scholarly journals Clumping and Viability of Bone Marrow Derived Mesenchymal Stromal Cells under Different Preparation Procedures: A Flow Cytometry-Based In Vitro Study

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Li-li Cui ◽  
Tuure Kinnunen ◽  
Johannes Boltze ◽  
Johanna Nystedt ◽  
Jukka Jolkkonen
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Mesenchymal Stromal Cells ◽  
Normal Saline ◽  
Stromal Cells ◽  
Pulse Width ◽  
In Vitro Study ◽  
High Viability ◽  
Storage Solutions

Complications of microocclusions have been reported after intra-arterial delivery of mesenchymal stromal cells. Hence, quantification and efficient limitation of cell clumps in suspension before transplantation is important to reduce the risk. We used a flow cytometry-based pulse-width assay to assess the effects of different cell suspension concentrations (0.2–2.0 × 106/mL), storage solutions (complete growth medium, Dulbecco’s phosphate-buffered saline, and normal saline), storage time in suspension (0–9 h), and freeze-thawing procedure on the clumping of rat bone marrow derived mesenchymal stromal cells (BMMSCs) and also evaluated cell viability at the same time. Surprisingly, increasing the cell concentration did not result in more cell clumps in vitro. Freshly harvested (fresh) cells in normal saline had significantly fewer cell clumps and also displayed high viability (>90%). A time-dependent reduction in viability was observed for cells in all three storage solutions, without any significant change in the clumping tendency except for cells in medium. Fresh cells were more viable than their frozen-thawed counterparts, and fresh cells in normal saline had fewer cell clumps. In conclusion, cell clumping and viability could be affected by different cell preparation procedures, and quantification of cell clumping can be conducted using the flow cytometry-based pulse-width assay before intra-arterial cell delivery.

GD2+ Selected Mesenchymal Stromal Cells (MSCs) Demonstrate a More Robust Proliferation and Differentiation Potential Compared to Unselected Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1919-1919
Author(s):  
Caridad Martinez ◽  
Ted J. Hofmann ◽  
Roberta Marino ◽  
Massimo Dominici ◽  
Edwin M. Horwitz
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Doubling Time ◽  
Blue Staining ◽  
Differentiation Potential ◽  
Proliferation And Differentiation

Abstract Human mesenchymal stromal cells (MSCs) are spindle-shape, plastic-adherent cells with capacity to differentiate to bone, cartilage, and fat. MSCs express fibroblast, endothelial, and lymphocyte antigens, e.g. CD105, CD73, CD90, and CD166 which are the cornerstone of phenotypic characterization of these cells. We recently showed that MSCs are the only bone marrow cell to express GD2, a neural ganglioside. Now, for the first time we show that GD2 may serve as the single, unique, and definitive marker of marrow and adipose derived MSCs that can be used to isolate GD2+ MSCs, which possess important biologic properties justifying prospective isolation. MSCs expression of GD2 is uniformly high on freshly isolated and culture-expanded cells. Using the Miltenyi AutoMACS® device and a monoclonal antibody recognizing GD2 (clone 14.G2A) we prospectively isolated a highly enriched MSC population from bone marrow MNCs. The selected fraction was >98% pure for GD2+ cells determined by flow cytometry. Light microscopy showed that the GD2-selected cells were smaller, thinner, and more spindle-like when attached to plastic compared to unselected MSCs which spread wider along the surface of the culture flask, the so-called “fried egg” appearance. The doubling time of GD2-selected MSCs was 30 hrs compared to 90 hrs for unselected cells representing a 3-fold greater growth rate. Cell cycle analysis by flow cytometry showed ∼80% of cells were in G0/G1 and ∼20% were in S/G2/M phases of the cell cycle in both populations. With the shorter doubling time, this data indicates that GD2-selected MSCs move through the cell cycle more rapidly than unselected cells. In accordance with this finding, electron microscopy showed few organelles in the GD2-selected cells, but increase lamellar bodies indicating overall less complexity, but consistent with a greater membrane turnover rate (cell division) than unselected MSCs. Moreover, flow cytometric analysis revealed an increased expression of receptors for bFGF and EFG, known mitogenic factor receptors for MSCs, compared to unselected MSCs. In vitro differentiation of GD2-selected MSCs showed a more robust osteoid matrix formation (osteoblast) and proteoglycan formation (chondroblast) assayed by semi-quantitative Alizarin Red and Alcian blue staining, respectively. Additionally, more GD2-selected MSCs differentiated to adipocytes than among unselected cells. Surprisingly, GD2 expression persisted on the in vitro human MSC-differentiated osteoblasts, chondroblasts, and adipocytes, in contrast to human bone-derived osteoblasts, adipose tissue, and cartilage which lacked GD2 expression. We conclude that GD2 is a unique, stably expressed surface MSC marker which can be used to prospectively isolate MSCs from marrow, GD2-selcted cells have a more robust in vitro proliferation and differentiation potential which may be valuable for cell therapy, and biologically, in vitro isolated MSCs may not represent the in vivo progenitor for bone, fat, or cartilage.

