Changes In Mesenchymal Stem Cells Research Articles

Paving the way to a neural fate – RNA signatures in naive and trans-differentiating mesenchymal stem cells

Mesenchymal Stem Cells (MSCs) derived from the embryonic mesoderm persist as a viable source of multipotent cells in adults and have a crucial role in tissue repair. One of the most promising aspects of MSCs is their ability to trans-differentiate into cell types outside of the mesodermal lineage, such as neurons. This characteristic positions MSCs as potential therapeutic tools for neurological disorders. However, the definition of a clear MSC signature is an ongoing topic of debate. Likewise, there is still a significant knowledge gap about functional alterations of MSCs during their transition to a neural fate. In this study, our focus is on the dynamic expression of RNA in MSCs as they undergo trans-differentiation compared to undifferentiated MSCs. To track and correlate changes in cellular signaling, we conducted high-throughput RNA expression profiling during the early time-course of human MSC neurogenic trans-differentiation. The expression of synapse maturation markers, including NLGN2 and NPTX1, increased during the first 24 h. The expression of neuron differentiation markers, such as GAP43 strongly increased during 48 h of trans-differentiation. Neural stem cell marker NES and neuron differentiation marker, including TUBB3 and ENO1, were highly expressed in mesenchymal stem cells and remained so during trans-differentiation. Pathways analyses revealed early changes in MSCs signaling that can be linked to the acquisition of neuronal features. Furthermore, we identified microRNAs (miRNAs) as potential drivers of the cellular trans-differentiation process. We also determined potential risk factors related to the neural trans-differentiation process. These factors include the persistence of stemness features and the expression of factors involved in neurofunctional abnormalities and tumorigenic processes. In conclusion, our findings contribute valuable insights into the intricate landscape of MSCs during neural trans-differentiation. These insights can pave the way for the development of safer treatments of neurological disorders.

Synergistic effect of mesenchymal stem cells and silver nanoparticles in the treatment of retinal degenerative diseases

Aims/Purpose: Retinal degenerative diseases represent one of the leading causes of visual loss. Mesenchymal stem cells (MSCs) provide potential option for the treatment of several type of pathologies, including ocular diseases. Moreover, silver nanoparticles (AgNPs) are currently widely used in various fields of medicine mainly due to their antibacterial and antiangiogenic properties. Several studies showed their beneficial but also harmful effect. However, there are limited information about the synergistic effect of AgNPs with MSC‐based therapies.Methods: Chronical retinal degeneration was induced in BALB/c mice by repeated intraperitoneal application of sodium iodate (NaIO3). MSCs, AgNPs or MSCs plus AgNPs were intravitreally injected 48 h before the last application of NaIO3. Retinas were isolated after additional 7 days and tested by RT‐PCR for the expression of genes for rhodopsin (photoreceptors), Iba‐1 (microglia/macrophages), IL‐1β and galectin‐3. The presence of MSCs stained with PKH‐26 was analysed by flow cytometry.Results: It was shown that MSCs and MSCs applied with AgNPs were still present in the retina on day 7 after application. In all treated groups, the expression of the gene for galectin‐3 was decreased in contrast to untreated control. However, co‐application of MSCs with AgNPs had limited effect on downregulation of Iba‐1 and IL‐1β in contrast to MSCs or AgNPs treated group.Conclusions: The application of MSCs and AgNPs modulated immune response in chronical retinal degeneration but co‐application of MSCs with AgNPs decreased immunoregulatory effects comparted with single‐component therapy. Thus, there is a need for more investigation focusing on potential changes in MSCs applied with AgNPs.

IL-33-Pretreated Mesenchymal Stem Cells Attenuate Acute Liver Failure by Improving Homing and Polarizing M2 Macrophages.

Mesenchymal stem cells (MSCs) are highly effective in the treatment of acute liver failure (ALF). The efficacy of MSCs is closely related to the inflammatory environment. Therefore, we investigated the functional changes of MSCs in response to interleukin-33 (IL-33) stimulation. The results showed that bone marrow mesenchymal stem cells (BMSCs) pretreated with IL-33 had increased CCR2 expression, targeted CCL2 in the injured liver tissue, and improved the migration ability. Under LPS stimulation, the NF-κB pathway of BMDM was activated, and its phenotype polarized to the M1-type, while BMSCs pretreated with IL-33 inhibited the NF-κB pathway and enhanced M2 macrophage polarization. The M2-type macrophages could further inhibit hepatocytes inflammation, reduce hepatocytes apoptosis, and promote hepatocytes repair. These results suggest that IL-33 can enhance the efficacy of BMSCs in ALF and provide a new strategy for cell therapy of liver diseases.

