Suitability of autologous serum for expanding rabbit adipose-derived stem cell populations

반재준,정진영,임우석,김만호,김세훈 대한수의학회 2012 Journal of Veterinary Science Vol.13 No.4

Adipose-derived stem cells (ASCs) are believed to have potential use for treating many illnesses. Most cells, including ASCs, are generally cultured in medium containing fetal bovine serum (FBS). However, FBS, which could induce an immune response or infection, is not recommended for clinical applications. In the present study, we evaluated the morphology, proliferation rate, and characterization of rabbit ASCs grown in medium containing autologous serum (AS) and compared these cells to ones cultured with FBS. Morphological changes were monitored by microscopy and flow cytometry. Proliferation rates were assessed with cell counting and ASC phenotypes were characterized by flow cytometry using representative surface markers (CD44 and CD45). Expression of epidermal growth factor, brain-derived neurotrophic factor, and vascular endothelial growth factor was measured by reverse transcription-polymerase chain reaction. Results of our study showed that ASCs had a greater expansion rate in AS without developing morphological heterogeneity than cells grown in FBS. AS-cultured ASCs expressed representative growth factors, CD44 but not CD45, similar to cells cultured in FBS. Expression levels of some growth factors were different between AS and FBS. In conclusion, our findings indicated that AS could potentially be used as a culture medium supplement for the expansion of autologous ASCs.

Influence of Storage Condition on Exosome Recovery

이미정,반재준,임우석,김만호 한국생물공학회 2016 Biotechnology and Bioprocess Engineering Vol.21 No.2

Most of mammalian cells release extracellular vesicles including exosomes which mediate intercellular communication by delivering a variety of molecules. Despite of their importance in normal physiology and disease progression, the standard criteria of storage condition is indefinite and controversial. Therefore, we investigated exosome’s recovery yield and stability by various storage conditions. To investigate the effect of short-term storage temperature on exosome stability, exosomes were incubated at temperatures ranging from -70 to 90°C for 30 min. Immunoblot results showed that all exosome-associated proteins incubated at 90°C were mostly degraded for a short period of time. To examine the effect of long-term storage, isolated exosomes were incubated for 10 days at from -70°C to room temperature (RT), and exosomal protein, RNA and exosome markers were examined. Protein and RNA amounts were most reduced at RT compared with -70 and 4°C. Incubation at 4°C and RT resulted in major loss of CD63, and decreasing level of HSP70 was shown at only RT. In addition, flow cytometry result showed that exosome population became more dispersed after RT incubation for 10 days compared with -70°C incubated or freshly isolated exosomes. In summary, our results indicate that different storage temperature and period influences recovery yield and morphology of exosome, and storage at below -70°C is the favorable condition for preservation of fresh exosomes for clinical application and basic researches.

Sun Ginseng Protects Endothelial Progenitor Cells From Senescence Associated Apoptosis

임우석,정진영,반재준,임지연,이순태,주건,김만호 고려인삼학회 2012 Journal of Ginseng Research Vol.36 No.1

Endothelial progenitor cells (EPC) are a population of cells that circulate in the blood stream. They play a role in angiogenesis and, therefore, can be prognostic markers of vascular repair. Ginsenoside Rg3 prevents endothelial cell apoptosis through the inhibition of the mitochondrial caspase pathway. It also affects estrogen activity, which reduces EPC senescence. Sun ginseng (SG),which is heat-processed ginseng, has a high content of ginsenosides. The purpose of this study was to investigate the protective effects of SG on senescence-associated apoptosis in EPCs. In order to isolate EPCs, mononuclear cells of human blood buffy coats were cultured and characterized by their uptake of acetylated low-density lipoprotein (acLDL) and their binding of Ulex europaeus agglutinin I (ulex-lectin). Flow cytometry with annexin-V staining was performed in order to assess early and late apoptosis. Senescence was determined by β-galactosidase (β-gal) staining. Staining with 4′-6-Diamidino-2-phenylindole verifi ed that most adherent cells (93±2.7%) were acLDL-positive and ulex-lectin-positive. The percentage of β-gal-positive EPCs was decreased from 93.8±2.0% to 62.5±3.6% by SG treatment. A fl uorescence-activated cell sorter (FACS) analysis showed that 4.9% of EPCs were late apoptotic in controls. Sun ginseng decreased the apoptotic cell population by 39% in the late stage of apoptosis from control baseline levels. In conclusion, these results show antisenescent and antiapoptotic effects of SG in human-derived EPCs, indicating that SG can enhance EPC-mediated repair mechanisms. Endothelial progenitor cells (EPC) are a population of cells that circulate in the blood stream. They play a role in angiogenesis and, therefore, can be prognostic markers of vascular repair. Ginsenoside Rg3 prevents endothelial cell apoptosis through the inhibition of the mitochondrial caspase pathway. It also affects estrogen activity, which reduces EPC senescence. Sun ginseng (SG),which is heat-processed ginseng, has a high content of ginsenosides. The purpose of this study was to investigate the protective effects of SG on senescence-associated apoptosis in EPCs. In order to isolate EPCs, mononuclear cells of human blood buffy coats were cultured and characterized by their uptake of acetylated low-density lipoprotein (acLDL) and their binding of Ulex europaeus agglutinin I (ulex-lectin). Flow cytometry with annexin-V staining was performed in order to assess early and late apoptosis. Senescence was determined by β-galactosidase (β-gal) staining. Staining with 4′-6-Diamidino-2-phenylindole verifi ed that most adherent cells (93±2.7%) were acLDL-positive and ulex-lectin-positive. The percentage of β-gal-positive EPCs was decreased from 93.8±2.0% to 62.5±3.6% by SG treatment. A fl uorescence-activated cell sorter (FACS) analysis showed that 4.9% of EPCs were late apoptotic in controls. Sun ginseng decreased the apoptotic cell population by 39% in the late stage of apoptosis from control baseline levels. In conclusion, these results show antisenescent and antiapoptotic effects of SG in human-derived EPCs, indicating that SG can enhance EPC-mediated repair mechanisms.

