A latent subset of human hematopoietic stem cells resists regenerative stress to preserve stemness.

Continuous supply of immune cells throughout life relies on the delicate balance in the hematopoietic stem cell (HSC) pool between long-term maintenance and meeting the demands of both normal blood production and unexpected stress conditions. Here we identified distinct subsets of human long-term (LT)-HSCs that responded differently to regeneration-mediated stress: an immune checkpoint ligand CD112lo subset that exhibited a transient engraftment restraint (termed latency) before contributing to hematopoietic reconstitution and a primed CD112hi subset that responded rapidly. This functional heterogeneity and CD112 expression are regulated by INKA1 through direct interaction with PAK4 and SIRT1, inducing epigenetic changes and defining an alternative state of LT-HSC quiescence that serves to preserve self-renewal and regenerative capacity upon regeneration-mediated stress. Collectively, our data uncovered the molecular intricacies underlying HSC heterogeneity and self-renewal regulation and point to latency as an orchestrated physiological response that balances blood cell demands with preserving a stem cell reservoir.

A stemness screen reveals C3orf54/INKA1 as a promoter of human leukemia stem cell latency.

There is a growing body of evidence that the molecular properties of leukemia stem cells (LSCs) are associated with clinical outcomes in acute myeloid leukemia (AML), and LSCs have been linked to therapy failure and relapse. Thus, a better understanding of the molecular mechanisms that contribute to the persistence and regenerative potential of LSCs is expected to result in the development of more effective therapies. We therefore interrogated functionally validated data sets of LSC-specific genes together with their known protein interactors and selected 64 candidates for a competitive in vivo gain-of-function screen to identify genes that enhanced stemness in human cord blood hematopoietic stem and progenitor cells. A consistent effect observed for the top hits was the ability to restrain early repopulation kinetics while preserving regenerative potential. Overexpression (OE) of the most promising candidate, the orphan gene C3orf54/INKA1, in a patient-derived AML model (8227) promoted the retention of LSCs in a primitive state manifested by relative expansion of CD34+ cells, accumulation of cells in G0, and reduced output of differentiated progeny. Despite delayed early repopulation, at later times, INKA1-OE resulted in the expansion of self-renewing LSCs. In contrast, INKA1 silencing in primary AML reduced regenerative potential. Mechanistically, our multidimensional confocal analysis found that INKA1 regulates G0 exit by interfering with nuclear localization of its target PAK4, with concomitant reduction of global H4K16ac levels. These data identify INKA1 as a novel regulator of LSC latency and reveal a link between the regulation of stem cell kinetics and pool size during regeneration.

Antibiotics prescriptions for pneumonia analyzed by claim information in Japan.

OBJECTIVE: Japan is an aging society, and pneumonia is the leading cause of death, but the suitability of antibiotics for treating community-acquired pneumonia (CAP) in Japan is not clear. The purpose of this study was to investigate antibacterial drugs for treating CAP according to age. MATERIALS AND METHODS: Using the Japanese national database from 2011 to 2014, we analyzed the usage of antibiotics for CAP according to age. RESULTS: The numbers of claim information were 9,386, and 70% of the patients were aged ≥ 75 years. Sulbactam/ampicillin (SBT/ABPC) or ceftriaxone (CTRX) was used in 60%, but broad-spectrum antibiotics, combination therapy, and anti-mycoplasma antibiotics were used in 15 - 28% of all age groups. The 30-day survival rate did not differ between SBT/ABPC or CTRX vs. others. There was no difference in 30-day mortality and risk in any group between the ages of 15 and 64 years. On the other hand, the use of anti-mycoplasma antibiotics reduced the 30-day mortality by 0.50 times (p < 0.01), and the use of two or more antibiotics increased the 30-day mortality by 1.45 times (p = 0.02) at age ≥ 65 years. CONCLUSION: Approximately half of the antibiotics used for CAP requiring hospitalization consisted of CTRX or SBT/ABPC as recommended by the Japanese Respiratory Society (JRS) guidelines. On the other hand, the usage of broad-spectrum antibiotics and combination therapy were relatively frequent at all ages, although their use does not always contribute to survival. Our data provide basic information for analyzing the outcome of pneumonia treatment in terms of an antimicrobial resistance action plan in Japan.

