scholarly journals Human Mesenchymal Stromal Cells Unveil an Unexpected Differentiation Potential toward the Dopaminergic Neuronal Lineage

2020 ◽  
Vol 21 (18) ◽  
pp. 6589
Author(s):  
Giulia Gaggi ◽  
Andrea Di Credico ◽  
Pascal Izzicupo ◽  
Francesco Alviano ◽  
Michele Di Mauro ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Dopaminergic Neurons ◽  
High Efficiency ◽  
Ethical Issues ◽  
Differentiation Potential ◽  
Homogeneous Population ◽  
Human Mesenchymal Stromal Cells ◽  
Midbrain Dopaminergic Neurons

Degeneration of dopaminergic neurons represents the cause of many neurodegenerative diseases, with increasing incidence worldwide. The replacement of dead cells with new healthy ones may represent an appealing therapeutic approach to these pathologies, but currently, only pluripotent stem cells can generate dopaminergic neurons with high efficiency. However, with the use of these cells arises safety and/or ethical issues. Human mesenchymal stromal cells (hFM-MSCs) are perinatal stem cells that can be easily isolated from the amniochorionic membrane after delivery. Generally considered multipotent, their real differentiative potential is not completely elucidated. The aim of this study was to analyze their stemness characteristics and to evaluate whether they may overcome their mesenchymal fate, generating dopaminergic neurons. We demonstrated that hFM-MSCs expressed embryonal genes OCT4, NANOG, SOX2, KLF4, OVOL1, and ESG1, suggesting they have some features of pluripotency. Moreover, hFM-MSCs that underwent a dopaminergic differentiation protocol gradually increased the transcription of dopaminergic markers LMX1b, NURR1, PITX3, and DAT. We finally obtained a homogeneous population of cells resembling the morphology of primary midbrain dopaminergic neurons that expressed the functional dopaminergic markers TH, DAT, and Nurr1. In conclusion, our results suggested that hFM-MSCs retain the expression of pluripotency genes and are able to differentiate not only into mesodermal cells, but also into neuroectodermal dopaminergic neuron-like cells.

scholarly journals Multi-pronged approach to human mesenchymal stromal cells senescence quantification with a focus on label-free methods

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Weichao Zhai ◽  
Jerome Tan ◽  
Tobias Russell ◽  
Sixun Chen ◽  
Dennis McGonagle ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Preclinical Model ◽  
Label Free ◽  
Current Standard ◽  
Differentiation Potential ◽  
Human Mesenchymal Stromal Cells ◽  
Endogenous Fluorophores

AbstractHuman mesenchymal stromal cells (hMSCs) have demonstrated, in various preclinical settings, consistent ability in promoting tissue healing and improving outcomes in animal disease models. However, translation from the preclinical model into clinical practice has proven to be considerably more difficult. One key challenge being the inability to perform in situ assessment of the hMSCs in continuous culture, where the accumulation of the senescent cells impairs the culture’s viability, differentiation potential and ultimately leads to reduced therapeutic efficacies. Histochemical $$\upbeta $$ β -galactosidase staining is the current standard for measuring hMSC senescence, but this method is destructive and not label-free. In this study, we have investigated alternatives in quantification of hMSCs senescence, which included flow cytometry methods that are based on a combination of cell size measurements and fluorescence detection of SA-$$\upbeta $$ β -galactosidase activity using the fluorogenic substrate, C$${_{12}}$$ 12 FDG; and autofluorescence methods that measure fluorescence output from endogenous fluorophores including lipopigments. For identification of senescent cells in the hMSC batches produced, the non-destructive and label-free methods could be a better way forward as they involve minimum manipulations of the cells of interest, increasing the final output of the therapeutic-grade hMSC cultures. In this work, we have grown hMSC cultures over a period of 7 months and compared early and senescent hMSC passages using the advanced flow cytometry and autofluorescence methods, which were benchmarked with the current standard in $$\upbeta $$ β -galactosidase staining. Both the advanced methods demonstrated statistically significant values, (r = 0.76, p $$\le $$ ≤ 0.001 for the fluorogenic C$${_{12}}$$ 12 FDG method, and r = 0.72, p $$\le $$ ≤ 0.05 for the forward scatter method), and good fold difference ranges (1.120–4.436 for total autofluorescence mean and 1.082–6.362 for lipopigment autofluorescence mean) between early and senescent passage hMSCs. Our autofluroescence imaging and spectra decomposition platform offers additional benefit in label-free characterisation of senescent hMSC cells and could be further developed for adoption for future in situ cellular senescence evaluation by the cell manufacturers.

