Ultrathin rubbery bio-optoelectronic stimulators for untethered cardiac stimulation.

Untethered electrical stimulation or pacing of the heart is of critical importance in addressing the pressing needs of cardiovascular diseases in both clinical therapies and fundamental studies. Among various stimulation methods, light illumination-induced electrical stimulation via photoelectric effect without any genetic modifications to beating cells/tissues or whole heart has profound benefits. However, a critical bottleneck lies in the lack of a suitable material with tissue-like mechanical softness and deformability and sufficient optoelectronic performances toward effective stimulation.

Soft Polyethylene Glycol Hydrogels Support Human PSC Pluripotency and Morphogenesis.

Lumenogenesis within the epiblast represents a critical step in early human development, priming the embryo for future specification and patterning events. However, little is known about the specific mechanisms that drive this process due to the inability to study the early embryo in vivo. While human pluripotent stem cell (hPSC)-based models recapitulate many aspects of the human epiblast, most approaches for generating these 3D structures rely on ill-defined, reconstituted basement membrane matrices.

MAGIK: A rapid and efficient method to create lineage-specific reporters in human pluripotent stem cells.

Precise insertion of fluorescent proteins into lineage-specific genes in human pluripotent stem cells (hPSCs) presents challenges due to low knockin efficiency and difficulties in isolating targeted cells. To overcome these hurdles, we present the modified mRNA (ModRNA)-based Activation for Gene Insertion and Knockin (MAGIK) method. MAGIK operates in two steps: first, it uses a Cas9-2A-p53DD modRNA with a mini-donor plasmid (without a drug selection cassette) to significantly enhance efficiency.

A drug-selectable acoustic reporter gene system for human cell ultrasound imaging.

A promising new field of genetically encoded ultrasound contrast agents in the form of gas vesicles has recently emerged, which could extend the specificity of medical ultrasound imaging. However, given the delicate genetic nature of how these genes are integrated and expressed, current methods of producing gas vesicle-expressing mammalian cell lines requires significant cell processing time to establish a clonal/polyclonal line that robustly expresses the gas vesicles sufficiently enough for ultrasound contrast. Here, we describe an inducible and drug-selectable acoustic reporter gene system that can enable gas vesicle expression in mammalian cell lines, which we demonstrate using HEK293T cells.

Engineered Artificial Human Neutrophils Exhibit Mature Functional Performance.

Neutrophils, a key innate immune component, are powerful effector leukocytes for mediating opposing effects on tumor progression and ameliorating pathogen infections. However, their short lifespan and complex purification process have limited neutrophil clinical applications. Here we combined genetic engineering technology with a nanodrug system to construct artificial neutrophils that display functions similar to those of native neutrophils.

Nuclear bodies protect phase separated proteins from degradation in stressed proteome.

RNA-binding proteins (RBPs) containing intrinsically disordered domains undergo liquid-liquid phase separation to form nuclear bodies under stress conditions. This process is also connected to the misfolding and aggregation of RBPs, which are associated with a series of neurodegenerative diseases. However, it remains elusive how folding states of RBPs changes upon the formation and maturation of nuclear bodies.

Engineered human pluripotent stem cell-derived natural killer cells with PD-L1 responsive immunological memory for enhanced immunotherapeutic efficacy.

Adoptive chimeric antigen receptor (CAR)-engineered natural killer (NK) cells have shown promise in treating various cancers. However, limited immunological memory and access to sufficient numbers of allogenic donor cells have hindered their broader preclinical and clinical applications. Here, we first assess eight different CAR constructs that use an anti-PD-L1 nanobody and/or universal anti-fluorescein (FITC) single-chain variable fragment (scFv) to enhance antigen-specific proliferation and anti-tumor cytotoxicity of NK-92 cells against heterogenous solid tumors.

Engineered anti-prostate cancer CAR-neutrophils from human pluripotent stem cells.

