Understanding the intricacies of cellular senescence in atherosclerosis: Mechanisms and therapeutic implications.

Cardiovascular disease is currently the largest cause of mortality and disability globally, surpassing communicable diseases, and atherosclerosis is the main contributor to this epidemic. Aging is intimately linked to atherosclerosis development and progression, however, the mechanism of aging in atherosclerosis is not well known. To emphasize the significant research on the involvement of senescent cells in atherosclerosis, we begin by outlining compelling evidence that indicates various types of senescent cells and SASP factors linked to atherosclerotic phenotypes. We subsequently provide a comprehensive summary of the existing knowledge, shedding light on the intricate mechanisms through which cellular senescence contributes to the pathogenesis of atherosclerosis. Further, we cover that senescence can be identified by both structural changes and several senescence-associated biomarkers. Finally, we discuss that preventing accelerated cellular senescence represents an important therapeutic potential, as permanent changes may occur in advanced atherosclerosis. Together, the review summarizes the relationship between cellular senescence and atherosclerosis, and inspects the molecular knowledge, and potential clinical significance of senescent cells in developing senescent-based therapy, thus providing crucial insights into their biology and potential therapeutic exploration.

Local delivery of stem cell spheroids with protein/polyphenol self-assembling armor to improve myocardial infarction treatment via immunoprotection and immunoregulation.

Stem cell therapies have shown great potential for treating myocardial infarction (MI) but are limited by low cell survival and compromised functionality due to the harsh microenvironment at the disease site. Here, we presented a Mesenchymal stem cell (MSC) spheroid-based strategy for MI treatment by introducing a protein/polyphenol self-assembling armor coating on the surface of cell spheroids, which showed significantly enhanced therapeutic efficacy by actively manipulating the hostile pathological MI microenvironment and enabling versatile functionality, including protecting the donor cells from host immune clearance, remodeling the ROS microenvironment and stimulating MSC's pro-healing paracrine secretion. The underlying mechanism was elucidated, wherein the armor protected to prolong MSCs residence at MI site, and triggered paracrine stimulation of MSCs towards immunoregulation and angiogenesis through inducing hypoxia to provoke glycolysis in stem cells. Furthermore, local delivery of coated MSC spheroids in MI rat significantly alleviated local inflammation and subsequent fibrosis via mediation macrophage polarization towards pro-healing M2 phenotype and improved cardiac function. In general, this study provided critical insight into the enhanced therapeutic efficacy of stem cell spheroids coated with a multifunctional armor. It potentially opens up a new avenue for designing immunomodulatory treatment for MI via stem cell therapy empowered by functional biomaterials.

Establishment and validation of an efficient method for the 3D culture of osteoclasts in vitro.

INTRODUCTION: Osteoclasts (OCs) play a crucial role in maintaining bone health. Changes in OC activity are linked to different bone diseases, making them an intriguing focus for research. However, most studies on OCs have relied on 2D cultures, limiting our understanding of their behavior. Yet, there's a lack of knowledge regarding platforms that effectively support osteoclast formation in 3D cultures. METHODS: In our investigation, we explored the capacity of collagen and GelMA hydrogels to facilitate osteoclast development in 3D culture settings. We assessed the osteoclast development by using different hydrogels and cell seeding strategies and optimizing cell seeding density and cytokine concentration. The osteoclast development in 3D cultures was further validated by biochemical assays and immunochemical staining. RESULTS: Our findings revealed that 0.3 % (w/v) collagen was conducive to osteoclast formation in both 2D and 3D cultures, demonstrated by increased multinucleation and higher TRAP activity compared to 0.6 % collagen and 5 % to 10 % (w/v) GelMA hydrogels. Additionally, we devised a "sandwich" technique using collagen substrates and augmented the initial macrophage seeding density and doubling cytokine concentrations, significantly enhancing the efficiency of OC culture in 3D conditions. Notably, we validated osteoclasts derived from macrophages in our 3D cultures express key osteoclast markers like cathepsin K and TRAP. CONCLUSIONS: To conclude, our study contributes to establishing an effective method for cultivating osteoclasts in 3D environments in vitro. This innovative approach not only promises a more physiologically relevant platform to study osteoclast behavior during bone remodeling but also holds potential for applications in bone tissue engineering. CLINICAL SIGNIFICANCE: This study introduces an efficient method for cultivating osteoclasts in 3D environments in vitro. It offers a more physiologically relevant platform to investigate osteoclast behavior and holds promise to advance research in bone biology and regenerative dentistry.

