Impact of Straight Stomach Reconstruction on Delayed Gastric Emptying and Nutritional Recovery After Pancreaticoduodenectomy.

BACKGROUND: The aim of this study was to evaluate the effectiveness of a modified reconstruction technique-anchored straight stomach reconstruction-in reducing the incidence of delayed gastric emptying (DGE) after pancreaticoduodenectomy (PD) and its impact on postoperative nutritional recovery. METHODS: A case series analysis of 125 consecutive PD patients was conducted: 104 of them had undergone anchored straight stomach reconstruction (SSR group) and the remaining 21 without (Non-SSR group). The incidence of DGE and the change in postoperative nutritional status (body weight and serum albumin level during 12 months post-surgery) were compared. RESULTS: The incidence of DGE in the SSR group (13%) was significantly lower than that in the Non-SSR group (33%) (P = .018); further the significant DGE (grade B or C) was only 5%. Comparison of nutritional status showed that SSR facilitated a prompt recovery of body weight and serum albumin level at 6 months after PD. At 12 months after surgery, body weight gain was significantly better in the SSR group than in the Non-SSR group (P = .006), and albumin level tended to be higher in the SSR group (P = .071). CONCLUSION: Straight stomach reconstruction is able to reduce DGE in patients after PD and also improves their postoperative nutritional recovery.

Secondary Publication: Proposal for Points of Consideration for Pluripotent Stem Cell Culture.

Human pluripotent stem cells, such as human embryonic stem cells and human induced pluripotent stem cells, are used in basic research and various applied fields, including drug discovery and regenerative medicine. Stem cell technologies have developed rapidly in recent years, and the supply of culture materials has improved. This has facilitated the culture of human pluripotent stem cells and has enabled an increasing number of researchers and bioengineers to access this technology. At the same time, it is a challenge to share the basic concepts and techniques of this technology among researchers and technicians to ensure the reproducibility of research results. Human pluripotent stem cells differ from conventional somatic cells in many aspects, and many points need to be considered in their handling, even for those experienced in cell culture. Therefore, we have prepared this proposal, "Points of Consideration for Pluripotent Stem Cell Culture," to promote the effective use of human pluripotent stem cells. This proposal includes seven items to be considered and practices to be confirmed before using human pluripotent stem cells. These are laws/guidelines and consent/material transfer agreements, diversity of pluripotent stem cells, culture materials, thawing procedure, media exchange and cell passaging, freezing procedure, and culture management. We aim for the concept of these points of consideration to be shared by researchers and technicians involved in the cell culture of pluripotent stem cells. In this way, we hope the reliability of research using pluripotent stem cells can be improved, and cell culture technology will advance.

Vasculature cells control neuroglial co-localization and synaptic connection in a central nervous system tissue mimic system.

Despite the development of neural tissue differentiation methods using a wide variety of stem cells and compartments, there is no standardized strategy for establishing synapses. As the neuronal network is developed in parallel with blood vessel angiogenesis in the central nervous system (CNS) from the embryonic period, we examined neuron-astrocyte-vasculature interactions to understand the effect of the vasculature on the development and stabilization of neurological morphogenesis. We generated a cellular co-culture module targeting the CNS that was embedded in a collagen-based extracellular matrix (ECM) gel. Our neuron-astrocyte-vascular complex module identified the neurological co-localization effect by endothelial cells, as well as the pericyte-induced improvement of synaptic connections. Furthermore, it was suggested that the PDGF, BDNF, IGF, and WNT/BMP pathways were upregulated in synaptic connections enhanced conditions, which are composed of neurexin. These results suggest that the integrity of the vasculature cells in the CNS is important for the establishment of neuronal networks and for synapse connection.

Effects of switching from liraglutide to semaglutide or dulaglutide in patients with type 2 diabetes: A randomized controlled trial.