IFN-γ and TNF-α Synergistically Induce Mesenchymal Stem/Stromal Cell Death Via RIPK1-Independent Necroptosis

Interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) are two vital inflammatory factors elevated in many diseases (e.g. aplastic anemia), and an inflammatory microenvironment can cause cell damage and may be involved in the pathogenesis of certain diseases. However, the precise mechanisms of cell dysfunction or impairment during the inflammatory process are not fully understood. IFN-γ alone or in combination with TNF-α have distinct effects on the immunoregulatory properties of bone marrow derived mesenchymal stem cells (MSCs), and they have been raised to be optimal prime factors to enhance the immunosuppressive capacity of MSCs engineered in vitro. Yet controversies remain with regards to the normal function maintenance of cells as they may be impaired after exposure to inflammatory factors. Here, we find that IFN-γ and TNF-α can synergistically induce cell dysfunction and death of MSCs via a RIPK1-independent manner of necroptosis. When MSCs are exposed to both IFN-γ and TNF-α at a broad range of concentrations used in previous studies for about 24 hours or more, its morphological features, biological functions, differentiation capacity and immunoregulatory properties are totally injured. First of all, IFN-γ and TNF-α synergistically induce distinct morphological changes of MSCs. When primed with both IFN-γ and TNF-α, MSCs show a gradual change from a fibroblastic-like spindle shape to an enlarged widespread morphology and the cells grow in a disordered way. And from a high power view, the cells looked like a flat spider web and the blurred cell membrane hints that these cells may have “ruptured”, but most notably, the immunofluorescence staining shows that the nuclei were intact. Secondly, MSCs are functionally impaired after exposure in regard to their capacities of proliferation and migration, which, however, are not associated with increased cell apoptosis or senescence. Furthermore, the adipocytes, osteocytes and chondrocytes differentiation capacities of MSCs are entirely impaired and the immunosuppressive efficacies of MSCs on T cells are also attenuated by the synergistic effect of IFN-γ and TNF-α, though these impaired MSCs retain normal capacity of releasing indoleamine 2,3-dioxygenase (IDO), an immunosuppressive mediator of human MSCs. Mechanistically, we initially find that the signaling of IFN-γ might be enhanced by TNF-α through increasing the abundance of its receptor IFN-γ receptor1 on the surface of MSCs, which is possible to be the interplay mechanism of the two cytokines. And when the injured cells are subjected to RNA-Sequencing, it reveals that those MSCs have undergone a unique cell death, which is necroptosis, a form of programmed cell death characterized by a necrotic morphology including cell swelling, membrane rupture, and releasing of cellular contents, such as inflammatory cytokines, damage-associated molecular patterns (DAMPs), and chemokines, thereby leading to a variety of inflammatory responses subsequently. Compared with the control groups, IFN-γ and TNF-α synergistically increased the expression level of major necroptotic signaling cascade RIPK1, RIPK3 and MLKL into a significant level, and further analysis reveals that all other genes associated with necroptosis (TRADD, TICAM1, ZBP1, TLR3, and TLR4) are significantly upregulated as well. Finally, the rescue experiments targeting signaling pathways of necroptosis show that the necroptotic process of MSCs induced by IFN-γ & TNF-α cannot be reversed by RIPK1 inhibitor (Necrostatin-1), yet RIPK3 and MLKL inhibitor (GSK872 and Necrosulfonamide) hold individual or collaborative effects of protection against it, suggesting the undergoing necroptosis in this setting is independent of RIPK1, and targeting downstream proteins of necroptotic signaling pathway is a promising strategy for inhibiting such a cell death. Collectively, our results disclose an inflammatory injury mechanism of human bone marrow derived MSCs based on its morphological and functional alterations in combination with RNA-Seq identification, and a firstly found necroptotic manner of cell death in MSCs sheds new light on revealing the MSC deficits in some inflammatory or immune-mediated diseases with expectations to innovate some potential therapeutics. Our findings also suggest that cell therapy based on MSCs primed with IFN-γ and TNF-α should take this inflammatory injury effect into consideration.

Herpes Simplex Virus Infection Alters the Immunological Properties of Adipose-Tissue-Derived Mesenchymal-Stem Cells.

