Mijung Lee

Low pH increases the yield of exosome isolation

Exosomes are the extracellular vesicles secreted by various cells. Exosomes mediate intercellular communication by delivering a variety of molecules between cells. Cancer cell derived exosomes seem to be related with tumor progression and metastasis. Tumor microenvironment is thought to be acidic and this low pH controls exosome physiology, leading to tumor progression. Despite the importance of microenvironmental pH on exosome, most of exosome studies have been performed without regard to pH. Therefore, the difference of exosome stability and yield of isolation by different pH need to be studied. In this research, we investigated the yield of total exosomal protein and RNA after incubation in acidic, neutral and alkaline conditioned medium. Representative exosome markers were investigated by western blot after incubation of exosomes in different pH. As a result, the concentrations of exosomal protein and nucleic acid were significantly increased after incubation in the acidic medium compared with neutral medium. The higher levels of exosome markers including CD9, CD63 and HSP70 were observed after incubation in an acidic environment. On the other hand, no exosomal protein, exosomal RNA and exosome markers have been detected after incubation in an alkaline condition. In summary, our results indicate that the acidic condition is the favorable environment for existence and isolation of exosomes.

Extracts of Adipose Derived Stem Cells Slows Progression in the R6/2 Model of Huntington's Disease

Stem cell therapy is a promising treatment for incurable disorders including Huntingtons disease (HD). Adipose-derived stem cell (ASC) is an easily available source of stem cells. Since ASCs can be differentiated into nervous stem cells, it has clinically feasible potential for neurodegenerative disease. In addition, ASCs secrete various anti-apoptotic growth factors, which improve the symptoms of disease from transplanted ASCs. Thus, cell-free extracts of ASCs (ASCs-E) could be a potential candidate for treatment of HD. Here, we investigated effects of ASCs-E on R6/2 HD mouse model and neuronal cells. In R6/2 HD model, injection of ASCs-E improved the performance in Rotarod test. ASCs-E also ameliorated striatal atrophy and mutant huntingtin aggregation in the striatum. In Western blot increased expressions of p-Akt, p-CREB and PGC1α were noted by injection of ASCs-E, when comparing to the R6/2 HD model. Neuro2A neuroblastoma cells treated with ASCs-E showed increased expression of p-CREB and PGC1α. In conclusion, ASCs-E delayed disease progression in animal model of HD by restoring of CREB-PGC1α pathway and could be a potential resource for treatment of HD.

Exosomes from adipose-derived stem cells ameliorate phenotype of Huntington's disease in vitro model

Huntingtons disease (HD) is a hereditary neurodegenerative disorder caused by the aggregation of mutant Huntingtin (mHtt). Adipose‐derived stem cells (ASCs) have a potential for use in the treatment of incurable disorders, including HD. ASCs secrete various neurotrophic factors and microvesicles, and modulate hostile microenvironments affected by disease through paracrine mechanisms. Exosomes are small vesicles that transport nucleic acid and protein between cells. Here, we investigated the therapeutic role of exosomes from ASCs (ASC‐exo) using in vitro HD model by examining pathological phenotypes of this model. Immunocytochemistry result showed that ASC‐exo significantly decreases mHtt aggregates in R6/2 mice‐derived neuronal cells. Western blot result further confirmed the reduction in mHtt aggregates level by ASC‐exo treatment. ASC‐exo up‐regulates PGC‐1, phospho‐CREB and ameliorates abnormal apoptotic protein level in an in vitro HD model. In addition, MitoSOX Red, JC‐1 and cell viability assay showed that ASC‐exo reduces mitochondrial dysfunction and cell apoptosis of in vitro HD model. These findings suggest that ASC‐exo has a therapeutic potential for treating HD by modulating representative cellular phenotypes of HD.

Adipose-derived stem cell exosomes alleviate pathology of amyotrophic lateral sclerosis in vitro

