scholarly journals Vertebral Bone Marrow-Derived Mesenchymal Stromal Cells from Osteoporotic and Healthy Patients Possess Similar Differentiation Properties In Vitro

2020 ◽  
Vol 21 (21) ◽  
pp. 8309
Author(s):  
El-Mustapha Haddouti ◽  
Thomas M. Randau ◽  
Cäcilia Hilgers ◽  
Werner Masson ◽  
Robert Pflugmacher ◽  
...  
Keyword(s):  
Osteogenic Differentiation ◽  
Mesenchymal Stromal Cells ◽  
Vertebral Body ◽  
Stromal Cells ◽  
Osteogenic Potential ◽  
Ion Release ◽  
Differentiation Potential ◽  
Phosphate Ion ◽  
Alp Activity ◽  
Immunomodulatory Capacity

Osteoporosis is a disease characterized by low bone mass and an increased risk of fractures. Although several cellular players leading to osteoporosis have been identified, the role of mesenchymal stromal cells (MSC) is still not fully elaborated. The aim of this study was, therefore, to isolate and characterize MSCs from vertebral body of healthy non-osteoporotic and osteoporotic patients, with a particular focus on their osteogenic differentiation potential. Isolated MSCs were characterized by their osteogenic, adipogenic, and chondrogenic differentiation, as well as surface marker expression, proliferation behavior, and immunomodulatory capacity. The mineralization process was confirmed using Alizarin Red S and alkaline phosphatase (ALP) stains and further evaluated by determining ALP activity, mineral deposition, and free phosphate ion release. MSCs from both healthy and osteoporotic patients showed common fibroblast-like morphology and similar proliferation behavior. They expressed the typical MSC surface markers and possessed immunomodulatory capacity. Both groups demonstrated solid trilineage differentiation potential; osteogenic differentiation was further confirmed by increased ALP activity, deposition of inorganic crystals, phosphate ion release, and expression of osteoblast marker genes. Overall, MSCs from osteoporotic and non-osteoporotic patients showed neither a difference in general MSC features nor in the detailed analysis regarding osteogenic differentiation. These data suggest that vertebral body MSCs from osteoporotic patients were not impaired; rather, they possessed full osteogenic potential compared to MSCs from non-osteoporotic patients.

scholarly journals The Identification of Marker Genes for Predicting the Osteogenic Differentiation Potential of Mesenchymal Stromal Cells

2021 ◽  
Vol 43 (3) ◽  
pp. 2157-2166
Author(s):  
Masami Kanawa ◽  
Akira Igarashi ◽  
Katsumi Fujimoto ◽  
Tania Saskianti ◽  
Ayumu Nakashima ◽  
...  
Keyword(s):  
Regenerative Medicine ◽  
Osteogenic Differentiation ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Marker Genes ◽  
Mrna Levels ◽  
Differentiation Potential ◽  
Alp Activity ◽  
Osteogenic Induction ◽  
Promising Source

Mesenchymal stromal cells (MSCs) have the potential to differentiate into a variety of mature cell types and are a promising source of regenerative medicine. The success of regenerative medicine using MSCs strongly depends on their differentiation potential. In this study, we sought to identify marker genes for predicting the osteogenic differentiation potential by comparing ilium MSC and fibroblast samples. We measured the mRNA levels of 95 candidate genes in nine ilium MSC and four fibroblast samples before osteogenic induction, and compared them with alkaline phosphatase (ALP) activity as a marker of osteogenic differentiation after induction. We identified 17 genes whose mRNA expression levels positively correlated with ALP activity. The chondrogenic and adipogenic differentiation potentials of jaw MSCs are much lower than those of ilium MSCs, although the osteogenic differentiation potential of jaw MSCs is comparable with that of ilium MSCs. To select markers suitable for predicting the osteogenic differentiation potential, we compared the mRNA levels of the 17 genes in ilium MSCs with those in jaw MSCs. The levels of 7 out of the 17 genes were not substantially different between the jaw and ilium MSCs, while the remaining 10 genes were expressed at significantly lower levels in jaw MSCs than in ilium MSCs. The mRNA levels of the seven similarly expressed genes were also compared with those in fibroblasts, which have little or no osteogenic differentiation potential. Among the seven genes, the mRNA levels of IGF1 and SRGN in all MSCs examined were higher than those in any of the fibroblasts. These results suggest that measuring the mRNA levels of IGF1 and SRGN before osteogenic induction will provide useful information for selecting competent MSCs for regenerative medicine, although the effectiveness of the markers is needed to be confirmed using a large number of MSCs, which have various levels of osteogenic differentiation potential.

