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Next-generation tools for multiplexed imaging have driven a new wave of innovation in understanding how single-cell function and tissue structure are interrelated. In previous work, we developed multiplexed ion beam imaging by time of flight, a highly multiplexed platform that uses secondary ion mass spectrometry to image dozens of antibodies tagged with metal reporters. As instrument throughput has increased, the breadth and depth of imaging data have increased as well. To extract meaningful information from these data, we have developed tools for cell identification, cell classification, and spatial analysis. In this review, we discuss these tools and provide examples of their application in various contexts, including ductal carcinoma in situ, tuberculosis, and Alzheimer's disease. We hope the synergy between multiplexed imaging and automated image analysis will drive a new era in anatomic pathology and personalized medicine wherein quantitative spatial signatures are used routinely for more accurate diagnosis, prognosis, and therapeutic selection. Expected final online publication date for the Annual Review of Pathology: Mechanisms of Disease, Volume 17 is January 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Description, Multiplex single-synapse events from multiple regions of Alzheimer’s disease brain revealed new alterations unique to humans., Synaptic molecular characterization is limited for Alzheimer’s disease (AD). Our newly invented mass cytometry–based method, synaptometry by time of flight (SynTOF), was used to measure 38 antibody probes in approximately 17 million single-synapse events from human brains without pathologic change or with pure AD or Lewy body disease (LBD), nonhuman primates (NHPs), and PS/APP mice. Synaptic molecular integrity in humans and NHP was similar. Although not detected in human synapses, Aβ was in PS/APP mice single-synapse events. Clustering and pattern identification of human synapses showed expected disease-specific differences, like increased hippocampal pathologic tau in AD and reduced caudate dopamine transporter in LBD, and revealed previously unidentified findings including increased hippocampal CD47 and lowered DJ1 in AD and higher ApoE in AD with dementia. Our results were independently supported by multiplex ion beam imaging of intact tissue. This highlights the higher depth and breadth of insight on neurodegenerative diseases obtainable through SynTOF.

Pediatric acute myeloid leukemia (AML) is a rare disease characterized by an accumulation of immature white blood cells in the bone marrow and/or peripheral blood. In some cases, it is thought that the predisposition to leukemia begins in utero. Often, leukemogenesis in children is caused by recurrent chromosomal translocations that result in fusion proteins that do not occur in AML in adults. In particular, NUP98-JARID1A is a recurrent fusion that occurs in an aggressive AML characterized by malignant megakaryoblasts. The prognosis for these patients is poor and there are few novel therapies on the horizon. Using human pluripotent stem cells, we can model this disease by recapitulating the events of leukemogenesis with the goal to better understand the pathogenesis of this disease and to identify better treatments for children. To this end, we generated human pluripotent cell lines that express the NUP98-JARID1A (NJ) fusion protein through a lentiviral expression system. These cells have been characterized in the pluripotent state by RNAseq and molecular and cell culture methods. The cell lines can be differentiated to the three germ layers, and we can focus on the myeloid lineage. Here we are using mass cytometry to analyze the differentiation phenotypes of these cells in comparison to both normal bone marrow stem and progenitor populations as well as primary patient samples. Induced pluripotent cells carrying the NJ fusion maintain their pluripotent state by the presence of surface markers and expression of pluripotent genes despite a higher sensitivity to stress and difficulties to culture them. These cells display abnormal mitosis showing multiple poles and bad segregation of chromosomes, which results in aberrant karyotypes. Interestingly, as NJ iPS cells differentiate to the three germ layers, NJ iPS demonstrate a substantial increase in specific mesodermal markers as CD34 and KDR, but without differences in ectoderm or endoderm specific markers. We will discuss comparisons between myeloid developmental populations from NJ iPS cells, wild-type iPS cells differentiated to the myeloid lineage and normal bone marrow. Finally, we compare differentiated NJ iPS leukemic cells to primary patient samples based on their phenotypic and signaling features as determined by CyTOF. In conclusion, we demonstrate an early model of NUP98-JARID1A fusion AML that recapitulates the genomic instability of this leukemia and demonstrates a deregulation of mesodermal development and further myeloid differentiation. NUP98 has been described with 30 other fusion partners, all related to leukemia, and this model may be used as a basis for understanding its role in leukemogenesis. Citation Format: Joan Domingo-Reines, Samuel Kimmey, Kausalia Vijayaragavan, Marc Bosse, Sean Bendall, Kara Davis, Veronica Ramos-Mejía. Validation of a model of pediatric leukemia based on pluripotent stem cells using mass cytometry [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B29.

