Recent Advances in Developmental Hematopoiesis: Diving Deeper With New Technologies.

The journey of a hematopoietic stem cell (HSC) involves the passage through successive anatomical sites where HSCs are in direct contact with their surrounding microenvironment, also known as niche. These spatial and temporal cellular interactions throughout development are required for the acquisition of stem cell properties, and for maintaining the HSC pool through balancing self-renewal, quiescence and lineage commitment. Understanding the context and consequences of these interactions will be imperative for our understanding of HSC biology and will lead to the improvement of in vitro production of HSCs for clinical purposes. The aorta-gonad-mesonephros (AGM) region is in this light of particular interest since this is the cradle of HSC emergence during the embryonic development of all vertebrate species. In this review, we will focus on the developmental origin of HSCs and will discuss the novel technological approaches and recent progress made to identify the cellular composition of the HSC supportive niche and the underlying molecular events occurring in the AGM region.

Advance in Human Epithelial-Derived Organoids Research.

Organoids have complex three-dimensional structures that exhibit functionalities and feature architectures similar to those of in vivo organs and are developed from adult stem cells, embryonic stem cells, and pluripotent stem cells through a self-organization process. Organoids derived from adult epithelial stem cells are the most mature and extensive. In recent years, using organoid culture techniques, researchers have established various adult human tissue-derived epithelial organoids, including intestinal, colon, lung, liver, stomach, breast, and oral mucosal organoids, all of which exhibit strong research and application prospects. Studies have shown that epithelial organoids are mainly applied in drug discovery, personalized drug response testing, disease mechanism research, and regenerative medicine. In this review, we mainly discuss current organoid culture systems and potential applications of this technique with human epithelial tissue.

A systematic review on thyroid organoid models: time-trend and its achievements.

Current in vitro models have played important roles in improving knowledge and understanding of cellular and molecular biology, but cannot exactly recapitulate the physiology of human tissues such as thyroid. In this article, we conducted a systematic review to present scientific and methodological time-trends of the reconstruction and generation of 3 D functional thyroid follicles and organoids for thyroid research in health and disease. "Web of Science (ISI)", "Scopus", "Embase", "Cochrane Library", and "PubMed" were systematically searched for papers published since 1950 to May 2020 in English language, using the predefined keywords. 212 articles were reviewed and finally 28 papers that met the inclusion and exclusion criteria were selected. Among the evidence for the examination of 3 D cell culture methods in thyroid research, there were only a few studies related to the organoid technology and its potential applications in understanding morphological, histological, and physiological characteristics of the thyroid gland and reconstructing this tissue. Besides, there was no study using organoids to investigate the tumorigenesis process of thyroid. Based on the results of this study, despite all the limitations and controversies, the exciting and promising organoid technology offers researchers a wide range of potential applications for more accurate modeling of thyroid in health and diseases and provides an excellent preclinical in vitro platform. In future, organoid technology can provide a better understanding of the molecular mechanisms of pathogenesis and tumorigenesis of thyroid tissue and more effective treatment for related disorders due to more accurate simulation of the thyroid physiology.

Advanced biomedical applications based on emerging 3D cell culturing platforms.

It is of great value to develop reliable in vitro models for cell biology and toxicology. However, ethical issues and the decreasing number of donors restrict the further use of traditional animal models in various fields, including the emerging fields of tissue engineering and regenerative medicine. The huge gap created by the restrictions in animal models has pushed the development of the increasingly recognized three-dimensional (3D) cell culture, which enables cells to closely simulate authentic cellular behaviour such as close cell-to-cell interactions and can achieve higher functionality. Furthermore, 3D cell culturing is superior to the traditional 2D cell culture, which has obvious limitations and cannot closely mimic the structure and architecture of tissues. In this study, we review several methods used to form 3D multicellular spheroids. The extracellular microenvironment of 3D spheroids plays a role in many aspects of biological sciences, including cell signalling, cell growth, cancer cell generation, and anti-cancer drugs. More recently, they have been explored as basic construction units for tissue and organ engineering. We review this field with a focus on the previous research in different areas using spheroid models, emphasizing aqueous two-phase system (ATPS)-based techniques. Multi-cellular spheroids have great potential in the study of biological systems and can closely mimic the in vivo environment. New technologies to form and analyse spheroids such as the aqueous two-phase system and magnetic levitation are rapidly overcoming the technical limitations of spheroids and expanding their applications in tissue engineering and regenerative medicine.