Prevalence of positive IGRAs and innate immune system in HIV-infected individuals in Japan.

INTRODUCTION: Human immunodeficiency virus (HIV) infected individuals are at increased risk of developing active tuberculosis (TB). TB incidence remains higher than in non-HIV subjects after antiretroviral therapy (ART) initiation. This study was conducted to estimate the prevalence of positive IGRA, reflecting latent tuberculosis infection and/or a history of active TB, in HIV-infected individuals after ART initiation in Japan. METHODS: Two IGRAs (Interferon (IFN)-γ release assays), QuantiFERON®-TB Gold Plus (QFT-Plus) and T-Spot®.TB (TSPOT), were used. We also analyzed the TB associated risk factors for the IGRAs results and the role of CD4+ T-cells, CD8+ T-cells and NK cells for producing IFN-γ. We also analyzed the risk factors for positive IGRA responses and the role of CD4+ T-cells, CD8+ T-cells and NK cells for producing IFN-γ. RESULTS: One hundred eight-four subjects were prospectively enrolled. Median age was 49 years. The positivity rates of QFT-Plus and TSPOT were 7.6% [95%CI 4.6-12.4] and 2.7% [95%CI 1.2-6.2], respectively, with significant difference. TB-associated risk factors and NK cells ≥300/µL were selected as independently significant factors by multivariate logistic regression. The NK cell count revealed significant linear regression with IFN-γ production responding to TB-specific antigens. CONCLUSIONS: The prevalence of positive IGRAs was 2.7%-7.6%. QFT-Plus would be practical for a higher positivity rate and reflect TB risk factors. The innate immune system, referring to IFN-γ production, plays an important role in the immune response to TB-specific antigens even after initiating ART.

Antibiotics for hospitalized children with community-acquired pneumonia in Japan: Analysis based on Japanese national database.

INTRODUCTION: Community-acquired pneumonia (CAP) is one of the most common causes of pediatric infection requiring hospitalization. Antimicrobial resistance due to the inappropriate use poses a threat worldwide. Our objective is to analyze and optimize the trends of antibiotics used for pediatric inpatients with CAP in a claims database provided by the Ministry of Health, Labour and Welfare. METHODS: Our database randomly sampled 10% of the hospitalized patients every October from 2011 to 2014. Patients aged <15 years in whom antibiotic therapy was initiated within two days of admission were listed. Subsequently, we investigated the antibiotics administered on the first day of prescription. RESULTS: A total of 4,831 antibiotics were prescribed for 3,909 patients. Many patients aged ≤ five years were treated with ß-lactams alone whereas many patients aged ≥ six years were treated with a single antibiotic, such as a macrolide, tetracycline, and quinolone, which covers atypical bacteria. Combination therapy was primarily used in children aged ≥ six years (nearly 30%); the main combination was a ß-lactam and non-ß-lactam covering atypical bacteria. Ampicillin-sulbactam was the most frequently prescribed ß-lactam in children of all ages other than infants. Ampicillin, however, was most often prescribed in infants, but its usage rate was low at other ages. CONCLUSIONS: Antibiotics were appropriately prescribed and were similar to that recommended in the 2011 guidelines for pediatric inpatients with CAP. However, combination therapy was frequently prescribed in children aged ≥ six years. According to the revised guidelines in 2017, ampicillin should be used more frequently for patients hospitalized with CAP.

Epidemiology and treatment outcome of pneumonia: Analysis based on Japan national database.

