Transcriptome Of Mesenchymal Stem Cells Research Articles

Evaluation of genetic response of mesenchymal stem cells to nanosecond pulsed electric fields by whole transcriptome sequencing.

Mesenchymal stem cells (MSCs) modulated by various exogenous signals have been applied extensively in regenerative medicine research. Notably, nanosecond pulsed electric fields (nsPEFs), characterized by short duration and high strength, significantly influence cell phenotypes and regulate MSCs differentiation via multiple pathways. Consequently, we used transcriptomics to study changes in messenger RNA (mRNA), long noncoding RNA (lncRNA), microRNA (miRNA), and circular RNA expression during nsPEFs application. To explore gene expression profiles and potential transcriptional regulatory mechanisms in MSCs pretreated with nsPEFs. The impact of nsPEFs on the MSCs transcriptome was investigated through whole transcriptome sequencing. MSCs were pretreated with 5-pulse nsPEFs (100 ns at 10 kV/cm, 1 Hz), followed by total RNA isolation. Each transcript was normalized by fragments per kilobase per million. Fold change and difference significance were applied to screen the differentially expressed genes (DEGs). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed to elucidate gene functions, complemented by quantitative polymerase chain reaction verification. In total, 263 DEGs were discovered, with 92 upregulated and 171 downregulated. DEGs were predominantly enriched in epithelial cell proliferation, osteoblast differentiation, mesenchymal cell differentiation, nuclear division, and wound healing. Regarding cellular components, DEGs are primarily involved in condensed chromosome, chromosomal region, actin cytoskeleton, and kinetochore. From aspect of molecular functions, DEGs are mainly involved in glycosaminoglycan binding, integrin binding, nuclear steroid receptor activity, cytoskeletal motor activity, and steroid binding. Quantitative real-time polymerase chain reaction confirmed targeted transcript regulation. Our systematic investigation of the wide-ranging transcriptional pattern modulated by nsPEFs revealed the differential expression of 263 mRNAs, 2 miRNAs, and 65 lncRNAs. Our study demonstrates that nsPEFs may affect stem cells through several signaling pathways, which are involved in vesicular transport, calcium ion transport, cytoskeleton, and cell differentiation.

Multi-omics analysis of human mesenchymal stem cells shows cell aging that alters immunomodulatory activity through the downregulation of PD-L1

Mesenchymal stem cells (MSCs) possess potent immunomodulatory activity and have been extensively investigated for their therapeutic potential in treating inflammatory disorders. However, the mechanisms underlying the immunosuppressive function of MSCs are not fully understood, hindering the development of standardized MSC-based therapies for clinical use. In this study, we profile the single-cell transcriptomes of MSCs isolated from adipose tissue (AD), bone marrow (BM), placental chorionic membrane (PM), and umbilical cord (UC). Our results demonstrate that MSCs undergo a progressive aging process and that the cellular senescence state influences their immunosuppressive activity by downregulating PD-L1 expression. Through integrated analysis of single-cell transcriptomic and proteomic data, we identify GATA2 as a regulator of MSC senescence and PD-L1 expression. Overall, our findings highlight the roles of cell aging and PD-L1 expression in modulating the immunosuppressive efficacy of MSCs and implicating perinatal MSC therapy for clinical applications in inflammatory disorders.

Extracellular vesicles from mesenchymal stem cells of dental pulp and adipose tissue display distinct transcriptomic characteristics suggestive of potential therapeutic targets.

Objective: Mesenchymal stem cells (MSCs) are isolated from various human tissues and used for therapy, in which beneficial effects are attributed mainly to mesenchymal stem cell-derived extracellular vesicles (MSC-EVs). Whereas MSCs of diverse tissue types share cardinal stem cell features, it is becoming evident that MSCs of each tissue type possess unique properties as well. For designing efficient stem cellbased therapies, it is crucial to understand the unique properties associated with MSCs and MSC-EVs of each tissue type. Such unique properties can be analyzed through transcriptomic approaches using comprehensive gene expression databases and sophisticated analytical tools. Here, we comparatively studied the transcriptomes in MSC-EVs of dental pulp and adipose tissue. Additionally, the transcriptomes of MSC-EVs were compared with the cellular transcriptomes of MSCs for the same tissue types. Methods: MSCs were cultured from human dental pulp and adipose tissue specimens. Conditioned culture media were collected to prepare MSC-EVs, from which RNAs were isolated and subjected to next-generation sequencing for transcriptomic analysis. Gene expression signatures in MSC-EVs of each tissue type were investigated using gene set analysis. Results: MSC-EVs obtained from dental pulp-derived MSCs showed distinct transcriptomic signatures of neurogenesis and neural retina development while MSC-EVs of adipose tissue-derived MSCs showed signatures of mitochondrial activity and skeletal system development. The transcriptomes of MSC-EVs resembled the cellular transcriptomes of MSCs, and the genes associated with neurogenesis were highly expressed in both MSCs and MSC-EVs of dental pulp. Adipose tissue-derived MSCs and MSC-EVs highly expressed genes associated with angiogenesis, hair growth, and dermal matrices. Conclusion: The clear and distinct signatures of neurogenesis and neural retina development in dental pulp-derived MSC-EVs imply neurodegenerative disorders and retinal diseases as putative therapeutic targets. In contrast, the transcripts in adipose tissue-derived MSC-EVs could be useful in rejuvenating the skin and musculoskeletal system. Further insights into MSC-EVs of divergent tissue types may expand the list of potential therapeutic targets.

