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 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.

scholarly journals Platelet Microparticles Accelerate Proliferation and Growth of Mesenchymal Stem Cells through Longevity-Related Genes

2021 ◽  
Vol 24 (8) ◽  
pp. 607-614
Author(s):  
Maryam Samareh Salavati Pour ◽  
Fatemeh Hoseinpour Kasgari ◽  
Alireza Farsinejad ◽  
Ahmad Fatemi ◽  
Gholamhossein Hassanshahi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Real Time ◽  
Real Time Pcr ◽  
Treated Group ◽  
Population Doubling ◽  
Control Group ◽  
Population Doubling Time ◽  
Longevity Genes

Background: Due to their self-renewal and differentiation ability, the mesenchymal stem cells (MSCs) have been studied extensively. However, the MSCs lifespan is restricted; they undergo several divisions in vitro that cause several alternations in cellular features and relatively lessens their application. Thus, this study was aimed to assess the effect of platelet-derived microparticles (PMPs), a valuable source of proteins, microRNAs (miRNAs), and growth factors, on the expression of hTERT, c-MYC, p16, p53, and p21 as the most important aging and cell longevity genes alongside with population doubling time (PDT) of PMP-treated cells in comparison to a control group. Methods: Umbilical cord MSCs (UC-MSCs) were used in this study, whereby they reached a confluency of 30%. MSCs were treated by PMPs (50 µg/mL), and then, PDT was determined for both groups. Quantitative expression of hTERT, c-MYC, p16, p53, and p21 was examined through quantitative real-time PCR at various intervals (i.e. after five and thirty days as well as freezing-thawing process). Results: Our results demonstrated that the treated group had a shorter PDT in comparison to the control group (P<0.050). The real-Time PCR data also indicated that PMPs were able to remarkably up-regulate hTERT and c-MYC genes expression while down-regulating the expression of p16, p21, and p53 genes (P<0.050), especially following five days of treatment. Conclusion: According to these data, it appears that PMPs are a safe and effective candidate for prolonging the lifespan of UC-MSCs; however, further investigations are needed to corroborate this finding.

Absence of P21 Gene Augments Proliferation of Mesenchymal Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3039-3039
Author(s):  
Anthony Y. Tsai ◽  
Stephanie C. Filice ◽  
Elizabeth H. Javazon ◽  
Andrea T. Badillo ◽  
Alan W. Flake
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Kinase Inhibitor ◽  
Adipogenic Differentiation ◽  
Population Doubling ◽  
Facs Analysis ◽  
Competition Assay ◽  
Cell Counts ◽  
Population Doublings

