scholarly journals Flow Cytometry Assisted Isolation of Adipose Tissue Derived Stem Cells

2017 ◽  
pp. 17-24 ◽  
Author(s):  
Umesh D. Wankhade ◽  
Sushil G. Rane
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Adipose Tissue

Abstract 248: Comparison Of Transplantation Of Bone Marrow-derived Stem Cells, Adipose-derived Stem Cells And Endometrium- Derived Stem Cells In The Infarcted Heart

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Xin Yang Hu ◽  
Kan Wang ◽  
Jian-an Wang
Keyword(s):  
Myocardial Infarction ◽  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Adipose Tissue ◽  
Cardiac Function ◽  
Control Cell ◽  
Intramyocardial Injection ◽  
Infarcted Heart

Background: A variety of adult stem cells have been transplanted into the infarcted heart to cure myocardial infarction(MI), however, comparative studies are lacking to show more suitable source of cells for transplantation. Mesenchymal stem cells hold promise for myocardial regeneration therapy. Derivation of these cells from the endometrium tissue might be easier compared to bone marrow and adipose tissue. However,the in vivo fate of endometrium stem cells (EnSCs) in the infarcted heart has never been compared directly to mesenchymal cells derived from bone marrow(BMMSCs) and adipose tissue(AdMSCs). Methods: EnSCs, AdMSCs and BMMSCs were isolated from healthy donors were characterized using flow cytometry for surface markers identification and microscopy for cell morphology. They were characterized with β-actin promoter driving firefly luciferase and green fluorescent protein (Fluc-GFP) double fusion reporter gene, and were characterized using flow cytometry, bioluminescence imaging (BLI) and luminometry. Cell proliferation was tested by CCK-8 kit, colony forming unit(CFU) was stained by crystal violet staining and apoptosis ratio were detected by TUNEL assay. Rat (n=8/group) underwent myocardial infarction followed by intramyocardial injection of 5х105 EnSCs, AdMSCs and BMMSCs, or saline (negative control). Cell survival was measured using BLI for 6 weeks and cardiac function was monitored by echocardiography and hemodynamics analysis. Ventricular morphology was assessed using histology. Results: EnSCs, AdMSCs and BMMSCs were CD29+, CD90+, CD105+, shared similar morphology, but EnSCs had best proliferation, colony-forming and anti-apoptosis activity of 3 types of MSCs. Cells expressed Fluc reporter genes in a number-dependent fashion, as confirmed by luminometry. After cardiac transplantation, transplantation of EnSCs was better capable of preserving ventricular function and dimensions than others, as confirmed by echo test, PV-loops and histology. Conclusions: This is the first study comparing the in in vitro and in vivo behavior of 3 types of MSCs in the infarcted heart. AdMSCs and BMMSCs do not tolerate well in the cardiac environment, resulting in more cell death andworse cardiac function than EnSCs groups.

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 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.

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.

202 IN VITRO DIFFERENTIATION OF ADULT PORCINE ADIPOSE-DERIVED STEM CELLS AFTER LABELING WITH PKH26 AND FLOW CYTOMETRY

2006 ◽  
Vol 18 (2) ◽  
pp. 209
Author(s):  
M. Mello ◽  
A. Lima ◽  
S. Malusky ◽  
S. Lane ◽  
M. Wheeler
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Adipose Tissue ◽  
Osteogenic Differentiation ◽  
Fluorescent Dye ◽  
Positive Staining ◽  
Adipose Derived Stem Cells ◽  
Differentiation Potential ◽  
Oil Red O

The purpose of this study was to investigate the possible effects of the fluorescent dye PKH26 and flow cytometry on adult porcine adipose-derived stem cells (ADSCs) after exposing them to adipogenic and osteogenic differentiation conditions. Adipose tissue was isolated from swine (11 months of age) and digested with 0.075% collagenase at 37�C for 90 min. The digested adipose tissue was centrifuged at 200g for 10 min to obtain a cell pellet. The pellet was re-suspended with DMEM, and the ADSCs were plated onto 75 cm2 flasks (5000-10 000 cells per cm2) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% gentamicin. Passage 3 cells were labeled with fluorescent dye (PKH26 red fluorescent cell linker kit; Sigma Chemical, St. Louis, MO, USA) and sorted by flow cytometry. After labeling and sorting, the sorted and unsorted (control group) cells were replated and exposed to adipogenic (1 �M dexamethasone, 0.5 mM isobutylmethylxantine, 10 �M insulin and 200�M indomethacin) and osteogenic (0.1 �M dexamethasone, 10 mM �-glycerophosphate, and 50�M ascorbic acid) differentiation conditions when the cells were 90% confluent. Cells were evaluated based on morphology and specific staining properties. Adipogenic differentiation was confirmed by oil red O-positive staining of large lipid vacuoles, and osteogenic differentiation by Von Kossa staining 2 weeks after initiation of differentiation. The frequency of oil red O-positive colonies in both sorted and unsorted group was similar (15.0% vs. 13.2%, respectively). The number of osteogenic nodules, confirmed by the presence of calcium by Von Kossa staining, in the sorted and unsorted group was 17 and 184 per flask, respectively. In conclusion, this study demonstrates that adult porcine adipose-derived stem cells maintain their differentiation potential after labeling with fluorescent dye and sorting by flow cytometry. This should allow for more rapid evaluation of the differentiation potential of ADSCs in vitro. This work was partially supported by the Council for Food and Agricultural Research (C-FAR) Sentinel Program, University of Illinois and CNPq, Brazil (M. Mello).

