[Show abstract][Hide abstract] ABSTRACT: Mesenchymal stromal/stem cells (MSCs) demonstrate immunomodulation capacity that has been implicated in the reduction of graft-versus-host disease. Accordingly, we herein investigated the capacity of MSCs derived from several tissue sources to modulate both proinflammatory (interferon [IFN] γ and tumor necrosis factor [TNF] α) and immunosuppressive cytokines (transforming growth factor [TGF] β and interleukin [IL] 10) employing xenogeneic human MSC-mixed lymphocyte reaction (MLR) test. Bone marrow-derived MSCs showed higher self-renewal capacity with relatively slow proliferation rate in contrast to adipose-derived MSCs which displayed higher proliferation rate. Except for the lipoprotein gene, there were no marked changes in osteogenesis- and adipogenesis-related genes following in vitro differentiation; however, the histological marker analysis revealed that adipose MSCs could be differentiated into both adipose and bone tissue. TGFβ and IL10 were detected in adipose MSCs and bone marrow MSCs, respectively. However, skin-derived MSCs expressed both IFNγ and IL10, which may render them sensitive to immunomodulation. The xenogeneic human MLR test revealed that MSCs had a partial immunomodulation capacity, as proliferation of activated and resting peripheral blood mononuclear cells was not affected, but this did not differ among MSC sources. MSCs were not tumorigenic when introduced into immunodeficient mice. We concluded that the characteristics of MSCs are tissue source-dependent and their in vivo application requires more in-depth investigation regarding their precise immunomodulation capacities.
Stem cell International 09/2015; · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Endometrial stromal cells (EMSCs) obtained from porcine uterus (n = 6) were positive for mesenchymal stem cell markers (CD29, CD44 and CD90), and negative for epithelial marker CD9 and hematopoietic markers CD34, CD45 analyzed by flow cytometry. Further the cells were positive for expression of mesenchymal markers, CD105, CD140b, and CD144 by PCR. Pluripotent markers OCT4, SOX2, and NANOG were positively expressed in EMSCs analyzed by Western blotting and PCR. Further, differentiation into adipocytes and osteocytes was confirmed by cytochemical staining and lineage specific gene expression by quantitative realtime-PCR. Adipocyte (FABP, LPL, AP2) and osteocyte specific genes (ON, BG, RUNX2) in differentiated EMSCs showed significant (p < 0.05) increase in expression compared to undifferentiated control cells. Neurogenic OPEN ACCESS Int. J. Mol. Sci. 2015, 16 10935 transdifferentiation of EMSCs exhibited distinctive dendritic morphology with axon projections and neuronal specific genes, NFM, NGF, MBP, NES, B3T and MAP2 and proteins, B3T, NFM, NGF, and TRKA were positively expressed in neuronal differentiated cells. Functional analysis of neuronal differentiated EMSCs displayed voltage-dependence and kinetics for transient outward K + currents (Ito), at holding potential of −80 mV, Na + currents and during current clamp, neuronal differentiated EMSCs was more negative than that of control EMSCs. Porcine EMSCs is a suitable model for studying molecular mechanism of transdifferentiation, assessment of electrophysiological properties and their efficiency during in vivo transplantation.
International Journal of Molecular Sciences 05/2015; 16(5):10934-10951. DOI:10.3390/ijms160510934 · 2.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The identification of stable reference genes is a prerequisite for ensuring accurate validation of gene expression, yet too little is known about stable reference genes of porcine MSCs. The present study was, therefore, conducted to assess the stability of reference genes in porcine MSCs derived from bone marrow (BMSCs), adipose (AMSCs), and skin (SMSCs) with their in vitro differentiated cells into mesenchymal lineages such as adipocytes, osteocytes, and chondrocytes. Twelve commonly used reference genes were investigated for their threshold cycle (Ct) values by qRT-PCR. The Ct values of candidate reference genes were analyzed by geNorm software to clarify stable expression regardless of experimental conditions. Thus, Pearson's correlation was applied to determine correlation between the three most stable reference genes (NF 3) and optimal number of reference genes (NF opt). In assessment of stability of reference gene across experimental conditions by geNorm analysis, undifferentiated MSCs and each differentiated status into mesenchymal lineages showed slightly different results but similar patterns about more or less stable rankings. Furthermore, Pearson's correlation revealed high correlation (í µí± > 0.9) between NF 3 and NF opt. Overall, the present study showed that HMBS, YWHAZ, SDHA, and TBP are suitable reference genes for qRT-PCR in porcine MSCs.