Abstract Bone marrow derived mesenchymal stem cells (MSCs) have the capacity to differentiate into cells of the various mesodermal lineages. Efficiency of engraftment, however, remains a significant obstacle in MSC transplantation. P21 is a G1 checkpoint regulator and cyclin-dependent kinase inhibitor. In its absence, hematopoieitc stem cell (HSC) proliferation increases under normal homeostatic conditions. The aim of the study is to evaluate the effects of p21 deficiency on the proliferative capacity of MSCs. Therefore, MSCs from p21 KO and wildtype mice were maintained in culture in hypoxic and normoxic conditions. Cell counts and respective days in cultures were recorded from which population doubling (PD) times were calculated and compared. 1:1 Competition assays were performed between p21 KO MSCs transduced with DsRed and eGFP transduced WT MSCs as well as between DsRed transduced p21 KO MSCs and non-transduced WT MSCs. Osteogenic, chondrogenic, and adipogenic differentiation assays were performed on both cell populations using standard protocols. Sca-1, CD34, CD45, CD90, MHC Class I and II and other surface antigens were assessed using FACS analysis. In normoxic conditions, the p21 KO MSCs went through 25.68 population doublings in 60 days versus 25.43 population doublings in 103 days for the p21 WT MSCs. Under hypoxic conditions, the KO MSC population doubled 23.3 times in 58 days versus 14.78 times in 60 days for the WT MSCs. All population doubling times were taken from passage 10 cells. When the DsRed transduced KO MSCs (96.6-98.5% DsRed+) was placed in a 1:1 competition assay with eGFP transduced WT MSCs (99.3-99.9% eGFP+) or non-transduced WT MSCs (99.9–100% eGFP−), the normoxic results are as shown in Table 1. Similarly, the competition assay result between DsRed transduced KO MSCs (86.9–94.1% DsRed+) and eGFP transduced WT MSCs (99.7–100% eGFP+) or non-transduced WT MSCs (100% eGFP−) for the hypoxic condition are shown in Table 2. Both the p21 KO and wildtype MSCs populations were able to differentiate into ostenogenic, adipogenic, and chondrogenic lineages. No significant surface marker differences were observed between the 2 populations on FACS analysis. Our results clearly showed that p21 deficient MSCs have increased proliferative ability in vitro compared to normal MSCs. These findings have implications for expansion of MSC populations in vitro , and for the enhancement of competitive capacity of MSCs following in vivo administration. Table 1 - Normoxic Competition Assay KO MSC (DsRed) WT MSC (eGFP) KO MSC (DsRed) WT MSC (none) results analyzed by FACS Day 6 64.2±3.7% 34.5±4.3% 70.3±0.4% 19.7±0.6% Day 11 84.0±4.5% 13.6±4.6% 92.0±0.8% 6.4±0.7% Table 2 - Hypoxic Competition Assay KO MSC (DsRed) WT MSC (eGFP) KO MSC (DsRed) WT MSC (none) results analyzed by FACS Day 4 70.3±1.7% 30.0±1.7% 83.0±0.8% 18.6±0.9% Day 8 75.7±0.7% 25.8±0.7% 81.3±1.2% 20.6±1.2%

scholarly journals Identification of WNT16 as a Predictable Biomarker for Accelerated Osteogenic Differentiation of Tonsil-Derived Mesenchymal Stem Cells In Vitro

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Yu-Hee Kim ◽  
Kyung-Ah Cho ◽  
Hyun-Ji Lee ◽  
Minhwa Park ◽  
Han Su Kim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Selection Criterion ◽  
Osteogenic Potential ◽  
Differentiation Potential ◽  
Real Time Quantitative Pcr ◽  
Single Cell Cloning

The application of mesenchymal stem cells (MSCs) for treating bone-related diseases shows promising outcomes in preclinical studies. However, cells that are isolated and defined as MSCs comprise a heterogeneous population of progenitors. This heterogeneity can produce variations in the performance of MSCs, especially in applications that require differentiation potential in vivo, such as the treatment of osteoporosis. Here, we aimed to identify genetic markers in tonsil-derived MSCs (T-MSCs) that can predict osteogenic potential. Using a single-cell cloning method, we isolated and established several lines of nondifferentiating (ND) or osteoblast-prone (OP) clones. Next, we performed transcriptome sequencing of three ND and three OP clones that maintained the characteristics of MSCs and determined the top six genes that were upregulated in OP clones. Upregulation of WNT16 and DCLK1 expression was confirmed by real-time quantitative PCR, but only WNT16 expression was correlated with the osteogenic differentiation of T-MSCs from 10 different donors. Collectively, our findings suggest that WNT16 is a putative genetic marker that predicts the osteogenic potential of T-MSCs. Thus, examination of WNT16 expression as a selection criterion prior to the clinical application of MSCs may enhance the therapeutic efficacy of stem cell therapy for bone-related complications, including osteoporosis.

