Publications by authors named "Benjamin Demaree"

11 Publications

  • Page 1 of 1

Constrained chromatin accessibility in PU.1-mutated agammaglobulinemia patients.

J Exp Med 2021 Jul 5;218(7). Epub 2021 May 5.

Department of Genetics, University of Alabama at Birmingham, Birmingham, AL.

The pioneer transcription factor (TF) PU.1 controls hematopoietic cell fate by decompacting stem cell heterochromatin and allowing nonpioneer TFs to enter otherwise inaccessible genomic sites. PU.1 deficiency fatally arrests lymphopoiesis and myelopoiesis in mice, but human congenital PU.1 disorders have not previously been described. We studied six unrelated agammaglobulinemic patients, each harboring a heterozygous mutation (four de novo, two unphased) of SPI1, the gene encoding PU.1. Affected patients lacked circulating B cells and possessed few conventional dendritic cells. Introducing disease-similar SPI1 mutations into human hematopoietic stem and progenitor cells impaired early in vitro B cell and myeloid cell differentiation. Patient SPI1 mutations encoded destabilized PU.1 proteins unable to nuclear localize or bind target DNA. In PU.1-haploinsufficient pro-B cell lines, euchromatin was less accessible to nonpioneer TFs critical for B cell development, and gene expression patterns associated with the pro- to pre-B cell transition were undermined. Our findings molecularly describe a novel form of agammaglobulinemia and underscore PU.1's critical, dose-dependent role as a hematopoietic euchromatin gatekeeper.
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http://dx.doi.org/10.1084/jem.20201750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8105723PMC
July 2021

Joint profiling of DNA and proteins in single cells to dissect genotype-phenotype associations in leukemia.

Nat Commun 2021 03 11;12(1):1583. Epub 2021 Mar 11.

Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA, USA.

Studies of acute myeloid leukemia rely on DNA sequencing and immunophenotyping by flow cytometry as primary tools for disease characterization. However, leukemia tumor heterogeneity complicates integration of DNA variants and immunophenotypes from separate measurements. Here we introduce DAb-seq, a technology for simultaneous capture of DNA genotype and cell surface phenotype from single cells at high throughput, enabling direct profiling of proteogenomic states in tens of thousands of cells. To demonstrate the approach, we analyze the disease of three patients with leukemia over multiple treatment timepoints and disease recurrences. We observe complex genotype-phenotype dynamics that illustrate the subtlety of the disease process and the degree of incongruity between blast cell genotype and phenotype in different clinical scenarios. Our results highlight the importance of combined single-cell DNA and protein measurements to fully characterize the heterogeneity of leukemia.
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http://dx.doi.org/10.1038/s41467-021-21810-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7952600PMC
March 2021

Networks of High Aspect Ratio Particles to Direct Colloidal Assembly Dynamics and Cellular Interactions.

Adv Funct Mater 2020 Nov;30(48)

Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco. San Francisco, CA 94158.

Injectable colloids that self-assemble into three-dimensional networks are promising materials for applications in regenerative engineering, as they create open systems for cellular infiltration, interaction, and activation. However, most injectable colloids have spherical morphologies, which lack the high material-biology contact areas afforded by higher aspect ratio materials. To address this need, injectable high aspect ratio particles (HARPs) were developed that form three-dimensional networks to enhance scaffold assembly dynamics and cellular interactions. HARPs were functionalized for tunable surface charge through layer-by-layer electrostatic assembly. Positively charged Chitosan-HARPs had improved particle suspension dynamics when compared to spherical particles or negatively charged HARPs. Chit-HARPs were used to improve the suspension dynamics and viability of MIN6 cells in three-dimensional networks. When combined with negatively charged gelatin microsphere (GelMS) porogens, Chit-HARPs reduced GelMS sedimentation and increased overall network suspension, due to a combination of HARP network formation and electrostatic interactions. Lastly, HARPs were functionalized with fibroblast growth factor 2 (FGF2) to highlight their use for growth factor delivery. FGF2-HARPs increased fibroblast proliferation through a combination of 3D scaffold assembly and growth factor delivery. Taken together, these studies demonstrate the development and diverse uses of high aspect ratio particles as tunable injectable scaffolds for applications in regenerative engineering.
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http://dx.doi.org/10.1002/adfm.202005938DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7687842PMC
November 2020

Single-cell mutation analysis of clonal evolution in myeloid malignancies.

Nature 2020 11 28;587(7834):477-482. Epub 2020 Oct 28.

Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Myeloid malignancies, including acute myeloid leukaemia (AML), arise from the expansion of haematopoietic stem and progenitor cells that acquire somatic mutations. Bulk molecular profiling has suggested that mutations are acquired in a stepwise fashion: mutant genes with high variant allele frequencies appear early in leukaemogenesis, and mutations with lower variant allele frequencies are thought to be acquired later. Although bulk sequencing can provide information about leukaemia biology and prognosis, it cannot distinguish which mutations occur in the same clone(s), accurately measure clonal complexity, or definitively elucidate the order of mutations. To delineate the clonal framework of myeloid malignancies, we performed single-cell mutational profiling on 146 samples from 123 patients. Here we show that AML is dominated by a small number of clones, which frequently harbour co-occurring mutations in epigenetic regulators. Conversely, mutations in signalling genes often occur more than once in distinct subclones, consistent with increasing clonal diversity. We mapped clonal trajectories for each sample and uncovered combinations of mutations that synergized to promote clonal expansion and dominance. Finally, we combined protein expression with mutational analysis to map somatic genotype and clonal architecture with immunophenotype. Our findings provide insights into the pathogenesis of myeloid transformation and how clonal complexity evolves with disease progression.
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http://dx.doi.org/10.1038/s41586-020-2864-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7677169PMC
November 2020

Multiplatform genomic profiling and magnetic resonance imaging identify mechanisms underlying intratumor heterogeneity in meningioma.

Nat Commun 2020 09 23;11(1):4803. Epub 2020 Sep 23.

Department of Neurological Surgery, University of California San Francisco, San Francisco, CA, 94143, USA.

Meningiomas are the most common primary intracranial tumors, but the molecular drivers of meningioma tumorigenesis are poorly understood. We hypothesized that investigating intratumor heterogeneity in meningiomas would elucidate biologic drivers and reveal new targets for molecular therapy. To test this hypothesis, here we perform multiplatform molecular profiling of 86 spatially-distinct samples from 13 human meningiomas. Our data reveal that regional alterations in chromosome structure underlie clonal transcriptomic, epigenomic, and histopathologic signatures in meningioma. Stereotactic co-registration of sample coordinates to preoperative magnetic resonance images further suggest that high apparent diffusion coefficient (ADC) distinguishes meningioma regions with proliferating cells enriched for developmental gene expression programs. To understand the function of these genes in meningioma, we develop a human cerebral organoid model of meningioma and validate the high ADC marker genes CDH2 and PTPRZ1 as potential targets for meningioma therapy using live imaging, single cell RNA sequencing, CRISPR interference, and pharmacology.
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http://dx.doi.org/10.1038/s41467-020-18582-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7511976PMC
September 2020

Microfluidic automated plasmid library enrichment for biosynthetic gene cluster discovery.

Nucleic Acids Res 2020 05;48(8):e48

Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA, USA.

Microbial biosynthetic gene clusters are a valuable source of bioactive molecules. However, because they typically represent a small fraction of genomic material in most metagenomic samples, it remains challenging to deeply sequence them. We present an approach to isolate and sequence gene clusters in metagenomic samples using microfluidic automated plasmid library enrichment. Our approach provides deep coverage of the target gene cluster, facilitating reassembly. We demonstrate the approach by isolating and sequencing type I polyketide synthase gene clusters from an Antarctic soil metagenome. Our method promotes the discovery of functional-related genes and biosynthetic pathways.
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http://dx.doi.org/10.1093/nar/gkaa131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7192590PMC
May 2020

Multiplatform Molecular Profiling Reveals Epigenomic Intratumor Heterogeneity in Ependymoma.

Cell Rep 2020 02;30(5):1300-1309.e5

Department of Radiation Oncology, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Neurological Surgery, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address:

Ependymomas exist within distinct genetic subgroups, but the molecular diversity within individual ependymomas is unknown. We perform multiplatform molecular profiling of 6 spatially distinct samples from an ependymoma with C11orf95-RELA fusion. DNA methylation and RNA sequencing distinguish clusters of samples according to neuronal development gene expression programs that could also be delineated by differences in magnetic resonance blood perfusion. Exome sequencing and phylogenetic analysis reveal epigenomic intratumor heterogeneity and suggest that chromosomal structural alterations may precede accumulation of single-nucleotide variants during ependymoma tumorigenesis. In sum, these findings shed light on the oncogenesis and intratumor heterogeneity of ependymoma.
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http://dx.doi.org/10.1016/j.celrep.2020.01.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7313374PMC
February 2020

Human intestinal spheroids cultured using Sacrificial Micromolding as a model system for studying drug transport.