Stem cell International 03/2015; 2015:14. DOI:10.1155/2015/235192 · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abstract The present study compared the potential of porcine bone marrow mesenchymal stem cells (pBMSCs) at different passages as nuclear transfer (NT) donors and the developmental efficiency of NT embryos from donor cells transfected with/without Oct4 and Sox2. Early-passage pBMSCs showed higher proliferation and expression of Oct4 and Sox2 and differentiation potential into mesenchymal lineages than middle- and late-passage pBMSCs. Cleavage rate did not differ among pBMSCs at different passages, but NT embryos with early-passage pBMSCs and middle-passage pBMSCs transfected with Oct4 (Oct4-pBMSCs) had significantly (p<0.05) higher blastocyst development than those with middle-passage pBMSCs. The incidence of apoptotic bodies in NT blastocysts from late-passage pBMSCs and Sox2-transfected middle-passage pBMSCs (Sox2-pBMSCs) was significantly (p<0.05) higher than others. The transcriptional levels of Oct4, Sox2, Nanog, Cdx2, Dnmt3a, and Igf2r genes were significantly (p<0.05) higher in Oct4- and Sox2-pBMSCs NT embryos. Middle-passage pBMSCs NT embryos revealed lower transcriptional levels of Bcl2 than others, except Sox2-pBMSCs NT embryos. The transcriptional level of Bax increased gradually in NT embryos derived from pBMSCs following extended passages and was significantly (p<0.05) higher in Sox2-pBMSCs NT embryos. Our results demonstrated that early-passage pBMSCs are more potent in expressing transcription factors and displayed higher differentiation ability, and middle-passage pBMSCs transfected with Oct4 improved the developmental efficiency of NT embryos, suggesting that high Oct4 expression cells are more efficient as NT donors.
[Show abstract][Hide abstract] ABSTRACT: The combination of nuclear transfer (NT) with secondary oocyte cytoplasm (ooplasm) transfer would provide an opportunity to understand the early nuclear and cytoplasmic interactions, mitochondrial functions and developmental capacity of embryos. The present study investigated the effect of ooplasm transfer on the development of porcine NT embryos. The primary cultures of fibroblasts derived from porcine ear skin was cultured in DMEM supplemented with 10% FBS until 3-4 passage, and employed as nuclear donors. After in vitro maturation of porcine oocytes collected from slaughterhouse, oocytes were removed their nuclei with 1st polar body and smallest ooplasm, and transferred donor cell into their perivitalline space (NT). To analyze the effect of ooplasm, oocytes were removed of additionally ~10% ooplasm and received the equivalent volume of secondary donor ooplasm (OT) from other oocytes. After fusion by electrical stimulation, both reconstructed embryos were compared the rates of cleavage, blastocyst development and total cell numbers with IVF counterparts. Although blastocyst development was significantly (P<0.05) higher in IVF than other reconstructed groups, it did not differ the rates of cleavage, blastocyst development and total cell numbers in OT and NT embryos. Furthermore, after 1 h post fusion, OT embryos showed no morphologically difference in further developmental stages, as compared to IVF and NT embryos. Gene expression studies at blastocyst stage by qRT-PCR in terms of apoptosis and stress related genes, BAK and HSP90 showed up-regulated in both reconstructed embryos compared to IVF. Expression of methylation and acetylation related genes, DNMT1, HDAC1 and HAT, revealed no difference among the groups, however, DNMT3a expression was significantly (P<0.05) higher in OT embryos. TFAM, MFN and Vdac, which represents mitochondrial activity, were significantly (P<0.05) up-regulated in NT and OT than IVF. In conclusion, NT and OT embryos present decreased embryo development and increased expression of apoptosis and mitochondrial activity than IVF embryos. However, when compared to general NT embryos, supplement of secondary ooplasm in OT embryo displayed no identifying attributes on embryo development and gene expression, except just methylation related gene expression.