scholarly journals 167 ISOLATION AND COMPARATIVE PROFILING OF HUMAN ADIPOSE-DERIVED ADULT STEM CELLS

2005 ◽  
Vol 17 (2) ◽  
pp. 234
Author(s):  
A. Boquest ◽  
A. Shahdadfar ◽  
K. Fronsdal ◽  
J. Brinchmann ◽  
P. Collas
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Flow Cytometry ◽  
Endothelial Cells ◽  
Adipose Tissue ◽  
Stem Cell ◽  
Regenerative Medicine ◽  
Cell Signaling ◽  
Proliferative Capacity ◽  
Stromal Stem Cells

The stromal compartment of mesenchymal tissues is thought to harbor stem cells that display extensive proliferative capacity and multilineage potential. However, despite their potential impact in the field of regenerative medicine, little is known about the biology of stromal stem cells prior to culture. After removing adipocytes and erythrocytes from collagenase digested human adipose tissue, we identified two cell populations using flow cytometry which shared expression of stem cell markers SH2 and CD34, but lacked the phenotypic characteristics of leukocytes (CD45−). However, they were found to be discernible based on CD31 expression, a marker for endothelial cells. Using CD31 conjugated magnetic beads, we separated these cells (CD45-CD31− and CD45-CD31+) from three patients and compared global gene expression profiles using an Affymetrix platform. The prominant feature of CD45-CD31+ cells was the up-regulation of genes associated with endothelial cells. By contrast, CD45-CD31− cells were found to overexpress transcripts involved in cell cycle quiescence and cell signaling elements including those of the WNT pathway thought to be important for maintaining the stem cell state. Upon culture in DMEM/F12 with 20% FCS, only CD45-CD31− cells were capable of adhering to plastic and forming colonies. These cells with fibroblastic morphology met the key criterion of stem cells, the ability to proliferate while retaining the capacity to differentiate into mature tissues. Under appropriate inductive conditions, they were found to exclusively form bone, cartilage, adipose and neuronal-like tissues in vitro. Clonal cell lines generated from individually cultured CD45-CD31− cells displayed multilineage and proliferative capacity, validating our conclusion that they are true stem cells and not simply committed progenitors. We then undertook extensive comparative profiling of CD45-CD31− cells with their cultured counterparts to examine changes that stromal stem cells undergo during culture. Except for the disappearance of CD34, flow cytometry analysis using 52 antibodies revealed little change in cell surface phenotype as a result of culture. However, comparative global gene profiling revealed extensive down-regulation of many genes during culture. These included cell cycle arresting genes, as expected, and genes encoding elements involved in cell signaling including those belonging to the tumor necrosis factor, interleukin, transforming growth factor and chemokine families. The consequences of these changes remain unknown, but ultimately may affect the potential use of adipose tissue stem cells in regenerative medicine.

scholarly journals Expression of CD34 marker in the adipose tissue derived stem cells

2018 ◽  
Vol 5 (1) ◽  
Author(s):  
Phuc Van Pham ◽  
Ngoc Bich Vu ◽  
Van Hong Tran
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Adipose Tissue ◽  
Hematopoietic Stem Cells ◽  
Adipose Derived Stem Cells ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Cd34 Expression

Introduction: Adipose-derived stem cells (ADSCs) are considered as mesenchymal stem cells (MSCs). Indeed, they display all characteristics of MSCs that compliant with the minimal criteria of MSCs suggested by Domonici et al. (2006). However, some recent studies showed that ADSCs contain the subpopulation that was positive with CD34 marker – a marker of hematopoietic stem cells. This study aimed to analyze and determine the expression of CD34 marker in ten samples of ADSCs obtained from 10 donors. Methods: All ADSC samples were isolated and expanded according to the published previous protocols. They were confirmed as the MSCs with some markers and differentiation potential, excepting the CD34 expression. Then they were cultured and analyzed the expression of CD34 by flow cytometry at passage 3, 5, 7 and 9. Results: The results showed that expression of CD34 in ADSCs was different between donors and their passages that accounted from 1.21% to 23.38%. Conclusion: These results suggested that ADSCs are not ‘truly” MSCs like MSCs from bone marrow.