Environmental fate of microplastics in alpine and canyon-type river-cascade reservoir systems: Large-scale investigation of the Yalong River in the eastern Qinghai-Tibet Plateau.

Reservoirs are regarded as potential collection sites for microplastics (MPs), and ample water resources in plateau regions provide favorable natural conditions for hydroelectric power generation. However, research on the impact of cascade reservoir construction in the plateau region on the fate of MPs within the watershed is limited. In this study, the Yalong River, an alpine canyon river in the eastern Qinghai-Tibet Plateau, was selected as the research area. This study explored the distribution of MPs at various depths in water, sediment, and riverbank soil as well as the formation of "MP communities" within the river-cascade reservoir system. Furthermore, the effects of dam construction on MPs' migration in different environments were analyzed. The results revealed that the abundance of MPs in the water and sediment within the cascade reservoir area (CRA) was significantly higher than that in the river area (RA) (P < 0.001). Additionally, the trend of increasing MPs in water with decreasing altitude was notably slower in CRA. Regarding shape, the proportion of fibers in the water within the CRA was significantly lower than that in the RA, with a smaller vertical migration rate in the water than in the sediment. The proportion of MPs < 500 µm in the water within the CRA was significantly higher than that in the RA. High-density MPs were notably deposited in the reservoir sediments. The analysis of the MP communities revealed that the construction of cascade dams led to relative geographical isolation between different sampling sites, reducing the similarity of MP communities in the CRA. This study established a theoretical foundation for understanding the impact of cascade dam construction on the fate characteristics of MPs and their potential risks in plateau areas.

Morphology-based deep learning enables accurate detection of senescence in mesenchymal stem cell cultures.

BACKGROUND: Cell senescence is a sign of aging and plays a significant role in the pathogenesis of age-related disorders. For cell therapy, senescence may compromise the quality and efficacy of cells, posing potential safety risks. Mesenchymal stem cells (MSCs) are currently undergoing extensive research for cell therapy, thus necessitating the development of effective methods to evaluate senescence. Senescent MSCs exhibit distinctive morphology that can be used for detection. However, morphological assessment during MSC production is often subjective and uncertain. New tools are required for the reliable evaluation of senescent single cells on a large scale in live imaging of MSCs. RESULTS: We have developed a successful morphology-based Cascade region-based convolution neural network (Cascade R-CNN) system for detecting senescent MSCs, which can automatically locate single cells of different sizes and shapes in multicellular images and assess their senescence state. Additionally, we tested the applicability of the Cascade R-CNN system for MSC senescence and examined the correlation between morphological changes with other senescence indicators. CONCLUSIONS: This deep learning has been applied for the first time to detect senescent MSCs, showing promising performance in both chronic and acute MSC senescence. The system can be a labor-saving and cost-effective option for screening MSC culture conditions and anti-aging drugs, as well as providing a powerful tool for non-invasive and real-time morphological image analysis integrated into cell production.

Integrative Analysis of Machine Learning and Molecule Docking Simulations for Ischemic Stroke Diagnosis and Therapy.

