Engineering stem cell niches in bioreactors

Stem cells, including embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells and amniotic fluid stem cells have the potential to be expanded and differentiated into various cell types in the body. Efficient differentiation of stem cells with the desired tissue-specific function is critical for stem cell-based cell therapy, tissue engineering, drug discovery and disease modeling. Bioreactors provide a great platform to regulate the stem cell microenvironment, known as “niches”, to impact stem cell fate decision. The niche factors include the regulatory factors such as oxygen, extracellular matrix (synthetic and decellularized), paracrine/autocrine signaling and physical forces (i.e., mechanical force, electrical force and flow shear). The use of novel bioreactors with precise control and recapitulation of niche factors through modulating reactor operation parameters can enable efficient stem cell expansion and differentiation. Recently, the development of microfluidic devices and microbioreactors also provides powerful tools to manipulate the stem cell microenvironment by adjusting flow rate and cytokine gradients. In general, bioreactor engineering can be used to better modulate stem cell niches critical for stem cell expansion, differentiation and applications as novel cell-based biomedicines. This paper reviews important factors that can be more precisely controlled in bioreactors and their effects on stem cell engineering.     

Stem cell mechanobiology

Stem cells are undifferentiated cells that are capable of proliferation, self‐maintenance and differentiation towards specific cell phenotypes. These processes are controlled by a variety of cues including physicochemical factors associated with the specific mechanical environment in which the cells reside. The control of stem cell biology through mechanical factors remains poorly understood and is the focus of the developing field of mechanobiology. This review provides an insight into the current knowledge of the role of mechanical forces in the induction of differentiation of stem cells. While the details associated with individual studies are complex and typically associated with the stem cell type studied and model system adopted, certain key themes emerge. First, the differentiation process affects the mechanical properties of the cells and of specific subcellular components. Secondly, that stem cells are able to detect and respond to alterations in the stiffness of their surrounding microenvironment via induction of lineage‐specific differentiation. Finally, the application of external mechanical forces to stem cells, transduced through a variety of mechanisms, can initiate and drive differentiation processes. The coalescence of these three key concepts permit the introduction of a new theory for the maintenance of stem cells and alternatively their differentiation via the concept of a stem cell ‘mechano‐niche’, defined as a specific combination of cell mechanical properties, extracellular matrix stiffness and external mechanical cues conducive to the maintenance of the stem cell population. J. Cell. Biochem. 112: 1–9, 2011. © 2010 Wiley‐Liss, Inc.     

Mechanoresponse of stem cells for vascular repair

Stem cells have shown great potential in vascular repair. Numerous evidence indicates that mechanical forces such as shear stress and cyclic strain can regulate the adhesion, proliferation, migration, and differentiation of stem cells via serious signaling pathways. The enrichment and differentiation of stem cells play an important role in the angiogenesis and maintenance of vascular homeostasis. In normal tissues, blood flow directly affects the microenvironment of vascular endothelial cells (ECs); in pathological status, the abnormal interactions between blood flow and vessels contribute to the injury of vessels. Next, the altered mechanical forces are transduced into cells by mechanosensors to trigger the reformation of vessels. This process occurs when signaling pathways related to EC differentiation are initiated. Hence, a deep understanding of the responses of stem cells to mechanical stresses and the underlying mechanisms involved in this process is essential for clinical translation. In this the review, we provide an overview of the role of stem cells in vascular repair, outline the performance of stem cells under the mechanical stress stimulation, and describe the related signaling pathways.     

Effects of shear stress on differentiation of stem cells into endothelial cells

Stem cell transplantation is an appealing potential therapy for vascular diseases and an indispensable key step in vascular tissue engineering. Substantial effort has been made to differentiate stem cells toward vascular cell phenotypes, including endothelial cells (ECs) and smooth muscle cells. The microenvironment of vascular cells not only contains biochemical factors that influence differentiation but also exerts hemodynamic forces, such as shear stress and cyclic strain. More recently, studies have shown that shear stress can influence the differentiation of stem cells toward ECs. A deep understanding of the responses and underlying mechanisms involved in this process is essential for clinical translation. This review highlights current data supporting the role of shear stress in stem cell differentiation into ECs. Potential mechanisms and signaling cascades for transducing shear stress into a biological signal are proposed. Further study of stem cell responses to shear stress will be necessary to apply stem cells for pharmacological applications and cardiovascular implants in the realm of regenerative medicine.     

