scholarly journals Characterization of mesenchymal stem cells derived from equine adipose tissue

2013 ◽  
Vol 65 (4) ◽  
pp. 939-945 ◽  
Author(s):  
A.M. Carvalho ◽  
A.L.M. Yamada ◽  
M.A. Golim ◽  
L.E.C. Álvarez ◽  
L.L. Jorge ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Adipose Tissue ◽  
Mhc Class Ii ◽  
Class Ii ◽  
Specific Staining ◽  
Alizarin Red ◽  
Lineage Differentiation ◽  
Mixed Breed

Stem cell therapy has shown promising results in tendinitis and osteoarthritis in equine medicine. The purpose of this work was to characterize the adipose-derived mesenchymal stem cells (AdMSCs) in horses through (1) the assessment of the capacity of progenitor cells to perform adipogenic, osteogenic and chondrogenic differentiation; and (2) flow cytometry analysis using the stemness related markers: CD44, CD90, CD105 and MHC Class II. Five mixed-breed horses, aged 2-4 years-old were used to collect adipose tissue from the base of the tail. After isolation and culture of AdMSCs, immunophenotypic characterization was performed through flow cytometry. There was a high expression of CD44, CD90 and CD105, and no expression of MHC Class II markers. The tri-lineage differentiation was confirmed by specific staining: adipogenic (Oil Red O), osteogenic (Alizarin Red), and chondrogenic (Alcian Blue). The equine AdMSCs are a promising type of adult progenitor cell for tissue engineering in veterinary medicine.

399 ISOLATION AND CHARACTERIZATION OF BOVINE MESENCHYMAL STEM CELLS DERIVED FROM ADIPOSE TISSUE

2010 ◽  
Vol 22 (1) ◽  
pp. 356 ◽  
Author(s):  
R. V. Sampaio ◽  
M. R. Chiaratti ◽  
F. F. Bressan ◽  
J. R. Sangalli ◽  
M. S. Miranda ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Adipose Tissue ◽  
Morphological Changes ◽  
Embryonic Stem ◽  
Success Rates ◽  
Specific Staining ◽  
Alizarin Red ◽  
Isolation And Characterization ◽  
Donor Cells

Stem cells have been widely used because of their multi-differentiation ability. Compared with embryonic stem cells, mesenchymal stem cells (MSC) are more easily sourced and cultured, besides being easily obtained from adult individuals. In this regard, bovine MSC is of great interest because of its wide application in basic and applied research, e.g. in somatic cell nuclear transfer (SCNT). Success rates of SCNT are expected to be enhanced with the use of MSC as donor cells because they exhibit a more undifferentiated condition when compared with the most widely used cell type, fibroblast. There are few reports on MSC referring to this species. Therefore, our aim was to isolate and culture adipose MSC from cattle. Mesenchymal stem cells were induced to differentiate into adipocytes and osteocytes to prove their multi-differentiation ability. A small piece of adipose tissue was sourced from the base of the tail of a cow and extensively washed in cold saline solution containing 5% antibiotic. Recovered tissue was minced and digested in 0.001% collagenase 1 and incubated for 3 h. Collagenase was inactivated in alpha minimal essential medium (MEM) supplemented with 15% FCS. The treated tissue was centrifuged and the pellet plated on plastic dishes in alpha MEM supplemented with 15% FCS and 1% antibiotic. Culture medium was replaced every 2 days, and cells were plated on new dishes before reaching 75% confluence. To test their resistance to grow after thawing, cells were frozen after each passage, thawed, and cultured. Cell growth was accompanied through several weeks, and cells were tested for differentiation ability after 3 passages, when adipocyte and osteocyte differentiation was accomplished using a standard protocol. Briefly, adipocyte differentiation was inducted in DMEM containing 2% FCS, isobutyl-methylxanthine, dexamethasone, insulin, and indomethacin. Osteocyte differentiation was carried out in alpha MEM without FCS containing ascorbate-2-phosphate, dexamethasone, and beta-glycerophosphate.To confirm differentiation, a tissue-specific staining was carried out using Oil Red for adipocyte staining and Alizarin Red for osteocyte staining. The cells adhered to plastic dishes shortly after plating, presented fibroblast-like morphology, and showed an exponential growth curve during the first 6 passages. The cells were capable of growing after thawing similarly to unthawed cells. After 1 week under differentiation protocols, standard morphological changes were observed in the cells. Mesenchymal stem cells subjected to differentiation into adipocytes showed an increase in their size, developed lipid-like vesicles, and stained positive for Oil Red. An increase in the cell size was also observed in the cells subjected to differentiation into osteocyte. Moreover, these cells stained positive for Alizarin Red. Altogether these results provide evidence that these cells are multi-potent MSC. Therefore, we conclude that the current methodology was efficient in isolating bovine MSC from adipose tissue, and the cells resemble those isolated from other mammals. Bovine MSC constitute a significant source of nuclear donor cells for future experiments in SCNT. Financial support was provided by FAPESPA and Cnpq.