Abstract 142: The EGFR signaling modulates in mesenchymal stem cells the expression of microRNAs involved in the interaction with breast cancer cells

Abstract Introduction. We previously demonstrated that the activation of the epidermal growth factor receptor (EGFR) in mesenchymal stem cells (MSCs) modulates the expression of a number of genes coding for secreted proteins that promote breast cancer cell progression. As different microRNAs (miRNAs) have been shown to be involved in the cross-talk between MSCs and tumor cells, we hypothesized that the EGFR might regulate in MSCs the expression of miRNAs that might affect breast cancer progression through paracrine circuits. To this aim, we analysed the whole small RNA transcriptome of MSCs stimulated or not with transforming growth factor α (TGFα), one of the main ligands of the EGFR. Materials and methods. Small RNA sequencing was performed using the SOLiD 5500xl System. The DAVID (Database for Annotation, Visualization and Integrated Discovery) database was used for pathway analysis. Exosomes containing miRNAs were isolated from conditioned media with the ExoQuick reagent. MiRNA expression levels in conditioned medium were analyzed by Real Time PCR. Anchorage-dependent growth assays and Boyden-chamber based colorimetric migration assays were used to assess the proliferation and the migration of breast cancer cells, respectively. Results. Following small RNA sequencing, we identified 36 miRNAs differentially expressed between untreated and TGFα-treated MSCs with a fold change (FC) <0.56 or FC ≥1.90 (Confidence Interval, CI, 95%) and a threshold of sequence counts≥150 in at least one sample. In particular, 18 miRNAs resulted up-regulated with FC≥1.90 and 18 miRNAs down-regulated with FC<0.56. Pathways analysis of the target genes of the differentially expressed miRNAs revealed a significant enrichment in different KEGG pathways involved in cancer development and progression, including TGFβ signaling, focal adhesion, Rap1 signaling, Hippo signaling, mTOR and RAS signaling. To study miRNAs involved in the TGFα-mediated interaction between MSCs and breast cancer cells, we collected conditioned media from MSCs and analysed the expression levels of selected miRNAs. Real Time PCR analysis revealed the presence of several miRNAs in the conditioned medium from MSCs, including miR-23c, miR379-3p and miR-432-5p. The biological activity of the identified miRNAs was explored in a panel of breast cancer cell lines. MiR-23c was found to regulate the proliferation and migration of triple negative MDA-MB-468 and MDA-MB-231 breast cancer cells. Conclusion. Collectively, our data suggest that the EGFR signaling regulate in MSCs a wide number of miRNAs that might be involved in breast cancer progression, providing novel information on the mechanisms that regulate the MSC-tumor cross-talk. Citation Format: Marianna Gallo, Marianeve Carotenuto, Cristin Roma, Francesca Bergantino, Pasqualino de Antonellis, Nicola Normanno, Antonella De Luca. The EGFR signaling modulates in mesenchymal stem cells the expression of microRNAs involved in the interaction with breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 142.

The Effect of Erythropoietin Treatment on Gene Expression Profile of Mesenchymal Stem Cells

