Genome-scale reconstructions of the mammalian secretory pathway predict metabolic costs and limitations of protein secretion.

In mammalian cells, >25% of synthesized proteins are exported through the secretory pathway. The pathway complexity, however, obfuscates its impact on the secretion of different proteins. Unraveling its impact on diverse proteins is particularly important for biopharmaceutical production. Here we delineate the core secretory pathway functions and integrate them with genome-scale metabolic reconstructions of human, mouse, and Chinese hamster ovary cells. The resulting reconstructions enable the computation of energetic costs and machinery demands of each secreted protein. By integrating additional omics data, we find that highly secretory cells have adapted to reduce expression and secretion of other expensive host cell proteins. Furthermore, we predict metabolic costs and maximum productivities of biotherapeutic proteins and identify protein features that most significantly impact protein secretion. Finally, the model successfully predicts the increase in secretion of a monoclonal antibody after silencing a highly expressed selection marker. This work represents a knowledgebase of the mammalian secretory pathway that serves as a novel tool for systems biotechnology.

Isolation and prolonged expansion of oral mesenchymal stem cells under clinical-grade, GMP-compliant conditions differentially affects "stemness" properties.

BACKGROUND: Development of clinical-grade cell preparations is central to meeting the regulatory requirements for cellular therapies under good manufacturing practice-compliant (cGMP) conditions. Since addition of animal serum in culture media may compromise safe and efficient expansion of mesenchymal stem cells (MSCs) for clinical use, this study aimed to investigate the potential of two serum/xeno-free, cGMP culture systems to maintain long-term "stemness" of oral MSCs (dental pulp stem cells (DPSCs) and alveolar bone marrow MSCs (aBMMSCs)), compared to conventional serum-based expansion. METHODS: DPSC and aBMMSC cultures (n = 6/cell type) were established from pulp and alveolar osseous biopsies respectively. Three culture systems were used: StemPro_MSC/SFM_XenoFree (Life Technologies); StemMacs_MSC/XF (Miltenyi Biotek); and α-MEM (Life Technologies) with 15% fetal bovine serum. Growth (population doublings (PDs)), immunophenotypic (flow cytometric analysis of MSC markers) and senescence (ß-galactosidase (SA-ß-gal) activity; telomere length) characteristics were determined during prolonged expansion. Gene expression patterns of osteogenic (ALP, BMP-2), adipogenic (LPL, PPAR-γ) and chondrogenic (ACAN, SOX-9) markers and maintenance of multilineage differentiation potential were determined by real-time PCR. RESULTS: Similar isolation efficiency and stable growth dynamics up to passage 10 were observed for DPSCs under all expansion conditions. aBMMSCs showed lower cumulative PDs compared to DPSCs, and when StemMacs was used substantial delays in cell proliferation were noted after passages 6-7. Serum/xeno-free expansion produced cultures with homogeneous spindle-shaped phenotypes, while serum-based expansion preserved differential heterogeneous characteristics of each MSC population. Prolonged expansion of both MSC types but in particular the serum/xeno-free-expanded aBMMSCs was associated with downregulation of CD146, CD105, Stro-1, SSEA-1 and SSEA-4, but not CD90, CD73 and CD49f, in parallel with an increase of SA-gal-positive cells, cell size and granularity and a decrease in telomere length. Expansion under both serum-free systems resulted in "osteogenic pre-disposition", evidenced by upregulation of osteogenic markers and elimination of chondrogenic and adipogenic markers, while serum-based expansion produced only minor changes. DPSCs retained a diminishing (CCM, StemPro) or increasing (StemMacs) mineralization potential with passaging, while aBMMSCs lost this potential after passages 6-7 under all expansion conditions. CONCLUSIONS: These findings indicate there is still a vacant role for development of qualified protocols for clinical-grade expansion of oral MSCs; a key milestone achievement for translation of research from the bench to clinics.

An assessment of the overexpression of BMP-2 in transfected human osteoblast cells stimulated by mineral trioxide aggregate and Biodentine.

