Advances in ex vivo expansion of hematopoietic stem and progenitor cells for clinical applications.

As caretakers of the hematopoietic system, hematopoietic stem cells assure a lifelong supply of differentiated populations that are responsible for critical bodily functions, including oxygen transport, immunological protection and coagulation. Due to the far-reaching influence of the hematopoietic system, hematological disorders typically have a significant impact on the lives of individuals, even becoming fatal. Hematopoietic cell transplantation was the first effective therapeutic avenue to treat such hematological diseases. Since then, key use and manipulation of hematopoietic stem cells for treatments has been aspired to fully take advantage of such an important cell population. Limited knowledge on hematopoietic stem cell behavior has motivated in-depth research into their biology. Efforts were able to uncover their native environment and characteristics during development and adult stages. Several signaling pathways at a cellular level have been mapped, providing insight into their machinery. Important dynamics of hematopoietic stem cell maintenance were begun to be understood with improved comprehension of their metabolism and progressive aging. These advances have provided a solid platform for the development of innovative strategies for the manipulation of hematopoietic stem cells. Specifically, expansion of the hematopoietic stem cell pool has triggered immense interest, gaining momentum. A wide range of approaches have sprouted, leading to a variety of expansion systems, from simpler small molecule-based strategies to complex biomimetic scaffolds. The recent approval of Omisirge, the first expanded hematopoietic stem and progenitor cell product, whose expansion platform is one of the earliest, is predictive of further successes that might arise soon. In order to guarantee the quality of these ex vivo manipulated cells, robust assays that measure cell function or potency need to be developed. Whether targeting hematopoietic engraftment, immunological differentiation potential or malignancy clearance, hematopoietic stem cells and their derivatives need efficient scaling of their therapeutic potency. In this review, we comprehensively view hematopoietic stem cells as therapeutic assets, going from fundamental to translational.

Systems bioengineering approaches for developmental toxicology.

Developmental toxicology is the field of study that examines the effects of chemical and physical agents on developing organisms. By using principles of systems biology and bioengineering, a systems bioengineering approach could be applied to study the complex interactions between developing organisms, the environment, and toxic agents. This approach would result in a holistic understanding of the effects of toxic agents on organisms, by considering the interactions between different biological systems and the impacts of toxicants on those interactions. It would be useful in identifying key biological pathways and mechanisms affected by toxic agents, as well as in the development of predictive models to assess potential risks of exposure to toxicants during development. In this review, we discuss the relevance of systems bioengineering to the field of developmental toxicity and provide up-to-date examples that illustrate the use of engineering principles for this application.

A Concise Review on Electrospun Scaffolds for Kidney Tissue Engineering.

Chronic kidney disease is one of the deadliest diseases globally and treatment methods are still insufficient, relying mostly on transplantation and dialysis. Engineering of kidney tissues in vitro from induced pluripotent stem cells (iPSCs) could provide a solution to this medical need by restoring the function of damaged kidneys. However, implementation of such approaches is still challenging to achieve due to the complexity of mature kidneys in vivo. Several strategies have been defined to obtain kidney progenitor endothelial and epithelial cells that could form nephrons and proximal tube cells, but these lack tissue maturity and vascularisation to be further implemented. Electrospinning is a technique that has shown promise in the development of physiological microenvironments of several tissues and could be applied in the engineering of kidney tissues. Synthetic polymers such as polycaprolactone, polylactic acid, and poly(vinyl alcohol) have been explored in the manufacturing of fibres that align and promote the proliferation and cell-to-cell interactions of kidney cells. Natural polymers including silk fibroin and decellularised extracellular matrix have also been explored alone and in combination with synthetic polymers promoting the differentiation of podocytes and tubular-specific cells. Despite these attempts, further work is still required to advance the applications of electrospun fibres in kidney tissue engineering and explore this technique in combination with other manufacturing methods such as bioprinting to develop more organised, mature and reproducible kidney organoids.

A Dynamic 3D Aggregate-Based System for the Successful Expansion and Neural Induction of Human Pluripotent Stem Cells.

The demand for large cell numbers for cellular therapies and drug screening applications requires the development of scalable platforms capable of generating high-quality populations of tissue-specific cells derived from human pluripotent stem cells (hPSCs). Here, we studied the ability of Gibco StemScale PSC Suspension Medium to promote the efficient expansion of hPSC cultures as aggregates grown in suspension. We tested human induced pluripotent stem cell (hiPSC) growth in 6-well plates (on orbital shaker platforms) and single-use vertical-wheel bioreactors for a total of three consecutive passages. Up to a 9-fold increase in cell number was observed over 5 days per passage, with a cumulative fold change up to 600 in 15 days. Additionally, we compared neural induction of hiPSCs by using a dual SMAD inhibition protocol with a commercially available neural induction medium, which can potentially yield more than a 30-fold change, including neural progenitor induction and expansion. This system can also be adapted toward the generation of floor plate progenitors, which yields up to an 80-fold change in cell number and generates FOXA2-positive populations. In summary, we developed platforms for hiPSC expansion and neural induction into different brain regions that provide scalability toward producing clinically relevant cell numbers.

