Speeding to pluripotency.

Finding a cell that reprograms in a nonstochastic manner without genetic manipulation has proven elusive. In this issue, Guo et al. report the identification of a cell defined by an ultrafast cycle whose progeny reprogram in a synchronous and rapid manner.

Retrospective Analysis of Blood Biomarkers of Neurological Injury in Human Cases of Viral Infection and Bacterial Sepsis.

BACKGROUND: Blood biomarkers of neurological injury could provide a rapid diagnosis of central nervous system (CNS) injury caused by infections. An FDA-approved assay for mild traumatic brain injury (TBI) measures glial fibrillary acidic protein (GFAP) and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), which signal astrocyte and neuronal injury, respectively. Here, we assessed the applicability of this biomarker assay for determining infection-induced brain injury. METHODS: We measured serum levels of GFAP and UCH-L1 retrospectively in serum samples from three study populations: 1) human cases infected with Venezuelan equine encephalitis virus (VEEV) and Madariaga virus (MADV) (n = 73), 2) human sepsis patients who were severely ill or diagnosed with encephalitis (n = 66), and 3) sepsis cases that were subsequently evaluated for cognitive impairment (n = 64). RESULTS: In the virus infection group, we found elevated GFAP for VEEV (p = 0.014) and MADV (p = 0.011) infections, which correlated with seizures (p = 0.006). In the bacterial sepsis group, GFAP was elevated in cases diagnosed with encephalitis (p = 0.0007) and correlated with headaches (p = 0.0002). In the bacterial sepsis cases with a later cognitive assessment, elevated GFAP (p = 0.0057) at study enrollment was associated with cognitive impairment six months later with a positive prognostic capacity of 79% (CI: 66-95%; p = 0.0068). CONCLUSIONS: GFAP and UCH-L1 levels measured using an FDA-approved assay for TBI may indicate brain injury resulting from viral or bacterial infections and could predict the development of neurological sequelae.

Precision symptom phenotyping identifies early clinical and proteomic predictors of distinct COVID-19 sequelae.

BACKGROUND: Post-COVID conditions (PCC) are difficult to characterize, diagnose, predict, and treat due to overlapping symptoms and poorly understood pathology. Identifying inflammatory profiles may improve clinical prognostication and trial endpoints. METHODS: 1,988 SARS-CoV-2 positive U.S. Military Health System beneficiaries with quantitative post-COVID symptom scores were included in this analysis. Among participants who reported moderate-to-severe symptoms on surveys collected 6-months post-SARS-CoV-2 infection, principal component analysis (PCA) followed by K-means clustering identified distinct clusters of symptoms. RESULTS: Three symptom-based clusters were identified: a sensory cluster (loss of smell and/or taste), a fatigue/difficulty thinking cluster, and a difficulty breathing/exercise intolerance cluster. Individuals within the sensory cluster were all outpatients during their initial COVID-19 presentation. The difficulty breathing cluster had a higher likelihood of obesity and COVID-19 hospitalization compared to those with no/mild symptoms at 6-months post-infection. Multinomial regression linked early post-infection D-dimer and IL-1RA elevation to fatigue/difficulty thinking, and elevated ICAM-1 concentrations to sensory symptoms. CONCLUSIONS: We identified three distinct symptom-based PCC phenotypes with specific clinical risk factors and early post-infection inflammatory predictors. With further validation and characterization, this framework may allow more precise classification of PCC cases and potentially improve the diagnosis, prognostication, and treatment of PCC.

Durability of SARS-CoV-2-Specific T-Cell Responses at 12 Months Postinfection.

