Localized Induction of Gene Expression in Embryonic Stem Cell Aggregates Using Holographic Optical Tweezers to Create Biochemical Gradients.

Three-dimensional (3D) cell models that mimic the structure and function of native tissues are enabling more detailed study of physiological and pathological mechanisms in vitro. We have previously demonstrated the ability to build and manipulate 3D multicellular microscopic structures using holographic optical tweezers (HOTs). Here, we show the construction of a precisely patterned 3D microenvironment and biochemical gradient model consisting of mouse embryoid bodies (mEBs) and polymer microparticles loaded with retinoic acid (RA), embedded in a hydrogel. We demonstrate discrete, zonal expression of the RA-inducible protein Stra8 within mEBs in response to release of RA from polymer microparticles, corresponding directly to the defined 3D positioning of the microparticles using HOTs. These results demonstrate the ability of this technology to create chemical microgradients at definable length scales and to elicit, with fidelity and precision, specific biological responses. This technique can be used in the study of in vitro microenvironments to enable new insights on 3D cell models, their cellular assembly, and the delivery of drug or biochemical molecules for engineering and interrogation of functional and morphogenic responses. Graphical abstract.

Bone extracellular matrix hydrogel enhances osteogenic differentiation of C2C12 myoblasts and mouse primary calvarial cells.

Hydrogel scaffolds derived from the extracellular matrix (ECM) of mammalian tissues have been successfully used to promote tissue repair in vitro and in vivo. The objective of this study was to evaluate the osteogenic potential of ECM hydrogels prepared from demineralized and decellularized bovine bone in the presence and absence of osteogenic medium. Culture of C2C12 and mouse primary calvarial cells (mPCs) on decellularized bone ECM (bECM) and demineralized bone matrix (DBM) gels resulted in increased expression of osteogenic gene markers, including a 3.6- and 13.4-fold increase in osteopontin and 15.7- and 27.1-fold increase in osteocalcin when mPCs were cultured upon bECM with basal and osteogenic media, respectively. bECM hydrogels stimulated the osteogenic differentiation of C2C12 and mPCs even in the absence of osteogenic medium. These results suggest that bECM hydrogel scaffolds may have great utility in future clinical applications for bone tissue engineering. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 900-908, 2018.

Precision assembly of complex cellular microenvironments using holographic optical tweezers.

The accurate study of cellular microenvironments is limited by the lack of technologies that can manipulate cells in 3D at a sufficiently small length scale. The ability to build and manipulate multicellular microscopic structures will facilitate a more detailed understanding of cellular function in fields such as developmental and stem cell biology. We present a holographic optical tweezers based technology to accurately generate bespoke cellular micro-architectures. Using embryonic stem cells, 3D structures of varying geometries were created and stabilized using hydrogels and cell-cell adhesion methods. Control of chemical microenvironments was achieved by the temporal release of specific factors from polymer microparticles positioned within these constructs. Complex co-culture micro-environmental analogues were also generated to reproduce structures found within adult stem cell niches. The application of holographic optical tweezers-based micromanipulation will enable novel insights into biological microenvironments by allowing researchers to form complex architectures with sub-micron precision of cells, matrices and molecules.

Comparison of osteogenic differentiation of embryonic stem cells and primary osteoblasts revealed by responses to IL-1ß, TNF-α, and IFN-γ.

There are well-established approaches for osteogenic differentiation of embryonic stem cells (ESCs), but few show direct comparison with primary osteoblasts or demonstrate differences in response to external factors. Here, we show comparative analysis of in vitro osteogenic differentiation of mouse ESC (osteo-mESC) and mouse primary osteoblasts. Both cell types formed mineralized bone nodules and produced osteogenic extracellular matrix, based on immunostaining for osteopontin and osteocalcin. However, there were marked differences in the morphology of osteo-mESCs and levels of mRNA expression for osteogenic genes. In response to the addition of proinflammatory cytokines interleukin-1ß, tumor necrosis factor-α, and interferon-γ to the culture medium, primary osteoblasts showed increased production of nitric oxide (NO) and prostaglandin E2 (PGE2) at early time points and decreases in cell viability. In contrast, osteo-mESCs maintained viability and did not produce NO and PGE2 until day 21. The formation of bone nodules by primary osteoblasts was reduced markedly after cytokine stimulation but was unaffected in osteo-mESCs. Cell sorting of osteo-mESCs by cadherin-11 (cad-11) showed clear osteogenesis of cad-11(+) cells compared to unsorted osteo-mESCs and cad-11(-) cells. Moreover, the cad-11(+) cells showed a significant response to cytokines, similar to primary osteoblasts. Overall, these results show that while osteo-mESC cultures, without specific cell sorting, show characteristics of osteoblasts, there are also marked differences, notably in their responses to cytokine stimuli. These findings are relevant to understanding the differentiation of stem cells and especially developing in vitro models of disease, testing new drugs, and developing cell therapies.

Early gene regulation of osteogenesis in embryonic stem cells.

The early gene regulatory networks (GRNs) that mediate stem cell differentiation are complex, and the underlying regulatory associations can be difficult to map accurately. In this study, the expression profiles of the genes Dlx5, Msx2 and Runx2 in mouse embryonic stem cells were monitored over a 48 hour period after exposure to the growth factors BMP2 and TGFß1. Candidate GRNs of early osteogenesis were constructed based on published experimental findings and simulation results of Boolean and ordinary differential equation models were compared with our experimental data in order to test the validity of these models. Three gene regulatory networks were found to be consistent with the data, one of these networks exhibited sustained oscillation, a behaviour which is consistent with the general view of embryonic stem cell plasticity. The work cycle presented in this paper illustrates how mathematical modelling can be used to elucidate from gene expression profiles GRNs that are consistent with experimental data.

Techniques for analysing pattern formation in populations of stem cells and their progeny.

BACKGROUND: To investigate how patterns of cell differentiation are related to underlying intra- and inter-cellular signalling pathways, we use a stochastic individual-based model to simulate pattern formation when stem cells and their progeny are cultured as a monolayer. We assume that the fate of an individual cell is regulated by the signals it receives from neighbouring cells via either diffusive or juxtacrine signalling. We analyse simulated patterns using two different spatial statistical measures that are suited to planar multicellular systems: pair correlation functions (PCFs) and quadrat histograms (QHs). RESULTS: With a diffusive signalling mechanism, pattern size (revealed by PCFs) is determined by both morphogen decay rate and a sensitivity parameter that determines the degree to which morphogen biases differentiation; high sensitivity and slow decay give rise to large-scale patterns. In contrast, with juxtacrine signalling, high sensitivity produces well-defined patterns over shorter lengthscales. QHs are simpler to compute than PCFs and allow us to distinguish between random differentiation at low sensitivities and patterned states generated at higher sensitivities. CONCLUSIONS: PCFs and QHs together provide an effective means of characterising emergent patterns of differentiation in planar multicellular aggregates.

Hyperpolarization of human mesenchymal stem cells in response to magnetic force.

Magnetic particle tagging techniques are currently being applied to tissue engineering applications such as controlled differentiation of mesenchymal stem cells (MSC). In order to define key mechanotransducers underpinning these applications, the electrophysiological responses of human MSCs (hMSC) have been investigated. Ferromagnetic microparticles were coated with L-arginyl-glycyl-L-aspartic acid in order to target the application of dynamic force (6 pN) directly to cell surface integrins. Human MSCs demonstrated cell membrane hyperpolarization responses after the application of force, mediated by BK channels and intracellular calcium release.