Intratumoral Cell Heterogeneity in Patient-Derived Glioblastoma Cell Lines Revealed by Single-Cell RNA-Sequencing.

Glioblastoma cell lines derived from different patients are widely used in tumor biology research and drug screening. A key feature of glioblastoma is the high level of inter- and intratumor heterogeneity that accounts for treatment resistance. Our aim was to investigate whether intratumor heterogeneity is maintained in cell models. Single-cell RNA sequencing was used to investigate the cellular composition of a tumor sample and six patient-derived glioblastoma cell lines. Three cell lines preserved the mutational profile of the original tumor, whereas three others differed from their precursors. Copy-number variation analysis showed significantly rearranged genomes in all the cell lines and in the tumor sample. The tumor had the most complex cell composition, including cancer cells and microenvironmental cells. Cell lines with a conserved genome had less diverse cellularity, and during cultivation, a relative increase in the stem-cell-derived progenitors was noticed. Cell lines with genomes different from those of the primary tumors mainly contained neural progenitor cells and microenvironmental cells. The establishment of cell lines without the driver mutations that are intrinsic to the original tumors may be related to the selection of clones or cell populations during cultivation. Thus, patient-derived glioblastoma cell lines differ substantially in their cellular profile, which should be taken into account in translational studies.

Modeling the profibrotic microenvironment in vitro: Model validation.

Establishing the molecular and cellular mechanisms of fibrosis requires the development of validated and reproducible models. The complexity of in vivo models challenges the monitoring of an individual cell fate, in some cases making it impossible. However, the set of factors affecting cells in vitro culture systems differ significantly from in vivo conditions, insufficiently reproducing living systems. Thus, to model profibrotic conditions in vitro, usually the key profibrotic factor, transforming growth factor beta (TGFß-1) is used as a single factor. TGFß-1 stimulates the differentiation of fibroblasts into myofibroblasts, the main effector cells promoting the development and progression of fibrosis. However, except for soluble factors, the rigidity and composition of the extracellular matrix (ECM) play a critical role in the differentiation process. To develop the model of more complex profibrotic microenvironment in vitro, we used a combination of factors: decellularized ECM synthesized by human dermal fibroblasts in the presence of ascorbic acid if cultured as cell sheets and recombinant TGFß-1 as a supplement. When culturing human mesenchymal stromal cells derived from adipose tissue (MSCs) under described conditions, we observed differentiation of MSCs into myofibroblasts due to increased number of cells with stress fibrils with alpha-smooth muscle actin (αSMA), and increased expression of myofibroblast marker genes such as collagen I, EDA-fibronectin and αSMA. Importantly, secretome of MSCs changed in these profibrotic microenvironment: the secretion of the profibrotic proteins SPARC and fibulin-2 increased, while the secretion of the antifibrotic hepatocyte growth factor (HGF) decreased. Analysis of transciptomic pattern of regulatory microRNAs in MSCs revealed 49 miRNAs with increased expression and 3 miRNAs with decreased expression under profibrotic stimuli. Bioinformatics analysis confirmed that at least 184 gene targets of the differently expressed miRNAs genes were associated with fibrosis. To further validate the developed model of profibrotic microenvironment, we cultured human dermal fibroblasts in these conditions and observed increased expression of fibroblast activation protein (FAPa) after 12 h of cultivation as well as increased level of αSMA and higher number of αSMA + stress fibrils after 72 h. The data obtained allow us to conclude that the conditions formed by the combination of profibrotic ECM and TGFß-1 provide a complex profibrotic microenvironment in vitro. Thus, this model can be applicable in studying the mechanism of fibrosis development, as well as for the development of antifibrotic therapy.

T-Cadherin Deficiency Is Associated with Increased Blood Pressure after Physical Activity.

T-cadherin is a regulator of blood vessel remodeling and angiogenesis, involved in adiponectin-mediated protective effects in the cardiovascular system and in skeletal muscles. GWAS study has previously demonstrated a SNP in the Cdh13 gene to be associated with hypertension. However, the role of T-cadherin in regulating blood pressure has not been experimentally elucidated. Herein, we generated Cdh13∆Exon3 mice lacking exon 3 in the Cdh13 gene and described their phenotype. Cdh13∆Exon3 mice exhibited normal gross morphology, life expectancy, and breeding capacity. Meanwhile, their body weight was considerably lower than of WT mice. When running on a treadmill, the time spent running and the distance covered by Cdh13∆Exon3 mice was similar to that of WT. The resting blood pressure in Cdh13∆Exon3 mice was slightly higher than in WT, however, upon intensive physical training their systolic blood pressure was significantly elevated. While adiponectin content in the myocardium of Cdh13∆Exon3 and WT mice was within the same range, adiponectin plasma level was 4.37-fold higher in Cdh13∆Exon3 mice. Moreover, intensive physical training augmented the AMPK phosphorylation in the skeletal muscles and myocardium of Cdh13∆Exon3 mice as compared to WT. Our data highlight a critically important role of T-cadherin in regulation of blood pressure and stamina in mice, and may shed light on the pathogenesis of hypertension.

