scholarly journals Purinergic responses of chondrogenic stem cells to dynamic loading

2013 ◽  
Vol 78 (12) ◽  
pp. 1865-1874 ◽  
Author(s):  
Ivana Gadjanski ◽  
Gordana Vunjak-Novakovic
Keyword(s):  
Stem Cells ◽  
Dynamic Loading ◽  
Lucifer Yellow ◽  
Atp Release ◽  
Human Mesenchymal Stem Cells ◽  
Cell Types ◽  
Flufenamic Acid ◽  
Release Assay ◽  
Extracellular Environment ◽  
Connexin Hemichannels

In habitually loaded tissues, dynamic loading can trigger ATP (adenosine 5?- triphosphate) release to extracellular environment, and result in calcium signaling via ATP binding to purine P2 receptors1. In the current study we have compared purinergic responses (ATP release) of two types of cells: bovine chondrocytes (bCHs) and human mesenchymal stem cells (hMSC) that were encapsulated in agarose and subjected to dynamic loading. Both cell types were cultured under chondrogenic conditions, and their responses to loading were evaluated by ATP release assay in combination with connexin (Cx)-sensitive fluorescent dye (Lucifer Yellow - LY) and a Cx-hemichannel blocker (Flufenamic acid - FFA). In response to dynamic loading, chondrogenic hMSCs released significantly higher amounts of ATP (5-fold) in comparison to the bCHs early in culture (day 2). Triggering of LY uptake in the bCHs and hMSCs by dynamic loading implies opening of the Cx-hemichannels. However, the number of LY-positive cells in hMSC-constructs was 2.5-fold lower compared to the loaded bCH-constructs, suggesting utilization of additional mechanisms of ATP release. Cx-reactive sites were detected in both bCHs and hMSCs-constructs. FFA application led to reduced ATP release both in bCHs and hMSCs, which confirms the involvement of connexin hemichannels, with more prominent effects in bCHs than in hMSCs, further implying the existence of additional mechanism of ATP release in chondrogenic hMSCs. Taken together, these results indicate stronger purinergic response to dynamic loading of chondrogenic hMSCs than primary chondrocytes, by activation of connexin hemichannels and additional mechanisms of ATP release.

scholarly journals Identification of Novel FNIN2 and FNIN3 Fibronectin-Derived Peptides That Promote Cell Adhesion, Proliferation and Differentiation in Primary Cells and Stem Cells

2021 ◽  
Vol 22 (6) ◽  
pp. 3042
Author(s):  
Eun Ju Lee ◽  
Khurshid Ahmad ◽  
Shiva Pathak ◽  
SunJu Lee ◽  
Mohammad Hassan Baig ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Culture ◽  
Cell Adhesion ◽  
3D Culture ◽  
Human Mesenchymal Stem Cells ◽  
Cell Types ◽  
Primary Cells ◽  
Cell Culture Techniques ◽  
Culture Techniques ◽  
Promote Cell Adhesion

In recent years, a major rise in the demand for biotherapeutic drugs has centered on enhancing the quality and efficacy of cell culture and developing new cell culture techniques. Here, we report fibronectin (FN) derived, novel peptides fibronectin-based intergrin binding peptide (FNIN)2 (18-mer) and FNIN3 (20-mer) which promote cell adhesion proliferation, and the differentiation of primary cells and stem cells. FNIN2 and 3 were designed based on the in silico interaction studies between FN and its receptors (integrin α5β1, αvβ3, and αIIbβ3). Analysis of the proliferation of seventeen-cell types showed that the effects of FNINs depend on their concentration and the existence of expressed integrins. Significant rhodamine-labeled FNIN2 fluorescence on the membranes of HeLa, HepG2, A498, and Du145 cells confirmed physical binding. Double coating with FNIN2 or 3 after polymerized dopamine (pDa) or polymerized tannic acid (pTA) precoating increased HBEpIC cell proliferation by 30–40 percent, suggesting FNINs potently affect primary cells. Furthermore, the proliferation of C2C12 myoblasts and human mesenchymal stem cells (MSCs) treated with FNINs was significantly increased in 2D/3D culture. FNINs also promoted MSC differentiation into osteoblasts. The results of this study offer a new approach to the production of core materials (e.g., cell culture medium components, scaffolds) for cell culture.

