Marta Barba

Adipose-Derived Mesenchymal Cells for Bone Regereneration: State of the Art

Adipose tissue represents a hot topic in regenerative medicine because of the tissue source abundance, the relatively easy retrieval, and the inherent biological properties of mesenchymal stem cells residing in its stroma. Adipose-derived mesenchymal stem cells (ASCs) are indeed multipotent somatic stem cells exhibiting growth kinetics and plasticity, proved to induce efficient tissue regeneration in several biomedical applications. A defined consensus for their isolation, classification, and characterization has been very recently achieved. In particular, bone tissue reconstruction and regeneration based on ASCs has emerged as a promising approach to restore structure and function of bone compromised by injury or disease. ASCs have been used in combination with osteoinductive biomaterial and/or osteogenic molecules, in either static or dynamic culture systems, to improve bone regeneration in several animal models. To date, few clinical trials on ASC-based bone reconstruction have been concluded and proved effective. The aim of this review is to dissect the state of the art on ASC use in bone regenerative applications in the attempt to provide a comprehensive coverage of the topics, from the basic laboratory to recent clinical applications.

Bone substitutes in orthopaedic surgery: from basic science to clinical practice

Bone substitutes are being increasingly used in surgery as over two millions bone grafting procedures are performed worldwide per year. Autografts still represent the gold standard for bone substitution, though the morbidity and the inherent limited availability are the main limitations. Allografts, i.e. banked bone, are osteoconductive and weakly osteoinductive, though there are still concerns about the residual infective risks, costs and donor availability issues. As an alternative, xenograft substitutes are cheap, but their use provided contrasting results, so far. Ceramic-based synthetic bone substitutes are alternatively based on hydroxyapatite (HA) and tricalcium phosphates, and are widely used in the clinical practice. Indeed, despite being completely resorbable and weaker than cortical bone, they have exhaustively proved to be effective. Biomimetic HAs are the evolution of traditional HA and contains ions (carbonates, Si, Sr, Fl, Mg) that mimic natural HA (biomimetic HA). Injectable cements represent another evolution, enabling mininvasive techniques. Bone morphogenetic proteins (namely BMP2 and 7) are the only bone inducing growth factors approved for human use in spine surgery and for the treatment of tibial nonunion. Demineralized bone matrix and platelet rich plasma did not prove to be effective and their use as bone substitutes remains controversial. Experimental cell-based approaches are considered the best suitable emerging strategies in several regenerative medicine application, including bone regeneration. In some cases, cells have been used as bioactive vehicles delivering osteoinductive genes locally to achieve bone regeneration. In particular, mesenchymal stem cells have been widely exploited for this purpose, being multipotent cells capable of efficient osteogenic potential. Here we intend to review and update the alternative available techniques used for bone fusion, along with some hints on the advancements achieved through the experimental research in this field.

Spinal Fusion in the Next Generation: Gene and Cell Therapy Approaches

Bone fusion represents a challenge in the orthopedics practice, being especially indicated for spine disorders. Spinal fusion can be defined as the bony union between two vertebral bodies obtained through the surgical introduction of an osteoconductive, osteoinductive, and osteogenic compound. Autogenous bone graft provides all these three qualities and is considered the gold standard. However, a high morbidity is associated with the harvest procedure. Intensive research efforts have been spent during the last decades to develop new approaches and technologies for successful spine fusion. In recent years, cell and gene therapies have attracted great interest from the scientific community. The improved knowledge of both mesenchymal stem cell biology and osteogenic molecules allowed their use in regenerative medicine, representing attractive approaches to achieve bone regeneration also in spinal surgery applications. In this review we aim to describe the developing gene- and cell-based bone regenerative approaches as promising future trends in spine fusion.

Genetic basis of single-suture synostoses: genes, chromosomes and clinical implications

BackgroundNon syndromic craniosynostoses are the most frequent craniofacial malformations worldwide. They represent a wide and heterogeneous group of entities, in which the dysmorphism may occur in a single (simple forms) or in multiple sutures (complex forms). Simple forms present a higher birth prevalence and are classified according to the involved suture and to the corresponding abnormal cranial shape: scaphocephaly (SC; sagittal suture), trigonocephaly (TC; metopic suture), anterior plagiocephaly (unilateral coronal suture), posterior plagiocephaly (unilateral lambdoid suture). They occur commonly as sporadic forms, although a familiar recurrence is sometimes observed, suggesting a mendelian inheritance. The genetic causes of simple craniosynostosis are still largely unknown, as mutations in common craniosynostosis-associated genes and structural chromosomal aberrations have been rarely found in these cases.AimsThis review is intended to dissect comprehensively the state-of-the art on the genetic etiology of single suture craniosynostoses, in the attempt to categorize all known disease-associated genes and chromosomal aberrations. Possible genotype/phenotype correlations are discussed as useful clues towards the definition of optimized clinical management flowcharts.

