Sung-Lim Lee

RELATIVE CONTRIBUTIONS OF BACTERIA, PROTOZOA, AND FUNGI TO IN VITRO DEGRADATION OF ORCHARD GRASS CELL WALLS AND THEIR INTERACTIONS

ABSTRACT To assess the relative contributions of microbial groups (bacteria, protozoa, and fungi) in rumen fluids to the overall process of plant cell wall digestion in the rumen, representatives of these groups were selected by physical and chemical treatments of whole rumen fluid and used to construct an artificial rumen ecosystem. Physical treatments involved homogenization, centrifugation, filtration, and heat sterilization. Chemical treatments involved the addition of antibiotics and various chemicals to rumen fluid. To evaluate the potential activity and relative contribution to degradation of cell walls by specific microbial groups, the following fractions were prepared: a positive system (whole ruminal fluid), a bacterial (B) system, a protozoal (P) system, a fungal (F) system, and a negative system (cell-free rumen fluid). To assess the interactions between specific microbial fractions, mixed cultures (B+P, B+F, and P+F systems) were also assigned. Patterns of degradation due to the various treatments resulted in three distinct groups of data based on the degradation rate of cell wall material and on cell wall-degrading enzyme activities. The order of degradation was as follows: positive and F systems > B system > negative and P systems. Therefore, fungal activity was responsible for most of the cell wall degradation. Cell wall degradation by the anaerobic bacterial fraction was significantly less than by the fungal fraction, and the protozoal fraction failed to grow under the conditions used. In general, in the mixed culture systems the coculture systems demonstrated a decrease in cellulolysis compared with that of the monoculture systems. When one microbial fraction was associated with another microbial fraction, two types of results were obtained. The protozoal fraction inhibited cellulolysis of cell wall material by both the bacterial and the fungal fractions, while in the coculture between the bacterial fraction and the fungal fraction a synergistic interaction was detected.

Characterization of Porcine Multipotent Stem/Stromal Cells Derived from Skin, Adipose, and Ovarian Tissues and Their Differentiation In Vitro into Putative Oocyte-Like Cells

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.

Enhanced cryosurvival of bovine blastocysts produced in vitro in serum-free medium.

AbstractPurpose: Culture systems affect the development of IVP embryos and consequently their cryosurvival potential. The viability of postthawed bovine IVP embryos developed from IVM/IVC medium in the presence or absence of serum was compared. Methods: Cumulus-oocyte complexes were matured in IVM medium supplemented with or without serum. Some oocytes were evaluated for nuclear maturation status and others were inseminated with semen. Presumptive zygotes were cultured in IVC medium supplemented with or without serum for 9 days. Blastocysts were cryopreserved with 1.5 M ethylene glycol in PBS. Results: No difference was observed in the nuclear maturation status and cleavage rates in both groups, but significantly (P < 0.05) higher in blastocyst rates in the serum-supplemented group. After freezing, survival of blastocysts was higher in the serum-free group. At 36 h culture after thawing, blastocysts developed without serum had significantly (P < 0.05) higher cell number than those cultured with serum. Conclusions: We conclude that serum-free culture system enhances the viability of frozen-thawed bovine embryos.

Cell source-dependent in vivo immunosuppressive properties of mesenchymal stem cells derived from the bone marrow and synovial fluid of minipigs

The in vitro differentiation and immunosuppressive capacity of mesenchymal stem cells (MSCs) derived from synovial fluid (SF-MSCs) and bone marrow extract (BM-MSCs) in an isogenic background of minipigs were comparatively analyzed in a collagen-induced arthritis (CIA) mouse model of rheumatoid arthritis (RA). The proliferation capacity and expression of pluripotent transcription factors (Oct3/4 and Sox2) were significantly (P<0.05) higher in SF-MSCs than in BM-MSCs. The differentiation capacity of SF-MSCs into adipocytes, osteocytes and neurocytes was significantly (P<0.05) lower than that of BM-MSCs, and the differentiation capacity of SF-MSCs into chondrocytes was significantly (P<0.05) higher than that of BM-MSCs. Systemic injection of BM- and SF-MSCs significantly (P<0.05) ameliorated the clinical symptoms of CIA mice, with SF-MSCs having significantly (P<0.05) higher clinical and histopathological recovery scores than BM-MSCs. Furthermore, the immunosuppressive properties of SF-MSCs in CIA mice were associated with increased levels of the anti-inflammatory cytokine interleukin (IL)-10, and decreased levels of the pro-inflammatory cytokine IL-1β and osteoclast-related sRANKL. In conclusion, SF-MSCs exhibited eminent pluripotency and differentiation capacity into chondrocytes, addition to substantial in vivo immunosuppressive capacity by elevating IL-10 and reducing IL-1β levels in CIA mice.

Characterisation and differentiation of porcine ovarian theca-derived multipotent stem cells.

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.

Comparison of Immunomodulation Properties of Porcine Mesenchymal Stromal/Stem Cells Derived from the Bone Marrow, Adipose Tissue, and Dermal Skin Tissue

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.

In vitro comparative analysis of human dental stem cells from a single donor and its neuronal differentiation potential evaluated by electrophysiology.

