Bone regeneration and stem cells.

?  Introduction ?  Bone fracture healing and healing problems ?  Biomaterial scaffolds and tissue engineering in bone formation -  Bone tissue engineering -  Biomaterial scaffolds -  Synthetic scaffolds -  Micro- and nanostructural properties of scaffolds -  Conclusion ?  Mesenchymal stem cells and osteogenesis -  Bone tissue -  Origin of osteoblasts -  Isolation and characterization of bone marrow derived MSC -  In vitro differentiation of MSC into osteoblast lineage cells -  In vivo differentiation of MSC into bone -  Factors and pathways controlling osteoblast differentiation of hMSC -  Defining the relationship between osteoblast and adipocyte differentiation from MSC -  MSC and sex hormones -  Effect of aging on osteoblastogenesis -  Conclusion ?  Embryonic, foetal and adult stem cells in osteogenesis -  Cell-based therapies for bone -  Specific features of bone cells needed to be advantageous for clinical use -  Development of therapeutic biological agents -  Clinical application concerns -  Conclusion ?  Platelet-rich plasma (PRP), growth factors and osteogenesis -  PRP effects in vitro on the cells involved in bone repair -  PRP effects on osteoblasts -  PRP effects on osteoclasts -  PRP effects on endothelial cells -  PRP effects in vivo on experimental animals -  The clinical use of PRP for bone repair -  Non-union -  Distraction osteogenesis -  Spinal fusion -  Foot and ankle surgery -  Total knee arthroplasty -  Odontostomatology and maxillofacial surgery -  Conclusion ?  Molecular control of osteogenesis -  TGF-β signalling -  FGF signalling -  IGF signalling -  PDGF signalling -  MAPK signalling pathway -  Wnt signalling pathway -  Hedgehog signalling -  Notch signalling -  Ephrin signalling -  Transcription factors regulating osteoblast differentiation -  Conclusion ?  Summary This invited review covers research areas of central importance for orthopaedic and maxillofacial bone tissue repair, including normal fracture healing and healing problems, biomaterial scaffolds for tissue engineering, mesenchymal and foetal stem cells, effects of sex steroids on mesenchymal stem cells, use of platelet-rich plasma for tissue repair, osteogenesis and its molecular markers. A variety of cells in addition to stem cells, as well as advances in materials science to meet specific requirements for bone and soft tissue regeneration by addition of bioactive molecules, are discussed.

Positive and negative regulation of T cell responses by fibroblastic reticular cells within paracortical regions of lymph nodes.

Fibroblastic reticular cells (FRC) form the structural backbone of the T cell rich zones in secondary lymphoid organs (SLO), but also actively influence the adaptive immune response. They provide a guidance path for immigrating T lymphocytes and dendritic cells (DC) and are the main local source of the cytokines CCL19, CCL21, and IL-7, all of which are thought to positively regulate T cell homeostasis and T cell interactions with DC. Recently, FRC in lymph nodes (LN) were also described to negatively regulate T cell responses in two distinct ways. During homeostasis they express and present a range of peripheral tissue antigens, thereby participating in peripheral tolerance induction of self-reactive CD8(+) T cells. During acute inflammation T cells responding to foreign antigens presented on DC very quickly release pro-inflammatory cytokines such as interferon γ. These cytokines are sensed by FRC which transiently produce nitric oxide (NO) gas dampening the proliferation of neighboring T cells in a non-cognate fashion. In summary, we propose a model in which FRC engage in a bidirectional crosstalk with both DC and T cells to increase the efficiency of the T cell response. However, during an acute response, FRC limit excessive expansion and inflammatory activity of antigen-specific T cells. This negative feedback loop may help to maintain tissue integrity and function during rapid organ growth.

TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration.