Due to the narrow therapeutic window and high mortality of ischemic stroke, it is of great significance to investigate its diagnosis and therapy. We employed weighted gene coexpression network analysis (WGCNA) to ascertain gene modules related to stroke and used the maSigPro R package to seek the time-dependent genes in the progression of stroke. Three machine learning algorithms were further employed to identify the feature genes of stroke. A nomogram model was built and applied to evaluate the stroke patients. We analyzed single-cell RNA sequencing (scRNA-seq) data to discern microglia subclusters in ischemic stroke. The RNA velocity, pseudo time, and gene set enrichment analysis (GSEA) were performed to investigate the relationship of microglia subclusters. Connectivity map (CMap) analysis and molecule docking were used to screen a therapeutic agent for stroke. A nomogram model based on the feature genes showed a clinical net benefit and enabled an accurate evaluation of stroke patients. The RNA velocity and pseudo time analysis showed that microglia subcluster 0 would develop toward subcluster 2 within 24 h from stroke onset. The GSEA showed that the function of microglia subcluster 0 was opposite to that of subcluster 2. AZ_628, which screened from CMap analysis, was found to have lower binding energy with Mmp12, Lgals3, Fam20c, Capg, Pkm2, Sdc4, and Itga5 in microglia subcluster 2 and maybe a therapeutic agent for the poor development of microglia subcluster 2 after stroke. Our study presents a nomogram model for stroke diagnosis and provides a potential molecule agent for stroke therapy.

ISX9 loaded thermoresponsive nanoparticles for hair follicle regrowth.

There is a high demand for an optimal drug delivery system to treat androgenetic alopecia. Topical application of ISX9, which is a neurogenesis inducer, has been found to stimulate hair follicle (HF) regrowth by upregulating the Wnt/ß-catenin signaling pathway, an essential pathway involved in initiating HF growth and development. In the present study, a temperature-sensitive, biopolymer-based, biocompatible, and eco-friendly drug-delivery system was synthesized. This system comprised chitosan-grafted poly(glycidyl methacrylate-co-N-isopropyl acrylamide) (Poly(GMA-co-NIPAAm)@CS-PGNCS) as the shell component and PF127 as the core polymer. The hydrophobic nature of the PF127 block copolymer efficiently dissolved the partially water-soluble drug, ISX9, and the thermos-responsive shell polymer effectively released the drug at a definite skin temperature. The optimized spherical nanoparticles demonstrated the lowest critical solution temperature (LCST) at 32 ± 2 °C with a diameter of 100-250 nm, which delivered encapsulated ISX9 with greater precision than topical ISX9. In a series of in vivo experiments, we demonstrated that ISX9-coated TBNPs upregulated the expression of ß-catenin, active ß-catenin, Wnt target genes, stemness marker genes, proliferating cell nuclear antigen, HF stem cell markers, and HF markers including VEGF, TGF, and IGF-1 more effectively than topical ISX9. These results suggest that TBNPs could be employed as a platform for effective transdermal delivery of various hydrophobic drugs.

Multi-omic analysis of mandibuloacral dysplasia type A patient iPSC-derived MSC senescence reveals miR-311 as a novel biomarker for MSC senescence.

Mandibuloacral dysplasia type A (MADA) is a rare genetic progeroid syndrome associated with lamin A/C (LMNA) mutations. Pathogenic mutations of LMNA result in nuclear structural abnormalities, mesenchymal tissue damage and progeria phenotypes. However, it remains elusive how LMNA mutations cause mesenchymal-derived cell senescence and disease development. Here, we established an in vitro senescence model using induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs) from MADA patients with homozygous LMNA p.R527C mutation. When expanded to passage 13 in vitro, R527C iMSCs exhibited marked senescence and attenuation of stemness potential, accompanied by immunophenotypic changes. Transcriptome and proteome analysis revealed that cell cycle, DNA replication, cell adhesion and inflammation might play important roles in senescence. In-depth evaluation of changes in extracellular vesicle (EV) derived iMSCs during senescence revealed that R527C iMSC-EVs could promote surrounding cell senescence by carrying pro-senescence microRNAs (miRNAs), including a novel miRNA called miR-311, which can serve as a new indicator for detecting chronic and acute mesenchymal stem cell (MSC) senescence and play a role in promoting senescence. Overall, this study advanced our understanding of the impact of LMNA mutations on MSC senescence and provided novel insights into MADA therapy as well as the link between chronic inflammation and aging development.

