55 INFLUENCE OF DONOR CELL TYPE ON THE DEVELOPMENT OF PORCINE NUCLEAR TRANSFER EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 136
Author(s):  
K. Lee ◽  
W. L. Fodor ◽  
Z. Machaty
Keyword(s):  
Stem Cells ◽  
Olfactory Bulb ◽  
Progenitor Cells ◽  
Nuclear Transfer ◽  
Cell Types ◽  
Blastocyst Stage ◽  
Blastocyst Formation ◽  
Developmental Potential ◽  
Differentiated Cells ◽  
Fetal Fibroblasts

Embryonic development after nuclear transfer is very low; the majority of cloned embryos do not survive the pre-implantation stage. Recent reports indicate that the characteristics of nuclear transfer embryos depend on the type of nuclear donor cells. It has been suggested that development after nuclear transfer improves if less differentiated cells are used as nuclear donors. The aim of the present study was to investigate the developmental potential of nuclear transfer embryos reconstructed using differentiated and non-differentiated cells. Two types of non-differentiated cells, skin stem cells and olfactory bulb progenitor cells, were used; fetal fibroblasts were used as differentiated control. Prior to nuclear transfer, the differentiated state of the cells was characterized by Oct-4 immunocytochemistry (Chemicon International, Inc., Temecula, CA, USA); Oct-4 is known to be expressed by pluripotent cells only. During nuclear transfer, the cells were transferred into the perivitelline space of in vitro-matured enucleated oocytes. After fusion, reconstructed oocytes were activated by an electrical pulse followed by incubation in 10 �g/mL cycloheximide and 5 �g/mL cytochalasin B for 5 h. The embryos were subsequently cultured in NCSU-23 medium for 6 days; their developmental data were recorded and compared by ANOVA. Non-differentiated cell types showed strong Oct-4 expression, whereas the marker protein was completely absent in fetal fibroblast cells. A total of 161 embryos were reconstructed using skin stem cells, 171 embryos from olfactory bulb progenitor cells, and 189 embryos from fibroblasts. Of the skin stem cell-derived embryos, 32.9% cleaved, and during subsequent culture, 5.6% developed to the morula/blastocyst stage. In the olfactory bulb progenitor cell group, 19.8% cleaved, and the percentage of embryos that developed to the morula/blastocyst stage was 4.7%. In the control group, 22.7% cleaved; the morula/blastocyst formation was 2.6%. Embryos reconstructed from skin stem cells showed superior cleavage rate compared to embryos from the other cell types (P < 0.05). Also, morula/blastocyst formation from skin stem cells was significantly higher than that from fetal fibroblasts (P < 0.05), and morula/blastocyst formation from olfactory bulb progenitor cell-derived embryos also tended to be higher compared to control embryos (P = 0.08). Furthermore, the formation of morulae/blastocysts per cleaved embryos was the highest in embryos reconstructed with olfactory bulb progenitor cells (23.5% vs. 17.0% using skin stem cells and 11.6% using fibroblasts) implying that embryos from olfactory bulb progenitor cells may have higher developmental potential in later stages of development. The results demonstrate that nuclei of different donor cells support development to various degrees and confirm previous reports that using non-differentiated cells as nuclear donors increases the efficiency of nuclear transfer in the pig.

52 EFFECT OF DONOR CELLS WITH VARIOUS DIFFERENTIATED STATUS ON THE DEVELOPMENTAL COMPETENCE OF PORCINE NUCLEAR TRANSFER EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 144
Author(s):  
J. G. Kim ◽  
E. J. Kang ◽  
M. K. Kim ◽  
S. Y. Choe ◽  
G. J. Rho
Keyword(s):  
Stem Cells ◽  
Nuclear Transfer ◽  
Statistical Significance ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Differentiation Potential ◽  
Developmental Potential ◽  
Differentiated Cells ◽  
Donor Cells ◽  
Fetal Fibroblasts

