Asterios S. Tsiftsoglou

Comparative analysis of in vitro osteo/odontogenic differentiation potential of human dental pulp stem cells (DPSCs) and stem cells from the apical papilla (SCAP).

OBJECTIVE The aim of this study was to compare the in vitro osteo/odontogenic differentiation potential of mesenchymal stem cells (MSCs) derived from the dental pulp (dental pulp stem cells - DPSCs) or the apical papilla (stem cells from the apical papilla - SCAP) of permanent developing teeth. DESIGN DPSCs and SCAP cultures were established from impacted third molars of young healthy donors at the stage of root development. Cultures were analysed for stem cell markers, including STRO-1, CD146, CD34 and CD45 using flow cytometry. Cells were then induced for osteo/odontogenic differentiation by media containing dexamethasone, KH(2)PO(4) and β-glycerophosphate. Cultures were analysed for morphology, growth characteristics, mineralization potential (Alizarin Red method) and differentiation markers (dentine sialophosphoprotein-DSPP, bone sialoprotein-BSP, osteocalcin-OCN, alkaline phosphatase-ALP), using immunocytochemistry and reverse transcriptase-polymerase chain reaction. RESULTS All DPSCs and SCAP cultures were positive for STRO-1, CD146 and CD34, in percentages varying according to cell type and donor, but negative for CD45. Both types of MSCs displayed an active potential for cellular migration, organization and mineralization, producing 3D mineralized structures. These structures progressively expressed differentiation markers, including DSPP, BSP, OCN, ALP, having the characteristics of osteodentin. SCAP, however, showed a significantly higher proliferation rate and mineralization potential, which might be of significance for their use in bone/dental tissue engineering. CONCLUSIONS This study provides evidence that different types of dental MSCs can be used in tissue engineering/regeneration protocols as an approachable stem cell source for osteo/odontogenic differentiation and biomineralization that could be further applied for stem cell-based clinical therapies.

Erythropoiesis: Model systems, molecular regulators, and developmental programs

Human erythropoiesis is a complex multistep developmental process that begins at the level of pluripotent hematopoietic stem cells (HSCs) at bone marrow microenvironment (HSCs niche) and terminates with the production of erythrocytes (RBCs). This review covers the basic and contemporary aspects of erythropoiesis. These include the: (a) cell‐lineage restricted pathways of differentiation originated from HSCs and going downward toward the blood cell development; (b) model systems employed to study erythropoiesis in culture (erythroleukemia cell lines and embryonic stem cells) and in vivo (knockout animals: avian, mice, zebrafish, and xenopus); (c) key regulators of erythropoiesis (iron, hypoxia, stress, and growth factors); (d) signaling pathways operating at hematopoietic stem cell niche for homeostatic regulation of self renewal (SCF/c‐kit receptor, Wnt, Notch, and Hox) and for erythroid differentiation (HIF and EpoR). Furthermore, this review presents the mechanisms through which transcriptional factors (GATA‐1, FOG‐1, TAL‐1/SCL/MO2/Ldb1/E2A, EKLF, Gfi‐1b, and BCL11A) and miRNAs regulate gene pattern expression during erythroid differentiation. New insights regarding the transcriptional regulation of α‐ and β‐globin gene clusters were also presented. Emphasis was also given on (i) the developmental program of erythropoiesis, which consists of commitment to terminal erythroid maturation and hemoglobin production, (two closely coordinated events of erythropoieis) and (ii) the capacity of human embryonic and umbilical cord blood (UCB) stem cells to differentiate and produce RBCs in culture with highly selective media. These most recent developments will eventually permit customized red blood cell production needed for transfusion.

Challenges with advanced therapy medicinal products and how to meet them

Advanced therapy medicinal products (ATMPs), which include gene therapy medicinal products, somatic cell therapy medicinal products and tissue-engineered products, are at the cutting edge of innovation and offer a major hope for various diseases for which there are limited or no therapeutic options. They have therefore been subject to considerable interest and debate. Following the European regulation on ATMPs, a consolidated regulatory framework for these innovative medicines has recently been established. Central to this framework is the Committee for Advanced Therapies (CAT) at the European Medicines Agency (EMA), comprising a multidisciplinary scientific expert committee, representing all EU member states and European Free Trade Association countries, as well as patient and medical associations. In this article, the CAT discusses some of the typical issues raised by developers of ATMPs, and highlights the opportunities for such companies and research groups to approach the EMA and the CAT as a regulatory advisor during development.

