Intermuscular fat is essential for enhancing the flavor and texture of cultured meat. Mesenchymal stem cells derived from intermuscular adipose tissues are a source of intermuscular fat. Therefore, as a step towards developing a platform to derive intermuscular fat from mesenchymal stem cells (MSCs) for insertion between myofibrils in cultured beef, an advanced protocol of intermuscular adipose tissue dissociation effective to the isolation of MSCs from intermuscular adipose tissues was developed in cattle. To accomplish this, physical steps were added to the enzymatic dissociation of intermuscular adipose tissues, and the MSCs were established from primary cells dissociated with physical step-free and step-added enzymatic dissociation protocols. The application of a physical step (intensive shaking up) at 5 minutes intervals during enzymatic dissociation resulted in the greatest number of primary cells derived from intermuscular adipose tissues, showed effective formation of colony forming units-fibroblasts (CFU-Fs) from the retrieved primary cells, and generated MSCs with no increase in doubling time. Thus, this protocol will contribute to the stable supply of good quality adipose-derived mesenchymal stem cells (ADMSCs) as a fat source for the production of marbled cultured beef.

Current studies have revealed the capacity of mesenchymal stem cells (MSCs) in term of immunomodulatory properties, and this distinct potential is downgraded according to the disease duration of patients-derived MSCs. In order to enhance the immunomodulatory and anti-tumorigenic properties of the rheumatoid arthritis (RA) joints-derived MSCs, we aggregate synovial fluid-derived MSCs from RA joints (RA-hMSCs) into 3D-spheroids by the use of hanging drop culture method. Cells were isolated from synovial fluids of RA joints with longstanding active status over 13 years. For aggregation of RA-hMSCs into 3D-spheroids, cells were plated in hanging drops in 30 μL of advanced DMEM (ADMEM) containing 25,000-30,000 cells/ drop and cultured for 48 h. To analyze the comparative immunomodulatory effects of 3D-spheroid and 2D monolayer cultured RA-hMSCs and then cells were cultured in ADMEM supplemented with 20% of synovial fluids of RA patients for 48 h and were evaluated by qRT-PCR for their expression of mRNA levels of inflammatory and antiinflammatory markers. Cellular aggregation of RA-hMSCs was observed and cells were aggregate into a single sphere. Following treatment of RA patient’s synovial fluids into the RA-hMSCs, spheroids formed RA-hMSCs showed significantly (p < 0.05) higher expression of TNFα stimulated gene/protein 6 (TSG-6) than the monolayer cultured RAhMSCs. Therefore, the 3D-spheroid culture methods of RA-hMSCs were more effective than 2D monolayer cultures in suppressing inflammatory response treated with 20% of RA-synovial fluids by expression of TNFα (TSG-6) according to the immune response and enhanced secretion of inflammatory factors.

Somatic cell nuclear transfer (SCNT) in pigs has been used as a very important tool to produce transgenic for the pharmaceutical protein, xenotransplantation, and disease model and basic research of cloned animals. However, the production efficiency of SCNT embryos is very low in pigs and miniature pigs. The type of donor cell is an important factor influencing the production efficiency of these cloned pigs. Here, we investigated the developmental efficiency of SCNT embryos to blastocysts and full term development using fetal fibroblasts (FF) and mesenchymal stem cells (MSCs) to identify a suitable cell type as donor cell. We isolated each MSCs and FF from the femoral region and fetus. Cultured donor cell was injected into matured embryos for cloning. After that, we transferred cloned embryos into surrogate mothers. In term of in vitro development, the SCNT embryos that used MSCs had significantly higher in cleavage rates than those of FF (81.5% vs. 72%) (p<0.05), but the blastocyst formation rates and apoptotic cell ratio was similar (15.1%, 6.18% vs. 20.8%, 9.32%). After embryo transferred to surrogates, nine and nineteen clone piglets were obtained from the MSCs and FF group, respectively, without significant differences in pregnancy and birth rate (50%, 40% vs. 52.3%, 45.4%) (p>0.05). Moreover, there was no significant difference in the corpus hemorrhagicum numbers of ovary, according to pregnancy, abortion, and delivery of surrogate mothers between MSCs and FF groups. Therefore, the MSCs and FF are useful donor cells for production of clone piglets through SCNT, and can be used as important basic data for improving the efficiency of production of transgenic clone pigs in the future.

