scholarly journals Novel Methods to Mobilize, Isolate, and Expand Mesenchymal Stem Cells

2021 ◽  
Vol 22 (11) ◽  
pp. 5728
Author(s):  
Cristiano P. Vieira ◽  
Taralyn M. McCarrel ◽  
Maria B. Grant
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Therapeutic Potential ◽  
Inflammatory Diseases ◽  
Embryoid Bodies ◽  
Surface Marker ◽  
Blood Collection ◽  
Surface Marker Expression ◽  
Marker Expression ◽  
Cellular Immune

Numerous studies demonstrate the essential role of mesenchymal stem cells (MSCs) in the treatment of metabolic and inflammatory diseases, as these cells are known to modulate humoral and cellular immune responses. In this manuscript, we efficiently present two novel approaches to obtain MSCs from equine or human sources. In our first approach, we used electro-acupuncture as previously described by our group to mobilize MSCs into the peripheral blood of horses. For equine MSC collection, culture, and expansion, we used the Miltenyi Biotec CliniMACS Prodigy system of automated cell manufacturing. Using this system, we were able to generate appoximately 100 MSC colonies that exhibit surface marker expression of CD105 (92%), CD90 (85%), and CD73 (88%) within seven days of blood collection. Our second approach utilized the iPSC embryoid bodies from healthy or diabetic subjects where the iPSCs were cultured in standard media (endothelial + mesoderm basal media). After 21 days, the cells were FACS sorted and exhibited surface marker expression of CD105, CD90, and CD73. Both the equine cells and the human iPSC-derived MSCs were able to differentiate into adipogenic, osteogenic, and chondrogenic lineages. Both methods described simple and highly efficient methods to produce cells with surface markers phenotypically considered as MSCs and may, in the future, facilitate rapid production of MSCs with therapeutic potential.

scholarly journals Shared Cell Surface Marker Expression in Mesenchymal Stem Cells and Adult Sarcomas

2012 ◽  
Vol 2 (1) ◽  
pp. 53-60 ◽  
Author(s):  
Stefan Wirths ◽  
Elke Malenke ◽  
Torsten Kluba ◽  
Simone Rieger ◽  
Martin R. Müller ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Cell Surface ◽  
Surface Marker ◽  
Cell Surface Marker ◽  
Surface Marker Expression ◽  
Marker Expression ◽  
Cell Surface Marker Expression

scholarly journals Comparison of Canine and Feline Adipose-Derived Mesenchymal Stem Cells/Medicinal Signaling Cells With Regard to Cell Surface Marker Expression, Viability, Proliferation, and Differentiation Potential

2021 ◽  
Vol 7 ◽  
Author(s):  
Metka Voga ◽  
Valerija Kovač ◽  
Gregor Majdic
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Surface Marker ◽  
Growth Media ◽  
Differentiation Potential ◽  
Surface Marker Expression ◽  
Proliferation And Differentiation ◽  
Marker Expression ◽  
In Cells

Remarkable immunomodulatory abilities of mesenchymal stem cells, also called multipotent mesenchymal stromal cells or medicinal signaling cells (MSCs), have entailed significant advances in veterinary regenerative medicine in recent years. Despite positive outcomes from MSC therapies in various diseases in dogs and cats, differences in MSC characteristics between small animal veterinary patients are not well-known. We performed a comparative study of cells' surface marker expression, viability, proliferation, and differentiation capacity of adipose-derived MSCs (ADMSCs) from dogs and domestic cats. The same growth media and methods were used to isolate, characterize, and culture canine and feline ADMSCs. Adipose tissue was collected from 11 dogs and 8 cats of both sexes. The expression of surface markers CD44, CD90, and CD34 was detected by flow cytometry. Viability at passage 3 was measured with the hemocytometer and compared to the viability measured by flow cytometry after 1 day of handling. The proliferation potential of MSCs was measured by calculating cell doubling and cell doubling time from second to eighth passage. Differentiation potential was determined at early and late passages by inducing cells toward adipogenic, osteogenic, and chondrogenic differentiation using commercial media. Our study shows that the percentage of CD44+CD90+ and CD34−/− cells is higher in cells from dogs than in cells from cats. The viability of cells measured by two different methods at passage 3 differed between the species, and finally, canine ADMSCs possess greater proliferation and differentiation potential in comparison to the feline ADMSCs.

