Enhancement of Dopaminergic Differentiation in Proliferating Midbrain Neuroblasts by Sonic Hedgehog and Ascorbic Acid

We analyzed the molecular mechanisms involved in the acquisition and maturation of dopaminergic (DA) neurons generated in vitro from rat ventral mesencephalon (MES) cells in the presence of mitogens or specific signaling molecules. The addition of basic fibroblast growth factor (bFGF) to MES cells in serum-free medium stimulates the proliferation of neuroblasts but delays DA differentiation. Recombinant Sonic hedgehog (SHH) protein increases up to three fold the number of tyrosine hydroxylase (TH)-positive cells and their differentiation, an effect abolished by anti-SHH antibodies. The expanded cultures are rich in nestin-positive neurons, glial cells are rare, all TH+ neurons are DA, and all DA and GABAergic markers analyzed are expressed. Adding ascorbic acid to bFGF/SHH-treated cultures resulted in a further five- to seven-fold enhancement of viable DA neurons. This experimental system also provides a powerful tool to generate DA neurons from single embryos. Our strategy provides an enriched source of MES DA neurons that are useful for analyzing molecular mechanisms controlling their function and for experimental regenerative approaches in DA dysfunction

Transcription factor KLF7 regulates differentiation of neuroectodermal and mesodermal cell lineages

Previous gene targeting studies in mice have implicated the nuclear protein Krüppel-like factor 7 (KLF7) in nervous system development while cell culture assays have documented its involvement in cell cycle regulation. By employing short hairpin RNA (shRNA)-mediated gene silencing, here we demonstrate that murine Klf7 gene expression is required for in vitro differentiation of neuroectodermal and mesodermal cells. Specifically, we show a correlation of Klf7 silencing with down-regulation of the neuronal marker microtubule-associated protein 2 (Map2) and the nerve growth factor (NGF) tyrosine kinase receptor A (TrkA) using the PC12 neuronal cell line. Similarly, KLF7 inactivation in Klf7-null mice decreases the expression of the neurogenic marker brain lipid-binding protein/fatty acid-binding protein 7 (BLBP/FABP7) in neural stem cells (NSCs). We also report that Klf7 silencing is detrimental to neuronal and cardiomyocytic differentiation of embryonic stem cells (ESCs), in addition to altering the adipogenic and osteogenic potential of mouse embryonic fibroblasts (MEFs). Finally, our results suggest that genes that are key for self-renewal of undifferentiated ESCs repress Klf7 expression in ESCs. Together with previous findings, these results provide evidence that KLF7 has a broad spectrum of regulatory functions, which reflect the discrete cellular and molecular contexts in which this transcription factor operates. © 2010 Elsevier Inc

Lmx1a-Dependent Activation of miR-204/211 Controls the Timing of Nurr1-Mediated Dopaminergic Differentiation

The development of midbrain dopaminergic (DA) neurons requires a fine temporal and spatial regulation of a very specific gene expression program. Here, we report that during mouse brain development, the microRNA (miR-) 204/211 is present at a high level in a subset of DA precursors expressing the transcription factor Lmx1a, an early determinant for DA-commitment, but not in more mature neurons expressing Th or Pitx3. By combining different in vitro model systems of DA differentiation, we show that the levels of Lmx1a influence the expression of miR-204/211. Using published transcriptomic data, we found a significant enrichment of miR-204/211 target genes in midbrain dopaminergic neurons where Lmx1a was selectively deleted at embryonic stages. We further demonstrated that miR-204/211 controls the timing of the DA differentiation by directly downregulating the expression of Nurr1, a late DA differentiation master gene. Thus, our data indicate the Lmx1a-miR-204/211-Nurr1 axis as a key component in the cascade of events that ultimately lead to mature midbrain dopaminergic neurons differentiation and point to miR-204/211 as the molecular switch regulating the timing of Nurr1 expression

Lmx1a-Dependent Activation of miR-204/211 Controls the Timing of Nurr1-Mediated Dopaminergic Differentiation

The development of midbrain dopaminergic (DA) neurons requires a fine temporal and spatial regulation of a very specific gene expression program. Here, we report that during mouse brain development, the microRNA (miR-) 204/211 is present at a high level in a subset of DA precursors expressing the transcription factor Lmx1a, an early determinant for DA-commitment, but not in more mature neurons expressing Th or Pitx3. By combining different in vitro model systems of DA differentiation, we show that the levels of Lmx1a influence the expression of miR-204/211. Using published transcriptomic data, we found a significant enrichment of miR-204/211 target genes in midbrain dopaminergic neurons where Lmx1a was selectively deleted at embryonic stages. We further demonstrated that miR-204/211 controls the timing of the DA differentiation by directly downregulating the expression of Nurr1, a late DA differentiation master gene. Thus, our data indicate the Lmx1a-miR-204/211-Nurr1 axis as a key component in the cascade of events that ultimately lead to mature midbrain dopaminergic neurons differentiation and point to miR-204/211 as the molecular switch regulating the timing of Nurr1 expression

miR-34b/c Regulates Wnt1 and Enhances Mesencephalic Dopaminergic Neuron Differentiation

