Background: Neuropathic pain (NP) is considered a clinically incurable condition despite various treatment options due to its diverse causes and complicated disease mechanisms. Since the early 2000s, multipotent human mesenchymal stem cells (hMSCs) have been used in the treatment of NP in animal models. However, the effects of hMSC injections have not been studied in chronic post-ischemia pain (CPIP) mice models. Here, we investigated whether intrathecal (IT) and intrapaw (IP) injections of hMSCs can reduce mechanical allodynia in CPIP model mice. Methods: Seventeen CPIP C57/BL6 mice were selected and randomized into four groups: IT sham (n = 4), IT stem (n = 5), IP sham (n = 4), and IP stem (n = 4). Mice in the IT sham and IT stem groups received an injection of 5 μL saline and 2 × 104 hMSCs, respectively, while mice in the IP sham and IP stem groups received an injection of 5 μL saline and 2 × 105 hMSCs, respectively. Mechanical allodynia was assessed using von Frey filaments from pre-injection to 30 days post-injection. Glial fibrillary acidic protein (GFAP) expression in the spinal cord and dorsal root ganglia were also evaluated. Results: IT and IP injections of hMSCs improved mechanical allodynia. GFAP expression was decreased on day 25 post-injection compared with the sham group. Injections of hMSCs improved allodynia and GFAP expression was decreased compared with the sham group. Conclusions: These results suggested that hMSCs may be also another treatment modality in NP model by ischemia-reperfusion.
Yi, TacGhee;Lee, Hyun-Joo;Cho, Yun-Kyoung;Jeon, Myung-Shin;Song, Sun U.
IMMUNE NETWORK
/
v.14
no.1
/
pp.54-65
/
2014
Bone marrow-derived mesenchymal stem cells (MSCs) are multipotent, with the ability to differentiate into different cell types. Additionally, the immunomodulatory activity of MSCs can downregulate inflammatory responses. The use of MSCs to repair injured tissues and treat inflammation, including in neuroimmune diseases, has been extensively explored. Although MSCs have emerged as a promising resource for the treatment of neuroimmune diseases, attempts to define the molecular properties of MSCs have been limited by the heterogeneity of MSC populations. We recently developed a new method, the subfractionation culturing method, to isolate homogeneous human clonal MSCs (hcMSCs). The hcMSCs were able to differentiate into fat, cartilage, bone, neuroglia, and liver cell types. In this study, to better understand the properties of neurally differentiated MSCs, gene expression in highly homogeneous hcMSCs was analyzed. Neural differentiation of hcMSCs was induced for 14 days. Thereafter, RNA and genomic DNA was isolated and subjected to microarray analysis and DNA methylation array analysis, respectively. We correlated the transcriptome of hcMSCs during neural differentiation with the DNA methylation status. Here, we describe and discuss the gene expression profile of neurally differentiated hcMSCs. These findings will expand our understanding of the molecular properties of MSCs and contribute to the development of cell therapy for neuroimmune diseases.
To improve the growth of human mesenchymal stem cells(hMSCs) under general cell culture conditions(20% $O_2$ and 5% $CO_2$), we examined the effect of s-allylcysteine(SAC), which is known as an antioxidant and the main component of aged-garlic extract, on hydrogen peroxide-induced cellular stress in hMSCs. We found that SAC blocked hydrogen peroxideinduced cell death and cellular apoptosis, but that SAC did not improve the growth of hMSCs during short-term culture. To evaluate the protective effect of SAC, we examined the endogenous expression of the antioxidant enzymes catalase (CAT), superoxide dismutase(SOD), and glutathione peroxidase(Gpx) in hMSCs. Hydrogen peroxide was found to downregulate the expression of CAT, SOD, and Gpx at the protein level. However, in the pre-treatment group of SAC, SAC inhibited the hydrogen peroxide-induced down-regulation of CAT, SOD, and Gpx. Unfortunately, treatment with SAC alone did not induce the up-regulation of antioxidant enzymes and the cell proliferation of hMSCs. Surprisingly, SAC improved cell growth in a single cell level culture of hMSCs. These results indicate that SAC may be involved in the preservation of the self-renewal capacity of hMSCs. Taken together, SAC improves the proliferation of hMSCs via inhibition of oxidative-stress-induced cell apoptosis through regulation of antioxidant enzymes. In conclusion, SAC may be an indispensable component in an in vitro culture system of human MSCs for maintaining self-renewal and multipotent characterization of human MSCs.