scholarly journals Identification of ALP+/CD73+ defining markers for enhanced osteogenic potential in human adipose-derived mesenchymal stromal cells by mass cytometry

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Daisy D. Canepa ◽  
Elisa A. Casanova ◽  
Eirini Arvaniti ◽  
Vinko Tosevski ◽  
Sonja Märsmann ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Osteogenic Differentiation ◽  
Single Cell ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Osteogenic Potential ◽  
Single Cell Level ◽  
Cellular Composition ◽  
Cell Level ◽  
Differentiation Potential

Abstract Background The impressive progress in the field of stem cell research in the past decades has provided the ground for the development of cell-based therapy. Mesenchymal stromal cells obtained from adipose tissue (AD-MSCs) represent a viable source for the development of cell-based therapies. However, the heterogeneity and variable differentiation ability of AD-MSCs depend on the cellular composition and represent a strong limitation for their use in therapeutic applications. In order to fully understand the cellular composition of MSC preparations, it would be essential to analyze AD-MSCs at single-cell level. Method Recent advances in single-cell technologies have opened the way for high-dimensional, high-throughput, and high-resolution measurements of biological systems. We made use of the cytometry by time-of-flight (CyTOF) technology to explore the cellular composition of 17 human AD-MSCs, interrogating 31 markers at single-cell level. Subcellular composition of the AD-MSCs was investigated in their naïve state as well as during osteogenic commitment, via unsupervised dimensionality reduction as well as supervised representation learning approaches. Result This study showed a high heterogeneity and variability in the subcellular composition of AD-MSCs upon isolation and prolonged culture. Algorithm-guided identification of emerging subpopulations during osteogenic differentiation of AD-MSCs allowed the identification of an ALP+/CD73+ subpopulation of cells with enhanced osteogenic differentiation potential. We could demonstrate in vitro that the sorted ALP+/CD73+ subpopulation exhibited enhanced osteogenic potential and is moreover fundamental for osteogenic lineage commitment. We finally showed that this subpopulation was present in freshly isolated human adipose-derived stromal vascular fractions (SVFs) and that could ultimately be used for cell therapies. Conclusion The data obtained reveal, at single-cell level, the heterogeneity of AD-MSCs from several donors and highlight how cellular composition impacts the osteogenic differentiation capacity. The marker combination (ALP/CD73) can not only be used to assess the differentiation potential of undifferentiated AD-MSC preparations, but also could be employed to prospectively enrich AD-MSCs from the stromal vascular fraction of human adipose tissue for therapeutic applications.

scholarly journals MiR-218 affects hypertrophic differentiation of human mesenchymal stromal cells during chondrogenesis via targeting RUNX2, MEF2C, and COL10A1

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Svitlana Melnik ◽  
Jessica Gabler ◽  
Simon I. Dreher ◽  
Nicole Hecht ◽  
Nina Hofmann ◽  
...  
Keyword(s):  
Mesenchymal Stromal Cells ◽  
Expression Profile ◽  
Stromal Cells ◽  
Chondrogenic Differentiation ◽  
Cartilage Tissue ◽  
Protein Accumulation ◽  
Differentiation Potential ◽  
Human Mesenchymal Stromal Cells ◽  
Alp Activity ◽  
Pulldown Assay

Abstract Background Human mesenchymal stromal cells (MSC) hold hopes for cartilage regenerative therapy due to their chondrogenic differentiation potential. However, undesirable occurrence of calcification after ectopic transplantation, known as hypertrophic degeneration, remains the major obstacle limiting application of MSC in cartilage tissue regeneration approaches. There is growing evidence that microRNAs (miRs) play essential roles in post-transcriptional regulation of hypertrophic differentiation during chondrogenesis. Aim of the study was to identify new miR candidates involved in repression of hypertrophy-related targets. Methods The miR expression profile in human articular chondrocytes (AC) was compared to that in hypertrophic chondrocytes derived from human MSC by microarray analysis, and miR expression was validated by qPCR. Putative targets were searched by in silico analysis and validated by miR reporter assay in HEK293T, by functional assays (western blotting and ALP-activity) in transiently transfected SaOS-2 cells, and by a miR pulldown assay in human MSC. The expression profile of miR-218 was assessed by qPCR during in vitro chondrogenesis of MSC and re-differentiation of AC. MSC were transfected with miR-218 mimic, and differentiation outcome was assessed over 28 days. MiR-218 expression was quantified in healthy and osteoarthritic cartilage of patients. Results Within the top 15 miRs differentially expressed between chondral AC versus endochondral MSC differentiation, miR-218 was selected as a candidate miR predicted to target hypertrophy-related genes. MiR-218 was downregulated during chondrogenesis of MSC and showed a negative correlation to hypertrophic markers, such as COL10A1 and MEF2C. It was confirmed in SaOS-2 cells that miR-218 directly targets hypertrophy-related COL10A1, MEF2C, and RUNX2, as a gain of ectopic miR-218 mimic caused drop in MEF2C and RUNX2 protein accumulation, with attenuation of COL10A1 expression and significant concomitant reduction of ALP activity. A miR pulldown assay confirmed that miR-218 directly targets RUNX2, MEF2C in human MSC. Additionally, the gain of miR-218 in human MSC attenuated hypertrophic markers (MEF2C, RUNX2, COL10A1, ALPL), although with no boost of chondrogenic markers (GAG deposition, COL2A1) due to activation of WNT/β-catenin signaling. Moreover, no correlation between miR-218 expression and a pathologic phenotype in the cartilage of osteoarthritis (OA) patients was found. Conclusions Although miR-218 was shown to target pro-hypertrophic markers MEF2C, COL10A1, and RUNX2 in human MSC during chondrogenic differentiation, overall, it could not significantly reduce the hypertrophic phenotype or boost chondrogenesis. This could be explained by a concomitant activation of WNT/β-catenin signaling counteracting the anti-hypertrophic effects of miR-218. Therefore, to achieve a full inhibition of the endochondral pathway, a whole class of anti-hypertrophic miRs, including miR-218, needs to be taken into consideration.

