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. Second, a deactivated Cas9 activator modRNA and a 'dead' guide RNA are used to temporarily activate the targeted gene, allowing for live cell sorting of targeted cells. Consequently, MAGIK eliminates the need for drug selection cassettes or labor-intensive single-cell colony screening, expediting precise gene editing. We showed MAGIK can be utilized to insert fluorescent proteins into various genes, including SOX17, NKX6.1, NKX2.5, and PDX1, across multiple hPSC lines. This underscores its robust performance and offers a promising solution for achieving knockin in hPSCs within a significantly shortened time frame.

Direct programming of human pluripotent stem cells into endothelial progenitors with SOX17 and FGF2.

Transcription factors (TFs) are pivotal in guiding stem cell behavior, including their maintenance and differentiation. Using single-cell RNA sequencing, we investigated TFs expressed in endothelial progenitors (EPs) derived from human pluripotent stem cells (hPSCs) and identified upregulated expression of SOXF factors SOX7, SOX17, and SOX18 in the EP population. To test whether overexpression of these factors increases differentiation efficiency, we established inducible hPSC lines for each SOXF factor and found only SOX17 overexpression robustly increased the percentage of cells expressing CD34 and vascular endothelial cadherin (VEC). Conversely, SOX17 knockdown via CRISPR-Cas13d significantly compromised EP differentiation. Intriguingly, we discovered SOX17 overexpression alone was sufficient to generate CD34+VEC+CD31- cells, and, when combined with FGF2 treatment, more than 90% of CD34+VEC+CD31+ EP was produced. These cells are capable of further differentiating into endothelial cells. These findings underscore an undiscovered role of SOX17 in programming hPSCs toward an EP lineage, illuminating pivotal mechanisms in EP differentiation.

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. Our drug-selectable construct design increases the stability and proportion of cells that successfully integrate all plasmids into their genome, thus reducing the amount of cell processing time required. Additionally, we demonstrate that our drug-selectable strategy forgoes the need for single-cell cloning and fluorescence-activated cell sorting, and that a drug-selected mixed population is sufficient to generate robust ultrasound contrast. Successful gas vesicle expression was optically and ultrasonically verified, with cells expressing gas vesicles exhibiting an 80% greater signal-to-noise ratio compared to negative controls and a 500% greater signal-to-noise ratio compared to wild-type HEK293T cells. This technology presents a new reporter gene paradigm by which ultrasound can be harnessed to visualize specific cell types for applications including cellular reporting and cell therapies.

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. K562 and HL60 human leukemia cells were engineered to express the human G protein-coupled receptor hM4Di. Compared to the parental cells, engineered hM4Di-K562 and hM4Di-HL60 cells exhibited excellent chemotaxis ability towards clozapine-N-oxide (CNO) and superior bacteria phagocytic behavior, resembling native neutrophils. The antibacterial ability of the hM4Di-K562 cells was further enhanced by loading them with the glycopeptide vancomycin via mesoporous silica nanoparticles (Nano@Van). Our proposed artificial cell engineering platform provides a new avenue to investigate the physiological properties of 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. Here, we describe SNAP-tag based imaging methods to visualize the folding states of RBPs in live cells via time-resolved quantitative microscopic analyses of their micropolarity and microviscosity. Using these imaging methods in conjunction with immunofluorescence imaging, we demonstrate that RBPs, represented by TDP-43, initially enters the PML nuclear bodies in its native state upon transient proteostasis stress, albeit it begins to misfolded during prolonged stress. Furthermore, we show that heat shock protein 70 co-enters the PML nuclear bodies to prevent the degradation of TDP-43 from the proteotoxic stress, thus revealing a previously unappreciated protective role of the PML nuclear bodies in the prevention of stress-induced degradation of TDP-43. In summary, our imaging methods described in the manuscript, for the first time, reveal the folding states of RBPs, which were previously challenging to study with conventional methods in nuclear bodies of live cells. This study uncovers the mechanistic correlations between the folding states of a protein and functions of nuclear bodies, in particular PML bodies. We envision that the imaging methods can be generally applied to elucidating the structural aspects of other proteins that exhibit granular structures under biological stimulus.

STAMP-Based Digital CRISPR-Cas13a for Amplification-Free Quantification of HIV-1 Plasma Viral Loads.