High throughput screening to investigate the interaction of stem cells with their extracellular microenvironment

Stem cells in vivo are housed within a functional microenvironment termed the “stem cell niche.” As the niche components can modulate stem cell behaviors like proliferation, migration and differentiation, evaluating these components would be important to determine the most optimal platform for their maintenance or differentiation. In this review, we have discussed methods and technologies that have aided in the development of high throughput screening assays for stem cell research, including enabling technologies such as the well-established multiwell/microwell plates and robotic spotting, and emerging technologies like microfluidics, micro-contact printing and lithography. We also discuss the studies that utilized high throughput screening platform to investigate stem cell response to extracellular matrix, topography, biomaterials and stiffness gradients in the stem cell niche. The combination of the aforementioned techniques could lay the foundation for new perspectives in further development of high throughput technology and stem cell research.     

Review of biophysical factors affecting osteogenic differentiation of human adult adipose-derived stem cells

Developing bone is subject to the control of a broad variety of influences in vivo. For bone repair applications, in vitro osteogenic assays are routinely used to test the responses of bone-forming cells to drugs, hormones, and biomaterials. Results of these assays are used to predict the behavior of bone-forming cells in vivo. Stem cell research has shown promise for enhancing bone repair. In vitro osteogenic assays to test the bone-forming response of stem cells typically use chemical solutions. Stem cell in vitro osteogenic assays often neglect important biophysical cues, such as the forces associated with regular weight-bearing exercise, which promote bone formation. Incorporating more biophysical cues that promote bone formation would improve in vitro osteogenic assays for stem cells. Improved in vitro osteogenic stimulation opens opportunities for “pre-conditioning” cells to differentiate towards the desired lineage. In this review, we explore the role of select biophysical factors—growth surfaces, tensile strain, fluid flow and electromagnetic stimulation—in promoting osteogenic differentiation of stem cells from human adipose. Emphasis is placed on the potential for physical microenvironment manipulation to translate tissue engineering and stem cell research into widespread clinical usage.     

Stem cells as probabilistic self-producing entities

Stem cells have the capacity both to self-renew and to give rise to differentiated progeny, and are vital to the organization of multicellular organisms. Stem cells raise a number of fundamental questions regarding lineage restriction and cellular differentiation, and they hold enormous promise for cell-based therapies. Here I propose a theoretical framework for stem cell biology based on the concepts of autopoiesis (self-production) and complementarity. I argue that stem cells are pivotal in the self-production of the organism and that we need complementary approaches to understand their probabilistic behavior. I discuss how this framework generates testable hypotheses regarding stem-cell functions.     

Cell adhesion and mechanical stimulation in the regulation of mesenchymal stem cell differentiation

Stem cells have been shown to have the potential to provide a source of cells for applications to tissue engineering and organ repair. The mechanisms that regulate stem cell fate, however, mostly remain unclear. Mesenchymal stem cells (MSCs) are multipotent progenitor cells that are isolated from bone marrow and other adult tissues, and can be differentiated into multiple cell lineages, such as bone, cartilage, fat, muscles and neurons. Although previous studies have focused intensively on the effects of chemical signals that regulate MSC commitment, the effects of physical/mechanical cues of the microenvironment on MSC fate determination have long been neglected. However, several studies provided evidence that mechanical signals, both direct and indirect, played important roles in regulating a stem cell fate. In this review, we summarize a number of recent studies on how cell adhesion and mechanical cues influence the differentiation of MSCs into specific lineages. Understanding how chemical and mechanical cues in the microenvironment orchestrate stem cell differentiation may provide new insights into ways to improve our techniques in cell therapy and organ repair.     

Stem cell niches in mammals

Stem cells safeguard tissue homeostasis and guarantee tissue repair throughout life. The decision between self-renewal and differentiation is influenced by a specialized microenvironment called stem cell niche. Physical and molecular interactions with niche cells and orientation of the cleavage plane during stem cell mitosis control the balance between symmetric and asymmetric division of stem cells. Here we highlight recent progress made on the anatomical and molecular characterization of mammalian stem cell niches, focusing particularly on bone marrow, tooth and hair follicle. The knowledge of the regulation of stem cells within their niches in health and disease will be instrumental to develop novel therapies that target stem cell niches to achieve tissue repair and re-establish tissue homeostasis.     