The proper functioning of mesenchymal stem cells (MSCs) is of paramount importance for the homeostasis of the body. Inflammation and infection can alter the function of MSCs, which can also affect the regenerative potential and immunological status of tissues. It is not known whether human herpes simplex viruses 1 and 2 (HSV1 and HSV2), well-known human pathogens that can cause lifelong infections, can induce changes in MSCs. In non-healing ulcers, HSV infection is known to affect deeper tissue layers. In addition, HSV infection can recur after initially successful cell therapies. Our aim was to study the response of adipose-derived MSCs (ADMSCs) to HSV infection in vitro. After confirming the phenotype and differentiation capacity of the isolated cells, we infected the cells in vitro with HSV1-KOS, HSV1-532 and HSV2 virus strains. Twenty-four hours after infection, we examined the gene expression of the cells via RNA-seq and RT-PCR; detected secreted cytokines via protein array; and determined autophagy via Western blot, transmission electron microscopy (TEM) and fluorescence microscopy. Infection with different HSV strains resulted in different gene-expression patterns. In addition to the activation of pathways characteristic of viral infections, distinct non-immunological pathways (autophagy, tissue regeneration and differentiation) were also activated according to analyses with QIAGEN Ingenuity Pathway Analysis, Kyoto Encyclopedia of Genes and Genome and Genome Ontology Enrichment. Viral infections increased autophagy, as confirmed via TEM image analysis, and also increased levels of the microtubule-associated protein light chain 3 (LC3B) II protein. We identified significantly altered accumulation for 16 cytokines involved in tissue regeneration and inflammation. Our studies demonstrated that HSV infection can alter the viability and immunological status of ADMSCs, which may have implications for ADMSC-based cell therapies. Alterations in autophagy can affect numerous processes in MSCs, including the inhibition of tissue regeneration as well as pathological differentiation.

The Cytokine Profile of Mesenchymal Stem Cells Changes with Culture Expansion

The Cytokine Profile of Mesenchymal Stem Cells Changes with Culture Expansion

Impact of Environmental and Epigenetic Changes on Mesenchymal Stem Cells during Aging

Many crucial epigenetic changes occur during early skeletal development and throughout life due to aging, disease and are heavily influenced by an individual's lifestyle. Epigenetics is the study of heritable changes in gene expression as the result of changes in the environment without any mutation in the underlying DNA sequence. The epigenetic profiles of cells are dynamic and mediated by different mechanisms, including histone modifications, non-coding RNA-associated gene silencing and DNA methylation. Given the underlining role of dysfunctional mesenchymal tissues in common age-related skeletal diseases such as osteoporosis and osteoarthritis, investigations into skeletal stem cells or mesenchymal stem cells (MSC) and their functional deregulation during aging has been of great interest and how this is mediated by an evolving epigenetic landscape. The present review describes the recent findings in epigenetic changes of MSCs that effect growth and cell fate determination in the context of aging, diet, exercise and bone-related diseases.

Metal Nanoparticles with Antimicrobial Properties: The Toxicity Response in Mouse Mesenchymal Stem Cells.

Some metal nanoparticles (NP) are characterized by antimicrobial properties with the potential to be used as alternative antibiotics. However, NP may negatively impact human organism, including mesenchymal stem cells (MSC), a cell population contributing to tissue growth and regeneration. To address these issues, we investigated the toxic effects of selected NP (Ag, ZnO, and CuO) in mouse MSC. MSC were treated with various doses of NP for 4 h, 24 h, and 48 h and multiple endpoints were analyzed. Reactive oxygen species were generated after 48 h CuO NP exposure. Lipid peroxidation was induced after 4 h and 24 h treatment, regardless of NP and/or tested dose. DNA fragmentation and oxidation induced by Ag NP showed dose responses for all the periods. For other NP, the effects were observed for shorter exposure times. The impact on the frequency of micronuclei was weak. All the tested NP increased the sensitivity of MSC to apoptosis. The cell cycle was most affected after 24 h, particularly for Ag NP treatment. In summary, the tested NP induced numerous adverse changes in MSC. These results should be taken into consideration when planning the use of NP in medical applications where MSC are involved.

Cytoskeletal Morphological Changes of Mesenchymal Stem Cells after Oxidant Damage and its Prevention by Thymoquinone

The functional integrity of the cytoskeleton of mesenchymal stem cells (MSCs) is essential for its differentiation into multiple cell lineages including adipocytes, chondrocytes, and osteoblasts. Abnormalities in the cytoskeletal proteins such as actin and microtubule can cause disrupted cell signalling and irregular movements of organelles leading to cell death. This study investigated cytoskeletal and nuclear morphological changes of the MSC due to oxidative damage by hydrogen peroxide (H2O2) and the possible prevention of these changes by the antioxidant thymoquinone (TQ). Bone marrow MSCs from Sprague Dawley rats were cultured and treated with different concentrations of H2O2 with or without TQ to observe the potential protective activity. Triple-label fluorescence immunocytochemistry was performed post-treatment to observe the nucleus, actin and microtubules using 4’,6-diamidino-2-phenylindole (DAPI), Alexa Fluor 488-labelled phalloidin and Cy3-labelled anti-tubulin antibody, respectively. The normal stem cell cytoskeleton demonstrated intact actin and microtubule structures along with normal appearance of the nucleus. However, oxidative damage by H2O2 caused a severe disruption of the cytoskeletal morphology of the actin and microtubule along with apoptosis and necrosis of the nucleus. Interestingly, both immunocytochemical and Fluorescence-Activated Cell Sorting (FACS) results showed that these morphological changes were prevented by TQ at low concentrations while higher concentrations of TQ were harmful. This study suggested that TQ could save MSCs from oxidative-induced cell death.

