scholarly journals Culture and characterization of various porcine integumentary-connective tissue-derived mesenchymal stromal cells to facilitate tissue adhesion to percutaneous metal implants

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Devaveena Dey ◽  
Nicholas G. Fischer ◽  
Andrea H. Dragon ◽  
Elsa Ronzier ◽  
Isha Mutreja ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Connective Tissue ◽  
Achilles Tendon ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Soft Tissues ◽  
Flexor Tendon ◽  
Cytoskeletal Proteins ◽  
Preclinical Research ◽  
Differentiation Potential

Abstract Background Transdermal osseointegrated prosthesis have relatively high infection rates leading to implant revision or failure. A principle cause for this complication is the absence of a durable impervious biomechanical seal at the interface of the hard structure (implant) and adjacent soft tissues. This study explores the possibility of recapitulating an analogous cellular musculoskeletal-connective tissue interface, which is present at naturally occurring integumentary tissues where a hard structure exits the skin, such as the nail bed, hoof, and tooth. Methods Porcine mesenchymal stromal cells (pMSCs) were derived from nine different porcine integumentary and connective tissues: hoof-associated superficial flexor tendon, molar-associated periodontal ligament, Achilles tendon, adipose tissue and skin dermis from the hind limb and abdominal regions, bone marrow and muscle. For all nine pMSCs, the phenotype, multi-lineage differentiation potential and their adhesiveness to clinical grade titanium was characterized. Transcriptomic analysis of 11 common genes encoding cytoskeletal proteins VIM (Vimentin), cell–cell and cell–matrix adhesion genes (Vinculin, Integrin β1, Integrin β2, CD9, CD151), and for ECM genes (Collagen-1a1, Collagen-4a1, Fibronectin, Laminin-α5, Contactin-3) in early passaged cells was performed using qRT-PCR. Results All tissue-derived pMSCs were characterized as mesenchymal origin by adherence to plastic, expression of cell surface markers including CD29, CD44, CD90, and CD105, and lack of hematopoietic (CD11b) and endothelial (CD31) markers. All pMSCs differentiated into osteoblasts, adipocytes and chondrocytes, albeit at varying degrees, under specific culture conditions. Among the eleven adhesion genes evaluated, the cytoskeletal intermediate filament vimentin was found highly expressed in pMSC isolated from all tissues, followed by genes for the extracellular matrix proteins Fibronectin and Collagen-1a1. Expression of Vimentin was the highest in Achilles tendon, while Fibronectin and Col1agen-1a1 were highest in molar and hoof-associated superficial flexor tendon bone marrow, respectively. Achilles tendon ranked the highest in both multilineage differentiation and adhesion assessments to titanium metal. Conclusions These findings support further preclinical research of these tissue specific-derived MSCs in vivo in a transdermal osseointegration implant model.

scholarly journals Expansion and characterization of bone marrow derived human mesenchymal stromal cells in serum-free conditions

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Samatha Bhat ◽  
Pachaiyappan Viswanathan ◽  
Shashank Chandanala ◽  
S. Jyothi Prasanna ◽  
Raviraja N. Seetharam
Keyword(s):  
Bone Marrow ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Population Doubling ◽  
Seeding Density ◽  
Population Doubling Time ◽  
Differentiation Potential ◽  
Serum Free ◽  
Safety Issues ◽  
Free Media

AbstractBone marrow-derived mesenchymal stromal cells (BM-MSCs) are gaining increasing importance in the field of regenerative medicine. Although therapeutic value of MSCs is now being established through many clinical trials, issues have been raised regarding their expansion as per regulatory guidelines. Fetal bovine serum usage in cell therapy poses difficulties due to its less-defined, highly variable composition and safety issues. Hence, there is a need for transition from serum-based to serum-free media (SFM). Since SFM are cell type-specific, a precise analysis of the properties of MSCs cultured in SFM is required to determine the most suitable one. Six different commercially available low serum/SFM with two different seeding densities were evaluated to explore their ability to support the growth and expansion of BM-MSCs and assess the characteristics of BM-MSCs cultured in these media. Except for one of the SFM, all other media tested supported the growth of BM-MSCs at a low seeding density. No significant differences were observed in the expression of MSC specific markers among the various media tested. In contrary, the population doubling time, cell yield, potency, colony-forming ability, differentiation potential, and immunosuppressive properties of MSCs varied with one another. We show that SFM tested supports the growth and expansion of BM-MSCs even at low seeding density and may serve as possible replacement for animal-derived serum.

