Selecting Monoclonal Cell Lineages from Somatic Reprogramming Using Robotic-Based Spatial-Restricting Structured Flow.

Somatic cell reprogramming generates induced pluripotent stem cells (iPSCs), which serve as a crucial source of seed cells for personalized disease modeling and treatment in regenerative medicine. However, the process of reprogramming often causes substantial lineage manipulations, thereby increasing cellular heterogeneity. As a consequence, the process of harvesting monoclonal iPSCs is labor-intensive and leads to decreased reproducibility. Here, we report the first in-house developed robotic platform that uses a pin-tip-based micro-structure to manipulate radial shear flow for automated monoclonal iPSC colony selection (~1 s) in a non-invasive and label-free manner, which includes tasks for somatic cell reprogramming culturing, medium changes; time-lapse-based high-content imaging; and iPSCs monoclonal colony detection, selection, and expansion. Throughput-wise, this automated robotic system can perform approximately 24 somatic cell reprogramming tasks within 50 days in parallel via a scheduling program. Moreover, thanks to a dual flow-based iPSC selection process, the purity of iPSCs was enhanced, while simultaneously eliminating the need for single-cell subcloning. These iPSCs generated via the dual processing robotic approach demonstrated a purity 3.7 times greater than that of the conventional manual methods. In addition, the automatically produced human iPSCs exhibited typical pluripotent transcriptional profiles, differentiation potential, and karyotypes. In conclusion, this robotic method could offer a promising solution for the automated isolation or purification of lineage-specific cells derived from iPSCs, thereby accelerating the development of personalized medicines.

Comparison of miRNA transcriptome of exosomes in three categories of somatic cells with derived iPSCs.

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) through epigenetic manipulation. While the essential role of miRNA in reprogramming and maintaining pluripotency is well studied, little is known about the functions of miRNA from exosomes in this context. To fill this research gap,we comprehensively obtained the 17 sets of cellular mRNA transcriptomic data with 3.93 × 1010 bp raw reads and 18 sets of exosomal miRNA transcriptomic data with 2.83 × 107 bp raw reads from three categories of human somatic cells: peripheral blood mononuclear cells (PBMCs), skin fibroblasts(SFs) and urine cells (UCs), along with their derived iPSCs. Additionally, differentially expressed molecules of each category were identified and used to perform gene set enrichment analysis. Our study provides sets of comparative transcriptomic data of cellular mRNA and exosomal miRNA from three categories of human tissue with three individual biological controls in studies of iPSCs generation, which will contribute to a better understanding of donor cell variation in functional epigenetic regulation and differentiation bias in iPSCs.

Decellularised spinal cord matrix manipulates glial niche into repairing phase via serglycin-mediated signalling pathway.

Astrocytes are the most abundant and widespread glial cells in the central nervous system. The heterogeneity of astrocytes plays an essential role in spinal cord injury (SCI) repair. Decellularised spinal cord matrix (DSCM) is advantageous for repairing SCI, but little is known regarding the exact mechanisms and niche alterations. Here, we investigated the DSCM regulatory mechanism of glial niche in the neuro-glial-vascular unit using single-cell RNA sequencing. Our single cell sequencing, molecular and biochemical experiments validated that DSCM facilitated the differentiation of neural progenitor cells through increasing the number of immature astrocytes. Upregulation of mesenchyme-related genes, which maintained astrocyte immaturity, causing insensitivity to inflammatory stimuli. Subsequently, we identified serglycin (SRGN) as a functional component of DSCM, which involves inducing CD44-AKT signalling to trigger human spinal cord-derived primary astrocytes (hspASCs) proliferation and upregulation of genes related to epithelial-mesenchymal transition, thus impeding astrocyte maturation. Finally, we verified that SRGN-COLI and DSCM had similar functions in the human primary cell co-culture system to mimic the glia niche. In conclusion, our work revealed that DSCM reverted astrocyte maturation and altered the glia niche into the repairing phase through the SRGN-mediated signalling pathway.

High-throughput "read-on-ski" automated imaging and label-free detection system for toxicity screening of compounds using personalised human kidney organoids.

Organoid models are used to study kidney physiology, such as the assessment of nephrotoxicity and underlying disease processes. Personalized human pluripotent stem cell-derived kidney organoids are ideal models for compound toxicity studies, but there is a need to accelerate basic and translational research in the field. Here, we developed an automated continuous imaging setup with the "read-on-ski" law of control to maximize temporal resolution with minimum culture plate vibration. High-accuracy performance was achieved: organoid screening and imaging were performed at a spatial resolution of 1.1 µm for the entire multi-well plate under 3 min. We used the in-house developed multi-well spinning device and cisplatin-induced nephrotoxicity model to evaluate the toxicity in kidney organoids using this system. The acquired images were processed via machine learning-based classification and segmentation algorithms, and the toxicity in kidney organoids was determined with 95% accuracy. The results obtained by the automated "read-on-ski" imaging device, combined with label-free and non-invasive algorithms for detection, were verified using conventional biological procedures. Taking advantage of the close-to-in vivo-kidney organoid model, this new development opens the door for further application of scaled-up screening using organoids in basic research and drug discovery.

The Dynamics of Metabolic Characterization in iPSC-Derived Kidney Organoid Differentiation via a Comparative Omics Approach.