AIMS The aim of this study was to find out a mesenchymal stem cells (MSCs) source from human dental tissues of the same donor (follicle, papilla and pulp), which exhibits higher neurogenic differentiation potential in vitro. MAIN METHODS MSCs were isolated from dental tissues (follicle, papilla and pulp) by digestion method. All MSCs were analyzed for pluripotent makers by western blot, cell surface markers by flow cytometry, adipo- and osteocytes markers by RT-qPCR. The neuronal differentiated MSCs were characterized for neuronal specific markers by RT-qPCR and immunofluorescence. Functional neuronal properties were analyzed by electrophysiology and synaptic markers expression. KEY FINDINGS All MSCs expressed pluripotent markers (Oct4, Sox2 and Nanog) and were found positive for mesenymal markers (CD44, CD90, CD105) while negative for hematopoietic markers (CD34 and CD45). Furthermore, MSCs were successfully differentiated into adipocytes, osteocytes and trans-differentiated into neuronal cells. Among them, dental pulp derived MSCs exhibits higher neurogenic differentiation potential, in term of expression of neuronal specific markers at both gene and protein level, and having higher Na(+) and K(+) current with the expression of synaptic markers. SIGNIFICANCE The three types of dental MSCs from a single donor broadly possessed similar cellular properties and can differentiate into neuronal cells; however, pulp derived MSCs showed higher neurogenic potential than the follicle and papilla, suggesting their use in future stem cells therapy for the treatment of neurodegenerative disorders.

Osteogenic differentiation of human mesenchymal stem cells promoted by the crude extracts of the mixture of Cortex mori radicis, Patrinia saniculaefolia

The present study investigated the effect of Cortex mori radicis (CMR) and Patrinia saniculaefolia (PS) on the osteogenic differentiation of the human mesenchymal stem cell. CMR and PS have been used as herbal medicine in traditional Korean medicine for a long time. Mesenchymal stem cells that can differentiate into adipocyte, chondrocyte and osteocyte recently issued as a therapeutic agent for degenerative disease. Here, mesenchymal stem cells isolated from synovial fluid of osteoarthritis patient, were cultured in specific media to differentiate into osteogenesis. Osteogenic differentiated mesenchymal stem cells were confirmed using Von Kossa and Arizarion Red S staining. And, the cells were divided into 4 groups: control group, CMR treated group, PS treated group and mixture treated group. To determine the effect of the herbal samples on the osteogenic mesenchymal stem cell, OCT4, SOX2 and NANOG mRNA expressions, known as the key maintenance factors of mesenchymal stem cell was measured using real-time Polymerase Chain Reaction (PCR). As a result, the maintenance factors, OCT4, SOX2 and NANOG, were more increased in cells treated by CMR, PS and the mixture of two herbs than control group. Therefore, we confirmed the enhancing effect of CMR, PS and their mixture on the osteogenic differentiation of mesenchymal stem cells which derived from the synovial fluid of osteoarthritis patient. This study demonstrates that the effect of the herbal samples on the osteogenic differentiation of human mesenchymal stem cell may have a possibility to be a therapeutic agent for the osteoarthritis patients.

Characterization and Evaluation of Neuronal Trans-Differentiation with Electrophysiological Properties of Mesenchymal Stem Cells Isolated from Porcine Endometrium

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 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.

Overexpression of Oct4 in porcine ovarian stem/stromal cells enhances differentiation of oocyte-like cells in vitro and ovarian follicular formation in vivo.

BackgroundRecent findings have revealed that the female gonad may have regenerative activity with having germ line stem cells in juveniles and adults. Application of these germ line stem cells could be an alternative therapy for reproductive disorders in regenerative medicine.MethodsTo enhance the potency of differentiation into oocyte-like cells (OLCs) and folliculogenesis, we overexpressed Oct4 in ovarian stem/stromal cell (OvSCs) and examined the cellular properties related to stemness and self-renewal ability and finally demonstrated the ability of in vitro differentiation and folliculogenesis.ResultsOvarian cortex included putative stem cells in terms of AP activity, cell cycle status, cell proliferation, expression of mesenchymal lineage surface markers and pluripotent transcriptional markers. Further, Oct4 transfected OvSCs (Oct4-OvSCs) were enhanced their AP activity and cell proliferation compared to OvSCs. The potential on in vitro differentiation into OLCs and in vivo folliculogenesis was also evaluated in OvSCs and Oct4-OvSCs, respectively. Oct4-OvSCs possessed higher oogenesis potential in vitro than OvSCs, in terms of expression of germ cell markers by RT-PCR and the number of OLCs. When OvSCs and Oct4-OvSCs were xeno-transplanted into infertile mice ovaries, the OvSCs transplantation induced new primary follicle formation and hormonal levels of estradiol and FSH remained similar to that of normal mice. However, Oct4-OvSCs possessed higher ability for folliculogenesis based on inducing developing follicles with thecal layer and granulosa cells and more similar estradiol level to normal mice.ConclusionsThese findings demonstrated that putative stem cells were present in ovarian cortex and exhibited differentiation ability into OLCs and folliculogenesis in vivo, and Oct4-overexpression enhanced these ability, suggesting their cellular models based on gene therapy in understanding the mechanisms of oogenesis and folliculogenesis, and finally in view of reproductive cell therapy.