University of Coimbra

8 ARTICLES PUBLISHED IN JoVE

Developmental Biology

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

Michael G. Daniel^1,2,3, Carlos-Filipe Pereira⁴, Jeffrey M. Bernitz^1,2,3, Ihor R. Lemischka^1,3,5, Kateri Moore^1,3

¹Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, ²The Graduate School of Biomedical Science, Icahn School of Medicine at Mount Sinai, ³Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, ⁴Center for Neuroscience and Cell Biology, University of Coimbra, ⁵Department of Pharmacology and Systems Therapeutics, Icahn School of Medicine at Mount Sinai

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

Biochemistry

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Xavier Morató^*1,2, Marc López-Cano^*1,2, Paula M. Canas³, Rodrigo A. Cunha³, Francisco Ciruela^1,2

¹Unitat de Farmacologia, Departament Patologia i Terapèutica Experimental, Facultat de Medicina, IDIBELL, Universitat de Barcelona, L'Hospitalet de Llobregat, ²Institut de Neurociències, Universitat de Barcelona, ³Center for Neurosciences of Coimbra, Institute of Biochemistry, Faculty of Medicine, University of Coimbra

Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments.

Cancer Research

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Mafalda Laranjo^*1,2,3,4, Maria João Carvalho^*1,2,3,4,5,6, Beatriz Serambeque^1,2,3, André Alves^2,7, Carlos Miguel Marto^1,2,3,4,8, Isabel Silva⁹, Artur Paiva^3,9,10, Maria Filomena Botelho^1,2,3,4,8

¹Institute of Biophysics, Faculty of Medicine, University of Coimbra, ²Coimbra Institute for Clinical and Biomedical Research (iCBR), area of Environment Genetics and Oncobiology (CIMAGO), Faculty of Medicine, University of Coimbra, ³CNC.IBILI/Center for Innovative Biomedicine and Biotechnology (CIBB), University of Coimbra, ⁴Clinical Academic Center of Coimbra (CACC), ⁵Universitary Clinic of Gynecology, Faculty of Medicine, University of Coimbra, ⁶Gynecology A Service, Coimbra Hospital and Universitary Center, ⁷Institute of Pharmacology & Experimental Therapeutics, Faculty of Medicine, University of Coimbra, ⁸Institute of Experimental Pathology, Faculty of Medicine, University of Coimbra, ⁹Cytometry Operational Management Unit, Clinical Pathology Service, Coimbra Hospital and Universitary Center, ¹⁰Polytechnic Institute of Coimbra, ESTESC-Coimbra Health School, Laboratory Biomedical Sciences

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

Developmental Biology

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

Rita Silvério-Alves^1,2,3, Andreia M. Gomes³, Ilia Kurochkin⁴, Kateri A. Moore^5,6, Carlos-Filipe Pereira^1,2,3

¹Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, ²Wallenberg Center for Molecular, Lund University, ³Center for Neuroscience and Cell Biology, University of Coimbra, ⁴Skolkovo Institute of Science and Technology, ⁵Department of Cell, Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, ⁶Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai

This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells.

Neuroscience

Isolation and Culture of Chick Ciliary Ganglion Neurons

Filipa J. Costa^*1, Marta S. Dias^*1, Rui O. Costa², Joana R. Pedro², Ramiro D. Almeida^1,2

¹Institute of Biomedicine, Department of Medical Sciences - iBiMED, University of Aveiro, ²CNC - Center for Neuroscience and Cell Biology, University of Coimbra

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system. Neuronal cultures of chick CG neurons were shown to be effective cell models in the study of nerve muscle interactions. We describe a detailed protocol for the dissection, dissociation and in vitro culture of CG neurons from chick embryos.

Biochemistry

Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment

Karina Garcia^1,2,3,4, André Alves^1,2,3, Teresa M. Ribeiro-Rodrigues^1,2,3, Flávio Reis^1,2,3, Sofia Viana^1,2,3,4

¹Institute of Pharmacology & Experimental Therapeutics & Coimbra Institute for Clinical and Biomedical Research (iCBR), Faculty of Medicine, University of Coimbra, ²Center for Innovative Biomedicine and Biotechnology (CIBB), University of Coimbra, ³Clinical Academic Center of Coimbra (CACC), ⁴Polytechnic Institute of Coimbra, Coimbra Health School

Herein, we demonstrate an optimized BODIPY 493/503 fluorescence-based protocol for lipid droplet characterization in liver tissue. Through the use of orthogonal projections and 3D reconstructions, the fluorophore allows for successful discrimination between microvesicular and macrovesicular steatosis and may represent a complementary approach to the classical histological protocols for hepatic steatosis assessment.

Biology

Valorization of the Red Seaweed Gracilaria gracilis Through a Biorefinery Approach

Alice Martins¹, Filipa R Pinto¹, Sónia Barroso¹, Tatiana Pereira¹, Teresa Mouga¹, Clélia Afonso¹, Marta V Freitas^1,2, Susete Pinteus¹, Rui Pedrosa¹, Maria M Gil¹

¹MARE-Marine and Environmental Sciences Centre/ARNET-Aquatic Research Network, ESTM, Polytechnic University of Leiria, ²MARE-Marine and Environmental Sciences Centre/ARNET-Aquatic Research Network, Department of Life Sciences, University of Coimbra

Here, we describe several protocols aiming at an integrated valorization of Gracilaria gracilis: wild species harvesting, in-house growth, and extraction of bioactive ingredients. The extracts' antioxidant, antimicrobial, and cytotoxic effects are evaluated, along with the nutritional and stability assessment of food enriched with whole seaweed biomass and pigments.

Bioengineering

Isolation of Adeno-Associated Viral Vectors Through a Single-Step and Semi-Automated Heparin Affinity Chromatography Protocol

Miguel M. Lopes^*1,2,3, Sara M. Lopes^*1,2,3, Rafael Baganha^1,2,4, Carina Henriques^1,2,5, Ana C. Silva^1,2,3, Diana D. Lobo^1,2,3, Luísa Cortes^1,2,3,6, Luís Pereira de Almeida^*1,2,4,5, Rui Jorge Nobre^*1,2,3,4

¹CNC-UC - Center for Neuroscience and Cell Biology, University of Coimbra, ²CIBB - Center for Innovative Biomedicine and Biotechnology, University of Coimbra, ³IIIUC - Institute for Interdisciplinary Research, University of Coimbra, ⁴ViraVector - Viral Vectors for Gene Transfer Core Facility, University of Coimbra, ⁵FFUC - Faculty of Pharmacy, University of Coimbra, ⁶MICC-CNC - Microscopy Imaging Center of Coimbra - CNC, University of Coimbra

This manuscript describes a detailed protocol for the generation and purification of adeno-associated viral vectors using an optimized heparin-based affinity chromatography method. It presents a simple, scalable, and cost-effective approach, eliminating the need for ultracentrifugation. The resulting vectors exhibit high purity and biological activity, proving their value in preclinical studies.