Induced pluripotent stem (iPS) cells may be of use in regenerative medicine. However, the low efficiency of reprogramming is a major impediment to the generation of patient-specific iPS cell lines. Here we report the first selection system for the isolation of human iPS cells. We developed the EOS (Early Transposon promoter and Oct-4 (Pou5f1) and Sox2 enhancers) lentiviral vector to specifically express in mouse and human embryonic stem cells but not in primary fibroblasts. The bicistronic EOS vector marked emerging mouse and human iPS cell colonies with EGFP, and we used puromycin selection to aid the isolation of iPS cell lines that expressed endogenous pluripotency markers. These lines differentiated into cell types from all three germ layers. Reporter expression was extinguished upon differentiation and therefore monitored the residual pluripotent cells that form teratomas. Finally, we used EOS selection to establish Rett syndrome-specific mouse and human iPS cell lines with known mutations in MECP2.

SummaryDuring human development, signals that govern lineage specification versus expansion of cells committed to a cell fate are poorly understood. We demonstrate that activation of canonical Wnt signaling by Wnt3a promotes proliferation of human embryonic stem cells (hESCs)—precursors already committed to the hematopoietic lineage. In contrast, noncanonical Wnt signals, activated by Wnt11, control exit from the pluripotent state and entry toward mesoderm specification. Unique to embryoid body (EB) formation of hESCs, Wnt11 induces development and arrangement of cells expressing Brachyury that coexpress E-cadherin and Frizzled-7 (Fzd7). Knockdown of Fzd7 expression blocks Wnt11-dependent specification. Our study reveals an unappreciated role for noncanonical Wnt signaling in hESC specification that involves development of unique mesoderm precursors via morphogenic organization within human EBs.

Voltage-gated Na(+) channels have an essential role in the biophysical properties of nociceptive neurons. Factors that regulate Na(+) channel function are of interest from both pathophysiological and therapeutic perspectives. Increasing evidence indicates that changes in expression or inappropriate modulation of these channels leads to electrical instability of the cell membrane and the inappropriate spontaneous activity that is observed following nerve injury, and that this might contribute to neuropathic pain. The role of Na(v) channels in nociception depends on modulation by factors such as auxiliary beta-subunits, cytoskeletal proteins and the phosphorylation state of neurons. In this review we describe the modulation of Na(v) channels on sensory neurons by auxiliary beta-subunits, protein kinases and cytoskeletal proteins.

1 Voltage-gated Na+ channels are transmembrane proteins that are essential for the propagation of action potentials in excitable cells. Nav1.7 and Nav1.8 dorsal root ganglion Na+ channels exhibit different kinetics and sensitivities to tetrodotoxin (TTX). We investigated the properties of both channels in the presence of lidocaine, a local anesthetic (LA) and class I anti-arrhythmic drug. 2 Nav1.7 and Nav1.8 Na+ channels were coexpressed with the beta1-subunit in Xenopus oocytes. Na+ currents were recorded using the two-microelectrode voltage-clamp technique. 3 Dose-response curves for both channels had different EC50 (dose producing 50% maximum current inhibition) (450 microm for Nav1.7 and 104 microm for Nav1.8). Lidocaine enhanced current decrease in a frequency-dependent manner. Steady-state inactivation of both channels was also affected by lidocaine, Nav1.7 being the most sensitive. Only the steady-state activation of Nav1.8 was affected while the entry of both channels into slow inactivation was affected by lidocaine, Nav1.8 being affected to a larger degree. 4 Although the channels share homology at DIV S6, the LA binding site, they differ in their sensitivity to lidocaine. Recent studies suggest that other residues on DI and DII known to influence lidocaine binding may explain the differences in affinities between Nav1.7 and Nav1.8 Na+ channels. 5 Understanding the properties of these channels and their pharmacology is of critical importance to developing drugs and finding effective therapies to treat chronic pain.