Exosome-Based Delivery of miR-124 in a Huntington’s Disease Model

이순태,임우석,반재준,이미정,정근화,이상건,주건,김만호 대한파킨슨병및이상운동질환학회 2017 Journal Of Movement Disorders Vol.10 No.1

Objective: Huntington’s disease (HD) is a genetic neurodegenerative disease that is caused by abnormal CAG expansion. Altered microRNA (miRNA) expression also causes abnormal gene regulation in this neurodegenerative disease. The delivery of abnormally downregulated miRNAs might restore normal gene regulation and have a therapeutic effect. Methods: We developed an exosome-based delivery method to treat this neurodegenerative disease. miR-124, one of the key miRNAs that is repressed in HD, was stably overexpressed in a stable cell line. Exosomes were then harvested from these cells using an optimized protocol. The exosomes (Exo-124) exhibited a high level of miR-124 expression and were taken up by recipient cells. Results: When Exo-124 was injected into the striatum of R6/2 transgenic HD mice, expression of the target gene, RE1-Silencing Transcription Factor, was reduced. However, Exo-124 treatment did not produce significant behavioral improvement.

Regulation of Wound Healing and Fibrosis by Hypoxia and Hypoxia-Inducible Factor-1

Robin J. Ruthenborg,반재준,Anum Wazir,Norihiko Takeda,Jung-whan Kim 한국분자세포생물학회 2014 Molecules and cells Vol.37 No.9

Wound healing is a complex multi-step process that requires spatial and temporal orchestration of cellular and non-cellular components. Hypoxia is one of the prominent microenvironmental factors in tissue injury and wound healing. Hypoxic responses, mainly mediated by a master transcription factor of oxygen homeostasis, hypoxia-inducible factor-1 (HIF-1), have been shown to be critically involved in virtually all processes of wound healing and remodeling. Yet, mechanisms underlying hypoxic regulation of wound healing are still poorly understood. Better understanding of how the wound healing process is regulated by the hypoxic microen¬vironment and HIF-1 signaling pathway will provide insight into the development of a novel therapeutic strategy for impaired wound healing conditions such as diabetic wound and fibrosis. In this review, we will discuss recent studies illuminating the roles of HIF-1 in physiologic and pathologic wound repair and further, the therapeutic potentials of HIF-1 stabilization or inhibition.

Ginsenoside Rc and Re Stimulate c-Fos Expression in MCF-7 Human Breast Carcinoma Cells

YoungJooLee,YoungRanJin,임원청,SangMiJi,JungYoonCho,반재준,이승기 대한약학회 2003 Archives of Pharmacal Research Vol.26 No.1

We have found that ginsenoside Rc and Re induce c-fos in MCF-7 human breast carcinoma cells at both the mRNA and protein levels. However, neither ginsenoside activated the expression of reporter gene under the control of AP-1/TPA response elements. We have also examined the possibility that ginsenoside Rc and Re act by binding to intracellular steroid hormone receptors that act as transcriptional factors in the nucleus in inducing c-fos mRNA in MCF7 human breast carcinoma cells. However, ginsenoside Rc and Re did not bind to glucocorticoid, androgen, estrogen, or retinoic acid receptors as examined by the transcription activation of the luciferase reporter genes in CV-1 cells that were transiently transfected with the corresponding steroid hormone receptors and hormone responsive luciferase reporter plasmids. These data demonstrate that ginsenoside Rc and Re act via other transcription factors and not via estrogen receptor in c-Fos expression.