Controlling cell–cell interactions using surface acoustic waves

The interactions between pairs of cells and within multicellular assemblies are critical to many biological processes such as intercellular communication, tissue and organ formation, immunological reactions, and cancer metastasis. The ability to precisely control the position of cells relative to one another and within larger cellular assemblies will enable the investigation and characterization of phenomena not currently accessible by conventional in vitro methods. We present a versatile surface acoustic wave technique that is capable of controlling the intercellular distance and spatial arrangement of cells with micrometer level resolution. This technique is, to our knowledge, among the first of its kind to marry high precision and high throughput into a single extremely versatile and wholly biocompatible technology. We demonstrated the capabilities of the system to precisely control intercellular distance, assemble cells with defined geometries, maintain cellular assemblies in suspension, and translate these suspended assemblies to adherent states, all in a contactless, biocompatible manner. As an example of the power of this system, this technology was used to quantitatively investigate the gap junctional intercellular communication in several homotypic and heterotypic populations by visualizing the transfer of fluorescent dye between cells.Multicellular systems rely on the interaction between cells to coordinate cell signaling and regulate cell functions. Understanding the mechanism and process of cell–cell interaction is critical to many physiological and pathological processes, such as embryogenesis, differentiation, cancer metastasis, immunological interactions, and diabetes (1–3). Despite significant advances in this field, to further understand how cells interact and communicate with each other, a robust, biocompatible method to precisely control the spatial and temporal association of cells and to create defined cellular assemblies is urgently needed (4). Although several methods have been used to pattern cells, limitations still exist for the demonstrated methods including those that make use of optical, electrical, magnetic, hydrodynamic, and contact printing technologies (5–9). Firstly, most of the methods require modification of the cell’s native state. The magnetic assembly method, for example, requires cells to be labeled with magnetic probes. Dielectrophoresis typically requires the use of a special medium (e.g., nonconductive) which may lack essential nutrients or have biophysical properties (such as the osmolality) that may adversely affect cell growth or physiology (6). Optical tweezers provide a label-free and contactless approach, but typically require high laser power to manipulate cells, leading to a high risk of cell damage (5). Secondly, the working principles of the existing technologies mostly preclude the combination of high precision and high throughput into a single device. It is difficult for high-throughput methods (such as magnetic assemblies) to achieve single-cell level precision, whereas the high-precision methods often require complex experimental setup to manipulate multiple cells simultaneously. Thirdly, most of the existing methodologies lack the ability to maintain cell assemblies in suspension, thereby limiting the application of these methods for the study of cell–cell and cell–matrix interactions.As an alternative to using optical, electrical, or magnetic forces to manipulate cells, it has been demonstrated that biological specimens can also be manipulated using acoustic forces (10–16). Acoustic force can be applied through either bulk acoustic waves (BAWs) or surface acoustic waves (SAWs). Compared with the conventional BAW-based approaches (12, 13), SAW-based approaches (14–16) are becoming increasingly important in applications in cell biology and medicine as SAWs allow simpler device fabrication and experimental setup, higher manipulation resolution and flexibility, and better compatibility with optical imaging systems (allowing use of transparent devices). Thus far, the SAW-based approach has been reported to be able to manipulate single cells (15), but it has not yet been demonstrated for controlling cell–cell distance and interactions. This is mainly due to the difficulties in achieving a sufficient level of regulation of pressure nodes, which is needed to control the position of the cells with a high degree of precision. In this study, we demonstrate a SAW device that can accurately and reproducibly control pressure nodes and perform various functions for cell–cell interaction studies. Through superposing two orthogonal standing SAWs with differential input frequencies, we achieved highly regulated dot-array configuration of pressure nodes that facilitate high-precision control of cell–cell interactions, rather than the net-array pressure node configurations used in previous SAW devices (14, 15). This acoustic tweezers cell-manipulation method does not require any modification of the growth conditions, allowing cells to be cultured in their native media. It is highly adaptable to the requirements of various applications and is capable of delivering both high precision (controlling intercellular distance at the micrometer scale) and high throughput (forming thousands of cell assemblies with tunable geometric configurations) in a single device. In addition, our method offers unprecedented flexibility over the control of cell assemblies. The geometry of cell assemblies can be finely tuned by changing the acoustic field. Moreover, the system is capable of holding cell assemblies in suspension at precise locations while assessing their biological functions without the use of permanent structures. These suspended cell assemblies can be allowed to settle to the surface to adhere and disperse. To demonstrate the power of this technology, we applied the system to explore gap junctional intercellular communication (GJIC) and quantitatively investigated various forms of functional intercellular communication by visualizing gap junctional dye exchange among coupled cells.     

When is a stem cell really a stem cell?

While bone marrow transplantation has long been established as an effective approach to the clinical management of a variety of malignant and nonmalignant diseases, the future application of pluripotent stem cells in transplant settings promises to deliver this therapy to a much broader range of indications. In this review, I summarize the emerging field of embryonic stem cell biology in the context of potential clinical applications and regulatory issues.     

