Caprine Endometrial Mesenchymal Stromal Stem Cell: Multilineage Potential, Characterization, and Growth Kinetics in Breeding and Anestrous Stages

The endometrial layer of the uterus contains a population of cells with similar characteristics of mesenchymal stem cells (MSCs). In the present study, caprine endometrial mesenchymal stromal stem cells (En-MSCs) characters and differentiation potential to chondrogenic, osteogenic, and adipogenic cell lines as well as their growth kinetics in breeding and anestrous stages were evaluated. En-MSCs were enzymatically isolated from endometrial layer of the uterus of adult goats and were cultured and subcultured until passage 4. The growth kinetics and population doubling time (PDT) of caprine En-MSCs in breeding and anestrous stages were determined. En-MSCs in passage 4 were used for the karyotyping and differentiation into chondrocytes, osteocytes, and adipocytes. The PDT in anestrus phase was 40.6 h and in cyclic goats was 53 h. En-MSCs were fibroblast-like in all passages. The number of chromosomes was normal (2n=60) with no chromosomal instability. Chondrogenic, osteogenic, and adipogenic differentiation of En-MSCs was confirmed by staining with Alcian blue, Alizarin red, and Oil Red O, respectively. Caprine En-MSCs demonstrated to be an alternative source of MSCs for cell therapy purposes in regenerative medicine.

Download Full-text

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

Stem Cells International ◽

10.1155/2016/2152435 ◽

2016 ◽

Vol 2016 ◽

pp. 1-15 ◽

Cited By ~ 99

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.

Download Full-text

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

10.21203/rs.3.rs-420611/v1 ◽

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.

Download Full-text

Characterization of deciduous teeth stem cells isolated from crown dental pulp

Vojnosanitetski pregled ◽

10.2298/vsp1408735d ◽

2014 ◽

Vol 71 (8) ◽

pp. 735-741 ◽

Cited By ~ 3

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.

Download Full-text

The effect of aging on the biological and immunological characteristics of periodontal ligament stem cells

10.21203/rs.3.rs-17276/v2 ◽

2020 ◽

Author(s):

Xiaoyu Li ◽

Bowen Zhang ◽

Hong Wang ◽

Xiaolu Zhao ◽

Zijie Zhang ◽

...

Keyword(s):

Stem Cells ◽

Periodontal Ligament ◽

Adipogenic Differentiation ◽

Annexin V ◽

Aging Treatment ◽

Cell Counting ◽

Periodontal Ligament Stem Cells ◽

Alizarin Red ◽

Differentiation Potential ◽

Elderly Individuals

Abstract Background: Periodontal ligament stem cells (PDLSCs) have many applications in the field of cytotherapy, tissue engineering, and regenerative medicine. However, the effect of age on the biological characteristics and immunoregulation of PDLSCs remains unclear.Methods: In this study, we compared PDLSCs isolated from young and elderly individuals. PDLSCs proliferation was analyzed by Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) staining, and apoptosis level was detected by Annexin V-PE/7-ADD staining. PDLSCs osteogenic/adipogenic differentiation potentials were assessed by alkaline phosphatase (ALP), Alizarin Red, Oil Red O staining and related quantitative analysis. PDLSCs immunoregulatory capacity was determined by EdU and Annexin V-PE/7-ADD staining. To explore its underlying mechanism, microarray, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), and western blot analyses were performed to detect differentially expressed genes and proteins in PDLSCs. Results: Our results demonstrated that with aging, the proliferation and osteogenic/adipogenic differentiation potential of PDLSCs decreased, whereas apoptosis of PDLSCs increased. Moreover, the immunosuppressive ability of PDLSCs decreased with aging. Compared with PDLSCs from young subjects, analysis of mRNA expression revealed an upregulation of CCND3 and RC3H2 , and a downregulation of Runx2, ALP, COL1A1, PPARγ2, CXCL12, FKBP1A, FKBP1B, NCSTN, P2RX7, PPP3CB, RIPK2, SLC11A1, and TP53 in those from elderly individuals. Furthermore, protein expression levels of Runx2, ALP, COL1A1, and PPARγ2 in the elderly group was decreased, whereas that of CCND3 increased. Conclusions: Taken together, aging influences biological and immunological characteristics of PDLSCs, and thus it is more appropriate to utilized PDLSCs from young individuals for tissue regeneration, post-aging treatment, and allotransplantation.