BACKGROUND: Characterizing the longevity and quality of cellular immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enhances understanding of coronavirus disease 2019 (COVID-19) immunity that influences clinical outcomes. Prior studies suggest SARS-CoV-2-specific T cells are present in peripheral blood 10 months after infection. Analysis of the function, durability, and diversity of cellular response long after natural infection, over a range of ages and disease phenotypes, is needed to identify preventative and therapeutic interventions. METHODS: We identified participants in our multisite longitudinal, prospective cohort study 12 months after SARS-CoV-2 infection representing a range of disease severity. We investigated function, phenotypes, and frequency of T cells specific for SARS-CoV-2 using intracellular cytokine staining and spectral flow cytometry, and compared magnitude of SARS-CoV-2-specific antibodies. RESULTS: SARS-CoV-2-specific antibodies and T cells were detected 12 months postinfection. Severe acute illness was associated with higher frequencies of SARS-CoV-2-specific CD4 T cells and antibodies at 12 months. In contrast, polyfunctional and cytotoxic T cells responsive to SARS-CoV-2 were identified in participants over a wide spectrum of disease severity. CONCLUSIONS: SARS-CoV-2 infection induces polyfunctional memory T cells detectable at 12 months postinfection, with higher frequency noted in those who experienced severe disease.

Sepsis endotypes identified by host gene expression across global cohorts.

BACKGROUND: Sepsis from infection is a global health priority and clinical trials have failed to deliver effective therapeutic interventions. To address complicating heterogeneity in sepsis pathobiology, and improve outcomes, promising precision medicine approaches are helping identify disease endotypes, however, they require a more complete definition of sepsis subgroups. METHODS: Here, we use RNA sequencing from peripheral blood to interrogate the host response to sepsis from participants in a global observational study carried out in West Africa, Southeast Asia, and North America (N = 494). RESULTS: We identify four sepsis subtypes differentiated by 28-day mortality. A low mortality immunocompetent group is specified by features that describe the adaptive immune system. In contrast, the three high mortality groups show elevated clinical severity consistent with multiple organ dysfunction. The immunosuppressed group members show signs of a dysfunctional immune response, the acute-inflammation group is set apart by molecular features of the innate immune response, while the immunometabolic group is characterized by metabolic pathways such as heme biosynthesis. CONCLUSIONS: Our analysis reveals details of molecular endotypes in sepsis that support immunotherapeutic interventions and identifies biomarkers that predict outcomes in these groups.

Sepsis is a life-threatening multi-organ failure caused by the body's immune response to infection. Clinical symptoms of sepsis vary from one person to another likely due to differences in host factors, infecting pathogen, and comorbidities. This difference in clinical symptoms may contribute to the lack of effective interventions for sepsis. Therefore, approaches tailored to targeting groups of patients who present similarly are of great interest. This study analysed a large group of sepsis patients with diverse symptoms using laboratory markers and mathematical analysis. We report four patient groups that differ by risk of death and immune response profile. Targeting these defined groups with tailored interventions presents an exciting opportunity to improve the health outcomes of patients with sepsis.

Gene expression signatures in blood from a West African sepsis cohort define host response phenotypes.

Our limited understanding of the pathophysiological mechanisms that operate during sepsis is an obstacle to rational treatment and clinical trial design. There is a critical lack of data from low- and middle-income countries where the sepsis burden is increased which inhibits generalized strategies for therapeutic intervention. Here we perform RNA sequencing of whole blood to investigate longitudinal host response to sepsis in a Ghanaian cohort. Data dimensional reduction reveals dynamic gene expression patterns that describe cell type-specific molecular phenotypes including a dysregulated myeloid compartment shared between sepsis and COVID-19. The gene expression signatures reported here define a landscape of host response to sepsis that supports interventions via targeting immunophenotypes to improve outcomes.

Direct and indirect contribution of human embryonic stem cell-derived hepatocyte-like cells to liver repair in mice.

