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Focus Area Development and Manufacturing of Cellular & Molecular Therapies

Modulation of the Stem Cell Niche Lab

PI: Cláudia Lobato da Silva

Deciphering the molecular and cellular crosstalk in the stem cell niche during tissue homeostasis and regeneration

Human hematopoietic stem/progenitor cells (HSPC) reside in the bone marrow (BM), which is a very complex tissue comprising various cell types namely mesenchymal stromal cells (MSC) along with their secreted cytokines, extracellular matrix (ECM) molecules and extracellular vesicles (EVs). A serum-free co-culture model previously established to expand/maintain human HSPC is being used to unveil the mechanisms underlying the hematopoietic supportive capacity of MSC obtained from healthy donors. In addition, cells from patients with idiopathic aplastic anemia will be used as a model system of a defective BM microenvironment. One important focus is on the instructive role of the MSC-derived extracellular matrix (ECM), by performing a comprehensive characterization of the hematopoietic supportive capacity of decellularized ECM (dECM). Moreover, MSC-derived EVs (including exosomes) retrieved from cultures of MSC and HSPC-MSC co-cultures are being thoroughly studied. Both MSC-derived dECM and EVs are expected to be (i) promising cell-free therapy candidates, as co-adjuvants in hematopoietic cell transplantation, and (ii) important BM niche-mimetics to support the ex-vivo expansion/maintenance of transplantable HSPC.
Also, with a particular focus on the ECM, novel bioengineering strategies are being developed to regenerate periodontal tissue by exploring compartmentalized systems to meet the different characteristics of the tissues involved in periodontal defects. dECM will be used to recreate the different niches of periodontium providing an opportunity to harness the properties of periodontal tissue ECM, by establishing a local niche at the tooth interface that can promote periodontal regeneration, particularly relevant for osteoporotic and older patients with compromised ECM.

Recapitulating niche cues to boost the potential therapeutic features of human stem/progenitor cells and their secretome

Engineering strategies are being developed to boost the therapeutic features of human mesenchymal stromal cells (MSC) and their secretome (i.e. extracellular vesicles (EVs) as exosomes) and secreted proteins) using cells obtained from different tissue sources (bone marrow, adipose tissue and umbilical cord matrix) through ex-vivo culture preconditioning recapitulating important niche cues (i.e. low oxygen, 3D, shear stress). One of the main focus has been on improving the angiogenic potential of these cells and their derived secretome (i.e. for therapeutic angiogenesis).
Another focus is on contributing to a better understanding of the potential therapeutic role of human MSC in cancer settings (i) as cell-based delivery systems of anti-cancer proteins (ii) by employing a cell-free approach, the use of MSC-derived EVs as drug-delivery-systems (DDS) of anti-cancer agents (e.g. p28 peptide). This work is being developed in collaboration with BSRG at iBB, where several cancer cell lines and 3D cancer cell models are available to study the interaction between MSC and tumour cells.
Finally, niche mimicry platforms are being established to support the ex-vivo proliferation of human skeletal muscle stem/progenitor cells and smooth muscle cells without losing their regenerative potential for Tissue Engineering applications targeting diseases affecting the muscular system (e.g. incontinence).

Transient gene control of the angiogenic properties of mesenchymal stromal cells and derived extracellular vesicles

The regulation of gene expression by transcription activation and/or inactivation of specific genes allows to transiently modify the properties of human mesenchymal stromal cells (MSC) towards the expansion and differentiation of these cells, but also to improve their intrinsic therapeutic features for applications in Cellular and Molecular Therapies, as well as Tissue Engineering. The target genes and associated regulatory sequences related to specific signalling pathways are delivered to cells by using minicircles, which are non-integrative, efficient and safe vectors. In this context, an ex-vivo gene therapy strategy is envisaged to transiently engineer MSC by transfection with rationally custom-designed minicircles encoding regulatory genes under control of specific sequences and complexed with nano/microparticles, which protect from nucleases attack and allow efficient trafficking to the nucleus. The research focus is to express or silence (RNAi/shRNA) genes involved in the control of the angiogenic properties of both MSC and derived extracellular vesicles towards anti-angiogenic or pro-angiogenic therapies.


