scholarly journals Comparison of the Migration Potential through Microperforated Membranes of CD146+ GMSC Population versus Heterogeneous GMSC Population

2021 ◽  
Vol 2021 ◽  
pp. 1-14
Author(s):  
Mohamed Al Bahrawy
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Doubling Time ◽  
Population Doubling ◽  
Stem Cell Population ◽  
Population Doubling Time ◽  
Homogeneous Population ◽  
Magnetic Sorting ◽  
Migration Dynamics ◽  
Lower Compartment

Background. Guided tissue regeneration (GTR) is a powerful modality for periodontal regeneration, but it blocks the periosteum and gingival stem cells (GMSCs), from supporting periodontal wound by the nutrients, growth factors, and regenerative cells. The microperforated membrane considered a rewarding solution for this major drawback; GMSCs can migrate through a GTR microperforated membrane toward a chemoattractant, with the blocking of other unfavorable epithelial cells and fibroblasts. In the absence of a sole marker for MSC, a homogeneous population of GMSC is difficult to isolate; using CD146 as confirmatory markers for MSC identification, testing the behaviour of such homogeneous population in migration dynamics was the question to answer in this study. Materials and Methods. GMSCs from healthy crown lengthening tissue was isolated ( n = 3 ), its stem cell nature was confirmed, CD146 and CD271 markers were confirmatory markers to confirm homogenous stem cell population, and magnetic sorting was used to isolate GMSC with CD146 markers. A homogenous CD146 population was compared to heterogeneous GMSCs of origin; the population doubling time and MTT test of the two populations were compared. Migration dynamics were examined in a transwell migration chamber through 8 μm perforated polycarbonic acid membrane, and 0.4 μm and 3 μm perforated collagen-coated polytetrafluoroethylene membrane (PTFE) and 10% fetal bovine serum (FBS) were the chemoattractants used in the lower compartment to induce cell migration, were incubated in a humidified environment for 24 hours, then migrated the cell in the lower compartment examined by a light and electron microscope. Results. GMSCs fulfilled all the minimal criteria of stem cells and showed low signal 10% for CD146 on average and extremely low signal 2% for CD271 on average. Magnetic sorting optimized the signal of CD146 marker to 55%. GMSC CD146 population showed nonstatistically significant shorter population doubling time. CD146 homogeneous population migrated cell numbers were statistically significant compared to the heterogeneous population, through 0.4 μm and 3 μm perforated collagen membrane and 8 μm perforated polycarbonate membrane. Scanning electron microscopy proved the migration of the cells. Conclusions. A subset of the isolated GMSC showed a CD146 marker, which is considered a dependable confirmatory marker for the stem cells. In terms of GMSC migration through the microperforated membrane, a homogeneous CD146 population migrates more statistically significant than a heterogeneous GMSC population.

scholarly journals Characterization of deciduous teeth stem cells isolated from crown dental pulp

2014 ◽  
Vol 71 (8) ◽  
pp. 735-741 ◽  
Author(s):  
Jasmina Debeljak-Martacic ◽  
Jelena Francuski ◽  
Tijana Luzajic ◽  
Nemanja Vukovic ◽  
Slavko Mojsilovic ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Dental Pulp ◽  
Doubling Time ◽  
Deciduous Teeth ◽  
Population Doubling ◽  
Population Doubling Time ◽  
Differentiation Potential