scholarly journals The Effects of Andrographolide on the Enhancement of Chondrogenesis and Osteogenesis in Human Suprapatellar Fat Pad Derived Mesenchymal Stem Cells

Molecules ◽  
2021 ◽  
Vol 26 (7) ◽  
pp. 1831
Author(s):  
Thitianan Kulsirirat ◽  
Sittisak Honsawek ◽  
Mariko Takeda-Morishita ◽  
Nuttanan Sinchaipanid ◽  
Wanvisa Udomsinprasert ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Adipogenic Differentiation ◽  
Human Mesenchymal Stem Cells ◽  
Fat Pad ◽  
Alizarin Red ◽  
Lineage Differentiation ◽  
Expression Of Genes ◽  
Dose Dependent ◽  
Oil Red O Staining

Andrographolide is a labdane diterpenoid herb, which is isolated from the leaves of Andrographis paniculata, and widely used for its potential medical properties. However, there are no reports on the effects of andrographolide on the human suprapatellar fat pad of osteoarthritis patients. In the present study, our goal was to evaluate the innovative effects of andrographolide on viability and Tri-lineage differentiation of human mesenchymal stem cells from suprapatellar fat pad tissues. The results revealed that andrographolide had no cytotoxic effects when the concentration was less than 12.5 µM. Interestingly, andrographolide had significantly enhanced, dose dependent, osteogenesis and chondrogenesis as evidenced by a significantly intensified stain for Alizarin Red S, Toluidine Blue and Alcian Blue. Moreover, andrographolide can upregulate the expression of genes related to osteogenic and chondrogenic differentiation, including Runx2, OPN, Sox9, and Aggrecan in mesenchymal stem cells from human suprapatellar fat pad tissues. In contrast, andrographolide suppressed adipogenic differentiation as evidenced by significantly diminished Oil Red O staining and expression levels for adipogenic-specific genes for PPAR-γ2 and LPL. These findings confirm that andrographolide can specifically enhance osteogenesis and chondrogenesis of mesenchymal stem cells from human suprapatellar fat pad tissues. It has potential as a therapeutic agent derived from natural sources for regenerative medicine.

296 IN VITRO DIFFERENTIATION OF PORCINE BONE MARROW-DERIVED MESENCHYMAL STEM CELLS INTO HEPATOCYTE-LIKE CELLS

2013 ◽  
Vol 25 (1) ◽  
pp. 295
Author(s):  
B. Mohana Kumar ◽  
W. J. Lee ◽  
Y. M. Lee ◽  
R. Patil ◽  
S. L. Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Class Ii ◽  
In Vitro Differentiation ◽  
Rt Pcr ◽  
Hepatic Differentiation ◽  
Alizarin Red ◽  
Differentiation Potential