Pneumonia is the third leading cause of death in Japan. Mortality increases at an accelerating rate in elderly patients aged ≥65 years. Elderly patients tend to have underlying conditions affecting pneumonia treatment. The national database (NDB) associated with medical services under Japanese universal health insurance is available for research purposes. Our NDB randomly sampled 10% of hospitalized patients every October from 2011 to 2014. In this NDB, we analyzed pneumonia epidemiology in patients aged ≥15 years and 30-day mortality in Japanese hospitals. This study also investigated the factors affecting treatment outcome. A total of 9386 patients were entered. The number of patients from age 65 years and older increased greatly, representing 85% of the total. The thirty-day mortality rate among all patients was 11.7%. Mortality rates at age 15-64, 65-74, 75-84, and ≥85 years were 9.5%, 12.0%, 8.3%, and 14.9%, respectively, showing significant differences (P < 0.001). The underlying conditions varied among age groups. Male gender, age, heart failure, chronic kidney disease (CKD), consciousness disorder, shock and respiratory failure are risk factors for 30-day mortality. Pneumonia develops mainly in people aged 65 years and older in Japan, and treatment outcome is generally poor in elderly patients. The underlying conditions were seen to affect the 30-day mortality rate. CURB-65 and ADROP, a modification of CURB-65 in Japan, have already estimated these risk factors, and heart failure and CKD might be additional factors for estimating pneumonia severity.

A retrospective observational study of antimicrobial treatment for non-tuberculous mycobacteria disease using a nationwide claims database in Japan.

The Macrolides (MLs), clarithromycin and azithromycin, are key drugs for non-tuberculous mycobacteria (NTM) diseases treatment. A three antibiotics regimen including MLs, rifampicin (RFP) and ethambutol (EB) has been recommended for the treatment of NTM diseases in ATS/IDSA guideline. However, anti-biotics are not necessarily prescribed in compliance with the guideline. Inappropriate regimens are risk of introducing MLs resistance. Therefore, we planned this study to evaluate the current Japanese NTM diseases treatment conditions. We used the national database (NDB) from 2011 to 2014. A total of 183 patients were entered into the study. The patients number increased at an accelerating rate in patients aged ≥55 years. Patients aged ≥55 years made up 91.3% of the total NTM diseases. Male and female patients were 61 and 122, respectively, a female/male ratio of 2.00. Clarithromycin, RFP, EB and fluoroquinolones were frequently prescribed, with the numbers of prescriptions being 125, 66, 57 and 45, respectively. The regimen of MLs, RFP and EB recommend by ATS/IDSA guideline 2007 was only followed by 25.1% of the patients. MLs monotherapy was as high as 30.6% of NTM diseases and would be a risk factor leading to an increase of MLs resistance and poor treatment outcome. Without effective NTM disease therapy, the increase of MLs-resistant NTM diseases would be a burden for Japanese health care facilities.

A retrospective observational study of antibiotics treatment for sepsis using a nationwide claim database in Japan.

Survival Sepsis Campaign (SSC) guidelines have recommended broad-spectrum antibiotics prescriptions to cover the possible pathogenic microorganisms. However, mortality from sepsis is still high, as about one quarter of cases are thought to result in death. We analyzed nationwide health claims data of universal health insurance systems in Japan. Our aim was to describe the antibiotics prescriptions and underlying conditions of Japanese sepsis patients. In addition, we analyzed the factors associated with 30-day mortality. A total of 1188 patients aged ≥15 years were entered, of which 80.1% were ≥65 years old. Broad-spectrum antibiotics were prescribed for 53.8%. Carbapenem, Piperacillin Tazobactam and Anti-pseudomonas Cephalosporin were prescribed for 30.8%, 13.0% and 12.2% of the patients, respectively. (Some patients were counted twice) The overall 30-day mortality rate was 21.3%. Risk factors associated with 30-day mortality were examined by Cox proportional hazards regression analysis. Age of ≥85 years, malignancy, chronic kidney disease (CKD), shock and respiratory failure were selected as risk factors, but broad-spectrum antibiotics was not included. Sepsis is mostly observed in those aged 65 years and over. The rates of broad-spectrum antibiotics were restricted, and antibiotics were also not necessarily prescribed on the basis of SSC guidelines. However, broad-spectrum antibiotics did not improve the treatment outcome. Aging and underlying conditions like malignancy, CKD, shock and respiratory failure were poor prognostic factors.

Microorganisms isolated at admission and treatment outcome in sputum smear-positive pulmonary tuberculosis.