The application of three-dimensional cell culture in clinical medicine.

Three-dimensional cell culture technology is a novel cell culture technology, which can simulate the growth state of cells in vivo by scaffolds or special devices. Cells can form tissues or organs in vitro. It combines some advantages of traditional cell experiments and animal model experiments. Because of its advantages, it is widely used in clinical medical research, including research on stem cell differentiation, research on cell behavior, migration and invasion, study on microenvironment, study on drug sensitivity and radio-sensitivity of tumor cells, etc. In this paper, the evolution and classification of three-dimensional cell culture are reviewed, also the advantages and shortages are compared. The application of three-dimensional cell culture in clinical medicine are summarized to provide an insight into translational medicine.

Microphysiological Systems: A Pathologist's Perspective.

High-throughput in vitro models lack human-relevant complexity, which undermines their ability to accurately mimic in vivo biologic and pathologic responses. The emergence of microphysiological systems (MPS) presents an opportunity to revolutionize in vitro modeling for both basic biomedical research and applied drug discovery. The MPS platform has been an area of interdisciplinary collaboration to develop new, predictive, and reliable in vitro methods for regulatory acceptance. The current MPS models have been developed to recapitulate an organ or tissue on a smaller scale. However, the complexity of these models (ie, including all cell types present in the in vivo tissue) with appropriate structural, functional, and biochemical attributes are often not fully characterized. Here, we provide an overview of the capabilities and limitations of the microfluidic MPS model (aka organs-on-chips) within the scope of drug development. We recommend the engagement of pathologists early in the MPS design, characterization, and validation phases, because this will enable development of more robust and comprehensive MPS models that can accurately replicate normal biology and pathophysiology and hence be more predictive of human responses.

Recent progress in induced pluripotent stem cell-derived cardiac cell sheets for tissue engineering.

The past decade has witnessed remarkable development in tissue engineering technologies and stem cells. Our lab has developed a novel technology - "cell sheet technology" for tissue engineering. After the confluent cells are cultured on an innovative temperature-responsive culture dish, the cells can be harvested as an intact sheet by lowering temperature. We have successfully created multiple cell sheet-based tissues for therapies of a vast variety of diseases, in particular, myocardial diseases. On the other side, the discovery of human induced pluripotent stem cells (hiPSC) enables stable production of defined tissue-specific cell types and thus makes it possible to regenerate tissues or even organs for clinical application and in vitro drug screening/disease modeling. Recently, we have combined cell sheet technology and hiPSC-derived cardiac cells for fabrication of functional human cardiac tissues. This review summarizes ongoing challenges in this field and our progresses in solving issues, such as large scale culture of hiPSC-derived cardiac cells, elimination of undifferentiated iPSCs to decrease the risk of tumor formation as well as myocardial tissue fabrication technologies.

Primary Cell-Derived Intestinal Models: Recapitulating Physiology.

The development of physiologically relevant intestinal models fueled by breakthroughs in primary cell-culture methods has enabled successful recapitulation of key features of intestinal physiology. These advances, paired with engineering methods, for example incorporating chemical gradients or physical forces across the tissues, have yielded ever more sophisticated systems that enhance our understanding of the impact of the host microbiome on human physiology as well as on the genesis of intestinal diseases such as inflammatory bowel disease and colon cancer. In this review we highlight recent advances in the development and usage of primary cell-derived intestinal models incorporating monolayers, organoids, microengineered platforms, and macrostructured systems, and discuss the expected directions of the field.

Platforms for Manufacturing Allogeneic, Autologous and iPSC Cell Therapy Products: An Industry Perspective.

As cell therapy processes mature from benchtop research protocols to industrial processes capable of manufacturing market-relevant numbers of doses, new cell manufacturing platforms are required. Here we give an overview of the platforms and technologies currently available to manufacture allogeneic cell products, such as mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs), and technologies for mass production of autologous cell therapies via scale-out. These technologies include bioreactors, microcarriers, cell separation and cryopreservation equipment, molecular biology tools for iPSC generation, and single-use controlled-environment systems for autologous cell production. These platforms address the challenges of manufacturing cell products in greater numbers while maintaining process robustness and product quality.

HSC Niche Biology and HSC Expansion Ex Vivo.