The effect of lncRNA MIR155HG-modified MSCs and exosome delivery to synergistically attenuate vein graft intimal hyperplasia

BackgroundThe mesenchymal stem cells (MSCs) were used to repair tissue injury. However, the treatment effect was not satisfactory. We investigated whether lncRNA MIR155HG could promote survival and migration of MSCs under oxidative stress, which mimics in vivo environments. Furthermore, we studied the protective effect of exosomes secreted by MSCs transfected with MIR155HG on endothelial cells. This study aimed to determine whether exploiting MSCs and exosomes modified with lncRNA MIR155HG would exert synergistic therapeutic effect to attenuate vein graft intimal hyperplasia more effectively.MethodsLentivirus containing lncRNA MIR155HG overexpressing vector was packaged and used to infect MSCs. Then, CCK-8 assay, flow cytometry, Transwell assay, and Elisa assay were used to assess the functional changes of MSCs with overexpressed MIR155HG (OE-MSCs). Furthermore, the associated pathways were screened by Western blot. MIR155HG-MSCs-derived exosomes (OE-exo) were collected and co-cultured with human umbilicus vein endothelial cell (HUVEC). We validated the protective effect of OE-exo on HUVEC. In vivo, both MSCs and exosomes modified with MIR155HG were injected into a vein graft rat model via tail vein. We observed MSCs homing and intimal hyperplasia of vein graft using a fluorescent microscope and histological stain.ResultsOur study found that lncRNA MIR155HG promoted proliferation, migration, and anti-apoptosis of MSCs. NF-κB pathway took part in the regulation process induced by MIR155HG. OE-exo could enhance the activity and healing ability of HUVEC and reduce apoptosis. In vivo, OE-MSCs had a higher rate of homing to vascular endothelium. The combined treatment with OE-MSCs and OE-exo protected vascular endothelial integrity, reduced inflammatory cell proliferation, and significantly attenuated intimal hyperplasia of vein graft.ConclusionLncRNA MIR155HG could promote the survival and activity of MSCs, and reduce the apoptosis of HUVECs using exosome delivery. Exploiting MSCs and exosomes modified with MIR155HG could attenuate vein graft intimal hyperplasia more effectively and maximize the surgical effect.

Sustainable Production and Activity Determination of Serum-Free Conditioned Medium from Menstrual Blood-Derived Endometrial Stem Cells.

Mesenchymal stem cells (MSCs) have exhibited great potential as a regenerative medicine, and MSC-derived paracrine effects, mainly including the secretion of various bioactive factors, play critical roles in MSC-based therapies. MSC-derived serum-free conditioned medium (MSC-CM) is defined as the secretome of MSC-derived bioactive factors and is considered a new cell-free therapeutic agent for disease treatment. However, the MSC-CM used in previous studies was prepared by a nearly disposable method that the MSCs were discarded after preparing MSC-CM, and the preparation time was variable; simultaneously, the viability changes of MSCs after MSC-CM preparation are still unknown. Therefore, this study takes MenSCs as a research project and aims to explore the suitable period of sustainable MenSC-CM preparation rather than using a disposable method. As expected, our results confirmed that MenSC-CM improves viability of both naïve targeted cells and H2O2-injured targeted cells, and suggested that 36h is suitable for sustainable MenSC-CM preparation in which the angiogenic factors almost reach to the peak. Simultaneously, the MenSCs used to prepare the MenSC-CM for 36h also maintained preferable cell viability and could be sustainably used for further MenSC-CM preparation. Moreover, the in vivo results further confirmed the improvement of MenSC-CM on promoting skin wound healing. Consequently, our results not only provide support for optimizing MSC-CM sustainable preparation based on various MSCs but also promote the comprehensive application of MenSCs in the clinic.

Preconditioned MSCs Alleviate Cerebral Ischemia-Reperfusion Injury in Rats by Improving the Neurological Function and the Inhibition of Apoptosis.

