organotypic-cultures-adult-human-cortex-as-an-ex-vivo-model-for-human

Organotypic Cultures of Adult Human Cortex as an <em>Ex vivo</em> Model for Human Stem Cell Transplantation and Validation

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the ...

Lund University

Reprogramming of somatic cells into pluripotency stem cell state has opened new opportunities in cell replacement therapy and disease modeling in a number of neurological disorders. It still remains unknown, however, to what degree the grafted human-induced pluripotent stem cells (hiPSCs) differentiate into a functional neuronal phenotype and if they integrate into the host circuitry. Here, we present a detailed characterization of the functional properties and synaptic integration of hiPSC-derived neurons grafted in an in vitro model of hyperexcitable epileptic tissue, namely organotypic hippocampal slice cultures (OHSCs), and in adult rats in vivo. The hiPSCs were first differentiated into long-term self-renewing neuroepithelial stem (lt-NES) cells, which are known to form primarily GABAergic neurons. When differentiated in OHSCs for 6 weeks, lt-NES cell-derived neurons displayed neuronal properties such as tetrodotoxin-sensitive sodium currents and action potentials (APs), as well as both spontaneous and evoked postsynaptic currents, indicating functional afferent synaptic inputs. The grafted cells had a distinct electrophysiological profile compared to host cells in the OHSCs with higher input resistance, lower resting membrane potential, and APs with lower amplitude and longer duration. To investigate the origin of synaptic afferents to the grafted lt-NES cell-derived neurons, the host neurons were transduced with Channelrhodopsin-2 (ChR2) and optogenetically activated by blue light. Simultaneous recordings of synaptic currents in grafted lt-NES cell-derived neurons using whole-cell patch-clamp technique at 6 weeks after grafting revealed limited synaptic connections from host neurons. Longer differentiation times, up to 24 weeks after grafting in vivo, revealed more mature intrinsic properties and extensive synaptic afferents from host neurons to the lt-NES cell-derived neurons, suggesting that these cells require extended time for differentiation/maturation and synaptogenesis. However, even at this later time point, the grafted cells maintained a higher input resistance. These data indicate that grafted lt-NES cell-derived neurons receive ample afferent input from the host brain. Since the lt-NES cells used in this study show a strong propensity for GABAergic differentiation, the host-to-graft synaptic afferents may facilitate inhibitory neurotransmitter release, and normalize hyperexcitable neuronal networks in brain diseases, for example, such as epilepsy.

Optogenetics reveal delayed afferent synaptogenesis on grafted human-induced pluripotent stem cell-derived neural progenitors.

Optogenetics is one of the most powerful tools in neuroscience, allowing for selective control of specific neuronal populations in the brain of experimental animals, including mammals. We report, for the first time, the application of optogenetic tools to human brain tissue providing a proof-of-concept for the use of optogenetics in neuromodulation of human cortical and hippocampal neurons as a possible tool to explore network mechanisms and develop future therapeutic strategies. 

Optogenetic control of human neurons in organotypic brain cultures.

Direct conversion of human somatic cells to induced neurons (iNs), using lineage-specific transcription factors has opened new opportunities for cell therapy in a number of neurological diseases, including epilepsy. In most severe cases of epilepsy, seizures often originate in the hippocampus, where populations of inhibitory interneurons degenerate. Thus, iNs could be of potential use to replace these lost interneurons. It is not known, however, if iNs survive and maintain functional neuronal properties for prolonged time periods in in vivo. We transplanted human fibroblast-derived iNs into the adult rat hippocampus and observed a progressive morphological differentiation, with more developed dendritic arborisation at six months as compared to one month. This was accompanied by mature electrophysiological properties and fast high amplitude action potentials at six months after transplantation. This proof-of-principle study suggests that human iNs can be developed as a candidate source for cell replacement therapy in temporal lobe epilepsy.

Directly Converted Human Fibroblasts Mature to Neurons and Show Long-Term Survival in Adult Rodent Hippocampus.

Astrocytes are emerging key players in neurological disorders. However, their role in disease etiology is poorly understood owing to inaccessibility of primary human astrocytes. Pluripotent stem cell-derived cells fail to mimic age and due to their clonal origin do not mimic genetic heterogeneity of patients. In contrast, direct conversion constitutes an attractive approach to generate human astrocytes that capture age and genetic diversity. We describe efficient direct conversion of human fibroblasts to functional induced astrocytes (iAs). Expression of the minimal combination Sox9 and Nfib generates iAs with molecular, phenotypic, and functional properties resembling primary human astrocytes. iAs could be obtained by conversion of fibroblasts covering the entire human lifespan. Importantly, iAs supported function of induced neurons obtained through direct conversion from the same fibroblast population. Fibroblast-derived iAs will become a useful tool to elucidate the biology of astrocytes and complement current in vitro models for studies of late-onset neurological disorders.

Article	Year
Optogenetics reveal delayed afferent synaptogenesis on grafted human-induced pluripotent stem cell-derived neural progenitors. Stem cells (Dayton, Ohio)\| PubMed ID: 25183299	2014
Optogenetic control of human neurons in organotypic brain cultures. Scientific reports\| PubMed ID: 27098488	2016
Directly Converted Human Fibroblasts Mature to Neurons and Show Long-Term Survival in Adult Rodent Hippocampus. Stem cells international\| PubMed ID: 29317869	2017
Transcription factor-based direct conversion of human fibroblasts to functional astrocytes. Stem cell reports\| PubMed ID: 35750047	2022

Natalia Avaliani

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