scholarly journals Jaw Periosteum-Derived Mesenchymal Stem Cells Regulate THP-1-Derived Macrophage Polarization

2021 ◽  
Vol 22 (9) ◽  
pp. 4310
Author(s):  
Fang He ◽  
Felix Umrath ◽  
Siegmar Reinert ◽  
Dorothea Alexander
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Macrophage Polarization ◽  
Polarization State ◽  
Osteonecrosis Of The Jaw ◽  
Surface Marker ◽  
Surface Marker Expression ◽  
Periosteal Cells ◽  
Immunomodulatory Properties ◽  
Marker Expression

Mesenchymal stem cells from bone marrow have powerful immunomodulatory capabilities. The interactions between jaw periosteal cells (JPCs) and macrophages are not only relevant for the application of JPCs in regenerative medicine, but this understanding could also help treating diseases like osteonecrosis of the jaw. In previous studies, we analyzed, for the first time, immunomodulatory features of 2D- and 3D-cultured JPCs. In the present work, the effects of JPCs on the polarization state of macrophages in contact coculture were analyzed. To improve the macrophage polarization study, different concentrations of PMA (5 nM, 25 nM, and 150 nM) or different medium supplementations (10% FBS, 10% hPL and 5% hPL) were compared. Further, in order to analyze the effects of JPCs on macrophage polarization, JPCs and PMA-stimulated THP-1 cells were cocultured under LPS/IFN-γ or IL-4/IL-13 stimulatory conditions. Surface marker expression of M1 and M2 macrophages were analyzed under the different culture supplementations in order to investigate the immunomodulatory properties of JPCs. Our results showed that 5 nM PMA can conduct an effective macrophage polarization. The analyses of morphological parameters and surface marker expression showed more distinct M1/M2 phenotypes over FBS supplementation when using 5% hPL during macrophage polarization. In the coculture, immunomodulatory properties of JPCs improved significantly under 5% hPL supplementation compared to other supplementations. We concluded that, under the culture condition with 5% hPL, JPCs were able to effectively induce THP-1-derived macrophage polarization.

Therapeutic Potential of Amniotic Fluid Derived Mesenchymal Stem Cells Based on their Differentiation Capacity and Immunomodulatory Properties

2019 ◽  
Vol 14 (4) ◽  
pp. 327-336 ◽  
Author(s):  
Carl R. Harrell ◽  
Marina Gazdic ◽  
Crissy Fellabaum ◽  
Nemanja Jovicic ◽  
Valentin Djonov ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Animal Models ◽  
Amniotic Fluid ◽  
Therapeutic Potential ◽  
Inflammatory Diseases ◽  
Therapeutic Effects ◽  
Differentiation Potential ◽  
Differentiation Capacity ◽  
Cell Based Therapy

Background: Amniotic Fluid Derived Mesenchymal Stem Cells (AF-MSCs) are adult, fibroblast- like, self-renewable, multipotent stem cells. During the last decade, the therapeutic potential of AF-MSCs, based on their huge differentiation capacity and immunomodulatory characteristics, has been extensively explored in animal models of degenerative and inflammatory diseases. Objective: In order to describe molecular mechanisms responsible for the therapeutic effects of AFMSCs, we summarized current knowledge about phenotype, differentiation potential and immunosuppressive properties of AF-MSCs. Methods: An extensive literature review was carried out in March 2018 across several databases (MEDLINE, EMBASE, Google Scholar), from 1990 to present. Keywords used in the selection were: “amniotic fluid derived mesenchymal stem cells”, “cell-therapy”, “degenerative diseases”, “inflammatory diseases”, “regeneration”, “immunosuppression”. Studies that emphasized molecular and cellular mechanisms responsible for AF-MSC-based therapy were analyzed in this review. Results: AF-MSCs have huge differentiation and immunosuppressive potential. AF-MSCs are capable of generating cells of mesodermal origin (chondrocytes, osteocytes and adipocytes), neural cells, hepatocytes, alveolar epithelial cells, insulin-producing cells, cardiomyocytes and germ cells. AF-MSCs, in juxtacrine or paracrine manner, regulate proliferation, activation and effector function of immune cells. Due to their huge differentiation capacity and immunosuppressive characteristic, transplantation of AFMSCs showed beneficent effects in animal models of degenerative and inflammatory diseases of nervous, respiratory, urogenital, cardiovascular and gastrointestinal system. Conclusion: Considering the fact that amniotic fluid is obtained through routine prenatal diagnosis, with minimal invasive procedure and without ethical concerns, AF-MSCs represents a valuable source for cell-based therapy of organ-specific or systemic degenerative and inflammatory diseases.