Mesenchymal stem cells (MSCs) have great application prospects in the treatment of ischemic injury. However, their long-time cultivation before transplantation and poor survival after transplantation greatly limit the therapeutic effect and applications. This study aimed to investigate whether MSCs under the ischemic microenvironment could improve their survival and better alleviate cerebral ischemic injury. Firstly, we used ischemic brain tissue to culture MSCs and evaluated the functional changes of MSCs. Then a middle cerebral artery occlusion (MCAO) model was induced in rats, and the pretreated MSCs were injected via the tail vein. The adhesive removal test, rotarod test, modified neurological severity score, and pathological analyses were applied to assess the rats’ neurological function. Then the expression of neuron and apoptosis related markers was detected. The results indicated that ischemic brain tissue pretreated MSCs promoted the proliferation and the release of the growth factors of MSCs. Meanwhile, in MCAO model rats, transplantation of pretreated MSCs enhanced the neurogenesis, attenuated behavioral changes, reduced infarct size, and inhibited apoptosis. The expression of B-cell lymphoma-2 (Bcl-2), brain-derived neurotrophic factor (BDNF), glial fibrillary acidic protein (GFAP), NF-L, and NeuN were increased, while BCL2-Associated X (Bax) and Caspase-3 decreased. Our results suggest that MSCs pretreatment with stroke brain tissue could be an effective strategy in treating cerebral ischemic injury.

Osteogenic differentiation of mesenchymal stem cells promotes c-Jun-dependent secretion of interleukin 8 and mediates the migration and differentiation of CD4+ T cells

BackgroundThe immune system and the skeletal system have complex interactions in the bone marrow and even in the joints, which has promoted the development of the concept of osteoimmunology. Some evidence has indicated that T cells and B cells contribute to the balance between the resorption and formation of bone. However, there has been little discussion on the regulation of CD4+ T lymphocytes by cells involved in bone metabolism. Mesenchymal stem cells (MSCs), which exert core functions related to immunoregulation and osteogenic differentiation, are crucial cells linked to both bone metabolism and the immune system. Previous studies have shown that the immunoregulatory capacity of MSCs changes following differentiation. However, it is still unclear whether the osteogenic differentiation of MSCs affects the migration and differentiation of CD4+ T cells.MethodsMSCs were cultured in growth medium or osteogenic medium for 10 days and then cocultured with CD4+ T cells. CD4+ T cell migration and differentiation were detected by flow cytometry. Further, gene expression levels of specific cytokines were analyzed by quantitative real-time PCR and enzyme-linked immunosorbent assays. A Proteome Profiler Human XL Cytokine Array Kit was used to analyze supernatants collected from MSCs. Alizarin red S staining and Alkaline phosphatase assay were used to detect the osteogenic differentiation of MSCs.ResultsHere, we found that the migration of CD4+ T cells was elevated, and the capacity to induce the differentiation of regulatory T (Treg) cells was weakened during MSC osteogenic differentiation, while the differentiation of T helper 1 (Th1), T helper 2 (Th2) and T helper 17 (Th17) cells was not affected. Further studies revealed that interleukin (IL)-8 was significantly upregulated during MSC osteogenic differentiation. Both a neutralizing antibody and IL-8-specific siRNA significantly inhibited the migration of CD4+ T cells and promoted the differentiation of Treg cells. Finally, we found that the transcription factor c-Jun was involved in regulating the expression of IL-8 and affected the osteogenic differentiation of MSCs, thereby mediating the migration and differentiation of CD4+ T cells.ConclusionThis study demonstrated that MSC osteogenic differentiation promoted c-Jun-dependent secretion of IL-8 and mediated the migration and differentiation of CD4+ T cells. These results provide a further understanding of the crosstalk between bone and the immune system and reveal information about the relationship between osteogenesis and inflammation in the field of osteoimmunology.

Hypoxia-Conditioned Mesenchymal Stem Cells in Tissue Regeneration Application.

Mesenchymal stem cells (MSCs) have been demonstrated as promising cell sources for tissue regeneration due to their capability of self-regeneration, differentiation, and immunomodulation. MSCs also exert extensive paracrine effects through release of trophic factors and extracellular vesicles (EVs). However, despite extended exploration of MSCs in preclinical studies, the results are far from satisfactory due to the poor engraftment and low level of survival after implantation. Hypoxia preconditioning has been proposed as an engineering approach to improve the therapeutic potential of MSCs. During in vitro culture, hypoxic conditions can promote MSC proliferation, survival, and migration through various cellular responses to the reduction of oxygen tension. The multilineage differentiation potential of MSCs is altered under hypoxia, with consistent reports of enhanced chondrogenesis. Hypoxia also stimulates the paracrine activities of MSCs and increases the production of secretome both in terms of soluble factors as well as EVs. The secretome from hypoxia-preconditioned MSCs play important roles in promoting cell proliferation and migration, enhancing angiogenesis while inhibiting apoptosis and inflammation. In this review, we summarize current knowledge of hypoxia-induced changes in MSCs and discuss the application of hypoxia-preconditioned MSCs as well as hypoxic secretome in different kinds of disease models. Impact statement Mesenchymal stem cells (MSCs) have been applied in numerous cell-based and secretome-based therapies for tissue regeneration. Hypoxic conditions enhance the function of MSCs by increasing proliferation, survival, homing, differentiation, and paracrine activities. A timely up-to-date comprehensive overview of the effect of low oxygen tension to MSC, with emphasis on the influence and molecular mechanism of hypoxia preconditioning toward MSC's functionality is provided, including the therapeutic use of hypoxia-preconditioned MSC as well as hypoxic secretome in various prove-of-concept disease models. This knowledge would contribute to future engineering of MSC culture conditions for improved translational application.

