In Vitro Repair of Meniscal Radial Tear With Hydrogels Seeded With Adipose Stem Cells and TGF-β3

2018 ◽  
Vol 46 (10) ◽  
pp. 2402-2413 ◽  
Author(s):  
Hiroshi Sasaki ◽  
Benjamin B. Rothrauff ◽  
Peter G. Alexander ◽  
Hang Lin ◽  
Riccardo Gottardi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transforming Growth Factor ◽  
Meniscal Repair ◽  
Explant Culture ◽  
Differentiation Potential ◽  
3 Dimensional ◽  
Institutional Review ◽  
Radial Tear ◽  
In Vitro Repair

Background: Radial tears of the meniscus are a common knee injury, frequently resulting in osteoarthritis. To date, there are no established, effective treatments for radial tears. Adipose-derived stem cells (ASCs) may be an attractive cell source for meniscal regeneration because they can be quickly isolated in large number and are capable of undergoing induced fibrochondrogenic differentiation mediated by transforming growth factor β3 (TGF-β3). However, the use of ASCs for meniscal repair is largely unexplored. Hypothesis: ASC-seeded hydrogels with preloaded TGF-β3 will improve meniscal healing of radial tears, as modeled in an explant model. Study Design: Controlled laboratory study. Methods: With an institutional review board–exempted protocol, human ASCs were isolated from the infrapatellar fat pads of 3 donors, obtained after total knee replacement, and characterized. ASCs were encapsulated in photocrosslinkable methacrylated gelatin hydrogels to form 3-dimensional constructs, which were placed into tissue culture. The effect of TGF-β3—whether preloaded into the hydrogel or added as a soluble medium supplement—on matrix-sulfated proteoglycan deposition in the constructs was evaluated. A meniscal explant culture model was used to simulate meniscal repair. Cylindrical-shaped explants were excised from the inner avascular region of adult bovine menisci, and a radial tear was modeled by cutting perpendicular to the meniscal main fibers to the length of the radius. Six combinations of hydrogels—namely, acellular and ASC-seeded hydrogels supplemented with preloaded TGF-β3 (2 µg/mL) or soluble TGF-β3 (10 ng/mL) and without supplement—were injected into the radial tear and stabilized by photocrosslinking with visible light. At 4 and 8 weeks of culture, healing was assessed through histology, immunofluorescence staining, and mechanical testing. Results: ASCs isolated from the 3 donors exhibited colony-forming and multilineage differentiation potential. Hydrogels preloaded with TGF-β3 and those cultured in soluble TGF-β3 showed robust matrix-sulfated proteoglycan deposition. ASC-seeded hydrogels promoted superior healing as compared with acellular hydrogels, with preloaded or soluble TGF-β3 further improving histological scores and mechanical properties. Conclusion: These findings demonstrated that ASC-seeded hydrogels preloaded with TGF-β3 enhanced healing of radial meniscal tears in an in vitro meniscal repair model. Clinical Relevance: Injection delivery of ASCs in a TGF-β3-preloaded photocrosslinkable hydrogel represents a novel candidate strategy to repair meniscal radial tears and minimize further osteoarthritic joint degeneration.

Mini Review; Differentiation of Human Pluripotent Stem Cells into Oocytes

2020 ◽  
Vol 15 (4) ◽  
pp. 301-307 ◽  
Author(s):  
Gaifang Wang ◽  
Maryam Farzaneh
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Human Pluripotent Stem Cells ◽  
Human Oocyte ◽  
Differentiation Potential ◽  
Cell Lineages ◽  
Cell Based Therapy ◽  
Induced Pluripotent

