Biology subjects

Kaestner, K. H.

Publications and source records attributed to Kaestner, K. H..

26 records · Page 2Linked to original sources

bioRxiv2021

Single-cell analysis of the human pancreas in type 2 diabetes using multi-spectral imaging mass cytometry

Type 2 diabetes mellitus (T2D) is a chronic age-related disorder characterized by hyperglycemia due to the failure of pancreatic beta cells to compensate for increased insulin demand, typically associated with peripheral insulin resistance. However, despite decades of research, the pathogenic mechanisms underlying T2D remain poorly defined. Imaging mass cytometry (IMC) enables multiplexed assessment of the abundance and subcellular localization of multiple proteins on the same tissue section. Herein, we utilized IMC with a panel of 34 antibodies to quantify key markers of pancreatic exocrine, islet, and immune cells as well as stromal components. We employed this panel to analyze over 2.1 million cells from 16 pancreata obtained from donors with T2D and 13 pancreata from age similar non-diabetic controls. In the T2D pancreata, we observed significant alterations in islet architecture, endocrine cell composition, and surprisingly immune cell constituents. Thus, both HLA-DR positive CD8 T cells and macrophages were enriched intra-islet in the T2D pancreas. These efforts demonstrate the utility of IMC to investigate complex events at the cellular level in order to provide new insights into the pathophysiology of T2D.

molecular biology↗

bioRxiv2020

SARS-CoV-2 Cell Entry Factors ACE2 and TMPRSS2 are Expressed in the Pancreas but Not in Islet Endocrine Cells

Summary/AbstractReports of new-onset diabetes and diabetic ketoacidosis in individuals with COVID-19 have led to the hypothesis that SARS-CoV-2, the virus that causes COVID-19, is directly cytotoxic to pancreatic islet {beta} cells. This would require binding and entry of SARS-CoV-2 into host {beta} cells via cell surface co-expression of ACE2 and TMPRSS2, the putative receptor and effector protease, respectively. To define ACE2 and TMPRSS2 expression in the human pancreas, we examined six transcriptional datasets from primary human islet cells and assessed protein expression by immunofluorescence in pancreata from donors with and without diabetes. ACE2 and TMPRSS2 transcripts were low or undetectable in pancreatic islet endocrine cells as determined by bulk or single cell RNA sequencing, and neither protein was detected in or {beta} cells from these donors. Instead, ACE2 protein was expressed in the islet and exocrine tissue microvasculature and also found in a subset of pancreatic ducts, whereas TMPRSS2 protein was restricted to ductal cells. The absence of significant ACE2 and TMPRSS2 co-expression in islet endocrine cells reduces the likelihood that SARS-CoV-2 directly infects pancreatic islet {beta} cells through these cell entry proteins.

cell biology↗

bioRxiv2020

Single cell resolution regulatory landscape of the mouse kidney highlights cellular differentiation programs and renal disease targets

Determining the epigenetic program that generates unique cell types in the kidney is critical for understanding cell-type heterogeneity during tissue homeostasis and injury response. Here, we profiled open chromatin and gene expression in developing and adult mouse kidneys at single cell resolution. We show critical reliance of gene expression on distal regulatory elements (enhancers). We define key cell type-specific transcription factors and major gene-regulatory circuits for kidney cells. Dynamic chromatin and expression changes during nephron progenitor differentiation demonstrated that podocyte commitment occurs early and is associated with sustained Foxl1 expression. Renal tubule cells followed a more complex differentiation, where Hfn4a was associated with proximal and Tfap2b with distal fate. Mapping single nucleotide variants associated with human kidney disease identified critical cell types, developmental stages, genes, and regulatory mechanisms. We provide a global single cell resolution view of chromatin accessibility of kidney development. The dataset is available via interactive public websites.

developmental biology↗

bioRxiv2020

Collapse of the hepatic gene regulatory network in the absence of FoxA factors

The FoxA transcription factors are critical for liver development through their pioneering activity, which initiates a highly complex regulatory network thought to become progressively resistant to the loss of any individual hepatic transcription factor via mutual redundancy. To investigate the dispensability of FoxA factors for maintaining this regulatory network, we ablated all FoxA genes in the adult mouse liver. Remarkably, loss of FoxA caused rapid hepatocyte dedifferentiation manifested by a massive reduction in the expression of key liver genes. Interestingly, expression of these genes was reduced back to the low levels of the fetal prehepatic endoderm stage, leading to necrosis and lethality within days. Mechanistically, we found FoxA proteins to be required for maintaining enhancer activity, chromatin accessibility, nucleosome positioning and binding by HNF4. Thus, the FoxA factors act continuously, guarding hepatic enhancer activity throughout life.

genetics↗

bioRxiv2019

In vivo screen identifies LXR agonism potentiates sorafenib killing of hepatocellular carcinoma