In this research we proceeded experiments to find the basis which make it possible to explain the physical and pathological process of Sasang constitutional medicine, in the way substituting hematopoietic-immune system(essence of life, blood, Ki and mental faculties : 精血氣神) Under these suppositions, the essence of life(精) is the multipotent stem cell which has the possibility to be specialized to any cell, the Ki(氣), blood(血) and mental faculties(神) are inferred that they are formed from specialized the essence of life(精), the blood(血) is the red blood cells and etc. that appears as the result of the genesis of circulation system. The Ki(氣) is from specialized basic immunity, the mental faculties(神) means long-term memories or combined immunity. Cytokines can act as specilaizing, growing factors and particiate in extremly combined procedure being controlled by both positive and negative specializing signals. Blood gathering was carried out in the morning and on empty stomach. The plasma was seperated and Erythropoietin, Stem cell factor, Granulocyte-colony stimulaing factor, Tumor necrosis factor, interlukin-3, Interleukin-6 were measured with ELISA kit. According to the result of post analysis by Duncan, each constitution is different in SCF(stem cell factor), IL-6(interleukin-6), EPO(erythropoietin). The value of Stem cell factor is high in order of Soumin(少陰人), Soyangin(少陽人), and Taeumin(太陰人), The value of interleukin-6 is high in Taeumin, Soumin, and Soyangin. Erythropoietin is high in order of Soumin, Soyangin, and Taeumin.
Human umbilical cord blood(HUCB) contains a rich source of hematopoietic stem cells, mesenchymal stem cells and endothelial cell precursors. Mesenchymal stem cells(MSCs) in HUCB are multipotent stem cells, differ from hematopoietic stem cells and can be differentiated into neural cells. We studied on transdifferentiation-promoting conditions in neural cells and cholinergic neuron induction of HUCB-derived MSCs. Neural differentiation was induced by addingdimethyl sulphoxide(DMSO) and butylated hydroxyanisole(BHA) in Dulbeco's Modified Essential Medium(DMEM) and fetal bovine serum(FBS). Differentiation of MSCs to cholinergic neurons was induced by combined treatment with basic fibroblast growth factor(bFGF), retinoic acid(RA) and sonic hedgehog(Shh). MSCs treated with DMSO and BHA rapidly assumed the morphology of multipolar neurons. Both immunocytochemistry and RT-PCR analysis indicated that the expression of a number of neural markers including $\beta$-tubulin III, GFAP and MBP, was markedly elevated during this acute differentiation. The differentiation rate was about $32.3{\pm}2.9%$ for $\beta$-tubulin III-positive cells, $11.0{\pm}0.9%$ for GFAP, and $9.4{\pm}1.0%$ for Gal-C. HUCB-MSCs treated combinatorially with bFGF, RA and Shh were differentiated into cholinergic neurons. After cholinergic neuronal differentiation, the $\beta$-tubulin III-positive cell population of total cells was $31.3{\pm}3.2%$ and of differentiated neuronal population, $70.0{\pm}7.8%$ was ChAT-positive showing 3 folds higher in cholinergic population than neural induction. Conclusively, HUCB-derived MSCs can be differentiated into neural and cholinergic neurons and these findings suggest that HUCB are alternative cell source of treatment for neurodegenerative diseases such as Alzheimer's disease.