Novel Potential Markers of Myofibroblast Differentiation Revealed by Single-Cell RNA Sequencing Analysis of Mesenchymal Stromal Cells in Profibrotic and Adipogenic Conditions.

Mesenchymal stromal cells (MSCs) are the key regulators of tissue homeostasis and repair after damage. Accumulating evidence indicates the dual contribution of MSCs into the development of fibrosis induced by chronic injury: these cells can suppress the fibrotic process due to paracrine activity, but their promoting role in fibrosis by differentiating into myofibroblasts has also been demonstrated. Many model systems reproducing fibrosis have shown the ability of peroxisome proliferator-activated receptor (PPAR) agonists to reverse myofibroblast differentiation. Thus, the differentiation of multipotent cells into myofibroblasts and adipocytes can be considered as processes that require the activation of opposite patterns of gene expression. To test this hypothesis, we analyzed single cell RNA-Seq transcriptome of human adipose tissue MSCs after stimulation of the myofibroblast or adipogenic differentiation and revealed several genes that changed their expression in a reciprocal manner upon these conditions. We validated the expression of selected genes by RT-PCR, and evaluated the upregulation of several relevant proteins using immunocytochemistry, refining the results obtained by RNA-Seq analysis. We have shown, for the first time, the expression of neurotrimin (NTM), previously studied mainly in the nervous tissue, in human adipose tissue MSCs, and demonstrated its increased gene expression and clustering of membrane receptors upon the stimulation of myofibroblast differentiation. We also showed an increased level of CHD3 (Chromodomain-Helicase-DNA-binding protein 3) in MSCs under profibrotic conditions, while retinol dehydrogenase-10 (RDH10) was detected only in MSCs after adipogenic induction, which contradicted the data of transcriptomic analysis and again highlights the need to validate the data obtained by omics methods. Our findings suggest the further analysis of the potential contribution of neurotrimin and CHD3 in the regulation of myofibroblast differentiation and the development of fibrosis.

Corrigendum: Declined adipogenic potential of senescent MSCs due to shift in insulin signaling and altered exosome cargo.

[This corrects the article DOI: 10.3389/fcell.2022.1050489.].

Declined adipogenic potential of senescent MSCs due to shift in insulin signaling and altered exosome cargo.

Multipotent mesenchymal stromal cells (MSCs) maintain cellular homeostasis and regulate tissue renewal and repair both by differentiating into mesodermal lineage, e.g., adipocytes, or managing the functions of differentiated cells. Insulin is a key physiological inducer of MSC differentiation into adipocytes, and disturbances in MSC insulin sensitivity could negatively affect adipose tissue renewal. During aging, regulation and renewal of adipose tissue cells may be disrupted due to the altered insulin signaling and differentiation potential of senescent MSCs, promoting the development of serious metabolic diseases, including metabolic syndrome and obesity. However, the potential mechanisms mediating the dysfunction of adipose-derived senescent MSC remains unclear. We explored whether aging could affect the adipogenic potential of human adipose tissue-derived MSCs regulated by insulin. Age-associated senescent MSCs (isolated from donors older than 65 years) and MSCs in replicative senescence (long-term culture) were treated by insulin to induce adipogenic differentiation, and the efficiency of the process was compared to MSCs from young donors. Insulin-dependent signaling pathways were explored in these cells. We also analyzed the involvement of extracellular vesicles secreted by MSCs (MSC-EVs) into the regulation of adipogenic differentiation and insulin signaling of control and senescent cells. Also the microRNA profiles of MSC-EVs from aged and young donors were compared using targeted PCR arrays. Both replicatively and chronologically senescent MSCs showed a noticeably decreased adipogenic potential. This was associated with insulin resistance of MSCs from aged donors caused by the increase in the basal level of activation of crucial insulin-dependent intracellular effectors ERK1/2 and Akt. To assess the impact of the paracrine cross-talk of MSCs, we analyzed microRNAs profile differences in MSC-EVs and revealed that senescent MSCs produced EVs with increased content of miRNAs targeting components of insulin-dependent signaling cascade PTEN, MAPK1, GAREM1 and some other targets. We also confirmed these data by differentiation of control MSCs in the presence of EVs from senescent cells and vice versa. Thus, aging attenuated the adipogenic potential of MSCs due to autocrine or paracrine-dependent induction of insulin resistance associated with the specific changes in MSC-EV cargo.

Platelet-Derived Growth Factor Induces SASP-Associated Gene Expression in Human Multipotent Mesenchymal Stromal Cells but Does Not Promote Cell Senescence.

