ReN VM spheroids in matrix: A neural progenitor three-dimensional in vitro model reveals DYRK1A inhibitors as potential regulators of radio-sensitivity

Abstract

Introduction: Pre-clinical testing of small molecules for therapeutic development across many pathologies relies on the use of in-vitro and in-vivo models. When designed and implemented well, these models serve to predict the clinical outcome as well as the toxicity of the evaluated therapies. The two-dimensional (2D) reductionist approach where cells are incubated in a mono-layer on hard plastic microtiter plates is relatively inexpensive but not physiologically relevant. In contrast, well developed and applied three dimensional (3D) in vitro models could be employed to bridge the gap between 2D in vitro primary screening and expensive in vivo rodent models by incorporating key features of the tissue microenvironment to explore differentiation, cortical development, cancers and various neuronal dysfunctions. These features include an extracellular matrix, co-culture, tension and perfusion and could replace several hundred rodents in the drug screening validation cascade. Methods: Human neural progenitor cells from middle brain (ReN VM, Merck Millipore, UK) were expanded as instructed by the supplier (Merck Millipore, UK), and then seeded in 96-well low-attachment plates (Corning, UK) to form multicellular spheroids followed by adding a Matrigel layer to mimic extracellular matrix around neural stem cell niche. ReN VM cells were then differentiated via EGF and bFGF deprivation for 7 days and were imaged at day 7. Radiotherapy was mimicked via gamma-radiation at 2Gy in the absence and presence of selected DYRK1A inhibitors Harmine, INDY and Leucettine 41 (L41). Cell viability was measured by AlamarBlue assay. Immunofluorescence staining was used to assess cell pluripotency marker SOX2 and differentiation marker GFAP. Results: After 7 days of differentiation, neuron early differentiation marker (GFAP, red) started to be expressed among the cells expressing neural stem cell marker SOX2 (green). Radiation treatment caused significant morphology change including the reduced viability of the spheroids. These spheroids also revealed sensitizing potential of DYRK1A inhibitors tested in this study, including Harmine, INDY and L41. Discussion & conclusions: Combined with the benefit of greatly reducing the issues associated with in vivo rodent models, including reducing numbers of animals used in a drug screening cascade, cost, ethics, and potential animal welfare burden, we feel the well-developed and applied 3D neural spheroid model presented in this study will provide a crucial tool to evaluate combinatorial therapies, optimal drug concentrations and treatment dosages.

Differentiated cultures of an immortalized human neural progenitor cell line do not replicate prions despite PrPC overexpression

“…By embedding ReN cells within Matrigel, they can also be differentiated as thin-3D cultures. We found ReN cells within these thin 3D cultures to self-arrange into spheroid-like clusters of cells ( Figure 1c ), similar to 3D neurospheroids produced by culturing ReN cells in low-attachment round-bottomed plates [ 33 ] or microwell chips [ 34 ]. The morphology of ReN progenitor cells was confirmed by immunofluorescence staining of NES at day 0 of differentiation ( Figure 1d ), while the morphology of ReN neurons and astrocytes was examined by staining for TUBB3 and GFAP at day 28 post-differentiation ( Figure 1e ).…”

Minimalised Three-Dimensional Human Midbrain In Vitro Model for Neurotoxicity Evaluation of DYRK1 Inhibitors

“…Modelling human brain tissue has historically been a challenge for the scientific research community and this can be attributed to many factors, including the complexity of brain architecture, the need to co-culture several specialized cell types and the dynamics of the brain microenvironment -including, but not limited to -the ECM [5]. Therefore, by reconstituting the 3D in vitro microenvironment for neural stem cells, these cells can adopt a more physiologically relevant and robust phenotype suitable for screening purposes [10], Shin, Yang [19], [27,28]. In this study, we chose the commercially available neural progenitor cell line ReN from the midbrain (ReN VM) to establish our platform, given their immortality, ability to be readily differentiated into neurons and glial cells and their rapid growth on laminin in the expansion stage [11].…”

“…Here we describe the minimalised but physiologically relevant format of a 3D in vitro model to evaluate the effects of a novel DYRK1A inhibitor K04179, using a human mid-brain originated neural progenitor cell line ReN VM. Compared with our previous ReN VM multicellular spheroid model [28], this study integrated Matrigel as an ECM replacement, but with a minimalised volume for cost and imaging conveniency, which is described as 'sandwich culture' here. We previously reported a similar sandwich model but with cancer cells and vascular endothelial cells during a shorter culture term [16].…”