scholarly journals In vitro Culture and Morphometry of Porcine Adipose Derived Mesenchymal Stem Cells (pAD-MSCs)

2022 ◽  
Author(s):  
Thokchom Shitarjit Singh ◽  
O.R. Sathyamoorthy ◽  
Soundian Eswari ◽  
Sabiha Hayath Basha ◽  
M. Parthiban
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Population Doubling ◽  
Population Doubling Time ◽  
Specific Cell ◽  
Stem Cell Markers ◽  
Trypan Blue Exclusion Method ◽  
Cell Markers ◽  
Positive Expression

Background: Mesenchymal stem cells are well known for their self-renewal capacity and ability to differentiate into multiple cell lineages. The aim of the study was to develop a simple technique for isolation of mesenchymal stem cells from porcine adipose tissue and to study the morphometric characteristics of porcine mesenchymal stem cells. Methods: Porcine adipose derived mesenchymal stem cells were isolated in vitro by using collagenase type II enzyme. Cell yield and viability of the cells were calculated by using trypan blue exclusion method using Neubauer’s chamber. Characterization of MSCs were done by using specific cell markers. The morphological changes, morphometry were analysed in culture using Leishman’s stain. The cell doubling (CD) and Population doubling time (PDT) were also calculated. Result: The isolated adherent cells start forming colony and demonstrated an elongated, round and spindle like fibroblastic morphology by day 1. Almost 80-90 per cent confluency was attained on day 8-9 after the initial seeding and was reduced to day 3-4 in the subsequent passages. RT-PCR reactions revealed positive expression of mesenchymal stem cell markers CD44, CD73 and negative expression of CD34, a hematopoietic cell surface marker. Immunocytochemistry also revealed positive expression for CD44 and negative for CD34. In morphometric studies, the cell length, nucleus length, cell width and nucleus width were increased between 24 and 48 hours in both P2 and P3.

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 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 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.

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.

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.

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.

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