Primary Ovarian Insufficiency (POI) is one of the main diseases causing female infertility that occurs in about 1% of women between 30-40 years of age. There are few effective methods for the treatment of women with POI. In the past few years, stem cell-based therapy as one of the most highly investigated new therapies has emerged as a promising strategy for the treatment of POI. Human pluripotent stem cells (hPSCs) can self-renew indefinitely and differentiate into any type of cell. Human Embryonic Stem Cells (hESCs) as a type of pluripotent stem cells are the most powerful candidate for the treatment of POI. Human-induced Pluripotent Stem Cells (hiPSCs) are derived from adult somatic cells by the treatment with exogenous defined factors to create an embryonic-like pluripotent state. Both hiPSCs and hESCs can proliferate and give rise to ectodermal, mesodermal, endodermal, and germ cell lineages. After ovarian stimulation, the number of available oocytes is limited and the yield of total oocytes with high quality is low. Therefore, a robust and reproducible in-vitro culture system that supports the differentiation of human oocytes from PSCs is necessary. Very few studies have focused on the derivation of oocyte-like cells from hiPSCs and the details of hPSCs differentiation into oocytes have not been fully investigated. Therefore, in this review, we focus on the differentiation potential of hPSCs into human oocyte-like cells.

scholarly journals Cell Source-Dependent In Vitro Chondrogenic Differentiation Potential of Mesenchymal Stem Cell Established from Bone Marrow and Synovial Fluid of Camelus dromedarius

Animals ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 1918
Author(s):  
Young-Bum Son ◽  
Yeon Ik Jeong ◽  
Yeon Woo Jeong ◽  
Mohammad Shamim Hossein ◽  
Per Olof Olsson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Synovial Fluid ◽  
Cartilage Regeneration ◽  
Cartilage Tissue ◽  
Chondrocyte Differentiation ◽  
Differentiation Potential ◽  
Proliferation Capacity

Mesenchymal stem cells (MSCs) are promising multipotent cells with applications for cartilage tissue regeneration in stem cell-based therapies. In cartilage regeneration, both bone marrow (BM-MSCs) and synovial fluid (SF-MSCs) are valuable sources. However, the cellular characteristics and chondrocyte differentiation potential were not reported in either of the camel stem cells. The in vitro chondrocyte differentiation competence of MSCs, from (BM and SF) sources of the same Camelus dromedaries (camel) donor, was determined. Both MSCs were evaluated on pluripotent markers and proliferation capacity. After passage three, both MSCs showed fibroblast-like morphology. The proliferation capacity was significantly increased in SF-MSCs compared to BM-MSCs. Furthermore, SF-MSCs showed an enhanced expression of transcription factors than BM-MSCs. SF-MSCs exhibited lower differentiation potential toward adipocytes than BM-MSCs. However, the osteoblast differentiation potential was similar in MSCs from both sources. Chondrogenic pellets obtained from SF-MSCs revealed higher levels of chondrocyte-specific markers than those from BM-MSCs. Additionally, glycosaminoglycan (GAG) content was elevated in SF-MSCs related to BM-MSCs. This is, to our knowledge, the first study to establish BM-MSCs and SF-MSCs from the same donor and to demonstrate in vitro differentiation potential into chondrocytes in camels.

scholarly journals Similar Features, Different Behaviors: A Comparative In Vitro Study of the Adipogenic Potential of Stem Cells from Human Follicle, Dental Pulp, and Periodontal Ligament

2021 ◽  
Vol 11 (8) ◽  
pp. 738
Author(s):  
Melissa D. Mercado-Rubio ◽  
Erick Pérez-Argueta ◽  
Alejandro Zepeda-Pedreguera ◽  
Fernando J. Aguilar-Ayala ◽  
Ricardo Peñaloza-Cuevas ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dental Pulp ◽  
Periodontal Ligament ◽  
Adipogenic Differentiation ◽  
In Vitro Study ◽  
Mrna Levels ◽  
Dental Tissue ◽  
Periodontal Ligament Stem Cells ◽  
Differentiation Potential

Dental tissue-derived mesenchymal stem cells (DT-MSCs) are a promising resource for tissue regeneration due to their multilineage potential. Despite accumulating data regarding the biology and differentiation potential of DT-MSCs, few studies have investigated their adipogenic capacity. In this study, we have investigated the mesenchymal features of dental pulp stem cells (DPSCs), as well as the in vitro effects of different adipogenic media on these cells, and compared them to those of periodontal ligament stem cells (PLSCs) and dental follicle stem cells (DFSCs). DFSC, PLSCs, and DPSCs exhibit similar morphology and proliferation capacity, but they differ in their self-renewal ability and expression of stemness markers (e.g OCT4 and c-MYC). Interestingly, DFSCs and PLSCs exhibited more lipid accumulation than DPSCs when induced to adipogenic differentiation. In addition, the mRNA levels of adipogenic markers (PPAR, LPL, and ADIPOQ) were significantly higher in DFSCs and PLSCs than in DPSCs, which could be related to the differences in the adipogenic commitment in those cells. These findings reveal that the adipogenic capacity differ among DT-MSCs, features that might be advantageous to increasing our understanding about the developmental origins and regulation of adipogenic commitment.

