Jeong Su Byeon

Chondrogenic potential and anti-senescence effect of hypoxia on canine adipose mesenchymal stem cells

Mesenchymal stem cells (MSCs) have the ability to differentiate into multi-lineage cells, which confers great promise for use in regenerative medicine. In this study, canine adipose MSCs (cAD-MSCs) were isolated from canine adipose tissue. These cells clearly represented stemness (Oct4, Sox2, and Nanog) and differentiation potential into the mesoderm (adipocytes, chondrocytes, and osteoblasts) at early passages. The aim of this study was to evaluate the effects of hypoxia on the differentiation potential into mesoderm, and the expression of anti-apoptotic genes associated with cell survival for the optimal culturing of MSCs. We observed that the proliferation of the cAD-MSCs meaningfully increased when cultured under hypoxic condition than in normoxic condition, during 7 consecutive passages. Also, we found that hypoxia strongly expressed anti-senescence related genes such as HDAC1 (histone deacetylase 1), DNMT1 (DNA (cytosine-5)-methyltransferase 1), Bcl-2 (inhibitor of apoptosis), TERT (telomerase reverse transcriptase), LDHA (lactate dehydrogenase A), SLC2A1 (glucose transporter), and DKC1 (telomere holoenzyme complex) and differentiation potential of cAD-MSCs into chondrocytes, than seen under the normoxic culture conditions. We also examined the multipotency of hypoxic conditioned MSCs using quantitative real-time RT-PCR. We found that the expression levels of stemness genes such as Oct-4, Nanog, and Sox-2 were increased in hypoxic condition when compared to the normoxic condition. Collectively, these results suggest that hypoxic conditions have the ability to induce proliferation of MSCs and augment their chondrogenic potential. This study suggests that cell proliferation of cAD-MSC under hypoxia could be beneficial, when considering these cells for cell therapies of canine bone diseases.

Effect of donor age on the proliferation and multipotency of canine adipose-derived mesenchymal stem cells

Research into adipose tissue-derived mesenchymal stem cells (AD-MSCs) has demonstrated the feasibility of their use in clinical applications due to their ease of isolation and abundance in adipose tissue. We isolated AD-MSCs from young and old dogs, and the cells were subjected to sequential sub-passaging from passage 1 (P1) to P7. Canine AD-MSCs (cAD-MSCs) were examined for proliferation kinetics, expression of molecules associated with self-renewal, expression of cell surface markers, and differentiation potentials at P3. Cumulative population doubling level was significantly higher in cAD-MSCs of young donors than in those of old donors. In addition, expressions of CD73, CD80, Oct3/4, Nanog, cell survival genes and differentiation potentials were significantly higher in young donors than in old donors. The present study suggests that donor age should be considered when developing cell-based therapies for clinical application of cAD-MSCs.

Extensive characterization of feline intra-abdominal adipose-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) isolated from various tissues have been well characterized for therapeutic application to clinical diseases. However, in contrast to MSCs from other animal species, the characteristics of feline MSCs have not been fully documented. In this study, we conducted extensive characterization of feline adipose tissue-derived MSCs (fAD-MSCs). Study fAD-MSCs were individually isolated from the intra-abdominal adipose tissues of six felines. The expression levels of cell surface markers and pluripotent markers were evaluated. Next, proliferation capacity was analyzed by performing cumulative population doubling level (CPDL) and doubling time (DT) calculation assays. Differentiation potentials of fAD-MSCs into mesodermal cell lineages were analyzed by examining specific staining and molecular markers. All fAD-MSCs positively expressed cell surface markers such as CD29, CD44, CD90, CD105, CD166, and MHC-I, while CD14, CD34, CD45, and CD73 were negatively expressed. The CPDL of the fAD-MSCs was maintained until passage 5 to 6 (P5 to P6), whereas DT increased after P3 to P4. Also, stem cell-specific pluripotent markers (Oct3/4, Nanog, and SSEA-4) were detected. Importantly, all fAD-MSCs demonstrated mesodermal differentiation capacity. These results suggest that fully characterized fAD-MSCs could be beneficial when considering the use of these cells in feline disease research.