Mesenchymal stem cells (MSC) are isolated from bone marrow or other tissues, and have properties of self renewal and multilineage differentiation ability. The current study investigated the in vitro differentiation potential of porcine bone marrow derived MSCs into hepatocyte-like cells. The MSC were isolated from the bone marrow of adult miniature pigs (7 months old, T-type, PWG Micro-pig®, PWG Genetics, Seoul, Korea) and adherent cells with fibroblast-like morphology were cultured on plastic. Isolated MSCs were positive for CD29, CD44, CD73, CD90, and vimentin, and negative for CD34, CD45, major histocompatibility complex-class II (MHC-class II), and swine leukocyte antigen-DR (SLA-DR) by flow cytometry analysis. Further, trilineage differentiation of MSC into osteocytes (alkaline phosphatase, von Kossa and Alizarin red), adipocytes (Oil Red O), and chondrocytes (Alcian blue) was confirmed. Differentiation of MSC into hepatocyte-like cells was induced with sequential supplementation of growth factors, cytokines, and hormones for 21 days as described previously (Taléns-Visconti et al. 2006 World J. Gastroenterol. 12, 5834–5845). Morphological analysis, expression of liver-specific markers, and functional assays were performed to evaluate the hepatic differentiation of MSC. Under hepatogenic conditions, MSC acquired cuboidal morphology with cytoplasmic granules. These hepatocyte-like cells expressed α-fetoprotein (AFP), albumin (ALB), cytokeratin 18 (CK18), cytochrome P450 7A1 (CYP7A1), and hepatocyte nuclear factor 1 (HNF-1) markers by immunofluorescence assay. In addition, the expression of selected markers was demonstrated by Western blotting analysis. In accordance with these features, RT-PCR revealed transcripts of AFP, ALB, CK18, CYP7A1, and HNF-1α. Further, the relative expression levels of these transcripts were analysed by quantitative RT-PCR after normalizing to the expression of the endogenous control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data were analysed statistically by one-way ANOVA using PASW statistics 18 (SPSS Inc., Chicago, IL, USA), and significance was considered at P < 0.05. The results showed that the relative expressions of selected marker genes in hepatocyte-like cells were significantly increased compared with that in untreated MSC. The generated hepatocyte-like cells showed glycogen storage as analysed by periodic acid-Schiff (PAS) staining. Moreover, the induced cells produced urea at Day 21 of culture compared with control MSC. In conclusion, our results indicate the potential of porcine MSC to differentiate in vitro into hepatocyte-like cells. Further studies on the functional properties of hepatocyte-like cells are needed to use porcine MSC as an ideal source for liver cell therapy and preclinical drug evaluation. This work was supported by Basic Science Research Program through the National Research Foundation (NRF), funded by the Ministry of Education, Science and Technology (2010-0010528) and the Next-Generation BioGreen 21 Program (No. PJ009021), Rural Development Administration, Republic of Korea.

scholarly journals Mesenchymal Stem Cells Immunosuppressed IL-22 in Patients with Immune Thrombocytopenia via Soluble Cellular Factors

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Mei Wu ◽  
Hongfeng Ge ◽  
Shue Li ◽  
Hailiang Chu ◽  
Shili Yang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Cell Proliferation ◽  
Mesenchymal Stem Cells ◽  
Immune Thrombocytopenia ◽  
Therapeutic Strategy ◽  
Alizarin Red ◽  
Cellular Factors ◽  
Oil Red O ◽  
Proliferation Suppression

Mesenchymal stem cells are immunoregulation cells. IL-22 plays an important role in the pathogenesis of immune thrombocytopenia. However, the effects of mesenchymal stem cells on IL-22 production in patients with immune thrombocytopenia remain unclear. Flow cytometry analyzed immunophenotypes of mesenchymal stem cells; differentiation of mesenchymal stem cells was observed by oil red O and Alizarin red S staining; cell proliferation suppression was measured with MTS; IL-22 levels of cell-free supernatants were determined by ELISA. Mesenchymal stem cells inhibited the proliferation of activated CD4+T cells; moreover, mesenchymal stem cells immunosuppressed IL-22 by soluble cellular factors but not PGE2. These results suggest that mesenchymal stem cells may be a therapeutic strategy for patients with immune thrombocytopenia.

B7-H1 Inhibits T Cell Proliferation Through MHC Class II in Human Mesenchymal Stem Cells

2014 ◽  
Vol 46 (5) ◽  
pp. 1638-1641 ◽  
Author(s):  
I.K. Jang ◽  
H.H. Yoon ◽  
M.S. Yang ◽  
J.E. Lee ◽  
D.-H. Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Proliferation ◽  
Mesenchymal Stem Cells ◽  
T Cell ◽  
Mhc Class Ii ◽  
Human Mesenchymal Stem Cells ◽  
Class Ii ◽  
T Cell Proliferation

scholarly journals Study on a 3D-Bioprinted Tissue Model of Self-Assembled Nanopeptide Hydrogels Combined With Adipose-Derived Mesenchymal Stem Cells

2021 ◽  
Vol 9 ◽  
Author(s):  
Guanzhou Zhou ◽  
Ailing Tian ◽  
Xin Yi ◽  
Lufeng Fan ◽  
Wenchong Shao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Primary Cultures ◽  
Mixed Solution ◽  
Control Groups ◽  
Alizarin Red ◽  
Tissue Model ◽  
Self Assembled ◽  
Experimental Group

