Heterologous survey of 130 DNA transposons in human cells highlights their functional divergence and expands the genome engineering toolbox.

Experimental studies on DNA transposable elements (TEs) have been limited in scale, leading to a lack of understanding of the factors influencing transposition activity, evolutionary dynamics, and application potential as genome engineering tools. We predicted 130 active DNA TEs from 102 metazoan genomes and evaluated their activity in human cells. We identified 40 active (integration-competent) TEs, surpassing the cumulative number (20) of TEs found previously. With this unified comparative data, we found that the Tc1/mariner superfamily exhibits elevated activity, potentially explaining their pervasive horizontal transfers. Further functional characterization of TEs revealed additional divergence in features such as insertion bias. Remarkably, in CAR-T therapy for hematological and solid tumors, Mariner2_AG (MAG), the most active DNA TE identified, largely outperformed two widely used vectors, the lentiviral vector and the TE-based vector SB100X. Overall, this study highlights the varied transposition features and evolutionary dynamics of DNA TEs and increases the TE toolbox diversity.

Dual Structural Design of Platinum-Nickel Hydrogels for Wearable Glucose Biosensing with Ultrahigh Stability.

Wearable glucose sensors are of great significance and highly required in mobile health monitoring and management but suffering from limited long-term stability and wearable adaptability. Here a simultaneous component and structure engineering strategy is presented, which involves Pt with abundant Ni to achieve three-dimensional, dual-structural Pt-Ni hydrogels with interconnected networks of PtNi nanowires and Ni(OH)2 nanosheets, showing prominent electrocatalytic activity and stability in glucose oxidation under neutral condition. Specifically, the PtNi(1:3) dual hydrogels shows 2.0 and 270.6 times' activity in the glucose electro-oxidation as much as the pure Pt and Ni hydrogels. Thanks to the high activity, structural stability, good flexibility, and self-healing property, the PtNi(1:3) dual gel-based non-enzymatic glucose sensing chip is endowed with high performance. It features a high sensitivity, an excellent selectivity and flexibility, and particularly an outstanding long-term stability over 2 months. Together with a pH sensor and a wireless circuit, an accurate, real-time, and remote monitoring of sweat glucose is achieved. This facile design of novel dual-structural metallic hydrogels sheds light to rationally develop new functional materials for high-performance wearable biosensors.

Effect of mattress bedding layer structure on pressure relief performance and subjective lying comfort.

Mattresses' pressure relief performance and comfort largely affect sleep quality. Mattress filling materials have been proven to affect the interface pressure distribution and comfort, but the effect of mattress structure is unclear. In this paper, the interface pressure distribution and subjective comfort of 10 subjects were assessed in the different bedding layer structures of mattresses, after mattress support performance was tested. The results show that the mattresses with bedding material hardness gradually increasing from the top layer to the bottom layer (BMH-ITTB) structure have a softer surface layer, a better support core layer, and higher fitness. This enables the mattress to achieve a better decompression effect. The low-pressure area (PAI≤0.67kPa) increased, while the high-pressure area (PAI≥2.67kPa and PAI≥4.00kPa), maximum pressure (P95), average pressure (P50), and pressure index (PI) decreased. This also enables the mattress to achieve higher subjective comfort scores.

New insight into the effect of riluzole on cadmium tolerance and accumulation in duckweed (Lemna turionifera).

Cadmium (Cd) damages plant photosynthesis, affects roots and leaves growth, and triggers molecular responses. Riluzole (RIL), which protected neuronal damage via inhibiting excess Glu release in animals, has been found to improve Cd tolerance in duckweed in this study. Firstly, RIL treatment alleviated leaf chlorosis by protecting chlorophyll and decreased root abscission under Cd stress. Secondly, RIL declines Cd accumulation by alleviating excess Glu release during Cd shock. RIL mitigate Glu outburst in duckweed during Cd stress by a decline in Glu in roots. The Cd2+ influx was repressed by RIL addition with Cd shock. Finally, differentially expressed genes (DEGs) of duckweed under Cd stress with RIL have been investigated. 2141 genes were substantially up-regulated and 3282 genes were substantially down-regulated with RIL addition. RIL down-regulates the genes related to the Glu synthesis, and genes related to DNA repair have been up-regulated with RIL treatment under Cd stress. These results provide new insights into the possibility of RIL to reduce Cd accumulation and increase Cd tolerance in duckweed, and lay the foundation for decreasing Cd accumulation in crops.