Inflammation participates in tissue repair through multiple mechanisms including directly regulating the cell fate of resident progenitor cells critical for successful regeneration. Upon surveying target cell types of the TNF ligand TWEAK, we observed that TWEAK binds to all progenitor cells of the mesenchymal lineage and induces NF-kappaB activation and the expression of pro-survival, pro-proliferative and homing receptor genes in the mesenchymal stem cells, suggesting that this pro-inflammatory cytokine may play an important role in controlling progenitor cell biology. We explored this potential using both the established C2C12 cell line and primary mouse muscle myoblasts, and demonstrated that TWEAK promoted their proliferation and inhibited their terminal differentiation. By generating mice deficient in the TWEAK receptor Fn14, we further showed that Fn14-deficient primary myoblasts displayed significantly reduced proliferative capacity and altered myotube formation. Following cardiotoxin injection, a known trigger for satellite cell-driven skeletal muscle regeneration, Fn14-deficient mice exhibited reduced inflammatory response and delayed muscle fiber regeneration compared with wild-type mice. These results indicate that the TWEAK/Fn14 pathway is a novel regulator of skeletal muscle precursor cells and illustrate an important mechanism by which inflammatory cytokines influence tissue regeneration and repair. Coupled with our recent demonstration that TWEAK potentiates liver progenitor cell proliferation, the expression of Fn14 on all mesenchymal lineage progenitor cells supports a broad involvement of this pathway in other tissue injury and disease settings.

Ochratoxin A at nanomolar concentration perturbs the homeostasis of neural stem cells in highly differentiated but not in immature three-dimensional brain cell cultures.

Ochratoxin A (OTA), a fungal contaminant of basic food commodities, is known to be highly cytotoxic, but the pathways underlying adverse effects at subcytotoxic concentrations remain to be elucidated. Recent reports indicate that OTA affects cell cycle regulation. Therefore, 3D brain cell cultures were used to study OTA effects on mitotically active neural stem/progenitor cells, comparing highly differentiated cultures with their immature counterparts. Changes in the rate of DNA synthesis were related to early changes in the mRNA expression of neural stem/progenitor cell markers. OTA at 10nM, a concentration below the cytotoxic level, was ineffective in immature cultures, whereas in mature cultures it significantly decreased the rate of DNA synthesis together with the mRNA expression of key transcriptional regulators such as Sox2, Mash1, Hes5, and Gli1; the cell cycle activator cyclin D2; the phenotypic markers nestin, doublecortin, and PDGFRα. These effects were largely prevented by Sonic hedgehog (Shh) peptide (500ngml(-1)) administration, indicating that OTA impaired the Shh pathway and the Sox2 regulatory transcription factor critical for stem cell self-renewal. Similar adverse effects of OTA in vivo might perturb the regulation of stem cell proliferation in the adult brain and in other organs exhibiting homeostatic and/or regenerative cell proliferation.

JNK controls the onset of mitosis of planarian stem cells and triggers apoptotic cell death required for regeneration and remodeling

Regeneration of lost tissues depends on the precise interpretation of molecular signals that control and coordinate the onset of proliferation, cellular differentiation and cell death. However, the nature of those molecular signals and the mechanisms that integrate the cellular responses remain largely unknown. The planarian flatworm is a unique model in which regeneration and tissue renewal can be comprehensively studied in vivo. The presence of a population of adult pluripotent stem cells combined with the ability to decode signaling after wounding enable planarians to regenerate a complete, correctly proportioned animal within a few days after any kind of amputation, and to adapt their size to nutritional changes without compromising functionality. Here, we demonstrate that the stress-activated c-jun-NH2-kinase (JNK) links wound-induced apoptosis to the stem cell response during planarian regeneration. We show that JNK modulates the expression of wound-related genes, triggers apoptosis and attenuates the onset of mitosis in stem cells specifically after tissue loss. Furthermore, in pre-existing body regions, JNK activity is required to establish a positive balance between cell death and stem cell proliferation to enable tissue renewal, remodeling and the maintenance of proportionality. During homeostatic degrowth, JNK RNAi blocks apoptosis, resulting in impaired organ remodeling and rescaling. Our findings indicate that JNK-dependent apoptotic cell death is crucial to coordinate tissue renewal and remodeling required to regenerate and to maintain a correctly proportioned animal. Hence, JNK might act as a hub, translating wound signals into apoptotic cell death, controlled stem cell proliferation and differentiation, all of which are required to coordinate regeneration and tissue renewal.

