/358 (57.8%) and 110/358 (30.7%), respectively. Although the in vitro developmental potential of porcine NT embryos derived from non-apoptotic/non-necrotic cumulus cells was significantly lower (P<0.001) than that of NT embryos reconstructed with adult dermal fibroblast cells, the obtained morula/blastocyst formation rates turned out to be considerably higher as compared to the rates reported by other investigators. Altogether, to our knowledge, the comprehensive research aimed at the determination of preimplantation developmental outcomes of cloned pig embryos produced using nuclear donor somatic cells of different provenance (cumulus oophorus cells or adult cutaneous fibroblast cells) that were vitally diagnosed for the lack of proapoptotic transformations in their plasma membranes has not yet been accomplished.

185 NONSURGICAL EMBRYO TRANSFER FOR PRODUCTION OF PIGS BY IN VITRO FERTILIZATION AND SOMATIC CELL NUCLEAR TRANSFER

2010 ◽  
Vol 22 (1) ◽  
pp. 251
Author(s):  
J.-G. Yoo ◽  
M.-R. Park ◽  
H.-N. Kim ◽  
Y.-G. Ko ◽  
J.-Y. Lee ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Embryo Transfer ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Fibroblast Cells ◽  
Vitro Fertilization ◽  
Miniature Pigs ◽  
Donor Cells

Instead of surgical embryo transfer (ET) in the pig, nonsurgical ET is a hopeful method to increase the efficiency of biotechnology applications such as cloning and transgenesis. In this study, we conducted surgical and nonsurgical ET methods after somatic cell nuclear transfer (SCNT) with MHC miniature pig cells to find out the best condition for production of cloned miniature pigs. Ovaries were obtained from prepubertal crossbred gilts at a local slaughterhouse. Oocytes were matured for 40 to 44 h at 38.5°C under 5% CO2 in air. As donor cells, fibroblast cells were cultured from ear skin tissue of 8-month-old MHC inbred miniature pigs. Fibroblast cells were cultured, passaged (3 to 8 passages), and used as donor cells for NT. After the enucleation and injection process, eggs were held in TCM-199. For fusion, 2 DC pulses of 1.2 kV cm-1 were applied for 30 μs. Both IVF and SCNT embryos were cultured in PZM-3 medium. After IVF, 84.9% (411/484) of embryos cleaved and 27.3% (132/484) of embryos reached the blastocyst stage. In the SCNT group, 80.8% (231/286) of eggs fused and 25.9% (60/286) of embryos developed to blastocysts. For surgical ET, approximately 200 SCNT embryos were transferred into oviducts of each synchronized recipient. For nonsurgical ET, embryos were cultured in PZM-3 for 6 days after SCNT and IVF, and then good quality blastocyst stage embryos were selected for ET. The pregnancy status of recipients at Day 30 was determined by ultrasound scanning. Using Day 30 of gestation as an endpoint, the nonsurgical ET method (47.3%, 9/19) had a similar pregnancy rate as the surgical ET method (56.5%, 13/23). Further study is needed to optimize the nonsurgical ET method especially for SCNT eggs. This work received grant support from the Agenda Program (no. 200901FHT010305535), Rural Development Administration, Republic of Korea.

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.

97 CLONED CALF PRODUCTION BY AGGREGATION OF SOMATIC CELL NUCLEAR-TRANSFERRED EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 166
Author(s):  
D. Yamaguchi ◽  
W. Suzuki ◽  
N. Adachi ◽  
S. Akagi ◽  
S. Watanabe ◽  
...  
Keyword(s):  
Somatic Cell ◽  
High Frequency ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Fibroblast Cells ◽  
Cell Cloning ◽  
Cell Stage ◽  
Cell Numbers ◽  
Donor Cells

