Cellular and molecular mechanisms responsible for the action of testosterone on human skeletal muscle. A basis for illegal performance enhancement

The popularity of testosterone among drug users is due to its powerful effects on muscle strength and mass. Important mechanisms behind the myotrophic effects of testosterone were uncovered both in athletes using steroids for several years and in short-term controlled studies. Both long-term and short-term steroid usage accentuates the degree of fibre hypertrophy in human skeletal muscle by enhancing protein synthesis. A mechanism by which testosterone facilitates the hypertrophy of muscle fibres is the activation of satellite cells and the promotion of myonuclear accretion when existing myonuclei become unable to sustain further enhancement of protein synthesis. Interestingly, long-term steroid usage also enhances the frequency of fibres with centrally located myonuclei, which implies the occurrence of a high regenerative activity. Under the action of testosterone, some daughter cells generated by satellite cell proliferation may escape differentiation and return to quiescence, which help to replenish the satellite cell reserve pool. However, whether long-term steroid usage induces adverse effects of satellite cells remains unknown. Testosterone might also favour the commitment of pluripotent precursor cells into myotubes and inhibit adipogenic differentiation. The effects of testosterone on skeletal muscle are thought to be mediated via androgen receptors expressed in myonuclei and satellite cells. Some evidence also suggests the existence of an androgen-receptor-independent pathway. Clearly, testosterone abuse is associated with an intense recruitment of multiple myogenic pathways. This provides an unfair advantage over non-drug users. The long-term consequences on the regenerative capacity of skeletal muscle are unknown.     

Stem cell ageing and apoptosis

Ageing has been defined as the process of deterioration of many body functions over the lifespan of an individual. In spite of the number of different theories about ageing, there is a general consensus in identifying ageing effects in a reduced capacity to regenerate injured tissues or organs and an increased propensity to infections and cancer. In recent years the stem cell theory of ageing has gained much attention. Adult stem cells residing in mammalian tissues are essential for tissue homeostasis and repair throughout adult life. With advancing age, the highly regulated molecular signalling necessary to ensure proper cellular, tissue, and organ homeostasis loses coordination and leads, as a consequence, to a compromised potential of regeneration and repair of damaged cells and tissues. Although a complete comprehension of the molecular mechanisms involved in stem cell ageing and apoptosis is far to be reached, recent studies are beginning to unravel the processes involved in stem cell ageing, particularly in adult skeletal muscle stem cells, namely satellite cells. Thus, the focus of this review is to analyse the relationship between stem cell ageing and apoptosis with a peculiar attention to human satellite cells as compared to haematopoietic stem cells. Undoubtedly, the knowledge of age-related changes of stem cells will help in understanding the ageing process itself and will provide novel therapeutic challenges for improved tissue regeneration.     

Adult mesenchymal stem cells in dental research: a new approach for tissue engineering

Many adult tissues contain a population of stem cells that have the ability to regenerate after trauma, disease or aging. Recently, there has been great interest in mesenchymal stem cells and their roles in maintaining the physiological structure of tissues. The studies on stem cells are thought to be very important and, in fact, it has been shown that this cell population can be expanded ex vivo to regenerate tissues not only of the mesenchymal lineage, such as intervertebral disc cartilage, bone and tooth-associated tissues, but also other types of tissues. Several studies have focused on the identification of odontogenic progenitors from oral tissues, and it has been shown that the mesenchymal stem cells obtained from periodontal ligament and dental pulp could have similar morphological and phenotypical features of the bone marrow mesenchymal cells. In fact a population of homogeneous human mesenchymal stem cells derived from periodontal ligament and dental pulp, and proliferating in culture with a well-spread morphology, can be recovered and characterized. Since these cells are considered as candidates for regenerative medicine, the knowledge of the cell differentiation mechanisms is imperative for the development of predictable techniques in implant dentistry, oral surgery and maxillo-facial reconstruction. Thus, future research efforts might be focused on the potential use of this cell population in tissue engineering. Further studies will be carried out to elucidate the molecular mechanisms involved in their maintenance and differentiation in vitro and in vivo.     