Rps6ka2 enhances iMSC chondrogenic differentiation to attenuate knee osteoarthritis through articular cartilage regeneration in mice.

Articular cartilage (AC) is most susceptible to degeneration in knee osteoarthritis (OA); however, the existing treatments for OA do not target the core link of the pathogenesis-"decreased tissue cell function activity and extracellular matrix (ECM) metabolic disorders" for effective intervention. iMSC hold lower heterogeneity and great promise in biological research and clinical applications. Rps6ka2 may play an important role in the iMSC to treat OA. In this study, the CRISPR/Cas9 gene editing Rps6ka2-/- iMSC were obtained. Effect of Rps6ka2 on iMSC proliferation and chondrogenic differentiation was evaluated in vitro. An OA model was constructed in mice by surgical destabilization of medial meniscus (DMM). The Rps6ka2-/- iMSC and iMSC were injected into the articular cavity twice-weekly for 8 weeks. In vitro experiments showed that Rps6ka2 could promote iMSC proliferation and chondrogenic differentiation. In vivo results further confirmed that Rps6ka2 could improve iMSC viability to promote ECM production to attenuate OA in mice.

Isoxazole 9 (ISX9), a small molecule targeting Axin, activates Wnt/ß-catenin signalling and promotes hair regrowth.

BACKGROUND AND PURPOSE: Isoxazole 9 (ISX9) is a neurogenesis-promoting small molecule compound that can up-regulate the expression of NeuroD1 and induce differentiation of neuronal, cardiac and islet endocrine progenitors. So far, the molecular mechanisms underlying the action of ISX9 still remain elusive. EXPERIMENTAL APPROACH: To identify a novel agonist of the Wnt/ß-catenin, a cell-based SuperTOPFlash reporter system was used to screen known-compound libraries. An activation effect of ISX9 on the Wnt/ß-catenin pathway was analysed with the SuperTOPFlash or SuperFOPFlash reporter system. Effects of ISX9 on Axin1/LRP6 interaction were examined using a mammalian two-hybrid system, co-immunoprecipitation, microscale thermophoresis, emission spectra and mass spectrometry assays. The expression of Wnt target and stemmness marker genes were evaluated with real-time PCR and immunoblotting. In vivo hair regeneration abilities of ISX9 were analysed by immunohistochemical staining, real-time PCR and immunoblotting in hair regrowth model using C57BL/6J mice. KEY RESULTS: In this study, ISX9 was identified as a novel agonist of the Wnt/ß-catenin pathway. ISX9 targeted Axin1 by covalently binding to its N-terminal region and potentiated the LRP6-Axin1 interaction, thereby resulting in the stabilization of ß-catenin and up-regulation of Wnt target genes and stemmness marker genes. Moreover, the topical application of ISX9 markedly promoted hair regrowth in C57BL/6J mice and induced hair follicle transition from telogen to anagen via enhancing Wnt/ß-catenin pathway. CONCLUSIONS AND IMPLICATIONS: Taken together, our study unravelled that ISX9 could activate Wnt/ß-catenin signalling by potentiating the association between LRP6 and Axin1, and may be a promising therapeutic agent for alopecia treatment.

Continuous estimation of multi-DOF movement from sEMG based on non-negative matrix factorization and L2 regulation.

Accurate continuous estimation of multi-DOF movement is crucial for simultaneous control of advanced myoelectric prosthetic. The decoupling of multi-DOF is a challenge for continuous estimation. In this paper, we propose a model combined non-negative matrix factorization (NMF) with Hadamard product and L2 regulation to suppress the non-active DOF and achieve the multi-DOF movement continuous estimation. The L2 regulation of non-active DOF activation coefficient was added to the object function of NMF with the benefit of Hadamard product. The angles were estimated by a linear combination of the activation coefficients. We performed a set of continuous estimation experiments for single-DOF and multi-DOF movements of wrist flexion/extend and hand open/close. The results illustrated that the novel model could suppress non-active DOF in single-DOF movement better than other methods based on muscle synergy theory. Moreover, we investigated the robustness of suppression effect and the similarity of synergy matrices at different speeds for NMF-based methods, and the results showed that the proposed method had a superior performance.