SSR 2014: Fertility: A Global Challenge,, Grand rapids, USA; 07/2014
[Show abstract][Hide abstract] ABSTRACT: Porcine models are of considerable interest in the field of biomedical research due their morphological, physiological, and developmental similarities with human (Fu et al., 2006; Vajta et al. 2007). Porcine mesenchymal stem cells (MSCs) have been isolated from many different tissues mainly because of their ability to differentiate into multilineages and also a potential source for regeneration therapy (Rho et al., 2009). Hence in our present study we isolated endometrial stromal cells from pubertal porcine uterus (n=6) by plating 500 cells/cm3 in Advanced Dulbecco’s modified essential medium (ADMEM) containing 10% heat inactivated fetal bovine serum (FBS) to obtain purified single suspensions of stromal cells. Isolated stromal cells were positive for mesenchymal stem cell markers CD29, CD44, CD90, CD105, CD140b, and CD144, negative for epithelial marker CD9, hematopoietic markers CD34 and CD45 analyzed by flow cytometry and PCR. Further, the stromal-MSCs were differentiated into adipocyte, and osteocyte confirmed by cytochemical staining and lineage specific gene expression by quantitative realtime-PCR. Neurogenic differentiation of stromal cells was carried out in neurogenic differentiation medium and verified their differentiation by morphological observation, immunofluorescence staining and quantitative realtime-PCR analysis of neuronal specific genes. Differentiated cells exhibited distinctive dendritic morphology and axon projections, and expressed neuronal specific genes, such as neurofilament, nerve growth factor, myelin basic protein and β-tubulin which were highly up-regulated compared to un-differentiated cells. We confirm that porcine uterus contain a population of stromal MSCs with great differentiation ability to multilineages and also a potential stem cells source for treatment of neurogenic disorder in the field of regeneration cell therapy.
SSR 2014: Fertility: A Global Challenge,, Grand rapids, USA; 07/2014
[Show abstract][Hide abstract] ABSTRACT: Mesenchymal stem cells (MSCs) were first isolated from bone marrow (BM) and their unique characteristics of self-renewal and differentiation into multi-lineage was attractive for regenerative medicine. However, adult BM-MSC have limited life span and variable differentiation potential depending on cell lines, which makes it difficult to obtain consistent results. Alternative source of MSC such as, umbilical cord blood (UCB), muscle, adipose tissue, joint and skin were evaluated in many studies. Among them UCB which can be collected without invasive techniques and has high proliferation capacity were of great research interest and also an excellent source of MSCs. Nevertheless, global gene expression profiling of UCB-MSC and BM-MSC after long-term culture still remain to be determined. In this present study, MSCs isolated from human BM extracts and UCB in each 5 replicates were cultured in defined medium and characterized for their cell surface CD markers, mesenchymal lineage differentiation potential, early transcription factors and evaluated for gene expression profiling using microarray analysis. UCB-MSCs positively expressed MSC markers CD73, CD90 and CD105, and steadily expressed transcriptional factors Oct4, Sox2 and Nanog. In addition these cells successfully differentiated into mesenchymal lineages, such as adipocyte, osteoblast and chondrocyte. However, the BM-MSCs ability of differentiation gradually declined from passage 9 and further. The cells from odd passages 1 to 19 were subjected to microarray analysis gene profiling, and totally 20,014 genes were investigated. Results from microarray analysis revealed that 9954 genes in BM-MSC and 61 genes in UCB-MSC were differentially expressed among different odd passages (FDR<0.05), and 31 genes were commonly expressed in both UCB-MSCs and BM-MSCs. Although the MSCs characteristics from both UCB and BM in early passage (till passage 9) were similar, but UCB-MSC displayed relatively consistent gene expression pattern and retained multilineage differentiation potential till late passages (passage 19). In conclusion, based on our data from gene expression profiling and differentiation capacity, UCB can be an excellent candidate source of MSCs for regenerative medicine.
