scholarly journals Neural differentiation of canine mesenchymal stem cells/multipotent mesenchymal stromal cells

2020 ◽  
Author(s):  
Sonja Prpar Mihevc ◽  
Vesna Kokondoska Grgich ◽  
Andreja Nataša Kopitar ◽  
Luka Mohorič ◽  
Gregor Majdic
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Glial Cells ◽  
Stromal Cells ◽  
Neural Differentiation ◽  
Neural Induction ◽  
Multipotent Mesenchymal Stromal Cells ◽  
Induction Medium ◽  
Neuronal Markers

Abstract Background: Ability of adipose tissue-derived multipotent mesenchymal stromal cells/mesenchymal stem cells (ASCs) to differentiate in neural lineages promises progress in the field of regenerative medicine especially for replacing damaged neuronal tissue in different neurological disorders. Reprogramming of ASCs can be induced by supplying growth medium with chemical neurogenic inductors and/or specific growth factors. We investigated the neural differentiation potential of canine ASCs using several growth media (KEM, NIMa, NIMb, NIMc) containing various combinations of neurogenic inductors: B27 supplement, valproic acid, forskolin, N2-supplement and retinoic acid. First the cells were preconditioned in proliferation medium, followed by induction of neuronal differentiation. Six canine ASCs cell lines were assessed, half from female and half from male donors. The cell morphology, growth dynamics, viability were observed along with expression of neuron and astrocyte specific markers, which were assessed by immunocytochemistry and flow cytometry. Results: After 3, 6 and 9 days, elongated neural-like cells with bipolar elongations were observed and some oval cells with light nuclei appeared. After three and nine days of neural induction, differentiation into neurons and glial cells was observed. Expression of neuronal markers tubulin beta III (TUBB3), neurofilament H (NF-H) and glial fibrillary acidic protein (GFAP) was observed by immunocytochemistry. High GFAP expression (between 70 and 90% of all cells) was detected after three days of growth in neural induction medium a (NIMa) by flow cytometry, and expression of adult neuronal markers NF-H and microtubule associated protein-2 (MAP2) was detected in around 25% of cells. After nine days of ASCs differentiation a drop in expression rates of all markers was detected. There were no differences between neural differentiation of ASCs isolated from female or male dogs. Conclusions: The differentiation repertoire of canine ASCs extends beyond mesodermal lineages. Using a defined neural induction medium the canine ASCs were able to transform to neural lineages, bearing markers of neuronal and glial cells and also displayed the typical neuronal morphology. Differentiated ASCs can be a source of neural cellular lineages for regenerative therapy of nerve damage and also could be applicable for modeling of neurodegenerative diseases.

scholarly journals Neural differentiation of canine mesenchymal stem cells/multipotent mesenchymal stromal cells

2019 ◽  
Author(s):  
Sonja Prpar Mihevc ◽  
Vesna Kokondoska Grgich ◽  
Andreja Nataša Kopitar ◽  
Luka Mohorič ◽  
Gregor Majdic
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Glial Cells ◽  
Stromal Cells ◽  
Neural Differentiation ◽  
Neural Induction ◽  
Multipotent Mesenchymal Stromal Cells ◽  
Induction Medium ◽  
Neuronal Markers

Abstract Background: Ability of adipose tissue-derived multipotent mesenchymal stromal cells/mesenchymal stem cells (ASCs) to differentiate in neural lineages promises progress in the field of regenerative medicine especially for replacing damaged neuronal tissue in different neurological disorders. Reprogramming of ASCs can be induced by supplying growth medium with chemical neurogenic inductors and/or specific growth factors. We investigated the neural differentiation potential of canine ASCs using several growth media (KEM, NIMa, NIMb, NIMc) containing various combinations of neurogenic inductors: B27 supplement, valproic acid, forskolin, N2-supplement and retinoic acid. First the cells were preconditioned in proliferation medium, followed by induction of neuronal differentiation. Six canine ASCs cell lines were assessed, half from female and half from male donors. The cell morphology, growth dynamics, viability were observed along with expression of neuron and astrocyte specific markers, which were assessed by immunocytochemistry and flow cytometry. Results: After 3, 6 and 9 days, elongated neural-like cells with bipolar elongations were observed and some oval cells with light nuclei appeared. After three and nine days of neural induction, differentiation into neurons and glial cells was observed. Expression of neuronal markers tubulin beta III (TUBB3), neurofilament H (NF-H) and glial fibrillary acidic protein (GFAP) was observed by immunocytochemistry. High GFAP expression (between 70 and 90% of all cells) was detected after three days of growth in neural induction medium a (NIMa) by flow cytometry, and expression of adult neuronal markers NF-H and microtubule associated protein-2 (MAP2) was detected in around 25% of cells. After nine days of ASCs differentiation a drop in expression rates of all markers was detected. There were no differences between neural differentiation of ASCs isolated from female or male dogs. Conclusions: The differentiation repertoire of canine ASCs extends beyond mesodermal lineages. Using a defined neural induction medium the canine ASCs were able to transform to neural lineages, bearing markers of neuronal and glial cells and also displayed the typical neuronal morphology. Differentiated ASCs can be a source of neural cellular lineages for regenerative therapy of nerve damage and also could be applicable for modeling of neurodegenerative diseases.

