Amranul Haque

Detecting transforming growth factor-β release from liver cells using an aptasensor integrated with microfluidics.

We developed a cell-culture/biosensor platform consisting of aptamer-modified Au electrodes integrated with reconfigurable microfluidics for monitoring of transforming growth factor-beta 1 (TGF-β1), an important inflammatory and pro-fibrotic cytokine. Aptamers were thiolated, labeled with redox reporters, and self-assembled on gold surfaces. The biosensor was determined to be specific for TGF-β1 with an experimental detection limit of 1 ng/mL and linear range extending to 250 ng/mL. Upon determining figures of merit, aptasensor was miniaturized and integrated with human hepatic stellate cells inside microfluidic devices. Reconfigurable microfluidics were developed to ensure that seeding of “sticky” stromal cells did not foul the electrode and compromise sensor performance. This microsystem with integrated aptasensors was used to monitor TGF-β1 release from activated stellate cells over the course of 20 h. The electrochemical response went down upon infusing anti-TGF-β1 antibodies into the microfluidic devices containing activated stellate cells. To further validate aptasensor responses, stellate cells were stained for markers of activation (e.g., alpha smooth muscle actin) and were also tested for presence of TGF-β1 using enzyme linked immunosorbent assay (ELISA). Given the importance of TGF-β1 as a fibrogenic signal, a microsystem with integrated biosensors for local and continuous detection of TGF-β1 may prove to be an important tool to study fibrosis of the liver and other organs.

The effect of recombinant E-cadherin substratum on the differentiation of endoderm-derived hepatocyte-like cells from embryonic stem cells

Generation of specific lineages of cells from embryonic stem (ES) cells is pre-requisite to use these cells in pre-clinical applications. Here, we developed a recombinant E-cadherin substratum for generation of hepatic progenitor populations at single cell level. This artificial acellular feeder layer supports the stepwise differentiation of ES cells to cells with characteristics of definitive endoderm, hepatic progenitor cells, and finally cells with phenotypic and functional characteristics of hepatocytes. The efficient differentiation of hepatic endoderm cells (approximately 55%) together with the absence of neuroectoderm and mesoderm markers suggests the selective induction of endoderm differentiation. The co-expression of E-cahderin and alpha-fetoprotein (approximately 98%) suggests the important role of E-cadherin as a surface marker for the enrichment of hepatic progenitor cells. With extensive expansion, approximately 92% albumin expressing cells can be achieved without any enzymatic stress and cell sorting. Furthermore, these mouse ES cell-derived hepatocyte-like cells showed higher morphological similarities to primary hepatocytes. In conclusion, we demonstrated that E-cadherin substratum can guide differentiation of ES cells into endoderm-derived hepatocyte-like cells. This recombinant extracellular matrix could be effectively used as an in vitro model for studying the mechanisms of early stages of liver development even at single cell level.

Microfluidic fabrication of bioactive microgels for rapid formation and enhanced differentiation of stem cell spheroids

UNLABELLED A major challenge in tissue engineering is to develop robust protocols for differentiating ES and iPS cells to functional adult tissues at a clinically relevant scale. The goal of this study is to develop a high throughput platform for generating bioactive, stem cell-laden microgels to direct differentiation in a well-defined microenvironment. We describe a droplet microfluidics system for fabricating microgels composed of polyethylene glycol and heparin, with tunable geometric, mechanical, and chemical properties, at kHz rates. Heparin-containing hydrogel particles sequestered growth factors Nodal and FGF-2, which are implicated in specifying pluripotent cells to definitive endoderm. Mouse ESCs were encapsulated into heparin microgels with a single dose of Nodal and FGF-2, and expressed high levels of endoderm markers Sox17 and FoxA2 after 5 days. These results highlight the use of microencapsulation for tailoring the stem cell microenvironment to promote directed differentiation, and may provide a straightforward path to large scale bioprocessing in the future. STATEMENT OF SIGNIFICANCE Multicellular spheroids and microtissues are valuable for tissue engineering, but fabrication approaches typically sacrifice either precision or throughput. Microfluidic encapsulation in polymeric biomaterials is a promising technique for rapidly generating cell aggregates with excellent control of microenvironmental parameters. Here we describe the microfluidic fabrication of bioactive, heparin-based microgels, and demonstrate the adsorption of heparin-binding growth factors for enhancing directed differentiation of embryonic stem cells toward endoderm. This approach also facilitated a ∼90-fold decrease in consumption of exogenous growth factors compared to conventional differentiation protocols.

