scholarly journals Phenotypic and Functional Properties of Porcine Dedifferentiated Fat Cells during the Long-Term CultureIn Vitro

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Xuewu Peng ◽  
Tongxing Song ◽  
Xiaoming Hu ◽  
Yuanfei Zhou ◽  
Hongkui Wei ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometric Analysis ◽  
Biological Properties ◽  
Population Doubling ◽  
Population Doubling Time ◽  
Long Term Culture ◽  
Multiple Differentiation ◽  
Dfat Cells

It has been proved that terminally differentiated mature adipocytes possess abilities to dedifferentiate into fibroblast-like progeny cells with self-renewal and multiple differentiation, termed dedifferentiated fat (DFAT) cells. However, the biological properties of DFAT cells during long-term culturein vitrohave not been elucidated. Here, we obtained fibroblast-like morphology of porcine DFAT cells by ceiling culture. During the dedifferentiation process, round mature adipocytes with single large lipid droplets changed into spindle-shaped cells accompanied by the adipogenic markersPPARγ,aP2,LPL, andAdiponectinsignificant downregulation. Flow cytometric analysis showed that porcine DFAT cells displayed similar cell-surface antigen profile to mesenchymal stem cells (MSCs). Furthermore, different passages of porcine DFAT cells during long-term culturein vitroretained high levels of cell viabilities (>97%), efficient proliferative capacity including population doubling time ranged from 20 h to 22 h and population doubling reached47.40±1.64by 58 days of culture. In addition, porcine DFAT cells maintained the multiple differentiation capabilities into adipocytes, osteoblasts, and skeletal myocytes and displayed normal chromosomal karyotypes for prolonged passaging. Therefore, porcine DFAT cells may be a novel model of stem cells for studying the functions of gene in the different biological events.

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 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.

scholarly journals Low Oxygen Tension Potentiates Proliferation and Stemness but Not Multilineage Differentiation of Caprine Male Germline Stem Cells

2021 ◽  
Author(s):  
Shiva Pratap Singh ◽  
Suresh Dinkar Kharche ◽  
Manisha Pathak ◽  
Ravi Ranjan ◽  
Yogesh Kumar Soni ◽  
...  
Keyword(s):  
Stem Cells ◽  
Ex Vivo ◽  
Growth Characteristics ◽  
Population Doubling ◽  
Population Doubling Time ◽  
Germline Stem Cells ◽  
Multilineage Differentiation ◽  
Low Oxygen ◽  
Differentiation Potential

Abstract The milieu of testicular germline stem cells (mGSCs) is characterized as low oxygen (O2) environment, whereas, there in-vitro expansion is typically performed under normoxia (20-21% O2). Here, we evaluated and compared the culture and multilineage differentiation characteristics of enriched (through differential platting and percoll density centrifugation) caprine mGSCs (cmGSCs) under hypoxic (5% O2) and normoxic (21% O2) culture conditions. For this, in addition to growth characteristics and population-doubling time (PDT); viability, proliferation, senescence, and expression of key-markers of adhesion (β-integrin and E-Cadherin) and stemness (OCT-4, THY-1 and UCHL-1) were evaluated and compared under normoxia and hypoxia. Moreover, the extent of multilineage differentiation (neurogenic, adipogenic, and chondrogenic differentiation) was assessed. The survival, viability and proliferation were significantly promoted and PDT was reduced (p < 0.05), thus yielding a higher number of viable cells with larger colonies under hypoxia. Furthermore, expression of stemness and adhesion markers was distinctly increased under lowered O2 condition. Conversely, the presence of differentiated regions and expression of differentiation specific key genes [C/EBPα (adipogenic), nestin and β-tubulin (neurogenic), and COL2A1 (chondrogenic)] were significantly (p < 0.05) reduced under hypoxic conditions. These data demonstrate that culturing cmGSCs under hypoxia augments the growth characteristics, and stemness but not the multilineage differentiation potential of cmGSCs as compared with normoxia. These data are important for the development of robust methodologies for ex-vivo expansion and lineage-committed differentiation of cmGSCs for clinical applications.

