Activity-dependent differentiation and fate-determination of induced human neurons

The research of diseases that affect the adult human brain is limited by a lack of appropriate model systems, as the biological aging process of our brain cells is of central importance here. Animal models have different physiologies than humans, and often only live for a few years. iPS cells offer an alternative: adult skin cells are converted into stem cells, which then can develop into nerve cells. However, these new nerve cells are rejuvenated, in that they have lost all memory of the age of their skin donor, and best resemble nerve cells before birth. We have established an alternative method that is based on the direct conversion of skin cells directly into nerve cells. This method allows to produce age-equivalent `adult` nerve cells: iNs. In previous and ongoing research projects, iNs have already tought us a lot about the aging process of our brains. However, there is still a lot of research to do to optimize iNs. Perhaps the most significant property of nerve cells in our brain is that they produce large numbers of tiny electrical discharges known as action potentials. These bits of information are used to send messages between the cells. The brain is made up of billions of nerve cells, each with remarkably sophisticated computing power. Nerve cells are connected to one another via an even larger number of nodes, so-called synapses. Synapses are extremely flexible, and many researchers believe that this is the basis for learning. However, also the ability of the nerve cells to process incoming information and to send action potentials is extremely adaptable. Being able to control this adaptability in a targeted manner would essentially mean that we could reprogram all computers in a gigantic network. Our research project aims precisely at that: using external stimuli to optimize the bioelectrical properties of human nerve cells; in other words: help them to `grow up`. To do this, we use flickering light pulses to which our genetically modified cells react like to natural incoming messages. We have copied the patterns of light impulses from natural messages of the human brain, and we will use them as `training units` for the nerve cells. We expect that nerve cells will always adjust their bioelectrical properties so that they can process the incoming messages most effectively. We also believe that adult iNs react very differently to certain training patterns than rejuvenated nerve cells from iPS cells. We believe that human nerve cells trained in this way will represent an even better model system for disease modeling, and also will represent candidate donor cells for clinical cell- replacement strategies in the future.

In this project, we have a comprehensive analysis of activity-dependent mechanisms that regulate the physiological and synaptic properties of human neurons obtained via two different cell-type genetic reprogramming methods. The methods of reprogramming human skin cells into nerve cells compared here are induced neurons (iNs) from induced pluripotent stem cells (iPSCs) on the one hand, and induced neurons (Fib -iNs) reprogramming directly from fibroblasts on the other. What is special about Fib-iNs is that they reflect the human age as well as the 'adult' cell status of the sonores. Therefore, it is expected that Fib-iNs also have different functional properties compared to 'embryonic-like' iPSC-Ns. We investigated this in the project by producing both iPSC-Ns and Fib-iNs from the same sonorers, the same skin cell cultures. The project used a combination of long-term optogenetic control of neuronal activity, in vitro electrophysiology and molecular biology. We were able to show that Fib-iNs from different old sonorers, and especially from patients, show peculiarities in their molecular and cellular, and especially in their metabolic structure. In addition, iPSC-Ns and Fib-iNs have different electrophysiological properties, which can be manipulated by optogenetic stimulation. After a short time, individual 'adult' Fib-iNs show very mature electrophysiological processional properties that are unknown in iPSC-Ns.