Therapeutic Properties of Superoxide Dismutase 3 and Mesenchymal Stromal Cells in Peripheral Ischemia

The aim of this study was to characterize the cellular mechanisms leading to the beneficial effect of anti-oxidative gene therapy and pro-angiogenic stem cell therapy in acute peripheral ischemia. Post-ischemic events aim to re-establish tissue blood perfusion, to clear cellular debris, and to regenerate lost tissue by differentiation of satellite cells into myoblasts. Although leukocytes have an essential role in clearing cellular debris and promoting angiogenesis, they also contribute to tissue injury through excessive ROS production. First, we investigated the therapeutic properties of extracellular superoxide dismutase (SOD3) gene transfer. SOD3 was shown to reduce oxidative stress, to normalize glucose metabolism, and to enhance cell proliferation in the ischemic muscle. Analysis of the mitogenic Ras-Erk1/2 pathway showed SOD3 mediated induction offering a plausible explanation for enhanced cell proliferation. In addition, SOD3 reduced NF-κB activity by enhancing IκBα expression thus leading to reduced expression of inflammatory cytokines and adhesion molecules with consequent reduction in macrophage infiltration. Secondly, we sought to determine the fate and the effect of locally transplanted mesenchymal stem/stromal cells (MSCs) in acute ischemia. We showed that a vast majority of the transplanted cells are cleared from the injury site within 24 hours after local transplantation. Despite rapid clearance, transplantation was able to temporarily promote angiogenesis and cell proliferation in the muscle. Lack of graft-derived growth factor expression suggests other than secretory function to mediate this observed effect. In conclusion, both SOD3 and MSCs could be utilized to alleviate peripheral ischemia induced tissue injury. We have described a previously unidentified growth regulatory role for SOD3, and suggest a novel mechanism whereby transplanted MSCs enhance the reparative potential of the recipient tissue through physical contacts.

Isolation and characterization of equine peripheral blood-derived multipotent mesenchymal stromal cells

The objective of the study was to isolate, cultivate and characterize equine peripheral blood-derived multipotent mesenchymal stromal cells (PbMSCs). Peripheral blood was collected, followed by the isolation of mononuclear cells using density gradient reagents, and the cultivation of adherent cells. Monoclonal mouse anti-horse CD13, mouse anti-horse CD44, and mouse anti-rat CD90 antibodies were used for the immunophenotypic characterization of the surface of the PbMSCs. These cells were also cultured in specific media for adipogenic and chondrogenic differentiation. There was no expression of the CD13 marker, but CD44 and CD90 were expressed in all of the passages tested. After 14 days of cell differentiation into adipocytes, lipid droplets were observed upon Oil Red O (ORO) staining. Twenty-one days after chondrogenic differentiation, the cells were stained with Alcian Blue. Although the technique for the isolation of these cells requires improvement, the present study demonstrates the partial characterization of PbMSCs, classifying them as a promising type of progenitor cells for use in equine cell therapy.

Secondary prevention of type 1 diabetes mellitus: stopping immune destruction and promoting ß-cell regeneration

Type 1 diabetes mellitus results from a cell-mediated autoimmune attack against pancreatic ß-cells. Traditional treatments involve numerous daily insulin dosages/injections and rigorous glucose control. Many efforts toward the identification of ß-cell precursors have been made not only with the aim of understanding the physiology of islet regeneration, but also as an alternative way to produce ß-cells to be used in protocols of islet transplantation. In this review, we summarize the most recent studies related to precursor cells implicated in the regeneration process. These include embryonic stem cells, pancreas-derived multipotent precursors, pancreatic ductal cells, hematopoietic stem cells, mesenchymal stem cells, hepatic oval cells, and mature ß-cells. There is controversial evidence of the potential of these cell sources to regenerate ß-cell mass in diabetic patients. However, clinical trials using embryonic stem cells, umbilical cord blood or adult bone marrow stem cells are under way. The results of various immunosuppressive regimens aiming at blocking autoimmunity against pancreatic ß-cells and promoting ß-cell preservation are also analyzed. Most of these regimens provide transient and partial effect on insulin requirements, but new regimens are beginning to be tested. Our own clinical trial combines a high dose immunosuppression with mobilized peripheral blood hematopoietic stem cell transplantation in early-onset type 1 diabetes mellitus.