Mesenchymal stem cells (MSCs) have been recognized as a therapeutic tool for various diseases due to its unique ability for tissue regeneration and immune regulation. However, poor survival during in vitro expansion and after being administrated in vivo limits its clinical uses. Accordingly, protocols for enhancing cell survivability is critical for establishing an efficient cell therapy is needed. CDDOMe is a synthetic C-28 methyl ester of 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid, which is known to stimulate nuclear factor erythroid 2-related factor 2 (Nrf2)- antioxidant response element (ARE) pathway. Herein, report that CDDO-Me promoted the proliferation of MSCs and increased colony forming units (CFU) numbers. No alteration in differentiation into tri-lineage mesodermal cells was found after CDDO-Me treatment. We observed that CDDO-Me treatment reduced the cell death induced by oxidative stress, demonstrated by the augment in the expression of Nrf2-downstream genes. Lastly, CDDO-Me led to the nuclear translocation of NRF2. Our data indicate that CDDO-Me can enhance the functionality of MSCs by stimulating cell survival and increasing viability under oxidative stress.

Mesenchymal stem cells (MSCs) are multipotent cells capable of replicating as undifferentiated cells, and thus hold therapeutic implications in field of regenerative medicine and reproductive biotechnology. In the present study, we compared the stem cell properties of bovine ear skin tissue (ESK)- and nasal mucosa (NM)-derived MSCs. Bovine ESK-MSCs and NM-MSCs were successfully isolated by collagenase digestion and maintained proliferative capacity during the 20 consecutive passages. Both ESK-MSCs and NM-MSCs showed similar morphology and expressed common cell surface markers (CD29, CD44, CD90, and CD105). Also, we compared differentiation potentials of bovine ESK-MSCs and NM-MSCs into osteogenic, adipogenic, and chondrogenic lineages through specific staining and quantitative real-time RT-PCR. As results, bovine ESK-MSCs and NM-MSCs could differentiate into mesodermal cell lineages. However, bovine ESK-MSCs and NM-MSCs exhibited difference in expression of differentiation-related specific markers. Specifically, NM-MSCs exhibited increased expression levels of osteocalcin, peroxisome proliferator-activated receptor gamma, and aggrecan compared to ESK-MSCs. Also, ESK-MSCs exhibited increased expression levels of collagen type I, II, and lipoprotein lipase compared to NM-MSCs. We suggest that the nasal mucosa of bovine could be used as a source of bovine MSCs.

Canine mammary tumors account for ~30% of all tumors in the female dogs and approximately 50% of the tumors are malignant. Exosomes have been the focus of great interest, as they appear to be involved in numerous important cellular processes. In this study, we examined the anti-tumor effects of canine mesenchymal stem cells-derived exosomes (MSC-exosomes) in an experimental murine mammary tumor model using canine mammary carcinoma cells, REM134. The MSC-exosomes were injected tumor site and tail vein of REM134 xenografted mice. We found that tumor size of the MSC-exosomes-treated group decreased compared to those of the only tumor group in REM134-driven tumorigenic mouse model. In addition, the MSC-exosomes-treated tumor group showed meaningfully reduced expression levels of the MMP-3, IL-1β, IL-6, and TNF-α compared to those in the tumor group. Specifically, we confirmed that the expression level of the CD133, potent cancer stem cell (CSC) markers, decreased in the MSC-exosomes-treated tumor group compared to the tumor group. This study suggests that the MSC-exosomes exhibited anti-tumor effects through downregulating CSC-related markers in the canine mammary tumor murine model. Further study is needed in the future, and we are conducting research on the detailed anti-tumor mechanism of the MSC-exosomes.