Background/Aim. The last decade has been profoundly marked by persistent attempts to use ex vivo expanded and manipulated mesenchymal stem cells (MSCs), as a tool in different types of regenerative therapy. In the present study we described immunophenotype and the proliferative and differentiation potential of cells isolated from pulp remnants of exfoliated deciduous teeth in the final phase of root resorption. Methods. The initial adherent cell population from five donors was obtained by the outgrowth method. Colony forming unit-fibroblast (CFU-F) assay was performed in passage one. Cell expansion was performed until passage three and all tests were done until passage eight. Cells were labeled for early mesenchymal stem cells markers and analysis have been done using flow cytometry. The proliferative potential was assessed by cell counting in defined time points and population doubling time was calculated. Commercial media were used to induce osteoblastic, chondrogenic and adipogenic differentiation. Cytology and histology methods were used for analysis of differentiated cell morphology and extracellular matrix characteristics. Results. According to immunophenotype analyses all undifferentiated cells were positive for the mesenchymal stem cell markers: CD29 and CD73. Some cells expressed CD146 and CD106. The hematopoietic cell marker, CD34, was not detected. In passage one, incidence of CFU-F was 4.7 ? 0.5/100. Population doubling time did not change significantly during cell subcultivation and was in average 25 h. After induction of differentiation, the multicolony derived cell population had a tri-lineage differentiation potential, since mineralized matrix, cartilage-like tissue and adipocytes were successfully formed after three weeks of incubation. Conclusion. Altogether, these data suggest that remnants of deciduous teeth dental pulp contained cell populations with mesenchymal stem cell-like features, with a high proliferation and trilineage differentiation potential and that these cultures are suitable for further in vitro evaluation of cell based therapies.

scholarly journals EXPRESSION OF PROTEIN CD73/CD90/CD105/CD34/CD45/CD11b/CD19/HLA-DR ON STEM CELLS FROM HUMAN FAT TISSUE, USING CYTOMETRY FLOW

2019 ◽  
Vol 12 (2) ◽  
pp. 133-141
Author(s):  
Imam Rosadi ◽  
Karina Karina ◽  
Iis Rosliana ◽  
Siti Sobariah ◽  
Irsyah Afini ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Doubling Time ◽  
Stromal Vascular Fraction ◽  
Population Doubling ◽  
Population Doubling Time ◽  
Surface Markers ◽  
Cell Surface Markers ◽  
Fat Tissue ◽  
Hla Dr

AbstrakSel punca merupakan sel yang dapat membelah dan berdiferensiasi menjadi sel jenis lainnya. Sel punca asal jaringan lemak potensial dikembangkan sebagai salah satu alternatif sel punca yang bersumber dari limbah sedot lemak manusia. Sel punca asal jaringan lemak akan mengekspresikan protein spesifik penanda permukaan CD73, CD90, CD105 dalam persentase yang tinggi dan CD34/CD45/CD11b/CD19/HLA-DR dalam persentase yang rendah. Studi ini bertujuan untuk memanfaatkan limbah sedot lemak manusia dengan melakukan isolasi sel punca asal jaringan lemak dan menguji protein penanda permukaan spesifik sel punca. Beberapa tahapan dalam studi ini adalah isolasi stromal vascular fraction (SVF) dan kultur sel punca asal jaringan lemak manusia, population doubling time (PDT) serta analisis protein penanda permukaan CD7Ee3, CD90, CD105, dan CD34/CD45/CD11b/CD19/HLA-DR pada pasase ke-1 dari 3 donor. Hasil dari studi ini menunjukkan bahwa sel dari jaringan lemak berhasil dikultur dengan durasi pembelahan sel adalah 3,3 hari. Sel mengekspresikan CD73 (99,79%), CD90 (94,17%), CD105 (48,75%), dan CD34/CD45/CD11b/CD19/HLA-DR (kurang dari 2%). Ekspresi CD105 yang rendah dari ketiga donor diduga berkaitan dengan tingkatan pasase sel yang digunakan. Berdasarkan hasil tersebut dapat disimpulkan bahwa sel punca asal jaringan lemak pasase ke-1 telah mengekspresikan ketiga marker protein penanda permukaan sel punca, yaitu CD73, CD90 dan CD105.Abstract Stem cells are cells that can divide into other different types of similar cells. Stem cells from fat tissue potential have been developed as an alternative stem cell from human liposuction. Stem cells from fat tissue will express high protein-specific markers on CD73, CD90, CD105 and CD34/CD45/CD11b/CD19/HLA-DR in a low percentage. This study aims to utilize human liposuction waste by isolating stem cells from fat tissue and testing protein-specific stem cell surface markers. Some stages in this study are isolation of stromal vascular fraction (SVF) and stem cell culture from human fat tissue, population doubling time (PDT) and protein analysis of surface markers CD73, CD90, CD105, and CD34/CD45/CD11b/CD19/HLA-DR on the 1st passage of 3 donors. The results of this study showed that cells from fat tissue were successfully cultured with cell division duration of 3.3 days. Cells expressed CD73 (99.79%), CD90 (94.17%), CD105 (48.75%), and CD34/CD45/CD11b/CD19/HLA-DR (less than 2%). The low expression of CD105 from all three donors is thought to be related to the level of cell passage used. Based on these results, it can be concluded that the stem cells from first passage fat tissue have expressed the three protein markers of stem cell surface markers, namely CD73, CD90 and CD105.

