Factor-induced reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is inefficient, complicating mechanistic studies. Here, we examined defined intermediate cell populations ...poised to becoming iPSCs by genome-wide analyses. We show that induced pluripotency elicits two transcriptional waves, which are driven by c-Myc/Klf4 (first wave) and Oct4/Sox2/Klf4 (second wave). Cells that become refractory to reprogramming activate the first but fail to initiate the second transcriptional wave and can be rescued by elevated expression of all four factors. The establishment of bivalent domains occurs gradually after the first wave, whereas changes in DNA methylation take place after the second wave when cells acquire stable pluripotency. This integrative analysis allowed us to identify genes that act as roadblocks during reprogramming and surface markers that further enrich for cells prone to forming iPSCs. Collectively, our data offer new mechanistic insights into the nature and sequence of molecular events inherent to cellular reprogramming.
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► Transcriptional analysis of iPSC formation reveals biphasic process ► c-Myc/Klf4 drive first phase whereas Oct4/Sox2/Klf4 drive second phase ► Bivalent genes form gradually whereas DNA methylation changes occur late ► Refractory cells can be rescued by elevated reprogramming factor expression
By sorting fibroblasts induced to reprogram to iPSCs, the molecular characteristics of cells in the early and late stages of reprogramming are revealed, as well as the attributes of the cells refractory to reprogramming. New genes that enhance reprogramming efficiency are identified.
Human brain organoids generated with current technologies recapitulate histological features of the human brain, but they lack a reproducible topographic organization. During development, spatial ...topography is determined by gradients of signaling molecules released from discrete signaling centers. We hypothesized that introduction of a signaling center into forebrain organoids would specify the positional identity of neural tissue in a distance-dependent manner. Here, we present a system to trigger a Sonic Hedgehog (SHH) protein gradient in developing forebrain organoids that enables ordered self-organization along dorso-ventral and antero-posterior positional axes. SHH-patterned forebrain organoids establish major forebrain subdivisions that are positioned with in vivo-like topography. Consistent with its behavior in vivo, SHH exhibits long-range signaling activity in organoids. Finally, we use SHH-patterned cerebral organoids as a tool to study the role of cholesterol metabolism in SHH signaling. Together, this work identifies inductive signaling as an effective organizing strategy to recapitulate in vivo-like topography in human brain organoids.
The classical way of defining enzyme inhibition has obscured the distinction between inhibitory effect and the inhibitor binding constant. This article examines the relationship between the simple ...binding curve used to define biomolecular interactions and the standard inhibitory term (1 + (
∕
)). By understanding how this term relates to binding curves which are ubiquitously used to describe biological processes, a modifier equation which distinguishes between inhibitor binding and the inhibitory effect, is examined. This modifier equation which can describe both activation and inhibition is compared to standard inhibitory equations with the development of global data fitting templates in Excel and via the global fitting of these equations to simulated and previously published datasets. In both cases, this modifier equation was able to match or outperform the other equations by providing superior fits to the datasets. The ability of this single equation to outperform the other equations suggests an over-complication of the field. This equation and the template developed in this article should prove to be useful tools in the study of enzyme inhibition and activation.
Aberrant inflammation in the CNS has been implicated as a major player in the pathogenesis of human neurodegenerative disease. We developed a new approach to derive microglia from human pluripotent ...stem cells (hPSCs) and built a defined hPSC-derived tri-culture system containing pure populations of hPSC-derived microglia, astrocytes, and neurons to dissect cellular cross-talk along the neuroinflammatory axis in vitro. We used the tri-culture system to model neuroinflammation in Alzheimer's disease with hPSCs harboring the APP
+/+ mutation and their isogenic control. We found that complement C3, a protein that is increased under inflammatory conditions and implicated in synaptic loss, is potentiated in tri-culture and further enhanced in APP
+/+ tri-cultures due to microglia initiating reciprocal signaling with astrocytes to produce excess C3. Our study defines the major cellular players contributing to increased C3 in Alzheimer's disease and presents a broadly applicable platform to study neuroinflammation in human disease.
