Samy Gobaa

Artificial niche microarrays for probing single stem cell fate in high throughput

To understand the regulatory role of niches in maintaining stem-cell fate, multifactorial in vitro models are required. These systems should enable analysis of biochemical and biophysical niche effectors in a combinatorial fashion and in the context of a physiologically relevant cell-culture substrate. We report a microengineered platform comprised of soft hydrogel microwell arrays with modular stiffness (shear moduli of 1–50 kPa) in which individual microwells can be functionalized with combinations of proteins spotted by robotic technology. To validate the platform, we tested the effect of cell-cell interactions on adipogenic differentiation of adherent human mesenchymal stem cells (MSCs) and the effect of substrate stiffness on osteogenic MSC differentiation. We also identified artificial niches supporting extensive self-renewal of nonadherent mouse neural stem cells (NSCs). Using this method, it is possible to probe the effect of key microenvironmental perturbations on the fate of any stem cell type in single cells and in high throughput.

3D niche microarrays for systems-level analyses of cell fate

The behaviour of mammalian cells in a tissue is governed by the three-dimensional (3D) microenvironment and involves a dynamic interplay between biochemical and mechanical signals provided by the extracellular matrix (ECM), cell–cell interactions and soluble factors. The complexity of the microenvironment and the context-dependent cell responses that arise from these interactions have posed a major challenge to understanding the underlying regulatory mechanisms. Here we develop an experimental paradigm to dissect the role of various interacting factors by simultaneously synthesizing more than 1,000 unique microenvironments with robotic nanolitre liquid-dispensing technology and by probing their effects on cell fate. Using this novel 3D microarray platform, we assess the combined effects of matrix elasticity, proteolytic degradability and three distinct classes of signalling proteins on mouse embryonic stem cells, unveiling a comprehensive map of interactions involved in regulating self-renewal. This approach is broadly applicable to gain a systems-level understanding of multifactorial 3D cell–matrix interactions.

Artificial three-dimensional niches deconstruct pancreas development in vitro

In the context of a cellular therapy for diabetes, methods for pancreatic progenitor expansion and subsequent differentiation into insulin-producing beta cells would be extremely valuable. Here we establish three-dimensional culture conditions in Matrigel that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the medium composition we generate either hollow spheres, which are mainly composed of pancreatic progenitors, or complex organoids that spontaneously undergo pancreatic morphogenesis and differentiation. The in vitro maintenance and expansion of pancreatic progenitors require active Notch and FGF signaling, thus recapitulating in vivo niche signaling interactions. Our experiments reveal new aspects of pancreas development, such as a community effect by which small groups of cells better maintain progenitor properties and expand more efficiently than isolated cells, as well as the requirement for three-dimensionality. Finally, growth conditions in chemically defined biomaterials pave the way for testing the biophysical and biochemical properties of the niche that sustains pancreatic progenitors.

Micropatterning of Hydrogels by Soft Embossing

Conventional in situ hydrogel micropatterning techniques work successfully for relatively stiff hydrogels, but they often result in locally damaged surfaces upon demolding in the case of soft and fragile polymer networks formed at low precursor concentration. To overcome this limitation, we have developed a versatile method, termed soft embossing, for the topographical micropatterning of fragile chemically cross-linked polymer hydrogels. Soft embossing is based on the imprinting of a microstructured template into a gel surface that is only partially cross-linked. Free functional groups continue to be consumed and upon complete cross-linking irreversibly confine the microstructure on the gel surface. Here we identify and optimize the parameters that control the soft embossing process and show that this method allows the fabrication of desired topographies with good fidelity. Finally, one of the produced gel micropatterns, an array of microwells, was successfully utilized forculturing and analyzing live single hematopoietic stem cells. Confining the stem cells to their microwells allowed for efficient quantification of their growth potential during in vitro culturing.

Diagnostic microchip to assay 3D colony-growth potential of captured circulating tumor cells.