Development of the endocrine pancreas and novel strategies for β-cell mass restoration and diabetes therapy

Diabetes mellitus represents a serious public health problem owing to its global prevalence in the last decade. The causes of this metabolic disease include dysfunction and/or insufficient number of β cells. Existing diabetes mellitus treatments do not reverse or control the disease. Therefore, β-cell mass restoration might be a promising treatment. Several restoration approaches have been developed: inducing the proliferation of remaining insulin-producing cells, de novo islet formation from pancreatic progenitor cells (neogenesis), and converting non-β cells within the pancreas to β cells (transdifferentiation) are the most direct, simple, and least invasive ways to increase β-cell mass. However, their clinical significance is yet to be determined. Hypothetically, β cells or islet transplantation methods might be curative strategies for diabetes mellitus; however, the scarcity of donors limits the clinical application of these approaches. Thus, alternative cell sources for β-cell replacement could include embryonic stem cells, induced pluripotent stem cells, and mesenchymal stem cells. However, most differentiated cells obtained using these techniques are functionally immature and show poor glucose-stimulated insulin secretion compared with native β cells. Currently, their clinical use is still hampered by ethical issues and the risk of tumor development post transplantation. In this review, we briefly summarize the current knowledge of mouse pancreas organogenesis, morphogenesis, and maturation, including the molecular mechanisms involved. We then discuss two possible approaches of β-cell mass restoration for diabetes mellitus therapy: β-cell regeneration and β-cell replacement. We critically analyze each strategy with respect to the accessibility of the cells, potential risk to patients, and possible clinical outcomes.

Neural stem cells and cell replacement therapy: making the right cells

The past few years have seen major advances in the field of NSC (neural stem cell) research with increasing emphasis towards its application in cell-replacement therapy for neurological disorders. However, the clinical application of NSCs will remain largely unfeasible until a comprehensive understanding of the cellular and molecular mechanisms of NSC fate specification is achieved. With this understanding will come an increased possibility to exploit the potential of stem cells in order to manufacture transplantable NSCs able to provide a safe and effective therapy for previously untreatable neurological disorders. Since the pathology of each of these disorders is determined by the loss or damage of a specific neural cell population, it may be necessary to generate a range of NSCs able to replace specific neurons or glia rather than generating a generic NSC population. Currently, a diverse range of strategies is being investigated with this goal in mind. In this review, we focus on the relationship between NSC specification and differentiation and discuss how this information may be used to direct NSCs towards a particular fate.

The secretome of alginate-encapsulated limbal epithelial stem cells modulates corneal epithelial cell proliferation

Limbal epithelial stem cells may ameliorate limbal stem cell deficiency through secretion of therapeutic proteins, delivered to the cornea in a controlled manner using hydrogels. In the present study the secretome of alginate-encapsulated limbal epithelial stem cells is investigated. Conditioned medium was generated from limbal epithelial stem cells encapsulated in 1.2% (w/v) calcium alginate gels. Conditioned medium proteins separated by 1-D gel electrophoresis were visualized by silver staining. Proteins of interest including secreted protein acidic and rich in cysteine, profilin-1, and galectin-1 were identified by immunoblotting. The effect of conditioned medium (from alginate-encapsulated limbal epithelial stem cells) on corneal epithelial cell proliferation was quantified and shown to significantly inhibit (Pcells, this protein may be responsible, at least in part, for inhibition of corneal epithelial cell proliferation. We conclude that limbal epithelial stem cells encapsulated in alginate gels may regulate corneal epithelialisation through secretion of inhibitory proteins.

Origin and regenerative potential of vertebrate mechanoreceptor-associated stem cells

Meissner corpuscles and Merkel cell neurite complexes are highly specialized mechanoreceptors present in the hairy and glabrous skin, as well as in different types of mucosa. Several reports suggest that after injury, such as after nerve crush, freeze injury, or dissection of the nerve, they are able to regenerate, particularly including reinnervation and repopulation of the mechanoreceptors by Schwann cells. However, little is known about mammalian cells responsible for these regenerative processes. Here we review cellular origin of this plasticity in the light of newly described adult neural crest-derived stem cell populations. We also discuss further potential multipotent stem cell populations with the ability to regenerate disrupted innervation and to functionally recover the mechanoreceptors. These capabilities are discussed as in context to cellularly reprogrammed Schwann cells and tissue resident adult mesenchymal stem cells.