Park, Soon-Jung;Jeon, Young-Joo;Kim, Ju-Mi;Shin, Jeong-Min;Chae, Jung-Il;Chung, Hyung-Min
Reproductive and Developmental Biology
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v.34
no.3
/
pp.143-151
/
2010
Pluripotency of human embryonic stem cell (hESC) is one of the most valuable ability of hESCs for applying cell therapy field, but also showing side effect, for example teratoma formation. When transplant multipotent stem cell, such as mesnchymal stem cell (MSC) which retains similar differentiation ability, they do not form teratoma in vivo, but there exist limitation of cellular source supply. Accordingly, differentiation of hESC into MSC will be promising cellular source with strong points of both hESC and MSC line. In this study, we described the derivation of MSC like cell population from feeder free cultured hESC (hESC-MSC) using direct differentiation system. Cells population, hESC-MSC and bone marrow derived MSC (BM-MSC) retained similar characteristics in vitro, such as morphology, MSC specific marker expression and differentiation capacity. At the point of differentiation of both cell populations, differentiation rate was slower in hESC-MSC than BM-MSC. As these reason, to verify differentially expressed molecular condition of both cell population which bring out different differentiation rate, we compare the molecular condition of hESC-MSC and BM-MSC using 2-D proteomic analysis tool. In the proteomic analysis, we identified 49 differentially expressed proteins in hESC-MSC and BM-MSC, and they involved in different biological process such as positive regulation of molecular function, biological process, cellular metabolic process, nitrogen compound metabolic process, macromolecule metabolic process, metabolic process, molecular function, and positive regulation of molecular function and regulation of ubiquitin protein ligase activity during mitotic cell cycle, cellular response to stress, and RNA localization. As the related function of differentially expressed proteins, we sought to these proteins were key regulators which contribute to their differentiation rate, developmental process and cell proliferation. Our results suggest that the expressions of these proteins between the hESC-MSC and BM-MSC, could give to us further evidence for hESC differentiation into the mesenchymal stem cell is associated with a differentiation factor. As the initial step to understand fundamental difference of hESC-MSC and BM-MSC, we sought to investigate different protein expression profile. And the grafting of hESC differentiation into MSC and their comparative proteomic analysis will be positively contribute to cell therapy without cellular source limitation, also with exact background of their molecular condition.
Human mesenchymal stem cells (hMSC) are multipotent stromal cells that have great potential to differentiate into a variety of cell types such as osteocytes, chondrocytes, and myocytes. Although there have been many studies on their clinical availability, little is known about how intracellular signals can be modulated by topographic features of the extracellular matrix (ECM). In this study, we investigated whether and how microwavy-patterned extracellular matrix (ECM) could affect the signaling activity of focal adhesion kinase (FAK), a key cellular adhesion protein. The fluorescence resonance energy transfer (FRET)-based FAK biosensor-transfected cells are incubated on microwavy-patterned surfaces and then platelet derived growth factor (PDGF) are treated to trigger FAK signals, followed by monitoring through live-cell FRET imaging in real time. As a result, we report that PDGF-induced FAK was highly activated in cells cultured on microwavy-patterned surface with L or M type, while inhibited by H type-patterned surface. In further studies, PDGF-induced FAK signals are regulated by functional support of actin filaments, microtubules, myosin-related proteins, suggesting that PDGF-induced FAK signals in hMSC upon microwavy surfaces are dependent on cytoskeleton (CSK)-actomyosin networks. Thus, our findings not only provide new insight on molecular mechanisms on how FAK signals can be regulated by distinct topographical cues of the ECM, but also may offer advantages in potential applications for regenerative medicine and tissue engineering.
BACKGROUND: Mesenchymal stromal cells (MSCs) are multipotent stem cells that can differentiate into several cell types. In addition, many studies have shown that MSCs modulate the immune response. However, little information is currently available regarding the maintenance of immunomodulatory characteristics of MSCs through passages. Therefore, we investigated and compared cytokine and gene expression levels from adipose (AD) and bone marrow (BM)-derived MSCs relevant to immune modulation from early to late passages. METHODS: MSC immunophenotype, growth characteristics, cytokine expressions, and gene expressions were analyzed. RESULTS: AD-MSCs and BM-MSCs had similar cell morphologies and surface marker expressions from passage 4 to passage 10. Cytokines secreted by AD-MSCs and BM-MSCs were similar from early to late passages. AD-MSCs and BM-MSCs showed similar immunomodulatory properties in terms of cytokine secretion levels. However, the gene expressions of tumor necrosis factor-stimulated gene (TSG)-6 and human leukocyte antigen (HLA)-G were decreased and gene expressions of galectin-1 and -3 were increased in both AD- and BM-MSCs with repeated passages. CONCLUSION: Our study showed that the immunophenotype and expression of immunomodulation-related cytokines of AD-MSCs and BM-MSCs immunomodulation through the passages were not significantly different, even though the gene expressions of both MSCs were different.