INTRODUCTION: Few studies have examined the effects of glucagon-like peptide-1 receptor agonist switching, particularly in Japanese patients. Therefore, we aimed to investigate the effects of switching from liraglutide to semaglutide or dulaglutide on blood glucose, body weight, and the occurrence of adverse effects in clinical practice. MATERIALS AND METHODS: This was an open-label, prospective, randomized, parallel-group controlled trial. Patients with type 2 diabetes treated with liraglutide (0.6 or 0.9 mg) at Yokosuka Kyosai Hospital in Japan were recruited from September 2020 to March 2022 and, after obtaining informed consent, randomly assigned to the semaglutide or dulaglutide group (1:1). Changes in the glycated hemoglobin level from baseline to weeks 8, 16, and 26 were evaluated post-treatment. RESULTS: Initially, 32 participants were enrolled, of whom 30 completed the study. Glycemic control was significantly better in the semaglutide group than in the dulaglutide group (-0.42 ± 0.49% vs -0.00 ± 0.34%, P = 0.0120). Body weight significantly decreased in the semaglutide group (-2.6 ± 3.6 kg, P = 0.0153), whereas no change was observed in the dulaglutide group (-0.1 ± 2.7 kg, P = 0.8432). We found a significant difference in body weight between the groups (P = 0.0469). The proportion of participants who reported adverse events was 75.0% and 18.8% in the semaglutide and dulaglutide groups, respectively. One patient in the semaglutide group had difficulty continuing treatment due to severe vomiting and weight loss. CONCLUSIONS: Switching from once-daily liraglutide to once-weekly semaglutide 0.5 mg significantly improved glycemic control and body weight compared with switching to once-weekly dulaglutide 0.75 mg.

Induction of angiogenesis and neural progenitor cells by basic fibroblast growth factor-releasing polyglycolic acid sheet following focal cerebral infarction in mice.

Biodegradable sheets loaded with basic fibroblast growth factor (bFGF) are prepared as novel bFGF-releasing systems from polyglycolic acid nonwoven fabric by oxygen plasma treatment followed by bFGF adsorption. In the present study, we investigated the therapeutic effects of this system on a focal cerebral infarction model (CB-17 mouse). A preliminary in vitro study showed that this system released bFGF in an acellular culture medium, thereby keeping the bFGF concentration in the medium at ≥5 ng/ml for a prolonged period of 7 days. The released bFGF from this system retained its biological activity to enhance endothelial tube formation in vitro. In a mouse model of subacute focal cerebral infarction, this system increased the expression of endogenous vascular endothelial growth factor in the peri-infarct cortex and subventricular zone, promoted angiogenesis in the striatum, and increased neural progenitor cells in the peri-infarct cortex. Thus, this bFGF-releasing system has the potential to be a novel therapeutic approach for cerebral infarction.

Periodic paralysis due to cumulative effects of rare variants in SCN4A with small functional alterations.

INTRODUCTION/AIMS: Mutations in the SCN4A gene encoding a voltage-gated sodium channel (Nav1.4) cause hyperkalemic periodic paralysis (HyperPP) and hypokalemic periodic paralysis (HypoPP). Typically, both HyperPP and HypoPP are considered as monogenic disorders caused by a missense mutation with a large functional effect. However, a few cases with atypical periodic paralysis phenotype have been caused by multiple mutations in ion-channel genes expressed in skeletal muscles. In this study we investigated the underlying pathogenic mechanisms in such cases. METHODS: We clinically assessed two families: proband 1 with HyperPP and proband 2 with atypical periodic paralysis with hypokalemia. Genetic analyses were performed by next-generation sequencing and conventional Sanger sequencing, followed by electrophysiological analyses of the mutant Nav1.4 channels expressed in human embryonic kidney 293T (HEK293T) cells using the whole-cell patch-clamp technique. RESULTS: In proband 1, K880del was identified in the SCN4A gene. In proband 2, K880del and a novel mutation, R1639H, were identified in the same allele of the SCN4A gene. Functional analyses revealed that the K880del in SCN4A has a weak functional effect on hNav1.4, increasing the excitability of the sarcolemma, which could represent a potential pathogenic factor. Although R1639H alone did not reveal functional changes strong enough to be pathogenic, Nav1.4 with both K880del and R1639H showed enhanced activation compared with K880del alone, indicating that R1639H may modify the hNav1.4 channel function. DISCUSSION: A cumulative effect of variants with small functional alterations may be considered as the underpinning oligogenic pathogenic mechanisms for the unusual phenotype of periodic paralysis.

Simultaneous presence of the hepato-spleno-mesenteric trunk, a common trunk for both inferior phrenic arteries, the left gastric artery, and a common hepatic artery that ran behind the portal vein in a patient with gastric cancer.