SSR 2014: Fertility: A Global Challenge,, Grand rapids, USA; 07/2014
[Show abstract][Hide abstract] ABSTRACT: In this study, the cellular properties and in vitro differentiation capacity of porcine ovarian theca-derived multipotent stem cells (TSCs) were examined. Isolated TSCs were expanded into a homogeneous population that had a typical fibroblast-shaped morphology and was positive for alkaline phosphatase activity. Cell cycle analysis indicated that TSCs had high proliferative potential. Flow cytometry analysis demonstrated expression of mesenchymal cell surface markers (CD29, CD44 and CD90) on TSCs. Among three pluripotent markers tested (OCT4, NANOG and SOX2), only SOX2 was expressed in TSCs at protein and mRNA levels. Cytochemical staining demonstrated that TSCs differentiated in vitro into osteocytes and adipocytes. Lineage specific transcripts expressed by differentiated osteocytes including osteonectin, osteocalcin and RUNX2. Lineage specific transcripts expressed by differentiated adipocytes included adipocyte fatty acid binding protein-2 (aP2) and peroxisome proliferator-activated receptor-γ2. Following induction in oogenesis media, TSCs exhibited sequential changes in morphology, resembling oocyte-like cells (OLCs), and expressed transcription factors (OCT4, NANOG and SOX2), oocyte-specific marker genes (GDF9B, C-MOS, DAZL, VASA, ZPC, SCP3 and STELLA) and the folliculogenesis marker follicular stimulating hormone receptor. These results indicated that TSCs derived from ovarian follicles are capable of differentiating into mesenchymal lineages and OLCs.
The Veterinary Journal 05/2013; 197(3). DOI:10.1016/j.tvjl.2013.04.011 · 1.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The present study compared the efficiency of somatic cell nuclear transfer (SCNT) using porcine oocytes that were denuded of their cumulus cells at different maturation time. In pre-denuded group, the cumulus cells from cumulus-oocyte complexes (COCs) were removed at 29 hr post in vitro maturation (hpm) and followed by further culture for 12 hr. In control group, as a commonly followed procedure, cumulus cells were removed from COCs at 41 hpm. The majority of porcine oocytes at 29 hpm were observed in metaphase of the first meiotic division (MI). At 41 hpm, no significant (P>0.05) differences were observed in nuclear maturation and mitochondrial distribution of oocytes between pre-denuded and control groups. However, in pre-denuded group oocytes, metaphase II (MII) plate and spindle were located closely as 'adjacent' to the first polar body (PB1), resulting in an increased enucleation rates than in control group oocytes by blind enucleation method. Following SCNT and parthenogenesis (PA) using pre-denuded group and control group oocytes, no significant (P>0.05) differences were observed with respect to the development, total cell number, incidence of apoptosis and the expression profile of developmentally important genes (Pou5f1, Dnmt1, Dnmt3a, Igf2r, Bax, Bcl2 and Glut1) at the blastocyst stage. In conclusion, the removal of cumulus cells at 29 hpm in porcine oocytes increased the enucleation rates through proper positioning of PB1 without compromising the quality of SCNT embryos during preimplantation development. Hence, this could be a valuable strategy to improve the SCNT efficiency in a porcine model.
The Japanese journal of veterinary research 11/2012; 60(4):191-203. · 0.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Canine mesenchymal stem cells (cMSCs) have generated a great interest as a promising source for cell based therapies. To understand the basic biological properties of cMSCs derived from bone marrow (cBM-MSCs), adipose tissue (cA-MSCs) and dermal skin(cDS-MSCs) from a single donor, the present study compared their alkaline phosphatase (AP) activity, expression of CD markers and stem cell transcription factors, differentiation ability into osteogenesis adipogenesis and chondrogenesis, in vivo ectopic bone formation, chromosomal stability, cell cycle status, telomere length and telomerase activity. Expressions of AP activity and transcription factors (Oct3/4, Nanog and Sox2) were either absent or extremely weak in all cMSCs. CD marker profile (CD45(-), 90(+) and 105(+)) and differentiation capacity were exhibited by all cMSCs, although cA-MSCs had enhanced cytochemical staining associated with lineage specific markers expression. In vivo bone formation of cMSCs was performed with demineralized bone matrix (DBM) by transplanting into the subcutaneous spaces of 9 weeks old BALB/c-nu mice, followed by radiographic and histological analysis after 1 and 2 months. cA-MSCs, and cDS-MSCs, which in contrast to the in vitro observations, also displayed higher in vivo osteogenic abilities than cBM-MSCs. Ploidy analysis showed that cells were diploid and contained no noticeable chromosomal abnormalities. Further, a relatively low percentage of cells were found at G1 phase in all cMSCs, especially in DS-MSCs. Regardless of varied tissue sources, cMSCs from a single donor showed no differences in telomere lengths (~18-19 kbp), but exhibited varied telomerase activity. The above results suggest that tissue specific cMSCs derived from a single donor possess slight differences in stem cell properties.