Fabrication and Intracellular Delivery of Doxorubicin/Carbonate Apatite Nanocomposites: Effect on Growth Retardation of Established Colon Tumor

In continuing search for effective treatments of cancer, the emerging model aims at efficient intracellular delivery of therapeutics into tumor cells in order to increase the drug concentration. However, the implementation of this strategy suffers from inefficient cellular uptake and drug resistance. Therefore, pH-sensitive nanosystems have recently been developed to target slightly acidic extracellular pH environment of solid tumors. The pH targeting approach is regarded as a more general strategy than conventional specific tumor cell surface targeting approaches, because the acidic tumor microclimate is most common in solid tumors. When nanosystems are combined with triggered release mechanisms in endosomal or lysosomal acidic pH along with endosomolytic capability, the nanocarriers demonstrated to overcome multidrug resistance of various tumors. Here, novel pH sensitive carbonate apatite has been fabricated to efficiently deliver anticancer drug Doxorubicin (DOX) to cancer cells, by virtue of its pH sensitivity being quite unstable under an acidic condition in endosomes and the desirable size of the resulting apatite-DOX for efficient cellular uptake as revealed by scanning electron microscopy. Florescence microscopy and flow cytometry analyses demonstrated significant uptake of drug (92%) when complexed with apatite nanoparticles. In vitro chemosensitivity assay revealed that apatite-DOX nanoparticles executed high cytotoxicity in several human cancer cell lines compared to free drugs and consequently apatite-DOX-facilitated enhanced tumor inhibitory effect was observed in colorectal tumor model within BALB/cA nude mice, thereby shedding light on their potential applications in cancer therapy.

The differentiation and isolation of mouse embryonic stem cells toward hepatocytes using galactose-carrying substrata

A simple culture system to achieve the differentiation of embryonic stem (ES) cells toward hepatocytes with high efficiency is crucial in providing a cell source for the medical application. In this study, we report the effect of a matrix-dependent enrichment of ES cell-derived hepatocytes using immobilized poly(N-p-vinylbenzyl-4-O-β-D-galactopyranosyl-D-gluconamide) (PVLA) with E-cadherin-IgG Fc (E-cad-Fc) as a galactose-carrying substratum. PVLA and E-cad-Fc were confirmed to be stably co-adsorbed onto polystyrene surface by quartz crystal microbalance (QCM). We showed that the E-cad-Fc/PVLA hybrid substratum was efficient in culturing primary hepatocytes and maintaining liver functions, on which the undifferentiated ES cells also maintained high proliferative capability. Furthermore, ES cell-derived hepatocytes on this hybrid matrix expressed elevated level of liver specific genes and functions together with early expression of definitive hepatocyte marker, asialoglycoprotein receptor (ASGPR). Finally, we isolated a high percentage of cells (about 60%) with ASGPR expression after re-seeding onto PVLA-coated surface, and observed the elimination of the poorly differentiated cells (Gata6(+) and Sox17(+)) and the ones toward another cell lineage (brachyury(+) and Pdx1(+)). The system uses a glycopolymer as an extracellular substratum for isolation and enrichment of ES cell-derived hepatocytes with adequate homogeneity and functionality.

Liver injury-on-a-chip: microfluidic co-cultures with integrated biosensors for monitoring liver cell signaling during injury

Tissue injury triggers complex communication between cells via secreted signaling molecules such as cytokines and growth factors. Discerning when and where these signals begin and how they propagate over time is very challenging with existing cell culture and analysis tools. The goal of this study was to develop new tools in the form of microfluidic co-cultures with integrated biosensors for local and continuous monitoring of secreted signals. Specifically, we focused on how alcohol injury affects TGF-β signaling between two liver cell types, hepatocytes and stellate cells. Activation of stellate cells happens early during liver injury and is at the center of liver fibrosis. We demonstrated that alcohol injury to microfluidic co-cultures caused significantly higher levels of stellate cell activation compared to conditioned media and transwell injury experiments. This highlighted the advantage of the microfluidic co-culture: placement of two cell types in close proximity to ensure high local concentrations of injury-promoting secreted signals. Next, we developed a microsystem consisting of five chambers, two for co-culturing hepatocytes with stellate cells and three additional chambers containing miniature aptamer-modified electrodes for monitoring secreted TGF-β. Importantly, the walls separating microfluidic chambers were actuatable; they could be raised or lowered to create different configurations of the device. The use of reconfigurable microfluidics and miniature biosensors revealed that alcohol injury causes hepatocytes to secrete TGF-β molecules, which diffuse over to neighboring stellate cells and trigger production of additional TGF-β from stellate cells. Our results lend credence to the emerging view of hepatocytes as active participants of liver injury. Broadly speaking, our microsystem makes it possible to monitor paracrine crosstalk between two cell types communicating via the same signaling molecule (e.g. TGF-β).

Impact of Nanotopography, Heparin Hydrogel Microstructures, and Encapsulated Fibroblasts on Phenotype of Primary Hepatocytes.