The celiac artery usually trifurcates into the common hepatic artery, splenic artery, and left gastric artery, but it is known to present several anatomical variations. In such cases, detailed knowledge of the variation is needed preoperatively to safely perform surgery. A 77-year-old woman was referred to our hospital for the treatment of gastric cancer. She had a triple anatomical variation: simultaneous presence of the hepato-spleno-mesenteric trunk, a common trunk for both inferior phrenic arteries and the left gastric artery, and a common hepatic artery that ran behind the portal vein. We detected this variation on routine preoperative multidetector computed tomography angiography, and safely and adequately performed laparoscopic distal gastrectomy.

Storable bFGF-Releasing Membrane Allowing Continuous Human iPSC Culture.

Basic fibroblast growth factor (bFGF) is a crucial supplement for culture media of human pluripotent stem cells. However, bFGF is extremely unstable under cell culture conditions, which makes frequent (generally every day) medium refreshment requisite. We recently developed a water-floatable, bFGF-releasing membrane via a simple bFGF adsorption process following oxygen plasma treatment by utilizing a polyethylene nonwoven fabric as an adsorbent. This membrane allowed sustained release of bFGF while floating on medium, thereby keeping the bFGF concentration in the medium sufficient for maintaining human-induced pluripotent stem cells (iPSCs) in a proliferative and pluripotent state for as long as 3 days. In this study, lyophilization was applied to the membrane to stabilize bFGF. The sustained bFGF-releasing function of the membrane was kept unchanged even after lyophilization and subsequent cryopreservation at -30 °C for 3 months. The cryopreserved membrane supported proliferation and colony formation of human iPSCs while retaining their viability and pluripotency in a medium-change-free continuous culture for 3 days. The present bFGF-releasing membrane is ready-to-use, storable for at least 3 months, and obviates daily medium refreshment. Therefore, it is a new and more practical bFGF supplement for culture media of human stem cells.

Relationship between basal sodium intake and the effects of dapagliflozin in albuminuric diabetic kidney disease.

We investigated the impact of basal dietary sodium intake on the dapagliflozin-induced changes in albuminuria and blood pressure (BP) measured at home in patients with diabetic kidney disease (DKD).This was a secondary analysis of the Y-AIDA Study, in which DKD patients with estimated glomerular filtration rate (eGFR) ≥ 45 ml/min/1.73 m2 and urinary albumin-to-creatinine ratio (UACR) ≥ 30 mg/g creatinine were administered dapagliflozin for 24 weeks, and dapagliflozin significantly improved albuminuria levels and home BP profiles. The effects on UACR, home-measured BP, and eGFR were compared between high- and low-sodium intake groups (HS and LS groups), which were created using baseline urinary sodium-to-creatinine ratio of 84 participants with available basal sodium-to-creatinine ratios. At baseline, clinic-/home-measured BPs, UACR, and eGFR, were comparable in the two groups. After 24 weeks, the reductions from baseline in ln-UACR were comparable in the two groups. In contrast, the reductions in evening home systolic BP and eGFR from baseline were larger in HS than in LS (BP: - 13 ± 2.08 vs. - 6 ± 1.88, P = 0.020; eGFR: - 3.33 ± 1.32 vs. 0.37 ± 1.29, P = 0.049). The home BP-lowering effects of dapagliflozin are larger in HS than LS, concomitant with a larger reduction in eGFR, suggesting a dapagliflozin-induced improvement in glomerular relative hyperfiltration in HS.

The impact of culture dimensionality on behavioral epigenetic memory contributing to pluripotent state of iPS cells.

Three-dimensional (3D) culture platforms have been explored to establish physiologically relevant cell culture environment and permit expansion scalability; however, little is known about the mechanisms underlying the regulation of pluripotency of human induced pluripotent stem cells (hiPSCs). This study elucidated epigenetic modifications contributing to pluripotency of hiPSCs in response to 3D culture. Unlike two-dimensional (2D) monolayer cultures, 3D cultured cells aggregated with each other to form ball-like aggregates. 2D cultured cells expressed elevated levels of Rac1 and RhoA; however, Rac1 level was significantly lower while RhoA level was persisted in 3D aggregates. Compared with 2D monolayers, the 3D aggregates also exhibited significantly lower myosin phosphorylation. Histone methylation analysis revealed remarkable H3K4me3 upregulation and H3K27me3 maintenance throughout the duration of 3D culture; in addition, we observed the existence of naïve pluripotency signatures in cells grown in 3D culture. These results demonstrated that hiPSCs adapted to 3D culture through alteration of the Rho-Rho kinase-phospho-myosin pathway, influencing the epigenetic modifications and transcriptional expression of pluripotency-associated factors. These results may help design culture environments for stable and high-quality hiPSCs.