scholarly journals Soft Substrate Maintains Proliferative and Adipogenic Differentiation Potential of human Mesenchymal Stem Cells on Long Term Expansion by Delaying Senescence

2018 ◽  
Author(s):  
Sanjay K. Kureel ◽  
Pankaj Mogha ◽  
Akshada Khadpekar ◽  
Vardhman Kumar ◽  
Rohit Joshi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Culture System ◽  
Human Mesenchymal Stem Cells ◽  
Population Doubling ◽  
Differentiation Potential ◽  
Lineage Differentiation ◽  
Poly Acrylamide

AbstractHuman mesenchymal stem cells (hMSCs), when cultured on tissue culture plate (TCP) for in vitro expansion, they spontaneously lose their proliferative capacity and multi-lineage differentiation potential. They also lose their distinct spindle morphology and become large and flat. After a certain number of population doubling, they enter into permanent cell cycle arrest, called senescence. This is a major roadblock for clinical use of hMSCs which demands large number of cells. A cell culture system is needed which can maintain the stemness of hMSCs over long term passages yet simple to use. In this study, we explore the role of substrate rigidity in maintaining stemness. hMSCs were serially passaged on TCP and 5 kPa poly-acrylamide gel for 20 population doubling. It was found that while on TCP, cell growth reached a plateau at cumulative population doubling (CPD) = 12.5, on 5 kPa gel, they continue to proliferate linearly till we monitored (CPD = 20). We also found that while on TCP, late passage MSCs lost their adipogenic potential, the same was maintained on soft gel. Cell surface markers related to MSCs were also unaltered. We demonstrated that this maintenance of stemness was correlated with delay in onset of senescence, which was confirmed by β-gal assay and by differential expression of vimentin, Lamin A and Lamin B. As preparation of poly-acrylamide gel is a simple, well established, and well standardized protocol, we believe that this system of cell expansion will be useful in therapeutic and research applications of hMSCs.One Sentence SummaryhMSCs retain their stemness when expanded in vitro on soft polyacrylamide gel coated with collagen by delaying senescence.Significance StatementFor clinical applications, mesenchymal stem cells (MSCs) are required in large numbers. As MSCs are available only in scarcity in vivo, to fulfill the need, extensive in vitro expansion is unavoidable. However, on expansion, they lose their replicative and multi-lineage differentiation potential and become senescent. A culture system that can maintain MSC stemness on long-term expansion, without compromising the stemness, is need of the hour. In this paper, we identified polyacrylamide (PAA) hydrogel of optimum stiffness that can be used to maintain stemness of MSCs during in vitro long term culture. Large quantity of MSCs thus grown can be used in regenerative medicine, cell therapy, and in treatment of inflammatory diseases.

Gelatin/Hydroxyapatite Scaffolds: Studies on Adhesion, Growth and Differentiation of Mesenchymal Stem Cells

2010 ◽  
Vol 93-94 ◽  
pp. 121-124
Author(s):  
Nuttapon Vachiraroj ◽  
Siriporn Damrongsakkul ◽  
Sorada Kanokpanont
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Bone Tissue Engineering ◽  
Bone Tissue ◽  
Calcium Content ◽  
Population Doubling ◽  
Population Doubling Time ◽  
3 Dimensional

In this work, we developed a 3-dimensional bone tissue engineering scaffold from type B gelatin and hydroxyapatite. Two types of scaffolds, pure gelatin (pI~5) (Gel) and gelatin/hydroxyapatite (30/70 wt./wt.) (Gel/HA), were prepared from concentrated solutions (5% wt./wt.) using foaming/freeze drying method. The results SEM revealed the interconnected-homogeneous pores of Gel and Gel/HA were 121  119 and 148  83m, respectively. Hydroxyapatite improved mechanical property of the gelatin scaffolds, especially at dry state. Compressive modulus of Gel and Gel/HA scaffolds were at 118±21.68 and 510±109.08 kPa, respectively. The results on in vitro cells culture showed that Gel/HA scaffolds promoted attachment of rat’s mesenchymal stem cells (MSC) to a 1.23 folds higher than the Gel scaffolds. Population doubling time (PDT) of MSC on Gel and Gel/HA scaffolds were 51.16 and 54.89 hours, respectively. In term of osteogenic differentiation, Gel/HA scaffolds tended to enhance ALP activity and calcium content of MSC better than those of the Gel scaffold. Therefore the Gel/HA scaffolds had a potential to be applied in bone tissue engineering.