Immunotherapy is a powerful technique where immune cells are modified to improve cytotoxicity against cancerous cells to treat cancers that do not respond to surgery, chemotherapy, or radiotherapy. Expressing chimeric antigen receptor (CAR) in immune cells, typically T lymphocytes, is a practical modification that drives an immune response against cancerous tissue. CAR-T efficacy is suboptimal in solid tumors due to the tumor microenvironment (TME) that limits T lymphocyte cytotoxicity.

CAR-neutrophil mediated delivery of tumor-microenvironment responsive nanodrugs for glioblastoma chemo-immunotherapy.

Glioblastoma (GBM) is one of the most aggressive and lethal solid tumors in human. While efficacious therapeutics, such as emerging chimeric antigen receptor (CAR)-T cells and chemotherapeutics, have been developed to treat various cancers, their effectiveness in GBM treatment has been hindered largely by the blood-brain barrier and blood-brain-tumor barriers. Human neutrophils effectively cross physiological barriers and display effector immunity against pathogens but the short lifespan and resistance to genome editing of primary neutrophils have limited their broad application in immunotherapy.

Chemically defined generation of human definitive hematopoietic stem and progenitor cells.

Here, we present a protocol to efficiently direct human pluripotent stem cells (hPSCs) into hematopoietic stem and progenitor cells (HSPCs) under a chemically defined, albumin-free system. We describe the induction of aorta-gonad-mesonephros-like hematopoiesis from hPSCs into SOX17+ hemogenic endothelium and then into CD34+CD45+ HSPCs via application of Wnt activator and TGFβ inhibitor, respectively. The generated HSPCs, characterized by flow cytometry and colony-forming unit assay, express definitive hematopoiesis markers and exhibit multilineage differentiation potential and the capacity to expand.

Robust genome editing via modRNA-based Cas9 or base editor in human pluripotent stem cells.

CRISPR systems have revolutionized biomedical research because they offer an unprecedented opportunity for genome editing. However, a bottleneck of applying CRISPR systems in human pluripotent stem cells (hPSCs) is how to deliver CRISPR effectors easily and efficiently. Here, we developed modified mRNA (modRNA)-based CRIPSR systems that utilized Cas9 and p53DD or a base editor (ABE8e) modRNA for the purposes of knocking out genes in hPSCs via simple lipid-based transfection.

Robust genome and RNA editing via CRISPR nucleases in PiggyBac systems.

CRISPR/Cas-mediated genome editing in human pluripotent stem cells (hPSCs) offers unprecedented opportunities for developing disease modeling, drug screening and cell-based therapies. To efficiently deliver the CRISPR components, here we developed two all-in-one vectors containing Cas9/gRNA and inducible Cas13d/gRNA cassettes for robust genome editing and RNA interference respectively. These vectors utilized the PiggyBac transposon system, which allows stable expression of CRISPR components in hPSCs.

Detection of SARS-CoV-2 with Solid-State CRISPR-Cas12a-Assisted Nanopores.

The outbreak of the SARS-CoV-2 caused the disease COVID-19 to spread globally. Specific and sensitive detection of SARS-CoV-2 facilitates early intervention and prevents the disease from spreading. Here, we present a solid-state CRISPR-Cas12a-assisted nanopore (SCAN) sensing strategy for the specific detection of SARS-CoV-2.

Generation of pancreatic progenitors from human pluripotent stem cells by small molecules.

Human pluripotent stem cell (hPSC)-derived pancreatic progenitors (PPs) provide promising cell therapies for type 1 diabetes. Current PP differentiation requires a high amount of Activin A during the definitive endoderm (DE) stage, making it economically difficult for commercial ventures. Here we identify a dose-dependent role for Wnt signaling in controlling DE differentiation without Activin A.

A dual cardiomyocyte reporter model derived from human pluripotent stem cells.

Cardiovascular diseases (CVD) remain the leading cause of death in the USA. Cardiomyocytes (CMs) derived from human pluripotent stem cells (hPSCs) provide a valuable cell source for regenerative therapy, disease modeling, and drug screening. Here, we established a hPSC line integrated with a mCherry fluorescent protein driven by the alpha myosin heavy chain (aMHC) promoter, which could be used to purify CMs based on the aMHC promoter activity in these cells.