[Show abstract][Hide abstract] ABSTRACT: We have characterized and compared the telomere length, telomerase, reverse transcriptase (RT) activity and expression of genes implicated in cancer and in pluripotency, in human mesenchymal stem cells (MSCs) derived from dental papilla tissue, umbilical cord matrix and adipose tissue and in cancer cells (MDA-MB-231, U-87 MG, and MCF-7). MRC-5 fetal fibroblasts and adult muscle cells were used as somatic cell controls. Telomere length was significantly (P<0.05) higher in MSCs and somatic cells (7.2-9.3 kb) than in cancer cell lines (3.9-6 kb). However, the relative telomerase activity (RTA) in the cancer cell lines was significantly (P<0.05) higher than that of MSCs and somatic cells. RTA tended to be slightly higher in MSCs but no significant differences were observed between some cancer cells and MSCs. However, RTA was not detected in somatic cells. Although differentially displayed, the expression of genes related to cancer (BCL-2, p53, NF-κB, TGF-β, VEGF) and transcription and pluripotency (OCT4, NANOG, STAT3, REX1) were commonly observed in MSCs and cancer cells. Thus, endogenous non-telomerase RTA might be a potential biological marker or regulator among MSCs and cancer cells. Further, by sharing the biological and molecular markers of self-renewal and proliferation with cancer cells, MSCs might play a contributory role as tissue resident stem cells in tumor development.
Cell and Tissue Research 06/2011; 345(1):149-61. DOI:10.1007/s00441-011-1191-9 · 3.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The present study evaluated the alkaline phosphatase activity, cell cycle stage, expression of markers and early transcriptional factors, and in vitro differentiation into selected cell lineages of porcine stem/stromal cells (SCs) isolated from skin (SSCs), adipose, and ovarian (OSCs) tissues. Skin and adipose SCs were isolated from a 6-month-old miniature pig, whereas OSCs were isolated from a newly born piglet. Isolated cells exhibited fibroblast-like cell population with significant renewal capacity and formed colonies by cells out-growth. All cells were positive for alkaline phosphatase activity and showed a relatively lower population at G0/G1 phase of the cell cycle. SCs derived from all tissues were strongly positive for cell surface markers, such as CD29, CD44, CD90, and vimentin. Further, relatively lower expression of cytokeratin and immunophenotype markers, such as major histocompatibility complex II (MHCII) and swine leukocyte antigen (SLA), was also observed. SCs derived from all tissues positively expressed the transcription factors, such as Oct-3/4, Nanog, and Sox-2. After induction, all SCs successfully differentiated into osteocytes and adipocytes and expressed the lineage specific marker genes. Further, cells from all tissues exhibited their potential for in vitro oogenesis with morphological changes and expression of markers during the germ-cell formation, namely Oct-4, growth differentiation factor 9b, c-Mos, Vasa, deleted in azoospermia-like gene, zona pellucida C, and follicle stimulating hormone receptor. Apart from basic features and selected lineage potential among all types of cells, OSCs possessed a greater ability to differentiate into the germ cell lineage in vitro.
Stem cells and development 02/2011; 20(8):1359-70. DOI:10.1089/scd.2010.0203 · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The present study compared the developmental ability of miniature pig embryos cloned with fetal fibroblasts (FFs), bone marrow-derived mesenchymal stem cells (MSCs) and differentiated (osteocytes, adipocytes and chondrocytes) MSCs. MSCs were isolated from an approximately 1-month-old female miniature pig (T-type, PWG Micro-pig((R)), PWG Genetics Korea). MSCs were differentiated into osteocytes, adipocytes and chondrocytes under controlled conditions and characterized by cell surface antigen profile using specific markers. These differentiated or undifferentiated MSCs, as well as FFs of miniature pig, were transferred into enucleated oocytes of domestic pigs. Data from 10 replicates involving 1567 cloned embryos were assessed in terms of developmental rates. The in vitro development rate to the blastocyst stage of embryos cloned with undifferentiated MSCs was significantly (P<0.05) higher than that of embryos cloned with differentiated MSCs and FFs. Surgical transfer of 523 two-cell stage embryos cloned with undifferentiated MSCs into five synchronized domestic pig recipients resulted in 5 cloned miniature pig offspring (1 stillborn and 4 viable) from 2 pregnant recipients. The results imply that MSCs might be multipotent and that they can be used to produce viable cloned miniature pigs that cannot be easily reproduced with differentiated somatic cells.