Sci Rep 2019 07 9;9(1):9936. Epub 2019 Jul 9.

Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA, USA.

In vitro models of the small intestine are crucial tools for the prediction of drug absorption. The Caco-2 monolayer transwell model has been widely employed to assess drug absorption across the intestine. However, it is now well-established that 3D in vitro models capture tissue-specific architecture and interactions with the extracellular matrix and therefore better recapitulate the complex in vivo environment. However, these models need to be characterized for barrier properties and changes in gene expression and transporter function. Here, we report that geometrically controlled self-assembling multicellular intestinal Caco-2 spheroids cultured using Sacrificial Micromolding display reproducible intestinal features and functions that are more representative of the in vivo small intestine than the widely used 2D transwell model. We show that Caco-2 cell maturation and differentiation into the intestinal epithelial phenotype occur faster in spheroids and that they are viable for a longer period of time. Finally, we were able to invert the polarity of the spheroids by culturing them around Matrigel beads allowing superficial access to the apical membrane and making the model more physiological. This robust and reproducible in vitro intestinal model could serve as a valuable system to expedite drug screening as well as to study intestinal transporter function.
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http://dx.doi.org/10.1038/s41598-019-46408-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6616551PMC
July 2019

Direct quantification of EGFR variant allele frequency in cell-free DNA using a microfluidic-free digital droplet PCR assay.

Methods Cell Biol 2018 1;148:119-131. Epub 2018 Nov 1.

Department of Bioengineering and Therapeutic Sciences, California Institute for Quantitative Biosciences, University of California, San Francisco, CA, United States; UC Berkeley-UCSF Graduate Program in Bioengineering, University of California, San Francisco, CA, United States; Chan Zuckerberg Biohub, San Francisco, CA, United States. Electronic address:

Analysis of liquid biopsy samples is a promising diagnostic intervention for noninvasive detection and monitoring of cancer genotypes. However, current methods used to assess mutation status are either costly, in the case of next-generation sequencing-based assays, or lacking in sensitivity, in the case of bulk quantitative PCR measurements. Digital droplet PCR (ddPCR) is at once a sensitive and low-cost method for detecting rare cancer mutations and measuring their variant allele frequency. In this chapter, we describe a method for conducting ddPCR assays without microfluidics in a process called "particle-templated emulsification" (PTE). Using hydrogel particles and a standard benchtop vortexer to rapidly emulsify large volumes, the method forgoes the specialized instrumentation required for conventional ddPCR assays and is capable of high experimental throughput. To assess the quantitative performance of the method, we apply PTE ddPCR to analysis of variant allele frequency in EGFR, a commonly mutated gene in lung adenocarcinomas.
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http://dx.doi.org/10.1016/bs.mcb.2018.10.002DOI Listing
January 2019

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing.

J Vis Exp 2018 05 23(135). Epub 2018 May 23.

Department of Bioengineering and Therapeutic Sciences, California Institute for Quantitative Biosciences, University of California, San Francisco; UC Berkeley-UCSF Graduate Program in Bioengineering, University of California, San Francisco; Chan Zuckerberg Biohub;

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing >50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.
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http://dx.doi.org/10.3791/57598DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6101372PMC
May 2018

Single-cell genome sequencing at ultra-high-throughput with microfluidic droplet barcoding.

Nat Biotechnol 2017 Jul 29;35(7):640-646. Epub 2017 May 29.

Department of Bioengineering and Therapeutic Sciences, California Institute for Quantitative Biosciences, University of California, San Francisco, California, USA.

The application of single-cell genome sequencing to large cell populations has been hindered by technical challenges in isolating single cells during genome preparation. Here we present single-cell genomic sequencing (SiC-seq), which uses droplet microfluidics to isolate, fragment, and barcode the genomes of single cells, followed by Illumina sequencing of pooled DNA. We demonstrate ultra-high-throughput sequencing of >50,000 cells per run in a synthetic community of Gram-negative and Gram-positive bacteria and fungi. The sequenced genomes can be sorted in silico based on characteristic sequences. We use this approach to analyze the distributions of antibiotic-resistance genes, virulence factors, and phage sequences in microbial communities from an environmental sample. The ability to routinely sequence large populations of single cells will enable the de-convolution of genetic heterogeneity in diverse cell populations.
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http://dx.doi.org/10.1038/nbt.3880DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5531050PMC
July 2017