Effect of Mechanical Forces on the Behavior of Dental Stem Cells: A Scoping Review of <i>In-Vitro</i> Studies

This article is a scoping review of the studies that assessed the effect of mechanical forces on the behavior of dental stem cells (DSCs). PubMed and Scopus searches were done for in-vitro studies evaluating the effect of tension, hydrostatic pressure (i.e., the pressure applied through an incompressible fluid), compression, simulated microgravity, and vibration on DSCs. The following factors were analyzed: osteogenic/odontogenic differentiation, proliferation, adhesion and migration. Articles were reviewed according to the Preferred Reporting Items for Systematic Reviews extension for scoping reviews (PRISMA-ScR) guideline. Included studies were evaluated based on the modified Consolidated Standards of Reporting Trials (CONSORT). A total of 18 studies published from 2008–2019 were included. Nine studies were focusing on Periodontal ligament Stem Cells (PDLSCs), eight studies on Dental Pulp Stem Cells (DPSCs) and one study on Stem Cells from Apical Papilla (SCAP). Results showed that tension, three-dimensional stress and simulated microgravity promoted the proliferation and osteogenic differentiation of PDLSCs. DPSCs proliferation increased after microgravity and tension exertion. In addition, dynamic hydrostatic pressure and compression promoted odontogenic differentiation of DPSCs. Besides, mechanical stimuli increased the osteogenic differentiation of DPSCs. One study analyzed the effect of carrier features on the response of DSCs to 3D-stress and showed that cells cultivated on scaffolds with 30% bioactive glass (BAG) had the highest osteogenic differentiation compared to other ratios of BAG. It has been shown that increasing the duration of tension (i.e., from 3 h to 24 h force application) enhanced the positive effect of force application on the osteogenic differentiation of DSCs. In conclusion, all types of mechanical forces except uniaxial tension increased the osteogenic/odontogenic differentiation of DSCs. In addition, the effect of mechanical stimulation on the proliferation of DSCs differs based on the type of stem cells and mechanical force.     

Stem cell niche: Dynamic neighbor of stem cells

Stem cell niche is a specialized and dynamic microenvironment around the stem cells which plays a critical role in maintaining the stemness properties of stem cells. Over the years, advancement in the research activity has revealed the various important aspects of stem cell niche including cell-cell interaction, cell-extracellular matrix interaction, a large number of soluble signaling factors and various biochemical and biophysical cues (such as oxygen tension, flow, and shear and pore size). Stem cells have the potential to be a powerful tool in regenerative medicine due to their self-renewal property and immense differentiation potential. Recent progresses in in vitro culture conditions of embryonic stem cells, adult stem cells and induced pluripotent stem cells have enabled the researchers to investigate and understand the role of the microenvironment in stem cell properties. The engineered artificial stem cell niche has led to a better execution of stem cells in regenerative medicine. Here we elucidate the key components of stem cell niche and their role in niche engineering and stem cell therapeutics.     

机械力及力学信号转导影响干细胞命运的研究进展

干细胞作为一种未分化的祖细胞,目前已被广泛应用于开展组织损伤修复、再生以及干细胞特异谱系分化的研究.大量研究表明,干细胞所处的微环境对调控干细胞的生长和分化具有重要作用,多种溶液介质、细胞外基质和信号通路等参与了干细胞命运的调控.尽管已有大量研究证明,溶液介质(如激素和生长因子)在干细胞的生长和分化中发挥重要作用,但近年来越来越多的研究表明,机械力及力学信号转导同样在干细胞自我更新、分化、衰老和凋亡等细胞生理过程中起到重要的作用.本文将对机械应力响应的细胞基础、生物力学及力学信号调控干细胞自我更新和分化,以及生物力学调控干细胞命运可能的作用机制几个方面加以综述.     

Shear Force at the Cell-Matrix Interface: Enhanced Analysis for Microfabricated Post Array Detectors

The interplay of mechanical forces between the extracellular environment and the cytoskeleton drives development, repair, and senescence in many tissues. Quantitative definition of these forces is a vital step in understanding cellular mechanosensing. Microfabricated post array detectors (mPADs) provide direct measurements of cell-generated forces during cell adhesion to extracellular matrix. A new approach to mPAD post labeling, volumetric imaging, and an analysis of post bending mechanics determined that cells apply shear forces and not point moments at the matrix interface. In addition, these forces could be accurately resolved from post deflections by using images of post tops and bases. Image analysis tools were then developed to increase the precision and throughput of post centroid location. These studies resulted in an improved method of force measurement with broad applicability and concise execution using a fully automated force analysis system. The new method measures cell-generated forces with less than 5% error and less than 90 seconds of computational time. Using this approach, we demonstrated direct and distinct relationships between cellular traction force and spread cell surface area for fibroblasts, endothelial cells, epithelial cells and smooth muscle cells.     

