Cláudia Correia

Process engineering of human pluripotent stem cells for clinical application

Human pluripotent stem cells (hPSCs), including embryonic and induced pluripotent stem cells, constitute an extremely attractive tool for cell therapy. However, flexible platforms for the large-scale production and storage of hPSCs in tightly controlled conditions are necessary to deliver high-quality cells in relevant quantities to satisfy clinical demands. Here we discuss the main principles for the bioprocessing of hPSCs, highlighting the impact of environmental factors, novel 3D culturing approaches and integrated bioreactor strategies for controlling hPSC culture outcome. Knowledge on hPSC bioprocessing accumulated during recent years provides important insights for the establishment of more robust production platforms and should potentiate the implementation of novel hPSC-based therapies.

Microencapsulation technology: a powerful tool for integrating expansion and cryopreservation of human embryonic stem cells.

The successful implementation of human embryonic stem cells (hESCs)-based technologies requires the production of relevant numbers of well-characterized cells and their efficient long-term storage. In this study, cells were microencapsulated in alginate to develop an integrated bioprocess for expansion and cryopreservation of pluripotent hESCs. Different three-dimensional (3D) culture strategies were evaluated and compared, specifically, microencapsulation of hESCs as: i) single cells, ii) aggregates and iii) immobilized on microcarriers. In order to establish a scalable bioprocess, hESC-microcapsules were cultured in stirred tank bioreactors. The combination of microencapsulation and microcarrier technology resulted in a highly efficient protocol for the production and storage of pluripotent hESCs. This strategy ensured high expansion ratios (an approximately twenty-fold increase in cell concentration) and high cell recovery yields (>70%) after cryopreservation. When compared with non-encapsulated cells, cell survival post-thawing demonstrated a three-fold improvement without compromising hESC characteristics. Microencapsulation also improved the culture of hESC aggregates by protecting cells from hydrodynamic shear stress, controlling aggregate size and maintaining cell pluripotency for two weeks. This work establishes that microencapsulation technology may prove a powerful tool for integrating the expansion and cryopreservation of pluripotent hESCs. The 3D culture strategy developed herein represents a significant breakthrough towards the implementation of hESCs in clinical and industrial applications.

Evidence for Divergent Evolution of Growth Temperature Preference in Sympatric Saccharomyces Species

The genus Saccharomyces currently includes eight species in addition to the model yeast Saccharomyces cerevisiae, most of which can be consistently isolated from tree bark and soil. We recently found sympatric pairs of Saccharomyces species, composed of one cryotolerant and one thermotolerant species in oak bark samples of various geographic origins. In order to contribute to explain the occurrence in sympatry of Saccharomyces species, we screened Saccharomyces genomic data for protein divergence that might be correlated to distinct growth temperature preferences of the species, using the dN/dS ratio as a measure of protein evolution rates and pair-wise species comparisons. In addition to proteins previously implicated in growth at suboptimal temperatures, we found that glycolytic enzymes were among the proteins exhibiting higher than expected divergence when one cryotolerant and one thermotolerant species are compared. By measuring glycolytic fluxes and glycolytic enzymatic activities in different species and at different temperatures, we subsequently show that the unusual divergence of glycolytic genes may be related to divergent evolution of the glycolytic pathway aligning its performance to the growth temperature profiles of the different species. In general, our results support the view that growth temperature preference is a trait that may have undergone divergent selection in the course of ecological speciation in Saccharomyces.

Robust Expansion of Human Pluripotent Stem Cells: Integration of Bioprocess Design With Transcriptomic and Metabolomic Characterization

Human embryonic stem cells (hESCs) have an enormous potential as a source for cell replacement therapies, tissue engineering, and in vitro toxicology applications. The lack of standardized and robust bioprocesses for hESC expansion has hindered the application of hESCs and their derivatives in clinical settings. We developed a robust and well‐characterized bioprocess for hESC expansion under fully defined conditions and explored the potential of transcriptomic and metabolomic tools for a more comprehensive assessment of culture system impact on cell proliferation, metabolism, and phenotype. Two different hESC lines (feeder‐dependent and feeder‐free lines) were efficiently expanded on xeno‐free microcarriers in stirred culture systems. Both hESC lines maintained the expression of stemness markers such as Oct‐4, Nanog, SSEA‐4, and TRA1‐60 and the ability to spontaneously differentiate into the three germ layers. Whole‐genome transcriptome profiling revealed a phenotypic convergence between both hESC lines along the expansion process in stirred‐tank bioreactor cultures, providing strong evidence of the robustness of the cultivation process to homogenize cellular phenotype. Under low‐oxygen tension, results showed metabolic rearrangement with upregulation of the glycolytic machinery favoring an anaerobic glycolysis Warburg‐effect‐like phenotype, with no evidence of hypoxic stress response, in contrast to two‐dimensional culture. Overall, we report a standardized expansion bioprocess that can guarantee maximal product quality. Furthermore, the “omics” tools used provided relevant findings on the physiological and metabolic changes during hESC expansion in environmentally controlled stirred‐tank bioreactors, which can contribute to improved scale‐up production systems.