Long noncoding RNA GAS5 alleviates the inflammatory response of human periodontal ligament stem cells by regulating the NF-κB signalling pathway.

OBJECTIVES: This study investigated the role of lncRNA growth arrest-specific transcript 5 (GAS5) in the inflammatory response of periodontal ligament stem cells (PDLSCs) during periodontitis with attempts to its possible mechanisms. MATERIALS AND METHODS: Gingiva samples were collected from healthy people and patients with periodontitis. The ligature-induced periodontitis model was established in mice. Cell transfection was utilized to knock down and overexpress GAS5 in PDLSCs. Quantitative real-time polymerase chain reaction (qRT-PCR) and fluorescence in situ hybridization were performed to detect the GAS5 expression. In combination with high-throughput sequencing technology, qRT-PCR, Western blotting, and immunofluorescence were performed to detect the effects of GAS5 on cytokines and proteins in the NF-κB pathway. RESULTS: GAS5 expression decreased in PDLSCs subjected to compressive force. GAS5 expression was downregulated in the gingiva tissues from patients with periodontitis. Consistent with the results of clinical samples, GAS5 expression decreased in the mouse ligature-induced periodontitis model. GAS5 expression was downregulated in PDLSCs under tumour necrosis factor (TNF)-α stimulation. Knockdown and overexpression of GAS5 increased and decreased the expression of cytokines induced by TNF-α in PDLSCs, respectively. The sequencing results showed that overexpressing GAS5 was related to genes in the NF-κB pathway. Overexpressing GAS5 alleviated p65 phosphorylation and inhibited the entry of p65 into the nucleus in the TNF-α activated NF-κB pathway, whereas GAS5 knockdown resulted in contrasting results. CONCLUSIONS: GAS5 alleviated the expression of cytokines in PDLSCs by inhibiting activation of the TNF-α-mediated NF-κB pathway. These findings provide new insight into the regulation of the PDLSCs inflammation response.

Linking oxidative DNA lesion 8-OxoG to tumor development and progression.

Cells of the aerobic metabolic organism are inevitably subjected to the damage from reactive oxygen species (ROS). ROS cause multiple forms of DNA damage, among which the oxidation product of guanine G 8-hydroxyguanine (8-oxoG) is the most frequent DNA oxidative damage, recognized by the specific glycosidase OGG1 that initiates the base excision repair pathway. If left unrepaired, 8-oxoG may pair with A instead of C, leading to a mutation of G: C to T: A during replication. Thus, the accumulation of 8-oxoG or the abnormal OGG1 repair is thought to affect gene function, which in turn leads to the development of tumor or aging-related diseases. However, a series of recent studies have shown that 8-oxoG tends to be produced in regulatory regions of the genome. 8-oxoG can be regarded as an epigenetic modification, while OGG1 is a specific reader of this information. Substrate recognition, binding or resection by OGG1 can cause DNA conformation changes or affect histone modifications, causing up-regulation or down-regulation of genes with different properties. Thus, in addition to the potential genotoxicity, the association of guanine oxidative damage with development of tumors is closely related to its aberrant initiation of gene expression through epigenetic mechanisms. In this review, we summarize the underlying mechanism of 8-oxoG and repair enzyme OGG1 in tumor development and progression, with aims to interpret the relationship between DNA oxidative damage and tumor from a new perspective, and provide new ideas and targets for tumor treatment.

Enzymatically inactive OGG1 binds to DNA and steers base excision repair toward gene transcription.

8-Oxoguanine DNA glycosylase1 (OGG1)-initiated base excision repair (BER) is the primary pathway to remove the pre-mutagenic 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Recent studies documented 8-oxoG serves as an epigenetic-like mark and OGG1 modulates gene expression in oxidatively stressed cells. For this new role of OGG1, two distinct mechanisms have been proposed: one is coupled to base excision, while the other only requires substrate binding of OGG1--both resulting in conformational adjustment in the adjacent DNA sequences providing access for transcription factors to their cis-elements. The present study aimed to examine if BER activity of OGG1 is required for pro-inflammatory gene expression. To this end, Ogg1/OGG1 knockout/depleted cells were transfected with constructs expressing wild-type (wt) and repair-deficient mutants of OGG1. OGG1's promoter enrichment, oxidative state, and gene expression were examined. Results showed that TNFα exposure increased levels of oxidatively modified cysteine(s) of wt OGG1 without impairing its association with promoter and facilitated gene expression. The excision deficient K249Q mutant was even a more potent activator of gene expression; whereas, mutant OGG1 with impaired substrate recognition/binding was not. These data suggested the interaction of OGG1 with its substrate at regulatory regions followed by conformational adjustment in the adjacent DNA is the primary mode to modulate inflammatory gene expression.

