Stem Cell Bioengineering

The Stem Cell Bioengineering Laboratory at IBB-CEBQ-IST aims at the development of highly controlled culture systems (e.g. bioreactors) for the ex-vivo expansion of stem cells and their controlled differentiation into specific cell types. As stem cells are rare, their isolation and expansion/differentiation in vitro significantly increase the cell population available for cellular therapies, highthroughput drug screening or tissue engineering. Development of ex-vivo culture conditions that facilitate the maintenance and expansion of long term transplantable stem cells, as well as their differentiated progeny, is a crucial component and a major challenge in stem cell research and therapy. Human hematopoietic stem cells (HSC), human mesenchymal stem cells (MSC), embryonic stem cells (ESC), human and mouse, and mouse neural stem cells (mNSC) are used as model systems.

Research Projects

Expansion of Human Hematopoietic Stem Cells in Co-culture with Human Mesenchymal Stem Cells

The main goal is the maximization of the ex-vivo expansion of human hematopoietic stem/progenitor cells (HSPC), especially those obtained from umbilical cord blood (UCB) (which contains a limited number of primitive cells) using human mesenchymal stem cells (MSC) as feeder cells, under serum-free conditions. Current research is focused on the understanding of the mechanisms underlying the hematopoietic supportive capacity of MSC combining proliferative, functional and proteomic analysis. In particular, the dependency on the direct contact between MSC and HSPC is currently under study. The elucidation of those mechanisms will have implications in terms of bioreactor design towards the maximization of human HSPC expansion in vitro. This project is developed in collaboration with Professor Sangeeta N. Bathia, MIT-Harvard, Boston, USA and Professor Graça Almeida-Porada from the University of Nevada, Reno, N.V., USA.

Clinical-scale Expansion of Human Mesenchymal Stem Cells

By combining a cross-disciplinary approach of Stem Cell Bioengineering and Experimental Hematology, it is aimed to identify optimal culture protocols for the clinical-scale production of human mesenchymal stem cells (MSC), while maintaining their multilineage and immunosuppressive capacities, for supplementation during hematopoietic stem cell transplantation. MSC are isolated from adult bone marrow (BM) and umbilical cord blood (UCB) (recently described as an alternative MSC source). The possibility of replacing currently used FBS by platelet lysates or special lots of animal serum approved by regulatory agencies is being addressed. The importance of using three-dimensional (3-D) matrices or scaffolds to sustain MSC cell adhesion and proliferation is also being evaluated and would be the basis for the rationale design of a bioreactor for the clinical-scale expansion of human MSC. This project has the collaboration of Professor Manuel Abecasis from Instituto Português de Oncologia de Lisboa Francisco Gentil, Lisboa, and Professor Hélder Trindade, from Centro de Histocompatibilidade do Sul, Lusotransplante.

Expansion of Embryonic Stem Cells and their Neural Commitment in Bioreactor Culture Systems

The Stem Cell Bioengineering Laboratory at IBB-CEBQ-IST also aims at the large-scale ex-vivo expansion of embryonic stem cells (ESC),human and mouse, as well as ESC-derived neural stem cells (NSC), from mouse, as starting material to generate mature cells (i.e. neurons) for potential use in regenerative medicine (e.g. treatment of neurological disorders), as well as for high throughput drug screening. The protocol for the expansion of both ESC and NSC is based on adherent monolayer culture, under serum-free conditions, without the confounding influences of cell aggregation as it happens in aggregate/embryoid body culture. The ultimate goal is to define an efficient, reproducible and cost-effective process for the production of ESC/NSC in a bioreactor system operating under highly controlled conditions, while maintaining their pluripotency/multipotency and multilineage differentiation potential. This project has the collaboration of Professor Robert Langer from MIT, Boston USA, Doctor Domingos Henrique from the Institute of Molecular Medicine (IMM) in Lisboa and Professor Jonathan Dordick from the Rensselaer Polytechnic Institute, Troy, N.Y., USA.

Gene Delivery Strategies for Stem Cell Expansion and Differentiation

The ultimate goal of this project is to increase hHSC, hMSCs and mNSCs expansion, using novel non-viral strategies to efficiently deliver to these cells plasmid vectors encoding for proteins involved in their proliferation and self renewal. Currently, several transfection protocols are being tested and transient expression of a reporter protein (GFP) is being evaluated using different plasmid constructs. Several biodegradable polymers, lipids and carbon-nanotubes will be tested in normal and reverse transfection methodologies. Moreover, a combined strategy of sorting and gene delivery system will also be developed for hHSC using immunomagnetoliposomes. Comparative studies will then be performed concerning the level and duration of protein expression and its effects on proliferative and differentiative potential of transfected cells.

Recombinant Protein Production for Stem Cell Research

This project is focused on the development of a platform for the expression and purification of human recombinant proteins used for stem cell research. The bioreactor scale conditions for animal cell culture and protein expression, as well as the establishment of a downstream process and structure stability studies, are currently being addressed.