Differentiation of human pluripotent stem cells to small brain-like structures known as brain organoids offers an unprecedented opportunity to model human brain development and disease. To provide a ...vascularized and functional in vivo model of brain organoids, we established a method for transplanting human brain organoids into the adult mouse brain. Organoid grafts showed progressive neuronal differentiation and maturation, gliogenesis, integration of microglia, and growth of axons to multiple regions of the host brain. In vivo two-photon imaging demonstrated functional neuronal networks and blood vessels in the grafts. Finally, in vivo extracellular recording combined with optogenetics revealed intragraft neuronal activity and suggested graft-to-host functional synaptic connectivity. This combination of human neural organoids and an in vivo physiological environment in the animal brain may facilitate disease modeling under physiological conditions.
Somatic cells can be inefficiently and stochastically reprogrammed into induced pluripotent stem (iPS) cells by exogenous expression of Oct4 (also called Pou5f1), Sox2, Klf4 and Myc (hereafter ...referred to as OSKM). The nature of the predominant rate-limiting barrier(s) preventing the majority of cells to successfully and synchronously reprogram remains to be defined. Here we show that depleting Mbd3, a core member of the Mbd3/NuRD (nucleosome remodelling and deacetylation) repressor complex, together with OSKM transduction and reprogramming in naive pluripotency promoting conditions, result in deterministic and synchronized iPS cell reprogramming (near 100% efficiency within seven days from mouse and human cells). Our findings uncover a dichotomous molecular function for the reprogramming factors, serving to reactivate endogenous pluripotency networks while simultaneously directly recruiting the Mbd3/NuRD repressor complex that potently restrains the reactivation of OSKM downstream target genes. Subsequently, the latter interactions, which are largely depleted during early pre-implantation development in vivo, lead to a stochastic and protracted reprogramming trajectory towards pluripotency in vitro. The deterministic reprogramming approach devised here offers a novel platform for the dissection of molecular dynamics leading to establishing pluripotency at unprecedented flexibility and resolution.
Human cortical organoids, three-dimensional neuronal cultures, are emerging as powerful tools to study brain development and dysfunction. However, whether organoids can functionally connect to a ...sensory network in vivo has yet to be demonstrated. Here, we combine transparent microelectrode arrays and two-photon imaging for longitudinal, multimodal monitoring of human cortical organoids transplanted into the retrosplenial cortex of adult mice. Two-photon imaging shows vascularization of the transplanted organoid. Visual stimuli evoke electrophysiological responses in the organoid, matching the responses from the surrounding cortex. Increases in multi-unit activity (MUA) and gamma power and phase locking of stimulus-evoked MUA with slow oscillations indicate functional integration between the organoid and the host brain. Immunostaining confirms the presence of human-mouse synapses. Implantation of transparent microelectrodes with organoids serves as a versatile in vivo platform for comprehensive evaluation of the development, maturation, and functional integration of human neuronal networks within the mouse brain.
Induced pluripotent stem cells (iPSCs) can be derived from somatic cells by ectopic expression of different transcription factors, classically Oct4 (also known as Pou5f1), Sox2, Klf4 and Myc ...(abbreviated as OSKM). This process is accompanied by genome-wide epigenetic changes, but how these chromatin modifications are biochemically determined requires further investigation. Here we show in mice and humans that the histone H3 methylated Lys 27 (H3K27) demethylase Utx (also known as Kdm6a) regulates the efficient induction, rather than maintenance, of pluripotency. Murine embryonic stem cells lacking Utx can execute lineage commitment and contribute to adult chimaeric animals; however, somatic cells lacking Utx fail to robustly reprogram back to the ground state of pluripotency. Utx directly partners with OSK reprogramming factors and uses its histone demethylase catalytic activity to facilitate iPSC formation. Genomic analysis indicates that Utx depletion results in aberrant dynamics of H3K27me3 repressive chromatin demethylation in somatic cells undergoing reprogramming. The latter directly hampers the derepression of potent pluripotency promoting gene modules (including Sall1, Sall4 and Utf1), which can cooperatively substitute for exogenous OSK supplementation in iPSC formation. Remarkably, Utx safeguards the timely execution of H3K27me3 demethylation observed in embryonic day 10.5-11 primordial germ cells (PGCs), and Utx-deficient PGCs show cell-autonomous aberrant epigenetic reprogramming dynamics during their embryonic maturation in vivo. Subsequently, this disrupts PGC development by embryonic day 12.5, and leads to diminished germline transmission in mouse chimaeras generated from Utx-knockout pluripotent cells. Thus, we identify Utx as a novel mediator with distinct functions during the re-establishment of pluripotency and germ cell development. Furthermore, our findings highlight the principle that molecular regulators mediating loss of repressive chromatin during in vivo germ cell reprogramming can be co-opted during in vitro reprogramming towards ground state pluripotency.