Amyotrophic lateral sclerosis (ALS) is a degenerative disorder that involves the death of motor neurons in the cortex, brain stem, and spinal cord. Adipose-derived stem cells (ADSCs) are considered as a perspective remedy for therapy of neurodegenerative diseases including ALS. Stem cells secrete various factors which can modulate a hostile environment, called paracrine effect. Exosomes are small extracellular vesicles containing cell derived factors and mediate paracrine effect of cells. Thus, exosomes from ADSCs (ADSC-exo) can be a potential candidate of therapeutic effects of stem cells. To investigate the effect of ADSC-exo on the cellular phenotypes of ALS, we used neuronal stem cells (NSCs), which can be differentiated into neuronal cells, isolated from wild type or G93A ALS mice model. ADSC-exo was treated to neuronal cells from G93A ALS mice model. Immunocytochemistry and dot-blot assay result showed that ADSC-exo alleviated aggregation of superoxide dismutase 1 (SOD1). Reduction of cytosolic SOD1 level by ADSC-exo was also confirmed by western blot. Mitochondria display various abnormalities in ALS and the decrease of phospho-CREB and PGC-1α were observed in the G93A cells. ADSC-exo treatment showed normalization of phospho-CREB/CREB ratio and PGC-1α expression level. Our results suggest that ADSC-exo modulates cellular phenotypes of ALS including SOD-1 aggregation and mitochondrial dysfunction, and can be a therapeutic candidate for ALS.

Wound healing potential of adipose tissue stem cell extract

Adipose tissue stem cells (ATSCs) are considered as a promising source in the field of cell therapy and regenerative medicine. In addition to direct cell replacement using stem cells, intercellular molecule exchange by stem cell secretory factors showed beneficial effects by reducing tissue damage and augmentation of endogenous repair. Delayed cutaneous wound healing is implicated in many conditions such as diabetes, aging, stress and alcohol consumption. However, the effects of cell-free extract of ATSCs (ATSC-Ex) containing secretome on wound healing process have not been investigated. In this study, ATSC-Ex was topically applied on the cutaneous wound and healing speed was examined. As a result, wound closure was much faster in the cell-free extract treated wound than control wound at 4, 6, 8 days after application of ATSC-Ex. Dermal fibroblast proliferation, migration and extracellular matrix (ECM) production are critical aspects of wound healing, and the effects of ATSC-Ex on human dermal fibroblast (HDF) was examined. ATSC-Ex augmented HDF proliferation in a dose-dependent manner and migration ability was enhanced by extract treatment. Representative ECM proteins, collagen type I and matrix metalloproteinase-1, are significantly up-regulated by treatment of ATSC-Ex. Our results suggest that the ATSC-Ex have improving effect of wound healing and can be the potential therapeutic candidate for cutaneous wound healing.

Influence of storage condition on exosome recovery

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.

Modulation of mitochondrial function by stem cell-derived cellular components.

Huntingtons disease (HD) is the most common hereditary neurodegenerative diseases, in which the loss of striatal neuron caused by the aggregation of mutant huntingtin protein (mHtt) is the main pathological feature. Our previous studies have demonstrated that human adipose stem cells (hASC) and its extracts can slow down the progression of HD in vitro and in vivo. hASC are readily accessible adult stem cells, and the cytosolic extracts contain a number of neurotrophic factors. Here, we further explored the role of the hASC extracts in neuronal death and mitochondrial function in HD. Our results showed that the hASC extracts prevent mHtt-induced cell toxicity and cell apoptosis. Moreover, the hASC extracts recovered mHtt-induced mitochondrial oxidative stress and reduced mitochondrial membrane potential. The hASC extracts blocked the interaction between p53 and mHtt, and decreased the endogenous p53 levels at both transcriptional and post-translational levels, resulting in the instability of p53 and increased neuronal survival. Taken together, these findings implicate protective roles of hASC extracts in mHtt-induced mitochondrial apoptosis, providing insights into the molecular mechanism of the hASC in the therapeutic strategy of HD.

Cytosolic Extract of Human Adipose Stem Cells Reverses the Amyloid Beta-Induced Mitochondrial Apoptosis via P53/Foxo3a Pathway

Human adipose stem cells (hASC) have therapeutic potential for the treatment of neurodegenerative disorders. Mitochondrial dysfunction is frequently observed in most neurodegenerative disorders, including Alzheimer’s disease. We explored the therapeutic potential of hASC cytosolic extracts to attenuate neuronal death induced by mitochondrial dysfunction in an Alzheimer’s disease (AD) in vitro models. Amyloid beta (Aβ) was used to induce cytotoxity in an immortal hippocampal cell line (HT22) and neuronal stem cells from the brain of TG2576 transgenic mice were also used to test the protective role of hASC cytosolic extracts. Cell viability and flow cytometry results demonstrated that the hASC extract prevents the toxicity and apoptosis in AD in vitro models. Moreover, JC-1 and MitoSoxRed staining followed by fluorescence microscopy and flow cytometry results showed that the hASC extract ameliorated the effect of Aβ-induced mitochondrial oxidative stress and reduced the mitochondrial membrane potential. Western blot result showed that hASC extract modulated mitochondria-associated proteins, such as Bax and Bcl2, and down-regulated cleaved caspase-3. In addition, hASC extract decreased Aβ generation and reversed up-regulated p53 and foxo3a protein level in AD in vitro model cell derived from TG2576 mice. Taken together, these findings implicate a protective role of the hASC extract in the Aβ-induced mitochondrial apoptosis via regulation of P53/foxo3a pathway, providing insight into the molecular mechanisms of hASC extract and a therapeutic strategy to ameliorate neuronal death induced by Aβ.