Exogenous Signaling Molecules Released from Aptamer-Functionalized Hydrogels Promote the Survival of Mesenchymal Stem Cell Spheroids.

Mesenchymal stem cells (MSCs) have a very low survival rate after in vivo delivery, which limits their great promise for treating human diseases. Various strategies have been studied to overcome this challenge. However, an overlooked but important potential is to apply exogenous signaling molecules as biochemical cues to promote MSC survival, presumably because it is well-known that MSCs themselves can release a variety of potent signaling molecules.

Sequence-Specific Recognition of HIV-1 DNA with Solid-State CRISPR-Cas12a-Assisted Nanopores (SCAN).

Nucleic acid detection methods are crucial for many fields such as pathogen detection and genotyping. Solid-state nanopore sensors represent a promising platform for nucleic acid detection due to its unique single molecule sensitivity and label-free electronic sensing. Here, we demonstrated the use of the glass nanopore for highly sensitive quantification of single-stranded circular DNAs (reporters), which could be degraded under the trans-cleavage activity of the target-specific CRISPR-Cas12a.

Fluorescent indicators for continuous and lineage-specific reporting of cell-cycle phases in human pluripotent stem cells.

Proper cell-cycle progression is essential for the self-renewal and differentiation of human pluripotent stem cells (hPSCs). The fluorescent ubiquitination-based cell-cycle indicator (FUCCI) has allowed the dual-color visualization of the G and S/G /M phases in various dynamic models, but its application in hPSCs is not widely reported. In addition, lineage-specific FUCCI reporters have not yet been developed to analyze complex tissue-specific cell-cycle progression during hPSC differentiation.

Heart regeneration with human pluripotent stem cells: Prospects and challenges.

Cardiovascular disease, ranging from congenital heart disease to adult myocardial infarction, is the leading cause of death worldwide. In pursuit of reliable cardiovascular regenerative medicine, human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), offer plenty of potential cell-based applications. HPSCs are capable of proliferating indefinitely in an undifferentiated state, and are also pluripotent, being able to differentiate into virtually any somatic cell types given specific stepwise cues, thus representing an unlimited source to generate functional cardiovascular cells for heart regeneration.

Sex-dependent VEGF expression underlies variations in human pluripotent stem cell to endothelial progenitor differentiation.

Human pluripotent stem cells (hPSCs) offer tremendous promise in tissue engineering and cell-based therapies because of their unique combination of two properties: pluripotency and a high proliferative capacity. To realize this potential, development of efficient hPSC differentiation protocols is required. In this work, sex-based differences are identified in a GSK3 inhibitor based endothelial progenitor differentiation protocol.

Beyond Purple Hearts: A Colorful Approach to Isolate Distinct Heart Cells from Human iPSCs.

In this issue of Cell Stem Cell, Zhang et al. (2019) describe a double-reporter iPSC line based on the expression of key cardiac transcription factors, TBX5 and NKX2.5, that delineates cardiac lineage specification in vitro and enables isolation of relatively pure chamber-specific cardiomyocytes, which are critical for drug screening, tissue engineering, and disease modeling.

Human beta cells generated from pluripotent stem cells or cellular reprogramming for curing diabetes.

Diabetes is a group of metabolic diseases characterized by aberrantly high blood glucose levels caused by defects in insulin secretion, its action, or both, which affects approximately 30.3 million people (9.4% of the population) in the United States.

An Ultrasensitive Calcium Reporter System via CRISPR-Cas9-Mediated Genome Editing in Human Pluripotent Stem Cells.

Genetically encoded calcium indicator (GCaMP) proteins have been reported for imaging cardiac cell activity based on intracellular calcium transients. To bring human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) to the clinic, it is critical to evaluate the functionality of CMs. Here, we show that GCaMP6s-expressing hPSCs can be generated and used for CM characterization.