Microfluidic technology has been successfully applied to isolate very rare tumor-derived epithelial cells (circulating tumor cells, CTCs) from blood with relatively high yield and purity, opening up exciting prospects for early detection of cancer. However, a major limitation of state-of-the-art CTC-chips is their inability to characterize the behavior and function of captured CTCs, for example to obtain information on proliferative and invasive properties or, ultimately, tumor re-initiating potential. Although CTCs can be efficiently immunostained with markers reporting phenotype or fate (e.g. apoptosis, proliferation), it has not yet been possible to reliably grow captured CTCs over long periods of time and at single cell level. It is challenging to remove CTCs from a microchip after capture, therefore such analyses should ideally be performed directly on-chip. To address this challenge, we merged CTC capture with three-dimensional (3D) tumor cell culture on the same microfluidic platform. PC3 prostate cancer cells were isolated from spiked blood on a transparent PDMS CTC-chip, encapsulated on-chip in a biomimetic hydrogel matrix (QGel™) that was formed in situ, and their clonal 3D spheroid growth potential was assessed by microscopy over one week in culture. The possibility to clonally expand a subset of captured CTCs in a near-physiological in vitro model adds an important element to the expanding CTC-chip toolbox that ultimately should improve prediction of treatment responses and disease progression.

High-throughput clonal analysis of neural stem cells in microarrayed artificial niches

To better understand the extrinsic signals that control neural stem cell (NSC) fate, here we applied a microwell array platform which allows high-throughput clonal analyses of NSCs, cultured either as neurospheres or as adherent clones, exposed to poly(ethylene glycol) (PEG) hydrogel substrates functionalized with selected signaling molecules. We analyzed by time-lapse microscopy and retrospective immunostaining the role of integrin and Notch ligands, two key NSC niche components, in altering the behavior of several hundred single stem cells isolated from a previously described Hes5::GFP reporter mouse. NSC self-renewal was increased by 1.5-fold upon exposure to covalently tethered Laminin-1 and fibronectin fragment 9-10 (FN(9-10)), where 60-65% of single cells proliferated extensively and remained Nestin positive. Tethering of the Notch ligand Jagged-1 induced activation of Notch signaling. While Jagged-1 alone increased cell survival and proliferation, no further increase in the clonogenic potential of Hes5::GFP cells was observed upon co-stimulation with Laminin-1 and Jagged-1. We believe that the bioengineering of such in vitro niche analogues is a powerful approach to elucidate single stem cell fate regulation in a well-controlled fashion.

Live mammalian cell arrays

High-content assays have the potential to drastically increase throughput in cell biology and drug discovery, but handling and culturing large libraries of cells such as primary tumor or cancer cell lines requires expensive, dedicated robotic equipment. We developed a simple yet powerful method that uses contact spotting to generate high-density nanowell arrays of live mammalian cells for the culture and interrogation of cell libraries.

Single-cell analyses identify bioengineered niches for enhanced maintenance of hematopoietic stem cells

The in vitro expansion of long-term hematopoietic stem cells (HSCs) remains a substantial challenge, largely because of our limited understanding of the mechanisms that control HSC fate choices. Using single-cell multigene expression analysis and time-lapse microscopy, here we define gene expression signatures and cell cycle hallmarks of murine HSCs and the earliest multipotent progenitors (MPPs), and analyze systematically single HSC fate choices in culture. Our analysis revealed twelve differentially expressed genes marking the quiescent HSC state, including four genes encoding cell–cell interaction signals in the niche. Under basal culture conditions, most HSCs rapidly commit to become early MPPs. In contrast, when we present ligands of the identified niche components such as JamC or Esam within artificial niches, HSC cycling is reduced and long-term multipotency in vivo is maintained. Our approach to bioengineer artificial niches should be useful in other stem cell systems.Haematopoietic stem cell (HSC) self-renewal is not sufficiently understood to recapitulate in vitro. Here, the authors generate gene signature and cell cycle hallmarks of single murine HSCs, and use identified endothelial receptors Esam and JamC as substrates to enhance HSC growth in engineered niches.

Artificial niche microarrays for identifying extrinsic cell-fate determinants

The complex cellular microenvironment plays an important role in determining cell fate. For example, stem cells located in a microenvironment termed niche integrate a wide variety of extrinsic cues to take distinct fate choices. Capturing this multiple-input/multiple-output system in vitro has proven to be very challenging. In order to address this issue, we developed and validated a microfabricated cellular array platform, termed artificial niche microarrays, which is capable of performing high-throughput single-cell assays under physiologically relevant conditions. The platform allows exposing cultured cells to differential signaling cues displayed on soft hydrogel substrates having variable stiffness. The behavior of the seeded cells can be readily quantified across over 2000 multivariate microenvironments. Here we describe a pipeline for performing multifactorial, image-based assays with these artificial niche microarrays. The procedure details the steps from microarray production, cell culture, cell phenotyping, data extraction to statistical analysis.