Colorimetric assessment of BCR-ABL1 transcripts in clinical samples via gold nanoprobes

AbstractGold nanoparticles functionalized with thiolated oligonucleotides (Au-nanoprobes) have been used in a range of applications for the detection of bioanalytes of interest, from ions to proteins and DNA targets. These detection strategies are based on the unique optical properties of gold nanoparticles, in particular, the intense color that is subject to modulation by modification of the medium dieletric. Au-nanoprobes have been applied for the detection and characterization of specific DNA sequences of interest, namely pathogens and disease biomarkers. Nevertheless, despite its relevance, only a few reports exist on the detection of RNA targets. Among these strategies, the colorimetric detection of DNA has been proven to work for several different targets in controlled samples but demonstration in real clinical bioanalysis has been elusive. Here, we used a colorimetric method based on Au-nanoprobes for the direct detection of the e14a2 BCR-ABL fusion transcript in myeloid leukemia patient samples without the need for retro-transcription. Au-nanoprobes directly assessed total RNA from 38 clinical samples, and results were validated against reverse transcription-nested polymerase chain reaction (RT-nested PCR) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The colorimetric Au-nanoprobe assay is a simple yet reliable strategy to scrutinize myeloid leukemia patients at diagnosis and evaluate progression, with obvious advantages in terms of time and cost, particularly in low- to medium-income countries where molecular screening is not routinely feasible. Graphical abstractGold nanoprobe for colorimetric detection of BCR-ABL1 fusion transcripts originating from the Philadelphia chromosome

Effective Hypothermic Storage of Human Pluripotent Stem Cell-Derived Cardiomyocytes Compatible With Global Distribution of Cells for Clinical Applications and Toxicology Testing

To fully explore the potential of human pluripotent stem cell‐derived cardiomyocytes (hPSC‐CMs), efficient methods for storage and shipment of these cells are required. Here, we evaluated the feasibility to cold store monolayers and aggregates of functional CMs obtained from different PSC lines using a fully defined clinical‐compatible preservation formulation and investigated the time frame that hPSC‐CMs could be subjected to hypothermic storage. We showed that two‐dimensional (2D) monolayers of hPSC‐CMs can be efficiently stored at 4°C for 3 days without compromising cell viability. However, cell viability decreased when the cold storage interval was extended to 7 days. We demonstrated that hPSC‐CMs are more resistant to prolonged hypothermic storage‐induced cell injury in three‐dimensional aggregates than in 2D monolayers, showing high cell recoveries (>70%) after 7 days of storage. Importantly, hPSC‐CMs maintained their typical (ultra)structure, gene and protein expression profile, electrophysiological profiles, and drug responsiveness.

Engineering Enriched Microenvironments with Gradients of Platelet Lysate in Hydrogel Fibers

Gradients of physical and chemical cues are characteristic of specific tissue microenvironments and contribute toward morphogenesis and tissue regeneration upon injury. Recent advances on microfluidics and hydrogel manipulation raised the possibility of generating biomimetic biomaterials enriched with bioactive factors and encapsulating cells following designs specifically tailored for a target application. The novelty of this work relies on the combination of methacrylated gellan gum (MeGG) with platelet lysate (PL), aiming to generate novel advanced 3D PL-enriched photo-cross-linkable hydrogels and overcoming the lack of adhesion sites provided by the native MeGG hydrogels. This combination takes advantage of the availability, enriched growth factor composition, and potential autologous application of PL while simultaneously preserving the ability provided by MeGG to tailor mechanical properties, protein release kinetics, and shape of the construct according to the desired goal. Incorporation of PL in the hydrogels significantly improved cellular adhesion and viability in the constructs. The use of microfluidic tools allowed the design of a fiber-like hydrogel incorporating a gradient of PL along the length of the fiber. These spatial protein gradients led to the viability and cell number gradients caused by maintenance of human umbilical vein endothelial cells (HUVECs) survival in the fibers toward the PL-enriched sections in comparison with the nonloaded MeGG sections of the fibers. Altogether, we propose a proof of concept strategy to design a PL gradient biomaterial with potential in tissue engineering approaches and analysis of cell-microenvironment interactions.

Bioprocessing of Human Pluripotent Stem Cells for Cell Therapy Applications

Human pluripotent stem cells (hPSCs), with their unique characteristics for indefinite proliferation and pluripotency, are an appealing source for cell replacement therapies, tissue engineering, drug discovery and in vitro toxicology. For the clinical implementation of these cells, there is the need for translating the culture protocols developed at research laboratories into validated bioprocesses that can guarantee reproducibility, scalability, standardization, robustness and safety.

3D aggregate culture improves metabolic maturation of human pluripotent stem cell derived cardiomyocytes

Three‐dimensional (3D) cultures of human pluripotent stem cell derived cardiomyocytes (hPSC‐CMs) hold great promise for drug discovery, providing a better approximation to the in vivo physiology over standard two‐dimensional (2D) monolayer cultures. However, the transition of CM differentiation protocols from 2D to 3D cultures is not straightforward. In this work, we relied on the aggregation of hPSC‐derived cardiac progenitors and their culture under agitated conditions to generate highly pure cardiomyocyte aggregates. Whole‐transcriptome analysis and 13C‐metabolic flux analysis allowed to demonstrate at both molecular and fluxome levels that such 3D culture environment enhances metabolic maturation of hiPSC‐CMs. When compared to 2D, 3D cultures of hiPSC‐CMs displayed down‐regulation of genes involved in glycolysis and lipid biosynthesis and increased expression of genes involved in OXPHOS. Accordingly, 3D cultures of hiPSC‐CMs had lower fluxes through glycolysis and fatty acid synthesis and increased TCA‐cycle activity. Importantly, we demonstrated that the 3D culture environment reproducibly improved both CM purity and metabolic maturation across different hPSC lines, thereby providing a robust strategy to derive enriched hPSC‐CMs with metabolic features closer to that of adult CMs.