Bioprinting is a novel technology that may help to overcome limitations associated with two-dimensional (2D) cell cultures and animal experiments, as it allows the production of three-dimensional ...(3D) tissue models composed of human cells. The present study describes the optimization of a bioink composed of alginate, gelatin and human extracellular matrix (hECM) to print human HepaRG liver cells with a pneumatic extrusion printer. The resulting tissue model was tested for its suitability for the study of transduction by an adeno-associated virus (AAV) vector and infection with human adenovirus 5 (hAdV5). We found supplementation of the basic alginate/gelatin bioink with 0.5 and 1 mg/mL hECM provides desirable properties for the printing process, the stability of the printed constructs, and the viability and metabolic functions of the printed HepaRG cells. The tissue models were efficiently transduced by AAV vectors of serotype 6, which successfully silenced an endogenous target (cyclophilin B) by means of RNA interference. Furthermore, the printed 3D model supported efficient adenoviral replication making it suitable to study virus biology and develop new antiviral compounds. We consider the approach described here paradigmatic for the development of 3D tissue models for studies including viral vectors and infectious viruses.
Penetration rates of foliar-applied polar solutes are highly variable and the underlying mechanisms are not yet fully understood. The contribution of stomata especially, is still a matter of debate. ...Thus, the size exclusion limits of the stomatal foliar uptake pathway, its variability and its transport capacity have been investigated. The size exclusion limits were analyzed by studying the penetration of water-suspended hydrophilic particles of two different sizes (43 nm or 1.1 μm diameter) into leaves of Vicia faba (L.). To avoid agglutination of the particles, plants were kept in water-saturated atmosphere. Penetration of the larger particles was never detected, whereas after 2 to 9 days, the smaller particles occasionally penetrated the leaf interior through stomatal pores. Permeability of stomata to Na₂-fluorescein along the leaf blade of Allium porrum (L.) was highly variable and not correlated with the position on the leaf. When evaporated residues of the foliar-applied solutions were rewetted repeatedly, approximately 60% of the previously penetrated stomata were penetrated again. The average rate constant of penetration of an individual stoma was in the same order of magnitude as typical rate constants reported for the cuticular pathway. The observed sparseness of stomatal penetration together with its high lateral variability but local and temporal persistency was taken as evidence that stomata contributing to uptake differ from non-penetrated ones in the wettability of their guard cell cuticle. These results show that the stomatal pathway is highly capacitive because of its large size exclusion limit above 10 nm and its high transport velocity, but at the same time the high variability renders this pathway largely unpredictable.
In modern biology, the correct identification of cell types is required for the developmental study of tissues and organs and the production of functional cells for cell therapies and disease ...modeling. For decades, cell types have been defined on the basis of morphological and physiological markers and, more recently, immunological markers and molecular properties. Recent advances in single-cell RNA sequencing have opened new doors for the characterization of cells at the individual and spatiotemporal levels on the basis of their RNA profiles, vastly transforming our understanding of cell types. The objective of this review is to survey the current progress in the field of cell-type identification, starting with the Human Cell Atlas project, which aims to sequence every cell in the human body, to molecular marker databases for individual cell types and other sources that address cell-type identification for regenerative medicine based on cell data guidelines.
