Background: Pluripotent stem cells (PSCs) are capable of differencing into various cell types in the body, providing them valuable for therapy of degenerative diseases. Patientspecific treatments using PSCs, such as mesenchymal stem cells in patient’s own body, may reduce the risk of immune rejection. Inducing the differentiation of PSCs into vascular endothelial cells (ECs) altering culture conditions or using specific growth factors is able to applied to the treatment of vascular diseases. The purpose of this study was to induce the differentiation of porcine epiblast stem cells (pEpiSCs), bone marrow-derived mesenchymal stem cells (pBM-MSCs) and adipose-derived mesenchymal stem cells (pAMSCs) into ECs and then examine the functionality of vascular ECs. Methods: Porcine pEpiSCs, pBM-MSCs and pA-MSCs were induced to differentiate into ECs on matrigel-coated plates in differentiation medium (EBM-2 + 50 ng/mL of VEGF) for 8 days. Cells differentiated from these stem cells were isolated using CD-31 positive (+) magnetic-activated cell sorting (MACS) and then proliferated in M199 medium. Evaluation of ECs differentiated from these stem cells was treated with capillary-like structure formation and three-dimensional spheroid sprouting assay. Results: Porcine pEpiSCs, pBM-MSCs and pA-MSCs showed similar expression of pluripotency-related genes (OCT-3/4. NANOG, SOX2). These stem cells were differentiated into vascular ECs, but showed different morphologies after the differentiation. Cells differentiated from pEpiSCs showed an elongated spindle-like morphology, whereas cells differentiated from pBM-MSCs showed a round pebble-like morphology. In the case of pA-MSCs, these two morphologies were mixed with each other. Additionally, vascular ECs differentiated from these stem cells showed different formation of capillary-like structure formation and three-dimensional spheroid sprouting assay. Conclusions: Cells differentiated from pEpiSCs, pBM-MSCs and pA-MSCs presented the functionality of different vascular ECs, demonstrating the potential of the excellent ECs differentiated from pEpiSCs.

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.

Mesenchymal stem cells (MSCs) are an attractive source for cell therapy, as they have the potential for differentiation into multi-lineage cells. Adipose tissue is a safe source due to its easy extraction and abundant resource, with minimal risk to the organ donor. In this study, we attempted to correlate the harvest yield and resulting multipotency of feline adipose tissue-derived mesenchymal stem cells (fAD-MSCs) in accordance with processing time. fAD-MSCs were individually isolated from the abdominal adipose tissues of 6 felines. They were divided into two groups, based on their processing times – Group 1: 0~1 day after adipose tissue harvesting; Group 2: more than 3 days after adipose tissue harvesting. In both groups, the proliferation capacity was analyzed using the cumulative population doubling level (CPDL) calculation assay. The expression levels of MSC-specific markers and differentiation potentials into mesodermal cell lineages were also evaluated. We observed that fAD-MSC isolation yields and CPDL were excellent in Group 1 compared with Group 2. We also found that the differentiation potential-specific genes (ACAN and OPN) were strongly expressed in Group 1 compared with Group 2. These results suggest that for the clinical treatments of feline diseases, fAD-MSCs should be isolated within 1 day after adipose tissue harvesting.

Bone fractures are most often seen in racetrack horses because of the high level of intensity in racing. These issues are the main cause of decreased performance in racehorses. Mesenchymal stem cells (MSCs) have been explored to improve intra-articular therapy in racehorses. MSCs are essential for the repair and regeneration of damaged tissues. In this study, the effect of intra-articular injection of MSCs in racehorses was investigated. Before accessing the MSC therapy, synovial fluids were obtained from the fracture site of racehorses, and adipose tissue was collected for MSC isolation. Using the MSC specific marker, adipose tissue-derived MSCs were identified. The racehorses received intra-articular injection of autologous MSCs (or allogeneic) (3 × 107 cells/3 mL). After 1 or 2 weeks, synovial fluids were collected from racehorses. To test the effect of MSC injection using ELISA, we analyzed inflammatory factors from the untreated samples compared to MSC-treated samples of racehorses. The level of pro-inflammatory factors (interleukin-1β and prostaglandin E2) was significantly decreased in synovial fluids of MSC-injected racehorses, compared to before accessing the MSC therapy, whereas, the level of anti-inflammatory factor (interleukin-10) was higher than prior to accessing the MSC therapy. Further studies are needed to investigate the anti-inflammatory mechanism of MSC in racehorses.