Adult stem cells are more desirable than somatic cells for nuclear transfer (NT) because of their easy reprogrammability to resemble the genome of the zygote (Zhu et al. 2004 Biol. Reprod. 70, 1088–1095). Mesenchymal stem cells (MSCs) are a heterogeneous population of uncommitted and lineage-committed cells and have a more flexible potential as donor cells for NT. The aim of this study was to compare the developmental potential of NT embryos using undifferentiated (MSCs) and differentiated cells in the same lineage (osteocyte, adipocyte, and chondrocyte) by assessing the cleavage and blastocyst rates. Fetal fibroblasts were used as NT control. MSCs obtained from the aspirated bone marrow of a neonatal pig were cultured in advanced-DMEM (ADMEM) supplemented with 5% FCS. The differentiation potential was demonstrated by culture of MSCs at passage 3 under the conditions that were favorable for adipogenic, osteogenic, and chondrogenic development (Pittenger et al. 1999 Science 284, 143–147). For NT, cells from passages 3–5 were transferred into the perivitelline space of enucleated MII oocytes that had been in vitro-matured after collection from slaughterhouse-derived ovaries. After fusion with a needle-type electrode, eggs were cultured in 7.5 µg mL−1 cytochalasin B for 3 h, and subsequently cultured in PZM-3 medium for 6 days. Statistical significance was tested using ANOVA with Bonferroni and Duncan tests. The results are presented in Table 1. The rates of cleavage and development to blastocyst stage of NT embryos varied among donor cell sources. Most eggs (92.2 ± 2.7%) cloned with MSCs cleaved, and 47.8% of eggs developed to the blastocyst stage. In contrast, NT eggs using differentiated MSCs—osteocytes, adipocytes, chondrocytes, and controls (fetal fibroblasts)—revealed significantly (P &lt; 0.05) lower cleavage (74.5, 63.4, 74.3, and 66.4%, respectively) and blastocyst development (33.7, 30.1, 36.5, and 25.5%, respectively) rates than those using undifferentiated MSCs. The results demonstrate that the genome of donor cells with different differentiated status supports embryonic development to various degrees, and multipotent MSCs might have a greater potential in producing viable cloned porcine embryos. Table 1.Development of NT embryos with undifferentiated and differentiated cells This work was supported by Grant No. R05-2004-000-10702-0 from KOSEF, Republic of Korea.

scholarly journals 112 THE PRESENCE OF LAMIN A/C ANTIGENS IN PORCINE EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 206
Author(s):  
K. Lee ◽  
W.L. Fodor ◽  
Z. Machaty
Keyword(s):  
Olfactory Bulb ◽  
Nuclear Envelope ◽  
Progenitor Cells ◽  
Nuclear Transfer ◽  
Type A ◽  
Lamin A ◽  
Primary Role ◽  
Nuclear Lamins ◽  
Differentiated Cells

Nuclear lamins are components of the nuclear lamina, and their primary role is to support the nuclear envelope and provide anchorage sites for the chromatin. While type B lamins are expressed in all cells, type A lamins (including lamins A and C) are developmentally regulated and expressed in differentiated cells only. There are conflicting results about the presence of lamin A/C in early mammalian embryos. Lamin A/C was found to localize in the nuclear envelope of bovine, pig, and mouse embryos, while recently it has been reported that early mouse and bovine embryos lacked lamin A/C antigens. It has also been suggested that the existence of lamin A/C in the pronuclei of mouse and bovine nuclear transfer embryos indicated faulty reprogramming. The aim of this study was to investigate the presence of lamin A/C in porcine embryos of different origins (in vivo, parthenogenetic, and nuclear transfer). Embryos of various developmental stages were collected from inseminated gilts. For the production of parthenogenetic embryos, mature oocytes were electroporated and cultured for up to seven days. Fibroblast cells served as differentiated controls; progenitor cells from the olfactory bulb of a porcine fetus were used as undifferentiated controls. Lamin A/C was visualized by immunocytochemistry. Olfactory bulb progenitor cells lacked lamin A/C (0 out of 50 cells showed staining) while all fibroblast nuclei (n = 50) reacted positively with the antibody. GV-stage oocytes, being terminally differentiated cells, also possessed lamin A/C antigens (30/30). Lamin A/C was not detectable in any of the mature oocytes examined (n = 30), but it was found in early cleavage-stage embryos [both in vivo (20/20) and parthenogenetic (30/30)] with the signal becoming weaker in blastocysts (15/15). After nuclear transfer, the lamin A/C signal from fibroblast nuclei disappeared (20/20), consistent with nuclear envelope breakdown. Later it became detectable again; all nuclear transfer embryos reconstructed with either fibroblast or progenitor cells displayed lamin A/C staining in their pronuclei and at all stages examined (n = 65). This suggests that recipient oocytes remodel the donor nuclei and reassemble the nuclear envelopes of both differentiated and undifferentiated cells with type A lamins. Activated oocytes (n = 30) and early embryos (n = 40) were then incubated in the presence of actinomycin D (an inhibitor of RNA polymerase II) or cycloheximide (a protein synthesis inhibitor) for 14 h. Lamin A/C assembly was not perturbed by either treatment, indicating that the assembly did not result from de novo gene transcription but rather from solubilized lamins already in the cytoplasm. The results imply that lamin A/C is present in early pig embryos and that its presence after nuclear transfer is not an indicator of erroneous reprogramming, unlike that reported in cattle and mouse.

