Future R&D

Stem cell research

Directed differentiation of stem cells towards a specific cell fate is a key issue for developmental biology and regenerative medicine. Naturally, as stem cells may differentiate into different lineages producing a diverse population of cells, it is essential to study the progeny at the resolution of the individual cell. In the case of neural differentiation, the identity and functional maturity of each cell can only be determined using functional parameters such as neurotransmitter release and specific gene expression. The Cell Retaining methodologies will be used to explore the effect of a large set of differentiation factors in controllable amounts and temporal sequences in a highly parallelized and automated fashion. The monitoring of cell differentiation will be performed by combined optical and electrochemical assays, as well as by transcriptome analysis. Specifically, we will study the differentiation of neurospheres into dopaminergic neurons as well as the differentiation of human limbal adult stem cells into neuronal lineages and pancreatic insulin-secreting beta-cells. The latter are of particular interest, since the production of autologous beta cells from adult stem cells could lead to a new cellular therapy of diabetes. The neurospheres, which are used routinely by one of our European academic collaborators, are composed of around 200 undifferentiated cells, obtained from rat brain at days 14-15 of embryogenesis. Their development can be directed toward becoming dopaminergic neuronal cells by a treatment with a complex mixture of various factors at an adequate time. The differentiation of neurospheres into dopaminergic (DA) neurons will be tested by a) direct microscopic observation of the morpho-type of the neurospheres; b) quantitative fluorescence analysis of a fluorescent promoter for tyrosine hydroxylase (TH, a dopamine neuron marker) and by catecholamine sensing electrodes; and c) transcriptome analysis of the neurospheres undergoing differentiation.


Traumatic and ischemic brain diseases

In order to enable the identification of new targets for therapeutic vascular protection in brain injury, brain endothelial cell dysfunction, that mediates brain injury aggravation, will be monitored. A recognized marker of the endothelium dysfunction is nitric oxide (NO) production, which is decreased, while superoxide anion production is increased.

A key challenge in studying endothelial cells from mammalian tissues is the cellular functional heterogeneity which compromises the ability to interpret data obtained from a large bulk of cells. While the molecular tools for the analysis of individual cells have been established in our group, functional assays that can deliver information on individual cell physiology are still missing. The use of the Cell Retaining methodology will allow the analysis of functional parameters in a large population of endothelial cells at the resolution of an individual cell. It will also facilitate the collection of individual cells for molecular assays after being tested for physiological parameters over time. As such, this approach is likely to deliver novel insights into endothelial cell pathophysiology. By using the present platform, we will study the hypoxic response on primary brain capillary and artery endothelial cells. Through NO and superoxide anion electrochemical and optical detection, this technology will allow, for the first time, to identify the individual cells that display the highest stress response. Such cells will be then collected on an individual basis using optical tweezers, and submitted to single-cell transcriptome analysis.


Vascular pathologies

The loss of endothelial barrier function and the subsequent increase in vascular permeability to low density lipoprotein (LDL) may be central events in the etiology of common and debilitating vascular pathologies such as migraine.

The production of free radicals by endothelial cells lining the vasculature within the central nervous system (CNS) has been associated with impaired endothelial barrier function. A direct link between endothelial free radical production and the release of inflammatory mediators by glial cells within the CNS has also been proposed.

The Cell Retainers will be used as a novel platform to facilitate the specific, sensitive and real-time analysis of free radical production by human middle cerebral artery endothelial cells (MCAECs). Free radical production will be stimulated by inflammatory mediators released from glial cells, co-cultured with MCAECs. The CR will enable the identification of endothelial cells that show high levels of free radical production, and could thus impair the endothelial barrier function. These cells will be selected, sub-cultured into monolayers and used in subsequent in vitro protein permeability studies.

The CR methodology will be also used in conjunction with specific endothelial membrane receptor antagonists to identify inflammatory mediators produced by glial cells that may have detrimental effects on the endothelial barrier function.