3D bioprinting of tissue units with mesenchymal stem cells, retaining their proliferative and differentiating potential, in polyphosphate-containing bio-ink

The three-dimensional (3D)-printing processes reach increasing recognition as important fabrication techniques to meet the growing demands in tissue engineering. However, it is imperative to fabricate 3D tissue units, which contain cells that have the property to be regeneratively active. In most bio-inks, a metabolic energy-providing component is missing. Here a formulation of a bio-ink is described, which is enriched with polyphosphate (polyP), a metabolic energy providing physiological polymer. The bio-ink composed of a scaffold (N,O-carboxymethyl chitosan), a hydrogel (alginate) and a cell adhesion matrix (gelatin) as well as polyP substantially increases the viability and the migration propensity of mesenchymal stem cells (MSC). In addition, this ink stimulates not only the growth but also the differentiation of MSC to mineral depositing osteoblasts. Furthermore, the growth/aggregate pattern of MSC changes from isolated cells to globular spheres, if embedded in the polyP bio-ink. The morphogenetic activity of the MSC exposed to polyP in the bio-ink is corroborated by qRT-PCR data, which show a strong induction of the steady-state-expression of alkaline phosphatase, connected with a distinct increase in the expression ratio between RUNX2 and Sox2. We propose that polyP should become an essential component in bio-inks for the printing of cells that retain their regenerative activity.

Oct4 downregulation-induced inflammation increases the migration and invasion rate of oral squamous cell carcinoma.

Inflammatory changes are involved in tumor cell proliferation, migration, and invasion. Tumor necrosis factor-α (TNF-α) and lipopolysaccharide (LPS) play important roles in inflammatory regulation during tumor development. Oct4 acts as a transcription factor that modulates inflammatory changes in mesenchymal stem cells. In this study, we explored the role of Oct4 in the invasion and migration of oral squamous cell carcinoma (OSCC) cells. LPS and TNF-α were used to treat the OSCC cell lines HN4 and CAL27 to induce inflammation. The generation of inflammatory cytokines, including TNF-α, interleukin (IL)-1β, and IL-6, was evaluated by enzyme-linked immunosorbent assay and real-time quantitative PCR. Western blot analysis was employed to detect the expression and phosphorylation of JNK1, p65, and STAT3, which are key modulators of inflammation. Wound scratch healing and transwell invasion assays were further used to determine the role of inflammation in the invasion and migration of OSCC cells. Robust inflammation was observed in HN4 and CAL27 cells treated with LPS and TNF-α. A marked increase in JNK1, p65, and STAT3 phosphorylation levels in OSCC cells was also detected after LPS and TNF-α treatment. The migration and invasion of HN4 and CAL27 cells were significantly boosted by stimulation with LPS and TNF-α. Furthermore, Oct4 mRNA and protein levels were significantly upregulated by stimulation with LPS and TNF-α. Silencing of Oct4 led to reduced inflammation and decreased levels of phosphorylated JNK1, p65, and STAT3 and impaired invasion and migration in LPS- and TNF-α-stimulated OSCC cells. Overall, LPS- and TNF-α-induced inflammation suppressed the migration and invasion of OSCC cells by upregulating Oct4 expression.

In vitro cellular and proteome assays identify Wnt pathway and CDKN2A-regulated senescence affected in mesenchymal stem cells from mice after a chronic LD gamma irradiation in utero