43 INFLUENCE OF DONOR CELL DIFFERENTIATION ON CLONING EFFICIENCY IN PIGS

2007 ◽  
Vol 19 (1) ◽  
pp. 140
Author(s):  
N. Hornen ◽  
W. A. Kues ◽  
A. Lucas-Hahn ◽  
B. Petersen ◽  
P. Hassel ◽  
...  
Keyword(s):  
Stem Cells ◽  
Nuclear Transfer ◽  
Primary Cultures ◽  
Donor Cell ◽  
Stem Cell Population ◽  
High Density Culture ◽  
Developmental Potential ◽  
Fetal Fibroblasts ◽  
Normal Offspring

We recently reported the discovery of a novel type of stem cells which could be derived from primary cultures of fibroblasts by high density culture (Kues et al. 2005 Biol. Reprod. 72, 1020–1028). The goal of the present study was to analyze the suitability of this specific stem cell population (fetal somatic stem cells, FSSCs) in NT and to test their ability to produce normal offspring upon transfer of cloned embryos. In the first of 4 experiments, FSSCs from isolated attached colonies were compared with fetal fibroblasts in their ability to form blastocysts upon use in NT. Fusion and cleavage rates were similar between the two groups [FSSCs: 75.3 � 10.5% (mean � SD) vs. 83.7 � 9.2%; fetal fibroblasts: 64.8 � 17.3% vs. 82.5 � 5.6%, respectively]. Blastocyst rate differed significantly between the two groups (6.4 � 3.5% vs. 24.9 � 8.6%). In the second experiment, FSSCs of 3 different sizes (&lt;14 �m, 15–20 �m, &gt;21 �m), obtained from dissociation of spheroids, were compared in their ability to form blastocysts upon use in NT. No differences were found among the 3 groups (fusion rates: 93.0 � 3.1 vs. 91.3 � 10.1 vs. 92.3 � 5.1; cleavage rates: 83.5 � 7.9 vs. 83.1 � 1.6 vs. 83.2 � 5.8; blastocyst rates: 15.3 � 7.9 vs. 17.6 � 6.8 vs. 10.4 � 2.7, respectively). In the third experiment, FSSCs 15–20 �m in size, derived from spheroids, were compared with fetal fibroblasts. No differences were detected between groups (fusion rates: 83.3� 7.3% vs. 86.8 � 5.3%; cleavage rates: 86.1 � 6.7% vs. 80.7 � 5.9%; blastocyst rates: 21.4 � 5.6% vs. 18.4 � 5.6%, respectively). In the final experiment, 70–100 nuclear transfer complexes cloned from FSSCs were transferred immediately after activation to prepubertal gilts to evaluate their in vivo developmental potential. Pregnancies were established in 3 of 7 recipients, which delivered 7 piglets, of which 3 piglets were vital and showed normal development. Four piglets were lost due to dystocia. These results show that FSSCs are able to generate cloned embryos, and pregnancies can be established and vital piglets can be produced.

