scholarly journals Bone Marrow Mesenchymal Stem Cells Are an Attractive Donor Cell Type for Production of Cloned Pigs As Well As Genetically Modified Cloned Pigs by Somatic Cell Nuclear Transfer

2013 ◽  
Vol 15 (5) ◽  
pp. 459-470 ◽  
Author(s):  
Zicong Li ◽  
Xiaoyan He ◽  
Liwen Chen ◽  
Junsong Shi ◽  
Rong Zhou ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Cell Type ◽  
Marrow Mesenchymal Stem Cells ◽  
Donor Cell Type

Use of Adult Mesenchymal Stem Cells Isolated from Bone Marrow and Blood for Somatic Cell Nuclear Transfer in Pigs

2006 ◽  
Vol 8 (3) ◽  
pp. 166-173 ◽  
Author(s):  
Renate Faast ◽  
Sharon J. Harrison ◽  
Luke F.S. Beebe ◽  
Stephen M. Mcilfatrick ◽  
Rodney J. Ashman ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Adult Mesenchymal Stem Cells

Effect of donor cell type, gender on efficiency of somatic cell nuclear transfer

2016 ◽  
Vol 231 ◽  
pp. S13
Author(s):  
Sezen Arat ◽  
Arzu Tas Caputcu ◽  
Mesut Cevik ◽  
Tolga Akkoc ◽  
Gaye Cetinkaya
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Cell Type ◽  
Donor Cell Type

257 EXPRESSION OF DEVELOPMENTAL GENES IN PORCINE NUCLEAR TRANSFER EMBRYOS RECONSTRUCTED WITH BONE MARROW MESENCHYMAL STEM CELLS

2006 ◽  
Vol 18 (2) ◽  
pp. 236
Author(s):  
B. Mohana Kumar ◽  
H.-F. Jin ◽  
J.-G. Kim ◽  
S. Balasubramanian ◽  
S.-Y. Choe ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Nuclear Transfer ◽  
Expression Profiles ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Marrow Mesenchymal Stem Cells

Abnormal gene expression is frequently observed in nuclear transfer (NT) embryos and is one of the suggested causes of the low success rates of this approach. Recent study has suggested that adult stem cells may be better donor cells for NT, as their less differentiated state may ease epigenetic reprogramming by the oocyte (Kato et al. 2004 Biol. Reprod. 70, 415-418). In the present study, we investigated the expression profile of some selected genes involved in the development of the pre-implantation embryos of in vivo- and NT-derived origin using bone marrow mesenchymal stem cells (MSCs) and porcine fetal fibroblasts (pFF) as donors. Isolated population of MSCs from porcine bone marrow were characterized by cell-surface antigen profile (CD13pos, CD105pos, CD45neg, and CD133neg) and by their extensive consistent differentiation to multiple mesenchymal lineages (adipocytic, osteocytic and chondrocytic) under controlled in vitro conditions (Pittenger et al. 1999 Science 284, 143-147). Primary cultures of pFF from a female fetus at <30 days of gestation were established. for NT, donor cells at 3-4 passages were employed. Embryos cloned from MSCs showed enhanced developmental potential compared to pFF cloned embryos, indicated by higher rates of blastocyst formation (15.3% � 4.8 and 9.0% � 3.9, respectively) and total cell number (31.5 � 7.2 and 20.5 � 5.4, respectively) in Day 7 blastocysts. Total RNA was extracted from pools (triplicates) of 10 embryos each of 8-cell, morula, and blastocyst stages of in vivo and NT origin using Dynabeads� mRNA DIRECT" kit (Dynal, Oslo, Norway). Reverse transcription was performed with a Superscript" III cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA). Real-time PCR was performed on a Light cycler� using FastStart DNA Master SYBR Green I (Roche Diagnostics, Mannheim, Germany). The expression profiles of genes involved in transcription (Oct-4, Stat3), DNA methylation (Dnmt1), de novo methylation (Dnmt3a), histone deacetylation (Hdac2), anti-apoptosis (Bcl-xL), and embryonic growth (Igf2r) were determined. The mRNA of H2a was employed to normalize the levels. Significant differences (P < 0.05) in the relative abundance of Stat3, Dnmt1, Dnmt3a, Bcl2, and Igf2r were observed in pFF NT embryos compared with in vivo-produced embryos, whereas embryos derived from MSCs showed expression patterns similar to those of in vivo-produced embryos. However, Oct-4 and Hdac2 revealed similar expression profiles in NT- and in vivo-produced embryos. These results indicate that MSC-derived NT embryos had enhanced embryonic development and their gene expression pattern more closely resembled that of in vivo-produced embryos. Hence, less differentiated MSCs may have a more flexible potential in improving the efficiency of the porcine NT technique. This work was supported by Grant No. R05-2004-000-10702-0 from KOSEF, Republic of Korea.