Reliable data on the effects of chronic prenatal exposure to low dose (LD) of ionizing radiation in humans are missing. There are concerns about adverse long-term effects that may persist throughout postnatal life of the offspring. Due to their slow cell cycle kinetics and life-long residence time in the organism, mesenchymal stem cells (MSCs) are more susceptible to low level genotoxic stress caused by extrinsic multiple LD events. The aim of this study was to investigate the effect of chronic, prenatal LD gamma irradiation to the biology of MSCs later in life. C3H mice were exposed in utero to chronic prenatal irradiation of 10 mGy/day over a period of 3 weeks. Two years later, MSCs were isolated from the bone marrow and analyzed in vitro for their radiosensitivity, for cellular senescence and for DNA double-strand break recognition after a second acute gamma-irradiation. In addition to these cellular assays, changes in protein expression were measured using HPLC–MS/MS and dysregulated molecular signaling pathways identified using bioinformatics. We observed radiation-induced proteomic changes in MSCs from the offspring of in utero irradiated mice (leading to ~ 9.4% of all detected proteins being either up- or downregulated) as compared to non-irradiated controls. The proteomic changes map to regulation pathways involved in the extracellular matrix, the response to oxidative stress, and the Wnt signaling pathway. In addition, chronic prenatal LD irradiation lead to an increased rate of in vitro radiation-induced senescence later in life and to an increased number of residual DNA double-strand breaks after 4 Gy irradiation, indicating a remarkable interaction of in vivo radiation in combination with a second acute dose of in vitro radiation. This study provides the first insight into a molecular mechanism of persistent MSC damage response by ionizing radiation exposure during prenatal time and will help to predict therapeutic safety and efficacy with respect to a clinical application of stem cells.

Deciphering the in vivo Dynamic Proteomics of Mesenchymal Stem Cells in Critical Limb Ischemia.

ObjectiveRegenerative therapy using mesenchymal stem cells (MSC) is a promising therapeutic method for critical limb ischemia (CLI). To understand how the cells are involved in the regenerative process of limb ischemia locally, we proposed a metabolic protein labeling method to label cell proteomes in situ and then decipher the proteome dynamics of MSCs in ischemic hind limb.Methods and ResultsIn this study, we overexpressed mutant methionyl-tRNA synthetase (MetRS), which could utilize azidonorleucine (ANL) instead of methionine (Met) during protein synthesis in MSCs. Fluorescent non-canonical amino-acid tagging (FUNCAT) was performed to detect the utilization of ANL in mutant MSCs. Mice with hindlimb ischemia (HLI) or Sham surgery were treated with MetRSmut MSCs or PBS, followed by i.p. administration of ANL at days 0, 2 6, and 13 after surgery. FUNCAT was also performed in hindlimb tissue sections to demonstrate the incorporation of ANL in transplanted cells in situ. At days 1, 3, 7, and 14 after the surgery, laser doppler imaging were performed to detect the blood reperfusion of ischemic limbs. Ischemic tissues were also collected at these four time points for histological analysis including HE staining and vessel staining, and processed for click reaction based protein enrichment followed by mass spectrometry and bioinformatics analysis. The MetRSmut MSCs showed strong green signal in cell culture and in HLI muscles as well, indicating efficient incorporation of ANL in nascent protein synthesis. By 14 days post-treatment, MSCs significantly increased blood reperfusion and vessel density, while reducing inflammation in HLI model compared to PBS. Proteins enriched by click reaction were distinctive in the HLI group vs. the Sham group. 34, 31, 49, and 26 proteins were significantly up-regulated whereas 28, 32, 62, and 27 proteins were significantly down-regulated in HLI vs. Sham at days 1, 3, 7, and 14, respectively. The differentially expressed proteins were more pronounced in the pathways of apoptosis and energy metabolism.ConclusionIn conclusion, mutant MetRS allows efficient and specific identification of dynamic cell proteomics in situ, which reflect the functions and adaptive changes of MSCs that may be leveraged to understand and improve stem cell therapy in critical limb ischemia.

Single-Cell Analysis of Mesenchymal Stem Cells Reveals Their Molecular and Functional Heterogeneity in Myeloma