scholarly journals 55IN VITRO DEVELOPMENT OF ENUCLEATED DOMESTIC CAT OOCYTES RECONSTRUCTED WITH SKIN FIBROBLASTS OF DOMESTIC AND LEOPARD CATS

2004 ◽  
Vol 16 (2) ◽  
pp. 149 ◽  
Author(s):  
C. Lorthongpanich ◽  
C. Laowtammathron ◽  
S. Muenthaisong ◽  
T. Vetchayan ◽  
M. Ketudat-Cairns ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Donor Cell ◽  
Cell Types ◽  
Blastocyst Stage ◽  
Skin Fibroblasts ◽  
Domestic Cat ◽  
Developmental Potential ◽  
A Cell ◽  
Vitro Development

The domestic cat is a valuable model for studies in assisted reproductive technology in felid species. Therefore, in this experiment we evaluated the in vitro developmental potential of enucleated domestic cat oocytes reconstructed with somatic cells from domestic and leopard cats. Skin fibroblasts were isolated from female domestic and leopard cats. The oocytes were collected by aspiration of follicles from ovaries that were superovulated with 200IU PMSG. In vitro-matured oocytes were enucleated and individual donor cells (diameter 14–16μm) were inserted into the perivitelline space of the enucleated oocyte. Fusion was performed at 26–27h post-maturation by placing a cell-oocyte couplet between both tips of the needle electrode and electrostimulating with a 2-DC pulse (30V, 30μs) in fusion medium containing 0.3M Mannitol+0.1mM MgCl2. Activation was performed 1 to 2h post-fusion by incubation in 7% ethanol at room temperature for 5min followed by cultured in 10μgmL−1 cycloheximide and 1.25μgmL−1 cytochalasin D at 38°C in 5% O2, 5% CO2, 90% N2 conditions. After activation, the reconstructed embryos were cultured in 100-μL droplets of Tyrode’s medium (Gomez et al., 2003 Theriogenology 60, 239–251.) supplemented with 0.3% BSA at 38°C in a 5% O2, 5% CO2, 90% N2 environment for 2d. Then, 8-cell embryos were cultured in 100-μL droplets of Tyrode’s medium supplemented with 10% FCS at 38°C in a 5% O2, 5% CO2, 90% N2environment for 5d. The cleavage rates of oocytes reconstructed with either donor cell types were not different. The percentages of blastocyst formation from parthenogenotes and nuclear transfer embryos derived from domestic cat fibroblasts (8/56, 14.3% and 7/51, 13.7%, respectively) were significantly higher than that for nuclear transfer embryos constructed with leopard cat fibroblasts (3/45, 6.7%). These results indicate that enucleated domestic cat oocytes reconstructed with skin fibroblasts of leopard cats can develop to the blastocyst stage. This experiment was supported by Suranaree University of Technology. Table 1 In vitro development of domestic cat oocytes reconstructed with domestic and leopard skin fibroblasts and parthenogenetic activation

Critical role of p63 in the development of a normal esophageal and tracheobronchial epithelium

2004 ◽  
Vol 287 (1) ◽  
pp. C171-C181 ◽  
Author(s):  
Yaron Daniely ◽  
Grace Liao ◽  
Darlene Dixon ◽  
R. Ilona Linnoila ◽  
Adriana Lori ◽  
...  
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Squamous Metaplasia ◽  
Critical Role ◽  
Basal Layer ◽  
Cell Types ◽  
Basal Cells ◽  
Ciliated Cells ◽  
Differentiated Cells ◽  
Unique Cell