AIM: To evaluate the effect of MTA and Biodentine on viability, osteogenic differentiation and BMP-2 expression in osteogenic cells. METHODOLOGY: Saos-2 cells were used as a model of osteoblastic cells. Overexpression of BMP-2 was induced by transfection of a CMV-driven plasmid construct including the human BMP-2 coding sequence, and stably transfected cells were selected. Cell viability was assessed by the mitochondrial dehydrogenase enzymatic (MTT) assay. The bioactivity of the materials was evaluated by the alkaline phosphatase (ALP) assay and detection of calcium deposits with alizarin red staining (ARS). The gene expression of BMP-2 and ALP was quantified with real-time PCR. Statistical analysis was performed with analysis of variance and Bonferroni or Tukey post-test (α = 0.05). RESULTS: Viability tests revealed that MTA and Biodentine were not cytotoxic at the higher dilution (1 : 8) to BMP-2-transfected cells. MTA and Biodentine exhibited the highest ALP activity when compared to the Saos-BMP-2-unexposed control group (P < 0.05). Cell exposure to Biodentine and MTA had a significant stimulatory effect on the formation of mineralized nodules (P < 0.05). The highest increase in BMP-2 gene expression was observed after 3 days of BMP-2-transfected cells exposure to MTA and Biodentine in non-osteogenic medium in relation to Saos-BMP-2-unexposed control cells (P < 0.05). Exposure of cells to MTA in osteogenic medium for 1 day increased ALP gene expression by approximately 1.3-fold in relation to Saos-BMP-2-unexposed control cells (P < 0.05). CONCLUSIONS: Both MTA and Biodentine showed biocompatibility and bioactivity in Saos-BMP-2 overexpressing cells. Biodentine had a significantly greater effect on mineralization than MTA. Both MTA and Biodentine enhanced BMP-2 mRNA expression in the transfected system. Both MTA and Biodentine are suitable materials to improve osteoblastic cell mineralization.

Nanoscale control of surface immobilized BMP-2: toward a quantitative assessment of BMP-mediated signaling events.

In this work we determine the impact of surface density of immobilized BMP-2 on intracellular signal transduction. We use block copolymer micellar nanolithography to fabricate substrates with precisely spaced and tunable gold nanoparticle arrays carrying single BMP-2 molecules. We found that the immobilized growth factor triggers prolonged and elevated Smad signaling pathway activation compared to the same amount of soluble protein. This approach is suitable for achieving controlled and sustained local delivery of BMP-2 and other growth factors.

New insights in osteogenic differentiation revealed by mass spectrometric assessment of phosphorylated substrates in murine skin mesenchymal cells.

BACKGROUND: Bone fractures and loss represent significant costs for the public health system and often affect the patients quality of life, therefore, understanding the molecular basis for bone regeneration is essential. Cytokines, such as IL-6, IL-10 and TNFα, secreted by inflammatory cells at the lesion site, at the very beginning of the repair process, act as chemotactic factors for mesenchymal stem cells, which proliferate and differentiate into osteoblasts through the autocrine and paracrine action of bone morphogenetic proteins (BMPs), mainly BMP-2. Although it is known that BMP-2 binds to ActRI/BMPR and activates the SMAD 1/5/8 downstream effectors, little is known about the intracellular mechanisms participating in osteoblastic differentiation. We assessed differences in the phosphorylation status of different cellular proteins upon BMP-2 osteogenic induction of isolated murine skin mesenchymal stem cells using Triplex Stable Isotope Dimethyl Labeling coupled with LC/MS. RESULTS: From 150 µg of starting material, 2,264 proteins were identified and quantified at five different time points, 235 of which are differentially phosphorylated. Kinase motif analysis showed that several substrates display phosphorylation sites for Casein Kinase, p38, CDK and JNK. Gene ontology analysis showed an increase in biological processes related with signaling and differentiation at early time points after BMP2 induction. Moreover, proteins involved in cytoskeleton rearrangement, Wnt and Ras pathways were found to be differentially phosphorylated during all timepoints studied. CONCLUSIONS: Taken together, these data, allow new insights on the intracellular substrates which are phosphorylated early on during differentiation to BMP2-driven osteoblastic differentiation of skin-derived mesenchymal stem cells.

Assessment of stability of surface anchors for antibacterial coatings and immobilized growth factors on titanium.

Titanium (Ti) has been functionalized with biomolecules for biomedical purposes. However, there is very limited information on the stability of such functionalities. Ti surface functionalized with carboxymethyl chitosan (CMCS) and bone morphogenetic protein 2 (BMP-2) has been reported to inhibit bacterial colonization while at the same time enhances osteoblast functions. In this work, three types of anchoring molecules, (3-aminopropyl) triethoxysilane (Silane), dopamine (DA), and polydopamine (PDA), were used for immobilizing the CMCS on Ti. The CMCS-modified surfaces were subjected to 70% ethanol treatment, autoclaving, and prolonged immersion in phosphate buffered saline (PBS). After the treatment procedures, the ability of the CMCS-modified substrates to inhibit colonization by Staphylococcus epidermidis (S. epidermidis) was assessed to evaluate the stability of the immobilized CMCS. The bacterial adhesion assays showed that the CMCS-DA- and CMCS-PDA-modified Ti remained stable after 70% ethanol treatment, autoclaving, and prolonged immersion in PBS, whereas the CMCS-Silane-modified Ti was less stable after autoclaving and prolonged immersion in PBS. The CMCS-DA- and CMCS-PDA-modified Ti substrates were functionalized with BMP-2 and used to support osteoblast growth. Evaluation of alkaline phosphatase (ALP) activity and calcium deposition from osteoblasts cultured on these substrates, which have been treated with 70% ethanol, or subjected to autoclaving, and prolonged immersion in PBS indicated that the immobilized BMP-2 on these surfaces retained its bioactivity.