Several distinct components of voltage-gated sodium current have been recorded from native dorsal root ganglion (DRG) neurons that display differences in gating and pharmacology. This study compares the electrophysiological properties of two peripheral nerve sodium channels that are expressed selectively in DRG neurons (Na(v)1.7 and Na(v)1.8). Recombinant Na(v)1.7 and Na(v)1.8 sodium channels were coexpressed with the auxiliary beta(1) subunit in Xenopus oocytes. In this system coexpression of the beta(1) subunit with Na(v)1.7 and Na(v)1.8 channels results in more rapid inactivation, a shift in midpoints of steady-state activation and inactivation to more hyperpolarizing potentials, and an acceleration of recovery from inactivation. The coinjection of beta(1) subunit also significantly increases the expression of Na(v)1.8 by sixfold but has no effect on the expression of Na(v)1.7. In addition, a great percentage of Na(v)1.8+beta(1) channels is observed to enter rapidly into the slow inactivated states, in contrast to Nav1.7+beta(1) channels. Consequently, the rapid entry into slow inactivation is believed to cause a frequency-dependent reduction of Na(v)1.8+beta(1) channel amplitudes, seen during repetitive pulsing between 1 and 2 Hz. However, at higher frequencies (>20 Hz) Na(v)1.8+beta(1) channels reach a steady state to approximately 42% of total current. The presence of this steady-state sodium channel activity, coupled with the high activation threshold (V(0.5) = -3.3 mV) of Na(v)1.8+beta(1), could enable the nociceptive fibers to fire spontaneously after nerve injury.

The cellular mechanism and target cell affected by stromal microenvironments in augmenting hematopoietic specification from pluripotent human embryonic stem cells (hESCs) has yet to be evaluated. Here, in contrast to aorta-gonad-mesonephros-derived S62 stromal cells, OP9 cells inhibit apoptosis and also augment the proliferation of hemogenic precursors prospectively isolated from human embryoid bodies. In addition, OP9 stroma supported cells within the primitive hematopoietic compartment by inhibiting apoptosis of CD45+CD34+ cells committed to the hematopoietic lineage, but have no effect on more mature blood (CD45+CD34−) cells. Inability of hESC-derived hematopoietic cells cocultured with OP9 stromal cells to engraft in both the adult and newborn NOD/SCID mice after intrafemoral and intrahepatic injection illustrated that although OP9 stromal cells augment hESC-derived hematopoiesis and progenitor output, this optimized environment does not confer or augment repopulating function of specified hematopoietic cells derived from hESCs. OP9 coculture also increases hematopoietic progenitors output from hemogenic precursors overexpressing HOXB4. Our study demonstrates that OP9 cells support both hemogenic precursors and their primitive hematopoietic progeny, thereby providing the first evidence toward understanding the cellular targets and mechanisms underlying the capacity of OP9 stromal cells to support hematopoiesis from ESCs and define the future steps required to achieve the global goal of generating bona fide human hematopoietic stem cells from ESC lines. Disclosure of potential conflicts of interest is found at the end of this article.