Comparative analysis of mesenchymal stem/stromal cells derived from human induced pluripotent stem cells and the cognate umbilical cord mesenchymal stem/stromal cells.

Mesenchymal stem/stromal cells (MSCs) show tremendous potential for regenerative medicine due to their self-renewal, multi-differentiation and immunomodulatory capabilities. Largely studies had indicated conventional tissue-derived MSCs have considerable limited expandability and donor variability which hinders further application. Induced pluripotent stem cell (iPSCs)-derived MSCs (iMSCs) have created exciting source for standardized cellular therapy. However, the cellular and molecular differences between iMSCs and the cognate tissue-derived MSCs remains poorly explored. In this study, we first successfully reprogrammed human umbilical cords-derived mesenchymal stem/stromal cells (UMSCs) into iPSCs by using the cocktails of mRNA. Subsequently, iPSCs were further differentiated into iMSCs in xeno-free induction medium. Then, iMSCs were compared with the donor matched UMSCs by assessing proliferative state, differentiation capability, immunomodulatory potential through immunohistochemical analysis, flow cytometric analysis, transcriptome sequencing analysis, and combine with coculture with immune cell population. The results showed that iMSCs exhibited high expression of MSCs positive-makers CD73, CD90, CD105 and lack expression of negative-maker cocktails CD34, CD45, CD11b, CD19, HLA-DR; also successfully differentiated into osteocytes, chondrocytes and adipocytes. Further, the iMSCs were similar with their parental UMSCs in cell proliferative state detected by the CCK-8 assay, and in cell rejuvenation state assessed by ß-Galactosidase staining and telomerase activity related mRNA and protein analysis. However, iMSCs exhibited similarity to resident MSCs in Homeobox (Hox) genes expression profile and presented better neural differentiation potential by activation of NESTIN related pathway. Moreover, iMSCs owned enhanced immunosuppression capacity through downregulation pools of pro-inflammatory factors, including IL6, IL1B etc. and upregulation anti-inflammatory factors NOS1, TGFB etc. signals. In summary, our study provides an attractive cell source for basic research and offers fundamental biological insight of iMSCs-based therapy.

Deciphering postnatal limb development at single-cell resolution.

The early postnatal limb developmental progression bridges embryonic and mature stages and mirrors the pathological remodeling of articular cartilage. However, compared with multitudinous research on embryonic limb development, the early postnatal stage seems relatively unnoticed. Here, a systematic work to portray the postnatal limb developmental landscape was carried out by characterization of 19,952 single cells from murine hindlimbs at 4 postnatal stages using single-cell RNA sequencing technique. By delineation of cell heterogeneity, the candidate progenitor sub-clusters marked by Cd34 and Ly6e were discovered in articular cartilage and enthesis, and three cellular developmental branches marked by Col10a1, Spp1, and Tnni2 were reflected in growth plate. The representative transcriptomes and developmental patterns were intensively explored, and the key regulation mechanisms as well as evolvement in osteoarthritis were discussed. Above all, these results expand horizons of postnatal limb developmental biology and reach the interconnections between limb development, remodeling, and regeneration.

Cleaved SPP1-rich extracellular vesicles from osteoclasts promote bone regeneration via TGFß1/SMAD3 signaling.