Mesenchymal Stem Cells (MSCs) are one of the most important elements of bone marrow microenvironment, which has a role in stromal support and stem cell differentiation. Exosomes are small vesicles that responsible from various cellular roles such as cell-cell communication and cell signaling, which may affect nearby and distant cells/tissues. MSCs have therapeutic importance because of their multipotency and immune modulation potentials also with their exosomes. Erythropoietin (Epo), produced by liver hepatocytes, is responsible for erythroid differentiation (erythropoiesis) in bone marrow. In addition, Epo treatment of several cell types including MSCs, showed therapeutic effects in various diseases. Objective:The aim of this study is to examine the effect of erythropoietin on bone marrow MSCs transcriptome and exosome derived miRNA profile. Materials and Methods:Effect of 3 different doses of Epo (1 IU/ml, 10 IU/ml and 100 IU/ml) for 48 hours on MSCs transcriptome profile was analyzed. The results illustrated that 10 IU/ml Epo treatment has the most effective concentration in terms of gene expression profile. Therefore, small RNA libraries targeting miRNA was analyzed with 10 IU/ml Epo treated versus non treated groups with next generation sequencing. Results:We found that Epo treatment slightly changed global gene expression profile. On the other hand, it was observed that Epo treated MSCs have different exosomal miRNA profile. Conclusion:Differentially expressed exosomal miRNAs may have therapeutic effects in different conditions. It will be important to perform further studies with in vitro models, mimicking different physiological conditions and diseases for MSCs and exosome biology.

A Comparative Study of Biological Characteristics and Transcriptome Profiles of Mesenchymal Stem Cells from Different Canine Tissues

Mesenchymal stem cells (MSCs) are the most promising seed cells for cell therapy. Comparing the biological and transcriptome gene characteristics of MSCs from different sources provides an important basis for the screening of clinically used cells. The main purpose of this experiment was to establish methods for the isolation and culture of MSCs from five different canine sources, including adipose tissue, bone marrow, umbilical cord, amniotic membrane, and placenta, and compare biological and transcriptome characteristics of MSCs, in order to provide a basis for the clinical application of canine MSCs. MSCs were isolated from Chinese pastoral dogs, and the following experiments were performed: (1) the third, sixth, and ninth generations of cells were counted, respectively, and a growth curve was plotted to calculate the MSC population doubling time; (2) the expression of CD34 and CD44 surface markers was studied by immunofluorescence; (3) the third generation of cells were used for osteogenetic and adipogenic differentiation experiments; and (4) MSC transcriptome profiles were performed using RNA sequencing. All of the five types of MSCs showed fibroblast-like adherent growth. The cell surface expressed CD44 instead of CD34; the third-generation MSCs had the highest proliferative activity. The average population doubling time of adipose mesenchymal stem cells (AD-MSCs), placenta mesenchymal stem cells (P-MSCs), bone marrow mesenchymal stem cells (BM-MSCs), umbilical cord mesenchymal stem cells (UC-MSCs), and amniotic mesenchymal stem cells (AM-MSCs) were 15.8 h, 21.2 h, 26.2 h, 35 h, and 41.9 h, respectively. All five types of MSCs could be induced to differentiate into adipocytes and osteoblasts in vitro, with lipid droplets appearing after 8 days and bone formation occurring 5 days after AD-MSC induction. However, the multilineage differentiation for the remaining of MSCs was longer compared to that of the AD-MSCs. The MSC transcriptome profiles showed that AD-MSC and BM-MSCs had the highest homology, while P-MSCs were significantly different compared to the other four types of MSCs. All the isolated MSCs had the main biological characteristics of MSCs. AD-MSCs had the shortest time for proliferation, adipogenesis, and osteogenic differentiation.

Human testis-derived embryonic stem cell-like cells are not pluripotent, but possess potential of mesenchymal progenitors

Spontaneous in vitro transition of undifferentiated spermatogonia into the pluripotent cell state has been achieved using neonatal and adult mouse testis tissue. In an effort to establish an analogous source of human patient-specific pluripotent stem cells, several research groups have described the derivation of embryonic stem cell-like cells from primary cultures of human testis. These cells are characterized in all studies as growing in compact colonies, expressing pluripotency-associated markers and possessing multilineage differentiation capabilities in vitro, but only one study claimed their ability to induce teratomas. This controversy initiated a debate about the pluripotent state and origin of human testis-derived ES-like cells (htES-like cells). htES-like cell colonies were obtained from primary testicular cultures of three individuals and selectively expanded using culture conditions known to support the propagation of blastocyst-derived human embryonic stem cells (ESCs), mouse epiblast stem cells and 'naïve' human ESCs. The stem cell properties of htES-like cells were subsequently assessed by testing the expression of ESC-specific markers, differentiation abilities in vitro and in vivo, and microarray profiling. The expression of pluripotency-associated markers in htES-like cells and their differentiation abilities differed significantly from those of ESCs. Gene expression microarray analysis revealed that htES-like cells possess a transcriptome distinct from human ESCs and fibroblasts, but closely resembling the transcriptome of mesenchymal stem cells (MSCs). The similarity to MSCs was confirmed by detection of SSEA4/CD146 expressing cells within htES-like colonies and efficient in vitro differentiation toward three mesodermal lineages (adipogenic, osteogenic, chondrogenic). Taken together, these results indicate that htES-like cells, in contrast to pluripotent stem cells derived from adult mouse testis, are not pluripotent and most likely not of germ cell but of mesenchymal origin.