Download Full-text

The effect of aging on the biological and immunological characteristics of periodontal ligament stem cells

10.21203/rs.3.rs-17276/v1 ◽

2020 ◽

Author(s):

Xiaoyu Li ◽

Bowen Zhang ◽

Hong Wang ◽

Xiaolu Zhao ◽

Zijie Zhang ◽

...

Keyword(s):

Stem Cells ◽

Periodontal Ligament ◽

Adipogenic Differentiation ◽

Annexin V ◽

Aging Treatment ◽

Cell Counting ◽

Periodontal Ligament Stem Cells ◽

Alizarin Red ◽

Differentiation Potential ◽

Elderly Individuals

Abstract Background Periodontal ligament stem cells (PDLSCs) have many applications in the field of cytotherapy, tissue engineering, and regenerative medicine. However, the effect of age on the biological characteristics and immunoregulation of PDLSCs remains unclear. Methods In this study, we compared PDLSCs isolated from young and elderly individuals. PDLSCs proliferation was analyzed by Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) staining, and apoptosis level was detected by Annexin V-PE/7-ADD staining. PDLSCs osteogenic/adipogenic differentiation potentials were assessed by alkaline phosphatase (ALP), Alizarin Red, Oil Red O staining and related quantitative analysis. PDLSCs immunoregulatory capacity was determined by EdU and Annexin V-PE/7-ADD staining. To explore its underlying mechanism, microarray, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), and western blot analyses were performed to detect differentially expressed genes and proteins in PDLSCs. Results Our results demonstrated that with aging, the proliferation and osteogenic/adipogenic differentiation potential of PDLSCs decreased, whereas apoptosis of PDLSCs increased. Moreover, the immunosuppressive ability of PDLSCs decreased with aging. Compared with PDLSCs from young subjects, analysis of mRNA expression revealed an upregulation of CCND3 and RC3H2 , and a downregulation of Runx2, ALP, COL1A1, PPARγ2, CXCL12, FKBP1A, FKBP1B, NCSTN, P2RX7, PPP3CB, RIPK2, SLC11A1, and TP53 in those from elderly individuals. Furthermore, protein expression levels of Runx2, ALP, COL1A1, and PPARγ2 in the elderly group was decreased, whereas that of CCND3 increased. Conclusions Taken together, aging influences biological and immunological characteristics of PDLSCs, and thus it is more appropriate to utilized PDLSCs from young individuals for tissue regeneration, post-aging treatment, and allotransplantation.

Download Full-text

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

Cell Transplantation ◽

10.1177/0963689720917341 ◽

2020 ◽

Vol 29 ◽

pp. 096368972091734 ◽

Cited By ~ 1

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.

Download Full-text

ID3013 Comparative characterization of murine Bone marrow mesenchymal stem cells cultured using two different supplements

Biomedical Research and Therapy ◽

10.15419/bmrat.v4is.233 ◽

2017 ◽

Vol 4 (S) ◽

pp. 25

Author(s):