Cured or completed cases in newly diagnosed sputum smear-positive pulmonary tuberculosis (TB) is 47.7% in Japan in 2016. Aging of TB patients and their underlying conditions could affect treatment outcome. We analyzed the association between the isolation of microorganisms from sputum at admission and the 180-day mortality rate of the sputum smear-positive pulmonary TB patients in Chiba-East Hospital in Japan. Total subjects were 761 (median age: 63 years). Sputum test for microorganisms was conducted in 708 patients. Microorganisms other than the normal oral flora were isolated in 128 cases (18.1%). Details of the isolated microorganisms were as follows: methicillin-resistant Staphylococcus aureus 23 cases, Klebsiella pneumoniae 17 cases, Pseudomonas aeruginosa 16 cases. Mortality was significantly elevated in the patients with those microorganisms than the others (39.8% vs. 10.2%) (P < 0.01). Fifty-one of 128 patients with those microorganisms died, and 10 of them died of infectious disease, which is the most frequent cause of deaths. The factors associated with the isolation of those microorganisms were as follows: respiratory failure (adjusted odds ratio (aOR):2.5 [95% confidence interval (CI) 1.3-4.7]), performance status 3 or 4 (aOR:2.9 [95% CI 1.6-5.4]), serum albumin <3.0 mg/dL (aOR:2.1 [95% CI 1.3-3.6], age of 65 years or older (aOR:2.0 [95% CI 1.2-3.4]). Those strains were isolated from one of sixth patients. Patients with those microorganisms did not always develop infectious diseases; however, treatment outcomes were poor, with higher mortality. The isolations of microorganisms were associated with various underlying conditions, leading to death. Thus, attention should be paid to TB patients with the above factors.

Prominin-1/CD133 expression as potential tissue-resident vascular endothelial progenitor cells in the pulmonary circulation.

Pulmonary vascular endothelial cells could contribute to maintain homeostasis in adult lung vasculature. "Tissue-resident" endothelial progenitor cells (EPCs) play pivotal roles in postnatal vasculogenesis, vascular repair, and tissue regeneration; however, their local pulmonary counterparts remain to be defined. To determine whether prominin-1/CD133 expression can be a marker of tissue-resident vascular EPCs in the pulmonary circulation, we examined the origin and characteristics of prominin-1/CD133-positive (Prom1(+)) PVECs considering cell cycle status, viability, histological distribution, and association with pulmonary vascular remodeling. Prom1(+) PVECs exhibited high steady-state transit through the cell cycle compared with Prom1(-) PVECs and exhibited homeostatic cell division as assessed using the label dilution method and mice expressing green fluorescent protein. In addition, Prom1(+) PVECs showed more marked expression of putative EPC markers and drug resistance genes as well as highly increased activation of aldehyde dehydrogenase compared with Prom1(-) PVECs. Bone marrow reconstitution demonstrated that tissue-resident cells were the source of >98% of Prom1(+) PVECs. Immunofluorescence analyses revealed that Prom1(+) PVECs preferentially resided in the arterial vasculature, including the resistant vessels of the lung. The number of Prom1(+) PVECs was higher in developing postnatal lungs. Sorted Prom1(+) PVECs gave rise to colonies and formed fine vascular networks compared with Prom1(-) PVECs. Moreover, Prom1(+) PVECs increased in the monocrotaline and the Su-5416 + hypoxia experimental models of pulmonary vascular remodeling. Our findings indicated that Prom1(+) PVECs exhibited the phenotype of tissue-resident EPCs. The unique biological characteristics of Prom1(+) PVECs predominantly contribute to neovasculogenesis and maintenance of homeostasis in pulmonary vascular tissues.

Vascular Repair by Tissue-Resident Endothelial Progenitor Cells in Endotoxin-Induced Lung Injury.

