Plasticity of Cells and Ex Vivo Production of Red Blood Cells

The supply of transfusable red blood cells (RBCs) is not sufficient in many countries. If transfusable RBCs could be produced abundantly from certain resources, it would be very useful. Our group has developed a method to produce enucleated RBCs efficiently from hematopoietic stem/progenitor cells present in umbilical cord blood. More recently, it was reported that enucleated RBCs could be abundantly produced from human embryonic stem (ES) cells. The common obstacle for application of these methods is that they require very high cost to produce sufficient number of RBCs that are applicable in the clinic. If erythroid cell lines (immortalized cell lines) able to produce transfusable RBCs ex vivo were established, they would be valuable resources. Our group developed a robust method to obtain immortalized erythroid cell lines able to produce mature RBCs. To the best of our knowledge, this was the first paper to show the feasibility of establishing immortalized erythroid progenitor cell lines able to produce enucleated RBCs ex vivo. This result strongly suggests that immortalized human erythroid progenitor cell lines able to produce mature RBCs ex vivo can also be established

Design of Coelenterazine Analogue to Reveal Bioluminescent Reaction of Human Serum Albumin

This chapter describes the design of an imidazopyrazinone-type luciferin named as HuLumino1 by us and investigation of its luminescence properties. This luciferin was designed to generate bioluminescence by human serum albumin (HSA) rather than by luciferase derived from luminous organisms. HuLumino1 was developed by modifying a methoxy-terminated alkyl chain to the C-6 position and eliminating a benzyl group at the C-8 position of coelenterazine. To clarify the basis of light emission by HSA, the detailed kinetic properties of the HuLumino1/HSA pair were investigated using a calibrated luminometer. The enzymatic oxidation of HuLumino1 was observed only in the presence of HSA. Results of HSA quantification experiments using HuLumino1 agreed with less than 5% differences with those of enzyme-linked immunosorbent assays, suggesting HuLumino1 could be used for quantitative analysis of HSA levels in serum samples without any pretreatments. These results demonstrate the advantages of the coelenterazine analogue as a bioluminescence reagent to detect non-labeled proteins, which generally do not function as enzymes

Creating New β-Globin-Expressing Lentiviral Vectors by High-Resolution Mapping of Locus Control Region Enhancer Sequences.

Hematopoietic stem cell gene therapy is a promising approach for treating disorders of the hematopoietic system. Identifying combinations of cis-regulatory elements that do not impede packaging or transduction efficiency when included in lentiviral vectors has proven challenging. In this study, we deploy LV-MPRA (lentiviral vector-based, massively parallel reporter assay), an approach that simultaneously analyzes thousands of synthetic DNA fragments in parallel to identify sequence-intrinsic and lineage-specific enhancer function at near-base-pair resolution. We demonstrate the power of LV-MPRA in elucidating the boundaries of previously unknown intrinsic enhancer sequences of the human β-globin locus control region. Our approach facilitated the rapid assembly of novel therapeutic βAS3-globin lentiviral vectors harboring strong lineage-specific recombinant control elements capable of correcting a mouse model of sickle cell disease. LV-MPRA can be used to map any genomic locus for enhancer activity and facilitates the rapid development of therapeutic vectors for treating disorders of the hematopoietic system or other specific tissues and cell types

Near-Infrared Imaging Polarimetry of the NGC 2071 Star Forming Region with SIRPOL

We have conducted deep JHKs imaging polarimetry of a ~8' x 8' area of the NGC 2071 star forming region. Our polarization data have revealed various infrared reflection nebulae (IRNe) associated with the central IR young star cluster NGC2071IR and identified their illuminating sources. There are at least 4 IRNe in NGC2071IR and several additional IRNe are identified around nearby young stars in the same field-of-view. Each illuminating source coincides with a known near-IR source except for IRS3, which is only a part of IRN2 and is illuminated by the radio source 1c. Aperture polarimetry of each cluster source is used to detect unresolved circumstellar disk/outflow systems. Aperture polarimetry of the other point-like sources within the field is made in this region for the first time. The magnetic field structures (from ~1 pc down to \~0.1 pc) are derived using both aperture polarimetry of the point-like sources and imaging polarimetry of the shocked H2 emission that is seen as the dominant knotty nebulae in the Ks band image; they are both of dichroic origin and the derived field directions are consistent with each other. The magnetic field direction projected on the sky is also consistent with that inferred from the 850 micron thermal continuum emission polarimetry of the central 0.2 pc region, but running roughly perpendicular (~75 degrees) to the direction of the large scale outflow. We argue that the field strength is too weak to align the outflow in the large scale field direction via magnetic braking.Comment: o appear in PASJ, 9 pages, 11 Postscript figure

CRISPR-Cas9 interrogation of a putative fetal globin repressor in human erythroid cells