Karuppiah Thilakavathy

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Culture Medium ◽

Population Doubling ◽

Population Doubling Time ◽

Differentiation Potential ◽

Comparative Characterization ◽

Medium Formulation

Preclinical studies on mesenchymal stem cells (MSC) have allowed the cells to be considered as a promising candidate for cellular therapy. The mouse is the most widely used species for studying the characteristics of MSC. In recent years, conflicting data were reported regarding growth kinetics, surface marker profile, differentiation capacity, genetic instability or malignant transformation and so forth, that may be a result of a range of factors. One of the factors probably is the culture medium formulation. Here we have made a comparative characterization of bone marrow-derived mesenchymal stem cells (mBM-MSC), under the same experimental conditions, cultured using two common supplements, fetal bovine serum (FBS) and MesenCultTM Stimulatory Supplement (MSS). mBM-MSC isolated from the tibias of C57BL/6 mice were cultured and expanded in Dulbecco’s Modified Eagle’s Medium supplemented with either 15% FBS or 15% MSS. Clonogenic potential, population doubling time, immunophenotyping, differentiation immunosuppression potentials and chromosome analysis of early and late passage of mBM-MSC were assessed. The findings showed that the immunophenotype and differentiation potential of mBM-MSC were similar when cultured using these supplements irrespective of passages. Variations were seen in clonogenic, growth, proliferation rate and immunosuppression potential of the mBM-MSC. This study also revealed that prolonged culture will disrupt their genetic stability regardless of the supplements used. The genetically mutated mBM-MSC were also found to maintain their stemness characteristics and immunosuppression potential. In conclusion, culture medium formulation causes variations in the cultured MSC and may influence downstream investigation findings.

Download Full-text

Comparative Assessment of Oral Mesenchymal Stem Cells Isolated from Healthy and Diseased Tissues

Microscopy and Microanalysis ◽

10.1017/s1431927615014749 ◽

2015 ◽

Vol 21 (5) ◽

pp. 1249-1263 ◽

Cited By ~ 5

Author(s):

Emöke Páll ◽

Adrian Florea ◽

Olga Soriţău ◽

Mihai Cenariu ◽

Adrian S. Petruţiu ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Cell Lines ◽

Specific Antigen ◽

Human Mesenchymal Stem Cells ◽

Culture Method ◽

Explant Culture ◽

Population Doubling ◽

Population Doubling Time ◽

Differentiation Potential

AbstractThe aim of the present study was to isolate human mesenchymal stem cells (MSCs) from palatal connective and periodontal granulation tissues and to comparatively evaluate their properties. MSCs were isolated using the explant culture method. Adherence to plastic, specific antigen makeup, multipotent differentiation potential, functionality, and ultrastructural characteristics were investigated. The frequency of colony-forming unit fibroblasts for palatal-derived mesenchymal stem cells (pMSCs) was significantly higher than that of granulation tissue-derived mesenchymal stem cells (gtMSCs). A significantly higher population doubling time and lower migration potential were recorded for gtMSCs than for pMSCs. Both cell lines were positive for CD105, CD73, CD90, CD44, and CD49f, and negative for CD34, CD45, and HLA-DR, but the level of expression was different. MSCs from both sources were relatively uniform in their ultrastructure. Generally, both cell lines possessed a large, irregular-shaped euchromatic nucleus, and cytoplasm rich in mitochondria, lysosomes, and endoplasmic reticulum. The periphery of the plasma membrane displayed many small filopodia. MSCs from both cell lines were successfully differentiated into osteogenic, adiopogenic, and chondrogenic lineages. Both healthy and diseased tissues may be considered as valuable sources of MSCs for regenerative medicine owing to the high acceptance and fewer complications during harvesting.

Download Full-text

Role of Jagged/Notch Signaling in the Cell Fate Determination of Bone Marrow Human Mesenchymal Stem Cells.

Blood ◽

10.1182/blood.v110.11.1923.1923 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1923-1923

Author(s):