A low pregnancy rate and high frequency of abortion are observed in bovine somatic cell cloning. It is suggested that one of the factors is the low cell number of nuclear-transferred (NT) embryos at the blastocyst stage compared with that of in vivo-derived embryos. We reported that aggregation of bovine NT embryos can develop to blastocysts with cell numbers that are equivalent to in vivo-derived embryos (Akagi et al. 2005 Reprod. Fertil. Dev. 17, 162 abst). In this study, we examined the in vivo development of aggregates of NT embryos after embryo transfer (ET). Following culture in serum-starved medium for 5 to 7 days, fibroblast cells were used as donor cells for NT. NT was performed as previously described (Akagi et al. 2003 Mol. Reprod. Dev. 66, 264–272). Eight-cell stage embryos on Day 2 or 16- to 32-cell stage embryos on Day 4 were used for embryo aggregation after removal of the zona pellucida. Three NT embryos were placed into the depression in a drop of TCM-199 with 50 µg mL−1 phytohemagglutinin for 20 min, and NT aggregates were then moved into the depression in a drop of IVD-101 (Akagi et al. 2005). On Day 7 after NT, ET was performed nonsurgically. Results are summarized in Table 1. Pregnancy rates obtained with the aggregated NT embryos tended to be high compared with the single NT embryos. All pregnant cows in the single NT embryos and 6 of 8 pregnant cows in the aggregated NT embryos were aborted within 90 days. One cow carrying an aggregate of 3 Day 2 NT embryos went to term, and one carrying an aggregate of 3 Day 4 NT embryos is still pregnant at more than 7 months. These results demonstrate that aggregates of 3 NT embryos can develop to term in cattle. Table 1.In vivo development of aggregated NT embryos

83 IN VITRO DEVELOPMENTAL ABILITIES OF PORCINE CLONED EMBRYOS RECONSTITUTED WITH CELL NUCLEI OF GENETICALLY TRANSFORMED FETAL FIBROBLASTS CYTOMETRICALLY DIAGNOSED ON CELL CYCLE AND APOPTOSIS

2007 ◽  
Vol 19 (1) ◽  
pp. 159
Author(s):  
M. Samiec ◽  
M. Skrzyszowska ◽  
M. Bochenek ◽  
D. Lipinski ◽  
R. Slomski
Keyword(s):  
Cell Cycle ◽  
High Efficiency ◽  
Fibroblast Cells ◽  
Apoptotic Cells ◽  
Facs Analysis ◽  
Developmental Potential ◽  
Fetal Fibroblast ◽  
Donor Cells ◽  
Fetal Fibroblasts

The important factor that determines the development of mammalian cloned embryos is structuro-functional quality of nuclear donor cells. Analysis of nuclear DNA (nDNA) content of somatic cells undergoing apoptosis has become one of the most common methods for single-parameter flow cytometric measurement of this process. Apoptosis assessment is performed by quantification of hypodiploid cells. The aim of our study was to examine the in vitro developmental potential of porcine nuclear transfer (NT) embryos reconstituted with non-apoptotic fetal fibroblast cells expressing the eGFP transgene. The nuclear donor cells were derived from cell line populations whose representative random samples had been analyzed on both cell cycle and apoptosis through non-vital nDNA fluorescent dyeing and flow cytometry (FACS). Frozen-thawed fibroblast cells, which had been cultured up to a total confluency after 2–4 passages, were used for the diagnostics. The cells were fixed in ice-cold 70% ethanol. Then, the fetal fibroblasts were exposed to nDNA extraction buffer for 5 min at room temperature, and incubated in DNA staining solution (propidium iodide and RNAse) for 30 min. After fluorescent labeling, the cells were analyzed in the flow cytometer by reading nDNA fluorescence in the red band. In vitro-matured oocytes were the source of recipient cells. Fibroblast cell–ooplast couplets were simultaneously fused and activated. Reconstructed embryos were cultured in NCSU-23/BSA/FBS medium for 6–7 days. The rates of cleavage and development to morula/blastocyst stages were examined on Days 2 and 6/7, respectively. FACS analysis revealed that, out of all of the diagnosed fetal fibroblast cells, 54.7% were cycling, and up to 45.3% were late-apoptotic. In turn, from among the normal (i.e. non-apoptotic) cells, 82.2% were at G0/G1 stages of cell cycle, 17.0% at the S stage, and 0.8% at G2/M stages. A total of 150 enucleated oocytes were successfully fused with non-apoptotic transgenic nuclear donor cells. Out of 150 cultured NT embryos, 123 (82.0%) were cleaved. The frequencies of cloned embryos that reached the morula and blastocyst stages yielded 53/150 (35.3%) and 37/150 (24.7%), respectively. In conclusion, the FACS analysis for mitotic cycle of 100%-confluent transgenic fetal fibroblasts confirmed the high efficiency of the cell cycle synchronization at G0/G1 phases. However, a contact inhibition method induced the high frequency of late-apoptotic cells. Moreover, the relatively high percentage of NT blastocysts was developed from oocytes reconstructed with eGFP transgenic fetal fibroblast cells. This research was supported by the State Committee for Scientific Research as a Solicited Project number PBZ-MIN-005/P04/2002/6 from year 2003 to year 2006.

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.

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