Objective: This study aimed to observe the cell growth status and multidirectional differentiation ability in a 3D-bioprinted tissue model of self-assembled nanopeptides and human adipose-derived mesenchymal stem cells (Ad-MSCs).Methods: Primary Ad-MSCs were isolated, cultured, and identified by flow cytometry. Tissue models were printed via 3D bioprinting technology using a “biological ink” consisting of a mixed solution of self-assembled nanopeptides and Ad-MSCs. Ad-MSCs were induced into osteogenic, adipogenic, and endothelial differentiation and compared with the control groups by staining.Results: The nanopeptide fiber was 10–30 nm in diameter and 200–500 nm in length under the atomic-force microscope. It had the characteristics of nano-scale materials. Flow cytometry showed that the isolated and cultured cells were positive for CD29 (98.51%), CD90 (97.87%), and CD166 (98.32%) but did not express CD31 (1.58%), CD34 (2.42%), CD45 (2.95%), or human leukocyte antigen (HLA)-DR (0.53%), consistent with the immunophenotype of Ad-MSCs. Then, a tissue model was printed using the biological ink, followed by induction of differentiation of Ad-MSCs within the tissue model. Alizarin red S staining showed the formation of calcium nodules in the osteogenesis induction experimental group, and oil red O stained lipid droplets in Ad-MSCs in the adipogenesis induction experimental group, whereas the two control groups were not stained.Conclusion: Ad-MSCs from primary cultures have the characteristics of stem cells. Self-assembled nanopeptide hydrogel is a good tissue engineering material that can serve as an extracellular matrix. Ad-MSCs in the 3D-printed tissue model using a biological ink consisting of a mixed solution of self-assembled nanopeptides and Ad-MSCs grew well and still had strong differentiation ability.

scholarly journals Comparative Analysis of Apoptotic Resistance of Mesenchymal Stem Cells Isolated from Human Bone Marrow and Adipose Tissue

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Gökhan Ertaş ◽  
Ertan Ural ◽  
Dilek Ural ◽  
Ayça Aksoy ◽  
Güliz Kozdağ ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Adipose Tissue ◽  
Serum Deprivation ◽  
Human Bone ◽  
Induced Apoptosis

Aim. Mesenchymal stem cells (MSCs) isolated from human bone marrow (hBM) and adipose tissue (hAT) are perceived as attractive sources of stem cells for cell therapy. The aim of this study was to compare MSCs from hBM and hAT for their immunocytochemistry staining and resistance to in vitro apoptosis. Methods. In our study, we investigated the antiapoptotic ability of these MSCs toward oxidative stress induced by hydrogen peroxide (H2O2) and serum deprivation. Results were assessed by MTT and flow cytometry. All experiments were repeated a minimum of three times. Results. Flow cytometry and MTT analysis revealed that hAT-MSCs exhibited a higher resistance toward H2O2-induced apoptosis (n=3, hBM-hAT viability H2O2  58.43±1.24–73.02±1.44, P<0.02) and to serum-deprivation-induced apoptosis at days 1 and 4 than the hBM-MSCs (n=3, hAT-hBM absorbance, resp., day 1: 0.305±0.027–0.234±0.015, P=0.029, day 4: 0.355±0.003–0.318±0.007, P=0.001, and day 7: 0.400±0.017–0.356±0.008, P=0.672). hAT-MSCs showed superior tolerance to oxidative stress triggered by 2 mmol/L H2O2 and also have superior antiapoptosis capacity toward serum-free culture. Conclusion. In this study we found that hAT-MSCs are more resistant to in vitro apoptosis.

scholarly journals Differentiation of mesenchymal stem cells derived from human bone marrow and subcutaneous adipose tissue into pancreatic islet-like clusters in vitro

2013 ◽  
Vol 18 (1) ◽  
Author(s):  
Dhanasekaran Marappagounder ◽  
Indumathi Somasundaram ◽  
Sudarsanam Dorairaj ◽  
Rajkumar Sankaran
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Adipose Tissue ◽  
Pancreatic Islet ◽  
Subcutaneous Adipose Tissue ◽  
Lineage Differentiation ◽  
Wide Range ◽  
Subcutaneous Adipose