Mechanical tugging force regulates the size of cell–cell junctions

Actomyosin contractility affects cellular organization within tissues in part through the generation of mechanical forces at sites of cell–matrix and cell–cell contact. While increased mechanical loading at cell–matrix adhesions results in focal adhesion growth, whether forces drive changes in the size of cell–cell adhesions remains an open question. To investigate the responsiveness of adherens junctions (AJ) to force, we adapted a system of microfabricated force sensors to quantitatively report cell–cell tugging force and AJ size. We observed that AJ size was modulated by endothelial cell–cell tugging forces: AJs and tugging force grew or decayed with myosin activation or inhibition, respectively. Myosin-dependent regulation of AJs operated in concert with a Rac1, and this coordinated regulation was illustrated by showing that the effects of vascular permeability agents (S1P, thrombin) on junctional stability were reversed by changing the extent to which these agents coupled to the Rac and myosin-dependent pathways. Furthermore, direct application of mechanical tugging force, rather than myosin activity per se, was sufficient to trigger AJ growth. These findings demonstrate that the dynamic coordination of mechanical forces and cell–cell adhesive interactions likely is critical to the maintenance of multicellular integrity and highlight the need for new approaches to study tugging forces.     

Detection of tumor stem cell markers in pancreatic carcinoma cell lines

IntroductionT he cell population of most tumors is hetero- geneous with regard to its proliferation capacity, apoptosis-resistance mechanisms, and ability to reconstitute the tumor upon xeno-transplantation. This phenomenon arises as the result of accumulation of multiple genetic and epigenetic changes. Current evidence suggests that only a few cells within the tumor, the cancer stem cells, possess unlimited proliferative capacity and give rise to tumors that phenotypically resemble their init…     

Impaired autophagy leads to abnormal dendritic cell–epithelial cell interactions

Background and aimsDendritic cells (DC) are key players in intestinal immunity, as these cells can direct the immune response to either a tolerogenic or an immunogenic phenotype. In the intestine, DC sample and process luminal antigens by protruding dendrites through the epithelial cell layer. At the same time barrier integrity is maintained through the continuous formation of tight junctions. Aberrations in these interactions may lead to altered antigen sampling and improper immune responses. We have recently shown that autophagy, a process implicated in the pathogenesis of Crohn's disease, regulates cellular interactions in the context of DC and T cells. In this study we aimed to determine whether autophagy also regulates DC–epithelial cell interactions and whether this influences the ensuing immune response.MethodsDC were generated from peripheral blood monocytes of healthy volunteers. For interaction studies, DC were co-cultured with intestinal epithelial cells on the baso-lateral side of a transwell insert. Modulation of autophagy was achieved using atg16l1 specific siRNA or pharmacological inhibitors. Intraepithelial protrusion of dendrites was determined by confocal microscopy. Luminal sampling and DC activation status were analyzed by flow cytometry. Protein expression was measured by immunoblotting and cytometric bead assay.ResultsAdhesion molecules E-cadherin and occludin partly localized to autophagosomes and increased autophagy resulted in decreased levels of these proteins. Reduced autophagy in either DC, epithelial cells or both resulted in the decreased formation of transepithelial protrusions by DC as well as a reduction in antigen sampling. Moreover, when autophagy was inhibited in the co-culture model, DC expressed increased levels of HLA-DR and costimulatory molecule CD86. Furthermore, decreased levels of autophagy resulted in lower IL-10 production by DC and these cells induced significantly more T-cell proliferation in an allogeneic mixed lymphocyte reaction.ConclusionsIn intestinal DC–epithelial cell interactions, autophagy deficiency leads to decreased antigen sampling, increased DC maturation and a more pro-inflammatory type of DC.     

New experimental markers for early detection of high-risk prostate cancer: role of cell–cell adhesion and cell migration

Today’s treatment and diagnosis of prostate cancer still exhibit major limitations. The search for new and additional prognostic markers is therefore still an actual field of interest. Potential markers involved in numerous biological processes in the tumor cell have been investigated intensively. For therapeutic interventions it is important to distinguish between harmless and aggressive disease in an early stage. Therefore the subject of this review is limited to markers associated with those functional processes, which discriminate early stage aggressive, metastatic cancer from harmless disease. Important processes in this respect are: altered cell adhesion and cellular migration. E-cadherin, N-cadherin, β-catenin, integrins, focal adhesion kinase, connexins and matrix metalloproteinases all appear promising biological markers associated with the early stage metastatic process in prostate cancer. Here we discuss their potential to become valid biological markers based on literature data. Thus far, none of these markers proved to be a valid individual marker by itself due to prostate cancer heterogeneity and transient expression. Analyzing a combination of the potential markers discussed in this review is expected to be a better approach toward discriminating high- from low-risk tumors in an early stage of prostate cancer.     