scholarly journals Augmenting emergency granulopoiesis with CpG conditioned mesenchymal stromal cells in murine neutropenic sepsis

2020 ◽  
Vol 4 (19) ◽  
pp. 4965-4979 ◽  
Author(s):  
Julie Ng ◽  
Fei Guo ◽  
Anna E. Marneth ◽  
Sailaja Ghanta ◽  
Min-Young Kwon ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Therapeutic Potential ◽  
Neutrophil Extracellular Trap ◽  
Differentiation Potential ◽  
Neutropenic Sepsis ◽  
Cpg Oligodeoxynucleotide ◽  
Increased Risk

Abstract Patients with immune deficiencies from cancers and associated treatments represent a growing population within the intensive care unit with increased risk of morbidity and mortality from sepsis. Mesenchymal stromal cells (MSCs) are an integral part of the hematopoietic niche and express toll-like receptors, making them candidate cells to sense and translate pathogenic signals into an innate immune response. In this study, we demonstrate that MSCs administered therapeutically in a murine model of radiation-associated neutropenia have dual actions to confer a survival benefit in Pseudomonas aeruginosa pneumo-sepsis that is not from improved bacterial clearance. First, MSCs augment the neutrophil response to infection, an effect that is enhanced when MSCs are preconditioned with CpG oligodeoxynucleotide, a toll-like receptor 9 agonist. Using cytometry by time of flight, we identified proliferating neutrophils (Ly6GlowKi-67+) as the main expanded cell population within the bone marrow. Further analysis revealed that CpG-MSCs expand a lineage restricted progenitor population (Lin−Sca1+C-kit+CD150−CD48+) in the bone marrow, which corresponded to a doubling in the myeloid proliferation and differentiation potential in response to infection compared with control. Despite increased neutrophils, no reduction in organ bacterial count was observed between experimental groups. However, the second effect exerted by CpG-MSCs is to attenuate organ damage, particularly in the lungs. Neutrophils obtained from irradiated mice and cocultured with CpG-MSCs had decreased neutrophil extracellular trap formation, which was associated with decreased citrullinated H3 staining in the lungs of mice given CpG-MSCs in vivo. Thus, this preclinical study provides evidence for the therapeutic potential of MSCs in neutropenic sepsis.

scholarly journals Comparative Analysis of Mesenchymal Stromal Cells Biological Properties

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Angela De Luca ◽  
Rosanna Verardi ◽  
Arabella Neva ◽  
Patrizia Benzoni ◽  
Elisabetta Crescini ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Adipose Tissue ◽  
Cell Therapy ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Subcutaneous Fat ◽  
Biological Properties ◽  
Human Mscs ◽  
Differentiation Potential ◽  
Tissue Samples

The stromal progenitors of mesodermal cells, mesenchymal stromal cells (MSCs), are a heterogeneous population of plastic adherent fibroblast-like cells with extensive proliferative capacity and differentiation potential. Human MSCs have now been isolated from various tissues including bone marrow, muscle, skin, and adipose tissue, the latter being one of the most suitable cell sources for cell therapy, because of its easy accessibility, minimal morbidity, and abundance of cells. Bone marrow and subcutaneous or visceral adipose tissue samples were collected, digested with collagenase if needed, and seeded in Iscove's medium containing 5% human platelet lysate. Nonadherent cells were removed after 2-3 days and the medium was replaced twice a week. Confluent adherent cells were detached, expanded, and analyzed for several biological properties such as morphology, immunophenotype, growth rate, senescence, clonogenicity, differentiation capacity, immunosuppression, and secretion of angiogenic factors. The results show significant differences between lines derived from subcutaneous fat compared to those derived from visceral fat, such as the higher proliferation rate of the first and the strong induction of angiogenesis of the latter. We are convinced that the identification of the peculiarities of MSCs isolated from different tissues will lead to their more accurate use in cell therapy.