Comprehensive characterization of human adipose tissue-derived stem cells expanded in vitro

Biologia ◽  
2013 ◽  
Vol 68 (4) ◽  
Author(s):  
Ľuboš Danišovič ◽  
Marcela Kuniaková ◽  
Zuzana Varchulová-Nováková ◽  
Martin Boháč ◽  
Ivan Varga ◽  
...  
Keyword(s):  
Stem Cells ◽  
Adipose Tissue ◽  
Growth Curve ◽  
Morphological Characteristics ◽  
Human Adipose Tissue ◽  
Population Doubling ◽  
Population Doubling Time ◽  
Tem Analysis ◽  
Hla Dr

AbstractAdipose tissue seems to be a rich and safe source of mesenchymal stem cells (MSCs). The present study was aimed to investigate the biological and morphological characteristics of human adipose tissue-derived stem cells (ATSCs). Light and transmission electron microscopy were used. Course of proliferation was analyzed by growth curve. Expression of surface antigens was assessed by flow cytometry. Chondrogenic potential was assessed by immunohistochemistry. Obtained results showed morphology typical of fibroblastoid cells. TEM analysis proved ultrastructural morphology similar to MSCs from other sources. ATSCs reflected their proteosynthetic and metabolic activity. Each cell had irregular shape of nucleus with noticeable nucleoli. Abundant cisterns of rough endoplasmic reticulum were present in their cytoplasm. Karyotype mapping showed normal count of human chromosomes (46,XX). The growth curve revealed high capability for proliferation and population doubling time was 27.36 hours. ATSCs were positive for CD13, CD29, CD44, CD73, CD90, CD105 and CD106, but did not express CD14, CD34, CD45 and HLA-DR. It was also proved that ATSCs underwent chondrogenic differentiation in vitro. On the basis of obtained results it should be emphasized that ATSCs are typical MSCs and after further investigations they may be used in tissue engineering and regenerative medicine.

Comparative Analysis of Biological and Functional Properties of Bone Marrow Mesenchymal Stromal Cells Expanded in Media with Different Platelet Lysate Content

2018 ◽  
Vol 205 (4) ◽  
pp. 226-239 ◽  
Author(s):  
Marijana Skific ◽  
Mirna Golemovic ◽  
Kristina Crkvenac-Gornik ◽  
Radovan Vrhovac ◽  
Branka Golubic Cepulic
Keyword(s):  
Bone Marrow ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Immunological Tolerance ◽  
Biological Properties ◽  
Platelet Lysate ◽  
Population Doubling ◽  
Population Doubling Time ◽  
Significant Difference

Due to their ability to induce immunological tolerance in the recipient, mesenchymal stromal cells (MSCs) have been utilized in the treatment of various hematological and immune- and inflammation-mediated diseases. The clinical application of MSCs implies prior in vitro expansion that usually includes the use of fetal bovine serum (FBS). The present study evaluated the effect of different platelet lysate (PL) media content on the biological properties of MSCs. MSCs were isolated from the bone marrow of 13 healthy individuals and subsequently expanded in three different culture conditions (10% PL, 5% PL, 10% FBS) during 4 passages. The cells cultured in different conditions had comparable immunophenotype, clonogenic potential, and differentiation capacity. However, MSC growth was significantly enhanced in the presence of PL. Cultures supplemented with 10% PL had a higher number of cumulative population doublings in all passages when compared to the 5% PL condition (p < 0.03). Such a difference was also observed when 10% PL and 10% FBS conditions were compared (p < 0.005). A statistically significant difference in population doubling time was determined only between the 10% PL and 10% FBS conditions (p < 0.005). Furthermore, MSCs cultured in 10% PL were able to cause a 66.9% reduction of mitogen-induced lymphocyte proliferation. Three chromosome aberrations were detected in PL conditions. Since two changes occurred in the same do nor, it is possible they were donor dependent rather than caused by the culture condition. These findings demonstrate that a 10% PL condition enables a higher yield of MSCs within a shorter time without altering MSC properties, and should be favored over the 5% PL condition.