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 Deregulated mito-nuclear communication alters chromatin plasticity and differentiation potential of mesenchymal stem cells upon ageing

2020 ◽  
Author(s):  
Andromachi Pouikli ◽  
Swati Parekh ◽  
Monika Maleszewska ◽  
Maarouf Baghdadi ◽  
Ignacio Tripodi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Culture Conditions ◽  
Osteogenic Potential ◽  
Acetyl Coa ◽  
Differentiation Potential ◽  
General Decline ◽  
Age Dependent ◽  
Transcriptional Output

ABSTRACTAgeing is accompanied by a general decline in the function of many cellular pathways, with metabolic alterations, epigenetic modifications, and stem cell exhaustion representing three important hallmarks of the ageing process. However, whether these pathways are causally or functionally related at a molecular level remains poorly understood. Here, we use bone marrow-derived mesenchymal stem cells (MSCs) isolated from young and old mice to address how age-dependent changes in metabolism and epigenetics are linked and how they impact on the ageing transcriptome and differentiation potential. Given that MSCs maintain specific age-associated properties even under prolonged culture conditions, such as the age-dependent decrease in osteogenic differentiation, they are an excellent model to investigate in vitro the connection of ageing hallmarks on a mechanistic level. In this study, we demonstrate that upon ageing, osteogenic potential of MSCs declines as a consequence of deregulated mito-nuclear communication, mediated by decreased levels of the citrate carrier (CiC). Age-dependent down-regulation of CiC results in acetyl-CoA trapping within mitochondria, hypo-acetylation of histones and chromatin compaction. Together, these changes lead to an altered transcriptional output and are responsible for the reduced differentiation capacity into osteoblasts. Strikingly, short-term supplementation of aged cells with acetate, an exogenous source for cytosolic acetyl-CoA production, rescues not only the age-associated reduction of histone acetylation, but also the osteogenesis defect, representing a potential target for in vitro MSC rejuvenation.

Culture Expanded Canine Mesenchymal Stem Cells Possess Osteochondrogenic Potential in Vivo and in Vitro

1997 ◽  
Vol 6 (2) ◽  
pp. 125-134 ◽  
Author(s):  
S. Kadiyala ◽  
R. G. Young ◽  
M. A. Thiede ◽  
S. P. Bruder
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Morphological Characteristics ◽  
Cartilage Regeneration ◽  
Population Doubling ◽  
Population Doubling Time ◽  
Human Mscs ◽  
And Cartilage

Mesenchymal Stem Cells (MSCs) possessing the capacity to differentiate into various cell types such as osteoblasts, chondrocytes, myoblasts, and adipocytes have been previously isolated from the marrow and periosteum of human, murine, lapine, and avian species. This study documents the existence of similar multipotential stem cells in canine marrow. The cells were isolated from marrow aspirates using a modification of techniques previously established for human MSCs (hMSCs), and found to possess similar growth and morphological characteristics, as well as osteochondrogenic potential in vivo and in vitro. On the basis of these results, the multipotential cells that were isolated and culture expanded are considered to be canine MSCs (cMSCs). The occurrence of cMSCs in the marrow was determined to be one per 2.5 × 104 nucleated cells. After enrichment of the cMSCs by centrifugation on a Per-coll cushion, the cells were cultivated in selected lots of serum. Like the hMSCs, cMSCs grew as colonies in primary culture and on replating, grew as a monolayer culture with very uniform spindle morphology. The population doubling time for these cMSCs was approximately 2 days. The morphology and the growth kinetics of the cMSCs were retained following repeated passaging. The osteogenic phenotype could be induced in the cMSC cultures by the addition of a synthetic glucocorticoid, dexamethasone. In these osteogenic cultures, alkaline phosphatase activity was elevated up to 10-fold, and mineralized matrix production was evident. When cMSCs were loaded onto porous ceramics and implanted in autologous canine or athymic murine hosts, copious amounts of bone and cartilage were formed in the pores of the implants. The MSC-mediated osteogenesis obtained by the implantation of the various MSC-loaded matrix combinations is the first evidence of osteogenesis in a canine model by implantation of culture expanded autologous stem cells. The identification and isolation of cMSCs now makes it feasible to pursue preclinical models of bone and cartilage regeneration in canine hosts.

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