Development and Regulation of Biosimilars: Current Status and Future Challenges

Biologic medicinal products developed via rDNA technology as recombinant protein-based medicines that have been in clinical use since the early 1980s as original biopharmaceuticals have greatly contributed to the therapy of severe metabolic and degenerative diseases. The recent expiration of the data protection or patents for most of them created opportunities for the development of copy versions of original biopharmaceuticals with similar biologic activity (termed biosimilars). Production of these new products is expected to meet worldwide demand, promote market competition, maintain the incentives for innovation, and sustain the healthcare systems. The licencing of these products, however, relies on the experience gained with the original biopharmaceuticals. Critical issues related to this class of medicinal products include their terminology (to avoid confusion with generics and non-innovator copy versions that have not been tested according to the biosimilar guidelines), manufacturing, and regulation. The European Union (EU) has been the first to establish a regulatory framework for marketing authorization application (MAA) and has named these products biosimilars, a term also recently adopted by the US FDA. Unlike the conventional, more common small molecular weight human medicines and chemical generics, protein-based medicines exhibit higher molecular weight, complexity in structure and function that can be affected by changes in the manufacturing process. Therefore, biosimilars represent a relatively heterogeneous class of medicinal products that make their regulation quite challenging. According to the current understanding in the EU, a biosimilar is a copy version of an already authorized biopharmaceutical (or reference product) with similar biologic activity, physicochemical characteristics, efficacy, and safety, based on a full comparability exercise at quality, preclinical and clinical level to ensure similar efficacy and safety. Guidance has been provided through several Committee for Medicinal Products for Human Use (CHMP) guidelines as well as individual scientific advice requested from the European Medicines Agency (EMA) by various companies for the development and regulation of biosimilars. This review is mainly focused on the current status of regulation of biosimilars in the EU as well as on future challenges lying ahead for the improvement of the requirements needed for the marketing authorization of biosimilars. Emphasis is given on the quality requirements concerning these medicinal products (biologics).

Structural and Functional Impairment of Mitochondria in Adriamycin-Induced Cardiomyopathy in Mice: Suppression of Cytochrome c Oxidase II Gene Expression

The use of adriamycin (ADR) in cancer chemotherapy has been limited due to its cumulative cardiovascular toxicity. Earlier observations that ADR interacts with mitochondrial cytochrome c oxidase (COX) and suppresses its enzyme activity led us to investigate ADRs action on the cardiovascular functions and heart mitochondrial morphology in Balb-c mice i.p. treated with ADR for several weeks. At various times during treatment, the animals were assessed for cardiovascular functions by electrocardiography and for heart tissue damage by electron microscopy. In parallel, total RNA was extracted from samples of dissected heart and analyzed by Northern blot hybridization to determine the steady-state level of three RNA transcripts encoded by the COXII, COXIII, and COXIV genes. Similarly, samples obtained from the liver of the same animals were analyzed for comparative studies. Our results indicated that 1) treatment of mice with ADR caused cardiovascular arrhythmias characterized by bradycardia, extension of ventricular depolarization time (tQRS), and failure of QRS at high concentrations (10-14 mg/kg body weight cumulative dose); 2) the heart mitochondria underwent swelling, fusion, dissolution, and/or disruption of mitochondrial cristae after several weeks of treatment. Such abnormalities were not observed in the mitochondria of liver tissue; and 3) among the three genes of COX enzyme examined, only COXII gene expression was suppressed by ADR treatment, mainly after 8 weeks in both heart and liver. Knowing that heart mitochondria represent almost 40% of heart muscle by weight, we conclude that the deteriorating effects of ADR on cardiovascular function involve mitochondrial structural and functional impairment.