Existing drug therapies for hepatocellular carcinoma (HCC), including sorafenib, extend patient survival by only three months. We sought to identify novel druggable targets for use in combination with sorafenib to increase its efficacy. We implemented an in vivo genetic screening paradigm utilizing a library of 43 genes-of-interest expressed in the context of repopulation of the injured livers of Fumarylacetoacetate Hydrolase-deficient (Fah-/-) mice, which led to highly penetrant HCC. We then treated mice with vehicle or sorafenib to discover genetic determinants of sensitivity and resistance. Liver X Receptor alpha (LXR) emerged as a potential target. To examine LXR agonism in combination with sorafenib treatment, we added varying concentrations of sorafenib and LXR agonist drugs to HCC cell lines. We performed transcriptomic analysis to elucidate the mechanisms of HCC death. Fah-/- mice injected with the screening library developed HCC tumor clones containing Myc cDNA plus various other cDNAs. Treatment with sorafenib resulted in sorafenib-resistant HCCs that were significantly depleted in Nr1h3 cDNA, encoding LXR, suggesting that LXR activation is incompatible with tumor growth in the presence of sorafenib treatment in vivo. The combination of sorafenib and LXR agonism led to enhanced cell death as compared to monotherapy in multiple HCC cell lines, due to reduced expression of cell cycle regulators and increased expression of genes associated with apoptosis. Combination therapy also enhanced cell death in a sorafenib-resistant primary human HCC cell line. Our novel in vivo screen led to the discovery that LXR agonist drugs potentiate the efficacy of sorafenib in treating HCC.

cancer biology↗

bioRxiv2019

A high-content in vivo screen to identify microRNA epistasis in the repopulating mouse liver

Liver regeneration is impaired in mice with hepatocyte-specific deficiencies in microRNA (miRNA) processing; yet the roles of individual miRNAs or their combinatorial effects in this process are largely unknown. In this study, we sought to identify miRNAs that regulate hepatocyte repopulation following toxic liver injury in a high-throughput manner using the Fah-/- mouse. We constructed plasmid pools encoding over 30,000 tough decoy (TuD) miRNA inhibitors designed to target hepatocyte miRNAs in a pairwise manner. Plasmid libraries were delivered to hepatocytes of Fah-/- mice at the time of liver injury via hydrodynamic tail vein injection and integrated transgene-containing transposons were quantified following repopulation via high-throughput sequencing. Changes in polysome-bound transcripts following miRNA inhibition were determined using translating ribosome affinity purification followed by high-throughput sequencing. Analysis of TuD abundance in hepatocyte genomic DNA and input plasmid pools identified several thousand miRNA inhibitors that were significantly altered following repopulation. We classified a subset of miRNA-binding sites (MBSs) as having strong effect on liver repopulation, thus implicating the targeted hepatocyte miRNAs as regulators of this process. Furthermore, we generated a high-content map of pairwise interactions between 171 MBSs and identified both synergistic and redundant effects. Our study highlights the power of higher-order screens to uncover miRNA functions that would go undetected by individual miRNA perturbations, and provides a new paradigm for the study of epistasis of miRNA activities.

systems biology↗

bioRxiv2019

A network of microRNAs acts to promote cell cycle exit and differentiation of human pancreatic endocrine cells

Pancreatic endocrine cell differentiation is orchestrated by transcription factors that operate in a gene regulatory network to activate endocrine lineage genes and repress lineage-inappropriate genes. MicroRNAs (miRNAs) are important modulators of gene expression, yet their role in endocrine cell differentiation has not been explored system-wide. Here we characterize miRNA-regulatory networks active in human endocrine cell differentiation by combining small RNA sequencing, miRNA overexpression experiments, and network modeling approaches. This analysis identifies Let-7g, Let-7a, miR-200a, and miR-375 as endocrine-enriched miRNAs with high impact on driving endocrine differentiation-associated gene expression changes. These miRNAs target different sets of transcription factors, which converge on a network of genes involved in cell cycle regulation. When expressed in human embryonic stem cell-derived pancreatic progenitors these miRNAs induce cell cycle exit and promote endocrine cell differentiation. Our study delineates the role of miRNAs in human endocrine cell differentiation and identifies miRNAs that could facilitate endocrine cell reprogramming.

developmental biology↗

bioRxiv2019

Comparative analysis of commercially available single-cell RNA sequencing platforms for their performance in complex human tissues

The past five years have witnessed a tremendous growth of single-cell RNA-seq methodologies. Currently, there are three major commercial platforms for single-cell RNA-seq: Fluidigm C1, Clontech iCell8 (formerly Wafergen) and 10x Genomics Chromium. Here, we provide a systematic comparison of the throughput, sensitivity, cost and other performance statistics for these three platforms using single cells from primary human islets. The primary human islets represent a complex biological system where multiple cell types coexist, with varying cellular abundance, diverse transcriptomic profiles and differing total RNA contents. We apply standard pipelines optimized for each system to derive gene expression matrices. We further evaluate the performance of each system by benchmarking single-cell data with bulk RNA-seq data from sorted cell fractions. Our analyses can be generalized to a variety of complex biological systems and serve as a guide to newcomers to the field of single-cell RNA-seq when selecting platforms.

genomics↗