Neuroectodermally converted human mesenchymal stromal cells provide cytoprotective effects on neural stem cells and inhibit their glial differentiation

Cytotherapy ◽  
2010 ◽  
Vol 12 (4) ◽  
pp. 491-504 ◽  
Author(s):  
Hans-Jörg Habisch ◽  
Stefan Liebau ◽  
Thomas Lenk ◽  
Albert C. Ludolph ◽  
Rolf Brenner ◽  
...  
Keyword(s):  
Stem Cells ◽  
Neural Stem Cells ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Glial Differentiation ◽  
Human Mesenchymal Stromal Cells

scholarly journals Interrupted reprogramming into induced pluripotent stem cells does not rejuvenate human mesenchymal stromal cells

2018 ◽  
Author(s):  
Carolin Göbel ◽  
Roman Goetzke ◽  
Thomas Eggermann ◽  
Wolfgang Wagner
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Pluripotent Stem Cells ◽  
Replicative Senescence ◽  
Chromosome 1 ◽  
Differentiation Potential ◽  
Pluripotency Factors ◽  
Induced Pluripotent

AbstractReplicative senescence hampers application of mesenchymal stromal cells (MSCs) because it limits culture expansion, impairs differentiation potential, and hinders reliable standardization of cell products. MSCs can be rejuvenated by reprogramming into induced pluripotent stem cells (iPSCs), which is associated with complete erasure of age- and senescence-associated DNA methylation (DNAm) patterns. However, this process is also associated with erasure of cell-type and tissue-specific epigenetic characteristics that are not recapitulated upon re-differentiation towards MSCs. In this study, we therefore followed the hypothesis that overexpression of pluripotency factors under culture conditions that do not allow full reprogramming might reset senescence-associated changes without entering a pluripotent state. MSCs were transfected with episomal plasmids and either successfully reprogrammed into iPSCs or cultured in different media with continuous passaging every week. Overexpression of pluripotency factors without reprogramming did neither prolong culture expansion nor ameliorate molecular and epigenetic hallmarks of senescence. Notably, transfection resulted in immortalization of one cell preparation with gain of large parts of the long arm of chromosome 1. Taken together, premature termination of reprogramming does not result in rejuvenation of MSCs and harbours the risk of transformation. This approach is therefore not suitable to rejuvenate cells for cellular therapy.

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 Long term maintenance of myeloid leukemic stem cells cultured with unrelated human mesenchymal stromal cells

2015 ◽  
Vol 14 (1) ◽  
pp. 95-104 ◽  
Author(s):  
Sawa Ito ◽  
A. John Barrett ◽  
Amalia Dutra ◽  
Evgenia Pak ◽  
Samantha Miner ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Leukemic Stem Cells ◽  
Human Mesenchymal Stromal Cells ◽  
Term Maintenance ◽  
Cells Cultured

scholarly journals Characteristics of Human Natal Stem Cells Cultured in Allogeneic Medium

2018 ◽  
Vol 29 (5) ◽  
pp. 427-434
Author(s):  
Jakub Suchánek ◽  
Klara Zoe Browne ◽  
Sherine Adel Nasry ◽  
Tereza Suchánková Kleplová ◽  
Nela Pilbauerová ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stromal Cells ◽  
Dental Pulp ◽  
Cellular Therapy ◽  
Platelet Rich Plasma ◽  
Differentiation Potential ◽  
Telomere Shortening ◽  
Human Mesenchymal Stromal Cells ◽  
Expansion Medium

Abstract Recently, human natal dental pulp stem cells (hNDP-SCs) have been characterized in vitro and it has been shown that they satisfy criteria defining human mesenchymal stromal cells (MSCs), as proposed by the International Society for Cellular Therapy. However, these results were reached in the presence of xenogeneic expansion medium, which has the potential to alter the cells’ functional capacity. To determine the validity of the previously reported hNDP-SCs characteristics for human cell therapy, we have cultured hNDP-SCs in allogeneic expansion medium. Two hNDP-SC lineages were isolated from vital natal teeth, donated by a healthy newborn female and cultured in 2% platelet rich plasma (PRP). Analysis of the phenotypic expressions, proliferation rates, viability, telomerase length and in vitro adipogenic, osteogenic and chondrogenic differentiation potentials of two hNDP-SCs lineages (Zn001 and Zn002) were performed. Both lineages displayed similar morphology, proliferation rates, adipogenic, chondrogenic and osteogenic differentiation potential. Telomere shortening by 41.0% and 13.49% occurred from 3rd till 14th passage for lineages Zn001 and Zn002 respectively. Viability of both lineages was higher than 90%. Flow cytometry demonstrated that both lineages were positive to the majority of tested markers, including markers, which were negatively, expressed when hNDP-SCs were cultured previously in xenogeneic medium. Using immune-cytochemistry the cells were shown to express beta III-tubulin, nestin, neurofilaments and Nanog. PRP used as allogeneic medium is suitable for cultivation of hNDP-SCs.

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 Differentiation capacity and maintenance of differentiated phenotypes of human mesenchymal stromal cells cultured on two distinct types of 3D polymeric scaffolds

2015 ◽  
Vol 7 (12) ◽  
pp. 1574-1586 ◽  
Author(s):  
A. M. Leferink ◽  
D. Santos ◽  
M. Karperien ◽  
R. K. Truckenmüller ◽  
C. A. van Blitterswijk ◽  
...  
Keyword(s):  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Differentiation Potential ◽  
Soluble Factors ◽  
3D Scaffolds ◽  
Differentiation Capacity ◽  
Human Mesenchymal Stromal Cells ◽  
Polymeric Scaffolds ◽  
Cells Cultured

This study shows that the classical validation of hMSC differentiation potential on 3D scaffolds might not be sufficient to ensure the maintenance of the cells functionality in the absence of differentiation inducing soluble factors.

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