Gene Expression Regulation underlying Osteo-, Adipo-, and Chondro-Genic Lineage Commitment of Human Mesenchymal Stem Cells

2013 ◽  
pp. 76-94 ◽  
Author(s):  
Ana M. Sotoca ◽  
Michael Weber ◽  
Everardus J. J. van Zoelen
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Gene Expression Regulation ◽  
Expression Profiles ◽  
Human Mesenchymal Stem Cells ◽  
Gene Expression Profiles ◽  
Stem Cell Differentiation ◽  
Cell Types ◽  
Lineage Commitment

Human mesenchymal stem cells have a high potential in regenerative medicine. They can be isolated from a variety of adult tissues, including bone marrow, and can be differentiated into multiple cell types of the mesodermal lineage, including adipocytes, osteocytes, and chondrocytes. Stem cell differentiation is controlled by a process of interacting lineage-specific and multipotent genes. In this chapter, the authors use full genome microarrays to explore gene expression profiles in the process of Osteo-, Adipo-, and Chondro-Genic lineage commitment of human mesenchymal stem cells.

scholarly journals Human Mesenchymal Stem Cells Display Reduced Expression of CD105 after Culture in Serum-Free Medium

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Peter Mark ◽  
Mandy Kleinsorge ◽  
Ralf Gaebel ◽  
Cornelia A. Lux ◽  
Anita Toelk ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Ex Vivo ◽  
Human Mesenchymal Stem Cells ◽  
Cell Types ◽  
Growth Media ◽  
Clinical Use ◽  
Differentiation Potential ◽  
Serum Free ◽  
The Impact

Human Mesenchymal Stem Cells (hMSCs) present a promising tool for regenerative medicine. However,ex vivoexpansion is necessary to obtain sufficient cells for clinical therapy. Conventional growth media usually contain the critical component fetal bovine serum. For clinical use, chemically defined media will be required. In this study, the capability of two commercial, chemically defined, serum-free hMSC growth media (MSCGM-CD and PowerStem) for hMSC proliferation was examined and compared to serum-containing medium (MSCGM). Immunophenotyping of hMSCs was performed using flow cytometry, and they were tested for their ability to differentiate into a variety of cell types. Although the morphology of hMSCs cultured in the different media differed, immunophenotyping displayed similar marker patterns (high expression of CD29, CD44, CD73, and CD90 cell surface markers and absence of CD45). Interestingly, the expression of CD105 was significantly lower for hMSCs cultured in MSCGM-CD compared to MSCGM. Both groups maintained mesenchymal multilineage differentiation potential. In conclusion, the serum-free growth medium is suitable for hMSC culture and comparable to its serum-containing counterpart. As the expression of CD105 has been shown to positively influence hMSC cardiac regenerative potential, the impact of CD105 expression onto clinical use after expansion in MSCGM-CD will have to be tested.

SDF-1-CXCR4 and HGF-c-met Axes Regulate Mobilization/Recruitment to Injured Tissue of Human Mesenchymal Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2331-2331 ◽  
Author(s):  
Bo-Ra Son ◽  
Dongling Zhao ◽  
Leah A. Marquez-Curtis ◽  
Neeta Shirvaikar ◽  
Mariusz Z. Ratajczak ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Human Mesenchymal Stem Cells ◽  
Cell Types ◽  
Therapeutic Tool ◽  
Chemotaxis Assay ◽  
Injured Tissue ◽  
Organ Tissue ◽  
Hepatocyte Growth