Mesenchymal stromal cells from amniotic fluid are less prone to senescence compared to those obtained from bone marrow: An in vitro study

2018 ◽  
Vol 233 (11) ◽  
pp. 8996-9006 ◽  
Author(s):  
Nicola Alessio ◽  
Caterina Pipino ◽  
Domitilla Mandatori ◽  
Pamela Di Tomo ◽  
Angela Ferone ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Amniotic Fluid ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
In Vitro Study ◽  
Vitro Study

scholarly journals Growth characteristics of human bone marrow mesenchymal stromal cells at cultivation on synthetic polyelectrolyte nanofilms in vitro

Heliyon ◽  
2021 ◽  
Vol 7 (3) ◽  
pp. e06517
Author(s):  
Lyudmila M. Mezhevikina ◽  
Dmitriy A. Reshetnikov ◽  
Maria G. Fomkina ◽  
Nurbol O. Appazov ◽  
Saltanat Zh. Ibadullayeva ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Growth Characteristics ◽  
Human Bone ◽  
Human Bone Marrow

Atelocollagen Sponge as a Stem Cell Implantation Scaffold for the Treatment of Scarred Vocal Folds

2010 ◽  
Vol 119 (11) ◽  
pp. 805-810 ◽  
Author(s):  
Satoshi Ohno ◽  
Shigeru Hirano ◽  
Ichiro Tateya ◽  
Shin-Ichi Kanemaru ◽  
Hiroo Umeda ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Stromal Cells ◽  
Fluorescent Protein ◽  
In Vitro Study ◽  
Vocal Folds ◽  
Green Fluorescent ◽  
Cell Implantation

Objectives: Treatment of vocal fold scarring remains a therapeutic challenge. Our group previously reported the efficacy of treating injured vocal folds by implantation of bone marrow—derived stromal cells containing mesenchymal stem cells. Appropriate scaffolding is necessary for the stem cell implant to achieve optimal results. Terudermis is an atelocollagen sponge derived from calf dermis. It has large pores that permit cellular entry and is degraded in vivo. These characteristics suggest that this material may be a good candidate for use as scaffolding for implantation of cells. The present in vitro study investigated the feasibility of using Terudermis as such a scaffold. Methods: Bone marrow—derived stromal cells were obtained from GFP (green fluorescent protein) mouse femurs. The cells were seeded into Terudermis and incubated for 5 days. Their survival, proliferation, and expression of extracellular matrix were examined. Results: Bone marrow—derived stromal cells adhered to Terudermis and underwent significant proliferation. Immunohistochemical examination demonstrated that adherent cells were positive for expression of vimentin, desmin, fibronectin, and fsp1 and negative for beta III tubulin. These findings indicate that these cells were mesodermal cells and attached to the atelocollagen fibers biologically. Conclusions: The data suggest that Terudermis may have potential as stem cell implantation scaffolding for the treatment of scarred vocal folds.

scholarly journals Evaluation of Browning Agents on the White Adipogenesis of Bone Marrow Mesenchymal Stromal Cells: A Contribution to Fighting Obesity

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 403
Author(s):  
Girolamo Di Maio ◽  
Nicola Alessio ◽  
Ibrahim Halil Demirsoy ◽  
Gianfranco Peluso ◽  
Silverio Perrotta ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
White Adipocyte ◽  
Drug Candidates ◽  
Fat Depots ◽  
Treatment Of Obesity ◽  
White Fat

Brown-like adipocytes can be induced in white fat depots by a different environmental or drug stimuli, known as “browning” or “beiging”. These brite adipocytes express thermogenin UCP1 protein and show different metabolic advantages, such as the ability to acquire a thermogenic phenotype corresponding to standard brown adipocytes that counteracts obesity. In this research, we evaluated the effects of several browning agents during white adipocyte differentiation of bone marrow-derived mesenchymal stromal cells (MSCs). Our in vitro findings identified two compounds that may warrant further in vivo investigation as possible anti-obesity drugs. We found that rosiglitazone and sildenafil are the most promising drug candidates for a browning treatment of obesity. These drugs are already available on the market for treating diabetes and erectile dysfunction, respectively. Thus, their off-label use may be contemplated, but it must be emphasized that some severe side effects are associated with use of these drugs.

scholarly journals Mesenchymal Stromal Cells Orchestrate Follicular Lymphoma Cell Niche Through the CCL2-Dependent Recruitment and Polarization of Monocytes

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1566-1566
Author(s):  
Fabien Guilloton ◽  
Gersende Caron ◽  
Cédric Ménard ◽  
Céline Pangault ◽  
Patricia Amé-Thomas ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Cell Growth ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Gene Set Enrichment Analysis ◽  
Cell Niche

