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.

Stem cell therapy is undoubtedly the most promising therapeutic approach for neurological disorders. Adipose tissue is ubiquitous and it can be easily harvested in large quantities under local anesthesia with little patient discomfort, making adipose tissue into the ideal large-scale source for research on clinical applications. In this study we monitored the neuronal cell differentiation potential of human adipocyte in the following condition; i) N2 medium containing 200 uM ascorbic acid (AA) and/or 10 uM flavonoid (F) and ⅱ) N2 medium containing AA and/or 10 ng/ml brain derived neurotrophic factor (BDNF) and/or, 200 ng/ml sonic hedgehog (SHH) plus 100 ng/ml fibroblast growth factor (FGF) 8. Adipose stem cells were cultured in above described differentiation condition for three weeks. RT-PCR analysis demonstrated that the mRNA levels of neuronal cell markers in differentiated adipose stem cells. Under the culture condition using N2 medium containing AA, the expression level of nestin (neural progenitor marker) m- RNA was high in all groups, while those of Neuro D, and LEP and FABP4 (adipocyte marker) mRNA were significantly decreased. Also, the addition of BDNF or SHH+FGF8 in N2 medium containing AA enhanced the neural cell differentiation from adipose stem cells, the expression level of Map2 (mature neuron) mRNA was increased, and that of TH (dopaminergic neuron marker) mRNA was high. In addition, we confirmed that the flavonoid addition has effect on the increase of Map2 expression. These results demonstrate that our designed culture condition has effect on the neural cell differentiation of adipose stem cells and this stimulatory effect may be further enhanced by transplantation.

While adipose-derived stem cell-conditioned medium (ADSC-CM) has been demonstrated to promote skin wound healing, the mechanism regulating this effect remains unelucidated. In this study, we aimed to investigate the role of Ell3 in the wound healing activity of ADSC-CM. In vitro analysis revealed that Ell3 suppression in ADSCs impairs the promotive activity of ADSC-CM on the proliferation and migration of mouse embryonic fibroblasts (MEF) and normal human dermal fibroblasts (NHDF). Consistently, the expression of MMP family genes, which regulate cell proliferation and migration, was significantly suppressed in MEF and NHDF treated with siEll3-transfected ADSC-CM. Proinflammatory cytokines, such as interleukin-1 and interleukin-6, were highly expressed in MEF treated with siEll3-transfected ADSC-CM. The wound healing activity of siEll3-transfected ADSC-CM was significantly lower than that of the control in vivo. Our results suggest that Ell3 may contribute to the inhibition of inflammatory response during skin wound healing.

Human adult stem cells have widely been examined for their clinical application including their wound healing effect in vivo. To function as therapeutic cells, however, cells must represent the ability of directed migration in response to signals. This study aimed to investigate the mechanism of platelet-derived growth factor (PDGF)-induced migration of the human abdominal adipose-derived stem cells (hADSCs) in vitro. A general matrix metalloproteinase (MMP) inhibitor or a MMP2 inhibitor significantly inhibited the PDGF-induced migration. PDGF treatment exhibited greater mRNA level and denser protein level of MMP1. The conditioned medium of PDGF-treated cells showed a caseinolytic activity of MMP1. Transfection of cells with siRNA against MMP1 significantly inhibited MMP1 expression, its caseinolytic activity, and cell migration following PDGF treatment. Phosphatidylinositol 3-kinase (PI3K) inhibitor reduced the migration by about 50% without affecting ERK and MLC proteins. Rho-associated protein kinase inhibitor mostly abolished the migration and MLC proteins. The results suggest that PDGF might signal hADSCs through PI3K, and MMP1 activity could play an important role in this PDGF-induced migration in vitro.

Previously we observed that human adipose-derived stem cells (hADSCs) could form aggregation during culture in the presence of human serum (HS). In the present study, we have examined if the aggregation might result from the cell migration and analyzed the difference of cell adhesivity after culture in various conditions. When cells were cultured in fetal bovine serum (FBS) alone, there was no morphological change. Similarly, cells pretreated with FBS for 1 day or cultured in a mixture of FBS and HS showed little change. In contrast, cells cultured in HS alone exhibited formation of cell-free area (spacing) and/or cell aggregation. When cells cultured in FBS or pretreated with FBS were treated with 0.06% trypsin, almost cells remained attached to the dish surfaces. In contrast, when cells cultured in HS alone were examined, most cells detached from the dish by the same treatment. Treatment of cells with forskolin, isobutylmethyl xanthine (IBMX) or LY294002 inhibited the formation of spacing whereas H89 or Y27632 showed little effect. When these cells were treated with 0.06% trypsin after culture, most cells detached from the dishes as cells cultured in HS alone did. However, cells treated with IBMX exhibited weaker adhesivity than HS alone. Based on these observations, it is suggested that HS treatment might decrease the adhesivity and induce three-dimensional migration of hADSCs, in the latter of which cAMP signaling could be involved.