Effects of HEMA and TEDGMA on the in vitro odontogenic differentiation potential of human pulp stem/progenitor cells derived from deciduous teeth

OBJECTIVES The aim of this study was to investigate the effects of HEMA and TEGDMA on the odontogenic differentiation potential of dental pulp stem/progenitor cells. METHODS Dental stem/progenitor cell cultures were established from pulp biopsies of human deciduous teeth of 1-3 year-old children (Deciduous Teeth Stem Cells-DTSCs). Cultures were characterized for stem cell markers, including STRO-1, CD146, CD34, CD45 using flow cytometry. Cytotoxicity was evaluated with the MTT assay. DTSCs were then induced for osteo/odontogenic differentiation by media containing dexamethasone, KH(2)PO(4),β-glycerophosphate and L-ascorbic acid phosphate in the presence of nontoxic concentrations of HEMA (0.05-0.5mM) and TEGDMA (0.05-0.25mM) for 3 weeks. Additionally, the effects of a single exposure (72 h) to higher concentrations of HEMA (2mM) and TEGDMA (1mM) were also evaluated. RESULTS DTSCs cultures were positive for STRO-1 (7.53±2.5%), CD146 (91.79±5.41%), CD34 (11.87±3.02%) and negative for CD45. In the absence of monomers cell migration, differentiation and production of mineralized dentin-like structures could be observed. Cells also progressively expressed differentiation markers, including dentin sialophosphoprotein-DSPP, bone sialoprotein-BSP, osteocalcin-OCN and alkaline phosphatase-ALP. On the contrary, long-term exposure to nontoxic concentrations of HEMA and TEGDMA significantly delayed the differentiation and mineralization processes of DTSCs, whereas, one time exposure to higher concentrations of these monomers almost completed inhibited mineral nodule formation. BSP, OCN, ALP and DSPP expression were also significantly down-regulated. SIGNIFICANCE These findings suggest that HEMA and TEGDMA can severely disturb the odontogenic differentiation potential of pulp stem/progenitor cells, which might have significant consequences for pulp tissue homeostasis and repair.

Protein secretion biotechnology using Streptomyces lividans: Large-scale production of functional trimeric tumor necrosis factor α

We evaluated the feasibility of large-scale production of biopharmaceuticals expressed as heterologous polypeptides from the Gram-positive bacterium Streptomyces lividans. As a model protein we used murine tumor necrosis factor alpha (mTNFalpha). mTNFalpha fused C-terminally to the secretory signal peptide of the subtilisin-inhibitor protein from Streptomyces venezuelae. Under appropriate fermentation conditions, significant amounts of mature mTNFalpha (80-120 mg/L) can be recovered from spent growth media. Efficient downstream processing allowing rapid purification of mTNFalpha from culture supernatants was developed. Importantly, the protein is recovered from the spent growth medium in its native trimeric state as judged by biophysical analysis. Further, mTNFalpha secreted by S. lividans is significantly more active in an in vitro apoptosis tissue culture assay than a corresponding polypeptide produced in Escherichia coli. This pilot study provides the first validation of S. lividans protein secretion as an alternative bioprocess for large-scale production of oligomeric proteins of potential therapeutic value.

Effects of hemin on apoptosis, suppression of cytochrome c oxidase gene expression, and bone-marrow toxicity induced by doxorubicin (adriamycin).

We have shown that hemin (iron-protoporphyrin IX) selectively counteracts doxorubicin (Adriamycin, ADR)-induced cytotoxicity on human leukemia K-562 cells by preventing ADR from inhibiting mitochondrial cytochrome c oxidase (COX), a novel target site for anthracyclines. Here, we investigated whether or not (a) treatment with ADR promotes apoptosis and represses the expression of two COX genes (one nuclear and one mitochondrial) in human K-562 cells in the absence and presence of hemin, and (b) injection of hemin preserves bone-marrow cellularity in ADR-myelosuppressed rats. Cultured K-562 cells were incubated with varying concentrations of ADR.HCl (0.2 microM to 5 microM) in the presence and absence of hemin (30 microM) and assessed for DNA degradation, as well as for expression of mitochondrial COXII and nuclear COXIV genes by RNA Northern blot hybridization analysis. In parallel, we investigated whether or not hemin injected i.p. in myelosuppressed rats affected ADR-induced bone-marrow cytotoxicity. These studies have shown the following: (a) ADR caused a dose- and time-dependent DNA fragmentation, characteristic of apoptosis, in K-562 cells; (b) hemin reduced the frequency of cell death caused by ADR: this effect was specific for ADR, because hemin failed to prevent apoptosis induced by methotrexate (MTX) in these cells; (c) ADR suppressed expression of COXIV and COXII genes, and exposure of ADR-treated K-562 cells to hemin did not reverse this suppression; and (d) i.p. injection of hemin in ADR-myelosuppressed rats improved bone-marrow cellularity, promoted colony formation (CFU-GM and CFU-F), and stromal cell outgrowth; moreover, hemin increased WBC counts depressed 12 days after ADR treatment. These studies indicate that hemin is a selective inhibitor of ADR-induced apoptosis of human leukemia cells and preserves bone-marrow cellularity in rats injected with ADR.