BACKGROUND & AIMS: Many studies of embryonic stem cells have investigated direct cell replacement of damaged tissues, but little is known about how donor cell-derived signals affect host tissue regeneration. We investigated the direct and indirect roles of human embryonic stem cell-derived cells in liver repair in mice. METHODS: To promote the initial differentiation of human embryonic stem cells into mesendoderm, we activated the ß-catenin signaling pathway with lithium; cells were then further differentiated into hepatocyte-like cells. The differentiated cells were purified by indocyanine green staining and laser microdissection and characterized by immunostaining, polymerase chain reaction, biochemical function, electron microscopy, and transplantation analyses. To investigate indirect effects of these cells, secreted proteins (secretomes) were analyzed by a label-free quantitative mass spectrometry. Carbon tetrachloride was used to induce acute liver injury in mice; cells or secreted proteins were administered by intrasplenic or intraperitoneal injection, respectively. RESULTS: The differentiated hepatocyte-like cells had multiple features of normal hepatocytes, engrafted efficiently into mice, and continued to have hepatic features; they promoted proliferation of host hepatocytes and revascularization of injured host liver tissues. Proteomic analysis identified proteins secreted from these cells that might promote host tissue repair. Injection of the secreted proteins into injured livers of mice promoted significant amounts of tissue regeneration without cell grafts. CONCLUSIONS: Hepatocyte-like cells derived from human embryonic stem cells contribute to recovery of injured liver tissues in mice, not only by cell replacement but also by delivering trophic factors that support endogenous liver regeneration.

New cell lines from mouse epiblast share defining features with human embryonic stem cells.

The application of human embryonic stem (ES) cells in medicine and biology has an inherent reliance on understanding the starting cell population. Human ES cells differ from mouse ES cells and the specific embryonic origin of both cell types is unclear. Previous work suggested that mouse ES cells could only be obtained from the embryo before implantation in the uterus. Here we show that cell lines can be derived from the epiblast, a tissue of the post-implantation embryo that generates the embryo proper. These cells, which we refer to as EpiSCs (post-implantation epiblast-derived stem cells), express transcription factors known to regulate pluripotency, maintain their genomic integrity, and robustly differentiate into the major somatic cell types as well as primordial germ cells. The EpiSC lines are distinct from mouse ES cells in their epigenetic state and the signals controlling their differentiation. Furthermore, EpiSC and human ES cells share patterns of gene expression and signalling responses that normally function in the epiblast. These results show that epiblast cells can be maintained as stable cell lines and interrogated to understand how pluripotent cells generate distinct fates during early development.

Point-of-care biomarker assay for rapid multiplexed detection of CRP and IP-10.

Rapid and accurate measurements of immune protein markers are essential for diagnosis and treatment in all clinical settings. The recent pandemic has revealed a stark need for developing new tools and assays that could be rapidly used in diverse settings and provide useful information to clinicians. Here, we describe the development and test application of a novel one-step CRP/IP-10 duplex assay for the LightDeck platform capable of delivering reproducible and accurate measurements in under eight minutes. We used the optimized assay to measure CRP and IP-10 levels in human blood and serum samples from healthy, SARS-CoV-2 (COVID-19) positive, and influenza-like illness (ILI) presenting patients. Our results agreed with previously published analyte levels and enabled us to make statistically significant comparisons relevant to multiple clinical parameters. Our duplex assay is a simple and powerful tool for aiding prognostic decision-making in diverse settings.

A machine learning approach identifies distinct early-symptom cluster phenotypes which correlate with hospitalization, failure to return to activities, and prolonged COVID-19 symptoms.

BACKGROUND: Accurate COVID-19 prognosis is a critical aspect of acute and long-term clinical management. We identified discrete clusters of early stage-symptoms which may delineate groups with distinct disease severity phenotypes, including risk of developing long-term symptoms and associated inflammatory profiles. METHODS: 1,273 SARS-CoV-2 positive U.S. Military Health System beneficiaries with quantitative symptom scores (FLU-PRO Plus) were included in this analysis. We employed machine-learning approaches to identify symptom clusters and compared risk of hospitalization, long-term symptoms, as well as peak CRP and IL-6 concentrations. RESULTS: We identified three distinct clusters of participants based on their FLU-PRO Plus symptoms: cluster 1 ("Nasal cluster") is highly correlated with reporting runny/stuffy nose and sneezing, cluster 2 ("Sensory cluster") is highly correlated with loss of smell or taste, and cluster 3 ("Respiratory/Systemic cluster") is highly correlated with the respiratory (cough, trouble breathing, among others) and systemic (body aches, chills, among others) domain symptoms. Participants in the Respiratory/Systemic cluster were twice as likely as those in the Nasal cluster to have been hospitalized, and 1.5 times as likely to report that they had not returned-to-activities, which remained significant after controlling for confounding covariates (P < 0.01). Respiratory/Systemic and Sensory clusters were more likely to have symptoms at six-months post-symptom-onset (P = 0.03). We observed higher peak CRP and IL-6 in the Respiratory/Systemic cluster (P < 0.01). CONCLUSIONS: We identified early symptom profiles potentially associated with hospitalization, return-to-activities, long-term symptoms, and inflammatory profiles. These findings may assist in patient prognosis, including prediction of long COVID risk.

