Conductive Biomaterials as Substrates for Neural Stem Cells Differentiation towards Neuronal Lineage Cells

The injuries and defects in the central nervous system are the causes of disability and death of an affected person. As of now, there are no clinically available methods to enhance neural structural regeneration and functional recovery of nerve injuries. Recently, some experimental studies claimed that the injuries in brain can be repaired by progenitor or neural stem cells located in the neurogenic sites of adult mammalian brain. Various attempts have been made to construct biomimetic physiological microenvironment for neural stem cells to control their ultimate fate. Conductive materials have been considered as one the best choices for nerve regeneration due to the capacity to mimic the microenvironment of stem cells and regulate the alignment, growth, and differentiation of neural stem cells. The review highlights the use of conductive biomaterials, e.g., polypyrrole, polyaniline, poly(3,4‐ethylenedioxythiophene), multi‐walled carbon nanotubes, single‐wall carbon nanotubes, graphene, and graphite oxide, for controlling the neural stem cells activities in terms of proliferation and neuronal differentiation. The effects of conductive biomaterials in axon elongation and synapse formation for optimal repair of central nervous system injuries are also discussed.     

Cultivation of Encapsulated Primary Human B Lymphocytes: A First Step toward a Bioartificial Germinal Center

Polyelectrolyte microcapsules based on sodium cellulose sulfate (SCS) and poly-diallyl-dimethyl-ammonium chloride (PDADMAC) have previously been proposed as a suitable ex vivo microenvironment for the cultivation and differentiation of primary human T lymphocytes. Here, the same system is investigated for the cultivation of human primary B cells derived from adult tonsillar tissue. Proliferation and differentiation into subtypes are followed and compared to suspension cultures of B cells from the same pool performed in parallel. Total cell expansion is somewhat lower in the capsules than in the suspension cultures. More importantly, however, the differentiation of the initially mainly memory B cells into various subtypes, in particular into plasma cell (PC), shows significant differences. Clearly, the microenvironment provided by the microcapsules is beneficial for an accelerated induction of a germinal center-like B cell phenotype and afterward supports the long-term survival of the PC cells. Then, varying the encapsulation conditions (i.e., presence of human serum and dedicated cytokines in the capsule core) provides a tool for finetuning the B cell response. Hence, this methodology is suggested to pave the way toward ex vivo development of human immune organoids.     

Hepatocyte organoids and cell transplantation: What the future holds

Historically, primary hepatocytes have been difficult to expand or maintain in vitro. In this review, we will focus on recent advances in establishing hepatocyte organoids and their potential applications in regenerative medicine. First, we provide a background on the renewal of hepatocytes in the homeostatic as well as the injured liver. Next, we describe strategies for establishing primary hepatocyte organoids derived from either adult or fetal liver based on insights from signaling pathways regulating hepatocyte renewal in vivo. The characteristics of these organoids will be described herein. Notably, hepatocyte organoids can adopt either a proliferative or a metabolic state, depending on the culture conditions. Furthermore, the metabolic gene expression profile can be modulated based on the principles that govern liver zonation. Finally, we discuss the suitability of cell replacement therapy to treat different types of liver diseases and the current state of cell transplantation of in vitro-expanded hepatocytes in mouse models. In addition, we provide insights into how the regenerative microenvironment in the injured host liver may facilitate donor hepatocyte repopulation. In summary, transplantation of in vitro-expanded hepatocytes holds great potential for large-scale clinical application to treat liver diseases.Subject terms: Adult stem cells, Self-renewal     

Laponite–Gelatin Nanofibrous Microsphere Promoting Human Dental Follicle Stem Cells Attachment and Osteogenic Differentiation for Noninvasive Stem Cell Transplantation

