Reliance on rodents for understanding pancreatic genetics, development and islet function could limit progress in developing interventions for human diseases such as diabetes mellitus. Similarities ...of pancreas morphology and function suggest that porcine and human pancreas developmental biology may have useful homologies. However, little is known about pig pancreas development. To fill this knowledge gap, we investigated fetal and neonatal pig pancreas at multiple, crucial developmental stages using modern experimental approaches. Purification of islet β-, α- and δ-cells followed by transcriptome analysis (RNA-seq) and immunohistology identified cell- and stage-specific regulation, and revealed that pig and human islet cells share characteristic features that are not observed in mice. Morphometric analysis also revealed endocrine cell allocation and architectural similarities between pig and human islets. Our analysis unveiled scores of signaling pathways linked to native islet β-cell functional maturation, including evidence of fetal α-cell GLP-1 production and signaling to β-cells. Thus, the findings and resources detailed here show how pig pancreatic islet studies complement other systems for understanding the developmental programs that generate functional islet cells, and that are relevant to human pancreatic diseases.
Prime editors have been delivered using DNA or RNA vectors. Here we demonstrate prime editing with purified ribonucleoprotein complexes. We introduced somatic mutations in zebrafish embryos with ...frequencies as high as 30% and demonstrate germline transmission. We also observed unintended insertions, deletions and prime editing guide RNA (pegRNA) scaffold incorporations. In HEK293T and primary human T cells, prime editing with purified ribonucleoprotein complexes introduced desired edits with frequencies of up to 21 and 7.5%, respectively.
Tumor evolution from a single cell into a malignant, heterogeneous tissue remains poorly understood. Here, we profile single-cell transcriptomes of genetically engineered mouse lung tumors at seven ...stages, from pre-neoplastic hyperplasia to adenocarcinoma. The diversity of transcriptional states increases over time and is reproducible across tumors and mice. Cancer cells progressively adopt alternate lineage identities, computationally predicted to be mediated through a common transitional, high-plasticity cell state (HPCS). Accordingly, HPCS cells prospectively isolated from mouse tumors and human patient-derived xenografts display high capacity for differentiation and proliferation. The HPCS program is associated with poor survival across human cancers and demonstrates chemoresistance in mice. Our study reveals a central principle underpinning intra-tumoral heterogeneity and motivates therapeutic targeting of the HPCS.
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•Lung cancer progression is accompanied by a stereotypic expansion of heterogeneity•Cell state heterogeneity arises largely independently of genetic variation•State transitions occur via an HPCS harboring high differentiation and growth capacity•The HPCS is drug resistant and portends poor patient survival across all cancers
Cellular states capable of promoting tumor progression and resisting therapies exist in heterogeneous tumors. Marjanovic et al. discover that a high-plasticity cell state common to mouse and human lung tumors drives cellular heterogeneity, is highly tumorigenic and drug resistant, and associates with poor patient prognosis.
In tumors, a subset of CD8+ T cells expressing the transcription factor TCF-1 drives the response to immune checkpoint blockade. We examined the mechanisms that maintain these cells in an ...autochthonous model of lung adenocarcinoma. Longitudinal sampling and single-cell sequencing of tumor-antigen specific TCF-1+ CD8+ T cells revealed that while intratumoral TCF-1+ CD8+ T cells acquired dysfunctional features and decreased in number as tumors progressed, TCF-1+ CD8+ T cell frequency in the tumor draining LN (dLN) remained stable. Two discrete intratumoral TCF-1+ CD8+ T cell subsets developed over time—a proliferative SlamF6+ subset and a non-cycling SlamF6− subset. Blocking dLN egress decreased the frequency of intratumoral SlamF6+ TCF-1+ CD8+ T cells. Conventional type I dendritic cell (cDC1) in dLN decreased in number with tumor progression, and Flt3L+anti-CD40 treatment recovered SlamF6+ T cell frequencies and decreased tumor burden. Thus, cDC1s in tumor dLN maintain a reservoir of TCF-1+ CD8+ T cells and their decrease contributes to failed anti-tumor immunity.
