Human pluripotent stem cell derived endothelial cells (hPSC-ECs) are of great value for studying and treating vascular diseases. However, manufacturing high quantity and quality hPSC-ECs with current cell culture technologies remains very challenging. Here, we report a novel method that can manufacture hPSC-ECs in scalable and cell-friendly microenvironments to address this challenge. Using this method, hPSCs are expanded and differentiated into ECs in microscale alginate hydrogel tubes. The hydrogel tubes protect cells from the highly variable hydrodynamic conditions and critical hydrodynamic stresses in the culture vessel and limit the cell mass less than the diffusion limits (of human tissue) to ensure efficient mass transport. The hydrogel tubes provide uniform and friendly microenvironments for cells to grow. This novel design leads to extremely high production efficiency. We showed that hPSC-ECs could be produced in 10 days with high viability (>90%), high purity (>80%) and high yield (∼5.0 × 10⁸ cells per mL of microspace). The yield is about 250 times that of the current-state-of-the-art. hPSC-ECs made in these hydrogel tubes had similar in vitro and in vivo functions to hPSC-ECs generated by conventional cell culture methods. This simple, scalable, efficient, defined and cost-effective technology will make hPSC-ECs broadly available and affordable for various biomedical applications.

Human pluripotent stem cell (hPSC)-derived endothelial cells (ECs) are promising cell sources for drug discovery, tissue engineering, and studying or treating vascular diseases. However, hPSC-ECs derived from different culture methods display different phenotypes. Herein, we made a detailed comparative study of hPSC-ECs from three different culture systems (e.g., 2D, 3D PNIPAAm-PEG hydrogel, and 3D alginate hydrogel cultures) based on our previous reports. We expanded hPSCs and differentiated them into ECs in three culture systems. Both 3D hydrogel systems could mimic an in vivo physiologically relevant microenvironment to protect cells from shear force and prevent cell agglomeration, leading to a high culture efficiency and a high volumetric yield. We demonstrated that hPSC-ECs produced from both hydrogel systems had similar results as 2D-ECs. The transcriptome analysis showed that PEG-ECs and alginate-ECs displayed a functional phenotype due to their higher gene expressions in vasculature development, extracellular matrix, angiogenesis, and glycolysis, while 2D-ECs showed a proliferative phenotype due to their higher gene expressions in cell proliferation. Taken together, both PEG- and alginate-hydrogel systems will significantly advance the applications of hPSC-ECs in various biomedical fields.

Human pluripotent stem cell derived brain organoids offer an unprecedented opportunity for various applications as in vitro model. Currently, human brain organoids as models have been used to understand virus-induced neurotoxicity.

Vascular smooth muscle cells (VSMCs), which provides structural integrity and regulates the diameter of vasculature, are of great potential for modeling vascular-associated diseases and tissue engineering. Here, we presented a detailed comparison of differentiating human pluripotent stem cells (hPSCs) into VSMCs (hPSCs-VSMCs) in four different culture methods, including 2-dimensional (2D) culture, 3-dimensional (3D) PNIPAAm-PEG hydrogel culture, 3-dimensional (3D) alginate hydrogel culture, and transferring 3-dimensional alginate hydrogel culture to 2-dimensional (2D) culture. Both hydrogel-based culture methods could mimic in vivo microenvironment to protect cells from shear force, and avoid cells agglomeration, resulting in the extremely high culture efficiency (e.g., high viability, high purity and high yield) compared with 2D culture. We demonstrated hPSC-VSMCs produced from hydrogel-based culture methods had better contractile phenotypes and the potential of vasculature formation. The transcriptome analysis showed the hPSC-VSMCs derived from hydrogel-based culture methods displayed more upregulated genes in vasculature development, angiogenesis and blood vessel development, extracellular matrix compared with 2D culture. Taken together, hPSC-VSMCs produced from hydrogel-based culture system could be applied in various biomedical fields, and further indicated the suitable development of alginate hydrogel for industrial production by taking all aspects into consideration.

Vascular smooth muscle cells (VSMCs) are of great value and are needed in large quantities for tissue engineering, drug screening, disease modeling and cell-based therapies. However, getting high quantity VSMCs remains a challenge. Here, we report a method for the scalable manufacturing of VSMCs from human pluripotent stem cells (hPSCs). hPSCs are expanded and differentiated into VSMCs in a three dimensional (3D) thermoreversible hydrogel. The hydrogel not only acts as a 3D scaffold for cells to grow, but also protects cells from hydrodynamic stresses in the culture vessel and prevents cells from excessive aggregation. Together, the hydrogel creates a cell-friendly microenvironment, leading to high culture efficiency. We show that VSMCs can be generated in 10 days with high viability (>90%), high purity (>80%) and high yield (∼2.0 × 10⁷ cells per mL hydrogel) in the hydrogel scaffold. The generated VSMCs have normal functions. Genome-wide gene expression analysis shows VSMCs made in the hydrogel (i.e. 3D-VSMCs) have higher expression of genes related to vasculature development and glycolysis compared to VSMCs made in the conventional 2D cultures (i.e. 2D-VSMCs), while 2D-VSMCs have higher expression of genes related to cell proliferation. This simple, defined and efficient method is scalable for manufacturing hPSC-VSMCs for various biomedical applications.

Currently, an inactivated vaccine has been widely used with encouraging results as a prophylactic agent against COVID-19 infection, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants. However, in vitro SARS-CoV-2 vaccine-specific immune features remain elusive, hindering the promotion of a third dose of the vaccine. Here, we present a detailed in vitro immune cellular response and large-scale multi-omics analysis for peripheral blood mononuclear cells (PBMCs) from participants vaccinated with CoronaVac (Sinovac Life Sciences, Beijing, China) and recovered participants from COVID-19. The mean titers of SARS-CoV-2 serum-neutralizing antibodies were significantly increased after the boosting immunization (Day 45) compared to the unimmunized state. We observed that type-1 helper T cells (Th1) tended to dominate after the first dose of vaccine, while humoral immune responses became dominant after the second dose due to the activation of type-2 helper T cell (Th2), memory B cells, and plasmablasts. T follicular helper cells (Tfh) involved in antibody production were activated after the first dose and were maintained for the observed time points. Single-cell RNA sequencing of PBMCs revealed specific changes in cell compositions and gene expression in immunized participants. Multi-omics analysis also demonstrated that CoronaVac-specific serum proteins, plasma metabolites, and plasma lipid changes were skewed to those changes in convalescent patients. Collectively, we provide a comprehensive understanding of CoronaVac-specific in vitro immune features.