Paneth cell与肠道健康

潘氏细胞(paneth cell, PC)是由肠道干细胞(intestinal stemcells,ISC)分化而来并定居于小肠隐窝底部的分泌性细胞,是肠道健康的重要"守卫者". PC可分泌多种抗菌肽和细胞因子以调节肠道稳态与免疫反应,还可以合成分泌多种生长因子维持ISC的生态位,调控ISC增殖分化,甚至可作为静态干细胞库,在肠组织受损时获得干细胞样特性促进组织再生,从而有助于修复受损的肠组织.此外, PC与一些影响肠道健康的疾病密切相关,如炎症性肠病(inflammatory bowel disease,IBD)和结直肠癌(colorectal cancer,CRC),对于PC的研究或许可以为这些疾病的治疗提供思路.由此可见, PC功能的正常在维持肠道生理作用中值得高度重视.     

Stem cell transplantation for mantle cell lymphoma: if, when and how?

Although the prognosis for mantle cell lymphoma (MCL) patients has improved in recent years, the outlook for those with advanced or recurrent disease remains poor. High-dose chemotherapy and autografting performed early in responding patients appears to be a method to extend progression-free survival (PFS) and overall survival (OS). The use of monoclonal antibody therapy added into the initial therapy and in the peritransplant period may improve on these results. Myeloablative allogeneic transplant appears to be a modality capable of providing curative therapy, but is plagued by a high treatment-related mortality, especially in older patients. Reduced-intensity conditioning allografting have fewer problems associated with the initial phase of transplant and hence may be preferred for those patients for whom an allograft is considered but have comorbid conditions or age issues that preclude a full allograft. Long-term results are lacking and the side effects associated with chronic GVHD may be as significant and debilitating. Trials designed to look at newly diagnosed patients with MCL examining the outcomes after planned autologous and allogeneic transplant as part of the initial management are needed to confirm the role of these various modalities in the overall therapy of this poor-outcome lymphoma.     

HLA-identical stem cell transplantation: is there an optimal CD34 cell dose?

A review of the published literature, supplemented with a recent analysis of Fred Hutchinson data, has been undertaken to investigate the association of infused CD34 cell dose with various clinical outcomes after HLA-identical transplantation. Separate assessments for unrelated vs related donors and the use of bone marrow or mobilized G-PBMC have been incorporated. The three primary findings are: (1) higher CD34 dose results in better neutrophil and platelet recovery in all settings; (2) high CD34 doses (>8 x 10(6)/kg) are associated with the development of more chronic GVHD when using related G-PBMC; (3) higher CD34 dose is correlated with improved survival after bone marrow transplantation, especially with unrelated donors. This is not seen when using G-PBMC. The data suggest that the CD34 content of the graft can have a significant impact on clinical outcome after allogeneic transplantation, but optimal dose is dependent on both donor type and stem cell source.     

Paediatric haematologists’ attitudes regarding haematopoietic cell transplantation as treatment for sickle cell disease

Beginning early in childhood, patients with sickle cell disease [SCD; a group of genetic haemoglobin disorders characterized by the sickle or HbS mutation (HBB E7V)] are at risk of life-threatening and debilitating health events. Despite the high morbidity and mortality of this disease, haematopoietic cell transplantation (HCT), a curative therapy for SCD, remains underutilized. A variety of factors, including the limited availability of suitable donors, play a role in this trend, but do not fully explain the low frequency with which this therapy is employed. The objective of this study was to identify paediatric haematologists’ attitudes about HCT as a treatment option for SCD, and to describe the impact of these attitudes on their practices of discussing HCT with families of children affected by this disease. A nationwide survey of paediatric haematologists in the United States was conducted between February and May 2016. Two hundred and eighty-seven surveys were included in the final analysis (response rate 20%). On average, respondents reported informing 42% of families about HCT as a treatment option (N = 248, 95% confidence interval: 38–46). Clinician attitudes about the cost and safety of HCT were associated with practices of discussing this therapy with families. These findings suggest that clinician attitudes and referral practices may play a role in the underutilization of this therapy in the SCD population.     

B cell homeostasis and plasma cell homing controlled by Krüppel-like factor 2

Krüppel-like factor 2 (KLF2) controls T lymphocyte egress from lymphoid organs by regulating sphingosin-1 phosphate receptor 1 (S1Pr1). Here we show that this is not the case for B cells. Instead, KLF2 controls homeostasis of B cells in peripheral lymphatic organs and homing of plasma cells to the bone marrow, presumably by controlling the expression of β(7)-integrin. In mice with a B cell-specific deletion of KLF2, S1Pr1 expression on B cells was only slightly affected. Accordingly, all splenic B cell subsets including B1 cells were present, but their numbers were increased with a clear bias for marginal zone (MZ) B cells. In contrast, fewer peyers patches harboring fewer B cells were found, and fewer B1 cells in the peritoneal cavity as well as recirculating B cells in the bone marrow were detected. Upon thymus-dependent immunization, IgG titers were diminished, and antigen-specific plasma cells were absent in the bone marrow, although numbers of antigen-specific splenic plasmablasts were normal. KLF2 plays also a role in determining the identity of follicular B cells, as KLF2-deficient follicular B cells showed calcium responses similar to those of MZ B cells and failed to down-regulate MZ B cell signature genes, such as CD21 and CXCR7.