Extracellular xenogeneic hemoglobin suppresses the capacity for C2C12 myoblast myogenic differentiation

Functional characteristics of satellite cells (SCs) that act as myogenesis initiators and have emerged as a promising target for cell therapy, are dependent on their microenvironment. The aim of this study was to investigate the effect of cell-free hemoglobin, as a part of the microenvironment of SCs, on their functional characteristics. The C2C12 cell line served as the experimental model of SCs; hemoglobin isolated from porcine (PHb) and bovine (BHb) slaughterhouse blood served as the experimental model for extracellular hemoglobin. The proliferation rate of C2C12 cells was assessed by the MTT test, migration capacity by the scratch assay, and myogenic differentiation capacity by histochemical staining and RT-PCR analysis of the expression of genes specific for myogenic lineage. The effect of hemoglobin on the proliferation and migration of C2C12 cells was dependent on its concentration and the animal species it was isolated from, but the effect of BHb was more prominent. Both PHb and BHb decreased the expression levels of myogenin and muscle specific creatine kinase at a 10 mu M concentration. While PHb had no effect on the morphometric parameters of C2C12 myotubes, BHb modified the area and length of C2C12 myotubes cultivated in DMEM/2% horse serum and DMEM/10% fetal calf serum. While PHb and BHb had no effect on heme oxygenase 1 (Hmox1) expression, they stimulated the expression of hypoxia-inducible factor 1-alpha (Hif1 alpha) at a concentration of 10 mu M. The mainly inhibitory effect of cell-free hemoglobin on myogenic differentiation suggests that it could be a relevant factor in the outcome of cell therapy of muscle injury

Modulation of Functional Characteristics of Mesenchymal Stromal Cells by Acellular Preparation of Porcine Hemoglobin

Exploring the potential usage of the acellular preparation of porcine hemoglobin (PHb) isolated from slaughterhouse blood as a cell culture media component, we have tested its effects on the functional characteristics of stromal cells of mesodermal origin. Human peripheral blood mesenchymal stromal cells (PB-MSCs) were used in this study as a primary cell model system, along with three mouse cell lines (ATDC5, MC3T3-E1, and 3T3-L1), which represent more uniform model systems. We investigated the effect of PHb at concentrations of 0.1, 1, and 10 μM on these cells’ proliferation, cycle, and clonogenic and migratory potential, and found that PHb’s effect depended on both the cell type and its concentration. At the lowest concentration used (0.1 μM), PHb showed the least evident impact on the cell growth and migration; hence, we analyzed its effect on mesenchymal cell multilineage differentiation capacity at this concentration. Even under conditions that induce a specific type of MSC differentiation (cultivation in particular differentiation media), PHb modulated chondrogenic, osteogenic, and adipogenic differentiation, making it a potential candidate for a supplement of MSC culture. Through a model of porcine hemoglobin, these findings also contribute to improving the knowledge of extracellular hemoglobin’s influence on MSCs in vivo

SMAD3 is essential for transforming growth factor-beta 1-induced urokinase type plasminogen activator expression and migration in transformed keratinocytes

Transforming growth factor-beta 1 (TGF-beta 1) stimulates the extracellular matrix degrading proteases expression and cell migration in order to enhance cancer cells malignancy. In the present study, we analysed the role of TGF-beta 1-induced Smad3 activation in the urokinase type plasminogen activator (uPA) production, as well as in cell migration and E-cadherin downregulation in transformed PDV keratinocyte cell line. TGF-beta 1 signalling was interfered by the chemical inhibitor of the TGF-beta 1-receptor 1 (ALK5), SB505124, and the specific Smad3 inhibitor, SiS3. Our results showed that TGF-beta 1 stimulates uPA expression directly through ALK5 activation. The inhibition of Smad3 strongly reduced the capacity of TGF-beta 1 to stimulate uPA expression, in parallel decreasing the uPA inhibitor plasminogen activator inhibitor type 1 (PAI-1) expression. In addition, the transient expression of dominant negative Smad3 mutant inhibited the TGF-beta 1-induced uPA promoter transactivation. Moreover, Smad3-/- mouse embryonic fibroblasts were refractory to the induction of uPA by TGF-beta 1. The inhibition of both ALK5 and Smad3 dramatically blocked the TGF-beta 1-stimulated E-cadherin downregulation, F-actin reorganisation and migration of PDV cells. Taken together, our results suggest that the TGF-beta 1-induced activation of Smad3 is the critical step for the uPA upregulation and E-cadherin downregulation, which are the key events preceding the induction of cell migration by TGF-beta 1 in transformed cells