Vascular disruption is one of the pathological hallmarks in acute respiratory distress syndrome. Bone marrow (BM)-derived circulating endothelial progenitor cells (EPCs) and lung tissue-resident EPCs have been considered to play a pivotal role in pulmonary vascular repair; however, which population is predominant in local pulmonary vasculogenesis remains to be clarified. We therefore examined the origin of EPCs participating in the regenerative process of pulmonary vascular endothelial cells (PVECs) in experimental acute respiratory distress syndrome. Lung samples from mice administered LPS intratracheally were investigated for cell dynamics and EPC functions. Quantitative flow cytometric analysis demonstrated that the number of PVECs decreased by roughly 20% on Day 1 and then recovered on Day 7 of LPS challenge. Bromodeoxyuridine-incorporation assays and immunofluorescence microscopy demonstrated that proliferating PVECs preferentially located in the capillary vessels. Experiments using BM chimera mice revealed that most of the regenerating PVECs were tissue-resident cells, and BM-derived cells hardly engrafted as PVECs. The population of circulating putative phenotypical EPCs decreased during the first week after LPS challenge. The regenerating PVECs were characterized by high colony-forming and vasculogenic capacities, intracellular reactive oxygen species scavenging and aldehyde dehydrogenase activites, and enhanced gene expression of Abcb1b (a drug-resistant gene), suggesting that the population of PVECs included tissue-resident EPCs activated during regenerative process of PVECs. The proliferating PVECs expressed CD34, Flk-1/KDR, and c-kit more strongly and Prom1/CD133 less strongly on the surface than nonproliferating PVECs. Our findings indicated that lung tissue-resident EPCs predominantly contribute to pulmonary vascular repair after endotoxin-induced injury.

Hypoxia-induced proliferation of tissue-resident endothelial progenitor cells in the lung.

Exposure to hypoxia induces changes in the structure and functional phenotypes of the cells composing the pulmonary vascular wall from larger to most peripheral vessels. Endothelial progenitor cells (EPCs) may be involved in vascular endothelial repair. Resident EPCs with a high proliferative potential are found in the pulmonary microcirculation. However, their potential location, identification, and functional role have not been clearly established. We investigated whether resident EPCs or bone marrow (BM)-derived EPCs play a major role in hypoxic response of pulmonary vascular endothelial cells (PVECs). Mice were exposed to hypoxia. The number of PVECs transiently decreased followed by an increase in hypoxic animals. Under hypoxic conditions for 1 wk, prominent bromodeoxyuridine incorporation was detected in PVECs. Some Ki67-positive cells were detected among PVECs after 1 wk under hypoxic conditions, especially in the capillaries. To clarify the origin of proliferating endothelial cells, we used BM chimeric mice expressing green fluorescent protein (GFP). The percentage of GFP-positive PVECs was low and constant during hypoxia in BM-transplanted mice, suggesting little engraftment of BM-derived cells in lungs under hypoxia. Proliferating PVECs in hypoxic animals showed increased expression of CD34, suggesting hypoxia-induced gene expression and cell surface antigen of EPC or stem/progenitor cells markers. Isolated PVECs from hypoxic mice showed colony- and tube-forming capacity. The present study indicated that hypoxia could induce proliferation of PVECs, and the origin of these cells might be tissue-resident EPCs.

A culture method with berbamine, a plant alkaloid, enhances CAR-T cell efficacy through modulating cellular metabolism.

Memory T cells demonstrate superior in vivo persistence and antitumor efficacy. However, methods for manufacturing less differentiated T cells are not yet well-established. Here, we show that producing chimeric antigen receptor (CAR)-T cells using berbamine (BBM), a natural compound found in the Chinese herbal medicine Berberis amurensis, enhances the antitumor efficacy of CAR-T cells. BBM is identified through cell-based screening of chemical compounds using induced pluripotent stem cell-derived T cells, leading to improved viability with a memory T cell phenotype. Transcriptomics and metabolomics using stem cell memory T cells reveal that BBM broadly enhances lipid metabolism. Furthermore, the addition of BBM downregulates the phosphorylation of p38 mitogen-activated protein kinase and enhanced mitochondrial respiration. CD19-CAR-T cells cultured with BBM also extend the survival of leukaemia mouse models due to their superior in vivo persistence. This technology offers a straightforward approach to enhancing the antitumor efficacy of CAR-T cells.

Mini-TCRs: Truncated T cell receptors to generate T cells from induced pluripotent stem cells.