37 Healthy Foals Produced Using Bone Marrow-Mesenchymal Stem Cells as Nuclear Donors in Horse Cloning

2018 ◽  
Vol 30 (1) ◽  
pp. 158
Author(s):  
R. Olivera ◽  
L. Moro ◽  
R. Jordan ◽  
C. Luzzani ◽  
S. Miriuka ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Umbilical Cord ◽  
Donor Cell ◽  
Control Group ◽  
Nuclear Reprogramming ◽  
Marrow Mesenchymal Stem Cells

Somatic cell nuclear transfer efficiency is based on the capacity of the donor cell to be reset and reprogrammed to an embryonic state. So, the less differentiated the donor cells are, the more easily they could be reprogrammed by a recipient cytoplasm. Failures on appropriate nuclear reprogramming frequently lead to abnormalities associated with the placenta, umbilical cord, birthweight, and limbs. In the present study, we evaluated the efficiency of bone marrow mesenchymal stem cells (BM-MSC) compared with adult fibroblasts (AF) as nuclear donors in horse cloning and evaluated both in vitro and in vivo development of the embryos generated. Moreover, we focused on comparing the health of the foals generated and on the presence of anatomical abnormalities in foals produced from the different treatments. Embryos produced by AI, recovered by uterine flushing, and transferred to recipient mares were used as controls. All variables were analysed by Fisher test (P < 0.05). The cloning procedure was performed according to Olivera et al. (2016 PLoS One 11, e0164049, 10.1371/journal.pone.0164049). Both cleavage and blastocyst rates were higher when MSC were used as nuclear donors (P < 0.05). Cleavage rates were 85.6% (3875/4527) v. 90.2% (3095/3432) and blastocyst rates were 10.9% (492/4527) and 18.1% (622/3432) for AF and MSC groups, respectively. In the AF group, 476 blastocysts were transferred to recipient mares (232 transfers), and in the MSC group, 594 blastocysts were transferred 297 transfers). In the AI control group, 88 embryos were transferred. Pregnancies were diagnosed by transrectal ultrasonography 15 days after embryo transfer in all the groups. Pregnancy rates were similar between both cloning groups (41/232, 17.7% and 37/297, 12.5%for AF and MSC, respectively), but higher in the AI group (71/88, 80.7%). However, significant differences were observed in the birth of viable offsprings among the cloning groups. Despite similar rates of foal delivery (AF, 17/41, 41.5%; MSC, 21/37, 56.7%), a higher proportion of viable foals were obtained from the MSC group (20/37, 54.1%) compared with the AF group (9/41, 22%; P < 0.05). Surprisingly, as in the AI group (63/63, 100%), all of the viable foals obtained using MSC (20/20, 100%) were considered normal and did not show abnormalities associated with cloning. In contrast, in the AF group, only 4/9 (44.4%) were considered normal foals. The defects present in the other 5 foals were related to flexural and angular limb deformities and umbilical cord malformations. These were corrected rapidly with standard treatments or, in the case of the umbilical cords, minor surgery. This study shows for the first time that BM-MSC can be used as nuclear donors in horse cloning and that the foals obtained are as healthy as those produced by AI, showing no abnormalities related to deficiencies in nuclear reprogramming.

Transplantation of Haplo-Identical Bone Marrow-Derived Mesenchymal Stem Cells Together with Hematopoietic Stem Cells To Promote Engraftment in Children. A Phase I/II Multicenter Study.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2911-2911
Author(s):  
Lynne Margaret van Velzen-Ball ◽  
Katarina Le Blanc ◽  
Arjen C. Lankester ◽  
Helene Roelofs ◽  
Maarten Egeler ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Calf Serum ◽  
Donor Cell ◽  
Patient Characteristics ◽  
Allogeneic Transplantation ◽  
Immune Recovery ◽  
Hematopoietic Stem ◽  
Marrow Mesenchymal Stem Cells