Activation of multipotent mesenchymal stromal cells (MSCs) is a central part of tissue response to damage. Platelet-derived growth factor (PDGF-BB), which is abundantly released in the damaged area, potently stimulates the proliferation and migration of MSCs. Recent evidence indicates that tissue injury is associated with the accumulation of senescent cells, including ones of MSC origin. Therefore, we hypothesized that PDGF-BB induces MSC senescence. To evaluate mechanisms of early activation of MSCs by PDGF-BB, we performed transcriptome profiling of human MSCs isolated from adipose tissue after exposure to PDGF-BB for 12 and 24 h. We demonstrated that PDGF-BB induced the expression of several genes encoding the components of senescence-associated secretory phenotype (SASP) in MSCs such as plasminogen activator inhibitor-1 (PAI-1), urokinase-type plasminogen activator and its receptor (uPA and uPAR), and some matrix metalloproteases. However, further experimental validation of transcriptomic data clearly indicated that PDGF-BB exerted mitogenic and pro-migratory effects on MSCs, and augmented their pro-angiogenic activity, but did not significantly stimulate MSC senescence.

Cell Sheets from Adipose Tissue MSC Induce Healing of Pressure Ulcer and Prevent Fibrosis via Trigger Effects on Granulation Tissue Growth and Vascularization.

We report a comparative study of multipotent mesenchymal stromal cells (MSC) delivered by injection, MSC-based cell sheets (CS) or MSC secretome to induce healing of cutaneous pressure ulcer in C57Bl/6 mice. We found that transplantation of CS from adipose-derived MSC resulted in reduction of fibrosis and recovery of skin structure with its appendages (hair and cutaneous glands). Despite short retention of CS on ulcer surface (3-7 days) it induced profound changes in granulation tissue (GT) structure, increasing its thickness and altering vascularization pattern with reduced blood vessel density and increased maturation of blood vessels. Comparable effects on GT vascularization were induced by MSC secretome, yet this treatment has failed to induce repair of skin with its appendages we observed in the CS group. Study of secretome components produced by MSC in monolayer or sheets revealed that CS produce more factors involved in pericyte chemotaxis and blood vessel maturation (PDGF-BB, HGF, G-CSF) but not sprouting inducer (VEGF165). Analysis of transcriptome using RNA sequencing and Gene Ontology mapping found in CS upregulation of proteins responsible for collagen binding and GT maturation as well as fatty acid metabolism enzymes known to be negative regulators of blood vessel sprouting. At the same time, downregulated transcripts were enriched by factors activating capillary growth, suggesting that in MSC sheets paracrine activity may shift towards matrix remodeling and maturation of vasculature, but not activation of blood vessel sprouting. We proposed a putative paracrine trigger mechanism potentially rendering an impact on GT vascularization and remodeling. Our results suggest that within sheets, MSC may change their functional state and spectrum of soluble factors that influence tissue repair and induce more effective skin healing inclining towards regeneration and reduced scarring.

Functional Heterogeneity of Protein Kinase A Activation in Multipotent Stromal Cells.

Multipotent stromal cells (MSC) demonstrate remarkable functional heterogeneity; however, its molecular mechanisms remain largely obscure. In this study, we explored MSC response to hormones, which activate Gs-protein / cyclic AMP (cAMP) / protein kinase A (PKA) dependent signaling, at the single cell level using genetically encoded biosensor PKA-Spark. For the first time, we demonstrated that about half of cultured MSCs are not able to activate the cAMP/PKA pathway, possibly due to the limited availability of adenylyl cyclases. Using this approach, we showed that MSC subpopulations responding to various hormones largely overlapped, and the share of responding cells did not exceed 40%. Using clonal analysis, we showed that signaling heterogeneity of MSC could be formed de novo within 2 weeks.

Secretome of Mesenchymal Stromal Cells Prevents Myofibroblasts Differentiation by Transferring Fibrosis-Associated microRNAs within Extracellular Vesicles.

Fibroblasts differentiation into myofibroblasts is a central event of tissue fibrosis. Multipotent mesenchymal stromal cells (MSCs) secretome can interfere with fibrosis development; despite precise underlying mechanisms remain unclear. In this study, we tested the hypothesis that MSC secretome can affect fibroblast' differentiation into myofibroblasts by delivering regulatory RNAs, including microRNAs to these cells. Using the model of transforming growth factor-beta (TGFbeta)-induced fibroblast differentiation into myofibroblasts, we tested the activity of human MSC secretome components, specifically extracellular vesicles (MSC-EV). We showed that MSC-EV down-regulated secretion of extracellular matrix proteins by fibroblasts as well as suppressed their contractility resulting in prevention as well as reversion of fibroblasts differentiation to myofibroblasts. High-throughput sequencing of RNAs extracted from MSC-EV has revealed many fibrosis-associated microRNAs. Fibroblast treatment with MSC-EV led to direct transfer of microRNAs, which resulted in the elevation of most prominent fibrosis-associated microRNAs, including microRNA-21 and microRNA-29c. Using MSC-EV transfection by antagomirs to these microRNAs we demonstrated their involvement in the suppression of fibroblast differentiation in our model. Taken together, MSC secretome can suppress fibrosis by prevention of fibroblast differentiation into myofibroblasts as well as induce de-differentiation of the latter by direct transfer of specific microRNAs.