The multikinase inhibitor Sorafenib enhances glycolysis and synergizes with glycolysis blockade for cancer cell killing

Although the only effective drug against primary hepatocarcinoma, the multikinase inhibitor Sorafenib (SFB) usually fails to eradicate liver cancer. Since SFB targets mitochondria, cell metabolic reprogramming may underlie intrinsic tumor resistance. To characterize cancer cell metabolic response to SFB, we measured oxygen consumption, generation of reactive oxygen species (ROS) and ATP content in rat LCSC (Liver Cancer Stem Cells) -2 cells exposed to the drug. Genome wide analysis of gene expression was performed by Affymetrix technology. SFB cytotoxicity was evaluated by multiple assays in the presence or absence of metabolic inhibitors, or in cells genetically depleted of mitochondria. We found that low concentrations (2.5–5 μM) of SFB had a relatively modest effect on LCSC-2 or 293 T cell growth, but damaged mitochondria and increased intracellular ROS. Gene expression profiling of SFB-treated cells was consistent with a shift toward aerobic glycolysis and, accordingly, SFB cytotoxicity was dramatically increased by glucose withdrawal or the glycolytic inhibitor 2-DG. Under metabolic stress, activation of the AMP dependent Protein Kinase (AMPK), but not ROS blockade, protected cells from death. We conclude that mitochondrial damage and ROS drive cell killing by SFB, while glycolytic cell reprogramming may represent a resistance strategy potentially targetable by combination therapies.

Molecular mechanisms underlying human adipose tissue-derived stromal cells differentiation into a hepatocyte-like phenotype

BACKGROUND Adipose tissue-derived stromal cells (ATSCs) hold great promises in regenerative medicine. In the last decade, several studies have reported the plasticity of ATSCs toward a hepatocyte-like phenotype. Nonetheless, the molecular mechanisms underlying the conversion from a mesenchymal to an epithelial phenotype remain poorly understood. AIM In this study, we compared the full genome expression profiles of ATSCs cultured for 4 weeks under pro-hepatogenic conditions to undifferentiated ATSCs, in order to depict the molecular events involved in ATSC hepatic transdifferentiation. METHODS Analysis was performed using the Affymetrix human focus arrays. Sets of differentially expressed genes were functionally categorized in order to understand which pathways drive the hepatic conversion and interesting targets were validated by Q-PCR. RESULTS ATSC-derived hepatocyte-like cells activate several genes associated with specific liver functions, including protein metabolism, innate immune response regulation, and biodegradation of toxic compounds. Furthermore, microarray analysis highlighted downregulation of transcripts associated with the mesenchymal lineage, while epithelial-related genes were overexpressed. CONCLUSION Our data suggest that the in vitro system used in this study drove ATSCs toward a hepatic conversion through a subtle regulation of molecular pathways controlling lineage commitment that promote mesenchymal-epithelial transition.

Mitochondrial network genes in the skeletal muscle of amyotrophic lateral sclerosis patients.

Recent evidence suggested that muscle degeneration might lead and/or contribute to neurodegeneration, thus it possibly play a key role in the etiopathogenesis and progression of amyotrophic lateral sclerosis (ALS). To test this hypothesis, this study attempted to categorize functionally relevant genes within the genome-wide expression profile of human ALS skeletal muscle, using microarray technology and gene regulatory network analysis. The correlation network structures significantly change between patients and controls, indicating an increased inter-gene connection in patients compared to controls. The gene network observed in the ALS group seems to reflect the perturbation of muscle homeostasis and metabolic balance occurring in affected individuals. In particular, the network observed in the ALS muscles includes genes (PRKR1A, FOXO1, TRIM32, ACTN3, among others), whose functions connect the sarcomere integrity to mitochondrial oxidative metabolism. Overall, the analytical approach used in this study offer the possibility to observe higher levels of correlation (i.e. common expression trends) among genes, whose function seems to be aberrantly activated during the progression of muscle atrophy.