Quantification of HIV RNA in plasma is critical for identifying the disease progression and monitoring the effectiveness of antiretroviral therapy. While RT-qPCR has been the gold standard for HIV viral load quantification, digital assays could provide an alternative calibration-free absolute quantification method. Here, we reported a Self-digitization Through Automated Membrane-based Partitioning (STAMP) method to digitalize the CRISPR-Cas13 assay (dCRISPR) for amplification-free and absolute quantification of HIV-1 viral RNAs. The HIV-1 Cas13 assay was designed, validated, and optimized. We evaluated the analytical performances with synthetic RNAs. With a membrane that partitions ∼100 nL of reaction mixture (effectively containing 10 nL of input RNA sample), we showed that RNA samples spanning 4 orders of dynamic range between 1 fM (∼6 RNAs) to 10 pM (∼60k RNAs) could be quantified as fast as 30 min. We also examined the end-to-end performance from RNA extraction to STAMP-dCRISPR quantification using 140 µL of both spiked and clinical plasma samples. We demonstrated that the device has a detection limit of approximately 2000 copies/mL and can resolve a viral load change of 3571 copies/mL (equivalent to 3 RNAs in a single membrane) with 90% confidence. Finally, we evaluated the device using 140 µL of 20 patient plasma samples (10 positives and 10 negatives) and benchmarked the performance with RT-PCR. The STAMP-dCRISPR results agree very well with RT-PCR for all negative and high positive samples with Ct < 32. However, the STAMP-dCRISPR is limited in detecting low positive samples with Ct > 32 due to the subsampling errors. Our results demonstrated a digital Cas13 platform that could offer an accessible amplification-free quantification of viral RNAs. By further addressing the subsampling issue with approaches such as preconcentration, this platform could be further exploited for quantitatively determining viral load for an array of infectious diseases.

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. In this study, we demonstrate that neutrophils differentiated from human pluripotent stem cells modified with AAVS1-inserted CAR constructs showed a robust cytotoxic effect against prostate-specific membrane antigen (PSMA) expressing LNCaP cells as a model for prostate cancer in vitro. Our results suggest that engineered CAR can significantly enhance the neutrophil anti-tumor effect, providing a new avenue in treating prostate cancers.

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. We next genetically engineer human pluripotent stem cells (hPSCs) with optimized CARs and differentiate them into functional dual CAR-NK cells. The tumor microenvironment responsive anti-PD-L1 CAR effectively promoted hPSC-NK cell proliferation and cytotoxicity through antigen-dependent activation of phosphorylated STAT3 (pSTAT3) and pSTAT5 signaling pathways via an intracellular truncated IL-2 receptor ß-chain (ΔIL-2Rß) and STAT3-binding tyrosine-X-X-glutamine (YXXQ) motif. Anti-tumor activities of PD-L1-induced memory-like hPSC-NK cells were further boosted by administering a FITC-folate bi-specific adapter that bridges between a programmable anti-FITC CAR and folate receptor alpha-expressing breast tumor cells. Collectively, our hPSC CAR-NK engineering platform is modular and could constitute a realistic strategy to manufacture off-the-shelf CAR-NK cells with immunological memory-like phenotype for targeted immunotherapy.

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. Here we genetically engineer human pluripotent stem cells with CRISPR/Cas9-mediated gene knock-in to express various anti-GBM CAR constructs with T-specific CD3ζ or neutrophil-specific γ-signaling domains. CAR-neutrophils with the best anti-tumor activity are produced to specifically and noninvasively deliver and release tumor microenvironment-responsive nanodrugs to target GBM without the need to induce additional inflammation at the tumor sites. This combinatory chemo-immunotherapy exhibits superior and specific anti-GBM activities, reduces off-target drug delivery and prolongs lifespan in female tumor-bearing mice. Together, this biomimetic CAR-neutrophil drug delivery system is a safe, potent and versatile platform for treating GBM and possibly other devastating diseases.

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 TGFß 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. For complete details on the use and execution of this protocol, please refer to Chang et al. (2022a, 2022b).1,2.

Fabricating 3-dimensional human brown adipose microtissues for transplantation studies.