A SAGE View of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) were initially defined by their capacity to differentiate into connective tissue cell lineages and support hematopoiesis. More recently, MSCs have demonstrated some degree of therapeutic efficacy in a broad range of diseases including neurological and auto-immune disorders, stroke, diabetes, and chronic inflammatory conditions. An emerging paradigm suggests that MSCs alter the tissue microenvironment via paracrine signaling to induce angiogenesis, alter immune cell function, block inflammation, and stimulate growth of host cells to affect tissue repair. However, these activities appear at odds with the term "mesenchymal stem cell", which by definition implies a rare cell population that through a process of self-renewal yields progenitors that differentiate hierarchically into connective tissue cell types to maintain tissue homeostasis. Analysis of the MSC transcriptome via serial analysis of gene expression (SAGE) revealed that populations express a diverse array of proteins that are important for mesoderm specification but that also regulate various biochemical processes important in bone and marrow, such as angiogenesis, hematopoiesis, cell communication, and neural activities. Moreover, different classes of these regulatory proteins were found to be expressed within distinct sub populations of MSCs. Therefore, MSC populations appear to be more heterogeneous than initially envisions. Evidence is provided that this functional heterogeneity contributes significantly to the therapeutic effects of MSCs.

Functional Heterogeneity of MSC Populations Provides Clues to Their Broad Therapeutic Efficacy

Mesenchymal stem cells (MSCs) have demonstrated therapeutic efficacy in a variety of disease models and human clinical trials. The widespread benefits of MSCs have recently been attributed to their capacity to secrete soluble factors that promote tissue repair by stimulating angiogenesis and suppressing inflammation, apoptosis, and immune cell function. However, these functional attributes are inconsistent with the designation of MSCs as stem cells, which by definition lie at the apex of a hierarchy of lineage specification and cellular differentiation. Previously, we catalogued the MSC transcriptome via serial analysis of gene expression to gain better insight into their basic biology. This analysis revealed that MSCs express a diverse range of proteins regulating angiogenesis, cell motility and communication, hematopoiesis, neural activities, immunity and defense. Furthermore, we reported that different classes of expressed regulatory proteins are restricted to distinct subpopulations of MSCs. Based on these findings we proposed that the molecular heterogeneity of the MSC transcriptome reflects the functional complexity of marrow stroma as an organ system in vivo and this complexity accounts for the broad therapeutic effects of MSCs. Examples are provided to illustrate how unique traits of MSC subpopulations from marrow may be exploited to develop more potent cellular vectors for therapy.

Anti‐inflammatory Properties of Murine Mesenchymal Stem Cells: Role of Interleukin 1 Receptor Antagonist

Mesenchymal stem cells (MSCs) are pluri‐potent cells derived from the bone marrow that possess the capacity to regulate the cellular microenvironment by production of various cytokines, chemokines, and matrix proteins. The objective of this study was to identify novel products secreted by MSCs that may modulate the inflammatory response. Interrogation of the murine MSC transcriptome, which we previously catalogued via serial analysis of gene (SAGE), revealed numerous SAGE tags corresponding to interleukin 1 receptor antagonist (IL1RN). Screening of a murine MSC cDNA library by PCR confirmed that cells expressed mRNAs encoding IL1RN. Furthermore, fractionation studies indicated that MSCs are the principal source of IL1RN in murine bone marrow. FACS analysis further revealed that expression of IL1RN was restricted to ∼24% of murine MSCs, which was confirmed by immunostaining. Enzyme‐linked immunosorbent assays (ELISA) demonstrated that murine MSCs secrete approximately 3ng/ml/cell of IL1RN protein. Studies conducted in vitro showed that media conditioned by MSCs was able to block in a dose dependent fashion the ability of IL‐1α to stimulate proliferation of a T cell line. This inhibitory effect of MSC conditioned media could be abrogated by a neutralizing anti‐IL1RN antibody, thereby demonstrating that the effect was specific to IL1RN secreted by cells. Collectively, these studies identify a unique subpopulation of murine MSCs that express high levels of IL1RN, which functions to antagonize the activity of IL‐1α This activity may account, in part, for the anti‐inflammatory effects of MSCs in vivo.