Fernando Ugarte ◽

Martin F. Ryser ◽

Sebastian Thieme ◽

Martin Bornhaeuser ◽

Sebastian Brenner

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Notch Signaling ◽

Osteogenic Differentiation ◽

Adipogenic Differentiation ◽

Hematopoietic Progenitors ◽

Alizarin Red ◽

Differentiation Potential ◽

Expression Levels

Abstract Notch, expressed on hematopoietic progenitors plays a crucial role in hematopoiesis. Mesenchymal stem cells (MSC) express both, Notch and its ligand Jagged and are known to support self renewal of hematopoietic progenitors via cell-cell contact and cytokine secretion. The Jagged/Notch signaling pathway has been implicated in the differentiation process of MSC, however it is not completely understood and current observations are contradictory. In order to analyze the effect of Notch signaling on human MSC differentiation we constructed lentiviral vectors that contained either the GFP-marker gene, hJagged1 IRES GFP, hNotch1 intracellular domain (NICD) IRES GFP or a gene fusion between dominant negative Mastermind1 (MAML1dn - inhibitor of Notch signaling) and the Cherry reporter gene. Primary hMSC that were obtained from bone marrow of 3 different donors were transduced with respective lentivirus vectors to greater than 98%. After exposure to adipogenic and osteogenic differentiation stimuli hMSC differentiation was quantified by Alizarin red or oil red staining, alkaline phosphatase (AP) activity and expression levels of adipogenic or osteogenic markers by Real-time PCR. Jagged1 transduced hMSC demonstrated enhanced calcium phosphate deposits and enhanced AP activity and expression levels in osteogenic differentiation medium, while adipogenic differentiation was strongly inhibited as quantified by oil red staining and low mRNA expression of genes upregulated during adipogenic differentiation (pprY, Fabp4). Similarly, overexpression of NICD induced strong and rapid osteogenic differentiation while inhibiting adipogenic differentiation and reducing cell viability. Moreover, NICD overexpression upregulates the expression of endogenous Jagged1 up to 5-fold. Inhibition of Notch signaling via overexpression of MAML1dn partially blocked the effect of hJagged1 and NICD in co-transduction experiments. In another approach MSC samples obtained from 20 donors with various osteogenic differentiation potential as measured by AP activity were analyzed for Notch1 and Jagged1 expression. While there was no correlation between AP activity and Notch1 levels we observed a significant positive correlation for AP activity and Jagged1 expression. In summary, our data strongly suggest that increased Jagged/Notch signaling enhances the osteogenic differentiation of hMSC while inhibiting their adipogenic fate.

Download Full-text

Proliferation And Differentiation Potential Of Canine Synovial Fluid Cells

Acta veterinaria ◽

10.1515/acve-2015-0005 ◽

2015 ◽

Vol 65 (1) ◽

pp. 66-78 ◽

Cited By ~ 1

Author(s):

FRANCUSKI V Jelena ◽

DEBELJAK MARTAČIĆ Jasmina ◽

RADOVANOVIĆ Anita ◽

ANDRIĆ Nenad ◽

SOURICE-PETIT Sophie ◽

...

Keyword(s):

Synovial Fluid ◽

Three Dimensional ◽

Population Doubling ◽

Blue Staining ◽

Population Doubling Time ◽

Alcian Blue Staining ◽

German Shepherd ◽

Alizarin Red ◽

Differentiation Potential

Abstract The aim of this study was to determine whether synovial fluid (SF) of dogs contains cells that have characteristics of MSCs and to describe their differentiation potential. SF adherent cells from 5 young German shepherd dogs (average 3.8 ± 0.9 years) were expanded (37°C, 5% CO2, humidified atmosphere) three weeks before their phenotype was characterized by flow-cytometry for the presence of CD90 and CD34. Population doubling time (PDT), number of CFU-F and adipogenic, osteogenic and chondrogenic potentials have been determined in vitro. In early passages PTD was 31 ± 10 hours and expansion fold after 3 sub cultivations (9 days) theoretically could be 372 ± 134. At P1, 0.55 ± 0.05% of SF cells had the ability to form CFU-F. Sixty-six percent of cells expressed CD90 and none of the cells expressed markers of hematopoietic cells. Oil Red O staining has shown accumulation of fat droplets in cells grown in adipogenic medium, while deposits of calcium in the osteogenic medium were evidenced with Alizarin red staining. SF cultured in hondrogenic and control medium in three-dimensional conditions formed a cartilage-like tissue. Alcian blue staining of pellets’ slides have shown a significant amount of glycosaminoglycans (GAGs) and immunohistochemistry analysis documented collagen type II expression. The amount of GAGs in pellets grown in both conditions showed no difference. SF cells in vitro exhibited osteogenic, adipogenic and chondrogenic differentiation potentials, suggesting the presence of different mesenchymal progenitors. These results also demonstrated that SF cells have a spontaneous chondrogenic potential that should be further explored for possible tissue engineering protocols.

Download Full-text