Abstract Human mesenchymal stem cells (MSC) have been shown to egress from the bone marrow (BM), circulate in peripheral blood (PB) and differentiate into many cell types, making them attractive as a potential therapeutic tool for organ/tissue regeneration. However the signals required for their mobilization into PB and their recruitment into injured sites are not fully understood. We previously reported that stromal-derived factor (SDF)-1 and hepatocyte growth factor (HGF) are upregulated at sites of tissue damage (Cancer Research2003; 63:7926; Leukemia2004; 18:29) and in this study we examined whether these factors mediate the migration of MSC. We investigated (i) the expression in MSC of CXCR4 and c-met, the cognate receptors of SDF-1 and HGF, (ii) whether they are functional after early and late passages (using a chemotaxis assay across fibronectin and the reconstituted basement membrane Matrigel), and (iii) whether MSC express matrix metalloproteinases (MMPs) known to facilitate mobilization and homing of stem cells. MSC were derived from human bone marrow (BM) or cord blood (CB) and maintained for up to 18 passages (in IMDM and 10–20% FCS) with monitoring of markers for cardiac (Nkx2.5/Csx, GATA-4 and MEF2-C), skeletal muscle (Myo-D and myogenin) and endothelial cells (VE-cadherin and VEGFR-2). We found that (i) CB and BM MSC strongly express CXCR4 and c-met transcripts for up to 15 passages, (ii) these receptors are functional as the MSC cells were chemotactic and chemoinvasive (across Matrigel) towards gradients of SDF-1 (100 ng/mL) or HGF (40 ng/mL), and (iii) CB and BM MSC express MMP-2 mRNA and secrete both latent and active forms of MMP-2. Moreover, we found that CB and BM MSC expressed mRNA for all three cardiac markers and the endothelial marker VE-cadherin, indicating their potential for heart regeneration. In conclusion, these results indicate that the SDF-1-CXCR4 and HGF-c-met axes are important signaling pathways in MSC mobilization and their trafficking in PB, and could be involved in recruitment of MSC to damaged tissues (e.g., myocardium).

scholarly journals Isolation of human testicular cells and co-culture with embryonic stem cells

Reproduction ◽  
2018 ◽  
Vol 155 (2) ◽  
pp. 151-164 ◽  
Author(s):  
Meenakshi Gaur ◽  
Cyril Ramathal ◽  
Renee A Reijo Pera ◽  
Paul J Turek ◽  
Constance M John
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Lucifer Yellow ◽  
Colorimetric Detection ◽  
Three Dimensional ◽  
Embryonic Stem ◽  
Cell Types ◽  
Testicular Cells ◽  
Polarized Secretion ◽  
Cell Progression

Our overall goal is to create a three-dimensional human cell-based testicular model for toxicological and spermatogenesis studies. Methods to purify the major somatic testicular cells, namely Leydig cells (LCs), peritubular myoid cells (PCs) and Sertoli cells (SCs), from rats, mice and guinea pigs have been reported. In humans, the isolation of populations enriched for primary LCs, PCs or SCs also have described. One objective of this study was to determine if populations of cells enriched for all three of these cell types can be isolated from testes of single human donors, and we were successful in doing so from testes of three donors. Testes tissues were enzymatically digested, gravity sedimented and Percoll filtered to isolate populations enriched for LCs, PCs and SCs. LCs and PCs were identified by colorimetric detection of the expression of prototypical enzymes. Division of PCs and SCs in culture has been reported. We observed that primary human LCs could divide in culture by incorporation of 5-ethynyl-2′-deoxyuridine. SCs were identified and their functionality was demonstrated by the formation of tight junctions as shown by the expression of tight junction proteins, increased transepithelial electrical resistance, polarized secretion of biomolecules and inhibition of lucifer yellow penetration. Furthermore, we found that human SC feeder layers could facilitate germ cell progression of human embryonic stem cells (hESCs) by microarray analysis of gene expression.

scholarly journals Mapping SOX9 transcriptional dynamics during multi-lineage differentiation of human mesenchymal stem cells

2021 ◽  
Author(s):  
Kannan Govindaraj ◽  
Sakshi Khurana ◽  
Marcel Karperien ◽  
Janine Nicole Post
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Transcription Factor ◽  
Single Cell ◽  
Adipogenic Differentiation ◽  
Human Mesenchymal Stem Cells ◽  
Cell Types ◽  
Differentiation Potential ◽  
Lineage Differentiation ◽  
Transcriptional Dynamics

The master transcription factor SOX9 is a key player during chondrocyte differentiation, cartilage development, homeostasis and disease. Modulation of SOX9 and its target gene expression is essential during chondrogenic, osteogenic and adipogenic differentiation of human mesenchymal stem cells (hMSCs). However, lack of sufficient knowledge about the signaling interplay during differentiation remains one of the main reasons preventing successful application of hMSCs in regenerative medicine. We previously showed that Transcription Factor - Fluorescence Recovery After Photobleaching (TF-FRAP) can be used to study SOX9 dynamics at the single cell level. We showed that changes in SOX9 dynamics are linked to its transcriptional activity. Here, we investigated SOX9 dynamics during differentiation of hMSCs into the chondrogenic, osteogenic and adipogenic lineages. We show that there are clusters of cells in hMSCs with distinct SOX9 dynamics, indicating that there are a number of subpopulations present in the heterogeneous hMSCs. SOX9 dynamics data at the single cell resolution revealed novel insights about its activity in these subpopulations (cell types). In addition, the response of SOX9 to differentiation stimuli varied in these subpopulations. Moreover, we identified donor specific differences in the number of cells per cluster in undifferentiated hMSCs, and this correlated to their differentiation potential.