Magnetic resonance imaging (MRI) based diffusion tensor imaging (DTI) can assess white matter (WM) integrity through several metrics, such as fractional anisotropy (FA), axial/radial diffusivities ...(AxD/RD), and mode of anisotropy (MA). Standard DTI is susceptible to the effects of extracellular free water (FW), which can be removed using an advanced free-water DTI (FW-DTI) model. The purpose of this study was to compare standard and FW-DTI metrics in the context of Alzheimer's disease (AD). Data were obtained from the Open Access Series of Imaging Studies (OASIS-3) database and included both healthy controls (HC) and mild-to-moderate AD. With both standard and FW-DTI, decreased FA was found in AD, mainly in the corpus callosum and fornix, consistent with neurodegenerative mechanisms. Widespread higher AxD and RD were observed with standard DTI; however, the FW index, indicative of AD-associated neurodegeneration, was significantly elevated in these regions in AD, highlighting the potential impact of free water contributions on standard DTI in neurodegenerative pathologies. Using FW-DTI, improved consistency was observed in FA, AxD, and RD, and the complementary FW index was higher in the AD group as expected. With both standard and FW-DTI, higher values of MA coupled with higher values of FA in AD were found in the anterior thalamic radiation and cortico-spinal tract, most likely arising from a loss of crossing fibers. In conclusion, FW-DTI better reflects the underlying pathology of AD and improves the accuracy of DTI metrics related to WM integrity in Alzheimer's disease.
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Over the past decade, DNA-encoded libraries (DELs) have emerged as a leading platform for small molecule drug discovery among pharmaceutical companies, biotech companies and academic ...drug hunters alike. This revolutionary technology has tremendous potential that is yet to be fully realized, as the exploration of therapeutically relevant chemical space is fueled by the ever-expanding repertoire of DNA-compatible reactions used to construct the libraries. Advances in direct coupling reactions, like photo-catalytic cross couplings, unique cyclizations such as the formation of 1,2,4–oxadiazoles, and new functional group transformations are valuable contributions to the DEL reaction toolkit, and indicate where future reaction development efforts should focus in order to maximize the productivity of DELs.
The chromatin state of pluripotency genes has been studied extensively in embryonic stem cells (ESCs) and differentiated cells, but their potential interactions with other parts of the genome remain ...largely unexplored. Here, we identified a genome-wide, pluripotency-specific interaction network around the Nanog promoter by adapting circular chromosome conformation capture sequencing. This network was rearranged during differentiation and restored in induced pluripotent stem cells. A large fraction of Nanog-interacting loci were bound by Mediator or cohesin in pluripotent cells. Depletion of these proteins from ESCs resulted in a disruption of contacts and the acquisition of a differentiation-specific interaction pattern prior to obvious transcriptional and phenotypic changes. Similarly, the establishment of Nanog interactions during reprogramming often preceded transcriptional upregulation of associated genes, suggesting a causative link. Our results document a complex, pluripotency-specific chromatin “interactome” for Nanog and suggest a functional role for long-range genomic interactions in the maintenance and induction of pluripotency.
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•The Nanog promoter forms a pluripotency-specific, genome-wide chromatin interactome•Nanog interactions in ESCs are enriched for active marks and pluripotency factors•Mediator and cohesin are essential for the maintenance of the Nanog interactome•Many Nanog interactions form before increased gene expression during reprogramming
The Nanog promoter interacts with the rest of the genome in an unexpectedly complex pluripotency-specific chromatin interaction network that depends on Mediator and cohesin.
Acute Onset of Diplopia and Nystagmus Saladi, Nikita; Anandarajah, Hari; Walsh, Ryan D
Journal of neuro-ophthalmology,
2024-Mar-01, Letnik:
44, Številka:
1
Journal Article
PDF