Amniotic membrane stem cells are considered as a good alternative to embryonic stem cells, but their use in clinical studies is still not common. Here, exosomes from canine amniotic membrane mesenchymal stem cells (cAmMSCexo) were used for dog sperm cryopreservation. Upon cryopreserved straws using cryoprotectant containing 0, 0.5, 1, or 2 μg/mL of cAmMSC-exo were thawed, motility and membrane integrity were analyzed. However, results showed no significant differences between the groups. We concluded that cAmMSC-exo with lower than 2 µg/mL have no effects on sperm cryopreservation, and further studies to get higher concentrations of cAmMSC-exo should be conducted for clinical application.

Mesenchymal stem cells (MSCs) have been widely used as donor cells for somatic cell nuclear transfer (SCNT) to increase the efficiency of embryo cloning. Since replicative senescence reduces the efficiency of embryo cloning in MSCs during in vitro expansion, transfection of telomerase reverse transcriptase (TERT) into MSCs has been used to suppress the replicative senescence. Here, TERT-transfected MSCs in comparison with early passage MSCs (eMSCs) and sham-transfected MSCs (sMSCs) were used to evaluate the effects of embryo cloning with SCNT in a porcine model. Cloned embryos from tMSC, eMSC, and sMSC groups were indistinguishable in their fusion rate, cleavage rate, total cell number, and gene expression levels of OCT4, SOX2 and NANOG during the blastocyst stage. The blastocyst formation rates of tMSC and sMSC groups were comparable but significantly lower than that of the eMSC group (p < 0.05). In contrast, tMSC and eMSC groups demonstrated significantly reduced apoptotic incidence (p < 0.05), and decreased BAX but increased BCL2 expression in the blastocyst stage compared to the sMSC group (p < 0.05). Therefore, MSCs transfected with telomerase reverse transcriptase do not affect the overall development of the cloned embryos in porcine SCNT, but enables to maintain embryo quality, similar to apoptotic events in SCNT embryos typically achieved by an early passage MSC. This finding offers a bioengineering strategy in improving the porcine cloned embryo quality.

Osteoarthritis is a disorder characterized by a loss of cartilage as common aging-associated disease in humans and animals. However, unlike human clinical trials, investigational studies in pet animals are constrained by a lack of interest and funds. In addition, pet owners would often prefer the lowest cost method to treat arthritis of pet animals. Here, we report the outstanding and inexpensive way to prepare chondrocytes for cartilage repair using rabbit adipose derived mesenchymal stem cells (MSCs). This study focused on the development and enhancement of pre-chondrogenic condensation under external electric fields even without additional growth factors. We found that highly compact structures were formed within 3 days in micromass cultures of rabbit MSCs under electrical stimulation (ES), showing increased COL2A1 gene expression compared with their control 3D micromass cultures and 2D monolayer cultures. We further found that ES enhanced the production of proteoglycan, a highly produced extracellular matrix component in chondrocytes. Collectively, these results provide the commercial potential of electrical stimulation driving chondrogenesis of mesenchymal stem cells for repair of cartilage, which is a budget-friendly regimen.

Electrical stimulation (ES) is known to guide the development and regeneration of many tissues. Use of low-frequency ES for therapeutic purposes has been increasing during the last decades. Mesenchymal stem cells (MSCs) represent an appealing alternative cell source for cartilage repair. There are studies that induce differentiation into cartilage cells by treating the growth factors in stem cells or altering the properties of stem cells by genetic modification. In this study, we exposed equine adipose tissue-derived MSCs (eAD-MSCs) to ES and assessed changes in the chondrogenic differentiation potential. The cells obtained from equine adipose tissue attached to culture plates and expanded in vitro. Flow cytometric analysis at third passage indicated that the cells were strongly positive for CD44, CD90, and CD105, but negative for CD13, CD34, and CD45. Next, ES was applied to eAD-MSCs cultured under condition of high-density micromass under ES of 10 V/cm, with duration of 10 ms and a frequency of 2.0 Hz for three days. Gene expression of chondrogenic markers such as collagen type II, Aggrecan, and Sox9 was analyzed at three days of ES. As a result, we observed the differentiation potential of eAD-MSCs into chondrocytes by specific ES in absence of exogenous growth factors. We also found that ES upregulated the expression of heat shock protein 70, which affects cartilage formation. This study may contribute to the differentiation of MSCs into chondrogenic lineage under specific ES condition.