scholarly journals Modulation of Adult Mesenchymal Stem Cells Activity by Toll-Like Receptors: Implications on Therapeutic Potential

2010 ◽  
Vol 2010 ◽  
pp. 1-9 ◽  
Author(s):  
Olga DelaRosa ◽  
Eleuterio Lombardo
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Therapeutic Potential ◽  
Inflammatory Diseases ◽  
Culture Conditions ◽  
Toll Like Receptors ◽  
Adult Mesenchymal Stem Cells ◽  
Special Relevance ◽  
Tlr Signalling

Mesenchymal stem cells (MSCs) are of special interest as therapeutic agents in the settings of both chronic inflammatory and autoimmune diseases. Toll-like receptors (TLR) ligands have been linked with the perpetuation of inflammation in a number of chronic inflammatory diseases due to the permanent exposure of the immune system to TLR-specific stimuli. Therefore, MSCs employed in therapy can be potentially exposed to TLR ligands, which may modulate MSC therapeutic potential in vivo. Recent results demonstrate that MSCs are activated by TLR ligands leading to modulation of the differentiation, migration, proliferation, survival, and immunosuppression capacities. However inconsistent results among authors have been reported suggesting that the source of MSCs, TLR stimuli employed or culture conditions play a role. Notably, activation by TLR ligands has not been reported to modulate the “immunoprivileged” phenotype of MSCs which is of special relevance regarding the use of allogeneic MSC-based therapies. In this review, we discuss the available data on the modulation of MSCs activity through TLR signalling.

scholarly journals Higher Chondrogenic Potential of Extracellular Vesicles Derived from Mesenchymal Stem Cells Compared to Chondrocytes-EVs In Vitro

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
Maryam Hosseinzadeh ◽  
Amir Kamali ◽  
Samaneh Hosseini ◽  
Mohamadreza Baghaban Eslaminejad
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Extracellular Vesicles ◽  
Surface Marker ◽  
Cellular Processes ◽  
Surface Marker Expression ◽  
Chondrogenic Potential ◽  
Cell Sources ◽  
Rabbit Bone

The inability of cartilage to self-repair necessitates an effective therapeutic approach to restore damaged tissues. Extracellular vesicles (EVs) are attractive options because of their roles in cellular communication and tissue repair where they regulate the cellular processes of proliferation, differentiation, and recruitment. However, it is a challenge to determine the relevant cell sources for isolation of EVs with high chondrogenic potential. The current study aims to evaluate the chondrogenic potential of EVs derived from chondrocytes (Cho-EV) and mesenchymal stem cells (MSC-EV). The EVs were separately isolated from conditioned media of both rabbit bone marrow MSCs and chondrocyte cultures. The isolated vesicles were assessed in terms of size, morphology, and surface marker expression. The chondrogenic potential of MSCs in the presence of different concentrations of EVs (50, 100, and 150 μg/ml) was evaluated during 21 days, and chondrogenic surface marker expressions were checked by qRT-PCR and histologic assays. The extracted vesicles had a spherical morphology and a size of 44.25 ± 8.89  nm for Cho-EVs and 112.1 ± 10.10  nm for MSC-EVs. Both groups expressed the EV-specific surface markers CD9 and CD81. Higher expression of chondrogenic specified markers, especially collagen type II (COL II), and secretion of glycosaminoglycans (GAGs) and proteoglycans were observed in MSCs treated with 50 and 100 μg/ml MSC-EVs compared to the Cho-EVs. The results from the use of EVs, particularly MSC-EVs, with high chondrogenic ability will provide a basis for developing therapeutic agents for cartilage repair.

