Retrospective identification of cell-intrinsic factors that mark pluripotency potential in rare somatic cells.

Pluripotency can be induced in somatic cells by the expression of OCT4, KLF4, SOX2, and MYC. Usually only a rare subset of cells reprogram, and the molecular characteristics of this subset remain unknown. We apply retrospective clone tracing to identify and characterize the rare human fibroblasts primed for reprogramming. These fibroblasts showed markers of increased cell cycle speed and decreased fibroblast activation. Knockdown of a fibroblast activation factor identified by our analysis increased the reprogramming efficiency. We provide evidence for a unified model in which cells can move into and out of the primed state over time, explaining how reprogramming appears deterministic at short timescales and stochastic at long timescales. Furthermore, inhibiting the activity of LSD1 enlarged the pool of cells that were primed for reprogramming. Thus, even homogeneous cell populations can exhibit heritable molecular variability that can dictate whether individual rare cells will reprogram or not.

Clinical interpretation of KCNH2 variants using a robust PS3/BS3 functional patch-clamp assay.

Long QT syndrome (LQTS), caused by the dysfunction of cardiac ion channels, increases the risk of sudden death in otherwise healthy young people. For many variants in LQTS genes, there is insufficient evidence to make a definitive genetic diagnosis. We have established a robust functional patch-clamp assay to facilitate classification of missense variants in KCNH2, one of the key LQTS genes. A curated set of 30 benign and 30 pathogenic missense variants were used to establish the range of normal and abnormal function. The extent to which variants reduced protein function was quantified using Z scores, the number of standard deviations from the mean of the normalized current density of the set of benign variant controls. A Z score of -2 defined the threshold for abnormal loss of function, which corresponds to 55% wild-type function. More extreme Z scores were observed for variants with a greater loss-of-function effect. We propose that the Z score for each variant can be used to inform the application and weighting of abnormal and normal functional evidence criteria (PS3 and BS3) within the American College of Medical Genetics and Genomics variant classification framework. The validity of this approach was demonstrated using a series of 18 KCNH2 missense variants detected in a childhood onset LQTS cohort, where the level of function assessed using our assay correlated to the Schwartz score (a scoring system used to quantify the probability of a clinical diagnosis of LQTS) and the length of the corrected QT (QTc) interval.

Retrospective identification of intrinsic factors that mark pluripotency potential in rare somatic cells.

Pluripotency can be induced in somatic cells by the expression of the four "Yamanaka" factors OCT4, KLF4, SOX2, and MYC. However, even in homogeneous conditions, usually only a rare subset of cells admit reprogramming, and the molecular characteristics of this subset remain unknown. Here, we apply retrospective clone tracing to identify and characterize the individual human fibroblast cells that are primed for reprogramming. These fibroblasts showed markers of increased cell cycle speed and decreased fibroblast activation. Knockdown of a fibroblast activation factor identified by our analysis led to increased reprogramming efficiency, identifying it as a barrier to reprogramming. Changing the frequency of reprogramming by inhibiting the activity of LSD1 led to an enlarging of the pool of cells that were primed for reprogramming. Our results show that even homogeneous cell populations can exhibit heritable molecular variability that can dictate whether individual rare cells will reprogram or not.

ClonoCluster: A method for using clonal origin to inform transcriptome clustering.

Clustering cells based on their high-dimensional profiles is an important data reduction process by which researchers infer distinct cellular states. The advent of cellular barcoding, however, provides an alternative means by which to group cells: by their clonal origin. We developed ClonoCluster, a computational method that combines both clone and transcriptome information to create hybrid clusters that weight both kinds of data with a tunable parameter. We generated hybrid clusters across six independent datasets and found that ClonoCluster generated qualitatively different clusters in all cases. The markers of these hybrid clusters were different but had equivalent fidelity to transcriptome-only clusters. The genes most strongly associated with the rearrangements in hybrid clusters were ribosomal function and extracellular matrix genes. We also developed the complementary tool Warp Factor that incorporates clone information in popular 2D visualization techniques like UMAP. Integrating ClonoCluster and Warp Factor revealed biologically relevant markers of cell identity.

ClampFISH 2.0 enables rapid, scalable amplified RNA detection in situ.

RNA labeling in situ has enormous potential to visualize transcripts and quantify their levels in single cells, but it remains challenging to produce high levels of signal while also enabling multiplexed detection of multiple RNA species simultaneously. Here, we describe clampFISH 2.0, a method that uses an inverted padlock design to efficiently detect many RNA species and exponentially amplify their signals at once, while also reducing the time and cost compared with the prior clampFISH method. We leverage the increased throughput afforded by multiplexed signal amplification and sequential detection to detect 10 different RNA species in more than 1 million cells. We also show that clampFISH 2.0 works in tissue sections. We expect that the advantages offered by clampFISH 2.0 will enable many applications in spatial transcriptomics.