Transplanting cell cultured brown adipocytes (BAs) represents a promising approach to prevent and treat obesity (OB) and its associated metabolic disorders, including type 2 diabetes mellitus (T2DM). However, transplanted BAs have a very low survival rate in vivo. The enzymatic dissociation during the harvest of fully differentiated BAs also loses significant cells. There is a critical need for novel methods that can avoid cell death during cell preparation, transplantation, and in vivo. Here, we reported that preparing BAs as injectable microtissues could overcome the problem. We found that 3D culture promoted BA differentiation and UCP-1 expression, and the optimal initial cell aggregate size was 100 µm. The microtissues could be produced at large scales via 3D suspension assisted with a PEG hydrogel and could be cryopreserved. Fabricated microtissues could survive in vivo for long term. They alleviated body weight and fat gain and improved glucose tolerance and insulin sensitivity in high-fat diet (HFD)-induced OB and T2DM mice. Transplanted microtissues impacted multiple organs, secreted protein factors, and influenced the secretion of endogenous adipokines. To our best knowledge, this is the first report on fabricating human BA microtissues and showing their safety and efficacy in T2DM mice. The proposal of transplanting fabricated BA microtissues, the microtissue fabrication method, and the demonstration of efficacy in T2DM mice are all new. Our results show that engineered 3D human BA microtissues have considerable advantages in product scalability, storage, purity, safety, dosage, survival, and efficacy.

Wnt signaling directs human pluripotent stem cells into vascularized cardiac organoids with chamber-like structures.

Heart diseases are leading cause of death around the world. Given their unique capacity to self-renew and differentiate into all types of somatic cells, human pluripotent stem cells (hPSCs) hold great promise for heart disease modeling and cardiotoxic drug screening. hPSC-derived cardiac organoids are emerging biomimetic models for studying heart development and cardiovascular diseases, but it remains challenging to make mature organoids with a native-like structure in vitro. In this study, temporal modulation of Wnt signaling pathway co-differentiated hPSCs into beating cardiomyocytes and cardiac endothelial-like cells in 3D organoids, resulting in cardiac endothelial-bounded chamber formation. These chambered cardiac organoids exhibited more mature membrane potential compared to cardiac organoids composed of only cardiomyocytes. Furthermore, a better response to toxic drugs was observed in chamber-contained cardiac organoids. In summary, spatiotemporal signaling pathway modulation may lead to more mature cardiac organoids for studying cardiovascular development and diseases.

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. ABE8e modRNA was employed to disrupt the splice donor site, resulting in defective splicing of the target transcript and ultimately leading to gene knockout. Using our modRNA CRISPR systems, we achieved 73.3% ± 11.2% and 69.6 ± 3.8% knockout efficiency with Cas9 plus p53DD modRNA and ABE8e modRNA, respectively, which was significantly higher than the plasmid-based systems. In summary, we demonstrate that our non-integrating modRNA-based CRISPR methods hold great promise as more efficient and accessible techniques for genome editing of hPSCs.

Engineering chimeric antigen receptor neutrophils from human pluripotent stem cells for targeted cancer immunotherapy.

Neutrophils, the most abundant white blood cells in circulation, are closely related to cancer development and progression. Healthy primary neutrophils present potent cytotoxicity against various cancer cell lines through direct contact and via generation of reactive oxygen species. However, due to their short half-life and resistance to genetic modification, neutrophils have not yet been engineered with chimeric antigen receptors (CARs) to enhance their antitumor cytotoxicity for targeted immunotherapy. Here, we genetically engineered human pluripotent stem cells with synthetic CARs and differentiated them into functional neutrophils by implementing a chemically defined platform. The resulting CAR neutrophils present superior and specific cytotoxicity against tumor cells both in vitro and in vivo. Collectively, we established a robust platform for massive production of CAR neutrophils, paving the way to myeloid cell-based therapeutic strategies that would boost current cancer-treatment approaches.

Chemically-defined generation of human hemogenic endothelium and definitive hematopoietic progenitor cells.

Human hematopoietic stem cells (HSCs), which arise from aorta-gonad-mesonephros (AGM), are widely used to treat blood diseases and cancers. However, a technique for their robust generation in vitro is still missing. Here we show temporal manipulation of Wnt signaling is sufficient and essential to induce AGM-like hematopoiesis from human pluripotent stem cells. TGFß inhibition at the stage of aorta-like SOX17+CD235a- hemogenic endothelium yielded AGM-like hematopoietic progenitors, which closely resembled primary cord blood HSCs at the transcriptional level and contained diverse lineage-primed progenitor populations via single cell RNA-sequencing analysis. Notably, the resulting definitive cells presented lymphoid and myeloid potential in vitro; and could home to a definitive hematopoietic site in zebrafish and rescue bloodless zebrafish after transplantation. Engraftment and multilineage repopulating activities were also observed in mouse recipients. Together, our work provided a chemically-defined and feeder-free culture platform for scalable generation of AGM-like hematopoietic progenitor cells, leading to enhanced production of functional blood and immune cells for various therapeutic applications.