IntroductionBone marrow (BM) mesenchymal stem cells (MSCs) are rare but heterogeneous cell populations, and both MSCs and their lineages mediate BM niches, hematopoiesis, and myeloma (MM) growth.The aim of the study was to assess variation in gene expression of unexpanded single MSCs generated from the same source, and study the heterogeneous effect on MM cell growth of MSCs lines generated from single MSCs or normal MSCs primed by MM cells. We also studied changes in MSCs during MM progression and identified MSC factors that mediate MM growth.MethodsCells collected from bone pieces following enzymatic digestion were cultured for 2-4 days. The adherent cells were trypsinized, replated to separate MSCs from macrophages and allowed to adhere overnight. Cells were then trypsinized and single MSCs were sorted into 96-well plates. Validation of single cell sorting and viability was assessed microscopically by staining for CFSE.Single MSCs were generated from two normal samples and two MM patients. Sorted single cells were subjected to qRT-PCR using primers of 34 genes implicated in MSC biology (e.g. CD73), cell cycle (e.g. KI67, CDKN2A), differentiation (e.g. RUNX2, PPARG, CYR61), transcription (e.g. FOXC1), and specific MSC genes we identified to be top overexpressed (e.g. POSTN) or underexpressed (e.g. IGFBP2, LEPR) in bone biopsy samples from high-risk MM patients. Also included were CD45 and CD34 to exclude hematopoietic cells, and GAPDH and ACTB as housekeeping genes. After excluding CD45+ cells and cells with low expression of housekeeping genes a total of 111 and 104 single MSCs from normal and MM subjects, respectively, were analyzed. Single MSCs expressed typical MSC markers such as CD73, CD90, FN1 and FAP.For generating MSC lines from the same source, single MSCs from each source were subjected to long-term culture supplemented with CM from whole BM and MSC cultures.For preparing primed MSCs, normal MSCs generated by standard method were cultured alones (unprimed MSCs) or cocultured with MM cells for 5 days (primed MSCs).The cultured and co-cultured cells were trypsinized, replated for 40 min followed by serial washing that attain >95% purified adherent MSCs. Primed and unprimed MSCs were subjected to gene expression profiling (GEP) and their CM used for proteomics and growth assays.ResultsSingle MSCs from the same source (i.e. normal and MM bones) heterogeneously expressed genes related to cell cycle, differentiation, and extracellular matrix. MM single MSCs had lower expression of markers of proliferation (e.g. KI67, p<0.005) and differentiation (e.g. RUNX2, p<0.03), and higher expression of the senescence marker CDKN2A (p<0.001).To further capture MSC heterogeneity, MSCs lines were generated from single MSCs and tested for their ability to support MM cells. Growth of individual MSCs varied in culture, most had a limited proliferation rate, and few were capable of generating MSC lines. MSC lines generated from the same source (4-10 lines from normal or MM subjects) had variable effects on growth of MM cells in co-cultures, or when their CM used on MM cell grown alone (p<0.0008 between lowest to highest growth stimulating lines).CM from primed MSCs stimulated the growth of primary MM cells compared to CM from unprimed MSCs in short-term culture (n=10, p<0.007) or 10-days co-couture with normal MSCs (n=6, p<0.002).Primed MM cells had reduced expression and secretion of factors related to the IGF1 pathways including IGFBP2, IGFBP3 and IGF2, whereas IGF1 was not impacted. Recombinant IGF1 promoted growth of BM-dependent MM cells in serum-free conditions, an effect that was significantly blocked by recombinant IGFBP2 (p<0.0003).IGFBP2 expression by GEP was lower in biopsy samples from MM patients (n=531) than normal subjects (n=68), lower in samples from high-risk than low-risk patients, and lower in focal lesion than random BM biopsies from the same patients (n=77). Immunohistochemistry showed high IGFBP2 protein expression by BM mesenchymal cells, particularly, small adipocytes, and changes in the proportion of IGFBP2+ cells correlated with the reduced gene expression in MM patients' biopsies, and with disease stage.ConclusionsThese findings suggest that MSCs from normal subjects and MM patients are heterogeneous population of cells, are deregulated as part of MM disease progression, and that production of factors such as IGFBP2 by these cells impacts bioavailability of IGF1. DisclosuresDavies:Bristol-Myers: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Morgan:Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Bristol Myers: Consultancy, Honoraria.

Comparative evaluation of proliferative potential and replicative senescence associated changes in mesenchymal stem cells derived from dental pulp and umbilical cord.

Mesenchymal stem cells (MSC) have been widely studied for tissue regeneration and cell-based therapy. MSC can be isolated from different body tissues while several biological waste sources like dental pulp, umbilical cord, cord derived blood, amniotic fluid or urine have also emerged as potential sources of MSCs. Specifically, isolation of MSCs from such non-conventional sources show promising outcomes due to the non-invasiveness of the extraction process and high proliferation capacity of the isolated MSC. However, these stem cells also exhibit the limitation of replicative senescence in long-term culture condition. Inter-cellular reactive oxygen species is an important contributor for inducing cellular senescence under long-term culture conditions. For translational application, it becomes imperative to compare the stem cells isolated from these sources for their senescence and proliferative properties. In this study, MSC were extracted from two different sources of biological waste materials-dental pulp and umbilical cord, and compared for their proliferation capacity and replicative senescence at different passage numbers (i.e. P2 and P6). Intracellular ROS production was significantly (p < 0.001) less in dental pulp stem cells culture in comparison to umbilical cord-derived stem cells at P6. The β-gal expression also showed significantly (p < 0.001) low expression in DPSC culture compared to that of UCSC at P6. The study indicates the source of stem cells influences the proliferation capacity as well as replicative senescence of MSCs. This study will thus pave the path of future research in selecting appropriate stem cell source for regenerative medicine application.