Identification of Endothelin-1 and NR4A2 as CD133-regulated genes in colon cancer cells

Several in vitro assays have been proposed to identify cancer stem cells (CSCs), including immunophenotyping, sphere assay and side population (SP) assay. CD133 antigen has been proposed as a CSC marker in colon cancer (CC). However, no functional data are available to date and conflicting results have been reported regarding its role as true CSC marker. Here we set out to identify a molecular signature associated with potential CSC. CD133+ cells isolated from the CaCo‐2 CC cell line were analysed by microarray molecular profiling compared to CD133− counterparts. Various differentially expressed genes were identified and the most relevant transcripts found to be over‐expressed in CD133+ cells were evaluated by quantitative RT‐PCR in the CD133+ fractions isolated from several CC cell lines. In the attempt to find a correlation between putative CSCs, isolated by means of CD133 immunophenotyping and the SP approach, we demonstrated a significant enrichment of CD133+ cells within the SP fraction of CC cells, and comparison of the gene expression profiles revealed that Endothelin‐1 (END‐1) and nuclear receptor subfamily 4, group A, member 2 (NR4A2) transcripts are highly expressed in both CD133+ and SP fractions of CC cells. Moreover, depletion of CD133 by siRNA induced a significant attenuation of END‐1 and NR4A2 expression levels in CaCo‐2 cells, while expression of all three molecules decreased during sodium butyrate‐induced differentiation. In conclusion, we have identified a molecular signature associated with potential CSCs and showed for the first time the existence of a functional relationship between CD133, END‐1 and NR4A2 expression in colon cancer cells. Copyright

Genetic advances in craniosynostosis

Craniosynostosis, the premature ossification of one or more skull sutures, is a clinically and genetically heterogeneous congenital anomaly affecting approximately one in 2,500 live births. In most cases, it occurs as an isolated congenital anomaly, that is, nonsyndromic craniosynostosis (NCS), the genetic, and environmental causes of which remain largely unknown. Recent data suggest that, at least some of the midline NCS cases may be explained by two loci inheritance. In approximately 25–30% of patients, craniosynostosis presents as a feature of a genetic syndrome due to chromosomal defects or mutations in genes within interconnected signaling pathways. The aim of this review is to provide a detailed and comprehensive update on the genetic and environmental factors associated with NCS, integrating the scientific findings achieved during the last decade. Focus on the neurodevelopmental, imaging, and treatment aspects of NCS is also provided.

Over-expression of hNGF in adult human olfactory bulb neural stem cells promotes cell growth and oligodendrocytic differentiation.

The adult human olfactory bulb neural stem/progenitor cells (OBNC/PC) are promising candidate for cell-based therapy for traumatic and neurodegenerative insults. Exogenous application of NGF was suggested as a promising therapeutic strategy for traumatic and neurodegenerative diseases, however effective delivery of NGF into the CNS parenchyma is still challenging due mainly to its limited ability to cross the blood–brain barrier, and intolerable side effects if administered into the brain ventricular system. An effective method to ensure delivery of NGF into the parenchyma of CNS is the genetic modification of NSC to overexpress NGF gene. Overexpression of NGF in adult human OBNSC is expected to alter their proliferation and differentiation nature, and thus might enhance their therapeutic potential. In this study, we genetically modified adult human OBNS/PC to overexpress human NGF (hNGF) and green fluorescent protein (GFP) genes to provide insight about the effects of hNGF and GFP genes overexpression in adult human OBNS/PC on their in vitro multipotentiality using DNA microarray, immunophenotyping, and Western blot (WB) protocols. Our analysis revealed that OBNS/PC-GFP and OBNS/PC-GFP-hNGF differentiation is a multifaceted process involving changes in major biological processes as reflected in alteration of the gene expression levels of crucial markers such as cell cycle and survival markers, stemness markers, and differentiation markers. The differentiation of both cell classes was also associated with modulations of key signaling pathways such MAPK signaling pathway, ErbB signaling pathway, and neuroactive ligand-receptor interaction pathway for OBNS/PC-GFP, and axon guidance, calcium channel, voltage-dependent, gamma subunit 7 for OBNS/PC-GFP-hNGF as revealed by GO and KEGG. Differentiated OBNS/PC-GFP-hNGF displayed extensively branched cytoplasmic processes, a significant faster growth rate and up modulated the expression of oligodendroglia precursor cells markers (PDGFRα, NG2 and CNPase) respect to OBNS/PC-GFP counterparts. These findings suggest an enhanced proliferation and oligodendrocytic differentiation potential for OBNS/PC-GFP-hNGF as compared to OBNS/PC-GFP.