223 COMPARISON OF NEURAL DIFFERENTIATION BETWEEN RAT AND CAT MESENCHYMAL STEM CELLS UNDER CHALLENGE BY THE SAME NEUROINDUCTION AGENT

2007 ◽  
Vol 19 (1) ◽  
pp. 228
Author(s):  
G. Z. Jin ◽  
X. F. Yu ◽  
S. J. Cho ◽  
G. Y. Park ◽  
Y. J. Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Neural Differentiation ◽  
Culture Media ◽  
Expression Profiles ◽  
Neural Induction ◽  
Induction Medium ◽  
Microscopy Imaging ◽  
Neuronal Markers ◽  
Post Induction

Bone marrow-derived mesenchymal stem cells (MSCs) have the potential to differentiate into a variety of cell types, including osteocytes, chondrocytes, and adipocytes. Moreover, MSCs have the capacity to differentiate into neural lineages. The present study aimed to investigate the differences between the expression profiles during neural differentiation of rat and cat MSCs. MSCs in the 4th passage, which were isolated from the femurs and tibias by standard methodology, were used in our study. Culture media was divided into pre-induction medium, which consisted of DMEM-HG, 10% fetal bovine serum, 10 ng mL-1 of basic fibroblast growth factor (bFGF), and 1 mM �-mercaptoethanol, for 24 h, and neuron induction medium, which was composed of DMEM-HG, 2% DMSO, 200 �M BHA, 25 mMKCl, 10 �M forskolin, 5 �g mL-1 insulin-transferrin-sodium selenite, and 20 ng mL-1 bFGF, for 24 h. Thereafter, cell morphology and growth traits were determined by light microscopy imaging and by examining the cell-surface antigen profile and differentiation repertoire by immunocytochemistry, respectively. Regarding the expression of 3 MSC-related surface antigens, rat cells were positive for CD18 and CD44, whereas cat cells expressed CD9 and CD44. Under proneurogenic conditions, rat neuron-like cells progressively increased at 30 min post-induction, peaked at 1 h, and gradually declined after 12 h. At 24 h after neural induction, there were still some neuron-like cells. Meanwhile, cat cells were expressed increasingly during the first hour, peaked at 2 to 3 h, were sustained for 8 h after neural induction, and then gradually declined.A few neuron-like cells remained until 24 h post-induction. In neural differentiation, rat MSCs were positive for �III-tubulin, NF-L, NF-M, NF-H, trkA, and vimentin; cat MSCs were negative for �III-tubulin and NF-H but positive for NF-L, NF-M, trkA, and vimentin. Both MSCs were negative for oligodendrocyte markers. Our results revealed that there is variation in neural differentiation sensitivity due to species type. Rat cells were more sensitive in response to neuroinduction agents, maintained their morphology for a longer time, and expressed relatively mature neuronal markers. On the contrary, the sensitivity of cat cells was weaker, and survival time was shorter compared to that of the rat cells. The cat cells expressed immature neuronal markers. The present data suggest a significant aspect of the culture of MSCs from higher-grade species. This work was supported by KOSEF (Grant ? M10525010001-05N2501-00110).

scholarly journals Neural differentiation of canine mesenchymal stem cells/multipotent mesenchymal stromal cells

2020 ◽  
Vol 16 (1) ◽  
Author(s):  
Sonja Prpar Mihevc ◽  
Vesna Kokondoska Grgich ◽  
Andreja Nataša Kopitar ◽  
Luka Mohorič ◽  
Gregor Majdič
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Neural Differentiation ◽  
Multipotent Mesenchymal Stromal Cells

scholarly journals Ghrelin peptide improves glial conditioned medium effects on neuronal differentiation of human adipose mesenchymal stem cells