A High-Throughput Screen to Identify Compounds Preserving Primary Human AML Stem Cells Ex-Vivo,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3587-3587
Author(s):  
Caroline Pabst ◽  
Josee Hebert ◽  
Guy Sauvageau
Keyword(s):  
Stem Cells ◽  
High Throughput ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Surface Marker ◽  
Leukemic Cells ◽  
High Throughput Screen ◽  
Self Renewal ◽  
Surface Marker Expression ◽  
Marker Expression

Abstract Abstract 3587 Acute myeloid leukemia (AML) is the second most frequent leukemia in adults with still high relapse rates supposed to be due to a rare population of leukemia stem cells (LSC) within the leukemic bulk not responding to conventional anti-cancer drugs. Engraftment of human leukemic cells in immunocompromised mice mimicking the human disease is considered to prove the existence of stem cells in the sample; however, the engraftment potential of primary human AML cells is usually quickly lost upon ex-vivo culture indicating that LSC have undergone differentiation or apoptosis and thus lost their stem cell properties. This lack of appropriate culture conditions enabling ex-vivo maintenance of primary human LSC is a major obstacle in the development of LSC targeted therapies. Our aim is, therefore, to identify conditions which allow the maintenance and expansion of these rare cells ex-vivo to understand their unique properties and to further elucidate cellular pathways underlying the self-renewal program of human primary LSC. We established a flow cytometry based high-throughput screen in which we tested more than 6,000 selected compounds with regards to their potential to expand primary human AML cells while preserving their phenotypic and functional characteristics. We considered alteration of surface marker expression, i.e. loss of CD34 and acquisition of markers of myeloid maturation initially not expressed on the cells, as indicator of differentiation implicating at the same time a loss of stem cells in the culture. We hypothesized that compounds that would be able to prevent these alterations in surface marker expression could have the potential to promote self-renewal of primary human LSC ex vivo. A normal-karyotype AML representing the most frequent cytogenetic group of AML in adults with FAB M1 morphology was chosen to perform the screen. Moreover, the initial CD34+CD15- phenotype allowed us to monitor changes in surface marker expression. Cells were plated in 384 well plates with a density of 5,000 cells per well. 6,160 compounds were tested with and without an inhibitor to aryl hydrocarbon receptor (AhR) and the vehicle DMSO was used as negative control resulting in a final DMSO concentration of ∼0.1% in each well, which had been proven to be not toxic to the cells. After 5 days of incubation the cells were analyzed on a BD LSRII high-throughput flow cytometer with regards to CD34 and CD15 surface expression. Compounds showing an increase of ≥45% of CD34+ and CD34+CD15- cells over DMSO compared to the AhR antagonist were selected for further evaluation. In a secondary screen we retested 32 compounds in 5 different concentrations ranging from 1:34 to 3x based on the initial screen concentration after 5 and 9 days of incubation and confirmed an increase ≥50% of relative and absolute CD34+CD15- leukemic cells after 5 days for 13 compounds and after 9 days for 7 compounds. We have initiated functional in-vitro and in-vivo experiments to validate these results and to prove the existence of LSC in cultures where compounds have been added. The ability of the newly identified compounds to preserve human LSC ex-vivo will be further evaluated using a collection of primary AML samples comprising different morphological and cytogenetic groups. Together, our observations document the feasibility of finding anti-differentiation (pro-self-renewal?) compounds of primary human AML cells that can be expanded ex-vivo. These studies represent a first key step in the identification of specific LSC targeting compounds. Disclosures: No relevant conflicts of interest to declare.

Mass Cytometric Analysis of AML Stem and Early Progenitor Cells Reveals Karyotype and Genotype-Specific Immunophenotypes That May Represent Targets for Antibody-Directed Therapies

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2380-2380
Author(s):  
Gregory K. Behbehani ◽  
Wendy J. Fantl ◽  
Bruno C Medeiros ◽  
Garry P. Nolan
Keyword(s):  
Stem Cells ◽  
Progenitor Cell ◽  
Expression Patterns ◽  
Fold Increase ◽  
Surface Marker ◽  
Leukemic Cells ◽  
Therapeutic Antibodies ◽  
Hematopoietic Stem ◽  
Surface Marker Expression ◽  
Marker Expression

