Pyramidal neurons induced from human stem cells integrate into the mouse brain.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Stem cells are a great scientific discovery of the last decades. Since researchers first cultured them in a lab, many scientists from all over the world focused on them because of the great potential they can have from a medical point of view.

But where is research on this topic?

The use of stem cells greatly contributed to the current knowledge on neuronal development and the modeling of some diseases.

The cerebral cortex is a great example of the complexity of the brain circuitry. Several different neuronal types are connected with the rest of the brain, enabling the different brain functions. The major part of cortical neurons is represented by pyramidal neurons and are organized in different layers.

Few years ago it has been shown that mouse embryonic stem cells (ESC) or mouse induced pluripotent stem cells (iPSC) can be induced in vitro to differentiate into cortical neurons. This step does not require the common treatment with specific molecules called morphogens, and grafting mouse ESC-derived pyramidal neurons in neonatal mouse brain allowed neurons to send projections similarly to the endogenous neurons, demonstrating that they were in fact cortical neurons. However, all these studies were performed in mouse and even if human stem cells were already induced into pyramidal neurons, it was not clear if their differentiation into pyramidal neurons followed the same pathway as in mouse. Moreover, the exact type of neurons generated was not certain, as it was never grafted in vivo, so the axonal and dendritic projections were never checked.

In this paper the authors show that 1) just like in mouse, human ESC go through all the steps of corticogenesis even in absence of morphogens, with the exact same sequential generation involved in the genesis of the different cortical neurons (eg one first stage where one kind of neurons are generated, second stage another kind of neurons and so on); and 2) after grafting ESC-induced human cortical neurons in the mouse neonatal brain, they integrate functionally in the host circuitry, enabling the study of human cortex development, and related diseases, in vivo.

In vitro differentiation of human ESC. In vitro, human ESC/iPSC not only differentiate into cortical neurons, but they also become mature neurons. Indeed, after culturing human ESC/iPSC for 15 days without morphogens, the cells show the typical morphology of pyramidal neurons and express specific markers, with a pattern of expression similar to what is observed in samples of fetal cortices. After two months in culture, they also express pre-synaptic and post-synaptic markers, suggesting the formation of mature synapses, results comparable with data obtained from human samples at 19 days of gestation.

In vitro functionality of human ESC-derived neurons. Furthermore, these neurons are functional, as shown by imaging calcium waves, that are associated with neuronal excitability, and by recording of series of action potentials (called firing) in response to electrical stimulation, compared to immature neurons that show instead only a single action potential upon stimulation.

Pyramidal neurons can be subdivided in different categories that compose the different cortical layers and that are generated at different time points during cortical development. For example, first pioneer neurons are generated, then layers VI and V, then layer IV and finally layers II and III. By analyzing the onset of the expression of the markers of each specific layer during the differentiation period, they show that corticogenesis from human ESC follows the one described for mouse, but it lasts longer (in vitro 80 days compared to the 20 days required for the mouse). This reminds the longer corticogenesis period described in vivo in humans compared to mice.

In vivo. Axonal and dendritic projections are a key point in the integration of cortical neurons, which enable the functioning of brain cortex.

Grafting. Human cortical neurons were differentiated from human ESC labeled with GFP (Green Fluorescent Protein), grafted into mouse neonatal cortex and analyzed at different time points (meaning different months) after transplant. By checking the expression of the specific markers of the different subtypes of cortical neurons, they were able to see that even in vivo the differentiation of grafted cells follows exactly the same pattern than in vitro (from 1 to 10 months after transplant, the time required for corticogenesis). Just like in vitro, neurons of all the six layers differentiate, as identified by markers of each specific layer.

Projection of axons. GFP-labeled grafted cells project to the right targets of cortical neurons: for instance, cells of layer VI projecting to the thalamus, axons of layer V to midbrain and hindbrain, and axons from layers II, III, V to ipsi and contralateral cortex (meaning axons that project respectively on the same side or on the opposite side of the brain). Very few projections are found in areas that were not supposed to be innervated by cortical neurons, such as eg the cerebellum. The number of projections of later differentiated neurons increases if the samples are analyzed later on during development, reflecting the timing of differentiation described in vitro and in vivo.

Dendrites and synaptogenesis. Nine months after transplant, the neurons display an elaborated dendritic arbor of a mature pyramidal neuron, with dendritic spines, and  markers of pre- and post-synaptic components: grafted neurons make synapses with the host, receive innervations from the thalamus of the host, and  are also myelinated by the host. Grafted neurons integrate into the host circuitry.  

Functional integration. The integration of grafted neurons into the circuitry of the host is finally checked by measuring the electrical properties of neurons in mouse brain slices. These neurons show the excitability typical of mature pyramidal neurons, responding to injection of depolarizing current with a train of action potentials (firing). In order to finally assess the functional integration and synaptic connection of grafted neurons with the host, they provide an electrical stimulus at a certain distance from the graft and they recorded the induced action potentials in the grafted neurons. The response of the grafted neuron to the stimulus is switched off by inhibiting synaptic components typical of mature pyramidal neurons (the GABA-ergic and glutamatergic signalization), suggesting that synaptic formation has occurred.

Conclusion. Pyramidal neurons can be induced by human ESC, and the differentiation process recapitulates the different stages that differentiating cells undergo in vivo. Furthermore, differentiated cells become mature pyramidal neurons and functionally integrate into the mouse circuitry.

 

GLOSSARY:

Cerebral cortex. Outer layer of the brain, with a crucial role in different brain functions.

Morphogens. molecules modulating cell morphogenesis during development.

Stem cells. Undifferentiated cells retaining the potential of differentiating in any possible cell type. ESC are Embryonic Stem Cells isolated from an embryo at the very early stages after in vitro fecundation. iPSC are Induced Pluripotent Stem Cells, deriving from adult cells that are de-differentiated, so brought back to an undifferentiated stem cell-like state, in order to be re-differentiated afterwards into different cell types.

Corticogenesis. Process of development of cortical neurons.

Synapsis. Structure at the contact between two neurons that allows communication between cells through the signal transmission. Dendrites and axons. Neuronal processes involved in carrying electrical information, respectively receiving and transmitting the signal.

Dendritic spines. Small protrusion on dendrites having a crucial role for synaptic function and plasticity, undergoing changes in size and shape following synaptic activity.

Myelin. Fatty sheath that wrap axons, insulating it electrically and allowing fast saltatory conduction of the electrical signal. Myelination is carried out by Schwann cells in the peripheral nervous system and oligodendrocytes in the central nervous system.

Depolarizing current. Current that changes the membrane potential of a cell to more positive values. A neuronal cell is normally at very negative membrane potentials (around -70/-80mV) and the injection of depolarizing current triggers an action potential, meaning a rapid and transitory change of membrane potential of a cell (up to +40/50mV). At the end of an action potential membrane potential goes back to resting values (-80mV). A repetitive series of action potential is called firing.

  

Reference: Pyramidal neurons derived from human pluripotent stem cells integrate efficiently into mouse brain circuits in vivo. Espuny-Camacho I, Michelsen KA, Gall D, Linaro D, Hasche A, Bonnefont J, Bali C, Orduz D, Bilheu A, Herpoel A, Lambert N, Gaspard N, Péron S, Schiffmann SN, Giugliano M, Gaillard A, Vanderhaeghen P. Neuron