Nanofibrous microspheres (NFM) are emerging as prominent next-generation biomimetic injectable scaffold system for stem cell delivery and different tissue regeneration where nanofibrous topography facilitates ECM-like stem cells niches. Addition of osteogenic bioactive nanosilicate platelets within NFM can provide osteoconductive cues to facilitate matrix mediated osteogenic differentiation of stem cells and enhance the efficiency of bone tissue regeneration. In this study, gelatin nanofibrous microspheres are prepared containing fluoride-doped laponite XL21 (LP) using the emulsion mediated thermal induce phase separation (TIPS) technique. Systematic studies are performed to understand the effect of physicochemical properties of biomimicking NFM alone and with different concentrations of LP on human dental follicle stem cells (hDFSCs), their cellular attachment, proliferation, and osteogenic differentiation. The study highlights the effect of LP nanosilicate with biomimicking nanofibrous injectable scaffold system aiding in enhancing stem cell differentiation under normal physiological conditions compared to NFM without LP. The laponite–NFM shows suitability as excellent injectable biomaterials system for stem cell attachment, proliferation and osteogenic differentiation for stem cell transplantation and bone tissue regeneration.     

Design of Polymeric Culture Substrates to Promote Proangiogenic Potential of Stem Cells

Stem cells are a promising cell source for regenerative medicine due to their differentiation and self‐renewal capacities. In the field of regenerative medicine and tissue engineering, a variety of biomedical technologies have been tested to improve proangiogenic activities of stem cells. However, their therapeutic effect is found to be limited in the clinic because of cell loss, senescence, and insufficient therapeutic activities. To address this type of issue, advanced techniques for biomaterial synthesis and fabrication have been approached to mimic proangiogenic microenvironment and to direct proangiogenic activities. This review highlights the types of polymers and design strategies that have been studied to promote proangiogenic activities of stem cells. In particular, scaffolds, hydrogels, and surface topographies, as well as insight into their underlying mechanisms to improve proangiogenic activities are the focuses. The strategy to promote angiogenic activities of hMSCs by controlling substrate repellency is introduced, and the future direction is proposed.     

Strategies for genetic manipulation of adult stem cell-derived organoids

Organoid technology allows the expansion of primary epithelial cells from normal and diseased tissues, providing a unique model for human (patho)biology. In a three-dimensional environment, adult stem cells self-organize and differentiate to gain tissue-specific features. Accessibility to genetic manipulation enables the investigation of the molecular mechanisms underlying cell fate regulation, cell differentiation and cell interactions. In recent years, powerful methodologies using lentiviral transgenesis, CRISPR/Cas9 gene editing, and single-cell readouts have been developed to study gene function and carry out genetic screens in organoids. However, the multicellularity and dynamic nature of stem cell-derived organoids also present challenges for genetic experimentation. In this review, we focus on adult gastrointestinal organoids and summarize the state-of-the-art protocols for successful transgenesis. We provide an outlook on emerging genetic techniques that could further increase the applicability of organoids and enhance the potential of organoid-based techniques to deepen our understanding of gene function in tissue biology.Subject terms: Mechanisms of disease, Genetic engineering, Mutagenesis, Experimental models of disease     

Role of stem cell niche in body aging processes

Replacement of damaged differentiated cells by new cells due to the asymmetric division of stem cells can provide unlimited tolerance to degeneration of human tissues, if multipotent cells themselves are “immortal.” However, stem cells exist in a particular microenvironment (stem cell niche) and feel their negative impact with age, which attenuates the proliferate capacity of stem cells and leads to incorrect differentiation or, instead, uncontrolled division (promotion of tumor genesis). As a result, stem cell injection to elder persons will be not efficient, because “young” stem cell will engraft to the “old” niche. To sufficient increasing of lifespan, it is necessary to modify stem niche signals by injection of an additional quantity of required growth factors in well-defined proportions.     

Reconstructing the differentiation niche of embryonic stem cells using biomaterials

The biochemical cues and topographical architecture of the extracellular environment extensively influence ES cell fate. The microenvironment surrounding the developing embryo presents these instructive cues in a complex and interactive manner in order to guide cell fate decisions. Current stem cell research aims to reconstruct this multifaceted embryonic niche to recapitulate development in vitro. This review focuses on 2D and 3D differentiation niches created from natural and synthetic biomaterials to guide the differentiation of ES cells toward specific lineages. Biomaterials engineered to present specific physical constraints are also reviewed for their role in differentiation.     