Hematopoietic stem cell (HSC) transplantation can restore a new functional hematopoietic system in recipients in cases where the system of the recipient is not functional or for example is leukemic. However, the number of available donor HSCs is often too low for successful transplantation. Expansion of HSCs and thus HSC self-renewal ex vivo would greatly improve transplantation therapy in the clinic. In vivo, HSCs expand significantly in the niche, but establishing protocols that result in HSC expansion ex vivo remains challenging. In this review we discuss current knowledge of niche biology, the intrinsic regulators of HSC self-renewal in vivo, and introduce novel niche-informed strategies of HSC expansion ex vivo.

Best practices for media selection for mammalian cells.

Cell culture medium is a complex mixture of nutrients and growth factors that, along with the physical environment, can either help or destroy your experiment or production run. Nutritional requirements differ with different cell types and functions, as do optimal pH and osmolality. As cell growth proceeds, different cells will utilize amino acids and other components at different rates. By controlling for ammonia, free radicals, heavy metal toxicity, pH shifts, fluctuations in osmolality, nutrient depletion, and chemical and biological contaminants, you will optimize the chances of success. The contribution of each component of the medium is essential for the maintenance of the cell type of interest. While some cell types, such as established human cancer cell lines, may be quite able to tolerate a range of media and supplements, many normal cells and stems cells are not. Optimization of each component may be required to successfully maintain the latter cell types. The procedures for selecting and optimizing cell culture media and supplements are presented.

Regulatory T Cells: Central Concepts from Ontogeny to Therapy.

The balanced differentiation of naive CD4+ T cells into either pro- or anti-inflammatory fates is a central regulator of immune homeostasis, dysregulation of which can lead to inflammatory disease or cancer. Accordingly, the development of diagnostics and therapeutics to measure and modulate this balance is of great interest. In this review, we focus on the predominant anti-inflammatory subset, regulatory T cells, discussing key concepts including development, function, antigen specificity, and lineage stability. In particular, we highlight how these notions are shaping the evolution of therapeutics, especially in the context of the transfusion medicine specialist, and identify several key areas that urgently need to be addressed.

Recent advances for spermatogonial stem cell transplantation in livestock.

At the foundation of spermatogenesis are the actions of spermatogonial stem cells (SSCs), and a remarkable feature of these cells is the capacity to regenerate spermatogenesis following transplantation into testes of a recipient male that lacks endogenous germline. This ability could be exploited in livestock production as a breeding tool to enhance genetic gain. A key element to success is derivation of culture conditions that support proliferation of SSCs to provide sufficient numbers of cells for transfer into multiple recipient males. Using methodology devised for rodent cells as a foundation, advances in culturing cattle SSCs have occurred over the past few years and efforts are underway to extend this capability to pig cells. Another critical component to SSC transplantation is generation of males with germline ablation but intact somatic support cell function that can serve as surrogate sires for donor-derived spermatogenesis in a natural mating scheme. Recent advances in pigs using gene editing technologies have demonstrated that knockout of a key male germ cell-specific gene, namely NANOS2, leads to male-specific germline ablation but otherwise normal physiology, including intact seminiferous tubules. Together with recent advances in culturing spermatogonia of higher-order mammals, the now efficient means of producing germline-ablated recipient males have brought the application of SSC transplantation in livestock as a production tool closer to reality than ever before.

Scaffold-free Tissue Formation Under Real and Simulated Microgravity Conditions.

Scaffold-free tissue formation in microgravity is a new method in regenerative medicine and an important topic in Space Medicine. In this MiniReview, we focus on recent findings in the field of tissue engineering that were observed by exposing cells to real microgravity in space or to devices simulating to at least some extent microgravity conditions on Earth (ground-based facilities). Under both conditions - real and simulated microgravity - a part of the cultured cells of various populations detaches from the bottom of a culture flask. The cells form three-dimensional (3D) aggregates resembling the organs from which the cells have been derived. As spaceflights are rare and extremely expensive, cell culture under simulated microgravity allows more comprehensive and frequent studies on the scaffold-free 3D tissue formation in some aspects, as a number of publications have proven during the last two decades. In this MiniReview, we summarize data from our own studies and work from various researchers about tissue engineering of multi-cellular spheroids formed by cancer cells, tube formation by endothelial cells and cartilage formation by exposing the cells to ground-based facilities such as the 3D Random Positioning Machine (RPM), the 2D Fast-Rotating Clinostat (FRC) or the Rotating Wall Vessel (RWV). Subsequently, we investigated self-organization of 3D aggregates without scaffolds pursuing to enhance the frequency of 3D formation and to enlarge the size of the organ-like aggregates. The density of the monolayer exposed to real or simulated microgravity as well as the composition of the culture media revealed an impact on the results. Genomic and proteomic alterations were induced by simulated microgravity. Under microgravity conditions, adherent cells expressed other genes than cells grown in spheroids. In this MiniReview, the recent improvements in scaffold-free tissue formation are summarized and relationships between phenotypic and molecular appearance are highlighted.