Abstract Abstract 1566 Accumulating evidence indicates that infiltrating stromal cells contribute directly and indirectly to tumor growth in a wide range of solid cancers and hematological malignancies. In follicular lymphoma (FL), malignant B cells are found admixed with heterogeneous lymphoid-like stromal cells within invaded lymph nodes and bone marrow (BM). In addition, in vitro functional studies have underlined that mesenchymal cells recruit malignant FL B cells and protect them from spontaneous and drug-induced apoptosis. In particular, we have previously demonstrated that mesenchymal stromal cells (MSC) efficiently support in vitro FL B-cell survival, especially after their engagement towards lymphoid differentiation through treatment with TNF-α and Lymphotoxin-α1β2 (TNF/LT) or after coculture with malignant B cells. However, the mechanisms of this supportive activity remain largely unknown. In this study, we used Affymetrix U133 Plus 2.0 microarrays, to compare the gene expression profile (GEP) of bone marrow-derived MSC (BM-MSC) obtained from 10 FL patients at diagnosis versus 6 age-matched healthy donors (HD). In these conditions, neither the CFU-F concentration in the BM nor the cumulative population doubling of BM-MSC significantly differed between HD and FL patients. Unsupervised analysis was able to perfectly segregate FL-MSC from HD-MSC and we identified, using supervised analyzes, a list of 408 probesets defining FL-MSC signature, including 320 nonredundant genes upregulated in FL-MSC compared to HD-MSC. We then defined the GEP of human lymphoid-like stroma using HD-MSC treated in vitro by TNF/LT and demonstrated, by a Gene Set Enrichment Analysis (GSEA) approach, that the FL-MSC signature is significantly enriched for genes associated with a lymphoid-like commitment. Interestingly, CCL2 was strongly overexpressed by FL-MSC, was upregulated in HD-MSC by coculture with malignant B cells, and was detected at a higher level in FL BM plasma compared to normal BM plasma (504.4 pg/mL [23.8-4413] versus 33.9 pg/mL [5-126.1]; P <.01). In agreement, FL-MSC triggered a more potent CCL2-dependent monocyte migration than HD-MSC. Moreover, FL-MSC and macrophages cooperated to sustain malignant B-cell growth through both protection from apoptosis and enhancement of cell proliferation. Finally, FL-MSC promoted monocyte differentiation towards a proangiogenic LPS-unresponsive phenotype close to that of tumor-associated macrophages. We unraveled a key role for the Notch pathway in this process and identified an overexpression of JAGGED1 in FL-MSC compared to HD-MSC. Altogether, these results highlight the complex role of FL stromal cells that promote direct tumor B-cell growth and orchestrate FL cell niche. The identification and characterization of this intricate network of cell interactions may provide novel therapeutic targets in this disease. Disclosures: No relevant conflicts of interest to declare.

Comparative Analysis of Biological and Functional Properties of Bone Marrow Mesenchymal Stromal Cells Expanded in Media with Different Platelet Lysate Content

2018 ◽  
Vol 205 (4) ◽  
pp. 226-239 ◽  
Author(s):  
Marijana Skific ◽  
Mirna Golemovic ◽  
Kristina Crkvenac-Gornik ◽  
Radovan Vrhovac ◽  
Branka Golubic Cepulic
Keyword(s):  
Bone Marrow ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Immunological Tolerance ◽  
Biological Properties ◽  
Platelet Lysate ◽  
Population Doubling ◽  
Population Doubling Time ◽  
Significant Difference

Due to their ability to induce immunological tolerance in the recipient, mesenchymal stromal cells (MSCs) have been utilized in the treatment of various hematological and immune- and inflammation-mediated diseases. The clinical application of MSCs implies prior in vitro expansion that usually includes the use of fetal bovine serum (FBS). The present study evaluated the effect of different platelet lysate (PL) media content on the biological properties of MSCs. MSCs were isolated from the bone marrow of 13 healthy individuals and subsequently expanded in three different culture conditions (10% PL, 5% PL, 10% FBS) during 4 passages. The cells cultured in different conditions had comparable immunophenotype, clonogenic potential, and differentiation capacity. However, MSC growth was significantly enhanced in the presence of PL. Cultures supplemented with 10% PL had a higher number of cumulative population doublings in all passages when compared to the 5% PL condition (p < 0.03). Such a difference was also observed when 10% PL and 10% FBS conditions were compared (p < 0.005). A statistically significant difference in population doubling time was determined only between the 10% PL and 10% FBS conditions (p < 0.005). Furthermore, MSCs cultured in 10% PL were able to cause a 66.9% reduction of mitogen-induced lymphocyte proliferation. Three chromosome aberrations were detected in PL conditions. Since two changes occurred in the same do nor, it is possible they were donor dependent rather than caused by the culture condition. These findings demonstrate that a 10% PL condition enables a higher yield of MSCs within a shorter time without altering MSC properties, and should be favored over the 5% PL condition.

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