2021 ◽  
Author(s):  
Cristina Russo ◽  
Giuliana Mannino ◽  
Martina Patanè ◽  
Nunziatina Laura Parrinello ◽  
Rosalia Pellitteri ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Conditioned Medium ◽  
Neural Differentiation ◽  
Conditioned Media ◽  
Glial Fibrillary Acid Protein ◽  
Ghrelin Receptor ◽  
Neuronal Markers ◽  
Adipose Mesenchymal Stem Cells ◽  
Marker Expression

AbstractThe influences of ghrelin on neural differentiation of adipose-derived mesenchymal stem cells (ASCs) were investigated in this study. The expression of typical neuronal markers, such as protein gene product 9.5 (PGP9.5) and Microtubule Associated Protein 2 (MAP2), as well as glial Fibrillary Acid Protein (GFAP) as a glial marker was evaluated in ASCs in different conditions. In particular, 2 µM ghrelin was added to control ASCs and to ASCs undergoing neural differentiation. For this purpose, ASCs were cultured in Conditioned Media obtained from Olfactory Ensheathing cells (OEC-CM) or from Schwann cells (SC-CM). Data on marker expression were gathered after 1 and 7 days of culture by fluorescence immunocytochemistry and flow cytometry. Results show that only weak effects were induced by the addition of only ghrelin. Instead, dynamic ghrelin-induced modifications were detected on the increased marker expression elicited by glial conditioned media. In fact, the combination of ghrelin and conditioned media consistently induced a further increase of PGP9.5 and MAP2 expression, especially after 7 days of treatment. The combination of ghrelin with SC-CM produced the most evident effects. Weak or no modifications were found on conditioned medium-induced GFAP increases. Observations on the ghrelin receptor indicate that its expression in control ASCs, virtually unchanged by the addition of only ghrelin, was considerably increased by CM treatment. These increases were enhanced by combining ghrelin and CM treatment, especially at 7 days. Overall, it can be assumed that ghrelin favors a neuronal rather than a glial ASC differentiation.

scholarly journals Isolation and Characterization of Human Synovial Fluid-Derived Mesenchymal Stromal Cells from Popliteal Cyst

2020 ◽  
Vol 2020 ◽  
pp. 1-15
Author(s):  
Fang Li ◽  
Jianglin Chen ◽  
Mengjia Gong ◽  
Yang Bi ◽  
Chengchen Hu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Synovial Fluid ◽  
Stromal Cells ◽  
Pediatric Patients ◽  
Cyst Fluid ◽  
Popliteal Cyst ◽  
Cell Marker ◽  
Related Proteins

Mesenchymal stem cells (MSCs) are multipotent progenitor cells in adult tissues. The aim of this study is to isolate and identify synovial fluid-derived mesenchymal stromal cells (SF-MSCs) from the popliteal cyst fluid of pediatric patients. SF-MSCs were collected from the popliteal cyst fluid of pediatric patients during cystectomy surgery. After cyst fluid extraction and adherent culturing, in vitro morphology, growth curve, and cell cycle were observed. The expression of stem cell surface markers was analyzed by flow cytometry, and expression of cell marker protein was detected by immunofluorescence. SF-MSCs were cultured in osteogenic, adipogenic, and chondrogenic differentiation medium. The differentiation potential of SF-MSCs was analyzed by alkaline phosphatase (Alizarin Red), Oil Red O, and Alcian blue. Antibody detection of human angiogenesis-related proteins was performed compared with bone marrow mesenchymal stem cells (BM-MSCs). The results show that SF-MSCs from the popliteal cyst fluid of pediatric patients showed a shuttle appearance and logarithmic growth. Flow cytometry analysis revealed that SF-MSCs were negative for hematopoietic lineage markers (CD34, CD45) and positive for MSC markers (CD44, CD73, CD90, and CD105). Interstitial cell marker (vimentin) and myofibroblast-like cell marker alpha-smooth muscle actin (α-SMA) were positive. These cells could differentiate into osteogenic, adipogenic, and chondrogenic lineages, respectively. Several types of human angiogenesis-related proteins were detected in the cell secretory fluid. These results show that we successfully obtained SF-MSCs from the popliteal cyst fluid of pediatric patients, which have the potential to be a valuable source of MSCs.