Interleukin-17 and Its Implication in the Regulation of Differentiation and Function of Hematopoietic and Mesenchymal Stem Cells

Adult stem cells have a great potential applicability in regenerative medicine and cell-based therapies. However, there are still many unresolved issues concerning their biology, and the influence of the local microenvironment on properties of stem cells has been increasingly recognized. Interleukin (IL-) 17, as a cytokine implicated in many physiological and pathological processes, should be taken into consideration as a part of a regulatory network governing tissue-associated stem cells' fate. This review is focusing on the published data on the effects of IL-17 on the properties and function of hematopoietic and mesenchymal stem cells and trying to discuss that IL-17 achieves many of its roles by acting on adult stem cells

Inflammatory niche: Mesenchymal stromal cell priming by soluble mediators

Mesenchymal stromal/stem cells (MSCs) are adult stem cells of stromal origin that possess self-renewal capacity and the ability to differentiate into multiple mesodermal cell lineages. They play a critical role in tissue homeostasis and wound healing, as well as in regulating the inflammatory microenvironment through interactions with immune cells. Hence, MSCs have garnered great attention as promising candidates for tissue regeneration and cell therapy. Because the inflammatory niche plays a key role in triggering the reparative and immunomodulatory functions of MSCs, priming of MSCs with bioactive molecules has been proposed as a way to foster the therapeutic potential of these cells. In this paper, we review how soluble mediators of the inflammatory niche (cytokines and alarmins) influence the regenerative and immunomodulatory capacity of MSCs, highlighting the major advantages and concerns regarding the therapeutic potential of these inflammatory primed MSCs. The data summarized in this review may provide a significant starting point for future research on priming MSCs and establishing standardized methods for the application of preconditioned MSCs in cell therapy

Adipogenesis in Different Body Depots and Tumor Development

Adipose tissue (AT) forms depots at different anatomical locations throughout the body, being in subcutaneous and visceral regions, as well as the bone marrow. These ATs differ in the adipocyte functional profile, their insulin sensitivity, adipokines' production, lipolysis, and response to pathologic conditions. Despite the recent advances in lineage tracing, which have demonstrated that individual adipose depots are composed of adipocytes derived from distinct progenitor populations, the cellular and molecular dissection of the adipose clonogenic stem cell niche is still a great challenge. Additional complexity in AT regulation is associated with tumor-induced changes that affect adipocyte phenotype. As an integrative unit of cell differentiation, AT microenvironment regulates various phenotype outcomes of differentiating adipogenic lineages, which consequently may contribute to the neoplastic phenotype manifestations. Particularly interesting is the capacity of AT to impose and support the aberrant potency of stem cells that accompanies tumor development. In this review, we summarize the current findings on the communication between adipocytes and their progenitors with tumor cells, pointing out to the co-existence of healthy and neoplastic stem cell niches developed during tumor evolution. We also discuss tumor-induced adaptations in mature adipocytes and the involvement of alternative differentiation programs

Matične ćelije opredeljene za granulocitno-monocitnu lozu kostne srži i periferne krvi svinja

