Asymmetric Division and Lineage Commitment at the Level of Hematopoietic Stem Cells: Inference from Differentiation in Daughter Cell and Granddaughter Cell Pairs

How hematopoietic stem cells (HSCs) commit to a particular lineage is unclear. A high degree of HSC purification enabled us to address this issue at the clonal level. Single-cell transplantation studies revealed that 40% of the CD34−/low, c-Kit+, Sca-1+, and lineage marker− (CD34−KSL) cells in adult mouse bone marrow were able, as individual cells, to reconstitute myeloid and B- and T-lymphoid lineages over the long-term. Single-cell culture showed that >40% of CD34−KSL cells could form neutrophil (n)/macrophage (m)/erythroblast (E)/megakaryocyte (M) (nmEM) colonies. Assuming that a substantial portion of long-term repopulating cells can be detected as nmEM cells within this population, we compared differentiation potentials between individual pairs of daughter and granddaughter cells derived in vitro from single nmEM cells. One of the two daughter or granddaughter cells remained an nmEM cell. The other showed a variety of combinations of differentiation potential. In particular, an nmEM cell directly gave rise, after one cell division, to progenitor cells committed to nm, EM, or M lineages. The probability of asymmetric division of nmEM cells depended on the cytokines used. These data strongly suggest that lineage commitment takes place asymmetrically at the level of HSCs under the influence of external factors

Zero Casimir Force in Axion Electrodynamics and New Force Search

We point out that there is a stable configuration of metal plates where the Casimir force is vanishing in axion electrodynamics. We consider a concrete setup involving Weyl semimetals, which hosts an axion-like effect on the electromagnetism, towards the measurement of the axionic effect on the Casimir force. Our setup realizes zero Casimir force between metals and may be useful for the search for new force mediated by light particles at the micrometer scale.Comment: 28 pages, 9 figure

An Assessment Rubric for a Resident Training Program in Surgery: A Single-Institution Experience

Using a Collaborative Action Research model, our research team established a one-month clinical resident training program for first- and second-year clinical residents. We created and implemented an assessment rubric to assess the residents’ progress toward independent practice in surgery, and thereby, to evaluate the program itself. The program included training in three areas: basic techniques and procedures in the operating room, surgical ward management, and academic activities. The rubric measured the residents’ performance according to three achievement levels: Level 1 (demonstration), Level 2 (active help) and Level 3 (passive help). The program and rubric implementation began in June 2019 and continued until March 2020, when the program outcomes and shortcomings were analyzed. Among nineteen clinical residents, a total of nine clinical residents participated in the study. Most participants reached achievement Level 3 for their performance of basic techniques in the operating room. Finally, we discussed ideas for improvement and drafted plans for an improved rubric to complete the action research cycle. Our research team found the rubric to be a useful tool in evaluating the status of the new clinical resident training program

An All-Recombinant Protein-Based Culture System Specifically Identifies Hematopoietic Stem Cell Maintenance Factors.

Hematopoietic stem cells (HSCs) are considered one of the most promising therapeutic targets for the treatment of various blood disorders. However, due to difficulties in establishing stable maintenance and expansion of HSCs in vitro, their insufficient supply is a major constraint to transplantation studies. To solve these problems we have developed a fully defined, all-recombinant protein-based culture system. Through this system, we have identified hemopexin (HPX) and interleukin-1α as responsible for HSC maintenance in vitro. Subsequent molecular analysis revealed that HPX reduces intracellular reactive oxygen species levels within cultured HSCs. Furthermore, bone marrow immunostaining and 3D immunohistochemistry revealed that HPX is expressed in non-myelinating Schwann cells, known HSC niche constituents. These results highlight the utility of this fully defined all-recombinant protein-based culture system for reproducible in vitro HSC culture and its potential to contribute to the identification of factors responsible for in vitro maintenance, expansion, and differentiation of stem cell populations

Stepwise development of Hematopoietic stem Cells from Embryonic Stem Cells

The cellular ontogeny of hematopoietic stem cells (HSCs) remains poorly understood because their isolation from and their identification in early developing small embryos are difficult. We attempted to dissect early developmental stages of HSCs using an in vitro mouse embryonic stem cell (ESC) differentiation system combined with inducible HOXB4 expression. Here we report the identification of pre-HSCs and an embryonic type of HSCs (embryonic HSCs) as intermediate cells between ESCs and HSCs. Both pre-HSCs and embryonic HSCs were isolated by their c-Kit(+)CD41(+)CD45(−) phenotype. Pre-HSCs did not engraft in irradiated adult mice. After co-culture with OP9 stromal cells and conditional expression of HOXB4, pre-HSCs gave rise to embryonic HSCs capable of engraftment and long-term reconstitution in irradiated adult mice. Blast colony assays revealed that most hemangioblast activity was detected apart from the pre-HSC population, implying the early divergence of pre-HSCs from hemangioblasts. Gene expression profiling suggests that a particular set of transcripts closely associated with adult HSCs is involved in the transition of pre-HSC to embryonic HSCs. We propose an HSC developmental model in which pre-HSCs and embryonic HSCs sequentially give rise to adult types of HSCs in a stepwise manner

Endomucin, a CD34-like sialomucin, marks hematopoietic stem cells throughout development

To detect as yet unidentified cell-surface molecules specific to hematopoietic stem cells (HSCs), a modified signal sequence trap was successfully applied to mouse bone marrow (BM) CD34−c-Kit+Sca-1+Lin− (CD34−KSL) HSCs. One of the identified molecules, Endomucin, is an endothelial sialomucin closely related to CD34. High-level expression of Endomucin was confined to the BM KSL HSCs and progenitor cells, and, importantly, long-term repopulating (LTR)–HSCs were exclusively present in the Endomucin+CD34−KSL population. Notably, in the yolk sac, Endomucin expression separated multipotential hematopoietic cells from committed erythroid progenitors in the cell fraction positive for CD41, an early embryonic hematopoietic marker. Furthermore, developing HSCs in the intraembryonic aorta-gonad-mesonephros (AGM) region were highly enriched in the CD45−CD41+Endomucin+ fraction at day 10.5 of gestation (E10.5) and in the CD45+CD41+Endomucin+ fraction at E11.5. Detailed analyses of these fractions uncovered drastic changes in their BM repopulating capacities as well as in vitro cytokine responsiveness within this narrow time frame. Our findings establish Endomucin as a novel cell-surface marker for LTR-HSCs throughout development and provide a powerful tool in understanding HSC ontogeny