The exosome of adipose-derived stem cells reduces β-amyloid pathology and apoptosis of neuronal cells derived from the transgenic mouse model of Alzheimer’s disease

Adipose-derived stem cells (ADSC) have a therapeutic potential for the treatment of neurodegenerative disorders such as Alzheimers disease (AD). Exosomes are extracellular vesicles secreted from various types of cells, and stem cell-derived exosomes are known to have beneficial effects in many diseases. Many studies have suggested that amyloid beta (Aβ) peptides have a pivotal role in AD progression, by mitochondrial dysfunction of neuronal cells. We examined the therapeutic potential of exosomes derived from ADSCs (ADSC-Exo) in preventing the disease phenotypes induced by the Aβ cascade in an AD in vitro model. Neuronal stem cells (NSCs) from the brains of TG2576 AD mice were used to examine the effects of ADSC-Exo on AD phenotypes. NSCs from AD mice can be grown as a neurosphere and differentiated. Differentiated NSCs of TG2576 mice showed increase of Aβ42 and Aβ40 levels, and Aβ42/40 ratio. Apoptotic molecules such as p53, Bax and caspase-3 were increased and Bcl2, an anti-apoptotic molecule, was decreased in AD cells compared with wild-type littermate cells. Lower viable cell population and higher necrotic cells were examined in AD neuronal cells. ELISA result showed that ADSC-Exo treatment resulted in reduced Aβ42 levels, Aβ40 levels, and the Aβ42/40 ratio of AD cells. Increased apoptotic molecules, p53, Bax, pro-caspase-3 and cleaved-caspase-3, and decreased Bcl-2 protein level were normalized by ADSC-Exo treatment. Flow cytometry analysis revealed that increased cell apoptosis of AD neuronal cells was reduced by ADSC-Exo. In addition, neurite growth, which is impaired by Aβ in the brains of patients with AD, was augmented by ADSC-Exo treatment. Taken together, these findings implicate the disease-modulating effects of ADSC-Exo in the transgenic mice-derived AD in vitro model, and ADSC-Exo can be a therapeutic source to ameliorate the progression of Aβ-induced neuronal death and AD.

Adipose-derived stem cells extract has a proliferative effect on myogenic progenitors

Finding an effective method to regenerate muscle is a growing issue in the orthopedic field. Platelet-rich plasma (PRP) has recently been considered for therapeutic use due to its capacity to induce proliferation of myogenic progenitor cells (MPCs). Adipose-derived stem cells (ASCs) and its extract are regarded as a promising treatment for various disorders within the orthopedic field but their therapeutic relevance in the muscle regeneration is poorly investigated. In this study, rabbit MPCs were cultured from the supraspinatus of rabbit and characterized by myogenic markers. To investigate the paracrine effect of ASCs on MPCs, coculture experiments were performed. In order to see the anabolic effect of ASC-extracts (ASC-ex) in MPCs, cell proliferation assays were performed and compared with the PRP-added condition. Coculture experiment showed ASCs had an anabolic paracrine effect on proliferation of MPCs. PRP had a positive effect on proliferation of MPCs when compared to the control (100 ± 7.4% vs 195.2 ± 19.2%, p < 0.001); however, ASC-ex promoted greater proliferation than the PRP condition (467.3 ± 38.7%, p < 0.001 compared with PRP). Similarly, in C2C12 cells, PRP showed an increased rate when compared to the control (100 ± 5.9% vs 205.1 ± 45.4%, p < 0.001), and treatment of ASC-ex showed dramatic increase in proliferation (335.9 ± 37.8%, p < 0.001 compared with PRP). ASC-ex had positive effect on expanding MPCs of rabbit and myoblast cell line, and its capacity to induce proliferation was notably stronger than that of PRP. In conclusion, the study suggests that rabbit ASC-ex have stronger proliferative effect on MPCs than rabbit PRP. Thus, ASC-ex could be a therapeutic candidate for muscle regeneration by activation of endogenous MPCs.