A novel silk/PES hybrid nanofibrous scaffold promotes the in vitro proliferation and differentiation of adipose‐derived mesenchymal stem cells into insulin producing cells

Using stem cells to replace the lost beta cells is a hopeful strategy in the treatment of diabetic patients. Furthermore, during stem cell culture and therapy, it is a need to use a substrate to act as a supportive matrix to mimic 3D in vivo microenvironment. Therefore, in this study, human adipose‐derived stem cells were used to differentiate into insulin‐producing cells (IPCs) on a silk/polyethersulfone (PES) scaffold. After exposing to the differentiation media, 2D and 3D (silk/PES) cultured cells were gradually aggregated and formed spherical shaped clusters. The viability of cells was comparable in both 3D and 2D culture. As the results of gene expression assay in both RNA and protein level showed, the differentiation efficiency was higher in 3D culture. Furthermore, ELISA revealed that the release of C‐peptide and insulin was higher in 3D than 2D culture. It seems that silk/PES nanofibrous hybrid scaffold could provide an appropriate matrix to mimic in vivo microenvironment and therefore increases the IPC differentiation potency of stem cells.     

Advances in Biomimetic Nanoparticles for Targeted Cancer Therapy and Diagnosis

Biomimetic nanoparticles have recently emerged as a novel drug delivery platform to improve drug biocompatibility and specificity at the desired disease site, especially the tumour microenvironment. Conventional nanoparticles often encounter rapid clearance by the immune system and have poor drug-targeting effects. The rapid development of nanotechnology provides an opportunity to integrate different types of biomaterials onto the surface of nanoparticles, which enables them to mimic the natural biological features and functions of the cells. This mimicry strategy favours the escape of biomimetic nanoparticles from clearance by the immune system and reduces potential toxic side effects. Despite the rapid development in this field, not much has progressed to the clinical stage. Thus, there is an urgent need to develop biomimetic-based nanomedicine to produce a highly specific and effective drug delivery system, especially for malignant tumours, which can be used for clinical purposes. Here, the recent developments for various types of biomimetic nanoparticles are discussed, along with their applications for cancer imaging and treatments.     

Recent Development of Bio-inspired Porous Materials for Catalytic Applications

Nature has served as a source of inspiration for chemists to design the catalysts with superior performance via mimicking their characteristics, such as active sites, reaction microenvironment and electron transfer behaviors. Over a few decades, many different types of materials have been widely used to construct the biomimetic catalysts. Among these materials, the porous materials have been demonstrated to be powerful platforms for constructing the biomimetic catalysts owing to their high surface area, controllable composition and easy functionalization. In this review, we briefly showed recent advances in the construction of various biomimetic porous catalysts using porous materials for the design of excellent catalysts. In addition, we also present the challenge and opportunities in this emerging field.     

Generation of Haploid Spermatids on Silk Fibroin-Alginate-Laminin-Based Porous 3D Scaffolds

In vitro production of sperm is a desirable idea for fertility preservation in azoospermic men and prepubertal boys suffering from cancer. In this study, a biocompatible porous scaffold based on a triad mixture of silk fibroin (SF), alginate (Alg), and laminin (LM) is developed to facilitate the differentiation of mouse spermatogonia stem cells (SSCs). Following SF extraction, the content is analyzed by SDS-PAGE and stable porous 3D scaffolds are successfully prepared by merely Alg, SF, and a combination of Alg-SF, or Alg-SF-LM through freeze-drying. Then, the biomimetic scaffolds are characterized regarding the structural and biological properties, water absorption capacity, biocompatibility, biodegradability, and mechanical behavior. Neonatal mice testicular cells are seeded on three-dimensional scaffolds and their differentiation efficiency is evaluated using real-time PCR, flow cytometry, immunohistochemistry. Blend matrices showed uniform porous microstructures with interconnected networks, which maintained long-term stability and mechanical properties better than homogenous structures. Molecular analysis of the cells after 21 days of culture showed that the expression of differentiation-related proteins in cells that are developed in composite scaffolds is significantly higher than in other groups. The application of a composite system can lead to the differentiation of SSCs, paving the way for a novel infertility treatment landscape in the future.     