Micropatterning of human embryonic stem cells dissects the mesoderm and endoderm lineages

Human pluripotent cells such as human embryonic stem cells (hESC) are a great potential source of cells for cell-based therapies; however, directing their differentiation into the desired cell types with high purity remains a challenge. The stem cell microenvironment plays a vital role in directing hESC fate and we have previously shown that manipulation of colony size in a serum- and cytokine-free environment controls self-renewal and differentiation toward the extraembryonic endoderm lineage. Here we show that, in the presence of bone morphogenetic protein 2 and activin A, control of colony size using a microcontact printing technology is able to direct hESC fate to either the mesoderm or the endoderm lineage. Large, 1200-μm-diameter colonies give rise to mesoderm, while small 200-μm colonies give rise to definitive endoderm. This study links, for the first time, cellular organization to pluripotent cell differentiation along the mesoderm and endoderm lineages.     

Influence of substrate composition on human embryonic stem cell differentiation and extracellular matrix production in embryoid bodies

Stem cells reside in specialized niches in vivo. Specific factors, including the extracellular matrix (ECM), in these niches are directly responsible for maintaining the stem cell population. During development, components of the stem cell microenvironment also control differentiation with precise spatial and temporal organization. The stem cell microenvironment is dynamically regulated by the cellular component, including stem cells themselves. Thus, a mechanism exists whereby stem cells modify the ECM, which in turn affects the fate of the stem cell. In this study, we investigated whether the type of ECM initially adsorbed to the culture substrate can influence the composition of the ECM deposited by human embryonic stem cells (hESCs) differentiating in embryoid bodies, and whether different ECM composition and deposition profiles elicit distinct differentiation fates. We have shown that the initial ECM environment hESCs are exposed to affects the fate decisions of those cells and that this initial ECM environment is constantly modified during the differentiation process. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:212–219, 2015     

Breast Cancer Cells Adapt Contractile Forces to Overcome Steric Hindrance

Cell migration through the extracellular matrix is governed by the interplay between cell-generated propulsion forces, adhesion forces, and resisting forces arising from the steric hindrance of the matrix. Steric hindrance in turn depends on matrix porosity, matrix deformability, cell size, and cell deformability. In this study, we investigate how cells respond to changes in steric hindrance that arise from altered cell mechanical properties. Specifically, we measure traction forces, cell morphology, and invasiveness of MDA-MB 231 breast cancer cells in three-dimensional collagen gels. To modulate cell mechanical properties, we either decrease nuclear deformability by twofold overexpression of the nuclear protein lamin A or we introduce into the cells stiff polystyrene beads with a diameter larger than the average matrix pore size. Despite this increase of steric hindrance, we find that cell invasion is only marginally inhibited, as measured by the fraction of motile cells and the mean invasion depth. To compensate for increased steric hindrance, cells employ two alternative strategies. Cells with higher nuclear stiffness increase their force polarity, whereas cells with large beads increase their net contractility. Under both conditions, the collagen matrix surrounding the cells stiffens dramatically and carries increased strain energy, suggesting that increased force polarity and increased net contractility are functionally equivalent strategies for overcoming an increased steric hindrance.     

Novel regulators revealed by profiling Drosophila testis stem cells within their niche

Stem cells are defined by the fact that they both self-renew, producing additional stem cells, and generate lineal descendants that differentiate into distinct functional cell types. In Drosophila, a small germline stem cell population is influenced by a complex microenvironment, the stem cell niche, which itself includes a somatic stem cell population. While stem cells are unique, their immediate descendants retain considerable stem cell character as they mitotically amplify prior to differentiation and can be induced to de-differentiate into stem cells. Despite their importance, very few genes are known that are expressed in the stem cells or their early amplifying daughters. We present here whole-genome microarray expression analysis of testes specifically enriched for stem cells, their amplifying daughters, and their niche. These studies have identified a number of loci with highly specific stem cell expression and provide candidate downstream targets of Jak/Stat self-renewal signaling. Furthermore, functional analysis for two genes predicted to be enriched has enabled us to define novel regulators of the germline lineage. The gene list generated in this study thus provides a potent resource for the investigation of stem cell identity and regulation from functional as well as evolutionary perspectives.     

果蝇生殖腺干细胞和它们的微环境

干细胞微环境是由容纳一个或多个干细胞,并控制干细胞自我更新和子代细胞产生的组织细胞以及细胞外基质组成。干细胞必须在微环境内才能增殖,才能保持自我更新的特性。通过对果蝇生殖腺干细胞微环境的结构及其产生的信号路径（该路径可以调节干细胞自我更新）的研究,发现微环境中支持细胞和它们发出的信号路径在调节干细胞的增殖和分化中起重要的作用。