Pharmacological inhibition of myostatin/TGF-β receptor/pSmad3 signaling rescues muscle regenerative responses in mouse model of type 1 diabetes

Aim:

To study the influence of acute experimental diabetes on the regenerative potential of muscle stem (satellite) cells in mice.

Methods:

Male C57BL/6 young mice were injected with a single dose of streptozotocin (STZ, 180 mg/kg, ip) to induce diabetes. The diabetic mice were treated with insulin (0.75 U/kg, ip), follistatin (12 μg/kg, im) or Alk5 inhibitor (5 μmol/L per kg, sc) once a day. On the first day when high glucose levels were found, cardiotoxin (CTX) was focally injected into tibialis anterior and gastronemius muscles of the mice. The muscles were harvested 3 d and 5 d after CTX injection, and myofibers and satellite cells were isolated. Quantitative ex-vivo and in-vivo assays of myogenic potential were used to evaluate the muscle regenerative responses.

Results:

The satellite cells from the diabetic mice 3 d after CTX injection fail to activate, and the repair of muscle deteriorates, resembling that observed in old control mice. Furthermore, the satellite cells have excessive levels of myostatin, TGF-β receptor 1, pSmad3 and the cell cycle inhibitor p15, while the level of TGF-β1 remain unchanged. Treatment of the diabetic mice with insulin rescued muscle regenerative responses, and restored the expression levels of myostatin, TGF-β receptor 1, pSmad3, and p15 to those similar of healthy controls. Treatment of the diabetic mice with the myostatin antagonist follistatin, or with the Alk5 inhibitor of TGF-β receptor 1 (which did not diminish the blood glucose levels) rescued muscle regenerative responses and attenuated the myostatin/TGFβ receptor/pSmad3 signaling.

Conclusion:

The muscle regenerative responses are incapacitated and repair of the tissue fails within hours after the initiation of hyperglycemia in a mouse model of type 1 diabetes, but stem cell function is rescued by insulin, as well as follistatin or an Alk5 inhibitor that blocks TGF-β receptor signaling.     

Regeneration of cartilage and bone by defined subsets of mesenchymal stromal cells--potential and pitfalls

Mesenchymal stromal cells, also referred to as mesenchymal stem cells, can be obtained from various tissues. Today the main source for isolation of mesenchymal stromal cells in mammals is the bone marrow. Mesenchymal stromal cells play an important role in tissue formation and organogenesis during embryonic development. Moreover, they provide the cellular and humoral basis for many processes of tissue regeneration and wound healing in infancy, adolescence and adulthood as well. There is increasing evidence that mesenchymal stromal cells from bone marrow and other sources including term placenta or adipose tissue are not a homogenous cell population. Only a restricted number of appropriate stem cells markers have been explored so far. But routine preparations of mesenchymal stromal cells contain phenotypically and functionally distinct subsets of stromal cells. Knowledge on the phenotypical characteristics and the functional consequences of such subsets will not only extend our understanding of stem cell biology, but might allow to develop improved regimen for regenerative medicine and wound healing and novel protocols for tissue engineering as well. In this review we will discuss novel strategies for regenerative medicine by specific selection or separation of subsets of mesenchymal stromal cells in the context of osteogenesis and bone regeneration. Mesenchymal stromal cells, which express the specific cell adhesion molecule CD146, also known as MCAM or MUC18, are prone for bone repair. Other cell surface proteins may allow the selection of chondrogenic, myogenic, adipogenic or other pre-determined subsets of mesenchymal stromal cells for improved regenerative applications as well.     

Development of therapies against neuromuscular diseases causing muscle atrophy]

Skeletal muscles become atrophied by muscular disorders such as muscular dystrophy, wasting and even aging. In addition to muscle atrophy, progressive muscle damage, inflammation and replacement of muscle fibers with fibrous and fatty tissues are observed in muscular dystrophy. Neuronal innervation is required for skeletal muscle, and muscles become atrophic when motor neurons are affected by neurodegenerative disorders such as amyotrophic lateral sclerosis. Restoring muscle mass and function lost by diseases such as muscular dystrophy and neurodegenerative disorders is important. There are three rational therapies for muscular dystrophy and related diseases: gene therapy, cell therapy and drug therapy. Gene therapies to replace the defective genes have been tried with various degrees of effectiveness. Multiple myogenic stem cells including satellite cells, bone marrow cells, muscle side population cells, muscle-derived stem cells and mesoangioblast have been characterized. Cell therapies using these stem cells are one of the promising therapies for neuromuscular diseases causing muscle atrophy. As pharmacological drug therapies, increasing skeletal muscle mass by myostatin inhibition is quite promising and will be applied clinically in the near future.     