Purpose: Adipose-derived stromal cells (ADSCs) are multipotent cells that have been found to promote wound healing through the process of angiogenesis and reepithelialization. Generally, it is well known that the antigenicity of ADSCs doesn't affect stem cell therapy. In this study, we investigated the effect of allogeneic ADSCs in the wound healing process by applying allogeneic ADSCs on the wound healing splint model of mice. Methods: Adipose tissue was harvested from the epididymal fat pads of BALB/c and C57BL/6 mice. Twenty four mice BALB/c were divided into three groups; control, isogeneic, and allogeneic groups. Two full thickness defects with 6 mm diameters were created on the back of BALB/c mice. $1{\times}10^6$ ADSCs from BALB/c mice were applied on the isogeneic group. In the allogeneic group, ADSCs from the C57BL/6 mice were applied. No cells were applied to the control group. The sizes of the wounds were evaluated in 3, 5, 7, 10, and 14 days after the wounds were applied, and tissues were harvested in 7 and 14 days for histological analysis. Results: Wound healing rates had showed significant increase in 10, and 14 days when the isogeneic group was compared to the control group, but the allogeneic group showed significantly decrease compared to the isogeneic group (p<0.05). Histological scores in the isogeneic group were significantly high, but significantly lower in the allogeneic group when compared to the isogeneic group in 2 weeks (p<0.05). In the isogeneic group, thick inflammatory cell infiltration with abundant capillaries were observed in 1 week, and thick epithelium with many large capillaries were observed in 2 weeks. Conclusion: When isogeneic ADSCs were applied to wounds, they presented a faster wound healing rate compared to controls and the allogeneic group. Unlike general stem cell therapy, these findings suggest that cell therapy targeted at enhancing wound healing may benefit from the use of ADSCs with identical antigenicity, as opposed to allogeneic or xenogenic ADSCs.
Park, Bong-Wook;Byun, June-Ho;Lee, Sung-Gyoon;Hah, Young-Sool;Kim, Deok-Ryong;Cho, Yeong-Cheol;Sung, Iel-Yong;Kim, Jong-Ryoul
Maxillofacial Plastic and Reconstructive Surgery
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v.28
no.6
/
pp.511-519
/
2006
Autogenous bone grafts have been considered the gold standard for maxillofacial bony defects. However, this procedure could entail a complicated surgical procedure as well as potential donor site morbidity. Possibly the best solution for bone-defect regeneration is a tissue engineering approach, i.e. the use of a combination of a suitable scaffold with osteogenic cells. A major source of osteogenic cells is the bone marrow. Bone marrow-derived mesenchymal stem cells are multipotent and have the ability to differentiate into osteoblastic, chondrocytic, and adipocytic lineage cells. However, the isolation of cells from bone marrow has someproblems when used in clinical setting. Bone marrow aspiration is sometimes potentially more invasive and painful procedure and carries of a risk of morbidity and infection. A minimally invasive, easily accessible alternative would be cells derived from periosteum. The periosteum also contains multipotent cells that have the potential to differentiate into osteoblasts and chondrocytes. In the present study, we evaluated the osteogenic activity and mineralization of cultured human periosteal-derived cells. Periosteal explants were harvested from mandibule during surgical extraction of lower impacted third molar. The periosteal cells were cultured in the osteogenic inductive medium consisting of DMEM supplemented with 10% fetal calf serum, 50g/ml L-ascorbic acid 2-phosphate, 10 nmol dexamethasone and 10 mM -glycerophosphate for 42 days. Periosteal-derived cells showed positive alkaline phosphatase (ALP) staining during 42 days of culture period. The formation of ALP stain showed its maximal manifestation at day 14 of culture period, then decreased in intensity during the culture period. ALP mRNA expression increased up to day 14 with a decrease thereafter. Osteocalcin mRNA expression appeared at day 7 in culture, after that its expression continuously increased in a time-dependent manner up to the entire duration of culture. Von Kossa-positive mineralization nodules were first present at day 14 in culture followed by an increased number of positive nodules during the entire duration of the culture period. In conclusion, our study showed that cultured human periosteal-derived cells differentiated into active osteoblastic cells that were involved in synthesis of bone matrix and the subsequent mineralization of the matrix. As the periosteal-derived cells, easily harvested from intraoral procedure such as surgical extraction of impacted third molar, has the excellent potential of osteogenic capacity, tissue-engineered bone using periosteal-derived cells could be the best choice in reconstruction of maxillofacial bony defects.
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