A technique for removing tumourigenic pluripotent stem cells using rBC2LCN lectin.

INTRODUCTION: Tumourigenesis attributed to residual undifferentiated cells in a graft is considered to be a significant issue in cell therapy using human pluripotent stem cells. To ensure the safety of regenerative medicine derived from pluripotent stem cells, residual undifferentiated cells must be eliminated in the manufacturing process. We previously described the lectin probe rBC2LCN, which binds harmlessly and specifically to the cell surface of human pluripotent stem cells. We report here a technique using rBC2LCN to remove pluripotent cells from a heterogenous population to reduce the chance of teratoma formation. METHODS: We demonstrate a method for separating residual tumourigenic cells using rBC2LCN-bound magnetic beads. This technology is a novel use of their previous discovery that rBC2LCN is a lectin that selectively binds to pluripotent cells. We optimize and validate a method to remove hPSCs from a mixture with human fibroblasts using rBC2LCN-conjugated magnetic beads. RESULTS: Cells with the potential to form teratoma could be effectively eliminated from a heterogeneous cell population with biotin-labelled rBC2LCN and streptavidin-bound magnetic beads. The efficiency was measured by FACS, ddPCR, and animal transplantation, suggesting that magnetic cell separation using rBC2LCN is quite efficient for eliminating hPSCs from mixed cell populations. CONCLUSIONS: The removal of residual tumourigenic cells based on rBC2LCN could be a practical option for laboratory use and industrialisation of regenerative medicine using human pluripotent stem cells.

rBC2LCN lectin as a potential probe of early-stage HER2-positive breast carcinoma.

The recombinant N-terminal domain of BC2L-C lectin (rBC2LCN) is useful for detecting not only human pluripotent stem cells but also some cancers. However, the cancer types and stages that can be detected by rBC2LCN remain unclear. In this study, we identified the human breast carcinoma subtypes and stages that can be detected by rBC2LCN. Compared with rBC2LCN-negative breast carcinoma cell lines, the rBC2LCN-positive cells expressed higher levels of human epidermal growth factor receptor 2 (HER2) and epithelial marker genes. Importantly, rBC2LCN histochemical staining of human breast carcinoma tissues demonstrated the utility of rBC2LCN in detecting breast carcinoma types that express HER2 and have not spread much in the early phase of growth. We conclude that rBC2LCN may have potential as a detection probe and a drug delivery vehicle to identify and treat early-stage HER2-positive breast carcinoma.

The effect of long-term past glycemic control on executive function among patients with type 2 diabetes mellitus.

OBJECTIVE: Patients with type 2 diabetes mellitus (T2DM) show more executive dysfunction than nondiabetics. However, how long poor glycemic control affects executive function remains unclear. Thus, we aimed to investigate the relationships in a cross-sectional study. METHODS: We studied 118 T2DM outpatients (age, ≥ 60 years; excluding history of stroke, dementia and severe hypoglycemia). HbA1c values were recorded every ≤ 12 weeks for ≥ 5 years. All patients underwent verbal-fluency tests (reflecting executive function) and Mini-Mental State Examination (MMSE). The correlation between past glycemic control values and both cognitive tests scores was investigated. As markers of past glycemic control, we used average hemoglobin A1c (HbA1c) values and glycemic control variability [coefficient of variation (CV) of HbA1c values (HbA1c-CV)]. RESULTS: Verbal-fluency tests scores correlated with HbA1c-CV, but not with average HbA1c values, after adjusting for age, years of education and sex. Verbal-fluency tests scores correlated with HbA1c-CV for the past 5 years, best compared with HbA1c-CV for past < 5 years. MMSE scores were also related to only HbA1c-CV for the past 3 years in an adjustment model. CONCLUSIONS: Five-year HbA1c variability affected executive function in T2DM patients, but not average HbA1c values. Long-term longitudinal studies may be required.

Application of a human mesoderm tissue elongation system in vitro derived from human induced pluripotent stem cells to risk assessment for teratogenic chemicals.