The trachea and esophagus originate from the foregut endoderm during early embryonic development. Their epithelia undergo a series of changes involving the differentiation of stem cells into unique cell types and ultimately forming the mature epithelia. In this study, we monitored the expression of p63 in the esophagus and the trachea during development and examined in detail morphogenesis in p63−/− mice. At embryonic day 15.5 (E15.5), the esophageal and tracheobronchial epithelia contain two to three layers of cells; however, only the progenitor cells express p63. These progenitor cells differentiate first into ciliated cells (p63−/β-tubulin IV+) and after birth into mature basal cells (p63+/K14+/K5+/BS-I-B4+). In the adult pseudostratified, columnar tracheal epithelium, K14+/K5+/BS-I-B4+ basal cells stain most intensely for p63, whereas ciliated and mucosecretory cells are negative. In stratified squamous esophageal epithelium and during squamous metaplasia in the trachea, cells in the basal layer stain strongest for p63, whereas p63 staining declines progressively in transient amplifying and squamous differentiated cells. Generally, p63 expression is restricted to human squamous cell carcinomas, and adenocarcinomas and Barrett's metaplasia do not stain for p63. Examination of morphogenesis in newborn p63−/− mice showed an abnormal persistence of ciliated cells in the esophagus. Significantly, in both tissues, lack of p63 expression results in the development of a highly ordered, columnar ciliated epithelium deficient in basal cells. These observations indicate that p63 plays a critical role in the development of normal esophageal and tracheobronchial epithelia and appears to control the commitment of early stem cells into basal cell progeny and the maintenance of basal cells.

scholarly journals The Urodele Limb Regeneration Blastema: The Cell Potential

2010 ◽  
Vol 10 ◽  
pp. 954-971 ◽  
Author(s):  
Kenyon S. Tweedell
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Cell Activation ◽  
Limb Regeneration ◽  
Cell Types ◽  
Dermal Fibroblasts ◽  
Specific Cell ◽  
Cell Potential ◽  
Developmental Potential ◽  
Regeneration Blastema

The developmental potential of the limb regeneration blastema, a mass of mesenchymal cells of mixed origins, was once considered as being pluripotent, capable of forming all cell types. Now evidence asserts that the blastema is a heterogeneous mixture of progenitor cells derived from tissues of the amputation site, with limited developmental potential, plus various stem cells with multipotent abilities. Many specialized cells, bone, cartilage, muscle, and Schwann cells, at the injury site undergo dedifferentiation to a progenitor state and maintain their cell lineage as they redifferentiate in the regenerate. Muscle satellite reserve stem cells that are active in repair of injured muscle may also dedifferentiate and contribute new muscle cells to the limb blastema. Other cells from the dermis act as multipotent stem cells that replenish dermal fibroblasts and differentiate into cartilage. The blastema primordium is a self-organized, equipotential system, but at the cellular level can compensate for specific cell loss. It is able to induce dedifferentiation of introduced exogenous cells and such cells may be transformed into new cell types. Indigenous cells of the blastema associated with amputated tissues may also transform or possibly transdifferentiate into new cell types. The blastema is a microenvironment that enables dedifferentiation, redifferentiation, transdifferentiation, and stem cell activation, leading to progenitor cells of the limb regenerate.

Advances of Stem Cell Therapy to Treat Heart Failure

Nano LIFE ◽  
2019 ◽  
Vol 09 (03) ◽  
pp. 1941002
Author(s):  
Yanbin Fu ◽  
Zhiying He ◽  
Chao Zhang
Keyword(s):  
Heart Failure ◽  
Stem Cells ◽  
Stem Cell ◽  
Cell Therapy ◽  
Progenitor Cells ◽  
Stem Cell Therapy ◽  
Cell Types ◽  
Differentiated Cells ◽  
Novel Strategy

Stem cell therapy is being developed as a promising novel strategy for the treatment of heart-associated diseases. Several types of cells such as skeletal myoblasts, bone marrow (BM) mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs), adipose stem cells (ADSCs), cardiac progenitor cells (CPCs), induced pluripotent stem cells (iPSCs) have been tested in pre-clinical and clinical cardiac repairing models. Fibroblasts, as terminally differentiated cells, could also be trans-differentiated into cardiomyocytes in vitro. In this review, we will summarize the recent advances of cell types, potential applications and challenges of stem cell therapy in the treatment of heart failure.