scholarly journals A Comparative Study of Biological Characteristics and Transcriptome Profiles of Mesenchymal Stem Cells from Different Canine Tissues

2019 ◽  
Vol 20 (6) ◽  
pp. 1485 ◽  
Author(s):  
Xiao-Shu Zhan ◽  
Saeed El-Ashram ◽  
Dong-Zhang Luo ◽  
Hui-Na Luo ◽  
Bing-Yun Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Umbilical Cord ◽  
Doubling Time ◽  
Biological Characteristics ◽  
Population Doubling ◽  
Population Doubling Time ◽  
Third Generation ◽  
The Third

Mesenchymal stem cells (MSCs) are the most promising seed cells for cell therapy. Comparing the biological and transcriptome gene characteristics of MSCs from different sources provides an important basis for the screening of clinically used cells. The main purpose of this experiment was to establish methods for the isolation and culture of MSCs from five different canine sources, including adipose tissue, bone marrow, umbilical cord, amniotic membrane, and placenta, and compare biological and transcriptome characteristics of MSCs, in order to provide a basis for the clinical application of canine MSCs. MSCs were isolated from Chinese pastoral dogs, and the following experiments were performed: (1) the third, sixth, and ninth generations of cells were counted, respectively, and a growth curve was plotted to calculate the MSC population doubling time; (2) the expression of CD34 and CD44 surface markers was studied by immunofluorescence; (3) the third generation of cells were used for osteogenetic and adipogenic differentiation experiments; and (4) MSC transcriptome profiles were performed using RNA sequencing. All of the five types of MSCs showed fibroblast-like adherent growth. The cell surface expressed CD44 instead of CD34; the third-generation MSCs had the highest proliferative activity. The average population doubling time of adipose mesenchymal stem cells (AD-MSCs), placenta mesenchymal stem cells (P-MSCs), bone marrow mesenchymal stem cells (BM-MSCs), umbilical cord mesenchymal stem cells (UC-MSCs), and amniotic mesenchymal stem cells (AM-MSCs) were 15.8 h, 21.2 h, 26.2 h, 35 h, and 41.9 h, respectively. All five types of MSCs could be induced to differentiate into adipocytes and osteoblasts in vitro, with lipid droplets appearing after 8 days and bone formation occurring 5 days after AD-MSC induction. However, the multilineage differentiation for the remaining of MSCs was longer compared to that of the AD-MSCs. The MSC transcriptome profiles showed that AD-MSC and BM-MSCs had the highest homology, while P-MSCs were significantly different compared to the other four types of MSCs. All the isolated MSCs had the main biological characteristics of MSCs. AD-MSCs had the shortest time for proliferation, adipogenesis, and osteogenic differentiation.

Iron Overload Impairs Hematopoiesis by Damaging MSCs Through ROS Signaling Pathway.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2097-2097
Author(s):  
Wenyi Lu ◽  
Mingfeng Zhao ◽  
Xiao Chai ◽  
Juanxia Meng ◽  
Fang Xie ◽  
...  
Keyword(s):  
Stem Cells ◽  
Iron Overload ◽  
Signaling Pathways ◽  
Signaling Pathway ◽  
Doubling Time ◽  
Iron Chelation ◽  
Extracellular Matrix Protein ◽  
Population Doubling ◽  
Population Doubling Time ◽  
Hematopoietic Stem