Long interspersed nuclear element-1 (L1 or LINE-1) is a highly abundant mobile genetic element in both humans and mice, comprising almost 20% of each genome. L1s are silenced by several mechanisms, ...as their uncontrolled expression has the potential to induce genomic instability. However, L1s are paradoxically expressed at high levels in differentiating neural progenitor cells. Using in vitro and in vivo techniques to modulate L1 expression, we report that L1s play a critical role in both human and mouse brain development by regulating the rate of neural differentiation in a reverse-transcription-independent manner.
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•L1s are dynamically expressed during neural differentiation•L1s are required to maintain neural progenitor pools in mice and humans•L1s regulate neural differentiation in a retrotransposition-independent manner
Toda et al. find that long interspersed nuclear element-1 (L1 or LINE-1) regulates the maintenance of neural progenitor pools during brain development. The study highlights unrecognized physiological roles for repetitive elements in the genome during brain development and their potential impact on brain evolution.
During embryogenesis, the dorsal roof plate and the ventral floor plate (FP) act as organizing centers to pattern the developing neural tube. Organizer-secreted morphogens provide signals that are ...interpreted via the graded expression of transcription factors. These factors establish a combinatorial code, which subsequently determines the fate of neuronal progenitors along the dorsoventral axis. To further separate the fates and promote distinct identities of the neural progenitors, mutual repression takes place among transcription factors expressed in progenitors situated along the dorsoventral axis. The molecular mechanisms acting in the developing spinal cord and underlying the segregation of the progenitor pool containing cells with a mixed FP/p3 fate into separate FP cells and V3 neurons are not fully understood. Using in vivo ectopic expression in chick, we found that Nato3 induces ectopic Foxa2-positive cells and indirectly downregulates Nkx2.2 expression. To examine the role of Nato3 in the FP, Foxa2-Nato3 signaling was blocked in Nato3 null mice and to a greater extent in Nato3 null/Foxa2 heterozygous bigenic mutants. Complementary to the findings obtained by gain of function in chick, the loss of function in mouse indicated that the segregation of the FP/p3 population into its derivatives was interrupted. Together, the data suggest that Nato3 is a novel determinant factor regulating the segregation of the FP and p3 identities, which is an essential step for establishing a definitive FP fate in the embryonic spinal cord.
Several mutations that cause Parkinson's disease (PD) have been identified over the past decade. These account for 15-25% of PD cases; the rest of the cases are considered sporadic. Currently, it is ...accepted that PD is not a single monolithic disease but rather a constellation of diseases with some common phenotypes. While rodent models exist for some of the PD-causing mutations, research on the sporadic forms of PD is lagging due to a lack of cellular models. In our study, we differentiated PD patient-derived dopaminergic (DA) neurons from the induced pluripotent stem cells (iPSCs) of several PD-causing mutations as well as from sporadic PD patients. Strikingly, we observed a common neurophysiological phenotype: neurons derived from PD patients had a severe reduction in the rate of synaptic currents compared to those derived from healthy controls. While the relationship between mutations in genes such as the SNCA and LRRK2 and a reduction in synaptic transmission has been investigated before, here we show evidence that the pathogenesis of the synapses in neurons is a general phenotype in PD. Analysis of RNA sequencing results displayed changes in gene expression in different synaptic mechanisms as well as other affected pathways such as extracellular matrix-related pathways. Some of these dysregulated pathways are common to all PD patients (monogenic or idiopathic). Our data, therefore, show changes that are central and convergent to PD and suggest a strong involvement of the tetra-partite synapse in PD pathophysiology.
Naïve and primed pluripotent states retain distinct molecular properties, yet limited knowledge exists on how their state transitions are regulated. Here, we identify Mettl3, an N6-methyladenosine ...(m6A) transferase, as a regulator for terminating murine naïve pluripotency. Mettl3 knockout preimplantation epiblasts and naïve embryonic stem cells are depleted for m6A in mRNAs, yet are viable. However, they fail to adequately terminate their naïve state and, subsequently, undergo aberrant and restricted lineage priming at the postimplantation stage, which leads to early embryonic lethality. m6A predominantly and directly reduces mRNA stability, including that of key naïve pluripotency-promoting transcripts. This study highlights a critical role for an mRNA epigenetic modification in vivo and identifies regulatory modules that functionally influence naïve and primed pluripotency in an opposing manner.
Early cell fate decisions demand rapid rewiring of transcriptional circuits. Stanton and colleagues report on enhancer‐dependent partnering of Oct4 with either Sox2 or Sox17 to switch from ...pluripotency to differentiation.
Brain organoids are three-dimensional neural aggregates derived from pluripotent stem cells through self-organization and recapitulate architectural and cellular aspects of certain brain regions. ...Brain organoids are currently a highly exciting area of research that includes the study of human brain development, function, and dysfunction in unprecedented ways. In this Review, we discuss recent discoveries related to the generation of brain organoids that resemble diverse brain regions. We provide an overview of the strategies to complement these primarily neuroectodermal models with cell types of non-neuronal origin, such as vasculature and immune cells. Recent transplantation approaches aiming to achieve higher cellular complexity and long-term survival of these models will then be discussed. We conclude by highlighting unresolved key questions and future directions in this exciting area of human brain organogenesis.
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