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•Longitudinal scRNA-seq of tumor-specific TCF-1+ CD8+ T cells in KP lung adenocarcinoma•Identified a proliferative Slamf6+ TCF-1+ T cell subset and a non-cycling SlamF6− subset•The lymph node contains a recruitable reservoir of functional TCF-1+ CD8+ T cells•Flt3L+CD40 boosts cDC1, increases TCF-1+CD8+ T cell frequencies, decreases tumor burden
In tumors, TCF-1+ CD8+ T cells drive the response to immune checkpoint blockade. Schenkel, Herbst et al. reveal that, in lung adenocarcinoma, a reservoir of TCF-1+ CD8+ T cells is maintained in tumor draining lymph nodes by conventional type I dendritic cell (cDC1). Decrease of these cDC1 as the tumor progresses contributes to failed anti-tumor immunity.
Developing systems to identify the cell type-specific functions regulated by genes linked to type 2 diabetes (T2D) risk could transform our understanding of the genetic basis of this disease. ...However, in vivo systems for efficiently discovering T2D risk gene functions relevant to human cells are currently lacking. Here we describe powerful interdisciplinary approaches combining Drosophila genetics and physiology with human islet biology to address this fundamental gap in diabetes research. We identify Drosophila orthologs of T2D-risk genes that regulate insulin output. With human islets, we perform genetic studies and identify cognate human T2D-risk genes that regulate human beta cell function. Loss of BCL11A, a transcriptional regulator, in primary human islet cells leads to enhanced insulin secretion. Gene expression profiling reveals BCL11A-dependent regulation of multiple genes involved in insulin exocytosis. Thus, genetic and physiological systems described here advance the capacity to identify cell-specific T2D risk gene functions.
About 10-20% of Kawasaki disease (KD) patients are resistant to the initial infusion of intravenous immunoglobin (IVIG). The aim of this study was to assess whether IVIG resistance in KD patients ...could be predicted using standard clinical and laboratory features.
Data were from two cohorts: a Korean cohort of 7101 KD patients from 2015 to 2017 and a cohort of 649 KD patients from San Diego enrolled from 1998 to 2021. Features included laboratory values, the worst Z-score from the initial echocardiogram or during hospitalization, and the five clinical KD signs at presentation.
Five machine learning models achieved a maximum median AUC of 0.711 IQR: 0.706-0.72 in the Korean cohort and 0.696 IQR: 0.609-0.722 in the San Diego cohort during stratified 10-fold cross-validation using significant laboratory features identified from univariate analysis. Adding the Z-score, KD clinical signs, or both did not considerably improve the median AUC in either cohort.
Using commonly measured clinical laboratory data alone or in conjunction with echocardiographic findings and clinical features is not sufficient to predict IVIG resistance. Further attempts to predict IVIG resistance will need to incorporate additional data such as transcriptomics, proteomics, and genetics to achieve meaningful predictive utility.
We demonstrated that laboratory, echocardiographic, and clinical findings cannot predict intravenous immunoglobin (IVIG) resistance to a clinically meaningful extent using machine learning in a homogenous Asian or ethnically diverse population of patients with Kawasaki disease (KD). Visualizing these features using uniform manifold approximation and projection (UMAP) is an important step to evaluate predictive utility in a qualitative manner. Further attempts to predict IVIG resistance in KD patients will need to incorporate novel biomarkers or other specialized features such as genetic differences or transcriptomics to be clinically useful.
Regulatory T cells (Tregs) can impair anti-tumor immune responses and are associated with poor prognosis in multiple cancer types. Tregs in human tumors span diverse transcriptional states distinct ...from those of peripheral Tregs, but their contribution to tumor development remains unknown. Here, we use single-cell RNA sequencing (RNA-seq) to longitudinally profile dynamic shifts in the distribution of Tregs in a genetically engineered mouse model of lung adenocarcinoma. In this model, interferon-responsive Tregs are more prevalent early in tumor development, whereas a specialized effector phenotype characterized by enhanced expression of the interleukin-33 receptor ST2 is predominant in advanced disease. Treg-specific deletion of ST2 alters the evolution of effector Treg diversity, increases infiltration of CD8+ T cells into tumors, and decreases tumor burden. Our study shows that ST2 plays a critical role in Treg-mediated immunosuppression in cancer, highlighting potential paths for therapeutic intervention.