METTL3-mediated m6A modification is required for cerebellar development.

N6-methyladenosine (m6A) RNA methylation is the most abundant modification on mRNAs and plays important roles in various biological processes. The formation of m6A is catalyzed by a methyltransferase complex including methyltransferase-like 3 (METTL3) as a key factor. However, the in vivo functions of METTL3 and m6A modification in mammalian development remain unclear. Here, we show that specific inactivation of Mettl3 in mouse nervous system causes severe developmental defects in the brain. Mettl3 conditional knockout (cKO) mice manifest cerebellar hypoplasia caused by drastically enhanced apoptosis of newborn cerebellar granule cells (CGCs) in the external granular layer (EGL). METTL3 depletion-induced loss of m6A modification causes extended RNA half-lives and aberrant splicing events, consequently leading to dysregulation of transcriptome-wide gene expression and premature CGC death. Our findings reveal a critical role of METTL3-mediated m6A in regulating the development of mammalian cerebellum.

Multi-domain Features of the Non-phase-locked Component of Interest Extracted from ERP Data by Tensor Decomposition.

The waveform in the time domain, spectrum in the frequency domain, and topography in the space domain of component(s) of interest are the fundamental indices in neuroscience research. Despite the application of time-frequency analysis (TFA) to extract the temporal and spectral characteristics of non-phase-locked component (NPLC) of interest simultaneously, the statistical results are not always expectedly satisfying, in that the spatial information is not considered. Complex Morlet wavelet transform is widely applied to TFA of event-related-potential (ERP) data, and mother wavelet (which should be firstly defined by center frequency and bandwidth (CFBW) before using the method to TFA of ERP data) influences the time-frequency results. In this study, an optimal set of CFBW was firstly selected from the number sets of CFBW, to further analyze for TFA of the ERP data in a cognitive experiment paradigm of emotion (Anger and Neutral) and task (Go and Nogo). Then tensor decomposition algorithm was introduced to investigate the NPLC of interest from the fourth-order tensor. Compared with the TFA results which only revealed a significant difference between Go and Nogo task condition, the tensor-based analysis showed significant interaction effect between emotion and task. Moreover, significant differences were found in both emotion and task conditions through tensor decomposition. In addition, the statistical results of TFA would be affected by the selected region of interest (ROI), whereas those of the proposed method were not subject to ROI. Hence, this study demonstrated that tensor decomposition method was effective in extracting NPLC, by considering spatial information simultaneously as the potential to explore the brain mechanisms related to experimental design.

The evolution of left ventricular pseudoaneurysm from the rupture of left ventricular free wall following acute myocardial infarction: a case report.

BACKGROUND: Left ventricular pseudoaneurysm is a very rare complication following acute myocardial infarction, which results from a free wall rupture. Hemopericardium and cardiac tamponade caused by rupture of the free wall after acute myocardial infarction are often fatal. It is difficult to fully document the evolution of left ventricular pseudoaneurysm resulted from acute myocardial infarction with conservative treatment. CASE PRESENTATION: Herein, we followed a 75-year-old female patient for 3 years. Recorded the evolution of the disease: acute lateral myocardial infarction - emergency reperfusion therapy - cardiac rupture - positive successful rescue - the pseudoaneurysm formation - maintaining conservative treatment - gradual enlargement of the pseudoaneurysm - thrombosis in pseudoaneurysm - thrombus filling with pseudoaneurysm - finally stabilized condition - the treatment of coronary revascularization. CONCLUSIONS: This case is reported here because of its scarcity, which provides provides us with a complete record of the entire evolution and an astonishing indication of the long-term prognosis of non-surgical treatment for pseudoventricular.

Weighted gene co-expression network analysis revealed key biomarkers associated with the diagnosis of hypertrophic cardiomyopathy.