A calibrated functional patch-clamp assay to enhance clinical variant interpretation in KCNH2-related long QT syndrome.

Modern sequencing technologies have revolutionized our detection of gene variants. However, in most genes, including KCNH2, the majority of missense variants are currently classified as variants of uncertain significance (VUSs). The aim of this study was to investigate the utility of an automated patch-clamp assay for aiding clinical variant classification in KCNH2. The assay was designed according to recommendations proposed by the Clinical Genome Sequence Variant Interpretation Working Group. Thirty-one variants (17 pathogenic/likely pathogenic, 14 benign/likely benign) were classified internally as variant controls. They were heterozygously expressed in Flp-In HEK293 cells for assessing the effects of variants on current density and channel gating in order to determine the sensitivity and specificity of the assay. All 17 pathogenic variant controls had reduced current density, and 13 of 14 benign variant controls had normal current density, which enabled determination of normal and abnormal ranges for applying evidence of moderate or supporting strength for VUS reclassification. Inclusion of functional assay evidence enabled us to reclassify 6 out of 44 KCNH2 VUSs as likely pathogenic. The high-throughput patch-clamp assay can provide moderate-strength evidence for clinical interpretation of clinical KCNH2 variants and demonstrates the value of developing automated patch-clamp assays for functional characterization of ion channel gene variants.

Cell type determination for cardiac differentiation occurs soon after seeding of human-induced pluripotent stem cells.

BACKGROUND: Cardiac differentiation of human-induced pluripotent stem (hiPS) cells consistently produces a mixed population of cardiomyocytes and non-cardiac cell types, even when using well-characterized protocols. We sought to determine whether different cell types might result from intrinsic differences in hiPS cells prior to the onset of differentiation. RESULTS: By associating individual differentiated cells that share a common hiPS cell precursor, we tested whether expression variability is predetermined from the hiPS cell state. In a single experiment, cells that shared a progenitor were more transcriptionally similar to each other than to other cells in the differentiated population. However, when the same hiPS cells were differentiated in parallel, we did not observe high transcriptional similarity across differentiations. Additionally, we found that substantial cell death occurs during differentiation in a manner that suggested all cells were equally likely to survive or die, suggesting that there is no intrinsic selection bias for cells descended from particular hiPS cell progenitors. We thus wondered how cells grow spatially during differentiation, so we labeled cells by expression of marker genes and found that cells expressing the same marker tended to occur in patches. Our results suggest that cell type determination across multiple cell types, once initiated, is maintained in a cell-autonomous manner for multiple divisions. CONCLUSIONS: Altogether, our results show that while substantial heterogeneity exists in the initial hiPS cell population, it is not responsible for the variability observed in differentiated outcomes; instead, factors specifying the various cell types likely act during a window that begins shortly after the seeding of hiPS cells for differentiation.

Treatment of Central Vertigo With Low Dose Olanzapine: Report of Two Cases.

Good treatments are available for many cases of vertigo due to a peripheral cause such as benign paroxysmal positional vertigo. Conversely, vertigo secondary to a central lesion remains a treatment challenge typically without good pharmacologic or other treatments. We have successfully treated two patients, the first to our knowledge, with central vertigo, one from brain injury, one after stroke, with low dose olanzapine which we found to quickly and dramatically resolve vertigo and permit functional normalization. In our two cases, we found that a low dose of olanzapine 2.5mg daily (typical dosing of olanzapine for the psychiatric disease is 5-20mg daily) caused vertigo to rapidly and dramatically remit. Interestingly, our two cases had different causes and possibly lesion locations.

Application of data science methods to identify school and home risk factors for asthma and allergy-related symptoms among children in New York.

OBJECTIVES: Few studies have comprehensively assessed multiple environmental exposures affecting children's health. This study applied machine-learning methods to evaluate how indoor environmental conditions at home and school contribute to asthma and allergy-related symptoms. METHODS: We randomly selected 10 public schools representing different socioeconomic statuses in New York State (2017-2019) and distributed questionnaires to students to collect health status and home-and school-environmental exposures. Indoor air quality was measured at school, and ambient particle exposures (PM2.5 and components) were measured using real-time personal monitors for 48 h. We used random forest model to identify the most important risk factors for asthma and allergy-related symptoms, and decision tree for visualizing the inter-relationships among the multiple risk factors with the health outcomes. RESULTS: The top contributing factors identified for asthma were family rhinitis history (relative importance: 10.40%), plant pollen trigger (5.48%); bedroom carpet (3.58%); environmental tobacco smoke (ETS) trigger symptom (2.98%); and ETS exposure (2.56%). For allergy-related symptoms, plant pollen trigger (10.88%), higher paternal education (7.33%), bedroom carpet (5.28%), family rhinitis history (4.78%), and higher maternal education (4.25%) were the strongest contributing factors. Conversely, primary heating with hot water radiator was negatively (-6.86%) associated with asthma symptoms. Younger children (<9 years old) with family history of rhinitis and carpeting in the bedroom were the prominent combined risk factors for asthma. Children jointly exposed to pollen, solvents, and carpeting in their home tended to have greater risks of allergy-related symptoms, even without family history of rhinitis. CONCLUSION: Family rhinitis history, bedroom carpet, and pollen triggers were the most important risk factors for both asthma and allergy-related symptoms. Our new findings included that hot-water radiator was related to reduced asthma symptoms, and the combination of young age, rhinitis history, and bedroom carpeting was related to increased asthma symptoms. Further studies are needed to confirm our findings.