Hypoxic preconditioning was shown to improve the therapeutic efficacy of bone marrow-derived multipotent mesenchymal stromal cells (MSCs) upon transplantation in ischemic tissue. Given the interest ...in clinical applications of umbilical cord blood-derived MSCs, we developed a specific hypoxic preconditioning protocol and investigated its anti-apoptotic and pro-angiogenic effects on cord blood MSCs undergoing simulated ischemia in vitro by subjecting them to hypoxia and nutrient deprivation with or without preceding hypoxic preconditioning. Cell number, metabolic activity, surface marker expression, chromosomal stability, apoptosis (caspases-3/7 activity) and necrosis were determined, and phosphorylation, mRNA expression and protein secretion of selected apoptosis and angiogenesis-regulating factors were quantified. Then, human umbilical vein endothelial cells (HUVEC) were subjected to simulated ischemia in co-culture with hypoxically preconditioned or naïve cord blood MSCs, and HUVEC proliferation was measured. Migration, proliferation and nitric oxide production of HUVECs were determined in presence of cord blood MSC-conditioned medium. Cord blood MSCs proved least sensitive to simulated ischemia when they were preconditioned for 24 h, while their basic behavior, immunophenotype and karyotype in culture remained unchanged. Here, "post-ischemic" cell number and metabolic activity were enhanced and caspase-3/7 activity and lactate dehydrogenase release were reduced as compared to non-preconditioned cells. Phosphorylation of AKT and BAD, mRNA expression of BCL-XL, BAG1 and VEGF, and VEGF protein secretion were higher in preconditioned cells. Hypoxically preconditioned cord blood MSCs enhanced HUVEC proliferation and migration, while nitric oxide production remained unchanged. We conclude that hypoxic preconditioning protects cord blood MSCs by activation of anti-apoptotic signaling mechanisms and enhances their angiogenic potential. Hence, hypoxic preconditioning might be a translationally relevant strategy to increase the tolerance of cord blood MSCs to ischemia and improve their therapeutic efficacy in clinical applications.
Mesenchymal stromal cells (MSC) have been intensively studied for innovative therapeutic applications. MSC in vitro are characterized by plastic-adherent proliferation, their specific immunophenotype ...and multipotency, whereas MSC progenitors in vivo are described as perivascular cells. Whether MSC progenitors acquire in vitro MSC characteristics upon in vitro culture is still unclear. This question can be experimentally accessed by analyzing changes in cellular properties that occur during the early in vitro culture phase, the MSC derivation phase. Here, we examined dynamics in morphology, proliferation, and expression of surface markers used for MSC characterization (such as CD34, CD105, CD146, and CD271) in tight kinetics during the MSC derivation phase of adipose tissue-derived MSC (AT-MSC). Using multiparametric flow cytometry, we identified 3 major ex vivo stromal vascular cell subsets: CD34+ CD146-CD271(+/-) adventitial stromal cell-like cells (AdSC), CD34- CD146+ CD271(+/-) pericyte-like cells (PC), and CD34+ CD31+ CD146+ endothelial cells. Of these subsets, only AdSC, but not PC gave rise to MSC under MSC culture conditions. At day 4 of culture, AdSC became fibroblastoid and upregulated CD105, CD146, and CD271. Following this phenotypic transition, AdSC commenced proliferation and downregulated CD34. In our study, we demonstrate that AdSC are more clonogenic AT-MSC progenitors than PC. Moreover, we, for the first time have dissected the phenotypic transitions from MSC progenitors to in vitro MSC during the MSC derivation phase using multiparametric flow cytometry. Hence, we propose a model describing how de novo acquisition of the typical MSC morphology by AdSC is accompanied by concerted regulation of surface marker expression upon in vitro culture.
Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder of adults, characterized by uncontrolled cysts formation that causes a gradual impairment of kidney function. We ...generated a human induced pluripotent stem cell (hiPSC) line from the urinary cells of a patient diagnosed with ADPKD using a non-integrating Epi5™ Episomal iPSC reprogramming strategy. Characterization of the cell line was performed regarding their undifferentiated status, differentiation potential, and quality control for karyotypic integrity, identity, and clearance of reprogramming vectors. The newly derived hiPSC line, namely BCRTi007-A, can be used in vitro for disease modeling of ADPKD as well as testing for novel therapeutic approaches.