Human Mesenchymal Stem Cells Differentiate Promptly into Tissue-Specific Cell Types without Cell Fusion, Mitochondrial or Membrane Vesicular Transfer in Fetal Sheep.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 436-436 ◽  
Author(s):  
Evan J. Colletti ◽  
Judith A. Airey ◽  
Esmail D. Zanjani ◽  
Christopher D. Porada ◽  
Graça Almeida-Porada
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Cell Fusion ◽  
Human Mesenchymal Stem Cells ◽  
Cell Types ◽  
Specific Cell ◽  
Type I ◽  
Fetal Sheep ◽  
Tissue Specific ◽  
The Brain

Abstract Despite the exciting reports regarding the ability of human mesenchymal stem cells (MSC) to differentiate into different cells of different organs, the mechanism by which this process occurs remains controversial. Several possible explanations have been put forth as an alternative to the existence of a true differentiation mechanism. We previously showed that MSC, at a single cell level, are able to differentiate into cells of different germ cell layers. In the present study, we investigated whether transfer of mitochondria or membrane-derived vesicles between cells and/or cell fusion participate in the events that lead to the change of phenotype of MSC upon transplantation (Tx). To this end, 54 sheep fetuses (55–60 gestational days) were Tx intra-peritoneally with Stro-1+,CD45−, Gly-A- MSC labeled prior to Tx with either CFSE, that irreversibly couples to both intracellular and cell-surface proteins, or DiD that efficiently labels all cell membranes and intracellular organelles, such as mitochondria. Evaluation of the recipients’ different organs started at 20h post-Tx and continued at 25,30,40,60 and 120h. MSC reached the liver at 25h post-Tx (0.033%±0.0) with maximal engraftment at 40h (0.13%±0.02). MSC were first detected in the lung (0.028%±0.0) and brain (0.034%±0.0) at 30h and 40h respectively. In the brain, engraftment peaked at 60 hours post-Tx (0.08%±0.0) and in the lung at 120h (0.09%±0.01). Normalization of the number of engrafted cells per tissue mass and number of Tx cells revealed that 26% of the Tx MSC reached the lung; 2% the liver; and 3% the brain. Since the decreasing number of CFSE+ and DiD+ cells detected after 120h could be due to cell division, Ki67 staining was performed and revealed that 85–95% of the engrafted cells proliferated upon lodging in the organs, and divided throughout the evaluation period. To determine MSC differentiative timeline, confocal microscopy was performed to assess whether CFSE+ or DiD+ cells expressed tissue-specific markers (MSC were negative for these markers prior to transplant) within the engrafted organs. In the liver at 25h post-Tx, all CFSE+ or DiD+ cells co-expressed alpha-fetoprotein, demonstrating the rapid switch from an MSC to a fetal hepatocyte-like phenotype. In the lung, co-localization of pro-surfactant protein and CFSE/DiD was first detected at 30h post-Tx, but cells remained negative for Caveolin1; a phenotype that is consistent with differentiation to a type II epithelial cell, but not to a more mature type I. In the brain, MSC expressed Tau promptly, but synaptophysin expression was not detected until 120h. In situ hybridization on serial sections using either a human- or sheep-specific probe, with simultaneous visualization of CFSE+ or DiD+ cells allowed us to show that no membrane or mitochondrial transfer had occurred, since none of the sheep cells contained CFSE or DiD, and all of the dye+ cells hybridized only to the human probe. Furthermore, this combined methodology enabled us to determine that differentiation to all of the different cell types had occurred in the absence of cell fusion. In conclusion, MSC engraft multiple tissues rapidly, undergo proliferation, and give rise to tissue-specific cell types in the absence of cellular fusion or the transfer of mitochondria or membrane vesicles.