scholarly journals Kinship of conditionally immortalized cells derived from fetal bone to human bone-derived mesenchymal stroma cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
S. Marozin ◽  
B. Simon-Nobbe ◽  
S. Irausek ◽  
L. W. K. Chung ◽  
G. Lepperdinger
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Human Bone ◽  
Multilineage Differentiation ◽  
Differentiation Potential ◽  
Osteoblast Cell Line ◽  
Age Related

AbstractThe human fetal osteoblast cell line (hFOB 1.19) has been proposed as an accessible experimental model for study of osteoblast biology relating to drug development and biomaterial engineering. For their multilineage differentiation potential, hFOB has been compared to human mesenchymal progenitor cells and used to investigate bone-metabolism in vitro. Hereby, we studied whether and to what extent the conditionally immortalized cell line hFOB 1.19 can serve as a surrogate model for bone-marrow derived mesenchymal stromal cells (bmMSC). hFOB indeed exhibit specific characteristics reminiscent of bmMSC, as colony formation, migration capacity and the propensity to grow as multicellular aggregates. After prolonged culture, in contrast to the expected effect of immortalization, hFOB acquired a delayed growth rate. In close resemblance to bmMSC at increasing passages, also hFOB showed morphological abnormalities, enlargement and finally reduced proliferation rates together with enhanced expression of the cell cycle inhibitors p21 and p16. hFOB not only have the ability to undergo multilineage differentiation but portray several important aspects of human bone marrow mesenchymal stromal cells. Superior to primary MSC and osteoblasts, hFOB enabled the generation of continuous cell lines. These provide an advanced basis for investigating age-related dysfunctions of MSCs in an in vitro 3D-stem cell microenvironment.

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.

scholarly journals Molecular and Functional Phenotypes of Human Bone Marrow-Derived Mesenchymal Stromal Cells Depend on Harvesting Techniques

2020 ◽  
Vol 21 (12) ◽  
pp. 4382 ◽  
Author(s):  
Sebastian Walter ◽  
Thomas Randau ◽  
Cäcilia Hilgers ◽  
El-Mustapha Haddouti ◽  
Werner Masson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Cellular Therapy ◽  
Donor Site ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Differentiation Potential ◽  
Significant Difference ◽  
Harvesting Techniques

Mesenchymal stromal cells (MSC) harvested in different tissues from the same donor exhibit different phenotypes. Each phenotype is not only characterized by a certain pattern of cell surface markers, but also different cellular functionalities. Only recently were different harvesting and processing techniques found to contribute to this phenomenon as well. This study was therefore set up to investigate proteomic and functional properties of human bone marrow-derived MSCs (hBM-MSC). These were taken from the same tissue and donor site but harvested either as aspirate or bone chip cultures. Both MSC populations were profiled for MSC markers defined by the International Society for Cellular Therapy (ISCT), MSC markers currently under discussion and markers of particular interest. While classic ISCT MSC markers did not show any significant difference between aspirate and outgrowth hBM-MSCs, our additional characterization panel revealed distinct patterns of differentially expressed markers. Furthermore, hBM-MSCs from aspirate cultures demonstrated a significantly higher osteogenic differentiation potential than outgrowth MSCs, which could be confirmed using a transcriptional approach. Our comparison of MSC phenotypes obtained by different harvesting techniques suggests the need of future standardized harvesting, processing and phenotyping procedures in order to gain better comparability in the MSC field.

scholarly journals A focus on allogeneic mesenchymal stromal cells as a versatile therapeutic tool for treating multiple sclerosis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ameneh Shokati ◽  
Abdorreza Naser Moghadasi ◽  
Mohsen Nikbakht ◽  
Mohammad Ali Sahraian ◽  
Seyed Asadollah Mousavi ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
Bone Marrow ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Autologous Bone Marrow ◽  
Cell Number ◽  
Differentiation Potential ◽  
Disease Modifying Drugs ◽  
Impact Reduction ◽  
Ms Therapy