Temporal Expression of Transcription Factor ID2 Improves Natural Killer Cell Differentiation from Human Pluripotent Stem Cells.

Natural killer (NK) cells are one type of innate lymphoid cells, and NK cell-based immunotherapy serves as a potentially curative therapy for cancers. However, the lack of reliable resources for a large amount of NK cells required for clinical infusion has limited the broader application of NK cells in targeted immunotherapy. Substantial effort has thus been made to generate NK-like cells from human pluripotent stem cells (hPSCs), but detailed molecular mechanisms regulating NK cell differentiation remain elusive, preventing us from developing robust strategies for NK cell production. Here, we genetically engineered hPSCs with inducible overexpression of transcription factors NFIL3, ID2, or SPI1 via CRISPR/Cas9-mediated gene knock-in and investigated their temporal roles during NK cell differentiation. Our results demonstrated ID2 overexpression significantly promoted NK cell generation compared with NFIL3 and SPI1 overexpression under a chemically defined, feeder-free culture condition. The resulting ID2 hPSC-derived NK cells exhibited various mature NK-specific markers and displayed effective tumor-killing activities, comparable to NK cells derived from wildtype hPSCs. Our study provides a new platform for efficient NK cell production, serving as a realistic off-the-shelf cell source for targeted cancer immunotherapy.

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 in vitro 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. The Cas9 vector PB-CRISPR exhibited high efficiency (up to 99%) of inducing gene knockout in both protein-coding genes and long non-coding RNAs. The other inducible Cas13d vector achieved extremely high efficiency in RNA knockdown (98% knockdown for CD90) with optimized gRNA designs. Taken together, our PiggyBac CRISPR vectors can serve as powerful toolkits for studying gene functions 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. We introduced a nanopore-sized counting method to measure the cleavage ratio of reporters, which is used as a criterion for positive/negative classification. A kinetic cleavage model was developed and validated to predict the reporter size distributions. The model revealed the trade-offs between sensitivity, turnaround time, and false-positive rate of the SARS-CoV-2 SCAN. With preamplification and a 30 min CRISPR Cas12a assay, we achieved excellent specificity against other common human coronaviruses and a limit of detection of 13.5 copies/µL (22.5 aM) of viral RNA at a confidence level of 95%. These results suggested that the SCAN could provide a rapid, sensitive, and specific analysis of SARS-CoV-2.

Laser-induced graphene non-enzymatic glucose sensors for on-body measurements.

Non-enzymatic glucose sensors outperform enzymatic ones in terms of cost, sensitivity, stability, and operating duration. Though highly sensitive, it is still desirable to further improve the sensitivity of non-enzymatic glucose sensors to detect a trace amount of glucose in sweat and other biofluids. Among the demonstrated effective approaches using bimetals or 3D porous structures, the porous laser-induced graphene (LIG) on flexible polymers showcases good conductivity and a simple fabrication process for the integration of sensing materials. The uniform electroless plating of the nickel and gold layer on LIG electrodes demonstrates significantly enhanced sensitivity and a large linear range for glucose sensing. The sensor with the porous LIG foam exhibits a high sensitivity of 1080 µA mM-1 cm-2, whereas a further increased sensitivity of 3500 µA mM-1 cm-2 is obtained with LIG fibers (LIGF). Impressively, a large linear range (0-30 mM) can be achieved by changing the bias voltage from 0.5 to 0.1 V due to the Au coating. Because the existing non-enzymatic glucose sensors are limited to use in basic solutions, their application in wearable electronics is elusive. In addition to the reduced requirement for the basic solution, this work integrates a porous encapsulating reaction cavity containing alkali solutions with a soft, skin-interfaced microfluidic component to provide integrated microfluidic non-enzymatic glucose sensors for sweat sampling and glucose sensing. The accurate glucose measurements from the human sweat and cell culture media showcase the practical utility, which opens up opportunities for the non-enzymatic glucose sensors in wearable electronics.