Effects of three-dimensional spheroid culture on equine mesenchymal stem cell plasticity

Mesenchymal stem cells (MSCs) are useful candidates for tissue engineering and cell therapy fields. We optimize culture conditions of equine adipose tissue-derived MSCs (eAD-MSCs) for treatment of horse fractures. To investigate enhancing properties of three-dimensional (3D) culture system in eAD-MSCs, we performed various sized spheroid formation and determined changes in gene expression levels to obtain different sized spheroid for cell therapy. eAD-MSCs were successfully isolated from horse tailhead. Using hanging drop method, spheroid formation was generated for three days. Quantitative real-time PCR was performed to analyze gene expression. As results, expression levels of pluripotent markers were increased depending on spheroid size and the production of PGE2 was increased in spheroid formation compared to that in monolayer. Ki-67 showed a remarkable increase in the spheroid formed with 2.0 × 105 cells/drop as compared to that in the monolayer. Expression levels of angiogenesis-inducing factors such as VEGF, IL-6, IL-8, and IL-18 were significantly increased in spheroid formation compared to those in the monolayer. Expression levels of bone morphogenesis-inducing factors such as Cox-2 and TGF-β1 were also significantly increased in spheroid formation compared to those in the monolayer. Expression levels of osteocyte-specific markers such as RUNX2, osteocalcin, and differentiation potential were also significantly increased in spheroid formation compared to those in the monolayer. Therefore, spheroid formation of eAD-MSCs through the hanging drop method can increases the expression of angiogenesis-inducing and bone morphogenesis-inducing factors under optimal culture conditions.

Immunomodulatory Effect of Mesenchymal Stem Cell-Derived Exosomes in Lipopolysaccharide-Stimulated RAW 264.7 Cells

성체줄기세포는 지방세포, 조골세포, 연골세포, 근육세포 등의 세포로 분화 가능한 다능성 세포로 조직 재생[9], 면역 조절 등 다양한 능력을 가지고 있는 것으로 알려져 있다. 이 러한 성체줄기세포는 기초 연구 분야 및 심근경색[17], 이식 편대숙주병[37], 대장염[35], 간부전[36], 신부전[23], 크론병 [33], 중추신경계 외상[15], 자가면역성 뇌척수염[47], 류마티 스성 관절염[50] 등 질병과 관련한 임상 연구 분야에서 면역 조절 기전에 대한 많은 연구들이 진행되고 있다[16]. 염증 반응은 미생물 감염, 내독소, 조직 손상과 같은 위해 성 자극을 정상적으로 회복하기 위한 필수적 방어 작용으로, 정상적인 염증 반응은 시간이 지남에 따라 염증 촉진성 매 개체(pro-inflammatory mediators)의 생성은 감소하고 항염 증성 매개체(anti-inflammatory mediators)는 증가됨으로 써 스스로 염증 반응 조절 과정을 가진다[26]. 이러한 염증 반응이 최근 암, 비만, 당뇨병 등 다양한 질환의 발병과 진 행에 있어 중요한 역할을 한다고 보고되면서 염증 반응 조 절을 통해 염증 관련 질환이 개선될 수 있는 가능성을 보 이게 되었다[19]. 이러한 염증 반응에 관여하여 중요한 역 Immunomodulatory Effect of Mesenchymal Stem Cell-Derived Exosomes in Lipopolysaccharide-Stimulated RAW 264.7 Cells Soo-Kyung Jung†, Mi Jeong Park†, Jienny Lee†, Jeong Su Byeon, Na-Yeon Gu, In-Soo Cho, and Sang-Ho Cha* Viral Disease Division, Animal and Plant Quarantine Agency, Gimcheon 39660, Republic of Korea