PhD holders

Cláudia Lobato da Silva, Deciphering the molecular and cellular crosstalk in the niche towards the ex-vivo expansion of stem/progenitor cells (Keywords: Stem cell niche, Cellular Therapies, Secretome, Extracellular matrix, Bioreactors)

Gabriel A. Monteiro, Transient gene control of the angiogenic properties of mesenchymal stromal cells and derived extracellular vesicles (Keywords: Gene Therapy, Gene expression, Minicircles, RNAi, shRNA, Extracellular vesicles)

Marta Monteiro Silva Carvalho, Decellularized extracellular matrix as a biomaterial strategy for periodontal tissue regeneration (Keywords: Biomimetic Scaffolds, Periodontal tissue, Stem cell niche, Dental and mesenchymal stromal cells, Extracellular matrix)

Ana Luísa Pécurto Cartaxo, Bioreactor-based culture of skeletal muscle cells attached to implantable biodegradable microcarriers for fecal incontinence treatment (Keywords: Bioreactors, Incontinence, Microcarriers, Skeletal muscle cells)

PhD students

Marília Maria Rodrigues da Silva, Genome engineering of mesenchymal stromal cells to express the anti-cancer protein azurin (Keywords: Mesenchymal stromal cells, Gene Delivery, Cancer)

Miguel de Almeida Fuzeta, Scalable production of mesenchymal stromal cell-derived extracellular vesicles and their application as drug delivery systems for cancer therapy (Keywords: Extracellular vesicles, Bioreactors, Drug delivery systems)

Sara Ventura Bucar, Bioengineering of the thymic microenvironment towards the scalable ex-vivo generation of functional human T cells from umbilical cord blood stem/progenitor cells (Keywords: Hematopoietic stem/progenitor cells, T cell differentiation, Thymic tissue decellularization)

André Dargen de Matos Branco, Design and Operation of a Prototype Bioreactor for the Expansion and In Situ Selection of Human Hematopoietic Stem/Progenitor Cells (Keywords: Hematopoietic stem/progenitor cells, Bioreactor design, Process optimization)

Cristiana da Silva Ulpiano, Mesenchymal stromal cells (MSC)-derived extracellular vesicles for the delivery of RNAi to modulate angiogenesis in malignancy (Keywords: Extracellular Vesicles, Minicircles, RNA interference)

Medical doctor (MD), PhD student

Isabel Bogalho Martins, Deciphering the cellular crosstalk in the bone marrow microenvironment of patients newly diagnosed with idiopathic acquired aplastic anemia towards the expansion of transplantable hematopoietic stem and progenitor cells. (Keywords: Adult’s idiopathic aplastic anemia, Bone marrow decellularization, ex-vivo expansion of hematopoietic stem/progenitor cells)

Master students

Catalina Cepeleaga, Breaking down mesenchymal stromal cells: uncovering the potential of decellularized extracellular matrix (Keywords: Mesenchymal stromal cells, extracellular matrix, decellularization)

Mariana Coelho Martins, Development of scalable platforms for the manufacturing of muscle cells towards Regenerative Medicine applications (Keywords: Muscle cells, ex-vivo expansion, Microcarriers)

Key collaborators

Miguel Prazeres (iBB/IST), Nuno Bernardes (iBB/IST), Manuel Abecasis (Instituto Português de Oncologia Francisco Gentil, Lisboa), António Fiarresga (Hospital de Santa Marta, Lisboa), Paulo Vale (Hospital CUF Descobertas, Lisboa), José João Mendes (Centro de Investigação Interdisciplinar Egas Moniz, Clinical Research Unit (CiiEM,CRU)), Carla Lilaia (Hospital São Francisco Xavier, Lisboa), João Forjaz Lacerda (iMM/FMUL), Domingos Henrique (iMM/FMUL), Gabriela Silva (CEDOC-UNL), Graça Almeida-Porada (Wake Forest Institute for Regenerative Medicine (WFIRM)), Deepak Vashishth (Rensselaer Polytechnic Institute (RPI)), Richard Day (University College London (UCL)), Kamilla Swiech (University of São Paulo), Sang Han (Research Center for Gene Therapy, Federal University of São Paulo, Brazil), Pedro Baptista (Aragon Health Sciences Institute, Zaragoza), Joost Sluijter/Pieter Vader (University Medical Center Utrecht) Carla Cardoso (Crioestaminal – Saúde e Tecnologia, SA), Mohan Vemuri (Thermo Fisher Scientific Inc.), William Milligan (AventaCell Biomedical Corp. Ltd), Ricardo Egea (Bionet Servicios Técnicos, S.L)