Variability within rare cell states enables multiple paths toward drug resistance.

Molecular differences between individual cells can lead to dramatic differences in cell fate, such as death versus survival of cancer cells upon drug treatment. These originating differences remain largely hidden due to difficulties in determining precisely what variable molecular features lead to which cellular fates. Thus, we developed Rewind, a methodology that combines genetic barcoding with RNA fluorescence in situ hybridization to directly capture rare cells that give rise to cellular behaviors of interest. Applying Rewind to BRAFV600E melanoma, we trace drug-resistant cell fates back to single-cell gene expression differences in their drug-naive precursors (initial frequency of ~1:1,000-1:10,000 cells) and relative persistence of MAP kinase signaling soon after drug treatment. Within this rare subpopulation, we uncover a rich substructure in which molecular differences among several distinct subpopulations predict future differences in phenotypic behavior, such as proliferative capacity of distinct resistant clones after drug treatment. Our results reveal hidden, rare-cell variability that underlies a range of latent phenotypic outcomes upon drug exposure.

Genetic screening for single-cell variability modulators driving therapy resistance.

Cellular plasticity describes the ability of cells to transition from one set of phenotypes to another. In melanoma, transient fluctuations in the molecular state of tumor cells mark the formation of rare cells primed to survive BRAF inhibition and reprogram into a stably drug-resistant fate. However, the biological processes governing cellular priming remain unknown. We used CRISPR-Cas9 genetic screens to identify genes that affect cell fate decisions by altering cellular plasticity. We found that many factors can independently affect cellular priming and fate decisions. We discovered a new plasticity-based mode of increasing resistance to BRAF inhibition that pushes cells towards a more differentiated state. Manipulating cellular plasticity through inhibition of DOT1L before the addition of the BRAF inhibitor resulted in more therapy resistance than concurrent administration. Our results indicate that modulating cellular plasticity can alter cell fate decisions and may prove useful for treating drug resistance in other cancers.

Parallel Social Information Processing Circuits Are Differentially Impacted in Autism.

Parallel processing circuits are thought to dramatically expand the network capabilities of the nervous system. Magnocellular and parvocellular oxytocin neurons have been proposed to subserve two parallel streams of social information processing, which allow a single molecule to encode a diverse array of ethologically distinct behaviors. Here we provide the first comprehensive characterization of magnocellular and parvocellular oxytocin neurons in male mice, validated across anatomical, projection target, electrophysiological, and transcriptional criteria. We next use novel multiple feature selection tools in Fmr1-KO mice to provide direct evidence that normal functioning of the parvocellular but not magnocellular oxytocin pathway is required for autism-relevant social reward behavior. Finally, we demonstrate that autism risk genes are enriched in parvocellular compared with magnocellular oxytocin neurons. Taken together, these results provide the first evidence that oxytocin-pathway-specific pathogenic mechanisms account for social impairments across a broad range of autism etiologies.

Targeted demethylation at the CDKN1C/p57 locus induces human ß cell replication.

The loss of insulin-secreting ß cells is characteristic among type I and type II diabetes. Stimulating proliferation to expand sources of ß cells for transplantation remains a challenge because adult ß cells do not proliferate readily. The cell cycle inhibitor p57 has been shown to control cell division in human ß cells. Expression of p57 is regulated by the DNA methylation status of the imprinting control region 2 (ICR2), which is commonly hypomethylated in Beckwith-Wiedemann syndrome patients who exhibit massive ß cell proliferation. We hypothesized that targeted demethylation of the ICR2 using a transcription activator-like effector protein fused to the catalytic domain of TET1 (ICR2-TET1) would repress p57 expression and promote cell proliferation. We report here that overexpression of ICR2-TET1 in human fibroblasts reduces p57 expression levels and increases proliferation. Furthermore, human islets overexpressing ICR2-TET1 exhibit repression of p57 with concomitant upregulation of Ki-67 while maintaining glucose-sensing functionality. When transplanted into diabetic, immunodeficient mice, the epigenetically edited islets show increased ß cell replication compared with control islets. These findings demonstrate that epigenetic editing is a promising tool for inducing ß cell proliferation, which may one day alleviate the scarcity of transplantable ß cells for the treatment of diabetes.