Bone remodeling is a tightly coupled process between bone forming osteoblasts (OBs) and bone resorbing osteoclasts (OCs) to maintain bone architecture and systemic mineral homeostasis throughout life. However, the mechanisms responsible for the coupling between OCs and OBs have not been fully elucidated. Herein, we first validate that secreted extracellular vesicles by osteoclasts (OC-EVs) promote osteogenic differentiation of mesenchymal stem cells (MSCs) and further demonstrate the efficacy of osteoclasts and their secreted EVs in treating tibial bone defects. Furthermore, we show that OC-EVs contain several osteogenesis-promoting proteins as cargo. By employing proteomic and functional analysis, we reveal that mature osteoclasts secrete thrombin cleaved phosphoprotein 1 (SPP1) through extracellular vesicles which triggers MSCs osteogenic differentiation into OBs by activating Transforming Growth Factor ß1 (TGFß1) and Smad family member 3 (SMAD3) signaling. In conclusion, our findings prove an important role of SPP1, present as cargo in OC-derived EVs, in signaling to MSCs and driving their differentiation into OBs. This biological mechanism implies a paradigm shift regarding the role of osteoclasts and their signaling toward the treatment of skeletal disorders which require bone formation.

Stem Cell-Based Therapeutic Strategies for Premature Ovarian Insufficiency and Infertility: A Focus on Aging.

Reproductive aging is on the rise globally and inseparable from the entire aging process. An extreme form of reproductive aging is premature ovarian insufficiency (POI), which to date has mostly been of idiopathic etiology, thus hampering further clinical applications and associated with enormous socioeconomic and personal costs. In the field of reproduction, the important functional role of inflammation-induced ovarian deterioration and therapeutic strategies to prevent ovarian aging and increase its function are current research hotspots. This review discusses the general pathophysiology and relative causes of POI and comprehensively describes the association between the aging features of POI and infertility. Next, various preclinical studies of stem cell therapies with potential for POI treatment and their molecular mechanisms are described, with particular emphasis on the use of human induced pluripotent stem cell (hiPSC) technology in the current scenario. Finally, the progress made in the development of hiPSC technology as a POI research tool for engineering more mature and functional organoids suitable as an alternative therapy to restore infertility provides new insights into therapeutic vulnerability, and perspectives on this exciting research on stem cells and the derived exosomes towards more effective POI diagnosis and treatment are also discussed.

Development of the T-ALLiPSC-based therapeutic cancer vaccines for T-cell acute lymphoblastic leukemia.

The T-cell acute lymphoblastic leukemia (T-ALL) is a kind of hematological malignancy in children. Despite the significant improvement in the cure rate of T-ALL upon treatment with chemotherapy regimens, steroids, and allotransplantation there are relapses. This study focuses on the tumor-specific therapeutic vaccines derived from the induced pluripotent stem cells (iPSC) to address the issue of T-ALL recurrence. Patient-derived tumor cells and healthy donor cells were reprogrammed into the iPSCs and the RNA-seq data of the T-ALL-iPSCs and H-iPSCs were analyzed. In vitro, the whole-cell lysate antigens of iPSCs were prepared to induce the dendritic cells (DC) maturation, which in turn stimulated the tumor-specific T cells to kill the T-ALL tumor cells (Jurkat, CCRF-CEM, MOLT-4). The cytotoxic T lymphocyte stimulated by the DC-loaded T-ALL-iPSC-derived antigens showed specific cytotoxicity against the T-ALL cells in vitro. In conclusion, the T-ALL-iPSC-based therapeutic cancer vaccine can elicit a specific anti-tumor effect on T-ALL.

CRISPR Guide RNA Library Screens in Human Induced Pluripotent Stem Cells.