Abstract Abstract 2097 Iron is a useful component of cytochromes, oxygen-binding molecules and some enzymes due to its capacity to accept and donate electrons readily. However, excessive iron accumulation can damage tissues and cells by catalyzing the conversion of superoxide and hydrogen peroxide to free radical species that can attack cellular membranes, proteins and DNA. Recent multiple data revealed that iron chelation therapy was effective in treating cytopenia in iron overload disease, which supported the idea that iron overload affected hematopoiesis in bone marrow(BM). Based on these findings, We demonstrated that iron overload suppressed hematopoiesis by inhibiting hematopoietic stem/progenitor cells and the effects could be restored by iron chelation or anti-oxidants(Zhao et al., Blood, 2010, 116:4247a). However, it is unclear whether iron overload can impair BM hematopoiesis by injuring the microenvironment. As an important component of the BM microenvironment, Mesenchymal stem cells (MSCs) secrete a large amount of cytokines and extracellular matrix protein which provides a favorable platform for the localization, self-renewal, and differentiation of hematopoietic stem cells. Here we hypothesize that iron overload impairs BM microenvironment by affecting the function and survival of MSCs which is mediated by ROS. In this study we first established an iron overload model of MSCs by adding ferric ammonium citrae (FAC) to the culture medium. To confirm this model, the labile iron pool (LIP) level of MSCs was detected using the calcein-AM method. We found that the LIP of MSCs was significantly higher than control and reached the highest level when cultured at 400μmol/L FAC for 12h. Next we analyzed whether iron overload can affect proliferation, apoptosis and function of MSCs by the following experiments. Firstly, the proliferation of MSCs was evaluated using population doubling time (DT). Under iron overload, the population doubling time (DT) of MSCs was 24.43± 2.72 hours, which was signifcantly longer than control(16.03± 2.31 hours; P=0.015). However, the difference wasn't significant after two passages (P=0.936). Possible explanation could be that the injury to MSCs is reversible following decreased concentration of iron after passaging. Secondly, the apoptosis of MSCs altered by iron overload was measured by staining Annexin V/PI, and we found the apoptosis rate was higher in the iron overload group(12.75±0.32%) than control (3.63±0.80%)(P<0.05). Finally, mono-nuclear cells were purified from umbilical cord blood and co-cultured with MSCs to assess the hematopoiesis-supportive function of MSCs. Iron overload group showed decreased hematopoietic support capacity than control. Taken together, these findings proved iron overload impaired hematopoietic microenvironment by decreasing proliferation, inducing apoptosis and injuring the hematopoietic support capacity of MSCs. We then explored the possible mechanism that may take part in this process. It has already been reported that iron overload may result in the generation of reactive oxygen species (ROS). Similarly, we found that ROS level of MSCs could be positively correlated with the concentration of FAC and reached its highest level when cultured at 400μmol/L FAC for 12h. Finally, Western blot analysis of whole cell lysates from umbilical derived MSC using antibodies recognizing known ROS-related signaling pathways revealed robust increases in phospho-p38, p53 in response to FAC compared with control, with inhibition of these signaling pathways noted in response to NAC or GSH at suitable dose, suggesting that antioxidant can inhibit ROS-induced signaling pathway in iron overload. In conclusion, Our finding indicates that iron overload can injure hematopoiesis by enhancing oxidative stress in MSC. Our data further suggests creatively that antioxidant and cytotherapy may be an effective method in curing deficient hematopoiesis in iron overload. Disclosures: No relevant conflicts of interest to declare.

Abstract 62: Pim-1 Engineering of Human CPCs Increases Telomere Length from Aged Patients with Heart Failure

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Sadia Mohsin ◽  
Mohsin Khan ◽  
Kathleen Wallach ◽  
Travis Cottage ◽  
Michael Mcgregor ◽  
...  
Keyword(s):  
Heart Failure ◽  
Stem Cell ◽  
Elderly Patients ◽  
Doubling Time ◽  
Telomerase Activity ◽  
Population Doubling ◽  
Population Doubling Time ◽  
Cardiac Progenitor Cells ◽  
Senescent Phenotype ◽  
Patients With Heart Failure