ClampFISH detects individual nucleic acid molecules using click chemistry-based amplification.

Methods for detecting single nucleic acids in cell and tissues, such as fluorescence in situ hybridization (FISH), are limited by relatively low signal intensity and nonspecific probe binding. Here we present click-amplifying FISH (clampFISH), a method for fluorescence detection of nucleic acids that achieves high specificity and high-gain (>400-fold) signal amplification. ClampFISH probes form a 'C' configuration upon hybridization to the sequence of interest in a double helical manner. The ends of the probes are ligated together using bio-orthogonal click chemistry, effectively locking the probes around the target. Iterative rounds of hybridization and click amplify the fluorescence intensity. We show that clampFISH enables the detection of RNA species with low-magnification microscopy and in RNA-based flow cytometry. Additionally, we show that the modular design of clampFISH probes allows multiplexing of RNA and DNA detection, that the locking mechanism prevents probe detachment in expansion microscopy, and that clampFISH can be applied in tissue samples.

Visualizing allele-specific expression in single cells reveals epigenetic mosaicism in an H19 loss-of-imprinting mutant.

Imprinting is a classic mammalian epigenetic phenomenon that results in expression from a single parental allele. Imprinting defects can lead to inappropriate expression from the normally silenced allele, but it remains unclear whether every cell in a mutant organism follows the population average, which would have profound implications for human imprinting disorders. Here, we apply a new fluorescence in situ hybridization method that measures allele-specific expression in single cells to address this question in mutants exhibiting aberrant H19/Igf2 (insulin-like growth factor 2) imprinting. We show that mutant primary embryonic mouse fibroblasts are comprised of two subpopulations: one expressing both H19 alleles and another expressing only the maternal copy. Only in the latter cell population is Igf2 expression detected. Furthermore, the two subpopulations are stable in that cells do not interconvert between the two expression patterns. Combined small input methylation analysis and transcriptional imaging revealed that these two mutant subpopulations exhibit distinct methylation patterns at their imprinting control regions. Consistently, pharmacological inhibition of DNA methylation reduced the proportion of monoallelic cells. Importantly, we observed that the same two subpopulations are also present in vivo within murine cardiac tissue. Our results establish that imprinting disorders can display striking single-cell heterogeneity in their molecular phenotypes and suggest that such heterogeneity may underlie epigenetic mosaicism in human imprinting disorders.

Epigenetics and imprinting in human disease.

Most genes are expressed from both parental chromosomes; however, a small number of genes in mammals are imprinted and expressed in a parent-of-origin specific manner. These imprinted genes play an important role in embryonic and extraembryonic growth and development, as well as in a variety of processes after birth. Many imprinted genes are clustered in the genome with the establishment and maintenance of imprinted gene expression governed by complex epigenetic mechanisms. Dysregulation of these epigenetic mechanisms as well as genomic mutations at imprinted gene clusters can lead to human disease.

Cell age-specific vulnerability of neurons to anesthetic toxicity.

OBJECTIVE: Anesthetics have been linked to widespread neuronal cell death in neonatal animals. Epidemiological human studies have associated early childhood anesthesia with long-term neurobehavioral abnormalities, raising substantial concerns that anesthetics may cause similar cell death in young children. However, key aspects of the phenomenon remain unclear, such as why certain neurons die, whereas immediately adjacent neurons are seemingly unaffected, and why the immature brain is exquisitely vulnerable, whereas the mature brain seems resistant. Elucidating these questions is critical for assessing the phenomenon's applicability to humans, defining the susceptible age, predicting vulnerable neuronal populations, and devising mitigating strategies. METHODS: This study examines the effects of anesthetic exposure on late- and adult-generated neurons in newborn, juvenile, and adult mice, and characterizes vulnerable cells using birth-dating and immunohistochemical techniques. RESULTS: We identify a critical period of cellular developmental during which neurons are susceptible to anesthesia-induced apoptosis. Importantly, we demonstrate that anesthetic neurotoxicity can extend into adulthood in brain regions with ongoing neurogenesis, such as dentate gyrus and olfactory bulb. INTERPRETATION: Our findings suggest that anesthetic vulnerability reflects the age of the neuron, not the age of the organism, and therefore may potentially not only be relevant to children but also to adults undergoing anesthesia. This observation further predicts differential heightened regional vulnerability to anesthetic neuroapoptosis to closely follow the distinct regional peaks in neurogenesis. This knowledge may help guide neurocognitive testing of specific neurological domains in humans following exposure to anesthesia, dependent on the individual's age during exposure.