The use of differentiating human induced pluripotent stem cells (hiPSCs) in mini-tissue organoids provides an invaluable resource for regenerative medicine applications, particularly in the field of disease modeling. However, most studies using a kidney organoid model, focused solely on the transcriptomics and did not explore mechanisms of regulating kidney organoids related to metabolic effects and maturational phenotype. Here, we applied metabolomics coupled with transcriptomics to investigate the metabolic dynamics and function during kidney organoid differentiation. Not only did we validate the dominant metabolic alteration from glycolysis to oxidative phosphorylation in the iPSC differentiation process but we also showed that glycine, serine, and threonine metabolism had a regulatory role during kidney organoid formation and lineage maturation. Notably, serine had a role in regulating S-adenosylmethionine (SAM) to facilitate kidney organoid formation by altering DNA methylation. Our data revealed that analysis of metabolic characterization broadens our ability to understand phenotype regulation. The utilization of this comparative omics approach, in studying kidney organoid formation, can aid in deciphering unique knowledge about the biological and physiological processes involved in organoid-based disease modeling or drug screening.

Human Urinal Cell Reprogramming: Synthetic 3D Peptide Hydrogels Enhance Induced Pluripotent Stem Cell Population Homogeneity.

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs), which have promising potential applications in regenerative medicine. However, the challenges of successful applications of human iPSCs for medical purposes are the low generation efficiency, heterogeneous colonies, and exposure to the animal-derived product Matrigel. We aimed to investigate whether human urinal cells could be efficiently reprogrammed into iPSCs in three-dimensional Puramatrix (3D-PM) compared to two-dimensional Matrigel (2D-MG) and to understand how this 3D hydrogel environment affects the reprogramming process. Human urinal cells were successfully reprogrammed into iPSCs in the defined synthetic animal-free 3D-PM. Interestingly, although the colony efficiency in 3D-PM was similar to that in 2D-MG (∼0.05%), the reprogrammed colonies in 3D-PM contained an iPSC population with significantly higher homogeneity, as evidenced by the pluripotent-like morphology and expression of markers. This was further confirmed by transcriptome profile analysis in bulk cells and at the single cell level. Moreover, the homogeneity of the iPSC population in 3D-PM colonies was correlated with the downregulation of integrin ß1 (ITGB1) and phosphorylated focal adhesion kinase (FAK). Collectively, 3D-PM provides an alternative approach for obtaining iPSCs with enhanced homogeneity. This work also unveiled the regulation of human somatic cell reprogramming via the extracellular microenvironment.

Application of polyhydroxyalkanoates nanoparticles as intracellular sustained drug-release vectors.

Polyhydroxybutyrate (PHB), co-polyesters of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx), and polylactic acid (PLA) were used to prepare nanoparticles with average sizes of 160, 250 and 150 nm, respectively. A lipid-soluble colorant, rhodamine B isothiocyanate (RBITC), was employed to study drug-release behaviors from these nanoparticles. A high RBITC drug-loading efficiency of over 75% was achieved with all PHA nanoparticles prepared. Macrophage endocytosis led to an intracellular RBITC drug sustained release over a period of at least 20 days for PHB and PHBHHx nanoparticles, while PLA nanoparticles and free drug lasted only 15 days and a week, respectively. Polymer properties and particle sizes showed little effect on drug-release behavior. This study showed for the first time that PHB and PHBHHx can be used effectively to achieve intracellular controlled drug releases.

A specific drug targeting system based on polyhydroxyalkanoate granule binding protein PhaP fused with targeted cell ligands.

Polyhydroxyalkanoates (PHA) is a family of intracellular biopolyesters produced by many bacteria. PHA granule binding protein PhaP is able to bind to hydrophobic polymers via strong hydrophobic interaction. A receptor-mediated drug delivery system was developed in this study based on PhaP. The system consists of PHA nanoparticles, PhaP and polypeptide or protein ligands fused to PhaP. The PHA nanoparticles were used to package mostly hydrophobic drugs; PhaP fused with ligands produced by over-expression of their corresponding genes in Pichia pastoris, or E. coli was able to attach to hydrophobic PHA nanoparticle. At the end, the ligands were able to pull the PhaP-PHA nanoparticles to the targeted cells with receptors recognized by the ligands. It was found in this study that the receptor-mediated drug specific delivery system ligand-PhaP-PHA nanoparticles were taken up by macrophages, hepatocellular carcinoma cell BEL7402 in vitro and liver, hepatocellular carcinoma cells in vivo, respectively, when the ligands were mannosylated human alpha1-acid glycoprotein (hAGP) and human epidermal growth factor (hEGF), respectively, which were able to bind to receptors of macrophages or hepatocellular carcinoma cells. The nanoparticle system was clearly visible in the targeted cells and organs (liver or tumor) under fluorescence microscopy when rhodamine B isothiocyanate (RBITC) was used as a delivery model drug due to the specific targeting effect created by specific ligand and receptor binding. The delivery system of hEGF-PhaP-nanoparticles carrying RBITC was found to be endocytosed by the tumor cells in tumorous model mice. Thus, the ligand-PhaP-PHA specific drug delivery system was proven effective both in vitro and in vivo.