Journal of Reproduction and Development 04/2010; 56(2):256-62. DOI:10.1262/jrd.09-196A · 1.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study was designed to evaluate the nuclear maturation and maturation promoting factor (MPF) level at different maturation times, and the effect of parthenogenetic activation on nuclear maturation in canine oocytes. Cumulus-oocyte complexes (COCs) were matured in TCM-199 supplemented with 10% fetal bovine serum, hormones, 0.57 mM cysteine, and 10 ng/ml epidermal growth factor for 72 hr at 38.5 degrees C. In Experiment 1, COCs at 0, 24, 48 and 72 hr of culture were assessed for nuclear maturation and MPF levels using histone H1 kinase activity assay. A significantly higher rate of oocytes at 72 hr than 0, 24 and 48 hr of culture developed to metaphase I-anaphase I and metaphase II. Relative abundance of histone H1 kinase activity of oocytes matured for 48 hr increased to ~1.5 x, with a marked increase to approximately 2.5 x for 72 hr, significantly higher than others. In Experiment 2, oocytes matured for 48 hr were parthenogenetically activated with 5 microm ionomycin for 5 min (Group 1) and followed by 10 microg/ml cycloheximide for 3 hr (Group 2), or no treatment (Control). Oocytes were then cultured for 24 hr and assessed for nuclear maturation. A significantly higher rate of oocytes in Group 1 developed to metaphase II than in Group 2 and the control. These results indicated that ionomycin treatment at 48 hr of in vitro maturation had a positive influence on oocyte progression to the metaphase II stage.
Journal of Veterinary Medical Science 03/2010; 72(7):887-92. DOI:10.1292/jvms.09-0563 · 0.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In vitro and in vivo osteogenesis of skin-derived mesenchymal stem cell-like cells (SDMSCs) with a demineralized bone (DMB) and fibrin glue scaffold were compared. SDMSCs isolated from the ears of adult miniature pigs were evaluated for the expression of transcriptional factors (Oct-4, Sox-2, and Nanog) and MSC marker proteins (CD29, CD44, CD90, and vimentin). The isolated SDMSCs were cocultured in vitro with a mixed DMB and fibrin glue scaffold in a nonosteogenic medium for 1, 2, and 4 weeks. Osteonectin, osteocalcin, and Runx2 were expressed during the culture period and reached maximum at 2 weeks after in vitro coculture. von Kossa-positive bone minerals were also noted in the cocultured medium at 4 weeks. Autogenous porcine SDMSCs (1 x 10(7)) labeled with a tracking dye, PKH26, were grafted into the maxillary sinus with a DMB and fibrin glue scaffold. In the contralateral side, only a scaffold was grafted without SDMSCs (control). In vivo osteogenesis was evaluated from two animals euthanized at 2 and 4 weeks after grafting. In vivo PKH26 staining was detected in all the specimens at both time points. Trabecular bone formation and osteocalcin expression were more pronounced around the grafted materials in the SDMSC-grafted group compared with the control group. New bone generation was initiated from the periphery to the center of the grafted material. The number of proliferating cells increased over time and reached a peak at 4 weeks in both in vivo and in vitro specimens. These findings suggest that autogenous SDMSC grafting with a DMB and fibrin glue scaffold can serve as a predictable alternative to bone grafting in the maxillary sinus floor.
Tissue Engineering Part A 09/2009; 16(3):815-27. DOI:10.1089/ten.TEA.2009.0439 · 4.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mesenchymal stem/progenitor cells (MPCs) were isolated from porcine umbilical cord blood (UCB) and their morphology, proliferation, cell cycle status, cell-surface antigen profile and expression of hematopoietic cytokines were characterized. Their capacity to differentiate in vitro into osteocytes, adipocytes and chondrocytes was also evaluated. Primary cultures of adherent porcine MPCs (pMPCs) exhibited a typical fibroblast-like morphology with significant renewal capacity and proliferative ability. Subsequent robust cell growth was indicated by the high percentage of quiescent (G0/G1) cells. The cells expressed the mesenchymal surface markers, CD29, CD49b and CD105, but not the hematopoietic markers, CD45 and CD133 and synthesized hematopoietic cytokines. Over 21 days of induction, the cells differentiated into osteocytes adipocytes and chondrocytes. The expression of lineage specific genes was gradually upregulated during osteogenesis, adipogenesis and chondrogenesis. We conclude that porcine umbilical cord blood contains a population of MPCs capable of self-renewal and of differentiating in vitro into three classical mesenchymal lineages.