scholarly journals Characterization of adipose tissue macrophages and adipose-derived stem cells in critical wounds

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e2824 ◽  
Author(s):  
Bong-Sung Kim ◽  
Pathricia V. Tilstam ◽  
Katrin Springenberg-Jung ◽  
Arne Hendrick Boecker ◽  
Corinna Schmitz ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Adipose Tissue ◽  
Stem Cell ◽  
Subcutaneous Adipose Tissue ◽  
Stem Cell Markers ◽  
Adipose Derived Stem Cells ◽  
Cell Markers ◽  
Adipose Tissue Macrophages ◽  
Subcutaneous Adipose

Background Subcutaneous adipose tissue is a rich source of adipose tissue macrophages and adipose-derived stem cells which both play a key role in wound repair. While macrophages can be divided into the classically-activated M1 and the alternatively-activated M2 phenotype, ASCs are characterized by the expression of specific stem cell markers. Methods In the present study, we have investigated the expression of common macrophage polarization and stem cell markers in acutely inflamed adipose tissue. Subcutaneous adipose tissue adjacent to acutely inflamed wounds of 20 patients and 20 healthy subjects were harvested and underwent qPCR and flow cytometry analysis. Results Expression levels of the M1-specific markers CD80, iNOS, and IL-1b were significantly elevated in inflammatory adipose tissue when compared to healthy adipose tissue, whereas the M2-specific markers CD163 and TGF-β were decreased. By flow cytometry, a significant shift of adipose tissue macrophage populations towards the M1 phenotype was confirmed. Furthermore, a decrease in the mesenchymal stem cell markers CD29, CD34, and CD105 was observed whereas CD73 and CD90 remained unchanged. Discussion This is the first report describing the predominance of M1 adipose tissue macrophages and the reduction of stem cell marker expression in acutely inflamed, non-healing wounds.

scholarly journals Mitochondrial Activity Regulates Skin-derived Mesenchymal Stem Cell Differentiation Into Brown Adipocyte Contributing to Hypertension

2020 ◽  
Author(s):  
Da Wen Xi ◽  
Dong Wen Chen ◽  
Hong Wei Sun ◽  
Xiao Xiang Li ◽  
Min Zhi Suo ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Stem Cells ◽  
Flow Cytometry ◽  
Adipose Tissue ◽  
Oxygen Consumption ◽  
Mitochondrial Activity ◽  
Ang Ii ◽  
Direct Role ◽  
Brown Adipose

Abstract Background: Brown adipocytes (BAs) are the major component of brown adipose tissue (BAT) that is closely related to systemic hypertension. BAs are derived from multiple progenitors including PDGFRα+ adipose-derived stem cells (ASCs). Skin-derived Mesenchymal Stem Cells (S-MSCs) have the capacity to differentiate into adipocytes. However, the differentiation of S-MSCs into BAs remains unexplored. We aim to study the ability and regulation mechanism of S-MSCs differentiation into BAs, and the direct role of BAT in blood pressure regulation. Methods: Protein expression was measured by Flow Cytometry or Western blotting, and gene mRNA levels were detected by real-time quantitative PCR (RT-PCR). For the BA differentiation of S-MSCs, S-MSCs were stimulated with a brown adipogenic cocktail containing insulin, IBMX, dexamethasone, triiodothyronine (T3), and rosiglitazone for the indicated periods. The oxygen consumption rate (OCR) was measured with an XF24 Extracellular Flux Analyzer. Mitochondrial mass was checked by flow cytometry and fluorescence staining. Hypertensive mouse model was induced in WT mice by infusion with angiotensin II (Ang II) and measured SBP using tail-cuff. The interscapular brown adipose tissue (iBAT)-deficiency mice were gotten by surgically removing the iBAT depot and allowed to recover for 6 days. Aorta, iBAT or heart tissue sections were examined by hematoxylin and eosin (HE) staining. Results: We found that S-MSCs isolated from the mouse dermis expressed the stem cell markers CD90/105 and PDGFRα, and readily differentiated into BAs. Mitochondrial biogenesis and oxygen consumption were markedly increased during BA differentiation of S-MSCs in vitro. Another, Ang II-induced hypertensive mice carried the change of iBAT to white adipose tissue (WAT), the enhanced Ang II-induced blood pressure and vascular remodeling were observed in BAT-deficient mice generated by surgically removing iBAT comparing with C57BL/6 (wild type-WT) mice. Conclusions: S-MSCs represent a useful in vitro model for differentiation of BAs regulated by mitochondrial activity, and are progenitors of BAs. This study indicates that PDGFRα+ S-MSCs could differentiate into BAs, and BAT plays a direct role in Ang II-induced hypertension and target organ remodeling.

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