scholarly journals The Casitas B Lineage Lymphoma (Cbl) Mutant G306E Enhances Osteogenic Differentiation in Human Mesenchymal Stromal Cells in Part by Decreased Cbl-mediated Platelet-derived Growth Factor Receptor α and Fibroblast Growth Factor Receptor 2 Ubiquitination

2011 ◽  
Vol 286 (27) ◽  
pp. 24443-24450 ◽  
Author(s):  
Nicolas Sévère ◽  
Hichem Miraoui ◽  
Pierre J. Marie
Keyword(s):  
Bone Marrow ◽  
Growth Factor ◽  
Osteogenic Differentiation ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Growth Factor Receptor ◽  
Receptor Expression ◽  
Osteogenic Potential ◽  
Specific Inhibition ◽  
B Lineage

Human bone marrow-derived mesenchymal stromal cells (hMSCs) have the capacity to differentiate into several cell types including osteoblasts and are therefore an important cell source for bone tissue regeneration. A crucial issue is to identify mechanisms that trigger hMSC osteoblast differentiation to promote osteogenic potential. Casitas B lineage lymphoma (Cbl) is an E3 ubiquitin ligase that ubiquitinates and targets several molecules for degradation. We hypothesized that attenuation of Cbl-mediated degradation of receptor tyrosine kinases (RTKs) may promote osteogenic differentiation in hMSCs. We show here that specific inhibition of Cbl interaction with RTKs using a Cbl mutant (G306E) promotes expression of osteoblast markers (Runx2, alkaline phosphatase, type 1 collagen, osteocalcin) and increases osteogenic differentiation in clonal bone marrow-derived hMSCs and primary hMSCs. Analysis of molecular mechanisms revealed that the Cbl mutant increased PDGF receptor α and FGF receptor 2 but not EGF receptor expression in hMSCs, resulting in increased ERK1/2 and PI3K signaling. Pharmacological inhibition of FGFR or PDGFR abrogated in vitro osteogenesis induced by the Cbl mutant. The data reveal that specific inhibition of Cbl interaction with RTKs promotes the osteogenic differentiation program in hMSCs in part by decreased Cbl-mediated PDGFRα and FGFR2 ubiquitination, providing a novel mechanistic approach targeting Cbl to promote the osteogenic capacity of hMSCs.

scholarly journals Pooling of Patient-Derived Mesenchymal Stromal Cells Reduces Inter-Individual Confounder-Associated Variation without Negative Impact on Cell Viability, Proliferation and Osteogenic Differentiation

Cells ◽  
2019 ◽  
Vol 8 (6) ◽  
pp. 633 ◽  
Author(s):  
Benedikt Widholz ◽  
Stefanos Tsitlakidis ◽  
Bruno Reible ◽  
Arash Moghaddam ◽  
Fabian Westhauser
Keyword(s):  
Osteogenic Differentiation ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Generation Time ◽  
Negative Impact ◽  
Biological Properties ◽  
Calcium Deposition ◽  
Quality Of Data ◽  
Alp Activity ◽  
The Individual