Extruded Nucleoli in Poorly Differentiated Dental Pulp Cells of Human Frontal Teeth

2018 ◽  
Author(s):  
Mugurel Constantin Rusu ◽  
Adelina Maria Jianu ◽  
Rodica Lighezan ◽  
Roxana Oancea ◽  
Vasile Sorin Manoiu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Dental Pulp ◽  
Cell Types ◽  
Differentiated Cells ◽  
Nucleolar Proteins ◽  
Human Dental Pulp ◽  
Pulp Cells ◽  
Bona Fide ◽  
Poorly Differentiated

All tissues are known to contain stem cells niches. The dental pulp stem cells (DPSCs) are highly proliferative and can differentiate to various cell types, including endothelial cells. We aimed to evaluate the ultrastructural characteristics of the human dental pulp niche corresponding to the frontal teeth. We found bona fide quiescent DPSCs with high nucleoplasmic ratios, as well as poorly differentiated cells with increased amount of cytoplasm, these later being indicated as dental pulp progenitor cells (DPPCs). Vasculogenic cords were built up by mitotic endothelial progenitor cells with a peculiar trait: giant nucleoli extruded within the cytoplasm. They were either partly embedded within the nuclei, case in which their adnuclear side was coated by marginal heterochromatin and the abnuclear side was coated by a thin rim of ribosomes, or were completely isolated from the nuclei, being covered by ribosomes and surrounded by cytoplasmic fragments of chromatin. To our knowledge such giant extruded nucleoli were not previously reported in any human stem niche or cell type, although similar evidence was gathered in other species as well as in plants. They relate perhaps to intermediate stages of differentiation within the dental niche, thus appearing as transient structures. Their role needs further investigations as it looks that not only nucleolar proteins are exported into the cytoplasm, but the entire nucleolus could be extruded.

scholarly journals 36COMPARISON OF THE DEVELOPMENTAL POTENTIAL OF CAPRINE NUCLEAR TRANSFER EMBRYOS DERIVED FROM IN VITRO AND IN VIVO MATURED OOCYTES

2004 ◽  
Vol 16 (2) ◽  
pp. 140
Author(s):  
Y. Echelard ◽  
E. Memili ◽  
S.L. Ayres ◽  
M. O'Coin ◽  
L.H. Chen ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Reproductive Tract ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Serum Starvation ◽  
Developmental Potential ◽  
Fetal Fibroblasts ◽  
Student’S T

The objective of this study was to compare the development to the blastocyst stage of reconstructed caprine nuclear transfer (NT) embryos derived from two sources of ova. In vivo oocytes were flushed from the oviduct of superovulated donors by exposing the reproductive tract via a small ventral laparotomy. In vitro oocytes were collected from ovaries supplied by an abattoir located in Purdue, IN. Oocytes were aspirated, cultured in maturation medium (M199 +10% goat serum, 3μgmL−1 LH, 3μgmL−1 FSH and 0.22mM sodium pyruvate), and shipped overnight (38°C, air). Donor cell preparation and NT procedures were as previously reported (Behboodi et al., 2001 Theriogenology 55, 254 abst). Donor cells were transfected female fetal fibroblasts that were synchronized by 4 days of serum starvation, followed by a 10-hour exposure to medium containing 10% FCS. Oocytes were enucleated, karyoplast-cytoplast couplets were reconstructed, fused and then activated simultaneously by a single electrical pulse. Couplets containing in vitro oocytes were incubated in the presence of 5μgmL−1 ionomycin after fusion. Fused couplets were co-cultured in TCM199 with 10% FCS and oviductal epithelial cells for 8–10 days (38°C, 5% CO2). Embryos that developed in vitro to the blastocyst stage were surgically transferred to recipients. Pregnancies were confirmed by ultrasonography. One live kid was delivered on Day 150 of gestation via elective C-section. Southern blotting analysis confirmed that it was derived from the transgenic donor cell line. These experiments show that in vivo matured oocytes not only better support caprine NT embryo development to the blastocyst stage, but also can result in live birth (table). Although fusion and cleavage rates were similar in the two groups, development to the blastocyst stage was significantly higher (Student’s t-test) in the group utilizing in vivo-matured oocytes. In conclusion, this is the first live goat produced from goat NT blastocysts developed in vitro. This suggests that in vivo matured oocytes may be superior to oocytes developed in vitro for generating live animals from NT blastocysts. Table 1