Modeling the Human Body on Microfluidic Chips.

Animals often fail to faithfully mimic human diseases and drug toxicities, and most in vitro models are not complex enough to recapitulate human body function and pathophysiology. Organ-on-chip culture technology, however, offers a promising tool for the study of tissue development and homeostasis, which has brought us one step closer to performing human experimentation in vitro. To recapitulate the complex functionality of multiple organs at once, their respective on-chip models can be linked to create a functional human body-on-chip platform. Here, we highlight the advantages and translational potentials of body-on-chip platforms in disease modeling, therapeutic development, and personalized medicine. We provide the reader with current limitations of the body-on-chip approach and new ideas to address the pending issues moving forwards.

Human Pluripotent Stem Cells: Applications and Challenges for Regenerative Medicine and Disease Modeling.

In recent years, human pluripotent stem (hPS) cells have started to emerge as a potential tool with application in fields such as regenerative medicine, disease modeling, and drug screening. In particular, the ability to differentiate human-induced pluripotent stem (hiPS) cells into different cell types and to mimic structures and functions of a specific target organ, resourcing to organoid technology, has introduced novel model systems for disease recapitulation while offering a powerful tool to provide a faster and reproducible approach in the process of drug discovery. All these technologies are expected to improve the overall quality of life of the humankind. Here, we highlight the main applications of hiPS cells and the main challenges associated with the translation of hPS cell derivatives into clinical settings and other biomedical applications, such as the costs of the process and the ability to mimic the complexity of the in vivo systems. Moreover, we focus on the bioprocessing approaches that can be applied towards the production of high numbers of cells as well as their efficient differentiation into the final product and further purification.

hiPSC-Based Model of Prenatal Exposure to Cannabinoids: Effect on Neuronal Differentiation.

Phytocannabinoids are psychotropic substances ofcannabis with the ability to bind endocannabinoid (eCB) receptors that regulate synaptic activity in the central nervous system (CNS). Synthetic cannabinoids (SCs) are synthetic analogs of Δ9-tetrahydrocannabinol (Δ9-THC), the psychotropic compound of cannabis, acting as agonists of eCB receptor CB1. SC is an easily available and popular alternative to cannabis, and their molecular structure is always changing, increasing the hazard for the general population. The popularity of cannabis and its derivatives may lead, and often does, to a child's exposure to cannabis both in utero and through breastfeeding by a drug-consuming mother. Prenatal exposure to cannabis has been associated with an altered rate of mental development and significant changes in nervous system functioning. However, the understanding of mechanisms of its action on developing the human CNS is still lacking. We investigated the effect of continuous exposure to cannabinoids on developing human neurons, mimicking the prenatal exposure by drug-consuming mother. Two human induced pluripotent stem cells (hiPSC) lines were induced to differentiate into neuronal cells and exposed for 37 days to cannabidiol (CBD), Δ9-THC, and two SCs, THJ-018 and EG-018. Both Δ9-THC and SC, at 10 µM, promote precocious neuronal and glial differentiation, while CBD at the same concentration is neurotoxic. Neurons exposed to Δ9-THC and SC show abnormal functioning of voltage-gated calcium channels when stimulated by extracellular potassium. In sum, all studied substances have a profound impact on the developing neurons, highlighting the importance of thorough research on the impact of prenatal exposure to natural and SC.

Strategies for the expansion of human induced pluripotent stem cells as aggregates in single-use Vertical-Wheel™ bioreactors.

BACKGROUND: Since their inception, human induced pluripotent stem cells (hiPSCs) have held much promise for pharmacological applications and cell-based therapies. However, their potential can only be realised if large numbers of cells can be produced reproducibly on-demand. While bioreactors are ideal systems for this task, due to providing agitation and control of the culture parameters, the common impeller geometries were not designed for the expansion of mammalian cells, potentially leading to sub-optimal results. RESULTS: This work reports for the first time the usage of the novel Vertical-Wheel single-use bioreactors for the expansion of hiPSCs as floating aggregates. Cultures were performed in the PBS MINI 0.1 bioreactor with 60 mL of working volume. Two different culture media were tested, mTeSR1 and mTeSR3D, in a repeated batch or fed-batch mode, respectively, as well as dextran sulfate (DS) supplementation. mTeSR3D was shown to sustain hiPSC expansion, although with lower maximum cell density than mTeSR1. Dextran sulfate supplementation led to an increase in 97 and 106% in maximum cell number when using mTeSR1 or mTeSR3D, respectively. For supplemented media, mTeSR1 + DS allowed for a higher cell density to be obtained with one less day of culture. A maximum cell density of (2.3 ± 0.2) × 106 cells∙mL- 1 and a volumetric productivity of (4.6 ± 0.3) × 105 cells∙mL- 1∙d- 1 were obtained after 5 days with mTeSR1 + DS, resulting in aggregates with an average diameter of 346 ± 11 µm. The generated hiPSCs were analysed by flow cytometry and qRT-PCR and their differentiation potential was assayed, revealing the maintenance of their pluripotency after expansion. CONCLUSIONS: The results here described present the Vertical-Wheel bioreactor as a promising technology for hiPSC bioprocessing. The specific characteristics of this bioreactor, namely in terms of the innovative agitation mechanism, can make it an important system in the development of hiPSC-derived products under current Good Manufacturing Practices.