In support of the European Union biosimilar framework

745 studies unnecessary. Using this approach, the developer may avoid repeating timeconsuming and costly parts of product development, as well as undue clinical testing. Furthermore, in the situation where slight clinical differences are observed in the pivotal clinical comparative study, a comprehensive comparability exercise, including analyses of relevant physicochemical and biological attributes (including functional assays), may provide valuable arguments that the (slight) clinical differences observed are compatible with the inherent variability of both the reference medicinal product and the biosimilar and not attributable to a ‘real’ difference. As such, this first part of the comparability exercise will potentially become more important for more complex biosimilars. Schellekens and Moors mention a failed biosimilar version of interferon-a-2a; indeed, the European Public Assessment Report3 delineates that differences in the clinical outcome counting against the product were not counterbalanced by a sufficiently firm reassurance on analytical, physicochemical and biological grounds supporting biosimilarity. Conducting a comparability exercise starting at the quality level is important during early development for a new biosimilar candidate. If relevant differences are detected at an early stage, a developer could reconsider the feasibility and applicability of the biosimilar route, as compared with a stand-alone development program, which requires an entirely different development strategy. Indeed, if relevant differences to the reference product are detected at the quality level, the reduced nonclinical and clinical development program, as described in the current Committee for Medicinal Products for Human Use (CHMP) guidelines4, may no longer be sufficient to ensure the safety and efficacy of the new candidate, and in some cases, such differences will even disqualify the molecule from entering the biosimilar route. Such knowledge and understanding of the candidate product could facilitate a go-no-go decision in early development In support of the European Union biosimilar framework

Multilevel targeting of hematopoietic stem cell self-renewal, differentiation and apoptosis for leukemia therapy

Human leukemias are considered clonal hematological malignancies initiated by chromosomal aberrations or epigenetic alterations occurring at the level of either pluripotent hematopoietic stem cells (HSCs) or early multipotent progenitors (MPPs). Leukemic cells are transformed, immortalized, actively proliferating cells that are still able to differentiate into cells resembling mature blood cells. Future therapies of leukemias require identification of molecular targets involved in hematopoiesis under normal and leukemic conditions and detailed understanding of the interactions between normal hematopoietic and leukemic cells within the bone marrow micro-environment. This review presents the basic aspects of hematopoiesis and highlights multilevel exploitable targets for leukemia therapy. These include HSC niche components, signaling pathways (SCF/c-kit-R, EPO-R-JAK2/STAT, Wnt, Notch, HOX), inducer-receptor interactions, superfine chromatin structure modifications, fused transcription factors, microRNAs and signaling of cell death through the Bcl-2 apoptotic switch (BH3-only proteins). The classes of therapeutics developed or being under development to eradicate human leukemias include novel antimetabolites, DNA hypomethylating agents, histone deacetylation inhibitors (HDACIs), retinoids and other inducers of differentiation, targeted monoclonal antibodies raised against cell surface proteins, pro-apoptotic receptor agonists (PARAs), BH3 peptidomimetics, cell cycle inhibitors, siRNAs and perhaps microRNAs. Some of these agents induce terminal differentiation while others promote cell cycle arrest and apoptosis in leukemia cells. At last but not least, this article describes the mechanisms of removal of damaged/harmful cells from organs since impairment in clearance of such cells can lead to autoimmune disorders by self-antigens.