Patient-derived mesenchymal stromal cells (MSCs) play a key role in bone tissue engineering. Various donor-specific factors were identified causing significant variability in the biological properties of MSCs impairing quality of data and inter-study comparability. These limitations might be overcome by pooling cells of different donors. However, the effects of pooling on osteogenic differentiation, proliferation and vitality remain unknown and have, therefore, been evaluated in this study. MSCs of 10 donors were cultivated and differentiated into osteogenic lineage individually and in a pooled setting, containing MSCs of each donor in equal parts. Proliferation was evaluated in expansion (assessment of generation time) and differentiation (quantification of dsDNA content) conditions. Vitality was visualized by a fluorescence-microscopy-based live/dead assay. Osteogenic differentiation was assessed by quantification of alkaline phosphatase (ALP) activity and extracellular calcium deposition. Compared to the individual setting, generation time of pooled MSCs was shorter and proliferation was increased during differentiation with significantly lower variances. Calcium deposition was comparable, while variances were significantly higher in the individual setting. ALP activity showed high variance in both groups, but increased comparably during the incubation period. In conclusion, MSC pooling helps to compensate donor-dependent variability and does not negatively influence MSC vitality, proliferation and osteogenic differentiation.

scholarly journals Impact of 3D cell culture on bone regeneration potential of mesenchymal stromal cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Mesude Bicer ◽  
Graeme S. Cottrell ◽  
Darius Widera
Keyword(s):  
Stem Cells ◽  
Cell Culture ◽  
Osteogenic Differentiation ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Treatment Options ◽  
3D Cell Culture ◽  
Bone Diseases ◽  
Differentiation Potential ◽  
Number Of Patients

AbstractAs populations age across the world, osteoporosis and osteoporosis-related fractures are becoming the most prevalent degenerative bone diseases. More than 75 million patients suffer from osteoporosis in the USA, the EU and Japan. Furthermore, it is anticipated that the number of patients affected by osteoporosis will increase by a third by 2050. Although conventional therapies including bisphosphonates, calcitonin and oestrogen-like drugs can be used to treat degenerative diseases of the bone, they are often associated with serious side effects including the development of oesophageal cancer, ocular inflammation, severe musculoskeletal pain and osteonecrosis of the jaw.The use of autologous mesenchymal stromal cells/mesenchymal stem cells (MSCs) is a possible alternative therapeutic approach to tackle osteoporosis while overcoming the limitations of traditional treatment options. However, osteoporosis can cause a decrease in the numbers of MSCs, induce their senescence and lower their osteogenic differentiation potential.Three-dimensional (3D) cell culture is an emerging technology that allows a more physiological expansion and differentiation of stem cells compared to cultivation on conventional flat systems.This review will discuss current understanding of the effects of different 3D cell culture systems on proliferation, viability and osteogenic differentiation, as well as on the immunomodulatory and anti-inflammatory potential of MSCs.

scholarly journals Osteogenic Potential of Mouse Adipose-Derived Stem Cells Sorted for CD90 and CD105 In Vitro

2014 ◽  
Vol 2014 ◽  
pp. 1-17 ◽  
Author(s):  
Maiko Yamamoto ◽  
Hidemi Nakata ◽  
Jia Hao ◽  
Joshua Chou ◽  
Shohei Kasugai ◽  
...  
Keyword(s):  
Stem Cells ◽  
Adipose Tissue ◽  
Osteogenic Differentiation ◽  
Stromal Cells ◽  
Surface Protein ◽  
Osteogenic Potential ◽  
Nodule Formation ◽  
Differentiation Potential ◽  
Cluster Of Differentiation

Adipose tissue-derived stromal cells, termed ASCs, play an important role in regenerative applications. They resemble mesenchymal stem cells owing to their inexhaustibility, general differentiation potential, and plasticity and display a series of cell-specific and cluster-of-differentiation (CD) marker profiles similar to those of other somatic stem cells. Variations in phenotypes or differentiation are intimately associated with CD markers. The purpose of our study was to exhibit distinct populations of ASCs with differing characteristics for osteogenic differentiation. The primary cell batch of murine-derived ASCs was extracted from subcutaneous adipose tissue and the cells were sorted for the expression of the surface protein molecules CD90 and CD105 using flow cytometry. Each cell population sorted for CD90 and CD105 was analyzed for osteogenic potency after cell culture. The results suggested that ASCs exhibit distinct populations with differing characteristics for osteogenic differentiation: unsorted ASCs stimulated comparable mineralized nodule formation as bone marrow stromal cells (BMSCs) in osteogenic medium and viral transfection for BMP2 accelerated the formation of mineralized nodules in CD90 and/or CD105 positive ASCs with observation of decrease in CD105 expression after 14 days. Future studies assessing different immunophenotypes of ASCs should be undertaken to develop cell-based tissue engineering.

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