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•Single-cell profiling of CD4 T cells along tumor development in a mouse model•Treg diversity shifts to a Klrg1+Areg+ (KA) effector phenotype in advanced tumors•Il1rl1 (encoding ST2)+ Tregs have higher expression of KA effector Treg genes•Treg-specific ST2 loss enhances CD8+ T cell infiltration and decreases tumor burden
Li et al. show in a genetic mouse model of lung adenocarcinoma that during tumor development regulatory T cell (Treg) diversity shifts from an interferon-responsive to a ST2-positive, Klrg1+Areg+ effector-like phenotype. Treg-specific deletion of ST2 alters Treg heterogeneity, increases tumor infiltration by CD8+ T cells, and decreases tumor burden.
Gene targeting studies in primary human islets could advance our understanding of mechanisms driving diabetes pathogenesis. Here, we demonstrate successful genome editing in primary human islets ...using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9). CRISPR-based targeting efficiently mutated protein-coding exons, resulting in acute loss of islet β-cell regulators, like the transcription factor PDX1 and the K
channel subunit KIR6.2, accompanied by impaired β-cell regulation and function. CRISPR targeting of non-coding DNA harboring type 2 diabetes (T2D) risk variants revealed changes in ABCC8, SIX2 and SIX3 expression, and impaired β-cell function, thereby linking regulatory elements in these target genes to T2D genetic susceptibility. Advances here establish a paradigm for genetic studies in human islet cells, and reveal regulatory and genetic mechanisms linking non-coding variants to human diabetes risk.
The objective of this study is to optimize the cryopreservation of dissociated islet cells and obtain functional cells that can be used in single-cell transcriptome studies on the pathology and ...treatment of diabetes. Using an iterative graded freezing approach we obtained viable cells after cooling in 10% dimethyl sulfoxide and 6% hydroxyethyl starch at 1°C/min to -40°C, storage in liquid nitrogen, rapid thaw, and removal of cryoprotectants by serial dilution. The expression of epithelial cell adhesion molecule declined immediately after thaw, but recovered after overnight incubation, while that of an endocrine cell marker (HPi2) remained high after cryopreservation. Patch-clamp electrophysiology revealed differences in channel activities and exocytosis of various islet cell types; however, exocytotic responses, and the biophysical properties of voltage-gated Na+ and Ca2+ channels, are sustained after cryopreservation. Single-cell RNA sequencing indicates that overall transcriptome and crucial exocytosis genes are comparable between fresh and cryopreserved dispersed human islet cells. Thus, we report an optimized procedure for cryopreserving dispersed islet cells that maintained their membrane integrity, along with their molecular and functional phenotypes. Our findings will not only provide a ready source of cells for investigating cellular mechanisms in diabetes but also for bio-engineering pseudo-islets and islet sheets for modeling studies and potential transplant applications.
The physiological functions of many vital tissues and organs continue to mature after birth, but the genetic mechanisms governing this postnatal maturation remain an unsolved mystery. Human ...pancreatic β cells produce and secrete insulin in response to physiological cues like glucose, and these hallmark functions improve in the years after birth. This coincides with expression of the transcription factors SIX2 and SIX3, whose functions in native human β cells remain unknown. Here, we show that shRNA-mediated
or
suppression in human pancreatic adult islets impairs insulin secretion. However, transcriptome studies revealed that
and
regulate distinct targets. Loss of
markedly impaired expression of genes governing β-cell insulin processing and output, glucose sensing, and electrophysiology, while
loss led to inappropriate expression of genes normally expressed in fetal β cells, adult α cells, and other non-β cells. Chromatin accessibility studies identified genes directly regulated by SIX2. Moreover, β cells from diabetic humans with impaired insulin secretion also had reduced
transcript levels. Revealing how
and
govern functional maturation and maintain developmental fate in native human β cells should advance β-cell replacement and other therapeutic strategies for diabetes.
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