Robust and highly efficient hiPSC generation from patient non-mobilized peripheral blood-derived CD34+ cells using the auto-erasable Sendai virus vector

Background: Disease modeling with patient-derived induced pluripotent stem cells (iPSCs) is a powerful tool forelucidating the mechanisms underlying disease pathogenesis and developing safe and effective treatments. Patientperipheral blood (PB) cells are used for iPSC generation in many cases since they can be collected with minimuminvasiveness. To derive iPSCs that lack immunoreceptor gene rearrangements, hematopoietic stem and progenitorcells (HSPCs) are often targeted as the reprogramming source. However, the current protocols generally requireHSPC mobilization and/or ex vivo expansion owing to their sparsity at the steady state and low reprogrammingefficiencies, making the overall procedure costly, laborious, and time-consuming.Methods: We have established a highly efficient method for generating iPSCs from non-mobilized PB-derivedCD34+ HSPCs. The source PB mononuclear cells were obtained from 1 healthy donor and 15 patients and werekept frozen until the scheduled iPSC generation. CD34+ HSPC enrichment was done using immunomagnetic beads,with no ex vivo expansion culture. To reprogram the CD34+-rich cells to pluripotency, the Sendai virus vectorSeVdp-302L was used to transfer four transcription factors: KLF4, OCT4, SOX2, and c-MYC. In this iPSC generationseries, the reprogramming efficiencies, success rates of iPSC line establishment, and progression time wererecorded. After generating the iPSC frozen stocks, the cell recovery and their residual transgenes, karyotypes, T cellreceptor gene rearrangement, pluripotency markers, and differentiation capability were examined.Results：We succeeded in establishing 223 iPSC lines with high reprogramming efficiencies from 15 patients with 8 different disease types. Our method allowed the rapid appearance of primary colonies (~ 8 days), all of which were expandable under feeder-free conditions, enabling robust establishment steps with less workload. After thawing, the established iPSC lines were verified to be pluripotency marker-positive and of non-T cell origin. A majority of the iPSC lines were confirmed to be transgene-free, with normal karyotypes. Their trilineage differentiation capability was also verified in a defined in vitro assay.Conclusion：This robust and highly efficient method enables the rapid and cost-effective establishment of transgene-free iPSC lines from a small volume of PB, thus facilitating the biobanking of patient-derived iPSCs and their use for the modeling of various diseases

Neutralization against Omicron sublineages (BA.2/BA.5/BQ.1.1/XBB/XBB.1.5) in bivalent BNT162b2-vaccinated HCWs with or without risk factors, or following BT infection with Omicron

Abstract SARS-CoV-2-BA.4/5-adapted-bivalent-BNT162b2-vaccine (bvBNT), developed in response to the recent emergence of immune-evasive Omicron-variants, has been given to individuals who completed at least 2-doses of the monovalent-BNT162b2-vaccine (mvBNT). In the present cohort study, we evaluated neutralization-titers (NT50s) against Wuhan-strain (SCoV2Wuhan) and Omicron-sublineages including BA.2/BA.5/BQ.1.1/XBB/XBB.1.5, and vaccine-elicited S1-binding-IgG in sera from participants-vaccinated with 5th-bvBNT following 4th-mvBNT. The 5th-bvBNT-dose elicited good protective-activity against SCoV2Wuhan with geometric-mean (gMean)-NT50 of 1966–2091, higher than the peak-values post-4th-mvBNT with no statistical significance, and favorable neutralization-activity against not only BA.5 but also BA.2, with ~ 3.2-/~ 2.2-fold greater gMean-NT50 compared to the peak-values post-4th-mvBNT-dose, in participants with or without risk factors. However, neutralization-activity of sera post-5th-bvBNT-dose was low against BQ.1.1/XBB/XBB.1.5. Interestingly, participants receiving bvBNT following breakthrough (BT) infection during Omicron-wave had significantly enhanced neutralization-activity against SCoV2Wuhan/BA.2/BA.5 with ~ 4.6-/~ 6.3-/~ 8.1-fold greater gMean-NT50, respectively, compared to uninfected participants receiving bvBNT. Sera from BT-infected-participants receiving bvBNT had enhanced neutralization-activity against BQ.1.1/XBB/XBB.1.5 by ~ 3.8-fold compared to those from the same participants post-4th-mvBNT-dose, and had enhanced gMean-NT50 ~ 5.4-fold greater compared to those of uninfected-participants’ sera post-bvBNT. These results suggest that repeated stimulation brought about by exposure to BA.5’s-Spike elicit favorable cross-neutralization-activity against various SARS-CoV-2-variants

Accumulation patterns of soil carbon and nitrogen in a Japanese oak (Quercus crispula) plantation in eastern Hokkaido : Analyses using continuous 0-40 years old stands

本研究は0年生から40年生のミズナラ造林地において下層種であるミヤコザサを含む植生構造の造林後の林齢によって生じる変化が，土壌の炭素と窒素の蓄積，またC/N比に与える影響を評価した。下層種現存量は造林後7年目に極大値となる一山型分布を示した。一方上層種は調査期間中増加し続け，8年目以降は全ての林分において上層種が下層種を上回っていた。葉面積指数の変化から林冠の閉鎖が造林後12年目に起こっていた。林齢による土壌の炭素と窒素の蓄積は両方とも粗大有機物の集積するO層での変化が最も大きく，表層，深層の鉱質土の順に変化が小さくなった。O層で観察された変化は全て林分発達初期に起こっており，炭素蓄積量は造林後10年，窒素蓄積量は20年目まで増加した。O層の炭素蓄積量，および窒素蓄積量は上層木の着葉量との間に正の相関が見られた。O層の窒素蓄積量とC/N比は，下層種の現存量との間にそれぞれ負の相関，正の相関関係が見られた。一方，鉱質土層の窒素蓄積量はササの細根量と負の相関にあった。これらの結果から，林齢の増加に伴う林冠閉鎖や下層種の増減といった植生構造の変化が土壌の炭素および窒素の蓄積に影響を及ぼしていると考えられた。This study assessed the effect of the change in forest structure including understory vegetation of Sasa nipponica on soil C and N accumulation from 0 to 40-yr Japanese oak (Quercus crispula) plantation chronosequence. The peak of understory biomass was at 7 years after afforestation. Biomass of the overstory species linearly increased, and the biomass of overstory species was more than that of understory species after 8-yr. According to leaf mass index change along with stand age, canopy closure was occurred about 12 years after afforestation. The ranges of temporal changes in both soil C and N accumulations increased from surface to deep soil layers. The changes in C and N accumulation of O layer were large during early stage of stand development, and C and N accumulations increased until 10 years and 20 years after afforestation, respectively. Overstory leaf mass positively correlated with C and N accumulations in O layer. Understory biomass negatively correlated with N accumulation in O layer and positively correlated with C/N ratio. The N accumulations both in mineral soil 0-4 and 4-8cm in depth were negatively correlated with fine root mass of S. nipponica. The results suggested that change in vegetation structure such as canopy closure by overstory and dynamics of understory biomass affected soil C and N accumulations