Controversial role of toll-like receptor 4 in adult stem cells

Adult or somatic stem cells are tissue-resident cells with the ability to proliferate, exhibit self-maintenance as well as to generate new cells with the principal phenotypes of the tissue in response to injury or disease. Due to their easy accessibility and their potential use in regenerative medicine, adult stem cells raise the hope for future personalisable therapies. After infection or during injury, they are exposed to broad range of pathogen or damage-associated molecules leading to changes in their proliferation, migration and differentiation. The sensing of such damage and infection signals is mostly achieved by Toll-Like Receptors (TLRs) with Toll-like receptor 4 being responsible for recognition of bacterial lipopolysaccharides (LPS) and endogenous danger-associated molecular patterns (DAMPs). In this review, we examine the current state of knowledge on the TLR4-mediated signalling in different adult stem cell populations. Specifically, we elaborate on the role of TLR4 and its ligands on proliferation, differentiation and migration of mesenchymal stem cells, hematopoietic stem cells as well as neural stem cells. Finally, we discuss conceptual and technical pitfalls in investigation of TLR4 signalling in stem cells.

Systemic delivery of adult stem cells improves cardiac function in spontaneously hypertensive rats

Heart regeneration after myocardial infarction (MI) can occur after cell therapy, but the mechanisms, cell types and delivery methods responsible for this improvement are still under investigation. In the present study, we evaluated the impact of systemic delivery of bone marrow cells (BMC) and cultivated mesenchymal stem cells (MSC) on cardiac morphology, function and mortality in spontaneously hypertensive rats (SHR) submitted to coronary occlusion. Female syngeneic adult SHR, submitted or not (control group; C) to MI, were treated with intravenous injection of MSC (MI + MSC) or BMC (MI + BM) from male rats and evaluated after 1, 15 and 30 days by echocardiography. Systolic blood pressure (SBP), functional capacity, histology, mortality rate and polymerase chain reaction for the Y chromosome were also analysed. Myocardial infarction induced a decrease in SBP and BMC, but not MSC, prevented this decrease. An improvement in functional capacity and ejection fraction (38 +/- 4, 39 +/- 3 and 58 +/- 2% for MI, MI + MSC and MI + BM, respectively; P < 0.05), as well as a reduction of the left ventricle infarcted area, were observed in rats from the MI + BM group compared with the other three groups. Treated animals had a significantly reduced lesion tissue score. The mortality rate in the C, MI + BM, MI + MSC and MI groups was 0, 0, 16.7 and 44.4%, respectively (P < 0.05 for the MI + MSC and MI groups compared with the C and MI + BM groups). The results of the present study suggest that systemic administration of BMC can improve left ventricular function, functional capacity and, consequently, reduce mortality in an animal model of MI associated with hypertension. We speculate that the cells transiently home to the myocardium, releasing paracrine factors that recruit host cells to repair the lesion.

Human Multipotent Mesenchymal Stromal Cells from Distinct Sources Show Different In Vivo Potential to Differentiate into Muscle Cells When Injected in Dystrophic Mice

Limb-girdle muscular dystrophies are a heterogeneous group of disorders characterized by progressive degeneration of skeletal muscle caused by the absence or deficiency of muscle proteins. The murine model of Limb-Girdle Muscular Dystrophy 2B, the SJL mice, carries a deletion in the dysferlin gene. Functionally, this mouse model shows discrete muscle weakness, starting at the age of 4-6 weeks. The possibility to restore the expression of the defective protein and improve muscular performance by cell therapy is a promising approach for the future treatment of progressive muscular dystrophies (PMD). We and others have recently shown that human adipose multipotent mesenchymal stromal cells (hASCs) can differentiate into skeletal muscle when in contact with dystrophic muscle cells in vitro and in vivo. Umbilical cord tissue and adipose tissue are known rich sources of multipotent mesenchymal stromal cells (MSCs), widely used for cell-based therapy studies. The main objective of the present study is to evaluate if MSCs from these two different sources have the same potential to reach and differentiate in muscle cells in vivo or if this capability is influenced by the niche from where they were obtained. In order to address this question we injected human derived umbilical cord tissue MSCs (hUCT MSCs) into the caudal vein of SJL mice with the same protocol previously used for hASCs; we evaluated the ability of these cells to engraft into recipient dystrophic muscle after systemic delivery, to express human muscle proteins in the dystrophic host and their effect in functional performance. These results are of great interest for future therapeutic application.