Research of HIF-1 Mediated SDF-1/CXCR4 Axis on the Directional Migration and Neural Differentiation of Mesenchymal Stem Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4699-4699
Author(s):  
Qin Yu ◽  
Jie Lin ◽  
Lizhen Liu ◽  
Peipei Li ◽  
XiaoBo Xuan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Western Blotting ◽  
Protein Level ◽  
Neural Differentiation ◽  
Directional Migration ◽  
Protein Levels ◽  
Cxcr4 Mrna ◽  
Cxcr4 Axis

Abstract Abstract 4699 Introduction: Mesenchymal stem cells (MSCs) are under study as therapeutic delivery agents that assist in the repair of damaged tissues. At present, the mechanisms of targeted therapy of MSCs are known to relate with the hypoxia-inducible factor-1 (HIF-1) and its regulated biological axis stromal cell-derived factor-1/chemokine receptor 4 (SDF-1/CXCR4). SDF-1 and CXCR4 have a wide distribution in various cells and tissues, which plays an important role in the development of immune system, circulatory system and central nervous system. We thus inferred that SDF-1/CXCR4 may participate in the differentiation as well as the migration of stem cells. Our research aims to explore the effect of HIF-1α and its mediated SDF-1/CXCR4 axis on the directional migration and neural differentiation of MSCs, which may lead to a break in the efficiency and target distribution of MSCs therapy. Method: (1) Influence of hypoxia, CXCR4 antagonist (AMD3100) and SDF-1α on the proliferation of rat Mesenchymal Stem Cells (rMSCs): rMSCs were isolated from bone marrow of rats, and expanded in vitro. The growth feature of rMSCs exposed to hypoxia (PO2=1%) or normoxia was identified by growth curve, while the effect of AMD3100 (5ug/ml) and SDF-1α (10ng/ml and 100ng/ml) on the proliferation ability of rMSCs was detected by cell counting kit-8. (2) Effect of hypoxia on the expression of HIF-1α, CXCR4 and SDF-1α: Firstly, we used RT-PCR, western blotting and flow cytometry to detect the expression of HIF-1α and CXCR4 mRNA and protein levels in rMSCs which treated with hypoxia for 0h, 6h, 12h, 24h, 48h and 72h. Secondly, HIF-1α, SDF-1α mRNA and protein level in the hippocampus of rats which suffered hypoxia- ischemia for 1d, 3d, 5d, 7d, 14d and 21d were also detected by the same assays. (3) Research of HIF-1α and SDF-1/CXCR4 axis on the migration of rMSCs: We first detected the change of CXCR4 mRNA and protein levels in rMSCs treated with AMD3100 (5ug/ml) and SDF-1α (10ng/ml) by RT-PCR, western blotting and flow cytometry, and then studied SDF-1/CXCR4 axis on the migration of rMSCs using Transwell assay. (4) Effect of HIF-1α and SDF-1/CXCR4 axis on the differentiation of rMSCs: protein level of NSE and GFAP as well as positive rate of neural-induced rMSCs which have been pretreated with AMD3100 (5ug/ml) were detected by western blotting and immunocytochemistry. Results: Persistent hypoxia promoted the proliferation of rMSCs, while AMD3100 and SDF-1α at the concentration mentioned above had no effect. Compared to normal control, the protein expression of HIF-1α in rMSCs increased in hypoxic condition while the mRNA of HIF-1α did not change. Furthermore, the mRNA and protein level of CXCR4 both increased in rMSCs exposed to hypoxia for 6h and 12h, and the results confirmed by flow cytometry. We found HIF-1α mRNA was stably expressed in hippocampus, and increased significantly in hypoxia-ischemia brain damaged (HIBD) rats in a time dependent manner, which reached the peak on 7d. As expected, SDF-1α mRNA in hippocampus of HIBD rats was higher than that of normal control group, which reached the peak on 7d (P<0.01) and stably expressed till 21d, while the protein level is mainly in concordance. Moreover, CXCR4 mRNA was extremely up-regulated in rMSCs treated with SDF-1α (10ng/ml), however, in 5 ug/ml AMD3100 treated rMSCs, which decreased markedly (P<0.01), and the results were confirmed by western blotting and flow cytometry assays (P<0.05). Transwell assay manifested that SDF-1α had obvious chemotaxis to rMSCs. Protein level and positive cell number of NSE and GFAP were extremely down-regulated in rMSCs which pretreated with 5ug/ml AMD3100. Conclusion: Increased expression of HIF-1α led to the up-regulation of SDF-1/CXCR4 axis, and rMSCs displayed chemotaxis migration ascribed to the receptor-ligand interactions of SDF-1α and CXCR4, suggesting that HIF-1 and its mediated SDF-1/CXCR4 axis are of great significant on the directional migration of rMSCs. We also showed that CXCR4 antagonisation reduced the neural differentiation capabilities of rMSCs, thus suggested that SDF-1/CXCR4 axis may deeply involve in the neural differentiation of rMSCs. Disclosures: No relevant conflicts of interest to declare.