Rationale: Adoptive transfer of human cardiac progenitor cells (hCPCs) can repair damaged myocardium and improve function in a pathologically challenged heart. Aging can exhaust hCPC pool and pose a challenge to effectively regenerating the damaged myocardium. There is a dire need to reverse the senescent phenotype of these hCPCs isolated from elderly patients to target large segment of population suffering from heart failure. Objective: Demonstrate that Pim-1 engineering of human cardiac progenitor cells (hCPCs) isolated from elderly patients with heart failure can increase the telomere lengths, telomerase activity, population doubling and reverse their senescent phenotype. Methods and Results: hCPCs positive for the putative stem cell marker c-kit were isolated from heart biopsy samples from patients undergoing Left Ventricular Assist Device (LVAD) implantation. hCPCs isolated from patients aged 75-85 years (n=3) with heart failure demonstrate decreased population doubling time concomitant with decreased telomere lengths measured by in situ hybridization and telomerase activity measured by TRAP assay compared to patients aged 60-73 years (n=3). hCPCs were engineered to express Pim-1-GFP (hCPCeP), a fused GFP version of the kinase by using a lentivirus expression system. The cell cycling time measured by population doubling is increased after over expression of Pim-1 in hCPCs isolated from old patients. Pim-1 modification significantly increased telomere lengths and telomerase activity indicative of reversal of senescent characteristics of aged hCPCs. Conclusion: Ex vivo gene delivery of Pim-1 enhances telomere length, telomerase activity and decreases population-doubling time. Modification of aged hCPCs from elderly patients augments their ability to regenerate damaged myocardium, making stem cell engineering a viable option to address current limitations associated with senescent phenotype of aged hCPCs.

scholarly journals Molecular Aspects of Adipose-Derived Stromal Cell Senescence in a Long-Term Culture: A Potential Role of Inflammatory Pathways

2020 ◽  
Vol 29 ◽  
pp. 096368972091734 ◽  
Author(s):  
Marta Pokrywczynska ◽  
Małgorzata Maj ◽  
Tomasz Kloskowski ◽  
Monika Buhl ◽  
Daria Balcerczyk ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Doubling Time ◽  
Population Doubling ◽  
Population Doubling Time ◽  
Proliferative Potential ◽  
Differentiation Potential ◽  
Long Term Culture ◽  
Stromal Stem Cells

Long-term culture of mesenchymal stromal/stem cells in vitro leads to their senescence. It is very important to define the maximal passage to which the mesenchymal stromal/stem cells maintain their regenerative properties and can be used for cellular therapies and construction of neo-organs for clinical application. Adipose-derived stromal/stem cells were isolated from porcine adipose tissue. Immunophenotype, population doubling time, viability using bromodeoxyuridine assay, MTT assay, clonogencity, β-galactosidase activity, specific senescence-associated gene expression, apoptosis, and cell cycle of adipose-derived mesenchymal stromal/stem cells (AD-MSCs) were analyzed. All analyses were performed through 12 passages (P). Decreasing viability and proliferative potential of AD-MSCs with subsequent passages together with prolonged population doubling time were observed. Expression of β-galactosidase gradually increased after P6. Differentiation potential of AD-MSCs into adipogenic, chondrogenic, and osteogenic lineages decreased at the end of culture (P10). No changes in the cell cycle, the number of apoptotic cells and expression of specific AD-MSC markers during the long-term culture were revealed. Molecular analysis showed increased expression of genes involved in activation of inflammatory response. AD-MSCs can be cultured for in vivo applications without loss of their properties up to P6.

scholarly journals Differences in the Osteogenic Differentiation Capacity of Omental Adipose-Derived Stem Cells in Obese Patients With and Without Metabolic Syndrome