The pig is widely used as a large animal model for biomedical research and could be an interesting experimental model for studies of the hematopoietic system and its response in physiological and pathological conditions. With the intention of using the pig as a large animal model in hematopoietic research, a clonal assay in methylcellulose was developed and standardized for detection of committed progenitors of the granulocyte-macrophage lineage from adult pig bone marrow and peripheral blood. Progenitor cells were stimulated to proliferate and differentiate in vitro by adding pig leukocyte conditioned medium (LCM) as a source of homologous growth factors. The number of CFU-GM (Colony Forming Unit -Granulocyte-Macrophage) directly depended on the concentration of LCM. The proliferative rate of CFU-GM progenitor cells was determined by the cytosine arabinoside suicide technique. The percentage of bone marrow and peripheral blood CFU-GM cells in S phase of the cell cycle was 34.7% and 22.2%, respectively. The data obtained regarding the number and characteristics of pig bone marrow and peripheral blood CFU-GM confirmed that the organization of the pig CFU-GM progenitor cell compartment is similar and comparable to that in miniature swine, other animal species and humans.Svinja je životinja koja se koristi kao model u različitim biomedicinskim istraživanjima, a mogia bi biti i interesantan model u ispitivanju fiziologije i patolofizioloških promena hematopoetskog sistema. U cilju razvoja eksperimentalnog modela svinje u istraživanju hematopoeze, stanadardizovan je esej za odredivanje i karakterizaciju opredeljenih matičnih ćelija granulocitno-monocitne loze iz kostne srži i krvi odrasle svinje. Stimulacija proliferacije i diferencijacije ovih matičnih ćelija postignuta je dodavanjem medijuma kondicioniranog leukocitima (LCM - Leukocyte conditioned medium) bogatog faktorima rasta. Broj CFU-GM (Colony forming unit- granulocyte-macrophage) je direktno zavisio od koncentracije LCM-a. Procenat CFU-GM ćelija u S fazi ćelijskog cikiusa odredjivan je tehnikom 'suicida' korišćenjem citozin arabinozida (Ara-C) i iznosio je 34.7% za CFU-GM iz kostne srži i 22.2% za CFU-GM iz periferne krvi. Podaci dobijeni za broj i karakteristike CFU-GM iz kostne srži i periferne krvi potvrđuju da je ovaj odeljak matičnih ćelija kod odraslih svinja organizovan na isti način kao i kod minijaturnih svinja, drugih vrsta životinja i ljudi

The Roles of Mesenchymal Stromal/Stem Cells in Tumor Microenvironment Associated with Inflammation

State of tumor microenvironment (TME) is closely linked to regulation of tumor growth and progression affecting the final outcome, refractoriness, and relapse of disease. Interactions of tumor, immune, and mesenchymal stromal/stem cells (MSCs) have been recognized as crucial for understanding tumorigenesis. Due to their outstanding features, stem cell-like properties, capacity to regulate immune response, and dynamic functional phenotype dependent on microenvironmental stimuli, MSCs have been perceived as important players in TME. Signals provided by tumor-associated chronic inflammation educate MSCs to alter their phenotype and immunomodulatory potential in favor of tumor-biased state of MSCs. Adjustment of phenotype to TME and acquisition of tumor-promoting ability byMSCs help tumor cells inmaintenance of permissive TME and suppression of antitumor immune response. Potential utilization of MSCs in treatment of tumor is based on their inherent ability to home tumor tissue that makes them suitable delivery vehicles for immune-stimulating factors and vectors for targeted antitumor therapy. Here, we review data regarding intrusive effects of inflammatory TME on MSCs capacity to affect tumor development through modification of their phenotype and interactions with immune system

Tumorigenic Aspects of MSC Senescence—Implication in Cancer Development and Therapy

As an organism ages, many physiological processes change, including the immune system. This process, called immunosenescence, characterized by abnormal activation and imbalance of innate and adaptive immunity, leads to a state of chronic low-grade systemic inflammation, termed inflammaging. Aging and inflammaging are considered to be the root of many diseases of the elderly, as infections, autoimmune and chronic inflammatory diseases, degenerative diseases, and cancer. The role of mesenchymal stromal/stem cells (MSCs) in the inflammaging process and the age-related diseases is not completely established, although numerous features of aging MSCs, including altered immunomodulatory properties, impeded MSC niche supporting functions, and senescent MSC secretory repertoire are consistent with inflammaging development. Although senescence has its physiological function and can represent a mechanism of tumor prevention, in most cases it eventually transforms into a deleterious (para-)inflammatory process that promotes tumor growth. In this review we are going through current literature, trying to explore the role of senescent MSCs in making and/or sustaining a microenvironment permissive to tumor development and to analyze the therapeutic options that could target this process