Toxic compounds from the mother's diet and medication in addition to genetic factors and infection during pregnancy remain risks for various congenital disorders and misbirth. To ensure the safety of food and drugs for pregnant women, establishment of an in vitro system that morphologically resembles human tissues has been long desired. In this study, we focused on dorsal mesoderm elongation, one of the critical early development events for trunk formation, and we established in vitro autonomous elongating tissues from human induced pluripotent stem cells (hiPSCs). This artificial tissue elongation is regulated by MYOSIN II and FGF signaling, and is diminished by methylmercury or retinoic acid (RA), similar to in vivo human developmental disabilities. Moreover, our method for differentiation of hiPSCs requires only a short culture period, and the elongation is cell number-independent. Therefore, our in vitro human tissue elongation system is a potential tool for risk assessment assays for identification of teratogenic chemicals via human tissue morphogenesis.

Template Activating Factor-I α Regulates Retroviral Silencing during Reprogramming.

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) is accompanied by dramatic changes in epigenetic programs, including silencing of endogenous and exogenous retroviruses. Here, we utilized replication-defective and persistent Sendai virus (SeVdp)-based vectors to monitor retroviral silencing during reprogramming. We observed that retroviral silencing occurred at an early reprogramming stage without a requirement for KLF4 or the YY1-binding site in the retroviral genome. Insertional chromatin immunoprecipitation (iChIP) enabled us to isolate factors assembled on the silenced provirus, including components of inhibitor of histone acetyltransferase (INHAT), which includes the SET/TAF-I oncoprotein. Knockdown of SET/TAF-I in mouse embryonic fibroblasts (MEFs) diminished retroviral silencing during reprogramming, and overexpression of template activating factor-I α (TAF-Iα), a SET/TAF-I isoform predominant in embryonic stem cells (ESCs), reinforced retroviral silencing by an SeVdp-based vector that is otherwise defective in retroviral silencing. Our results indicate an important role for TAF-Iα in retroviral silencing during reprogramming.

The beneficial effects of a muscarinic agonist on pancreatic ß-cells.

The brain and nervous system play an important role in pancreatic ß-cell function. This study investigated the role of muscarinic agonists or acetylcholine, which is the major neurotransmitter in the vagal nerve, in regulating pancreatic ß-cell mass and glucose homeostasis. Administration of the muscarinic agonist bethanechol increased insulin secretion and improved glucose tolerance in insulin-receptor substrate 2 (IRS2)-knockout (IRS-2-/-) mice and diet-induced obesity mice. Oral administration of bethanechol increased ß-cell mass and proliferation in wild-type mice, but not IRS-2-/- mice. The muscarinic agonist also increased the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into islets isolated from wild-type mice and pancreatic ß-cell line MIN6. The phosphorylation of protein kinase B (Akt) induced by oral administration of bethanechol was observed in wild-type mice, but not IRS-2-/- mice. The secretion of glucagon-like peptide-1 (GLP-1) was also stimulated by bethanechol in wild-type mice, and a GLP-1 antagonist partially inhibited the bethanechol-induced increase in ß-cell mass. These results suggest that the muscarinic agonist exerted direct and indirect effects on ß-cell proliferation that were dependent on the IRS-2/Akt pathway. The bethanechol-stimulated release of GLP-1 may be indirectly associated with ß-cell proliferation.

Independent evolution for sex determination and differentiation in the DMRT family in animals.

Some DMRT family genes including arthropod dsx, nematode mab-3, and vertebrate dmrt1 are involved in sex determination and/or differentiation in bilaterian animals. Although there have been some reports about evolutionary analyses of the family by using its phylogenetic trees, it is still undecided as to whether these three sex determination-related genes share orthologous relationships or not. To clarify this question, we analyzed evolutional relationships among the family members in various bilaterians by using not only phylogenetic tree analysis, but also synteny analysis. We found that only four genes, dmrt2a/2b, dmrt3, dmrt4/ 5 and dmrt93B were commonly present in invertebrate bilateria. The syntenies of dmrt2a/2b-dmrt3 and dmrt4/5-dmrt93B are conserved before and after two rounds of whole genome duplication in the ancestral vertebrate. Importantly, this indicates that dmrt1 must have appeared in the common vertebrate ancestor. In addition, dmrt1, dsx, or mab-3 formed each different cluster at a distance in our phylogenetic tree. From these findings, we concluded that the three sex determination-related genes, dmrt1, dsx, and mab-3 have no orthologous relationships, and suggested independent evolution for sex determination and differentiation in the DMRT gene family. Our results may supply clues about why sex-determining systems have diverged during animal evolution.