Allogeneic T cell platforms utilizing induced pluripotent stem cell (iPSC) technology exhibit significant promise for the facilitation of adoptive immunotherapies. While mature T cell receptor (TCR) signaling plays a crucial role in generating T cells from iPSCs, the introduction of exogenous mature TCR genes carries a potential risk of causing graft-versus-host disease (GvHD). In this study, we present the development of truncated TCRα and TCRß chains, termed mini-TCRs, which lack variable domains responsible for recognizing human leukocyte antigen (HLA)-peptide complexes. We successfully induced cytotoxic T lymphocytes (CTLs) from iPSCs by employing mini-TCRs. Combinations of TCRα and TCRß fragments were screened from mini-TCR libraries based on the surface localization of CD3 proteins and their ability to transduce T cell signaling. Consequently, mini-TCR-expressing iPSCs underwent physiological T cell development, progressing from the CD4 and CD8 double-positive stage to the CD8 single-positive stage. The resulting iPSC-derived CTLs exhibited comparable cytokine production and cytotoxicity in comparison to that of full-length TCR-expressing T lymphocytes when chimeric antigen receptors (CARs) were expressed. These findings demonstrate the potential of mini-TCR-carrying iPSCs as a versatile platform for CAR T cell therapy, offering a promising avenue for advancing adoptive immunotherapies.

TGF-beta as a candidate bone marrow niche signal to induce hematopoietic stem cell hibernation.

Hematopoietic stem cells (HSCs) reside in a bone marrow niche in a nondividing state from which they occasionally are aroused to undergo cell division. Yet, the mechanism underlying this unique feature remains largely unknown. We have recently shown that freshly isolated CD34-KSL hematopoietic stem cells (HSCs) in a hibernation state exhibit inhibited lipid raft clustering. Lipid raft clustering induced by cytokines is essential for HSCs to augment cytokine signals to the level enough to re-enter the cell cycle. Here we screened candidate niche signals that inhibit lipid raft clustering, and identified that transforming growth factor-beta (TGF-beta) efficiently inhibits cytokine-mediated lipid raft clustering and induces HSC hibernation ex vivo. Smad2 and Smad3, the signaling molecules directly downstream from and activated by TGF-beta receptors were specifically activated in CD34-KSL HSCs in a hibernation state, but not in cycling CD34+KSL progenitors. These data uncover a critical role for TGF-beta as a candidate niche signal in the control of HSC hibernation and provide TGF-beta as a novel tool for ex vivo modeling of the HSC niche.

Drug Fever Due to Favipiravir Administration for the Treatment of a COVID-19 Patient.

A 55-year-old Japanese man was hospitalized with the novel coronavirus disease 2019 (COVID-19). On the 14th day after the start of favipiravir administration, the patient developed a fever with a temperature of 38.1°C. His pulse rate also became elevated to 128 bpm, so relative bradycardia was not suspected. Since he was in good overall health and no concomitant symptoms and signs were apparent, we considered it to be drug fever due to favipiravir. After the completion of favipiravir treatment, the patient's temperature normalized within 24 hours. We herein report this case of drug fever caused by favipiravir.

Possibility of deterioration of respiratory status when steroids precede antiviral drugs in patients with COVID-19 pneumonia: A retrospective study.

INTRODUCTION: Coronavirus disease (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2. Although most patients with COVID-19 develop asymptomatic or mild disease, some patients develop severe disease. The effectiveness of various therapeutic agents, including antiviral drugs, steroids, and anti-inflammatories for COVID-19, have been being confirmed. The effect of administering steroids in early disease is unclear. This study therefore aimed to evaluate the effectiveness and risk of exacerbation of steroids administered preceding antiviral drugs in patients with COVID-19 pneumonia. METHODS: This retrospective, single-center, observational study included consecutive patients with COVID-19 between March 2020 and March 2021. Patients were divided into a steroids-first group and antiviral-drugs-first group. Mortality, duration of hospitalization, incidence rate and duration of intensive care unit (ICU) admission, intubation, and extracorporeal membrane oxygenation (ECMO) induction of the two groups were compared. RESULTS: A total of 258 patients were admitted during the study period. After excluding patients who received symptomatic treatment only, who were taking immunosuppressive drugs, or who were administered antiviral drugs only, 68 patients were included in the analysis, 16 in the steroids-first group and 52 in the antiviral-drugs-first group. The rate of intubation, ICU admission and ECMO induction were significantly higher in the steroids-first group than in the antiviral-drugs-first group (81.3% vs. 33.3, p<0.001, 75.0% vs. 29.4%, p = 0.001, and 31.3% vs. 7.8%, p = 0.017, respectively). Furthermore, patients who received steroids within ten days after starting antiviral drugs had significantly lower rates of ICU admission, intubation, and ECMO induction. (81.3% vs. 42.9% p = 0.011, 75.0% vs. 37.1% p = 0.012, and 31.3% vs. 8.6% p = 0.039, respectively). CONCLUSIONS: Administering steroids prior to antiviral drugs soon after symptom onset can aggravate disease severity. When administration of steroids is considered soon after symptom onset, it may be safer to initiate antiviral drugs first.