Biological resources including proteins, cells, and tissues were confronted with both safe and stable preservation for practical use in biotechnological industry. Particularly, cell therapy for regenerative engineering is needed to restricted regulation and accurate preservation. Therefore, this study was investigated improved conditions of mesenchymal stem cells from human umbilical cord (hUCs) or aspirated adipose tissues (hATs) for clinical cell banks. Both cells were isolated according to standard operation procedure of Hurim BioCell Inc. and analyzed the inherent characteristics in passage 4. To compare the ability of experimental groups after cryopreservation, proliferation ability using calculated values and cytomorphological patterns of each experimental step were analyzed. Also proteins such as ice-binding protein or caspase inhibitor were applied to add the preservation medium of hUCs or hATs. Result of preservation solution with 20% serum was considered a positive group. Recovery rate and expansion results showed specific dosage and cell type-dependent differences in the experimental group. Chromosomal stability and multipotency of hUCs or hATs were expressed stable pattern after cryopreservation using advanced medium. As a result, these additives could be substituted for xenogenic sources in banking of hUCs or hATs.

Human eyelid adipose-derived stem cells (hEAs) and amniotic mesenchymal stem cells (hAMs) are very valuable sources for the cell therapeutics. Both types of cells have a great proliferating ability in vitro and a multipotency to differentiate into adipocytes, osteoblasts and chondrocytes. In the present study, we evaluated their stem cell characteristics after long-time cryopreservation for 6, 12 and 24 months. When frozen-thawed cells were cultivated in vitro, their cumulative cell number and doubling time were similar to freshly prepared cells. Also they expressed stem cell-related genes of SCF, NANOG, OCT4, and TERT, ectoderm-related genes of NCAM and FGF5, mesoderm/endoderm-related genes of CK18 and VIM, and immune-related genes of HLA-ABC and 2M. Following differentiation culture in appropriate culture media for 2-3 weeks, both types of cells exhibited well differentiation into adipocyte, osteoblast, and chondrocyte, as revealed by adipogenic, osteogenic or chondrogenic-specific staining and related genes, respectively. In conclusion, even after long-term storage hEAs and hAMs could maintain their stem cell characteristics, suggesting that they might be suitable for clinical application based on stem cell therapy.

본 실험은 transforming growth factor-(TGF-)이 첨가된 chondrogenic induction medium(CIM)을 이용하여 인간지방조직에서 유래된 중간엽 줄기세포(human adipose tissue-derived mesenchymal stem cells, ATMSCs)의 연골형성능과 gelatin-chondroitin-glucosamine scaffold(GCG-scaffold)에 접종시킨 ATMSCs의 연골형성능을 알아

본 실험은 bioceramic을 첨가하여 만든 다공성 poly D,L-lactic-co-glycolic acid(PLGA)-scaffold가 인간 지방조직에서 유래된 중간엽 줄기세포(human adipose tissue derived mesenchymal stem cells, ATMSCs)의 골 형성과정에 효과적인지를 알아보고자 수행하였다. ATMSCs를 well plate에 접종하여 골형성 유도(osteogenic induction, OI) 배양액으로