AbstractAlthough stem cells are present in various adult tissues and body fluids, bone marrow has been the most popular source of stem cells for treatment of a wide range of diseases. Recent results for stem cells from adipose tissue have put it in a position to compete for being the leading therapeutic source. The major advantage of these stem cells over their counterparts is their amazing proliferative and differentiation potency. However, their pancreatic lineage transdifferentiation competence was not compared to that for bone marrow-derived stem cells. This study aims to identify an efficient source for transdifferentiation into pancreatic islet-like clusters, which would increase potential application in curative diabetic therapy. The results reveal that mesenchymal stem cells (MSC) derived from bone marrow and subcutaneous adipose tissue can differentiate into pancreatic islet-like clusters, as evidenced by their islet-like morphology, positive dithizone staining and expression of genes such as Nestin, PDX1, Isl 1, Ngn 3, Pax 4 and Insulin. The pancreatic lineage differentiation was further corroborated by positive results in the glucose challenge assay. However, the results indicate that bone marrow-derived MSCs are superior to those from subcutaneous adipose tissue in terms of differentiation into pancreatic islet-like clusters. In conclusion, bone marrow-derived MSC might serve as a better alternative in the treatment of diabetes mellitus than those from adipose tissue.

scholarly journals Human Adipose-Derived Mesenchymal Stem Cells Cryopreservation and Thawing Decreaseα4-Integrin Expression

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Ana Carolina Irioda ◽  
Rafael Cassilha ◽  
Larissa Zocche ◽  
Julio Cesar Francisco ◽  
Ricardo Correa Cunha ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Adipose Tissue ◽  
Phenotypic Expression ◽  
Human Adipose Tissue ◽  
Integrin Expression ◽  
Alizarin Red ◽  
Differentiation Potential ◽  
Colony Forming Units ◽  
Before And After

Aim. The effects of cryopreservation on adipose tissue-derived mesenchymal stem cells are not clearly documented, as there is a growing body of evidence about the importance of adipose-derived mesenchymal stem cells for regenerative therapies. The aim of this study was to analyze human adipose tissue-derived mesenchymal stem cells phenotypic expression (CD34, CD45, CD73, CD90, CD105, and CD49d), colony forming unit ability, viability, and differentiation potential before and after cryopreservation.Materials and Methods. 12 samples of the adipose tissue were collected from a healthy donor using the liposuction technique. The cell isolation was performed by enzymatic digestion and then the cells were cultured up to passage 2. Before and after cryopreservation the immunophenotype, cellular viability analysis by flow cytometer, colony forming units ability, differentiation potential into adipocytes and osteoblasts as demonstrated by Oil Red O and Alizarin Red staining, respectively.Results. The immunophenotypic markers expression was largely preserved, and their multipotency was maintained. However, after cryopreservation, the cells decreasedα4-integrin expression (CD49d), cell viability, and number of colony forming units.Conclusions. These findings suggest that ADMSC transplanted after cryopreservation might compromise the retention of transplanted cells in the host tissue. Therefore, further studies are warranted to standardize protocols related to cryopreservation to attain full benefits of stem cell therapy.

scholarly journals Use of microfiltered vs only disaggregated mesenchymal stem cells from adipose tissue in regenerative medicine

2020 ◽  
Vol 51 (3) ◽  
pp. 152-157
Author(s):  
Fabiano Svolacchia ◽  
Lorenzo Svolacchia
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Adipose Tissue ◽  
Bioactive Peptides ◽  
Clinical Use ◽  
Adult Mesenchymal Stem Cells ◽  
Epidermal Regeneration ◽  
Inflammatory Component ◽  
Number Of Cells

Background: Clinical use of adult mesenchymal stem cells (MSCa) in medicine and regenerative surgery is constantly evolving. Adipose tissue-derived stem cells (ADSc) are capable of inducing the production of new extracellular matrix (ECM), deposition of new collagen and early revascularisation. Methods: Flow cytometry was performed for 2 mL of cell colonies harvested from adipose tissue (AT). Comparation has been made of at disaggregated only and the same at disaggregated and microfiltered at 50 mm, 100 mm and 200 mm. Signs of inflammation after dermo-epidermal regeneration session through the mesotherapy method were observed and compared. Results: Even after filtration, significant number of ADSc was collected. An increase in the size of the filter did not always translate into an increase in the number of cells that were found in the microfiltrate. In the non-filtered at disaggregated in both cases, highest number of cells was found, as expected, but at the expense of more pronounced inflammation. Sampling with the 16 Gauge needle produces superior results compared to the cannula in all cases. Conclusion: With this method in medicine and regenerative surgery it will be easier to exploit the growth factors, mRNA, MicroRNA, lipids and bioactive peptides emitted in the MSCa signalling micro-vesicles as they are isolated from the inflammatory component.

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