Abstract Introduction: Leukemic stem cells (LSCs) are recognized as important mediators of leukemia relapse. Thus, therapeutic antibodies are in development to target antigens present on these cells. The success of this approach relies on a detailed understanding of the surface marker expression patterns of LSCs. To address this, we applied multi-parametric single-cell mass cytometry (MCM) to deeply profile the surface marker expression of all major immunophenotypic populations in bone marrow aspirates (BM) from patients with acute myeloid leukemia (AML). Methods: BMs from 41 leukemia patients (30 AML, 4 APL, 2 high-risk MDS, 5 AML in CR) and 5 healthy donors and were processed immediately after aspiration (<1min) and stored for pooled analysis with two overlapping 39-antibody MCM panels (50 markers total). All samples were barcoded, such that 20 samples (leukemia and healthy) could be combined into a single tube for simultaneous antibody staining and analysis, resulting in high precision (coefficient of variation = 10-20%). Results: Distinct AML subtype-specific patterns of cell frequencies across immunophenotypic populations were detected. Patients with core binding factor (CBF) AML (n=5) and those with adverse-risk karyotpic abnormalities (n=6) exhibited the greatest expansion of immunophenotypic hematopoietic stem cells (HSCs) and early progenitors (MPP, CMP), while patients with APL (n=4) and FLT3-ITD normal karyotype AML (NK-AML; n=11) exhibited reduced expansion of early progenitors and expansion in more mature myeloid progenitors (GMP, myelo-monoblasts). As a result of barcoding, high resolution (2-3 fold changes) measurement of surface marker expression detected multiple aberrancies across almost all identifiable immunophenotypic populations. Specifically, several genotype- and karyotype-specific trends in aberrant marker expression were observed in hematopoietic stem and progenitor cell populations (HSPCs; CD34+CD38low). FLT3-ITD samples were characterized by increased CD7, CD33, CD123, CD45, CD321, and CD99, as well as decreased CD34, CD117, and CD38. FLT3wt NK-AML samples (n=6) were characterized by increased CD99 as well as decreased CD71, CD47, CD34, and CD45. Adverse-risk karyotype samples (n=6) were characterized by increased CD99 and decreased CD47. All p values were significant (ranging from 0.02 to 4.5x10-7). While all 36 samples (AML or APL) displayed immunophenotypic abnormalities within the CD34+CD38low gated population (3.6 abnormalities on average), there were 9 samples in which an unambiguous separation into normal and leukemic populations was observed among the cells in this gate. Interestingly, among these samples, some markers were aberrantly high in leukemic cells of one subtype, and aberrantly low in leukemic cells of another, e.g. HLA-DR was extremely high in HSPCs of sample #22 (MLL rearrangement), and aberrantly low in all 4 APL samples. CD99 was most consistently elevated in the AML samples (28/36), but was normal in 6 samples, including a majority of those with t(8;21). These results suggest that therapeutic antibodies directed against molecules such as CD33 may be less effective for AML subtypes such as FLT3wt NK-AML (~40% decrease in CD33), and more effective for other subtypes such as FLT3-ITD NK-AML (7-fold increase in CD33). HSPCs from patients with FLT3-ITD mutations also displayed a 20-fold increase in CD123, which could also be therapeutically targeted. These findings, combined with our recent observation that HSPCs from FLT3-ITD NK-AML patients have an extremely low S-phase fraction, may provide a mechanistic basis for the improved disease-free survival recently reported for FLT3-ITD NK-AML patients treated with fractionated gemtuzumab ozogamicin in combination with standard therapy. Conclusions: We have developed an innovative MCM approach for the analysis of hematologic malignancies and demonstrated that different genotypic and karyotypic subsets of AML are highly varied in their immunophenotypic properties, particularly within the stem and progenitor cell compartment. These data also suggest that antigens that distinguish AML stem cells from normal stems cells are likely to be karyotype and genotype-specific. These findings have important implications for the design of therapeutic strategies in AML. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures Behbehani: Fluidigm: Consultancy. Medeiros:Agios: Consulting - Ad board Other. Nolan:Fluidigm, Inc: Consultancy, Equity Ownership.

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