Mesenchymal stem cells (MSCs) has been reported as multipotent progenitor cells that can be expanded rapidly in vitro and differentiated into multiple mesodermal cell type. Human MSCs have been reported to be associated with neural differentiation especially in the cholinergic phenotype in several neural system. In this study, We investigated the ability of MSCs derived human aipose tissue to differentiation into neural cells expressing Islet-1 and further differentiates into cholinergic neurons in cholinergic differentiation media. Immunocytochemistry was performed to detect the expression of Islet-1 and demonstrate characteristic of neurons and cholinergic neurons. Islet-1 was massively detected in the induction stage. Following cholinergic differentiation from Islet-1-expressing MSCs, Cholinergic neuron marker ChAT was higly expressed. Also we examined the neuroprotective effects and neural differentiation of transplanted human adipose tissue-derived mesenchymal stem cells (AT-MSCs) in ischemic stroke. For transplantation, after 3days after MCAO. animal were divided into 2 group: Group A : injected phosphate buffered saline (PBS;5 ㎕ n=10), Group B: transplanted AT-MSCs (5×105 cells, n=10). Each animal received an injection into the right penumbra region (from bregma : AP;－1.3 ㎜, ML;－4.0 ㎜, DV;－5.9 ㎜). In all animals, behavior test were performed at 1, 3, 6, 9, 12, 15 days after MCAO, that was conducted by investigators who were blined to the experimental groups. mNSS test demonstrated that motor, sensory, and balance behavior were impaired after MCAO ischemic insult. Ischemic rats that received AT-MSCs exhibited significantly improved functional performance compared with PBS injected animals and histological analysis revealed that transplanted AT-MSCs expressed marker for neuron. These results suggest that AT-MSCs can be differentiated into neuron especially in cholinergic neuron and may be a potential source of treatment for neurodegenerative disease such as stroke.

Mesenchymal stem cells constitute an potential cellular source to promote brain regeneration with Parkinson's disease. Mesenchymal stem cells have significant advantages over other stem cell types and greater potential for immediate clinical application. The purpose of this study was to investigate whether hMSCs from the human adipose tissue could be induced to differentiate into dopaminergic cells and to assess the developmental potential of hMSC for selectively replacing the midbrain dopamine neurons lost in Parkinson's disease in vitro and in vivo. MSCs were cultured under conditions that promote differentiation of dopaminergic neuron. Using media that include SHH, FGF8, and GDNF. the MSCs were induced in vitro to become dopaminergic neurons. The expressions of the LIM homeobox transcription factor 1, alpha (Lmx1a), tyrosine hydroxylase(TH) proteins were determined by immunofluorescence. Lmx1a has been shown sufficient to confer neurogenic activity on mesencephalic floor plate cells and to determine a mesencephalic dopaminergic neurons fate. This result suggests that hMSCs have the ability to differfentiate into dopaminergic neurons. hMSCs were then transplanted into the striatal in a rat model of Parkinson's disease. The rats were unilaterally lesioned in the substantia nigra with 6-hydroxydopamine and were tested for rotational apomorphine-induced behavior. Following differentiation of dopaminergic neuron, cells displayed dopaminergic morphology and that they expressed dopaminergic marks genes. Finally transplantation of hMSCs into the striatal of Parkinsonian rats resulted in improvement of their behavioral deficits by apomorphine-induced rotational behavior. The hMSCs transplanted rats were proved to be better than compared with the transplantation of PBS. Immunohistochemical analysis of grafted brains revealed that abundant hMSCs survived from the grafts and some of them displayed dopaminergic marks. Our results indicate that hMSC may serve as a good cell source for the treatment of neurodegenerative diseases and have high potential for being used in multiple applications. This cellular approach might become a restorative therapy in Parkinson's disease.

최근 골수와 혈액으로 유래된 중간엽 줄기세포와 비슷한 능력을 가지는 것으로 알려진 지방 유래 중간엽줄기세포가 새로운 세포 치료제로 떠오르고 있다. 하지만 줄기세포를 이용하여 치료하려는 질병은 나이가 들어감에 따라 발병하는 퇴행성 질환들이 대부분인데, 노화가 진행됨에 따라 줄기세포의 능력이 차이가 있다고 알려져 있다. 이에 본 연구에서는 노화가 일어남에 따라 발생되는 신경성 질환을 자가 유래 지방 중간엽 줄기세포를 이용하여 치료함에 있어서 노화가 진행됨에

손상된 뇌신경조직내에서 신경줄기세포로부터 새로운 신경세포로의 분화가 상당히 제한되어 있어 이것이 손상된 뇌신경조직의 복구가 잘 이루어지지 않는 원인이라 여겨지고 있다. 본 연구에서는 세포배양을 통해 지방조직 중간엽 줄기세포를 도파민성 신경세포와 콜린성 신경세포로 분화를 유도하였다. 중간엽 줄기세포를 신경세포로 분화시키기 위해 N2배양액에 bFGF, EGF, dimethyl sulphoxide (DMSO)와 butylated hydroxyanisole (