Screening tools for predicting mortality of adults with suspected sepsis: an international sepsis cohort validation study.

OBJECTIVES: We evaluated the performance of commonly used sepsis screening tools across prospective sepsis cohorts in the USA, Cambodia and Ghana. DESIGN: Prospective cohort studies. SETTING AND PARTICIPANTS: From 2014 to 2021, participants with two or more SIRS (Systemic Inflammatory Response Syndrome) criteria and suspected infection were enrolled in emergency departments and medical wards at hospitals in Cambodia and Ghana and hospitalised participants with suspected infection were enrolled in the USA. Cox proportional hazards regression was performed, and Harrell's C-statistic calculated to determine 28-day mortality prediction performance of the quick Sequential Organ Failure Assessment (qSOFA) score ≥2, SIRS score ≥3, National Early Warning Score (NEWS) ≥5, Modified Early Warning Score (MEWS) ≥5 or Universal Vital Assessment (UVA) score ≥2. Screening tools were compared with baseline risk (age and sex) with the Wald test. RESULTS: The cohorts included 567 participants (42.9% women) including 187 participants from Kumasi, Ghana, 200 participants from Takeo, Cambodia and 180 participants from Durham, North Carolina in the USA. The pooled mortality was 16.4% at 28 days. The mortality prediction accuracy increased from baseline risk with the MEWS (C-statistic: 0.63, 95% CI 0.58 to 0.68; p=0.002), NEWS (C-statistic: 0.68; 95% CI 0.64 to 0.73; p<0.001), qSOFA (C-statistic: 0.70, 95% CI 0.64 to 0.75; p<0.001), UVA score (C-statistic: 0.73, 95% CI 0.69 to 0.78; p<0.001), but not with SIRS (0.60; 95% CI 0.54 to 0.65; p=0.13). Within individual cohorts, only the UVA score in Ghana performed better than baseline risk (C-statistic: 0.77; 95% CI 0.71 to 0.83; p<0.001). CONCLUSIONS: Among the cohorts, MEWS, NEWS, qSOFA and UVA scores performed better than baseline risk, largely driven by accuracy improvements in Ghana, while SIRS scores did not improve prognostication accuracy. Prognostication scores should be validated within the target population prior to clinical use.

Rapid, Low-Complexity, Simultaneous Bacterial Group Identification and Antimicrobial Susceptibility Testing Performed Directly on Positive Blood Culture Bottles Using Chromogenic Agar.

The use of positive blood culture bottles for direct disk diffusion susceptibility testing (dDD), together with chromogenic culture limited to groups of pathogens for antimicrobial susceptibility testing interpretation may provide a means for laboratories-in-development to introduce rapid abbreviated blood culture testing. We assessed the performance of dDD on Chromatic MH agar using contrived positive blood culture bottles and compared findings with current standard practice. Furthermore, we characterized the growth of 24 bacterial and 3 yeast species on Chromatic MH agar with the aid of rapid spot tests for same-day identification. The coefficient of variation for reproducibility of dDD of four reference strains in 4 to 10 replicates (238 data points) ranged from 0% to 16.3%. Together with an additional 10 challenge isolates, the overall categorical agreement was 91.7% (351 data points). The following bacteria were readily identifiable: cream/white Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus pyogenes; turquoise Streptococcus agalactiae, enterococci, Listeria monocytogenes; mauve Escherichia coli, Shigella sonnei, Citrobacter freundii; dark-blue Klebsiella and Enterobacter; green Pseudomonas aeruginosa; and brown Proteus. Clear colonies were seen with Salmonella, Acinetobacter, Burkholderia, and Yersinia enterocolitica (turns pink). Our study suggests that Chromatic MH for dDD may show promise as a rapid, clinically useful presumptive method for overnight simultaneous identification and antimicrobial susceptibility testing. However, there is a need to optimize the medium formulation to allow the recovery of Streptococcus pneumoniae and Haemophilus influenzae.