Myocardial regeneration: Roles of stem cells and hydrogels

Heart failure remains the leading cause of morbidity and mortality. Recently, it was reported that the adult heart has intrinsic regenerative capabilities, prompting a great wave of research into applying cell-based therapies, especially with skeletal myoblasts and bone marrow-derived cells, to regenerate heart tissues. While the mechanism of action for the observed beneficial effects of bone marrow-derived cells remains unclear, new cell candidates are emerging, including embryonic stem (ES) and introduced pluripotent stem (iPS) cells, as well as cardiac stem cells (CSCs) from adult hearts. However, the very low engraftment efficiency and survival of implanted cells prevent cell therapy from turning into a clinical reality. Injectable hydrogel biomaterials based on hydrophilic, biocompatible polymers and peptides have great potential for addressing many of these issues by serving as cell/drug delivery vehicles and as a platform for cardiac tissue engineering. In this review, we will discuss the application of stem cells and hydrogels in myocardial regeneration.     

Regenerative and immunomodulatory potential of mesenchymal stem cells

In the past few years, mesenchymal stem cells (MSCs) have come into the limelight because of their multi-lineage stem cell potential, which retains some aspects of embryonic stem cells, and because of their characteristic immunoregulatory functions exerted on different immune effector cells. The regenerative and immunomodulatory potential of MSCs has been used to support hemopoietic stem cell engraftment; to repair or regenerate damaged or mutated tissues, such as bone, cartilage, myocardial or hepatic tissues; to interfere with neoplastic cell growth by transfecting MSCs with anti-neoplastic molecules; and to modulate autoimmune reactions such as collagenopathies, multiple sclerosis and graft versus host disease. Thus, MSCs appear to be a very promising tool for regenerative and immunoregulatory cell therapy.     

Small molecules and future regenerative medicine

Recent advances in stem cell biology may make possible new approaches for the treatment of a number of diseases including cardiovascular disease, neurodegenerative disease, musculoskeletal disease, diabetes and cancer. These approaches could involve cell replacement therapy and/or drug treatment to stimulate the body's own regenerative capabilities by promoting survival, migration/homing, proliferation, and differentiation of endogenous stem/progenitor cells. However, such approaches will require identification of renewable cell sources of engraftable functional cells, an improved ability to manipulate their proliferation and differentiation, as well as a better understanding of the signaling pathways that control their fate. Cell-based phenotypic and pathway-specific screens of synthetic small molecules and natural products have historically provided useful chemical ligands to modulate and/or study complex cellular processes, and recently provided a number of small molecules that can be used to selectively regulate stem cell fate and developmental signaling pathways. Such molecules will likely provide new insights into stem cell biology, and may ultimately contribute to effective medicines for tissue repair and regeneration.     

Myocardial regeneration with stem cells: pharmacological possibilities for efficacy enhancement.

Cell therapy through the application of stem or progenitor cells to regenerate and repair damaged myocardium has gone from the bench to clinical trial. The functional benefits observed in clinical trials are, however, moderate, and many challenges need to be overcome before cell therapy can be put into widespread clinical use. Better understanding of the signals and molecules that promote cell proliferation, differentiation, engraftment and survival may lead to development of pharmacological agents that enhance the capacity of stem/progenitor cells for myocardial regeneration. The present review will provide a critical analysis of the merits and limitations of different populations of stem/progenitor cells and discuss the potential of pharmacological strategies for enhancing the efficacy of cell therapy for myocardial regeneration.     