43 PRODUCTION OF PORCINE EMBRYOS BY NUCLEAR TRANSFER OF BONE MARROW MESENCHYMAL STEM CELLS

2006 ◽  
Vol 18 (2) ◽  
pp. 130
Author(s):  
H.-F. Jin ◽  
B. Mohana Kumar ◽  
J.-G. Kim ◽  
H.-J. Song ◽  
S. Balasubramanian ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Nuclear Transfer ◽  
Total Cell ◽  
Differentiation Potential ◽  
Developmental Potential ◽  
Cell Numbers ◽  
Donor Cells ◽  
Fetal Fibroblasts

Recent experimental evidence indicates that adult stem cells are more desirable than somatic cells for nuclear transfer (NT) because of their easy reprogrammability to resemble the genome of the zygote (Zhu et al. 2004 Biol. Reprod. 71, 1890-1897). Mesenchymal stem cells (MSCs) are a heterogeneous population of uncommitted and lineage-committed cells and may have a more flexible potential as donor cells for NT. The aim of this study was to characterize an isolated population of porcine MSCs from bone marrow and to compare the developmental potential of cloned (IVF) embryos with MSCs and porcine fetal fibroblasts (pFFs) by assessing the cleavage and blastocyst rate, total cell numbers, inner cell mass (ICM) ratio and apoptosis. MSCs were obtained from the aspirated bone marrow of 6-8-month-old pigs. Cells were centrifuged, resuspended, and plated in advanced-DMEM (ADMEM) supplemented with 5% fetal bovine serum (FBS). The differentiation potential was demonstrated by culture of MSCs (passage 3) under conditions that were favorable for adipogenic, osteogenic, and chondrogenic development (Pittenger et al. 1999 Science 284, 143-147). Oil red O staining revealed that MSCs produced lipid droplets after incubation in adipogenic media. Following osteoinduction, MSCs exhibited robust alkaline phosphatase activity and cells later transformed into mineralized nodules as demonstrated by von Kossa staining. Histological staining of proteoglycan indicated chondrogenic differentiation. Cumulus-oocyte complexes were matured, fertilized, and cultured by the following method (Abeydeera et al. 2000 Theriogenology 54, 787-797). NT embryos were produced as described by Kim et al. (2005 Mol. Rep. Dev. 70, 308-313). Cleavage rate was significantly (P < 0.05) higher in IVF embryos than in NT embryos derived from MSCs and pFFs (84.5% � 4.6 vs. 52.2% � 5.4 and 50.8% � 5.2, respectively). However, blastocyst rates in IVF embryos and NT embryos derived from MSCs (20.6% � 2.5 and 18.5% � 3.0) did not differ but these rates were significantly (P < 0.05) higher than that for NT embryos derived from pFFs (9.5% � 2.1). Total cell numbers and the ratio of ICM to total cells among embryos developed in NT from MSCs (29.4 � 5.2 and 0.38 � 0.08, respectively) were significantly (P < 0.05) higher than for those from pFFs (22.6 � 5.5 and 0.18 � 0.12, respectively). Proportions of TUNEL-positive cells in NT embryos from pFFs (12.8 � 2.5) were significantly (P < 0.05) higher than in those from MSCs (8.6 � 1.8) and in IVF embryos (4.6 � 1.5). The results clearly demonstrate that multipotent bone marrow MSCs can make a suitable alternative to fibroblasts as donor cells and have a greater potential for producing viable cloned porcine embryos. This work was supported by Grant No. R05-2004-000-10702-0 from KOSEF, Republic of Korea.

Sign in / Sign up

Export Citation Format

Share Document