Assessing the use of a micro-sampling device for measuring blood protein levels in healthy subjects and COVID-19 patients.

BACKGROUND: Venous phlebotomy performed by trained personnel is critical for patient diagnosis and monitoring of chronic disease, but has limitations in resource-constrained settings, and represents an infection control challenge during outbreaks. Self-collection devices have the potential to shift phlebotomy closer to the point of care, supporting telemedicine strategies and virtual clinical trials. Here we assess a capillary blood micro-sampling device, the Tasso Serum Separator Tube (SST), for measuring blood protein levels in healthy subjects and non-hospitalized COVID-19 patients. METHODS: 57 healthy controls and 56 participants with mild/moderate COVID-19 were recruited at two U.S. military healthcare facilities. Healthy controls donated Tasso SST capillary serum, venous plasma and venous serum samples at multiple time points, while COVID-19 patients donated a single Tasso SST serum sample at enrolment. Concentrations of 17 protein inflammatory biomarkers were measured in all biospecimens by Ella multi-analyte immune-assay. RESULTS: Tasso SST serum protein measurements in healthy control subjects were highly reproducible, but their agreements with matched venous samples varied. Most of the selected proteins, including CRP, Ferritin, IL-6 and PCT, were well-correlated between Tasso SST and venous serum with little sample type bias, but concentrations of D-dimer, IL-1B and IL-1Ra were not. Self-collection at home with delayed sample processing was associated with significant concentrations differences for several analytes compared to supervised, in-clinic collection with rapid processing. Finally, Tasso SST serum protein concentrations were significantly elevated in in non-hospitalized COVID-19 patients compared with healthy controls. CONCLUSIONS: Self-collection of capillary blood with micro-sampling devices provides an attractive alternative to routine phlebotomy. However, concentrations of certain analytes may differ significantly from those in venous samples, and factors including user proficiency, temperature control and time lags between specimen collection and processing need to be considered for their effect on sample quality and reproducibility.

Distinct blood inflammatory biomarker clusters stratify host phenotypes during the middle phase of COVID-19.

The associations between clinical phenotypes of coronavirus disease 2019 (COVID-19) and the host inflammatory response during the transition from peak illness to convalescence are not yet well understood. Blood plasma samples were collected from 129 adult SARS-CoV-2 positive inpatient and outpatient participants between April 2020 and January 2021, in a multi-center prospective cohort study at 8 military hospitals across the United States. Plasma inflammatory protein biomarkers were measured in samples from 15 to 28 days post symptom onset. Topological Data Analysis (TDA) was used to identify patterns of inflammation, and associations with peak severity (outpatient, hospitalized, ICU admission or death), Charlson Comorbidity Index (CCI), and body mass index (BMI) were evaluated using logistic regression. The study population (n = 129, 33.3% female, median 41.3 years of age) included 77 outpatient, 31 inpatient, 16 ICU-level, and 5 fatal cases. Three distinct inflammatory biomarker clusters were identified and were associated with significant differences in peak disease severity (p < 0.001), age (p < 0.001), BMI (p < 0.001), and CCI (p = 0.001). Host-biomarker profiles stratified a heterogeneous population of COVID-19 patients during the transition from peak illness to convalescence, and these distinct inflammatory patterns were associated with comorbid disease and severe illness due to COVID-19.

COVID-19 Outcomes Among US Military Health System Beneficiaries Include Complications Across Multiple Organ Systems and Substantial Functional Impairment.