Electrospun scaffolds for stem cell engineering

Stem cells interact with and respond to a myriad of signals emanating from their extracellular microenvironment. The ability to harness the regenerative potential of stem cells via a synthetic matrix has promising implications for regenerative medicine. Electrospun fibrous scaffolds can be prepared with high degree of control over their structure creating highly porous meshes of ultrafine fibers that resemble the extracellular matrix topography, and are amenable to various functional modifications targeted towards enhancing stem cell survival and proliferation, directing specific stem cell fates, or promoting tissue organization. The feasibility of using such a scaffold platform to present integrated topographical and biochemical signals that are essential to stem cell manipulation has been demonstrated. Future application of this versatile scaffold platform to human embryonic and induced pluripotent stem cells for functional tissue repair and regeneration will further expand its potential for regenerative therapies.     

Micromechanical control of cell and tissue development: implications for tissue engineering

Tissue engineering approaches for repair of diseased or lost organs will require the development of new biomaterials that guide cell behavior and seamlessly integrate with living tissues. Previous approaches to engineer artificial tissues have focused largely on optimization of scaffold polymer chemistry and selection of appropriate biochemical additives (e.g., growth factors, adhesive ligands) to provide effective developmental control. However, recent work has shown that micromechanical forces and local variations of extracellular matrix (ECM) elasticity at the microscale regulate cell and tissue development both in vitro and in vivo. The micromechanical properties of the host tissue microenvironment also play a critical role in control of stem cell lineage switching. Here we discuss how new understanding of the fundamental role that mechanical forces play in tissue development might be leveraged to facilitate the development of new types of biomimetic materials for regenerative medicine, with a focus on the design of injectable materials that can target to injury sites, recruit stem cells and direct cellular self-assembly to regenerate functional tissues and organs in situ.     

Morphological and cytofluorimetric analysis of adult mesenchymal stem cells expanded ex vivo from periodontal ligament

Many adult tissues contain a population of stem cells that have the ability of regeneration after trauma, disease or aging. Recently, there has been great interest in mesenchymal stem cells and their roles in maintaining the physiological structure of tissues, and their studies have been considered very important and intriguing, after having shown that this cell population can be expanded ex vivo to regenerate tissues not only of the mesenchymal lineage, such as intervertebral disc cartilage, bone, tooth-associated tissue, cardiomyocytes, but also to differentiate into cells derived from other embryonic layers, including neurons. Currently, different efforts have been focused on the identification of odontogenic progenitors from oral tissues. In this study we isolated and characterized a population of homogeneous human mesenchymal stem cells proliferating in culture with an attached well-spread morphology derived from periodontal ligament, a tissue of ectomesenchymal origin, with the ability to form a specialized joint between alveolar bone and tooth. The adherent cells were harvested and expanded ex vivo under specific conditions and analysed by FACScan flow cytometer and morphological analysis was carried out by light, scanning and transmission electron microscopy. Our results displayed highly evident cells with a fibroblast-like morphology and a secretory apparatus, probably indicating that the enhanced function of the secretory apparatus of the mesenchymal stem cells may be associated with the secretion of molecules that are required to survive and proliferate. Moreover, the presence in periodontal ligament of CD90, CD29, CD44,CD166, CD 105, CD13 positive cells, antigens that are also identified as stromal precursors of the bone marrow, indicate that the periodontal ligament may turn out to be a new efficient source of the cells with intrinsic capacity to self-renewal, high ability to proliferate and differentiate, that can be utilized for a new approach to regenerative medicine and tissue engineering.     

Novel strategies of regenerative medicine using chemical compounds

Many diseases and/or physical defects due to injury result in the loss of specialized cells within organ systems and lead to organ system dysfunction. The ultimate goal of cell-based therapies is to regenerate and restore normal function. Populations of embryonic, fetal, adult stem cells and inducible pluripotent stem cells generated by reprogramming of adult cells show promise for the treatment of a variety of diseases. In addition, the recent advancements in adult stem cell biology in both normal and pathological conditions have led to the identification of some intrinsic and extrinsic factors that govern the decision between self renewal versus differentiation of tissue-resident adult stem cells. This is of primary importance for the design of an approach of stem cell-based therapy focused on their in vivo modulation by conventional chemical and biological therapeutics capable to stimulate endogenous cell regeneration. Such therapeutics can act in vivo to promote cell survival, proliferation, differentiation, reprogramming and homing of stem cells or can modulate their niches. In this review, we will highlight the burst of recent literature on novel perspectives of regenerative medicine and their possible clinical applications.