Improved home BP profile with dapagliflozin is associated with amelioration of albuminuria in Japanese patients with diabetic nephropathy: the Yokohama add-on inhibitory efficacy of dapagliflozin on albuminuria in Japanese patients with type 2 diabetes study (Y-AIDA study).

BACKGROUND: The Y-AIDA study was designed to investigate the renal- and home blood pressure (BP)-modulating effects of add-on dapagliflozin treatment in Japanese individuals with type 2 diabetes mellitus (T2DM) and albuminuria. METHODS: We conducted a prospective, multicenter, single-arm study. Eighty-six patients with T2DM, HbA1c 7.0-10.0%, estimated glomerular filtration rate (eGFR) ≥ 45 mL/min/1.73 m2, and urine albumin-to-creatinine ratio (UACR) ≥ 30 mg/g creatinine (gCr) were enrolled, and 85 of these patients were administered add-on dapagliflozin for 24 weeks. The primary and key secondary endpoints were change from baseline in the natural logarithm of UACR over 24 weeks and change in home BP profile at week 24. RESULTS: Baseline median UACR was 181.5 mg/gCr (interquartile range 47.85, 638.0). Baseline morning, evening, and nocturnal home systolic/diastolic BP was 137.6/82.7 mmHg, 136.1/79.3 mmHg, and 125.4/74.1 mmHg, respectively. After 24 weeks, the logarithm of UACR decreased by 0.37 ± 0.73 (P < 0.001). In addition, changes in morning, evening, and nocturnal home BP from baseline were as follows: morning systolic/diastolic BP - 8.32 ± 11.42/- 4.18 ± 5.91 mmHg (both P < 0.001), evening systolic/diastolic BP - 9.57 ± 12.08/- 4.48 ± 6.45 mmHg (both P < 0.001), and nocturnal systolic/diastolic BP - 2.38 ± 7.82/- 1.17 ± 5.39 mmHg (P = 0.0079 for systolic BP, P = 0.0415 for diastolic BP). Furthermore, the reduction in UACR after 24 weeks significantly correlated with an improvement in home BP profile, but not with changes in other variables, including office BP. Multivariate linear regression analysis also revealed that the change in morning home systolic BP was a significant contributor to the change in log-UACR. CONCLUSIONS: In Japanese patients with T2DM and diabetic nephropathy, dapagliflozin significantly improved albuminuria levels and the home BP profile. Improved morning home systolic BP was associated with albuminuria reduction. Trial registration The study is registered at the UMIN Clinical Trials Registry (UMIN000018930; http://www.umin.ac.jp/ctr/index-j.htm ). The study was conducted from July 1, 2015 to August 1, 2018.

mRNA and miRNA expression profiles in an ectoderm-biased substate of human pluripotent stem cells.

The potential applications of human pluripotent stem cells, embryonic stem (ES) cells, and induced pluripotent stem (iPS) cells in cell therapy and regenerative medicine have been widely studied. The precise definition of pluripotent stem cell status during culture using biomarkers is essential for basic research and regenerative medicine. Culture conditions, including extracellular matrices, influence the balance between self-renewal and differentiation. Accordingly, to explore biomarkers for defining and monitoring the pluripotent substates during culture, we established different substates in H9 human ES cells by changing the extracellular matrix from vitronectin to Matrigel. The substate was characterised by low and high expression of the pluripotency marker R-10G epitope and the mesenchymal marker vimentin, respectively. Immunohistochemistry, induction of the three germ layers, and exhaustive expression analysis showed that the substate was ectoderm-biased, tended to differentiate into nerves, but retained the potential to differentiate into the three germ layers. Further integrated analyses of mRNA and miRNA microarrays and qPCR analysis showed that nine genes (COL9A2, DGKI, GBX2, KIF26B, MARCH1, PLXNA4, SLC24A4, TLR4, and ZHX3) were upregulated in the ectoderm-biased cells as ectoderm-biased biomarker candidates in pluripotent stem cells. Our findings provide important insights into ectoderm-biased substates of human pluripotent stem cells in the fields of basic research and regenerative medicine.