Distinctive properties of stem cells are not autonomously achieved, and recent evidence points to a level of external control from the microenvironment. Here, we demonstrate that self-renewal and pluripotent properties of human embryonic stem (ES) cells depend on a dynamic interplay between human ES cells and autologously derived human ES cell fibroblast-like cells (hdFs). Human ES cells and hdFs are uniquely defined by insulin-like growth factor (IGF)- and fibroblast growth factor (FGF)-dependence. IGF 1 receptor (IGF1R) expression was exclusive to the human ES cells, whereas FGF receptor 1 (FGFR1) expression was restricted to surrounding hdFs. Blocking the IGF-II/IGF1R pathway reduced survival and clonogenicity of human ES cells, whereas inhibition of the FGF pathway indirectly caused differentiation. IGF-II is expressed by hdFs in response to FGF, and alone was sufficient in maintaining human ES cell cultures. Our study demonstrates a direct role of the IGF-II/IGF1R axis on human ES cell physiology and establishes that hdFs produced by human ES cells themselves define the stem cell niche of pluripotent human stem cells.

Publisher Summary Hematopoiesis provides a well-defined system for studying molecular and cellular processes that control growth and development. The hematopoietic system consists of a heterogeneous array of cells ranging from rare primitive hematopoietic stem cells to large numbers of mature cells. Studies from amphibians, chicks, fish, and mammals show that blood cell emergence is established through waves of distinct hematopoietic compartments arising from distinct anatomical sites. In many of these animal embryogenesis models, initial emergence of blood cells is recognized in the extra embryonic yolk sac which principally sustains primitive erythropoiesis. The induction and support of these various hematopoietic compartments during human embryoid body (EB) development is influenced by different cytokines and growth factors. The use of cytokines, including SCF, FIt3L, IL-3, IL-6, G-CSF, VEGF, and the ventral mesoderm inducer BMP-4, have been shown to promote the differentiation of a highly enriched hematopoietic progenitor population that possess a CD45+CD34+ phenotype similar to the first definitive hematopoietic cell detected within the wall of the dorsal aorta of human embryos. This chapter provides techniques commonly employed by laboratory to differentiate phenotypically and functionally assay the hematopoietic cells derived from human EBs.

The nociceptive C-fibers of the dorsal root ganglion express several sodium channel isoforms that associate with one or more regulatory beta-subunits (beta1-beta4). To determine the effects of individual and combinations of the beta-subunit isoforms, we co-expressed Nav1.8 in combination with these beta-subunits in Xenopus oocytes. Whole-cell inward sodium currents were recorded using the two-microelectrode voltage clamp method. Our studies revealed that the co-expression beta1 alone or in combination with other beta-subunits enhanced current amplitudes, accelerated current decay kinetics, and negatively shifted the steady-state curves. In contrast, beta2 alone and in combination with beta1 altered steady-state inactivation of Nav1.8 to more depolarized potentials. Co-expression of beta3 shifted steady-state inactivation to more depolarized potentials; however, combined beta1beta3 expression caused no shift in channel availability. The results in this study suggest that the functional behavior of Nav1.8 will vary depending on the type of beta-subunit that expressed under normal and disease states.

Mammalian cells poorly express rNa(v)1.8 channels. In contrast, rNa(v)1.7 dorsal root ganglion channels have 90-fold higher peak Na(+) current densities. We investigated the role of rNa(v)1.7 and rNa(v)1.8 carboxy-termini in modulating the expression of rNa(v)1.7 and rNa(v)1.8 channels in tsA201 cells. Mutations in the ubiquitination site of the C-terminus did not improve rNa(v)1.8 current levels. However, rNa(v)1.8 chimeras containing the entire or the proximal portion of the rNa(v)1.7 C-terminus expressed 3.2-fold and 4.8-fold higher peak current densities, respectively, than parent rNa(v)1.8 channels. We conclude that the two Na(+) channels may have different endoplasmic reticulum processing signals.