BACKGROUND: We evaluated clinical outcomes, functional burden, and complications 1 month after coronavirus disease 2019 (COVID-19) infection in a prospective US Military Health System (MHS) cohort of active duty, retiree, and dependent populations using serial patient-reported outcome surveys and electronic medical record (EMR) review. METHODS: MHS beneficiaries presenting at 9 sites across the United States with a positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) test, a COVID-19-like illness, or a high-risk SARS-CoV-2 exposure were eligible for enrollment. Medical history and clinical outcomes were collected through structured interviews and International Classification of Diseases-based EMR review. Risk factors associated with hospitalization were determined by multivariate logistic regression. RESULTS: A total of 1202 participants were enrolled. There were 1070 laboratory-confirmed SARS-CoV-2 cases and 132 SARS-CoV-2-negative participants. In the first month post-symptom onset among the SARS-CoV-2-positive cases, there were 212 hospitalizations, 80% requiring oxygen, 20 ICU admissions, and 10 deaths. Risk factors for COVID-19-associated hospitalization included race (increased for Asian, Black, and Hispanic compared with non-Hispanic White), age (age 45-64 and 65+ compared with <45), and obesity (BMI≥30 compared with BMI<30). Over 2% of survey respondents reported the need for supplemental oxygen, and 31% had not returned to normal daily activities at 1 month post-symptom onset. CONCLUSIONS: Older age, reporting Asian, Black, or Hispanic race/ethnicity, and obesity are associated with SARS-CoV-2 hospitalization. A proportion of acute SARS-CoV-2 infections require long-term oxygen therapy; the impact of SARS-CoV-2 infection on short-term functional status was substantial. A significant number of MHS beneficiaries had not yet returned to normal activities by 1 month.

Epiblast stem cells contribute new insight into pluripotency and gastrulation.

Gastrulation is the defining feature of metazoan development where it serves to apportion seemingly equivalent, pluripotent cells to specific fates. The three embryonic germ layers generated during gastrulation from the pluripotent epiblast including ectoderm, mesoderm, and definitive endoderm, contain the progenitors required to build all of the tissues of the developing organism. As a result, there is great interest in understanding the events that coordinate gastrulation. Because developing embryos in placental mammals are relatively inaccessible, stem cells are widely used for experimental and biochemical interrogation of these processes. Epiblast stem cells (EpiSCs) are grown from the post-implantation epiblast, which is the most proximal pluripotent tissue to the early somatic and germ cell precursors. Because EpiSCs can be propagated indefinitely in vitro as a stable state that recapitulates the properties of the post-implantation epiblast, they are uniquely positioned to provide novel insight into the developmental window where somatic and germ cell lineages are first established. Here we discuss the nature of EpiSCs and their significance in understanding gastrulation and cell specification in relationship to other pluripotent cell culture models.

Efficient serum-free derivation of oligodendrocyte precursors from neural stem cell-enriched cultures.

Oligodendrocytes derived in the laboratory from stem cells have been proposed as a treatment for acute and chronic injury to the central nervous system. Platelet-derived growth factor (PDGF) receptor alpha (PDGFRalpha) signaling is known to regulate oligodendrocyte precursor cell numbers both during development and adulthood. Here, we analyze the effects of PDGFRalpha signaling on central nervous system (CNS) stem cell-enriched cultures. We find that AC133 selection for CNS progenitors acutely isolated from the fetal cortex enriches for PDGF-AA-responsive cells. PDGF-AA treatment of fibroblast growth factor 2-expanded CNS stem cell-enriched cultures increases nestin(+) cell number, viability, proliferation, and glycolytic rate. We show that a brief exposure to PDGF-AA rapidly and efficiently permits the derivation of O4(+) oligodendrocyte-lineage cells from CNS stem cell-enriched cultures. The derivation of oligodendrocyte-lineage cells demonstrated here may support the effective use of stem cells in understanding fate choice mechanisms and the development of new therapies targeting this cell type.

Identification and characterization of cell type-specific and ubiquitous chromatin regulatory structures in the human genome.