Reprogramming of Oncogene Expression in Gingival Mesenchymal Stem Cells Following Long-Term Culture In Vitro

2017 ◽  
Vol 19 (3) ◽  
pp. 159-170 ◽  
Author(s):  
Agnese Gugliandolo ◽  
Thangavelu Soundara Rajan ◽  
Domenico Scionti ◽  
Francesca Diomede ◽  
Placido Bramanti ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Culture In Vitro ◽  
Long Term Culture ◽  
Oncogene Expression

scholarly journals The Influence of Aging on the Regenerative Potential of Human Adipose Derived Mesenchymal Stem Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-15 ◽  
Author(s):  
Monika Marędziak ◽  
Krzysztof Marycz ◽  
Krzysztof A. Tomaszewski ◽  
Katarzyna Kornicka ◽  
Brandon Michael Henry
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Adipogenic Differentiation ◽  
Population Doubling ◽  
Osteogenic Potential ◽  
Population Doubling Time ◽  
Joint Diseases ◽  
Differentiation Potential ◽  
Age Related

Tissue regeneration using human adipose derived mesenchymal stem cells (hASCs) has significant potential as a novel treatment for many degenerative bone and joint diseases. Previous studies have established that age negatively affects the proliferation status and the osteogenic and chondrogenic differentiation potential of mesenchymal stem cells. The aim of this study was to assess the age-related maintenance of physiological function and differentiation potential of hASCs in vitro. hASCs were isolated from patients of four different age groups: (1) >20 years (n=7), (2) >50 years (n=7), (3) >60 years (n=7), and (4) >70 years (n=7). The hASCs were characterized according to the number of fibroblasts colony forming unit (CFU-F), proliferation rate, population doubling time (PDT), and quantified parameters of adipogenic, chondrogenic, and osteogenic differentiation. Compared to younger cells, aged hASCs had decreased proliferation rates, decreased chondrogenic and osteogenic potential, and increased senescent features. A shift in favor of adipogenic differentiation with increased age was also observed. As many bone and joint diseases increase in prevalence with age, it is important to consider the negative influence of age on hASCs viability, proliferation status, and multilineage differentiation potential when considering the potential therapeutic applications of hASCs.

scholarly journals qPCR analysis of mesenchymal stem cell marker expression during the long-term culture of canine adipocyte derived stem cells

2020 ◽  
Vol 8 (4) ◽  
pp. 139-145
Author(s):  
Rut Bryl ◽  
Claudia Dompe ◽  
Maurycy Jankowski ◽  
Katarzyna Stefańska ◽  
Afsaneh Golkar Narenji ◽  
...  
Keyword(s):  
Stem Cells ◽  
Molecular Mechanisms ◽  
Cultured Cells ◽  
Stem Cell Marker ◽  
Pcr Analysis ◽  
Marker Genes ◽  
Long Term Culture ◽  
Marker Expression

AbstractDue to its availability and accessibility, adipose tissue has been the subject of various studies in many different medical fields and is believed to be a useful source of stem cells. The ability of ASCs to differentiate towards different cell lineages, with possibility of directing this differentiation, increases their possible clinical applications, and they have been widely employed in multiple therapies and treatment of different pathologies. However, a deeper understanding of the molecular mechanisms underlying the ASCs osteoblastic and chondrocyte differentiation may lead to novel applications treating a multitude of different bone-related diseases through techniques more likely meeting worldwide consensus. In this study, the RT-qPCR method was used to determine the changes in expression of ASC specific markers (CD105, CD73, CD14, CD34, CD90 and CD45) before and after long-term (14-day) in vitro cultures. To confirm the identity of the investigated cells, flow cytometry was used to evaluate the presence of positive (CD44, CD90) and negative (CD45, CD34) ASC markers. Overall, the results of the PCR analysis showed a significant change in expression of most of the marker genes, indicating significant changes in the cultured cells caused by their long-term culture, potentially altering their original stem-like characteristics.Running title: ASC marker expression during long-term in vitro culture

Sign in / Sign up

Export Citation Format

Share Document