The differentiation of dopaminergic neurons requires concerted action of morphogens and transcription factors acting in a precise and well-defined time window. Very little is known about the potential role of microRNA in these events. By performing a microRNA-mRNA paired microarray screening, we identified miR-34b/c among the most upregulated microRNAs during dopaminergic differentiation. Interestingly, miR-34b/c modulates Wnt1 expression, promotes cell cycle exit, and induces dopaminergic differentiation. When combined with transcription factors ASCL1 and NURR1, miR-34b/c doubled the yield of transdifferentiated fibroblasts into dopaminergic neurons. Induced dopaminergic (iDA) cells synthesize dopamine and show spontaneous electrical activity, reversibly blocked by tetrodotoxin, consistent with the electrophysiological properties featured by brain dopaminergic neurons. Our findings point to a role for miR-34b/c in neuronal commitment and highlight the potential of exploiting its synergy with key transcription factors in enhancing in vitro generation of dopaminergic neurons.Peer reviewe

Induction of immune response after SARS-CoV-2 mRNA BNT162b2 vaccination in healthcare workers

[no abstract available

Biodegradable polymeric systems for controlled release of growth factors in regenerative medicine

With advances in biotechnology, bioengineering, and chemistry, a wide variety of new, more potent and specific active molecules are being created. Because of common problems such as low solubility, high potency, and/or poor stability of many of these new molecules, the means of drug delivery can impact efficacy and potential for commercialization as much as the nature of the molecule itself. the controlled release systems (DDS’s) are one of the most promising technologies to solve these problems because offer several potential advantages over traditional methods of administration. First, drug release rates can be tailored to the needs of a specific application; for example, providing a constant rate of delivery or pulsatile release. Second, DDS provide protection of drugs, especially proteins, that are otherwise rapidly destroyed by the body. Finally, DDS can increase patient comfort and compliance by replacing frequent (e.g., daily) doses with infrequent (once per month or less) injection. the last twenty years the DDS in addition to being used for the release of drugs are also used in the field of tissue engineering for controlled release of specific molecules, such as growth factors or cytokines. In the first chapter of this thesis the drug delivery systems have been investigate for the control of the concentration of signal molecules within tridimensional scaffold for the induction of angiogenesis in bone regeneration processes. To achieve a good bone tissue regeneration is essential to have a three dimensional structure, called scaffold, with specific physical and mechanical properties, such as high porosity, high interconnected pore network, high biocompatibility but especially capable of releasing in a controlled way bioactive molecules such as proangiogenic factors (VEGF). For this reason we have developed a bottom up approach founded on the assembly of building blocks by solvent induced microparticle sintering to realize multifunctional polymer scaffolds with predefined pore dimension and fully percolative pathway, able to include interspersing DDS for the release of angiogenic factor or similar molecules. This approach offers the possibility to realize scaffolds that not only meet all the requirements in terms of controlled microstructure, chemical stability and mechanical response necessary to support neo-tissue growth, but also provide a guidance of cell and tissue processes by presenting bioactive agents in a predefined chrono-programmed manner. In the second chapter of this thesis has been developed intraocular drug delivery systems for the controlled release of growth factors for the treatment of different ocular diseases. The traditional treatment of these pathologies consists of daily administration of drugs but, the main limit of these conventional treatments is bound to the fact that blood levels of the drug falling into the therapeutic range are obtained right after administration. In other words, there is a risk that the drug action lasts too little to have a significant effect on the patient. To overcome this issues and to optimize the clinical performance we have developed PLGA micro e nanoparticles system for the controlled release of specific factor: Nerve Growth Factor (NGF) to humor vitreous. This factor has been shown to have a protective effect on the cells of the visual system, opening interesting therapeutic prospects for degenerative diseases currently no effective treatments, such as the age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa and glaucoma

Determinants of Normalized Bohemian Upper Hessemberg Matrices

A matrix is Bohemian if its elements are taken from a finite set of integers. We enumerate all possible determinants of Bohemian upper Hessenberg matrices with subdiagonal fixed to one, and consider the special case of families of matrices with only zeros on the main diagonal, whose determinants proved to be related to a generalization of Fibonacci numbers. Several conjectures recently stated by Corless and Thornton follow from our results