Stem Cell Engineering Research Group (SCERG)

 

The Stem Cell Engineering Research Group (SCERG) at iBB - Institute for Bioengineering and Biosciences of Instituto Superior Técnico, Universidade de Lisboa, Portugal aims at the development of cell production systems for the ex-vivo expansion of stem cells and/or their controlled differentiation into specific cell types, as well as their integration with bioseparation and high resolution purification technologies, to generate the large numbers of specific and high quality stem/progenitor cell subsets needed for Precision and Regenerative Medicine settings.

 

The Stem Cell Engineering Research Group (SCERG) is a recently created group within the Institute for Bioengineering and Biosciences (iBB) at Instituto Superior Técnico, University of Lisbon, Portugal, with roots in the former Stem Cell Bioengineering and Regenerative Medicine Laboratory (SCBL-RM).

The SCERG at iBB aims at the development of cell production systems for the ex-vivo expansion of stem cells and/or their controlled differentiation into specific cell types, as well as their integration with bioseparation and high resolution purification technologies, to generate the large numbers of specific and high quality stem/progenitor cell subsets needed for Precision and Regenerative Medicine settings.

SCERG has established an international track record in the Stem Cell Engineering field [WTEC-NSF/NIST/NIH International Assessment of R&D in Stem Cell Engineering report, page 240 (2013); WTEC-NSF International Assessment of Research in Biological Engineering and Manufacturing report, page 123 (2015)], as assessed by WTEC in US on behalf of NSF, NIST and NIH. The success of stem cell manufacturing relies on safe, robust and reproducible culture conditions and cost-effective processes, including bioreactor design, bioseparation, microscale technology and process control, combined with systems biology. The development of efficient, scalable and cost-effective production processes for Stem Cells is expected to boost their applications in Cellular and Gene Therapy, Tissue Engineering, high-throughput drug screening, toxicological testing and stem cell research.

Human hematopoietic stem/progenitor cells and mesenchymal stem/stromal cells, as well as human pluripotent stem cells (both embryonic stem cells and induced pluripotent stem cells) and their neural and cardiac derivatives have been used as model systems.

The organizational structure of SCERG relies on six Research Programs:

 

Bioreactor and Microcarrier Technologies for Stem Cell expansion and differentiation SCERG has greatly expanded the understanding of stem cell expansion and differentiation in bioreactors and pioneered the use of microcarrier technology for multipotent, and pluripotent stem cells. Animal-free microcarrier-based stirred culture systems, envisaging xeno-free culture systems for scalable cell production, have been established for human mesenchymal stem/stromal cells, and human induced pluripotent stem cells.
- Microcarriers are coated with specific ECM molecules and peptides potentiating cell adhesion and growth in feeder-free and xeno-free conditions. This is expected to have a major impact on mimicking the stem cell niche and therefore maximizing cell growth/differentiation.
- Large-scale expansion and differentiation of stem cells in controlled microcarrier-based bioreactors are addressed through rational reaction engineering approaches for a systematic analysis of stem cells production, including kinetic studies of cell growth/metabolism, hydrodynamics/mass transfer effects on cell phenotype, oxygen tension control and feeding regimen, namely continuous perfusion of culture medium to allow a more precise control over the concentrations of soluble compounds (nutrients, toxic metabolites, growth factors). The operation of novel disposable bioreactors, with low shear stress has been optimized for the scalable xeno-free microcarrier-based cultivation of hiPSC. Innovative bioreactor designs towards the maximization of cell yield has also been performed for HSC expansion.

 

Clinical Manufacturing of Stem Cell-based Therapies

The robust and scalable cell manufacturing towards the cost-effective delivery of safe and potent cell-based products (either autologous or allogeneic) relies on process engineering tools to understand the impact of cellular features (biological, biochemical, etc) on cell product function and performance, and how do process variables influence the critical quality attributes of the cell product. At SCERG we are developing the innovative manufacturing of two ex-vivo expanded cell-based products:
- umbilical cord blood (UCB)-derived hematopoietic stem/progenitor cells for potential application in hemato-oncological settings. Current research is focused on (i) the definition of optimal culture conditions namely concerning cytokine combinations supplementing xeno(geneic)-/serum-free culture media, enrichment procedures and initial cell concentrations, as well as oxygen tension; and (ii) the understanding of the mechanisms underlying the hematopoietic supportive capacity of human mesenchymal stem/stromal cells in a co-culture setting.
- mesenchymal stem/stromal cells (MSC) from different tissue sources for immunomodulation-based therapies. Human MSC from adult bone marrow (BM), adipose tissue (AT), umbilical cord matrix (UCM) and synovial membrane (SM) are studied. Proliferation kinetics and metabolism of human MSC cultured ex-vivo under different oxygen tensions have been thoroughly studied along consecutive passaging. Culture protocols are being optimized for the efficient isolation and expansion of MSC from the different sources, namely focusing the use of xeno-/serum-free culture conditions. In addition, a proteomic analysis platform established in collaboration with BSRG is being employed to understand how the ex-vivo culture process affects MSC features at the proteome level.