Endocrinology ◽  
2015 ◽  
Vol 156 (12) ◽  
pp. 4492-4501 ◽  
Author(s):  
Wilfredo Oliva-Olivera ◽  
Antonio Leiva Gea ◽  
Said Lhamyani ◽  
Leticia Coín-Aragüez ◽  
Juan Alcaide Torres ◽  
...  
Keyword(s):  
Metabolic Syndrome ◽  
Stem Cells ◽  
Nicotinamide Adenine Dinucleotide ◽  
Doubling Time ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Normal Weight ◽  
Population Doubling ◽  
Osteogenic Potential ◽  
Population Doubling Time ◽  
Mrna Expression Levels

Multiple studies have suggested that the reduced differentiation capacity of multipotent adipose tissue-derived mesenchymal stem cells (ASCs) in obese subjects could compromise their use in cell therapy. Our aim was to assess the osteogenic potential of omental ASCs and to examine the status of the isolated CD34negative-enriched fraction of omental-derived ASCs from subjects with different metabolic profiles. Omental ASCs from normal-weight subjects and subjects with or without metabolic syndrome were isolated, and the osteogenic potential of omental ASCs was evaluated. Additionally, osteogenic and clonogenic potential, proliferation rate, mRNA expression levels of proteins involved in redox balance, and fibrotic proteins were examined in the CD34negative-enriched fraction of omental-derived ASCs. Both the omental ASCs and the CD34negative-enriched fraction of omental ASCs from subjects without metabolic syndrome have a greater osteogenic potential than those from subjects with metabolic syndrome. The alkaline phosphatase and osteonectin mRNA were negatively correlated with nicotinamide adenine dinucleotide phosphate oxidase-2 mRNA and the mRNA expression levels of the fibrotic proteins correlated positively with nicotinamide adenine dinucleotide phosphate oxidase-5 mRNA and the homeostasis model assessment. Although the population doubling time was significantly higher in subjects with a body mass index of 25 kg/m2 or greater, only the CD34negative-enriched omental ASC fraction in the subjects with metabolic syndrome had a higher population doubling time than the normal-weight subjects. The osteogenic, clonogenic, fibrotic potential, and proliferation rate observed in vitro suggest that omental ASCs from subjects without metabolic syndrome are more suitable for therapeutic osteogenic applications than those from subjects with metabolic syndrome.

scholarly journals Stem Cells from Human Exfoliated Deciduous Teeth – Isolation, Long Term Cultivation and Phenotypical Analysis

2010 ◽  
Vol 53 (2) ◽  
pp. 93-99 ◽  
Author(s):  
Jakub Suchánek ◽  
Benjamín Víšek ◽  
Tomáš Soukup ◽  
Sally Kamal El-Din Mohamed ◽  
Romana Ivančaková ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Dental Pulp ◽  
Biological Properties ◽  
Permanent Teeth ◽  
Deciduous Teeth ◽  
Population Doubling ◽  
Population Doubling Time ◽  
Human Exfoliated Deciduous Teeth

Aims: Our aims were to isolate stem cells from human exfoliated deciduous teeth (SHED), to cultivate them in vitro and to investigate their basic biological properties, phenotype and to compare our findings with dental pulp stem cells (DPSC) isolated from permanent teeth. Methods: Dental pulp was gently evacuated from exfoliated teeth. After enzymatic dissociation of dental pulp, SHED were cultivated in modified cultivation media for mesenchymal adult progenitor cells containing 2 % FCS and supplemented with growth factors and insulin, transferrin, sodium (ITS) supplement. Cell viability and other biological properties were examined using a Vi-Cell analyzer and a Z2-Counter. DNA analyses and phenotyping were performed with flow cytometry. Results: We were able to cultivate SHED over 45 population doublings. Our results showed that SHED cultivated under same conditions as DPSC had longer average population doubling time (41.3 hrs for SHED vs. 24.5 hrs for DPSC). Phenotypic comparison of cultivated SHED to that of cultivated DPSC showed differential expression CD29, CD44, CD71, CD117, CD166. During long-term cultivation, SHED did not showed any signs of degeneration or spontaneous differentiation. Conclusions: We isolated stem cells from exfoliated teeth. In comparison to DPSC, SHED proliferation rate was about 50% slower, and SHED showed slightly different phenotype. These cells may be extremely useful for stem cell tissue banking, further stem cell research and future therapeutic applications.

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