Advanced therapy medicinal products (ATMPs) comprising cell therapy, gene therapy, and tissue-engineered products, offer a multitude of novel therapeutic approaches to a wide range of severe and ...debilitating diseases. To date, several advanced therapies have received marketing authorization for a variety of indications. However, some products showed disappointing market performance, leading to their withdrawal. The available evidence for quality, safety, and efficacy at product launch can play a crucial role in their market success. To evaluate the sufficiency of evidence in submissions of advanced therapies for marketing authorization and to benchmark them against more established biological products, we conducted a matched comparison of the regulatory submissions between ATMPs and other biologicals. We applied a quantitative assessment of the regulatory objections and divergence from the expected data requirements as indicators of sufficiency of evidence and regulatory flexibility, respectively. Our results demonstrated that product manufacturing was challenging regardless of the product type. Advanced therapies displayed critical deficiencies in the submitted clinical data. The submitted non-clinical data packages benefited the most from regulatory flexibility. Additionally, ATMP developers need to comply with more commitments in the post-approval phase, which might add pressure on market performance. Mitigating such observed deficiencies in future product development, may leverage their potential for market success.
This study is the first to compare cell and gene therapies’ regulatory submissions to other biological submissions. We observed that at the time of product launch, cell and gene therapies suffer more evidential shortcomings, which increase the risk of market failure and hinder their integration in routine clinical practice.
The human pluripotent stem cell registry (hPSCreg), accessible at http://hpscreg.eu, is a public registry and data portal for human embryonic and induced pluripotent stem cell lines (hESC and hiPSC). ...Since their first isolation the number of hESC lines has steadily increased to over 3000 and new iPSC lines are generated in a rapidly growing number of laboratories as a result of their potentially broad applicability in biomedicine and drug testing. Many of these lines are deposited in stem cell banks, which are globally established to store tens of thousands of lines from healthy and diseased donors. The Registry provides comprehensive and standardized biological and legal information as well as tools to search and compare information from multiple hPSC sources and hence addresses a translational research need. To facilitate unambiguous identification over different resources, hPSCreg automatically creates a unique standardized name for each cell line registered. In addition to biological information, hPSCreg stores extensive data about ethical standards regarding cell sourcing and conditions for application and privacy protection. hPSCreg is the first global registry that holds both, manually validated scientific and ethical information on hPSC lines, and provides access by means of a user-friendly, mobile-ready web application.
The human pluripotent stem cell (hPSC) research landscape is rapidly evolving. To assess possible novel trends in hPSC usage, we analyzed experimental hPSC research published from 2014 to 2016 and ...compared our data with those of earlier periods. The number of papers describing experimental work involving hPSCs increased further with clear differences in the scientific impact of publications from different countries. Our results confirm the leading position of US-based hPSC research, although to a lesser degree than observed previously. Our data reveal that research into human induced pluripotent stem cells alone surpassed human embryonic stem cell (hESC) research by 2015 and rapidly grew after that. We also report on continuing and even slightly growing research activities in the hESC field as well as on a generally declining rate of the generation of new hESC lines. An increasing portion of new hESC lines represents disease-specific and clinical-grade cell lines. The previously noted usage of only a few early established hESC lines in the vast majority of scientific work is sustained. We also provide a comprehensive overview on clinical trials on the basis of hPSCs. We find that the vast majority of those trials are based on hESC-derived cell products that were generated from an only limited number of relatively old cell lines.
•There are marked differences in the impact of hPSC research from different countries•Few very old hESC lines are most frequently used in recent years•hPSC-based clinical trials performed so far mainly use hESC-derived cell products
Guhr et al. show that there are marked differences in the impact of recent hPSC research from different countries. The hESC line usage patterns remained mainly unchanged. The authors provide a comprehensive overview on clinical trials involving hPSC-derived cell products and find that these trials are mainly based on hESCs.
We describe the generation of two human induced pluripotent stem cell (iPSC) lines derived from peripheral blood mononuclear cells (PBMCs) using a non-integrative episomal reprogramming strategy. The ...first cell line was derived from a NF1 patient with the genetic variant c.1466A>G (BCRTi011-A) which leads to a cryptic splice site and aberrant splicing. The second one was created from a healthy relative of first-degree (BCRTi010-A). The generated iPSC lines were shown to have tri-lineage differentiation potential, a normal karyotype, and expression of pluripotent markers. Both iPSC lines provide a powerful tool for in vitro disease modeling and therapy development.
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