Towards Multi-Organoid Systems for Drug Screening Applications.

A low percentage of novel drug candidates succeed and reach the end of the drug discovery pipeline, mainly due to poor initial screening and assessment of the effects of the drug and its metabolites over various tissues in the human body. For that, emerging technologies involving the production of organoids from human pluripotent stem cells (hPSCs) and the use of organ-on-a-chip devices are showing great promise for developing a more reliable, rapid and cost-effective drug discovery process when compared with the current use of animal models. In particular, the possibility of virtually obtaining any type of cell within the human body, in combination with the ability to create patient-specific tissues using human induced pluripotent stem cells (hiPSCs), broadens the horizons in the fields of drug discovery and personalized medicine. In this review, we address the current progress and challenges related to the process of obtaining organoids from different cell lineages emerging from hPSCs, as well as how to create devices that will allow a precise examination of the in vitro effects generated by potential drugs in different organ systems.

A scale out approach towards neural induction of human induced pluripotent stem cells for neurodevelopmental toxicity studies.

Stem cell's unique properties confer them a multitude of potential applications in the fields of cellular therapy, disease modelling and drug screening fields. In particular, the ability to differentiate neural progenitors (NP) from human induced pluripotent stem cells (hiPSCs) using chemically-defined conditions provides an opportunity to create a simple and straightforward culture platform for application in these fields. Here, we demonstrated that hiPSCs are capable of undergoing neural commitment inside microwells, forming characteristic neural structures resembling neural rosettes and further give rise to glial and neuronal cells. Furthermore, this platform can be applied towards the study of the effect of neurotoxic molecules that impair normal embryonic development. As a proof of concept, the neural teratogenic potential of the antiepileptic drug valproic acid (VPA) was analyzed. It was verified that exposure to VPA, close to typical dosage values (0.3 to 0.75 mM), led to a prevalence of NP structures over neuronal differentiation, as confirmed by analysis of the expression of neural cell adhesion molecule, as well as neural rosette number and morphology assessment. The methodology proposed herein for the generation and neural differentiation of hiPSC aggregates can potentially complement current toxicity tests such as the humanized embryonic stem cell test for the detection of teratogenic compounds that can interfere with normal embryonic development.

Scaling up a chemically-defined aggregate-based suspension culture system for neural commitment of human pluripotent stem cells.

The demand of high cell numbers for applications in cellular therapies and drug screening requires the development of scalable platforms capable to generating highly pure populations of tissue-specific cells from human pluripotent stem cells. In this work, we describe the scaling-up of an aggregate-based culture system for neural induction of human induced pluripotent stem cells (hiPSCs) under chemically-defined conditions. A combination of non-enzymatic dissociation and rotary agitation was successfully used to produce homogeneous populations of hiPSC aggregates with an optimal (140 µm) and narrow distribution of diameters (coefficient of variation of 21.6%). Scalable neural commitment of hiPSCs as 3D aggregates was performed in 50 mL spinner flasks, and the process was optimized using a factorial design approach, involving parameters such as agitation rate and seeding density. We were able to produce neural progenitor cell cultures, that at the end of a 6-day neural induction process contained less than 3% of Oct4-positive cells and that, after replating, retained more than 60% of Pax6-positive neural cells. The results here presented should set the stage for the future generation of a clinically relevant number of human neural progenitors for transplantation and other biomedical applications using controlled, automated and reproducible large-scale bioreactor culture systems.

Spatial and temporal control of cell aggregation efficiently directs human pluripotent stem cells towards neural commitment.

3D suspension culture is generally considered a promising method to achieve efficient expansion and controlled differentiation of human pluripotent stem cells (hPSCs). In this work, we focused on developing an integrated culture platform for expansion and neural commitment of hPSCs into neural precursors using 3D suspension conditions and chemically-defined culture media. We evaluated different inoculation methodologies for hPSC expansion as 3D aggregates and characterized the resulting cultures in terms of aggregate size distribution. It was demonstrated that upon single-cell inoculation, after four days of culture, 3D aggregates were composed of homogenous populations of hPSC and were characterized by an average diameter of 139 ± 26 µm, which was determined to be the optimal size to initiate neural commitment. Temporal analysis revealed that upon neural specification it is possible to maximize the percentage of neural precursor cells expressing the neural markers Sox1 and Pax6 after nine days of culture. These results highlight our ability to define a robust method for production of hPSC-derived neural precursors that minimizes processing steps and that constitutes a promising alternative to the traditional planar adherent culture system due to a high potential for scaling-up.