Osteoarthritis occurs when the cartilage that gradually deteriorates as common aging-associated disease in humans and animals. There is no cure, but the treatments are available to manage to relieve pain through medication such as steroids. Growing interest has been focused on the role of cell-based therapies using mesenchymal stem cells (MSCs). In addition, mesenchymal stem cells can be isolated from almost adult tissues and known for their potential of becoming cartilage. Clinical and experimental studies indicate that the development of treatment using stem cells is double-edged sword involving a possibility such as tumorigenesis. This study focused on the electrical features during articular cartilage development and hypothesized that external electric fields promote pre-chondrogenic condensation without concern relating to genetic modification or exogenous factors. Here, it has been reported that exogenous direct electric fields drive pre-chondrogenic condensation which is the stage where cartilage formation begins by condensation of stem cells and cartilage cells in the microenvironment of the joint. Time-dependent observations also support the contribution of electrical stimulation (ES) to induce gradual aggregation of MSCs into highly compact structures within 3 days. Collectively, our findings provide the potential of electrical stimulation-driven chondrogenesis of mesenchymal stem cells in the absence of exogenous factors for repair of cartilage defects.

Herbal medicine has been the basis for medical treatments through much of human history, and such traditional medicine is still widely practiced today. Modern medicine makes use of many plant-derived compounds as the basis for pharmaceutical drugs. In traditionally, Achyranthes aspera, Safflower (Carthamus tinctorius) seed and Acanthopanax senticosus have been used for the treatment and prevention of bone-related diseases. In this study, we investigated the pharmacological effect of mixture of Achyranthes aspera, Safflower (Carthamus tinctorius) seed and Acanthopanax senticosus and the other herbs. Two types of enzymes were used to enhance the extraction components of amino acid, mineral content, free sugar, and flavor recovery in extracting natural herbal mixtures(NME). We evaluated regulation of osteogenic differentiation in human bone marrow mesenchymal stem cells using alkaline phosphatase staining, alizarin red S staining and RT-PCR. The CCK-8 assay indicated that NME had no cytotoxicity but increased cell survival. In addition, NME promoted the mineralization and expression of osteogenic differention marker genes in human bone marrow mesenchymal stem cells. Therefore, NME has an effect of promoting proliferation and osteogenic differentiation of human mesenchymal stem cell.

Mesenchymal stem cells (MSCs) are capable of differentiating into mesenchymal tissue such as bone, cartilage, muscle, and adipose, and have been isolated and characterized from various species. Deer adipose tissue-derived MSCs (dAD-MSCs) have not been studied and deer bone marrow-derived MSCs (dBM-MSCs) have not been fully characterized. In this study, we firstly isolated MSCs from deer tissues and then compared characteristics of dAD-MSCs and dBM-MSCs. dAD-MSCs and dBM-MSCs exhibited significant increase in proliferation under low-glucose DMEM culture condition during 20 and 10 passages consecutive passages, respectively. Both cells expressed cell surface markers such as CD73, CD90, and CD105, but did not express CD34 and CD45. Two types of cells expressed stemness markers (Oct4, Sox2, and Nanog) and exhibited differentiation potential into mesodermal lineages. Both cells exhibited osteogenic and chondrogenic differentiation potential, but poor adipogenic differentiation potential. Specifically, dAD-MSCs have a greater capacity for chondrogenic differentiation potential compared to dBM-MSCs. Collectively, we successfully isolated dAD-MSCs from deer for the first time. This study suggests that adipose tissue of deer could be used as a source of deer MSCs.

Mesenchymal stem cells (MSCs) are multipotent cells capable of replicating as undifferentiated cells, and have the potential of differentiating into mesodermal lineages. Goats are commonly used as animal models for bone tissue engineering to test the potential of stem cells for bone regeneration. Goat MSCs isolated from bone marrow (BM) or adipose tissue (AD) should be evaluated using in vitro assays, prior to their application in a tissue engineering project. In this study, we compared the stem cell properties of MSCs derived from goat AD, BM and ear skin tissue (ESK). As results, BM and ESK-MSCs exhibited a spindle-shaped morphology comparable to that of AD-MSCs. Especially, BM-MSCs could be cultured for significantly longer periods and exhibited the greatest expansion capacity, whereas AD-MSCs had the shortest culture time and lowest growth rate. Also, we compared differentiation potentials of AD, BM and ESK-MSCs into adipogenic, chondrogenic, and osteogenic lineages through specific staining and quantitative real-time RT-PCR. Collectively, we successfully isolated ESK-MSCs from goat for the first time. This study suggests that adult skin tissue of goat could be used as a source of goat MSCs. Further studies are needed to show the more information for establishment and fully characterization of goat ESK-MSCs.