scholarly journals Bone Anabolic Effects of Soluble Si:In VitroStudies with Human Mesenchymal Stem Cells and CD14+ Osteoclast Precursors

2016 ◽  
Vol 2016 ◽  
pp. 1-12 ◽  
Author(s):  
J. Costa-Rodrigues ◽  
S. Reis ◽  
A. Castro ◽  
M. H. Fernandes
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Plasma Levels ◽  
Human Mesenchymal Stem Cells ◽  
Cell Types ◽  
Osteoclast Precursors ◽  
Wide Range ◽  
Basal Plasma ◽  
Important Player ◽  
And Function

Silicon (Si) is indispensable for many cellular processes including bone tissue metabolism. In this work, the effects of Si on human osteogenesis and osteoclastogenesis were characterized. Human mesenchymal stem cells (hMSC) and CD14+ stem cells, as osteoblast and osteoclast precursors, were treated with a wide range of Si concentrations, covering the physiological plasma levels. Si promoted a dose-dependent increase in hMSC proliferation, differentiation, and function, at levels similar to the normal basal plasma levels. Additionally, a decrease in the expression of the osteoclastogenic activators M-CSF and RANKL was observed. Also, Si elicited a decrease in osteoclastogenesis, which became significant at higher concentrations, as those observed after meals. Among the intracellular mechanisms studied, an upregulation of MEK and PKC signalling pathways was observed in both cell types. In conclusion, Si appears to have a direct positive effect on human osteogenesis, at basal plasma levels. On the other hand, it also seemed to be an inhibitor of osteoclastogenesis, but at higher concentrations, though yet in the physiological range. Further, an indirect effect of Si on osteoclastogenesis may also occur, through a downregulation of M-CSF and RANKL expression by osteoblasts. Thus, Si may be an important player in bone anabolic regenerative approaches.

scholarly journals SIRT1 Inhibition Affects Angiogenic Properties of Human MSCs

2014 ◽  
Vol 2014 ◽  
pp. 1-12 ◽  
Author(s):  
Botti Chiara ◽  
Caiafa Ilaria ◽  
Coppola Antonietta ◽  
Cuomo Francesca ◽  
Miceli Marco ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Molecular Mechanisms ◽  
Human Mesenchymal Stem Cells ◽  
Cell Types ◽  
Therapeutic Angiogenesis ◽  
Induced Response ◽  
Human Mscs ◽  
Hypoxic Conditions ◽  
And Migration

Human mesenchymal stem cells (hMSCs) are attractive for clinical and experimental purposes due to their capability of self-renewal and of differentiating into several cell types. Autologous hMSCs transplantation has been proven to induce therapeutic angiogenesis in ischemic disorders. However, the molecular mechanisms underlying these effects remain unclear. A recent report has connected MSCs multipotency to sirtuin families, showing that SIRT1 can regulate MSCs function. Furthermore, SIRT1 is a critical modulator of endothelial angiogenic functions. Here, we described the generation of an immortalized human mesenchymal bone marrow-derived cell line and we investigated the angiogenic phenotype of our cellular model by inhibiting SIRT1 by both the genetic and pharmacological level. We first assessed the expression of SIRT1 in hMSCs under basal and hypoxic conditions at both RNA and protein level. Inhibition of SIRT1 by sirtinol, a cell-permeable inhibitor, or by specific sh-RNA resulted in an increase of premature-senescence phenotype, a reduction of proliferation rate with increased apoptosis. Furthermore, we observed a consistent reduction of tubule-like formation and migration and we found that SIRT1 inhibition reduced the hypoxia induced accumulation of HIF-1α protein and its transcriptional activity in hMSCs. Our findings identify SIRT1 as regulator of hypoxia-induced response in hMSCs and may contribute to the development of new therapeutic strategies to improve regenerative properties of mesenchymal stem cells in ischemic disorders through SIRT1 modulation.

Effect of uniaxial stretch on morphology and cytoskeleton of human mesenchymal stem cells: static vs. dynamic loading

2011 ◽  
Vol 56 (5) ◽  
pp. 259-265 ◽  
Author(s):  
Zahra Goli-Malekabadi ◽  
Mohammad Tafazzoli-Shadpour ◽  
Mohsen Rabbani ◽  
Mohsen Janmaleki
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Dynamic Loading ◽  
Human Mesenchymal Stem Cells
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