Sickle Cell Disease and beta-thalassemia, which are caused by defective or deficient adult beta-globin (HBB) respectively, are the most common serious genetic blood diseases in the world. Persistent expression of the fetal beta-like globin, also known gamma-globin, can ameliorate both disorders by serving in place of the adult beta-globin as a part of the fetal hemoglobin tetramer (HbF). Here we use CRISPR-Cas9 gene editing to explore a potential gamma-globin silencer region upstream of the delta-globin gene identified by comparison of naturally-occurring deletion mutations associated with up-regulated gamma-globin. We find that deletion of a 1.7 kb consensus element or select 350 bp sub-regions from bulk populations of cells increases levels of HbF. Screening of individual sgRNAs in one sub-region revealed three single guides that caused increases gamma-globin expression. Deletion of the 1.7 kb region in HUDEP-2 clonal sublines, and in colonies derived from CD34+ hematopoietic stem/progenitor cells (HSPCs), does not cause significant up-regulation of gamma-globin. These data suggest that the 1.7 kb region is not an autonomous gamma-globin silencer, and thus by itself is not a suitable therapeutic target for gene editing treatment of beta-hemoglobinopathies.Peer reviewe

Identifying spawning events in the Japanese flounder Paralichthys olivaceus from depth time-series data

Vertical swimming events (VSEs) of the Japanese flounder, Paralichthys olivaceus, recorded by high-frequency depth data loggers, were analysed to identify spawning events. In total 25,907 VSEs from 10 adult fish were classified into 4 clusters using a k-means method. VSEs in a specific cluster (cluster-S) characterised by accelerated vertical swimming were identified as possible spawning events. Both the descent (0.43 ± 0.22 body length s− 1) and ascent rates (0.43 ± 0.24 body length s− 1) of VSEs in cluster-S were more than 4 times faster than in any other VSE. Our analyses indicated that 4 individuals exhibited the spawning events during the recording periods. The estimated spawning frequency ranged from 0.74 to 0.90 events day− 1. These values were comparable to those obtained in other field and laboratory studies. The spawning condition of fish at the time of recapture was confirmed by separate histological and anatomical observations, which supported the cluster analysis results. These results suggest that a clustering technique can be successfully applied to identify spawning behaviour from time-depth data of free-swimming flatfishes that exhibit vertical swimming movements

Rapid and Sensitive Assessment of Globin Chains for Gene and Cell Therapy of Hemoglobinopathies

The β-hemoglobinopathies sickle cell anemia and β-thalassemia are the focus of many gene-therapy studies. A key disease parameter is the abundance of globin chains because it indicates the level of anemia, likely toxicity of excess or aberrant globins, and therapeutic potential of induced or exogenous β-like globins. Reversed-phase high-performance liquid chromatography (HPLC) allows versatile and inexpensive globin quantification, but commonly applied protocols suffer from long run times, high sample requirements, or inability to separate murine from human β-globin chains. The latter point is problematic for in vivo studies with gene-addition vectors in murine disease models and mouse/human chimeras. This study demonstrates HPLC-based measurements of globin expression (1) after differentiation of the commonly applied human umbilical cord blood-derived erythroid progenitor-2 cell line, (2) in erythroid progeny of CD34+ cells for the analysis of clustered regularly interspaced short palindromic repeats/Cas9-mediated disruption of the globin regulator BCL11A, and (3) of transgenic mice holding the human β-globin locus. At run times of 8 min for separation of murine and human β-globin chains as well as of human γ-globin chains, and with routine measurement of globin-chain ratios for 12 nL of blood (tested for down to 0.75 nL) or of 300,000 in vitro differentiated cells, the methods presented here and any variant-specific adaptations thereof will greatly facilitate evaluation of novel therapy applications for β-hemoglobinopathies

Sex-inducing effects toward planarians widely present among parasitic flatworms

Summary Various parasitic flatworms infect vertebrates for sexual reproduction, often causing devastating diseases in their hosts. Consequently, flatworms are of great socioeconomic and biomedical importance. Although the cessation of parasitic flatworm sexual reproduction is a major target of anti-parasitic drug design, little is known regarding bioactive compounds controlling flatworm sexual maturation. Using the planarian Dugesia ryukyuensis, we observed that sex-inducing substances found in planarians are also widespread in parasitic flatworms, such as monogeneans and flukes (but not in tapeworms). Reverse-phase HPLC analysis revealed the sex-inducing substance(s) eluting around the tryptophan retention time in the fluke Calicophoron calicophorum, consistent with previous studies on the planarian Bipalium nobile, suggesting that the substance(s) is likely conserved among flatworms. Moreover, six of the 18 ovary-inducing substances identified via transcriptome and metabolome analyses are involved in purine metabolism. Our findings provide a basis for understanding and modifying the life cycles of various parasitic flatworms.journal articl