본 연구는 사람의 다양한 세포주를 이용하여 활성산소종(과산화수소수)이 세포의 노화에 미치는 영향을 비교 조사하였다. 여러 농도의 과산화수소수에 세포주를 일주일 동안 배양하여 MTT 방법으로 과산화수소수에 대한 세포 성장의 반억제농도를 구하였다. 그 결과, 50대에서 유래하는 피부 섬유아세포와 10대의 노화 유도 피부 섬유아세포와 비교하여 10대에서 유래하는 피부 섬유아세포에서 과산화수소수에 대한 반억제농도의 값이 유의적으로 더 높았고, 10대의 피부 섬유아세포보다는 10대의 여러 조직 기원하는 성체줄기세포에서 반억제농도의 값이 유의적으로 더 높게 관찰되었다. 또한, 50 ppm 과산화수소수를 1주일 동안 처리한 후, 50대의 피부 섬유아세포에서 다른 세포주에 비해 세포 성장이 현저히 억제되었고, 노화 관련 베타-갈락토시다아제의 활성이 증가되는 것을 관찰하였다. 또한, 활성산소의 세포 독성을 중화시키는 두 유전자, 글루타티온 과산화효소(GPX)와 카탈라아제(CAT)의 발현을 각 세포주에서 조사하였을 때, CAT의 발현은 모든 세포주에서 대체로 낮았지만, GPX 유전자의 발현이 50 대의 피부 섬유아세포보다 10대의 피부 섬유아세포와 성체줄기세포에서 현저히 높게 발현되는 것을 관찰하였다. 이상의 결과에서 활성산소는 세포 노화를 유도하고, GPX의 발현이 높은 10대의 피부 섬유아세포와 줄기세포보다는 50대의 피부 섬유아세포와 노화된 피부 섬유아세포에서 활성산소종에 대해 더 큰 민감성을 가지고 있는 것을 알 수 있었다.

The objective of this study was to identify the proteins actively involved in the protection and repair of damaged cells, secreted by canine adipose derived mesenchymal stem cells (AT-MSCs) into the conditioned media. For this purpose, conditioned media (CM) was recovered from passage three stage canine AT-MSCs and skin fibroblasts cultured in serum free media after 24, 48 and 72 h. The extraction of exosomes was performed from 10-20 ml of CM using total exosome isolation kit. The isolated exosomes were then subjected to western analysis for the identification of annexin-I, annexin-II, histone H3 and dysferlin proteins. Results demonstrated the expression of proteins in the conditioned media isolated from canine AT-MSCs reflecting their potential in reducing the extent of damage at cellular levels. In conclusion, the conditioned media derived from canine AT-MSCs can be helpful in restoring the normal structure of cells both in vivo and in vitro conditions.