OBJECTIVE: To reveal the molecular mechanism underlying the pathogenesis of HCM and find new effective therapeutic strategies using a systematic biological approach. METHODS: The WGCNA algorithm was applied to building the co-expression network of HCM samples. A sample cluster analysis was performed using the hclust tool and a co-expression module was constructed. The WGCNA algorithm was used to study the interactive connection between co-expression modules and draw a heat map to show the strength of interactions between modules. The genetic information of the respective modules was mapped to the associated GO terms and KEGG pathways, and the Hub Genes with the highest connectivity in each module were identified. The Wilcoxon test was used to verify the expression level of hub genes between HCM and normal samples, and the "pROC" R package was used to verify the possibility of hub genes as biomarkers. Finally, the potential functions of hub genes were analyzed by GSEA software. RESULTS: Seven co-expression modules were constructed using sample clustering analysis. GO and KEGG enrichment analysis judged that the turquoise module is an important module. The hub genes of each module are RPL35A for module Black, FH for module Blue, PREI3 for module Brown, CREB1 for module Green, LOC641848 for module Pink, MYH7 for module Turquoise and MYL6 for module Yellow. The results of the differential expression analysis indicate that MYH7 and FH are considered true hub genes. In addition, the ROC curves revealed their high diagnostic value as biomarkers for HCM. Finally, in the results of the GSEA analysis, MYH7 and FH highly expressed genes were enriched with the "proteasome" and a "PPAR signaling pathway," respectively. CONCLUSIONS: The MYH7 and FH genes may be the true hub genes of HCM. Their respective enriched pathways, namely the "proteasome" and the "PPAR signaling pathway," may play an important role in the development of HCM.

The establishment of methods for free PAR generation and PAR reader detection.

Poly (ADP-ribose) polymerase 1 (PARP1) is a DNA damage sensor that catalyzes the poly (ADP-ribose) (PAR) onto a variety of target proteins, such as histones, DSB repair factors and PARP1 itself under consumption of NAD+. Besides, PARP1 can affect a variety of proteins in noncovalent modification manner to carry out specific cellular functions. Here, we established a method to generate non-radiolabeled free PAR by PARG moderately cleaving PAR from autoPARylated PARP1, and utilized dot-blot assay to determine the interaction between free PAR and interested proteins. The methods to generate free PAR and detect the noncovalent interactions between proteins and free PAR are nonradioactive and convenient, which will facilitate the studies to explore the significance of PAR reading in various biological processes.

Decreasing the Carrier Concentration of ZrNiSn: An Opposite Way to the Best N-Type Half-Heusler Thermoelectrics.

N-type ZrNiSn-based alloys reach a record thermoelectric figure of merit zT ≈1.2 by increasing the carrier concentration to 4-5 × 1020 cm-3 . In this work, It is reported that a comparable zT can also be realized in trace Ru-doped ZrNiSn-based alloy at even lower temperature by decreasing the carrier concentration. Compared to the previously reported Co doping, the doping of Ru results in a more effective reduction in carrier concentration, and thus higher Seebeck coefficient, lower electronic thermal conductivity, and enhanced thermoelectric performance. The electronic specific heat coefficient of the ZrNi1- x Rux Sn sample remains constant with increasing Ru content, indicating no obvious change in the density of states effective mass. Theoretical calculations show that the doping of Ru has negligible effect on the bottom of conduction band. The lattice thermal conductivity is further reduced by alloying Ti and Hf at the Zr site, and the bipolar diffusion is suppressed by doping of 0.5 at.% Sb. As a result, Ti0.25 Zr0.5 Hf0.25 Ni0.99 Ru0.01 Sn0.995 Sb0.005 reaches not only a zT value of 1.1 at 773 K but also a record average zT value of 0.8 in 300 to 873 K, demonstrating the effectiveness of trace Ru doping on boosting the thermoelectric performance of ZrNiSn-based alloys.

Metagenomic next-generation sequencing for diagnosis of infectious encephalitis and meningitis: a retrospective study of 90 patients.