Abstract Graft failure or rejection is an identified problem in haplo-identical or second attempt transplantation. In the bone marrow, mesenchymal stem cells (MSCs) have been identified and shown in animal models to enhance hematopoietic stem cell (HSC) engraftment. Advances in techniques and higher quality culture components have allowed the development of an MSC expansion procedure resulting in sufficient MSCs for clinical application. Adult studies have suggested that co-transplantation of MSCs and HSCs in the HLA-identical setting, is feasible and safe. Here we present the first combined clinical experience within the EBMT MSC expansion consortium with respect to co-transplantation of haplo-identical MSCs in the pediatric allogeneic transplantation setting. Six weeks before the SCT, 50 cc of bone marrow are sampled under sterile conditions. Density gradient-separated MNCs are plated into tissue culture flasks in low-glucose DMEM supplemented with 10% fetal calf serum and incubated at 370C with 5% CO2. At 70% confluence, the cells are trypsinized and re-plated at 4x103 cells/ cm2 until the target dose of 1–2 x106/kg recipient body weight is obtained. Enrichment and expansion of MSCs is performed under GMP conditions in nationally accredited laboratories. MSCs (either fresh or cryopreserved) are administered i.v. 4 hours before infusion of donor HSCs. To date 4 children have undergone co-transplantation. Patient characteristics are summarized in the table. All toxicities associated with the procedure were documented, as were the engraftment kinetics and immune recovery. The study was carried out with approval of the respective local ethical committees. The data indicate that expansion and co-transplantation of MSCs is feasible and well tolerated. While the study is ongoing initial engraftment and immune recovery data (compared to historical data) is encouraging and to date there have been no episodes of graft rejection or severe adverse events related to this treatment. Patient characteristics Patient demographics HSC characteristics MSC characteristics UPN Gender Age Diagnosis Donor CD34 dose (106/kg) Donor Cell dose (106/kg) 1 M 15 ANLL mother-haplo 16 mother 1.5 2 M 2 XLPD father-haplo 20 father 1.9 3 M 2 XLPD father-haplo 18.7 father 1.56 4 M 8 SAA URD (2 loci mismatched) 2.65 mother 1.0

293 EFFECT OF MEDIUM TYPE FOR CULTURE OF ADIPOSE-DERIVED MESENCHYMAL STEM CELLS ON PRE-IMPLANTATION DEVELOPMENT OF CLONED EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 294
Author(s):  
G. A. Kim ◽  
H. J. Oh ◽  
J. Kim ◽  
T. H. Lee ◽  
J. H. Lee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Culture Medium ◽  
Culture Media ◽  
Formation Rate ◽  
Donor Cells

Mesenchymal stem cells (MSC) have been known as useful donor cells for somatic cell nuclear transfer (SCNT). It has been suggested that the culture condition of donor cells causes different results on preimplantation development of SCNT embryos. In this study, we investigated the patterns of gene expression of adipose-derived mesenchymal stem cells (ad-MSC) in different culture media (DMEM and RKME), and examined the effect of ad-MSC, with the gene expression changed, used as donor cells on the preimplantation development of cloned embryos. Canine ad-MSC were isolated from fat tissue of 3-year-old female beagle and were cultured in DMEM supplemented with 10% fetal bovine serum (MSC-DMEM) and RKME (MSC-MSC) provided from RNL Bio Corp. (Seoul, Korea). Total RNA was extracted from ad-MSC cultured in each culture medium. After synthesising cDNA of each sample, quantitative RT-PCR was done according to the Takara Bio Inc. guidelines and using the 7300 Real Time PCR Cycler System (Applied Biosystems, Carlsbad, CA, USA). The level of all tested gene transcription was normalized to β-actin expression levels. The relative quantification of gene expression was analysed by the 2–ΔΔCt method. The data from all experiments were analysed by Student’s t-test using a statistical analysis GraphPad Prism 4.02 (GraphPad Software Inc., San Diego, CA, USA). Significance was determined at P < 0.05. The stemness, the reprogramming-related gene expression level of donor cells of MSC-DMEM and MSC-MSC were compared. In order to confirm the effect of MSC cultured in 2 different culture media on somatic cell nuclear transfer, we performed interspecies somatic cell nuclear transfer (iSCNT). The enucleated bovine oocytes were injected, respectively, with donor cells of MSC-DMEM and MSC-MSC, and were fused by electrofusion. The iSCNT embryos were cultured in modified SOF at 38.5°C for 7 days in an atmosphere of 5% CO2 and 5% O2, and the developmental ability of iSCNT embryos was observed under the microscope. The MSC-MSC contained a significantly higher amount of Sox2, Nanog, Oct4, Stella, HDAC1, DNMT1, and MeCP2 than the MSC-DMEM, whereas the amount of Rex1 was not different in either MSC-MSC or MSC-DMEM. In the development ability of iSCNT embryos, MSC-DMEM embryos resulted in a 16-cell embryo formation rate that was higher than that of MSC-MSC embryos (9.09 and 5.30%, respectively; P < 0.05). However, the blastocyst formation rate was not different between MSC-DMEM embryos and MSC-MSC embryos (4.5 and 3.2%, respectively; P > 0.05). These results demonstrate that the gene expression of ad-MSC can be modified, by culture media, into a state where reprogramming is easily done. Even so, ad-MSC with gene expression changed by culture medium did not influence the developmental ability of blastocysts. In conclusion, the alteration of gene-related stemness and reprogramming in canine ad-MSC would not be able to effectively control reprogramming in SCNT. This study was supported by RDA (#PJ0089752012), RNL Bio (#550-20120006), IPET (#311062-04-1-SB010), Research Institute for Veterinary Science, and Nestlé Purina Korea.

Sign in / Sign up

Export Citation Format

Share Document