Effects of Self-Assembling Peptide RADA16-І Hydrogel on Neural Differentiation of Bone Marrow Mesenchymal Stem Cells

2014 ◽  
Vol 998-999 ◽  
pp. 238-242 ◽  
Author(s):  
Yan Sheng Liao ◽  
Li Deng ◽  
Xiao Qing Gao ◽  
Chao Xian Yang
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Neural Differentiation ◽  
Fluorescent Protein ◽  
Morphological Changes ◽  
Control Group ◽  
Induction Medium ◽  
Self Assembling ◽  
Marrow Mesenchymal Stem Cells

Objective: To establish composite culture system of rat bone marrow mesenchymal stem cells (BMSCs) and self-assembling peptide hydrogel RADA16-І, and to investigate the effect of RADA16-І hydrogel on neural differentiation of BMSCs. Methods: BMSCs were isolated, cultivated and labeled with green fluorescent protein (GFP), then they were inoculated on glass coverslips or in RADA16-І solution to form control group and RADA16-І group respectively. The morphological changes of BMSCs induced by neural induction medium were observed, and GFAP, NeuN and Map-2 expressions of BMSCs in each group were detected with immunofluorescence. Results: The induced BMSCs presented neuron-like change, and the rates of GFAP and NF-200 positive cells in RADA16-І group were higher than that in control group (P < 0.05). Conclusion: Self-assembling peptide RADA16-І hydrogel can promote neural differentiation of BMSCs, and which may be used as scaffold material on BMSCs transplantation for treatment of nervous system diseases.

Mesenchymal Stem Cells/Multipotent Mesenchymal Stromal Cells (MSCs)

2012 ◽  
Vol 11 (4) ◽  
pp. 244-253 ◽  
Author(s):  
Ji-Ping Zou ◽  
Sha Huang ◽  
Yan Peng ◽  
Hong-Wei Liu ◽  
Biao Cheng ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Multipotent Mesenchymal Stromal Cells

scholarly journals Phenotypical Characterization and Neurogenic Differentiation of Rabbit Adipose Tissue-Derived Mesenchymal Stem Cells

Genes ◽  
2021 ◽  
Vol 12 (3) ◽  
pp. 431
Author(s):  
Mária Tirpáková ◽  
Jaromír Vašíček ◽  
Andrea Svoradová ◽  
Andrej Baláži ◽  
Marián Tomka ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Genetic Information ◽  
Mrna Level ◽  
Gene Bank ◽  
Neuronal Markers ◽  
Digital Droplet Pcr ◽  
Similar Phenotype ◽  
Droplet Pcr

Although the rabbit is a frequently used biological model, the phenotype of rabbit adipose-derived mesenchymal stem cells (rAT-MSCs) is not well characterized. One of the reasons is the absence of specific anti-rabbit antibodies. The study aimed to characterize rAT-MSCs using flow cytometry and PCR methods, especially digital droplet PCR, which confirmed the expression of selected markers at the mRNA level. A combination of these methods validated the expression of MSCs markers (CD29, CD44, CD73, CD90 and CD105). In addition, cells were also positive for CD49f, vimentin, desmin, α-SMA, ALDH and also for the pluripotent markers: NANOG, OCT4 and SOX2. Moreover, the present study proved the ability of rAT-MSCs to differentiate into a neurogenic lineage based on the confirmed expression of neuronal markers ENO2 and MAP2. Obtained results suggest that rAT-MSCs have, despite the slight differences in marker expression, the similar phenotype as human AT-MSCs and possess the neurodifferentiation ability. Accordingly, rAT-MSCs should be subjected to further studies with potential application in veterinary medicine but also, in case of their cryopreservation, as a source of genetic information of endangered species stored in the gene bank.

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