可注射水凝胶及其在再生医学领域的应用

近年来,由工程生物材料制成的可注射治疗剂正变得越来越流行,并推动传统的临床实践走向微创化.可注射水凝胶由于其可调控的物理及化学特性、可控的降解性能、高含水量以及在微创方式下实现递送的能力,在组织工程和药物递送领域中变得越来越重要.研究者们已开发出例如原位交联水凝胶、大孔水凝胶、水凝胶微粒、动态交联水凝胶等一系列性能独特的可注射水凝胶.通过调控水凝胶的固含量和交联密度,并引入适当的共价或非共价相互作用,例如静电相互作用、疏水相互作用等,这些水凝胶可在注射过程中实现生物活性分子的递送.同时,可注射水凝胶亦可用于细胞的递送,提供细胞培养所需的三维环境,并通过调控力学性能、化学修饰、生物功能化修饰等手段调控细胞黏附、增殖、分化等行为.本文旨在回顾近年来可注射水凝胶的设计和制备的相关进展,以及其在再生医学中的应用,并对该领域存在的挑战和潜力进行了展望.     

Biomimetic Macroporous PCL Scaffolds for Ex Vivo Expansion of Cord Blood‐Derived CD34+ Cells with Feeder Cells Support

Ex vivo expansion of hematopoietic stem cells (HSCs) with most current methods can hardly satisfy clinical application requirement. While in vivo, HSCs efficiently self‐renew in niche where they interact with 3D extracellular matrix and stromal cells. Therefore, co‐cultures of CD34⁺ cells and mesenchyme stem cells derived from human amniotic membrane (hAMSCs) on the basis of biomimetic macroporous three‐dimensional (3D) poly(ε‐caprolactone) (PCL) scaffolds are developed, where scaffolds and hAMSCs are applied to mimic structural and cellular microenvironment of HSCs. The influence of scaffolds, feeder cells, and contact manners on expansion and stemness maintenance of CD34⁺ cells is investigated in this protocol. Biomimetic scaffolds‐dependent co‐cultures of CD34⁺ cells and hAMSCs can effectively promote the expansion of CD34⁺ cells; meanwhile, indirect contact is superior to direct contact. The combination of biomimetic scaffolds and hAMSCs represents a new strategy for achieving clinical‐scale ex vivo expansion of CD34⁺ cells.

     

Permselectivity Replication of Artificial Glomerular Basement Membranes in Nanoporous Collagen Multilayers

Basement membranes (BMs) play important roles in many biological functions such as tissue regeneration, cancer proliferation, nutrient/drug delivery, breathing, and many others. While there are many theoretical models, adequate experimental analogs of BMs describing basic physicochemical properties of BM, such as diffusion and permselectivity are not available. Taking BMs found in glomerulus of kidneys as an example, adequate reproduction of their permselectivity requires biomimetic membranes with submicron thickness, high uniformity, nanoscale porosity, and size-selective permeability. Artificial kidney BMs were assembled from poly(acrylic acid) and collagen using layer-by-layer (LBL) assembly technology and display multiple structural similarities with glomerular BMs. Diffusional transport through the artificial BMs faithfully replicate cut-off parameters of kidney membranes. Their utilization in understanding of unique diffusion processes in kidneys, in vitro studies of blood clearance time of small drugs/nanoscale drug carriers and design of more complex organoids including live cells for cancer proliferation studies is anticipated.     

Microtopography based on inverse opal structures regulates the behavior of bone marrow‐derived mesenchymal stem cells

Physical cues from the extracellular microenvironment play an important role in regulating cell behavior, such as adhesion, migration, and differentiation. Many studies have shown that different physical parameters (eg, stiffness and topography) could modulate the in vitro differentiation of mesenchymal stem cells (MSCs), which had multilineage differentiation potential and could be easily isolated from various tissues such as bone marrow, adipose tissue, and the umbilical cord. However, the underlying mechanism of the topographical influence on MSCs and the detailed cell‐substrate interaction remain unclear. Here, we present oriented elliptical inverse opal structures for regulating the morphology and alignment of bone marrow‐derived MSCs. The inverse opal structures were made through a convenient bottom‐up approach of self‐assembly, which is facile and cost effective. MSCs cultured on the oriented structures were highly aligned and extended highly oriented thick lamellipodia. Moreover, the oriented substrates cracked along the lateral boundary of the cells, suggesting that a strong cell‐substrate interaction was induced by the response of MSCs to the oriented topography. These features of the oriented elliptical topography indicated their promising value in stem cell research and tissue engineering.     