AbstractMultiple sclerosis (MS) is a central nervous system (CNS) chronic illness with autoimmune, inflammatory, and neurodegenerative effects characterized by neurological disorder and axonal loss signs due to myelin sheath autoimmune T cell attacks. Existing drugs, including disease-modifying drugs (DMD), help decrease the intensity and frequency of MS attacks, inflammatory conditions, and CNS protection from axonal damage. As they cannot improve axonal repair and show side effects, new therapeutic options are required. In this regard, due to their neuroprotection properties, immunomodulatory effects, and the ability to differentiate into neurons, the transplantation of mesenchymal stromal cells (MSCs) can be used for MS therapy. The use of adipose-derived MSCs (AdMSCs) or autologous bone marrow MSCs (BMSCs) has demonstrated unexpected effects including the invasive and painful isolation method, inadequate amounts of bone marrow (BM) stem cells, the anti-inflammatory impact reduction of AdMSCs that are isolated from fat patients, and the cell number and differentiation potential decrease with an increase in the age of BMSCs donor. Researchers have been trying to search for alternate tissue sources for MSCs, especially fetal annexes, which could offer a novel therapeutic choice for MS therapy due to the limitation of low cell yield and invasive collection methods of autologous MSCs. The transplantation of MSCs for MS treatment is discussed in this review. Finally, it is suggested that allogeneic sources of MSCs are an appealing alternative to autologous MSCs and could hence be a potential novel solution to MS therapy.

scholarly journals Vessel Wall-Derived Mesenchymal Stromal Cells Share Similar Differentiation Potential and Immunomodulatory Properties with Bone Marrow-Derived Stromal Cells

2020 ◽  
Vol 2020 ◽  
pp. 1-16
Author(s):  
Zoltán Veréb ◽  
Anett Mázló ◽  
Attila Szabó ◽  
Szilárd Póliska ◽  
Attila Kiss ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mesenchymal Stromal Cells ◽  
Saphenous Vein ◽  
Stromal Cells ◽  
Vessel Wall ◽  
Gene Expression Pattern ◽  
Differentiation Potential ◽  
Inflammatory Conditions ◽  
Immunomodulatory Properties

Purpose. This study is aimed at investigating the phenotype, differentiation potential, immunomodulatory properties, and responsiveness of saphenous vein vessel wall-derived mesenchymal stromal cells (SV-MSCs) to various TLR ligands and proinflammatory cytokines, as well as comparing their features to those of their bone marrow-derived counterparts (BM-MSCs). Methods. SV-MSCs were isolated by enzymatic digestion of the saphenous vein vessel wall. Phenotype analysis was carried out by flow cytometry and microscopy, whereas adipogenic, chondrogenic, and osteogenic differentiation potentials were tested in in vitro assays. For comparative analysis, the expression of different stemness, proliferation, and differentiation-related genes was determined by Affymetrix gene array. To compare the immunomodulatory properties of SV-MSCs and BM-MSCs, mixed lymphocyte reaction was applied. To investigate their responses to various activating stimuli, MSCs were treated with TLR ligands (LPS, PolyI:C) or proinflammatory cytokines (TNFα, IL-1β, IFNγ), and the expression of various early innate immune response-related genes was assessed by qPCR, while secretion of selected cytokines and chemokines was measured by ELISA. Results. The isolated SV-MSCs were able to differentiate into bone, fat, and cartilage cells/direction in vitro. SV-MSCs expressed the most important MSC markers (CD29, CD44, CD73, CD90, and CD105) and shared almost identical phenotypic characteristics with BM-MSCs. Their gene expression pattern and activation pathways were close to those of BM-MSCs. SV-MSCs showed better immunosuppressive activity inhibiting phytohemagglutinin-induced T lymphocyte proliferation in vitro than BM-MSCs. Cellular responses to treatments mimicking inflammatory conditions were comparable in the bone marrow- and saphenous vein-derived MSCs. Namely, similar to BM-MSCs, SV-MSCs secreted increased amount of IL-6 and IL-8 after 12- or 24-hour treatment with LPS, PolyI:C, TNFα, or IL-1β, compared to untreated controls. Interestingly, a different CXCL-10/IP-10 secretion pattern could be observed under inflammatory conditions in the two types of MSCs. Conclusion. Based on our results, cells isolated from saphenous vein vessel wall fulfilled the ISCT’s (International Society for Cellular Therapy) criteria for multipotent mesenchymal stromal cells, and no significant differences in the phenotype, gene expression pattern, and responsiveness to inflammatory stimuli could be observed between BM-MSCs and SV-MSCs, while the latter cells have more potent immunosuppressive activity in vitro. Further functional assays have to be performed to reveal whether SV-MSCs could be useful for certain regenerative therapeutic applications or tissue engineering purposes.

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