High-throughput CRISPR guide RNA (gRNA) library screen, that is, CRISPR/Cas9 screen, enables the unbiased identification of gene functions in a variety of biological processes. Typical pooled CRISPR/Cas9 screen couples a gRNA library and a guided Cas9 or dCas9 endonuclease to target specific gene loci, and then systematically uncover the causal link between candidate genes and observed cellular phenotypes via gRNA depletion or enrichment in screens. Here, we describe a detailed method of puromycin (PURO) concentration titration and lentiviral CRISPR gRNA library titration in Cas9 expressing monoclonal human iPSC line (Cas9+MNhiPSC) prior to performing the screens, conducting pooled CRISPR gRNA library screens in Cas9+MNhiPSC, genomic DNA extraction from the selected cell subpopulation and sequencing library preparation as well as next generation sequencing (NGS) to generate gRNA read counts. In CRISPR/Cas9 screen, we aim for 30% transduction efficiency (i.e., multiplicity of infection = 0.3) to ensure most of infected cells receive only one gRNA. The principles in this method can be applied to CRISPR perturbation (knockout, activation, repression or base editing) screens with other CRISPR gRNA libraries across many other cell models and other species.

A single-cell transcriptome of mesenchymal stromal cells to fabricate bioactive hydroxyapatite materials for bone regeneration.

The osteogenic microenvironment of bone-repairing materials plays a key role in accelerating bone regeneration but remains incompletely defined, which significantly limits the application of such bioactive materials. Here, the transcriptional landscapes of different osteogenic microenvironments, including three-dimensional (3D) hydroxyapatite (HA) scaffolds and osteogenic medium (OM), for mesenchymal stromal cells (MSCs) in vitro were mapped at single-cell resolution. Our findings suggested that an osteogenic process reminiscent of endochondral ossification occurred in HA scaffolds through sequential activation of osteogenic-related signaling pathways, along with inflammation and angiogenesis, but inhibition of adipogenesis and fibrosis. Moreover, we revealed the mechanism during OM-mediated osteogenesis involves the ZBTB16 and WNT signaling pathways. Heterogeneity of MSCs was also demonstrated. In vitro ossification of LRRC75A+ MSCs was shown to have better utilization of WNT-related ossification process, and PCDH10+ MSCs with superiority in hydroxyapatite-related osteogenic process. These findings provided further understanding of the cellular activity modulated by OM conditions and HA scaffolds, providing new insights for the improvement of osteogenic biomaterials. This atlas provides a blueprint for research on MSC heterogeneity and the osteogenic microenvironment of HA scaffolds and a database reference for the application of bioactive materials for bone regeneration.

Generation of Monoclonal iPSC Lines with Stable Cas9 Expression and High Cas9 Activity.

CRISPR/Cas9-mediated gene editing has been rapidly and widely applied in many organisms for delicate genetic manipulation, including human-induced pluripotent stem cells (iPSCs). Gene editing in human iPSCs is promising for genetics and biomedical research due to that gene-edited iPSC still possesses the potential to be differentiated into any cell lineages. In many cases, the generation of Cas9 expressing cell lines is a prerequisite toward performing successful editing of multiplex genes of interest. Here, we describe a simple, effective method to generate stable Cas9 expressing human iPSCs with high Cas9 activity. In this method, stable Cas9 expressing monoclonal human iPSC lines were generated through lentiviral transduction of Cas9 cassette, followed by blasticidin selection and subcloning with low seeding density. After colonies isolation and expansion, a BFP-GFP reporter assay was applied to validate the Cas9 activities of multiple monoclonal lines by flow cytometry (FACS). These Cas9 expressing human iPSCs generated by our method are single cell-derived monoclonal lines with homogenous population and Cas9 activity of up to 99%.

Induced Pluripotent Stem Cells Detection via Ensemble Yolo Network.

Induced pluripotent stem cells (iPSCs) have huge potential in regenerative medicine research and industrial applications. However, building automatic method without using cell staining technique for iPSCs identification is an important challenge. To improve the efficiency of producing iPSCs, we build an accurate and noninvasive iPSCs colonies detection method via ensemble Yolo network based on the self-collected bright-field microscopy images. Meanwhile, test-time augmentation (TTA) is leveraged to further improve the detection result of our iPSCs colonies detection method. Extensive experimental results on our dataset demonstrate that our method obtains quite favorable detection performance with the highest F1 score of 0.867 and the highest mean average precision score of 0.898, which outperforms most mainstream methods.