 

Bioprocessing of Human Pluripotent Stem Cells for Regenerative and Precision Medicine

The development of Integrated Bioprocesses is a crucial demand to overcome the main technological barriers that presently limit the application of human Pluripotent Stem Cells (PSC), embryonic (ESC) and induced pluripotent (iPSC), and their derivatives in Regenerative and Precision Medicine. This progress is expected to leverage the emergence of alternative therapies and the development of new personalized tissue models for disease modelling and drug discovery. Bioprocessing approaches that are being developed at SCERG include:
- Scalable expansion of hiPSC while maintaining their pluripotency. In particular, xeno-free culture systems are being explored as a means to improve the reproducibility and robustness of the bioprocess and to facilitate further translation of hiPSC-derived products into clinical applications;
- Scalable integrated expansion and controlled neural and cardiac differentiation of hiPSCs by culturing these cells as 3D aggregates in suspension. This culture platform is being used namely for production of neural precursors under chemically-defined conditions, by performing the spatial and temporal control of cell aggregation;
- Downstream processing methodologies for hiPSC and their differentiated derivatives and their integration with the scalable expansion and differentiation of hiPSC. Separation of hiPSC from microcarriers after expansion is performed namely using dissolvable microcarriers. Cell separation methods based on affinity principles are being focused for purification of hiPSC derivatives after the process of differentiation. This includes the depletion of tumorigenic hPSC that remain in the bioprocess after neural differentiation;
- Development of standardized culture platforms for production of neural and cardiac tissues from healthy and patient-specific hiPSCs for disease modelling and drug screening. A chemically-defined monolayer-based culture system for neural differentiation of patient-specific hiPSC is being explored for modelling of Rett Syndrome. Size-controlled aggregates of hiPSC-derived neural precursors are being produced in microwells for high-throughput neurotoxicology assays.

 

Gene Delivery Strategies to Modulate Stem Cell Function

The transient expression of genes encoded by minicircles enables the overexpression and/or prolonged expression of specific signaling molecules to modulate cell survival, proliferation or differentiation, foreseeing the maximization of the therapeutic potential of stem cells for applications in Cellular and Gene Therapy, as well as Tissue Engineering. Minicircles are safe and effective because are small molecules devoid of specific DNA sequences that may trigger gene silencing and the attack of the immune system.
At SCERG, gene induced modification of human mesenchymal stem/stromal cells (MSC) is pursued towards the maximization of the expansion and differentiation potential of these cells, but also to improve their intrinsic therapeutic features. In this context, an ex vivo gene therapy strategy is envisaged to engineer MSC from different human sources with minicircles in order to secrete modulating proteins and exosomes that will alter the behavior of targeting cells.
Minicircle non-viral vectors are rationally engineered to increase ex vivo MSC transfection, extend transgene expression and facilitate purification (collaboration with BERG). Minicircle gene delivery by microporation and by nano- and microparticles are being developed to obtain more efficient gene delivery strategies. Additionally, the composition of the cell secretome that contains several types of signaling molecules, including microRNAs, may be modified by minicircle-encoded genes and used to alter the response of target cells.

 

Designing Biomaterials and Devices for Stem Cell Engineering

Stem cells respond to different biochemical and physical stimuli, which impact on their expansion, differentiation and secretory profile when used as therapeutic agents, in disease models or for drug screening. At SCERG, we aim to develop and apply tailor-made platforms, comprising engineered biomaterials and designed devices, in the field of Regenerative and Precision Medicine. Synthetic, natural and hybrid polymers have been used in the construction of scaffolds to provide cues to direct cell organization and function. Electrospinning is currently being used to produce nanofiber matrices able to mimic structural and topological aspects of the extracellular matrix, promoting cell-cell and cell-material interactions. Extrusion of tridimensional structures is being explored to build scaffolds with highly interconnected pores at microscale and prototyping devices. Cell encapsulation or cultivation in hydrogels promotes tridimensional interaction of single cells or spheroids in softer and highly hydrated matrices. Additionally, we also aim to develop polymeric matrices able to provide time dependent stimuli to the cells at physiologic conditions. Devices are being designed and prototyped to control hydrodynamic and mechanic stimuli to cells cultured on scaffolds. We are particularly interested in the use electrical conductive polymers to provide electrical stimulation to cells, promoting cell differentiation, organization and communication. Decellularization strategies are also being developed to produce acellular bioscaffolds for Tissue Engineering settings, including the use of bioscaffold-derived soluble products.

 

Stem Cell Biosystems Engineering

Stem Cell Biosystems Engineering is an innovative approach for studying the mechanisms that modulate the pluripotency of human stem cells, and aims at contributing to a better understanding of the molecular and cellular events that regulate stem cell function . As model systems, we will employ in vitro platforms to study the cellular events involved in lineage commitment and further maturation of stem cells. The influence of microenvironmental stimuli on pluripotency will be investigated due to their capacity to modulating intracellular signaling pathways, and based on phenotypic and global analyses, modeling approaches will be used to uncover complex interactions between different signaling input. The ultimate goal is to develop predictive models of signaling networks from such raw data, study the dynamic regulation of pluripotency circuit components, and gain new insights into the regulatory mechanisms underlying multilineage cell specification, ultimately enabling novel tools for Regenerative and Precision Medicine, including stem cell-based therapies, and thus positively impacting health and welfare.

 

The Stem Cell Bioengineering program is developed in the Taguspark Campus, with 300 m2 of research laboratories, including 2 cell culture laboratories equipped with CO2 incubators, laminar flow hoods and cell culture systems and a GMP clean room.

 




 

 

Research Units