Molecules and Cells 01/2008; 24(3):343-50. · 2.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The objective of the present study was to develop a procedure for isolating pure populations of round spermatid(s) (RS) by Percoll density gradient from bull testes. Bull testes were de-capsulated and testicular tissues were dissociated enzymatically to recover RS. After being filtered through a 20 microm nylon mesh, the cells were centrifuged at 650 x g for 25 min through the discontinuous Percoll density gradients (20, 35, 40, 45 and 90% Percoll solution). Isolated cells were analyzed by microscopic observation for survivability and apoptosis. In Experiment 1, both microscopic observation and DNA analysis by flow cytometry showed that approximately 40% of cells collected from 35% Percoll gradient were presumptive RS, whereas in 40% Percoll gradient, mostly primary spermatocytes were observed. Experiment 2 compared the effect of 35% Percoll density isolation on the incidence of apoptosis and necrosis in fresh and frozen-thawed cells to those of untreated cells. The percentage (mean+/-S.E.M.) of necrosis in cells collected from 35% Percoll gradient was less (P<0.05) than in untreated and frozen-thawed cells from 35% Percoll gradient (11.7+/-3.1% compared with 26.3+/-2.0% and 53.5+/-1.3%, respectively), but the rate of apoptosis did not differ (1.2+/-0.49% compared with 2.5+/-0.8% and 0.9+/-0.04%, respectively). The proportional data (mean+/-S.E.M.) of live cells in Percoll treated group were greater (P<0.05) than in untreated and frozen-thawed cells from the 35% Percoll gradient (86.7+/-3.26% compared with 70.8+/-2.73% and 41.9+/-1.69%, respectively). Experiment 3 compared the development rates of embryos injected with RS isolated from fresh and frozen-thawed cells collected with the 35% Percoll gradient to those of untreated cells, and parthenotes as control. There were no significant (P>0.05) differences in the rates of cleavage and blastocyst development between untreated fresh cells and fresh cells collected from the 35% Percoll gradient (75.4 and 10.5% compared with 82.4 and 12.8%). However, there were lesser (P<0.05) cleavage and blastocyst rates in frozen-thawed cells from the 35% Percoll gradient (51.6 and 6.3%) and parthenotes (60.7 and 4.1%) were observed. These results suggest that isolation of presumptive RS by 35% Percoll density gradient is effective in eliminating apoptotic and early necrotic cells. However, the use of RS in improving the developmental potential of embryos merits further studies.
[Show abstract][Hide abstract] ABSTRACT: The timing between round spermatid(s) (RS) injection and oocyte activation are critical for spermatid remodeling and embryo development in intracytoplasmic injection of round spermatid (ROSI) procedure. The objective of the present study was to develop an appropriate oocyte activation method for producing developmentally competent bovine embryos reconstructed with RS. Embryos reconstructed by ROSI were compared with three activation treatments for the rates of pronuclear formation, development and ploidy. RS were isolated from bull testes by Percoll density gradients. Matured oocytes were divided into three activation groups. In Group 1, oocytes were activated with ionomycin (5 microM, 5 min) before ROSI. In Group 2, oocytes were activated with ionomycin after ROSI. In Group 3, oocytes were activated twice with ionomycin before and after ROSI. All the eggs were then incubated in cycloheximide (CHX, 10 microg/mL) for 5 h and cultured in CR1aa medium for up to 8 days. Three methods of oocyte activation were also compared for the activation and development of parthenotes. Activation rates among the groups were 70-79% and did not differ. Cleavage rates in parthenotes were significantly (P < 0.05) higher in Group 3 than in Groups 1 and 2, but blastocyst rates did not differ among the groups. In ROSI embryos, the rates of cleavage and development into blastocysts were significantly (P < 0.05) greater in Group 3 (82.3% and 13.1%) than in Groups 1 and 2 (53.7, 5.8% and 64.2, 1.7%, respectively). Ploidy analysis by examining the metaphase spreads of ROSI blastocysts displayed greater numbers of diploid chromosomal complements. These results suggest that intracytoplasmic RS injection combined with repeated ionomycin activation followed by CHX treatment is more efficient for producing developmentally competent embryos.