Effect of Substrate Elasticity on In Vitro Aging of Human Mesenchymal Stem Cells

2012 ◽  
Vol 1498 ◽  
pp. 39-45
Author(s):  
Courtney E. LeBlon ◽  
Caitlin R. Fodor ◽  
Tony Zhang ◽  
Xiaohui Zhang ◽  
Sabrina S. Jedlicka
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Elastic Modulus ◽  
Myogenic Differentiation ◽  
Human Mesenchymal Stem Cells ◽  
Immunofluorescent Staining ◽  
Differentiation Potential ◽  
Gene And Protein Expression ◽  
The Impact

ABSTRACTHuman mesenchymal stem cells (hMSCs) were routinely cultured on tissue-culture polystyrene (TCPS) to investigate the in vitro aging and cell stiffening. hMSCs were also cultured on thermoplastic polyurethane (TPU), which is a biocompatible polymer with an elastic modulus of approximately 12.9MPa, to investigate the impact of substrate elastic modulus on cell stiffening and differentiation potential. Cells were passaged over several generations on each material. At each passage, cells were subjected to osteogenic and myogenic differentiation. Local cell elastic modulus was measured at every passage using atomic force microscopy (AFM) indentation. Gene and protein expression was examined using qRT-PCR and immunofluorescent staining, respectively, for osteogenic and myogenic markers. Results show that the success of myogenic differentiation is highly reliant on the elastic modulus of the undifferentiated cells. The success of osteogenic differentiations is most likely somewhat dependent on the cell elastic modulus, as differentiations were more successful in earlier passages, when cells were softer.

scholarly journals High glucose induces early senescence in adipose-derived stem cells by accelerating p16 and mTOR

2019 ◽  
Vol 6 (6) ◽  
pp. 3213-3221
Author(s):  
Hieu Liem Pham ◽  
Phuc Van Pham
Keyword(s):  
Stem Cells ◽  
High Glucose ◽  
Glucose Concentration ◽  
Culture Medium ◽  
Diabetic Patients ◽  
Adipose Derived Stem Cells ◽  
Differentiation Potential ◽  
Normal Glucose

Introduction: The senescence of stem cells is the primary reason that causes aging of stem cell-containing tissues. Some hypotheses have suggested that high glucose concentration in diabetic patients is the main factor that causes senescence of cells in those patients. This study aimed to evaluate the effects of high glucose concentrations on the senescence of adipose-derived stem cells (ADSCs). Methods: ADSCs were isolated and expanded from human adipose tissues. They were characterized and confirmed as mesenchymal stem cells (MSCs) by expression of surface markers, their shape, and in vitro differentiation potential. They were then cultured in 3 different media- that contained 17.5 mM, 35 mM, or 55 mM of D-glucose. The senescent status of ADSCs was recorded by the expression of the enzyme beta-galactosidase, cell proliferation, and doubling time. Real-time RT-PCR was used to evaluate the expression of p16, p21, p53 and mTOR. Results: The results showed that high glucose concentrations (35 mM and 55 mM) in the culture medium induced senescence of human ADSCs. The ADSCs could progress to the senescent status quicker than those cultured in the lower glucose-containing medium (17.5 mM). The senescent state was related to the up-regulation of p16 and mTOR genes. Conclusion: These results suggest that high glucose in culture medium can trigger the expression of p16 and mTOR genes which cause early senescence in ADSCs. Therefore, ADSCs should be cultured in low glucose culture medium, or normal glucose concentration, to extend their life in vitro as well as in vivo.  

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.

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