Hepatocytes, the main epithelial cell type in the liver, perform most of the biochemical functions of the liver. Thus, maintenance of a primary hepatocyte phenotype is crucial for investigations of in vitro drug metabolism, toxicity, and development of bioartificial liver constructs. Here, we report the impact of topographic cues alone and in combination with soluble signals provided by encapsulated feeder cells on maintenance of the primary hepatocyte phenotype. Topographic features were 300 nm deep with pitches of either 400, 1400, or 4000 nm. Hepatocyte cell attachment, morphology and function were markedly better on 400 nm pitch patterns compared with larger scale topographies or planar substrates. Interestingly, topographic features having biomimetic size scale dramatically increased cell adhesion whether or not substrates had been precoated with collagen I. Albumin production in primary hepatocytes cultured on 400 nm pitch substrates without collagen I was maintained over 10 days and was considerably higher compared to albumin synthesis on collagen-coated flat substrates. In order to investigate the potential interaction of soluble cytoactive factors supplied by feeder cells with topographic cues in determining cell phenotype, bioactive heparin-containing hydrogel microstructures were molded (100 μm spacing, 100 μm width) over the surface of the topographically patterned substrates. These hydrogel microstructures either carried encapsulated fibroblasts or were free of cells. Hepatocytes cultured on nanopatterned substrates next to fibroblast carrying hydrogel microstructures were significantly more functional than hepatocytes cultured on nanopatterned surfaces without hydrogels or stromal cells significantly elevated albumin expression and cell junction formation compared to cells provided with topographic cues only. The simultaneous presentation of topographic biomechanical cues along with soluble signaling molecules provided by encapsulated fibroblasts cells resulted in optimal functionality of cultured hepatocytes. The provision of both topographic and soluble signaling cues could enhance our ability to create liver surrogates and inform the development of engineered liver constructs.

Artificial extracellular matrix for embryonic stem cell cultures: a new frontier of nanobiomaterials

Abstract Nanobiomaterials can play a central role in regenerative medicine and tissue engineering by facilitating cellular behavior and function, such as those where extracellular matrices (ECMs) direct embryonic stem (ES) cell morphogenesis, proliferation, differentiation and apoptosis. However, controlling ES cell proliferation and differentiation using matrices from natural sources is still challenging due to complex and heterogeneous culture conditions. Moreover, the systemic investigation of the regulation of self-renewal and differentiation to lineage specific cells depends on the use of defined and stress-free culture conditions. Both goals can be achieved by the development of biomaterial design targeting ECM or growth factors for ES cell culture. This targeted application will benefit from expansion of ES cells for transplantation, as well as the production of a specific differentiated cell type either by controlling the differentiation in a very specific pathway or by elimination of undesirable cell types.

Functional imaging of neuron–astrocyte interactions in a compartmentalized microfluidic device

Traditional approaches in cultivating neural cells in a dish without orienting their interactions have had only limited success in revealing neural network properties. To enhance the experimental capabilities of studying neural circuitry in vitro, we designed an experimental system combining concepts of micropatterned surfaces, microfluidic devices and genetically encoded biosensors. Micropatterning was used to position neurons and astrocytes in defined locations and guide interactions between the two cell types. Microfluidic chambers were placed atop micropatterned surfaces to allow delivery of different pharmacological agents or viral vectors to the desired cell types. In this device, astrocytes and neurons communicated through grooves molded into the floor of the microfluidic device. By combining microfluidics with genetically encoded calcium indicators as functional readouts, we further demonstrated the utility of this device for analyzing neuron–neuron and neuron–astrocyte interactions in vitro under both healthy and pathophysiological conditions. We found that both spontaneous and evoked calcium dynamics in astrocytes can be modulated by interactions with neurons. In the future, we foresee employing the microdevices described here for studying mechanisms of neurological disorders.

The effects of artificial E-cadherin matrix-induced embryonic stem cell scattering on paxillin and RhoA activation via α-catenin

Mechanical forces have been shown to affect stem cell behavior in a large array of ways. However, our understanding of how these mechanical cues may regulate the behavior of embryonic stem cells (ESCs) remains in its infancy. Here, we aim to clarify the effect of cell scattering on the regulation of Rho family GTPases Rac1 and RhoA as well as paxillin. Allowing ESCs to spread and scatter on a synthetically designed E-cadherin substratum causes phosphorylation of paxillin on consensus phosphorylation sites leading to activation of Rac1 and inactivation of RhoA. By culturing cells in presence of RhoA activator or growing cells to a highly confluent state reverses the effect of cell scattering phenotype. Knockdown of E-cadherin-adapter protein α-catenin revealed that it negatively affects paxillin phosphorylation and up-regulates RhoA activity in compact cellular aggregates. Collectively these results indicate that cell scattering might cause a conformational change of α-catenin limiting its capacity to inhibit paxillin phosphorylation that causes an increase in Rac1 activation and RhoA deactivation. Understanding how synthetically designed extracellular matrix affect ESC signaling through mechanical cues brings a new aspect for stem cell engineers to develop technologies for controlling cell function.