일반적으로 세포·조직 및 장기이식 성공 예측은 수여자와 공여자간의 백혈구항원 일치도이고, 불일치 시 심각한 거부 반응을 유발함으로 세포치료제로 사용할 때 우선적으로 백혈구 항원일치도가 고려된다. 그러나 중간엽줄기세포(Mesenchymal Stem Cells, MSCs)는 다른 체세포와 비교하여 상대적으로 낮은 MHC I 항원발현과, 극히 낮은 MHC II 항원을 가지고 있으므로 동종세포치료제로서 주목을 받고 있다. 따라서 본 연구에서는 개 모델에서 MSCs 의 동종세포치료제로서 효능을 예측하기 위해 선행연구로 백혈구 항원(Dog Leukocyte Antigen, DLA)형 및 가계도내 일치도와 유전적다형성(Polymorphism) 을 분석하였다. DLA 분석을 위해 한가계도의 비글(Covance Beagles) 4 두(모견 1 두, 자견 3 두)로 부터 전혈을 채취하고, 밀도구배를 이용하여 백혈구만을 분리 후 DNA들을 각각 추출하였다. DLA 분석은 ClassII 유전자(DLA-DQA, DLA-DQB, DLA-DRB)에서 엑손 2 영역(약 300bp)을 증폭하고 Direct Sequencing 을 통해 밝혀진 염기서열을 NCBI Blast 와 IPD(Immuno Polymorphism Database)를 기반으로 하여 Universal nomenclature 에 따라 유전자형을 판독 하였다. 그 결과 DLA-DQA(022:01/022:01)와 DLA-DQB(107:01/102:01)는 4마리 모두 유전자형이 동일하였으나, DLA-DQB 는 각각 046:01/022:02, 03701/022:02, 00201/022:02, 03701/022:02 로 차이를 보였다. 이 결과를 통해 모견과 자견이 공통적으로 가지는 일배체형(Haplotype)은 DLA–DQA*022:01, DLA-DQB*022:02, DLA-DRB*102:01 이었음을 확인할 수 있었다. 그리고 일부 유전자의 염기서열에서 99% 유사도를 보이는 후보군들이 4 개씩 검색되었는데 이는 단일염기다형성(SNP)에 기인한 유전적다형성(Polymorphism)이 매우 높다는 선행보고들과 유사한 결과를 보였다. 본 실험결과는 향후 DLA 의 일치군과 비 일치군의 개중간엽줄기세포와 말초혈액단핵구세포(PBMC)들의 공배양을 통해 동종세포치료제 연구에 사용될 예정이다. * 본 성과물은 농촌진흥청 반려동물 연구사업(세부과제명 : 반려견에서 DLA 일치하는 줄기세포의 체외 치료능 평가, 세부과제 번호 : PJ013957022018)의 지원에 의해 이루어짐.

Induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (iMSCs) serve as a unique source for cell therapy. We investigated whether exosomes from iMSCs promote the proliferation of human keratinocytes (HaCaT) and human dermal fibroblasts (HDFs). iPSCs were established from human Wharton’s jelly MSCs and were allowed to differentiate into iMSCs. Exosomes were collected from the culture supernatant of MSCs (MSC-exo) and iMSCs (iMSC-exo), and their characteristics were investigated. Both exosome types possessed basic characteristics of exosomes and were taken up by skin cells in vitro and in vivo. A significant increase in HaCaT proliferation was observed with iMSC-exo, although both exosomes increased the viability and cell cycle progression in HaCaT and HDFs. No significant difference was observed in the closure of wound scratch and the expression of reparative genes between cells treated with the two exosome types. Both exosomes enhanced the secretion of collagen in HaCaT and HDFs; however, an increase in fibronectin level was observed only in HaCaT, and this effect was better with iMSC-exo treatment. Only iMSC-exo increased the phosphorylation of extracellular signal-regulated kinase (ERK)-1/2. Our results indicate that iMSC-exo promote the proliferation of skin cells by stimulating ERK1/2 and highlight the application of iMSCs for producing exosomes.

Stem cells have special properties, such as self-renewal, proliferation, and the multilineage differentiation. Generally, stem cells are categorized into embryonic stem cells (ESCs), adult stem cells (ASCs), and induced pluripotent stem cells (iPSCs). Mesenchymal stem cells (MSCs) are a type of ASCs with a multipotent property. MSCs are easily isolated from various tissues and organs in the human body and can differentiation into multiple lineages, such as bone, cartilage, fat, and muscles. Compared to ESCs and iPSCs, MSCs possess less proliferation and differentiation capacities, therefore, a much scientific concern is concerned toward promoting the proliferation and the differentiation potency of MSCs. There are various methods to achieve this goal such as the treatment of various types of small molecules or culturing on specific peptides. Producing of high-quality MSCs with enhanced proliferation and differentiation capacities will definitely be a useful tool for stem cell-mediated tissue regeneration and the further clinical application.