Engineering of human induced pluripotent stem cells via human artificial chromosome vectors for cell therapy and disease modeling.

Genetic engineering of induced pluripotent stem cells (iPSCs) holds great promise for gene and cell therapy as well as drug discovery. However, there are potential concerns regarding the safety and control of gene expression using conventional vectors such as viruses and plasmids. Although human artificial chromosome (HAC) vectors have several advantages as a gene delivery vector, including stable episomal maintenance and the ability to carry large gene inserts, the full potential of HAC transfer into iPSCs still needs to be explored. Here, we provide evidence of a HAC transfer into human iPSCs by microcell-mediated chromosome transfer via measles virus envelope proteins for various applications, including gene and cell therapy, establishment of versatile human iPSCs capable of gene loading and differentiation into T cells, and disease modeling for aneuploidy syndrome. Thus, engineering of human iPSCs via desired HAC vectors is expected to be widely applied in biomedical research.

TFEB-mediated endolysosomal activity controls human hematopoietic stem cell fate.

It is critical to understand how human quiescent long-term hematopoietic stem cells (LT-HSCs) sense demand from daily and stress-mediated cues and then transition into bioenergetically active progeny to differentiate and meet these cellular needs. However, the demand-adapted regulatory circuits of these early steps of hematopoiesis are largely unknown. Here we show that lysosomes, sophisticated nutrient-sensing and signaling centers, are regulated dichotomously by transcription factor EB (TFEB) and MYC to balance catabolic and anabolic processes required for activating LT-HSCs and guiding their lineage fate. TFEB-mediated induction of the endolysosomal pathway causes membrane receptor degradation, limiting LT-HSC metabolic and mitogenic activation, promoting quiescence and self-renewal, and governing erythroid-myeloid commitment. In contrast, MYC engages biosynthetic processes while repressing lysosomal catabolism, driving LT-HSC activation. Our study identifies TFEB-mediated control of lysosomal activity as a central regulatory hub for proper and coordinated stem cell fate determination.

The Transition from Quiescent to Activated States in Human Hematopoietic Stem Cells Is Governed by Dynamic 3D Genome Reorganization.

Lifelong blood production requires long-term hematopoietic stem cells (LT-HSCs), marked by stemness states involving quiescence and self-renewal, to transition into activated short-term HSCs (ST-HSCs) with reduced stemness. As few transcriptional changes underlie this transition, we used single-cell and bulk assay for transposase-accessible chromatin sequencing (ATAC-seq) on human HSCs and hematopoietic stem and progenitor cell (HSPC) subsets to uncover chromatin accessibility signatures, one including LT-HSCs (LT/HSPC signature) and another excluding LT-HSCs (activated HSPC [Act/HSPC] signature). These signatures inversely correlated during early hematopoietic commitment and differentiation. The Act/HSPC signature contains CCCTC-binding factor (CTCF) binding sites mediating 351 chromatin interactions engaged in ST-HSCs, but not LT-HSCs, enclosing multiple stemness pathway genes active in LT-HSCs and repressed in ST-HSCs. CTCF silencing derepressed stemness genes, restraining quiescent LT-HSCs from transitioning to activated ST-HSCs. Hence, 3D chromatin interactions centrally mediated by CTCF endow a gatekeeper function that governs the earliest fate transitions HSCs make by coordinating disparate stemness pathways linked to quiescence and self-renewal.