Interleukin 1 receptor antagonist mediates the antiinflammatory and antifibrotic effect of mesenchymal stem cells during lung injury

Mesenchymal stem cells (MSCs) have been exploited as cellular vectors to treat a wide array of diseases but the mechanisms responsible for their therapeutic effect remain indeterminate. Previously, we reported that MSCs inhibit bleomycin (BLM)-induced inflammation and fibrosis within the lungs of mice. Interrogation of the MSC transcriptome identified interleukin 1 receptor antagonist (IL1RN) as a potential mediator of this effect. Fractionation studies indicated that MSCs are the principal source of IL1RN in murine bone marrow and that its expression is restricted to a unique subpopulation of cells. Moreover, MSC-conditioned media was shown to block proliferation of an IL-1alpha-dependent T cell line and inhibit production of TNF-alpha by activated macrophages in vitro. Studies conducted in mice revealed that MSC administration was more effective than recombinant IL1RN delivered via adenoviral infection or osmotic pumps in inhibiting BLM-induced increases in TNF-alpha, IL-1alpha, and IL1RN mRNA in lung, IL1RN protein in bronchoalveolar lavage (BAL) fluid, and trafficking of lymphocytes and neutrophils into the lung. Therefore, MSCs protect lung tissue from BLM-induced injury by blocking TNF-alpha and IL-1, two fundamental proinflammatory cytokines in lung. Identification of IL1RN-expressing human MSC subpopulations may provide a novel cellular vector for treating chronic inflammatory diseases in humans.

Functional Network Analysis of the Transcriptomes of Mesenchymal Stem Cells Derived from Amniotic Fluid, Amniotic Membrane, Cord Blood, and Bone Marrow

Using high-density oligonucleotide microarrays and functional network analyses, we examined whether MSCs derived from four different origins exhibited unique gene expression profiles individually and then compared the gene expression profiles of all MSCs with those of fetal organs. Our results indicated that within each group of MSCs from the same origin, the variability of the gene expression levels was smaller than that between groups of different origins. Functional genomic studies revealed the specific roles of MSCs from different origins. Our results suggest that amniotic fluid MSCs may initiate interactions with the uterus by upregulating oxytocin and thrombin receptors. Amniotic membrane MSCs may play a role in maintaining homeostasis of fluid and electrolytes by regulating the networks of endothelin, neprilysin, bradykinin receptors, and atrial natriuretic peptide. Cord blood MSCs may be involved in innate immune systems as the neonatal defense system against the earliest encountered pathogens. Adult bone marrow MSCs may be an important source not only of all blood lineages but also of bone formation. However, in spite of the different gene expression profiles seen in MSCs derived from different origins, a set of core gene expression profiles was preserved in these four kinds of MSCs. The core signature transcriptomes of all MSCs, when contrasted against those of fetal organs, included genes involved in the regulation of extracellular matrix and adhesion, transforming growth factor-beta receptor signaling, and the Wnt signaling pathways. Disclosure of potential conflicts of interest is found at the end of this article.

Human mesenchymal stem cell subpopulations express a variety of neuro-regulatory molecules and promote neuronal cell survival and neuritogenesis

Mesenchymal stem cells (MSCs) transplanted at sites of nerve injury are thought to promote functional recovery by producing trophic factors that induce survival and regeneration of host neurons. To evaluate this phenomenon further, we quantified in human MSCs neurotrophin expression levels and their effects on neuronal cell survival and neuritogenesis. Screening a human MSC cDNA library revealed expressed transcripts encoding BDNF and β-NGF but not NT-3 and NT-4. Immunostaining demonstrated that BDNF and β-NGF proteins were restricted to specific MSC subpopulations, which was confirmed by ELISA analysis of 56 separate subclones. Using a co-culture assay, we also demonstrated that BDNF expression levels correlated with the ability of MSC populations or subclones to induce survival and neurite outgrowth in the SH-SY5Y neuroblastoma cell line. However, these MSC-induced effects were only partially inhibited by a neutralizing anti-BDNF antibody. MSCs were also shown to promote neurite outgrowth within dorsal root ganglion explants despite secreting 25-fold lower level of β-NGF required exogenously to produce a similar effect. Interrogation of the human MSC transcriptome identified expressed mRNAs encoding various neurite-inducing factors, axon guidance and neural cell adhesion molecules. Moreover, a subset of these transcripts was shown to correlate with BDNF expression in MSC subclones. Collectively, these studies reveal the existence of MSC subpopulations that co-express neurotrophins and other potent neuro-regulatory molecules, which contribute to MSC-induced effects on neuronal cell survival and nerve regeneration. These subpopulations may represent more potent vectors for treating a variety of neurological disorders.