An immunophenotype-coupled transcriptomic atlas of human hematopoietic progenitors.

Analysis of the human hematopoietic progenitor compartment is being transformed by single-cell multimodal approaches. Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) enables coupled surface protein and transcriptome profiling, thereby revealing genomic programs underlying progenitor states. To perform CITE-seq systematically on primary human bone marrow cells, we used titrations with 266 CITE-seq antibodies (antibody-derived tags) and machine learning to optimize a panel of 132 antibodies. Multimodal analysis resolved >80 stem, progenitor, immune, stromal and transitional cells defined by distinctive surface markers and transcriptomes. This dataset enables flow cytometry solutions for in silico-predicted cell states and identifies dozens of cell surface markers consistently detected across donors spanning race and sex. Finally, aligning annotations from this atlas, we nominate normal marrow equivalents for acute myeloid leukemia stem cell populations that differ in clinical response. This atlas serves as an advanced digital resource for hematopoietic progenitor analyses in human health and disease.

Assay optimization for the objective quantification of human multilineage colony-forming units.

Colony-forming unit (CFU) assays are a powerful tool in hematopoietic research because they allow researchers to functionally test the lineage potential of individual stem and progenitor cells. Assaying for lineage potential is important for determining and validating the identity of progenitor populations isolated by methods such as fluorescence-activated cell sorting (FACS). However, current methods for CFU assays are limited in their ability to robustly assay multipotent progenitors with the ability to differentiate down the myeloid, erythroid, and megakaryocytic lineages because of the lack of specific growth factors necessary for certain lineage outputs. In addition, manual counting of colony types is subjective resulting in user to user variability in assessments of cell types based on colony and cell morphologies. We demonstrate that the addition of granulocyte colony-stimulating factor (G-CSF), macrophage (M)-CSF, and granulocyte-macrophage (GM)-CSF into a collagen-based MegaCult medium containing IL-3, IL-6, SCF, EPO, and TPO allows for the differentiation of common myeloid progenitors into expected proportions of colonies containing granulocytic (G), monocytic (M), erythroid (E), and megakaryocytic (Mk) cells. Additionally, we demonstrate an objective method using in situ immunofluorescence (IF) with anti-CD66b, anti-CD14, anti-CD235a, and anti-CD41 to detect G, M, E, and Mk cells, respectively. IF stained colonies can be analyzed individually at a microscope or using high-throughput microscopy. Thus, our improvements to the culture conditions and method for assay readout increase the accuracy, reproducibility, and throughput of the myeloid CFU assay.

Effect of CRISPR Knockout of AXIN1 or ARID1A on Proliferation and Migration of Porcine Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is an aggressive disease lacking effective treatment. Animal models of HCC are necessary for preclinical evaluation of the safety and efficacy of novel therapeutics. Large animal models of HCC allow testing image-guided locoregional therapies, which are widely used in the management of HCC. Models with precise tumor mutations mimicking human HCC provide valuable tools for testing precision medicine. AXIN1 and ARID1A are two of the most frequently mutated genes in human HCC. Here, we investigated the effects of knockout of AXIN1 and/or ARID1A on proliferation, migration, and chemotherapeutic susceptibility of porcine HCC cells and we developed subcutaneous tumors harboring these mutations in pigs. Gene knockout was achieved by CRISPR/Cas9 and was validated by Next Generation Sequencing. AXIN1 knockout increased the migration of porcine HCC cells but did not alter the cell proliferation. Knockout of ARID1A increased both the proliferation and migration of porcine HCC cells. Simultaneous knockout of AXIN1 and ARID1A increased the migration, but did not alter the proliferation of porcine HCC cells. The effect of gene knockout on the response of porcine HCC cells to two of the most commonly used systemic and locoregional HCC treatments was investigated; sorafenib and doxorubicin, respectively. Knockout of AXIN1 and/or ARID1A did not alter the susceptibility of porcine HCC cells to sorafenib or doxorubicin. Autologous injection of CRISPR edited HCC cells resulted in development of subcutaneous tumors in pigs, which harbored the anticipated edits in AXIN1 and/or ARID1A. This study elucidates the effects of CRISPR-mediated knockout of HCC-associated genes in porcine HCC cells, and lays the foundation for development and utilization of genetically-tailored porcine HCC models for in vivo testing of novel therapeutic approaches in a clinically-relevant large animal model.

Sperm Flagellar 1 Binds Actin in Intestinal Epithelial Cells and Contributes to Formation of Filopodia and Lamellipodia.

BACKGROUND & AIMS: Sperm flagellar 1 (also called CLAMP) is a microtubule-associated protein that regulates microtubule dynamics and planar cell polarity in multi-ciliated cells. We investigated the localization and function of sperm flagellar 1, or CLAMP, in human intestinal epithelia cells (IECs). METHODS: We performed studies with SKCO-15 and human intestinal enteroids established from biopsies from different intestinal segments (duodenal, jejunum, ileal, and colon) of a single donor. Enteroids were induced to differentiation after incubation with growth factors. The distribution of endogenous CLAMP in IECs was analyzed by immunofluorescence microscopy using total internal reflection fluorescence-ground state depletion and confocal microscopy. CLAMP localization was followed during the course of intestinal epithelial cell polarization as cells progressed from flat to compact, confluent monolayers. Protein interactions with endogenous CLAMP were determined in SKCO-15 cells using proximity ligation assays and co-immunoprecipitation. CLAMP was knocked down in SKCO-15 monolayers using small hairpin RNAs and cells were analyzed by immunoblot and immunofluorescence microscopy. The impact of CLAMP knock-down in migrating SKCO-15 cells was assessed using scratch-wound assays. RESULTS: CLAMP bound to actin and apical junctional complex proteins but not microtubules in IECs. In silico analysis predicted the calponin-homology domain of CLAMP to contain conserved amino acids required for actin binding. During IEC polarization, CLAMP distribution changed from primarily basal stress fibers and cytoplasm in undifferentiated cells to apical membranes and microvilli in differentiated monolayers. CLAMP accumulated in lamellipodia and filopodia at the leading edge of migrating cells in association with actin. CLAMP knock-down reduced the number of filopodia, perturbed filopodia polarity, and altered the organization of actin filaments within lamellipodia. CONCLUSIONS: CLAMP is an actin-binding protein, rather than a microtubule-binding protein, in IECs. CLAMP distribution changes during intestinal epithelial cell polarization, regulates the formation of filopodia, and appears to assist in the organization of actin bundles within lamellipodia of migrating IECs. Studies are needed to define the CLAMP domains that interact with actin and whether its loss from IECs affects intestinal function.