Relevance of the sterilization-induced effects on the properties of different hydroxyapatite nanoparticles and assessment of the osteoblastic cell response.

Hydroxyapatite (Hap) is a calcium phosphate with a chemical formula that closely resembles that of the mineral constituents found in hard tissues, thereby explaining its natural biocompatibility and wide biomedical use. Nanostructured Hap materials appear to present a good performance in bone tissue applications because of their ability to mimic the dimensions of bone components. However, bone cell response to individual nanoparticles and/or nanoparticle aggregates lost from these materials is largely unknown and shows great variability. This work addresses the preparation and characterization of two different Hap nanoparticles and their interaction with osteoblastic cells. Hap particles were produced by a wet chemical synthesis (WCS) at 37°C and by hydrothermal synthesis (HS) at 180°C. As the ultimate in vivo applications require a sterilization step, the synthesized particles were characterized 'as prepared' and after sterilization (autoclaving, 120°C, 20 min). WCS and HS particles differ in their morphological (size and shape) and physicochemical properties. The sterilization modified markedly the shape, size and aggregation state of WCS nanoparticles. Both particles were readily internalized by osteoblastic cells by endocytosis, and showed a low intracellular dissolution rate. Concentrations of WCS and HS particles less than 500 µg ml(-1) did not affect cell proliferation, F-actin cytoskeleton organization and apoptosis rate and increased the gene expression of alkaline phosphatase and BMP-2. The two particles presented some differences in the elicited cell response. In conclusion, WCS and HS particles might exhibit an interesting profile for bone tissue applications. Results suggest the relevance of a proper particle characterization, and the interest of an individual nanoparticle targeted research.

Assessment of immunization against recombinant human bone morphogenetic protein-2 (dibotermin alpha) on reproduction and development in rabbits.

BACKGROUND: To determine if the fetus was affected by maternal antibodies to BMP-2, the antibody response and developmental effects in fetuses from does immunized against recombinant human BMP-2 were evaluated. METHODS: Female New Zealand White rabbits received four intramuscular injections (on premating days 1, 8, 22, and 43 [3 days before mating]) of saline and adjuvant (TiterMax(®) Gold [control]) or recombinant human BMP-2 (2 mg/dose) and adjuvant (treated). On GD 29, fetuses were examined, and maternal and fetal anti-BMP-2 titer levels and neutralizing activity were assessed. RESULTS: Anti-BMP-2 antibodies were detected in 17 of 18 treated does (127 of 151 fetuses), and low levels were detected in 2 of 16 control does (no fetal exposure observed). In general, levels of fetal anti-BMP-2 antibodies were similar to those in the does, and pregnancy did not boost the immune response to BMP-2. There were no effects of immunization or anti-BMP-2 antibody titer levels on embryo-fetal viability, fetal weight, or fetal external, visceral, or skeletal development. Only a small number of fetuses (n = 4) displayed detectable neutralizing anti-BMP-2 antibodies, but there were no treatment-related effects in those fetuses. CONCLUSIONS: The lack of embryo-fetal effects may be due to dosage effects of neutralizing anti-BMP-2 antibodies, timing of exposure (stage and duration) to neutralizing anti-BMP-2 antibodies, and/or redundancy of effects of the various BMPs.

[Assessment of the expression profile during the entochondrostosis of vascular endothelial growth factor in bone morphogenetic protein 2 induced osteogenesis].

OBJECTIVES: To examine the gene expression profile of bone morphogenetic protein 2 (BMP-2) and vascular endothelial growth factor (VEGF) during entochondrostosis of mice and explore the expression rules and effects between BMP-2 and VEGF, and to detect the expression of VEGF in BMP-2 induced entochondrostosis in vivo. METHODS: cDNA microarray technique with 34,000 genes was used to analyze the gene expression profiles during entochondrostosis in the limbs of mice embryo from E10 to E14. Pathway analysis of BMP-2 and VEGF was performed with GCOS1.2 software. An experimental model of femoral muscular pouch in 20 mice was adopted. The expression of VEGF was examined by in situ hybridization method and immunohistochemical method in BMP-2 induced entochondrostosis in vivo. RESULTS: The expression signals of VEGF mRNA and VEGF appeared in cytoplasm during condensation of mesenchymal cell. As the mesenchymal cells differentiated into precartilage, the expression signals decreased in mesenchymal cells, but increased in chondrocytes and kept getting denser in the process of cartilage maturity. The peak expression of VEGF mRNA and VEGF in the experimental group appeared on the 14th day, accompanied by numerous hypertrophic chondrocytes. When mature cartilage calcified and new bone trabecula formed, the expression of VEGF mRNA and VEGF decreased in chondrocytes, but still expressed moderately in the osteoblasts and osteocytes. CONCLUSIONS: The finding reveals a complex pattern of gene coexpression of BMP-2 and VEGF during the critical period of entochondrostosis. It's feasible for the clinical application of BMP-2 in orthopedics.