Black soybean teata is helpful to preventing obesity through enhancing energy expenditure and suppressing accumulation in mesenteric adipose tissue. The ethanol testa-extract of Cheongja #3 black soybean (ETCBS) is also have similar effects on obesity. So far, it is not clear whether the ethanol testa extract of black soybean can have effect on the characters of subcutaneous adipose stem cells such as proliferation, activity, and adipogenicity. The doubling time was different between subcutaneous adipose-derived stem (ADS) and visceral ADS cells. By the in vitro culture and passage, the doubling time was increased both of them. The shape was not different between groups and their passages were not cause the change of shapes. In the case of visceral ADS cells, the doubling time was 62.3 h or 40.3 h in control or high fat diet administrated mice, respectively, but not modified in subcutaneous ADS cells. ETCBS administration caused of increased the doubling time from 62.3 h to 84.2 h. ETCBS had suppressive effects on the cellular activity of subcutaneous ADS cells. The intensity of Oil Red O staining was very faint in 100 and 200 mg/mL ETCBS treated groups. The amounts of accumulated triglyceride were also significantly low in 100 and 200 mg/mL treated groups. From these results we know that the doubling times and the effects of ETCBS are different by the anatomical origin of ADS cells. It also suggested that ETCBS may suppress the differentiation of subcutaneous ADS cells into the precursors and maturing of adipocytes.

Previously we have shown that human abdominal adipose derived-stem cells (ADSCs) could aggregate during the high-density culture in the presence of human serum (HS). In the present study, we observed that human cord blood serum (CBS) and follicular fluid (HFF) also induced aggregation. Similarly, porcine serum could induce aggregation whereas bovine and sheep sera induced little aggregation. qRT-PCR analyses demonstrated that, compared to FBS-cultured ADSCs, HScultured cells exhibited higher level of mRNA expression of CLDN3, -6, -7, -15, and -16 genes among the tight junction proteins. ADSCs examined at the time of aggregation by culture with HS, BSA, HFF, CBS, or porcine serum showed significantly higher level of mRNA expression of JAM2 among JAM family members. In contrast, cells cultured in FBS, bovine serum or sheep serum, showed lower level of JAM2 expression. Immunocytochemical analyses demonstrated that the aggregates of HS-cultured cells (HS-Agg) showed intense staining against the anti-JAM2 antibody whereas neither nonaggregated cells (HS-Ex) nor FBS-cultured cells exhibited weak staining. Western blot results showed that HS-Agg expressed JAM2 protein more prominently than HS-Ex and FBS-cultured cells, both of latter reveled weaker intensity. These results suggest that the aggregation property of ADSCs during high-density culture would be dependent on the specific components of serum, and that JAM2 molecule could play a role in the animal sera-induced aggregation in vitro.

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.

Fetal bovine serum (FBS) is the most frequently used serum for the cultivation of mammalian cells. However, since animal-derived materials might not be appropriate due to safety issues, allogeneic human serum (HS) has been used to replace FBS, particularly for the culture of human cells. While there has been a debate about the advantages of HS, its precise effect on human adult stem cells have not been clarified. The present study aimed to investigate the effect of HS on the human eyelid adipose stem cells (HEACs) in vitro. When HEACs were cultivated in a medium containing 10% HS, many cells moved into several spots and aggregated there. The phenomenon was observed as early as 9 days following 10% HS treatment, and 12 days following 5% HS plus 5% FBS treatment. However, the aggregation was never observed when the same cells were cultivated with 10% FBS or bovine serum albumin. To examine whether cell density might affect the aggregation, cells were seeded with different densities on 12-well dish. Until the beginning of aggregation, cells seeded at low densities exhibited the longest culture period of 16 days whereas cells seeded at high densities showed the shortest period of 9 days to form aggregation. The number of cells was as the least for the low density group, and as the greatest for the high density group. When human cord blood serum or normal bovine serum was examined for the same effect on HEACs, interestingly, cord blood serum induced the aggregation of cells whereas bovine serum treatment has never induced. When cells were cultivated with 10% HS for 9 days, they were obtained and analyzed by RT-PCR. Compared to FBS-cultivated HEACs, HS-cultivated HEACs did not express VIM, and less expressed GATA4, PALLD. On the other hand, HS-cultivated HEACs expressed MAP2 more than FBS-cultivated HEACs. In conclusion, human adult stem cells could move and form aggregates by the treatment with human body fluids.

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.

Mesenchymal stem cells (MSC) are of great interest for cell-based therapies and tissue engineering approaches, as these cells are capable for extensive self-renewal and display a multilineage differentiation potential. Clinical application of these cells for degenerative and age-related diseases has been accumulating. However, preparation of MSC before the onset of the diseases, it needs to develop the cryopreservation method. Most cryopreservation methods include fetal bovine serum (FBS) which is essential for effective cryopreservation. Yet it should not be used clinically because of the potential risk of infection. In the present study, we investigated whether human serum albumin (HSA), human serum (HS), and knockout serum replacement (KSR) can be used as an alternative of FBS for cryopreservation of human adipose derived stem cells (hADSC). Cells cryopreserved with 9% HSA showed much higher viability after thawing compared with cells frozen with 5% or 1% HSA. Cells cryopreserved with 90% HS or KSR exhibited greater viability than cells frozen with 25% and 5% HS or KSR, respectively. Viability of cells frozen with 9% HSA, 90% HS or 90% KSR was comparable to that with 90% FBS. Morphology and proliferation ability of these cells were not affected by cryopreservation when compared the freshly obtained cells. Cryopreserved hADSC expressed transcription factor genes including Oct3/4, Nanog, Nestin and Sox2, which are related to the self-renewal of stem cells. Flow cytometric analyses showed that both fresh and cryopreserved hADSC were positive for the antigens of HLA-ABC, CD44, CD73, CD90, and CD105, CD166, and negative for HLA-DR, CD31, and CD34. Similar to fresh cells, cryopreserved hADSC could differentiate into mesodermal lineages, adipogenic, osteogenic, or chondrogenic cells. These results suggest that 9% HSA, 90% HS or 90% KSR can be used to replace FBS during successful cryopreservation of hADSC.

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