Voltage-gated Na+channels (VGSC) are transmembrane proteins that are essential for the initiation and propagation of action potentials in neuronal excitability. Because neurons express a mixture of Na+channel isoforms and protein kinase C (PKC) isozymes, the nature of which channel is being regulated by which PKC isozyme is not known. We showed that DRG VGSC Nav1.7 (TTX-sensitive) and Nav1.8 (TTX-resistant), expressed in Xenopus oocytes were differentially regulated by protein kinase A (PKA) and PKC isozymes using the two-electrode voltage-clamp method. PKA activation resulted in a dose-dependent potentiation of Nav1.8 currents and an attenuation of Nav1.7 currents. PKA-induced increases (Nav1.8) and decreases (Nav1.7) in peak currents were not associated with shifts in voltage-dependent activation or inactivation. The PKA-mediated increase in Nav1.8 current amplitude was prevented by chloroquine, suggesting that cell trafficking may contribute to the changes in Nav1.8 current amplitudes. A dose-dependent decrease in Nav1.7 and Nav1.8 currents was observed with the PKC activators phorbol 12-myristate, 13-acetate (PMA) and phorbol 12,13-dibutyrate. PMA induced shifts in the steady-state activation of Nav1.7 and Nav1.8 channels by 6.5 and 14 mV, respectively, in the depolarizing direction. The role of individual PKC isozymes in the regulation of Nav1.7 and Nav1.8 was determined using PKC-isozyme-specific peptide activators and inhibitors. The decrease in the Nav1.8 peak current induced by PMA was prevented by a specific ϵPKC isozyme peptide antagonist, whereas the PMA effect on Nav1.7 was prevented by ϵPKC and βIIPKC peptide inhibitors. The data showed that Nav1.7 and Nav1.8 were differentially modulated by PKA and PKC. This is the first report demonstrating a functional role for ϵPKC and βIIPKC in the regulation of Nav1.7 and Nav1.8 Na+channels. Identification of the particular PKC isozymes(s) that mediate the regulation of Na+channels is essential for understanding the molecular mechanism involved in neuronal ion channel regulation in normal and pathological conditions.

Voltage-gated Na channels comprise four homologous domains each consisting of six transmembrane segments (S1-S6) linked by loops. The linkers between segments S5 and S6 in each domain (P-loops), denoted as SS1-SS2, form the pore of the channel. It is believed that the SS1 region of the P-loops dips into, while the SS2 region exits out of the membrane. We have reported previously that residues A728 and D730 (in SS1 of domain II) contribute to the external vestibule of the pore of the rat skeletal muscle Na channel (Na(v)1.4). In this study, we examined the role of a conserved neighbouring tryptophan residue at position 736 (W736) in the pore formation. The W736 residue of Na(v)1.4 was replaced by a cysteine using site-directed mutagenesis. Complementary RNAs encoding the wild-type and mutant channels were injected into Xenopus laevis oocytes and macroscopic Na(+) currents measured using the two-microelectrode voltage-clamp technique. The W736C mutant showed increased channel sensitivity to externally applied Cd(2+) and methanethiosulphonate-ethyltrimethylammonium (MTSET). Furthermore, micromolar concentrations of Cd(2+) reduced single-channel current amplitude in the Na(v)1.4/W736C mutant without affecting its voltage dependence. However, only small differences in tetrodotoxin and micro-conotoxin GIIIA affinity were observed between the wild-type and mutant channels. Replacing Na(+) with other cations - K(+), Li(+), Cs(+) or NH(4)(+) - did not change the ion permeation sequence of the Na(v)1.4/W736C mutant channel. The results suggest that W736 contributes to the formation of the pore, close to the mouth of the channel, but is not part of the selectivity filter.

Embryonic cell fate decisions require an integrated symphony of growth factors presented in a specific sequence and in discreet locations within the developing embryo. Because of species-specific organization of cellular topology in utero, growth factor regulation may differ among organisms. Accordingly, a better understanding of species-conserved growth factor requirements necessitates the need for precise modeling of cell differentiation and movement during construction of definitive embryonic tissues. Ideally, this model would allow control of growth factor production to experimentally dissect the relationship between growth factor signal and cell fate decision.