3D cell culture models and organ-on-a-chip: Meet separation science and mass spectrometry

In vitro derived simplified 3D representations of human organs or organ functionalities are predicted to play a major role in disease modeling, drug development, and personalized medicine, as they complement traditional cell line approaches and animal models. The cells for 3D organ representations may be derived from primary tissues, embryonic stem cells or induced pluripotent stem cells and come in a variety of formats from aggregates of individual or mixed cell types, self-organizing in vitro developed “organoids” and tissue mimicking chips. Microfluidic devices that allow long-term maintenance and combination with other tissues, cells or organoids are commonly referred to as “microphysiological” or “organ-on-a-chip” systems. Organ-on-a-chip technology allows a broad range of “on-chip” and “off-chip” analytical techniques, whereby “on-chip” techniques offer the possibility of real time tracking and analysis. In the rapidly expanding tool kit for real time analytical assays, mass spectrometry, combined with “on-chip” electrophoresis, and other separation approaches offer attractive emerging tools. In this review, we provide an overview of current 3D cell culture models, a compendium of current analytical strategies, and we make a case for new approaches for integrating separation science and mass spectrometry in this rapidly expanding research field.     

High Content Screening for Compounds that Induce Early Stages of Human Embryonic Stem Cell Differentiation

Embryonic stem cells, due to their self-renewal and pluripotency properties, can be used to repair damaged tissues and as an unlimited source of differentiated cells. Although stem cells represent an important opportunity for cell based therapy and small molecules screening (in the context of drug or target discovery) many drawbacks are still preventing their widespread use. One of the most significant limitations is related to the complexity, as well as the reliability, of current protocols driving stem cells into any homogeneously differentiated cellular population. In this respect there is a strong demand for molecular agents promoting differentiation and thereby enabling robust, efficient and safe production of differentiated cells. In order to identify novel molecules that enhance early stages of differentiation, we developed an image based high content screening (HCS) approach using human embryonic stem cells (hESC). In our approach, we took advantage of custom image mining software specifically adapted for the selection of stem cell differentiation agents and the rejection of false positive hits. As a proof of concept ～3500 small molecules originating from commercial libraries were screened and a number of molecules of interests were identified. These molecules show stem cell differentiation properties comparable to the phenotypic signature obtained with the reference compound retinoic acid.     

Human neural stem cell growth and differentiation in a gradient-generating microfluidic device

This paper describes a gradient-generating microfluidic platform for optimizing proliferation and differentiation of neural stem cells (NSCs) in culture. Microfluidic technology has great potential to improve stem cell (SC) cultures, whose promise in cell-based therapies is limited by the inability to precisely control their behavior in culture. Compared to traditional culture tools, microfluidic platforms should provide much greater control over cell microenvironment and rapid optimization of media composition using relatively small numbers of cells. Our platform exposes cells to a concentration gradient of growth factors under continuous flow, thus minimizing autocrine and paracrine signaling. Human NSCs (hNSCs) from the developing cerebral cortex were cultured for more than 1 week in the microfluidic device while constantly exposed to a continuous gradient of a growth factor (GF) mixture containing epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2) and platelet-derived growth factor (PDGF). Proliferation and differentiation of NSCs into astrocytes were monitored by time-lapse microscopy and immunocytochemistry. The NSCs remained healthy throughout the entire culture period, and importantly, proliferated and differentiated in a graded and proportional fashion that varied directly with GF concentration. These concentration-dependent cellular responses were quantitatively similar to those measured in control chambers built into the device and in parallel cultures using traditional 6-well plates. This gradient-generating microfluidic platform should be useful for a wide range of basic and applied studies on cultured cells, including SCs.