BACKGROUND: Infections of the central nervous system (CNS) are potentially life-threatening and can cause serious morbidity. We evaluated the clinical value of metagenomic next-generation sequencing (mNGS) in the diagnosis of infectious encephalitis and meningitis and explored the factors affecting the results of mNGS. METHODS: Patients with suspected cases of encephalitis or meningitis who presented in Northern Jiangsu People's Hospital from 1 March 2018 to 30 September 2022 were collected. Demographic, historical, and clinical information were obtained, and cerebrospinal fluid (CSF) samples were treated with mNGS. The pathogen was identified using National Center for Biotechnology Information (NCBI) GenBank sequence data. RESULTS: Ninety-six patients were screened and finally 90 subjects enrolled. Of the 90 enrolled cases, 67 (74.4%) were diagnosed with central nervous system infections, which included 48 cases (71.6%) of viral infection, 11 (12.2%) of bacterial infection, 5 (7.5%) of mycobacterium tuberculosis, 2 (3.0%) of fungal infection, and 1 (1.5%) of rickettsia infection. From these cases, mNGS identified 40 (44.4%) true-positive cases, 3 (3.3%) false-positive case, 22 (24.4%) true-negative cases, and 25 (27.8%) false-negative cases. The sensitivity and specificity of mNGS were 61.5% and 88%, respectively. mNGS of CSF could show a higher positive rate in patients with marked CSF abnormalities, including elevated protein concentrations and monocyte counts. CONCLUSION: mNGS of CSF is an effective method for detecting infectious encephalitis and meningitis, and the results should be analyzed combined with conventional microbiological testing results.

Parkinson's disease patients with absence of normal dipping status were more vulnerable to cognitive impairment from the early stages.

INTRODUCTION: The objective of this study was to determine the characteristics of cognitive function in Parkinson's disease (PD) patients with different dipping statuses. METHODS: Consecutive PD patients were recruited for this study. All participants underwent 24-h ambulatory blood pressure monitor (ABPM). Corresponding scales were employed to evaluate both motor and non-motor symptoms. The subjects were categorized into reverse, reduced, normal, and extreme dipping groups based on dipping patterns. Additionally, they were divided into early and non-early stage groups according to the disease duration being more than 5 years. RESULTS: The proportions of the four dipping groups in the early and non-early stage groups exhibited no significant differences. The Montreal Cognitive Assessment (MoCA) scores in the reverse group were significantly lower than those in the normal dipping group (16.2 ± 5.8 vs 21.1 ± 6.1,P = 0.003). The attention as well as delayed recall scores in the reverse dipping group were significant lower than those in the normal dipping group (P = 0.042; P < 0.001). The multivariate linear regression analysis revealed that absence of normal dipping was an independent risk factor (OR = -2.252; P = 0.027) for MoCA scores for PD patients. CONCLUSIONS: PD patients with absence of normal dipping status were more vulnerable to cognitive impairment from the early stages of the disease. The 24-h ABPM is recommended for early detection of abnormal dipping status and identification of individuals at risk for cognitive decline.

'Off the shelf' immunotherapies: Generation and application of pluripotent stem cell-derived immune cells.

In recent years, great strides have been made toward the development of immune cell-based therapies in the treatment of refractory malignancies. Primary T cells and NK cells armed with chimeric antigen receptors have achieved tremendous clinical success especially in patients with leukaemia and lymphoma. However, the autologous origin of these effector cells means that a single batch of laboriously engineered cells treats only a certain patient, leading to high cost, ununiform product quality, and risk of delay in treatment, and therefore results in restricted accessibility of these therapies to the overwhelming majority of the patients. Addressing these tricky obstacles calls for the development of universal immune cell products that can be provided 'off the shelf' in a large amount. Pluripotent stem cells (PSCs), owing to their unique capacity of self-renewal and the potential of multi-lineage differentiation, offer an unlimited cell source to generate uniform and scalable engineered immune cells. This review discusses the major advances in the development of PSC-derived immune cell differentiation approaches and their therapeutic potential in treating both hematologic malignancies and solid tumours. We also consider the potency of PSC-derived immune cells as an alternative therapeutic strategy for other diseases, such as autoimmune diseases, fibrosis, infections, et al.

Portable visual and electrochemical detection of hydrogen peroxide release from living cells based on dual-functional Pt-Ni hydrogels.