The identification of regulatory elements from different cell types is necessary for understanding the mechanisms controlling cell type-specific and housekeeping gene expression. Mapping DNaseI hypersensitive (HS) sites is an accurate method for identifying the location of functional regulatory elements. We used a high throughput method called DNase-chip to identify 3,904 DNaseI HS sites from six cell types across 1% of the human genome. A significant number (22%) of DNaseI HS sites from each cell type are ubiquitously present among all cell types studied. Surprisingly, nearly all of these ubiquitous DNaseI HS sites correspond to either promoters or insulator elements: 86% of them are located near annotated transcription start sites and 10% are bound by CTCF, a protein with known enhancer-blocking insulator activity. We also identified a large number of DNaseI HS sites that are cell type specific (only present in one cell type); these regions are enriched for enhancer elements and correlate with cell type-specific gene expression as well as cell type-specific histone modifications. Finally, we found that approximately 8% of the genome overlaps a DNaseI HS site in at least one the six cell lines studied, indicating that a significant percentage of the genome is potentially functional.

Concise Review: Reprogramming, Behind the Scenes: Noncanonical Neural Stem Cell Signaling Pathways Reveal New, Unseen Regulators of Tissue Plasticity With Therapeutic Implications.

UNLABELLED: Interest is great in the new molecular concepts that explain, at the level of signal transduction, the process of reprogramming. Usually, transcription factors with developmental importance are used, but these approaches give limited information on the signaling networks involved, which could reveal new therapeutic opportunities. Recent findings involving reprogramming by genetic means and soluble factors with well-studied downstream signaling mechanisms, including signal transducer and activator of transcription 3 (STAT3) and hairy and enhancer of split 3 (Hes3), shed new light into the molecular mechanisms that might be involved. We examine the appropriateness of common culture systems and their ability to reveal unusual (noncanonical) signal transduction pathways that actually operate in vivo. We then discuss such novel pathways and their importance in various plastic cell types, culminating in their emerging roles in reprogramming mechanisms. We also discuss a number of reprogramming paradigms (mouse induced pluripotent stem cells, direct conversion to neural stem cells, and in vivo conversion of acinar cells to ß-like cells). Specifically for acinar-to-ß-cell reprogramming paradigms, we discuss the common view of the underlying mechanism (involving the Janus kinase-STAT pathway that leads to STAT3-tyrosine phosphorylation) and present alternative interpretations that implicate STAT3-serine phosphorylation alone or serine and tyrosine phosphorylation occurring in sequential order. The implications for drug design and therapy are important given that different phosphorylation sites on STAT3 intercept different signaling pathways. We introduce a new molecular perspective in the field of reprogramming with broad implications in basic, biotechnological, and translational research. SIGNIFICANCE: Reprogramming is a powerful approach to change cell identity, with implications in both basic and applied biology. Most efforts involve the forced expression of key transcription factors, but recently, success has been reported with manipulating signal transduction pathways that might intercept them. It is important to start connecting the function of the classic reprogramming genes to signaling pathways that also mediate reprogramming, unifying the sciences of signal transduction, stem cell biology, and epigenetics. Neural stem cell studies have revealed the operation of noncanonical signaling pathways that are now appreciated to also operate during reprogramming, offering new mechanistic explanations.

7q11.23 dosage-dependent dysregulation in human pluripotent stem cells affects transcriptional programs in disease-relevant lineages.

Cell reprogramming promises to make characterization of the impact of human genetic variation on health and disease experimentally tractable by enabling the bridging of genotypes to phenotypes in developmentally relevant human cell lineages. Here we apply this paradigm to two disorders caused by symmetrical copy number variations of 7q11.23, which display a striking combination of shared and symmetrically opposite phenotypes--Williams-Beuren syndrome and 7q-microduplication syndrome. Through analysis of transgene-free patient-derived induced pluripotent stem cells and their differentiated derivatives, we find that 7q11.23 dosage imbalance disrupts transcriptional circuits in disease-relevant pathways beginning in the pluripotent state. These alterations are then selectively amplified upon differentiation of the pluripotent cells into disease-relevant lineages. A considerable proportion of this transcriptional dysregulation is specifically caused by dosage imbalances in GTF2I, which encodes a key transcription factor at 7q11.23 that is associated with the LSD1 repressive chromatin complex and silences its dosage-sensitive targets.