Regenerative Medicine: Solution in Sight.

The retina, like other central nervous system tissues, has poor regenerative properties in humans. Therefore, diseases that cause retinal cell loss, such as Age-related macular degeneration (AMD), retinitis pigmentosa (RP), Leber congenital amaurosis, Usher syndrome, glaucoma, and diabetic retinopathy, typically result in permanent visual impairment. Stem cell technologies have revolutionized our ability to produce neural cells in abundant supply. Much stem cell research effort is focused on producing the required cell types for cell replacement, or to generate disease-in-a-dish models to elucidate novel disease mechanisms for therapeutic development. Here we review the recent advances in stem cell studies relevant to producing RPE and retinal cells, and highlight future directions.

Drug Discovery Goes Three-Dimensional: Goodbye to Flat High-Throughput Screening?

Immortalized cells, generated from two-dimensional cell culture techniques, are widely used in compound screening, lead optimization, and drug candidate selection. However, such cells lack many characteristics of cells in vivo. This could account for the high failure rates of lead candidates in clinical evaluation. New approaches from cell biology, materials science, and bioengineering are increasing the utility of three-dimensional (3D) culture. These approaches have become more compatible with automation and, thus, provide more physiologically relevant cells for high-throughput/high-content screening, notably in oncology drug discovery. Techniques range from simple 3D spheroids, comprising one or more cell types, to complex multitissue organoids cultured in extracellular matrix gels or microfabricated chips. Furthermore, each approach can be applied to stem cells, such as induced pluripotent stem cells, thereby providing additional phenotypic relevance and the exciting potential to enable screening in disease-specific cell types.

Human embryonic stem cell cultivation: historical perspective and evolution of xeno-free culture systems.

Human embryonic stem cells (hESC) have emerged as attractive candidates for cell-based therapies that are capable of restoring lost cell and tissue function. These unique cells are able to self-renew indefinitely and have the capacity to differentiate in to all three germ layers (ectoderm, endoderm and mesoderm). Harnessing the power of these pluripotent stem cells could potentially offer new therapeutic treatment options for a variety of medical conditions. Since the initial derivation of hESC lines in 1998, tremendous headway has been made in better understanding stem cell biology and culture requirements for maintenance of pluripotency. The approval of the first clinical trials of hESC cells for treatment of spinal cord injury and macular degeneration in 2010 marked the beginning of a new era in regenerative medicine. Yet it was clearly recognized that the clinical utility of hESC transplantation was still limited by several challenges. One of the most immediate issues has been the exposure of stem cells to animal pathogens, during hESC derivation and during in vitro propagation. Initial culture protocols used co-culture with inactivated mouse fibroblast feeder (MEF) or human feeder layers with fetal bovine serum or alternatively serum replacement proteins to support stem cell proliferation. Most hESC lines currently in use have been exposed to animal products, thus carrying the risk of xeno-transmitted infections and immune reaction. This mini review provides a historic perspective on human embryonic stem cell culture and the evolution of new culture models. We highlight the challenges and advances being made towards the development of xeno-free culture systems suitable for therapeutic applications.

New trends in biotechnological production of rosmarinic acid.

Rosmarinic acid (RA), an ester of caffeic acid and 3,4-dihydroxyphenyl lactic acid, is widely distributed in the plant kingdom. Interest in it is growing due to its promising biological activities, including cognitive-enhancing effects and slowing the development of Alzheimer's disease, cancer chemoprotection or anti-inflammatory activity, among others. In order to meet the increasing demand for this compound, several biotechnological approaches to its production based on plant cell and hairy root cultures have been developed. Empirical strategies are currently being combined with metabolic engineering tools to increase RA production in plant cell platforms in a more rational way. Discussed here are the latest advances in the field, together with recent trends in plant biotechnology, such as the application of single use technology and the use of biosensors in downstream processes.