It is important to monitor the intra-/extracellular concentration of hydrogen peroxide (H2O2) in biological processes. However, miniaturized devices that enable portable and accurate H2O2 measurement are still in their infancy because of the difficulty of developing facile sensing strategies and highly integrated sensing devices. In this work, portable H2O2 sensors based on Pt-Ni hydrogels with excellent peroxidase-like and electrocatalytic activities are demonstrated. Thus, simple and sensitive H2O2 sensing is achieved through both colorimetric and electrochemical strategies. The as-fabricated H2O2 sensing chips exhibit favorable performance, with low detection limits (0.030 µM & 0.15 µM), wide linearity ranges (0.10 µM-10.0 mM & 0.50 µM-5.0 mM), outstanding long-term stability (up to 60 days), and excellent selectivity. With the aid of an M5stack development board, portable visual and electrochemical H2O2 sensors are successfully constructed without complicated and expensive equipment or professional operators. When applied to the detection of H2O2 released from HeLa cells, the results obtained by the developed sensors are in good agreement with those from an ultraviolet‒visible spectrophotometer (UV‒vis) (1.97 µM vs. 2.08 µM) and electrochemical station (1.77 µM vs. 1.84 µM).

High-efficiency targeted transgene integration via primed micro-homologues.

Due to the difficulties in precisely manipulating DNA repair pathways, high-fidelity targeted integration of large transgenes triggered by double-strand breaks is inherently inefficient. Here, we exploit prime editors to devise a robust knock-in (KI) strategy named primed micro-homologues-assisted integration (PAINT), which utilizes reverse-transcribed single-stranded micro-homologues to boost targeted KIs in different types of cells. The improved version of PAINT, designated PAINT 3.0, maximizes editing efficiency and minimizes off-target integration, especially in dealing with scarless in-frame KIs. Using PAINT 3.0, we target a reporter transgene into housekeeping genes with editing efficiencies up to 80%, more than 10-fold higher than the traditional homology-directed repair method. Moreover, the use of PAINT 3.0 to insert a 2.5-kb transgene achieves up to 85% KI frequency at several therapeutically relevant genomic loci, suggesting its potential for clinical applications. Finally, PAINT 3.0 enables high-efficiency non-viral genome targeting in primary T cells and produces functional CAR-T cells with specific tumor-killing ability. Thus, we establish that the PAINT method is a powerful gene editing tool for large transgene integrations and may open new avenues for cell and gene therapies and genome writing technologies.

High-Strength Cell Sheets and Vigorous Hydrogels from Mesenchymal Stem Cells Derived from Human Embryonic Stem Cells.

Natural cell derivates, including cell sheets (CSs) and matrix gels, have opened new opportunities to probe questions in tissue engineering and regenerative medicine. However, the potential of CSs and hydrogels generated by current protocols is still limited by the challenges of heterogeneity and weak mechanical properties. Here, we developed a 21 day long-term serum-free culture system for human embryonic stem cell (hESC)-derived immunity-and-matrix-regulatory cells (IMRCs). The CSs formed with IMRCs (IMRC-CSs) have a much greater secretion capacity for the extracellular matrix (ECM) and stronger mechanical properties than umbilical cord-derived MSCs, with a ten thousand-fold increase in elastin, a higher elastic modulus of 1500 kPa, a thicker structure of 20.59 µm, and a higher fiber count per square millimeter. The IMRC-CSs could promote corneal chemical injury repair and could be turned into injectable temperature-sensitive hydrogels for uterine adhesion repair via a decellularization process. In summary, we have established a high-strength CS platform using human pluripotent stem cells for the first time, providing a facile and scalable engineering approach for regenerative medicine.

A scalable culture system incorporating microcarrier for specialised mesenchymal stem cells from human embryonic stem cells.

Mesenchymal stromal cells (MSCs) derived from human embryonic stem cells (hESCs) are a desirable cell source for cell therapy owing to their capacity to be produced stably and homogeneously in large quantities. However, a scalable culture system for hPSC-derived MSCs is urgently needed to meet the cell quantity and quality requirements of practical clinical applications. In this study, we developed a new microcarrier with hyaluronic acid (HA) as the core material, which allowed scalable serum-free suspension culture of hESC-derived MSCs (IMRCs). We used optimal microcarriers with a coating collagen concentration of 100 â€‹µg/mL or concave-structured surface (cHAMCs) for IMRC amplification in a stirred bioreactor, expanding IMRCs within six days with the highest yield of over one million cells per milliliter. In addition, the harvested cells exhibited high viability, immunomodulatory and regenerative therapeutic promise comparable to monolayer cultured MSCs while showing more increased secretion of extracellular matrix (ECM), particularly collagen-related proteins. In summary, we have established a scalable culture system for hESC-MSCs, providing novel approaches for future cell therapies.