Engineered hematopoietic and immune cells derived from human pluripotent stem cells.

For the past decade, significant advances have been achieved in human hematopoietic stem cell (HSC) transplantation for treating various blood diseases and cancers. However, challenges remain with the quality control, amount, and cost of HSCs and HSC-derived immune cells. The advent of human pluripotent stem cells (hPSCs) may transform HSC transplantation and cancer immunotherapy by providing a cost-effective and scalable cell source for fundamental studies and translational applications. In this review, we discuss the current developments in the field of stem cell engineering for hematopoietic stem and progenitor cell (HSPC) differentiation and further differentiation of HSPCs into functional immune cells. The key advances in stem cell engineering include the generation of HSPCs from hPSCs, genetic modification of hPSCs, and hPSC-derived HSPCs for improved function, further differentiation of HPSCs into functional immune cells, and applications of cell culture platforms for hematopoietic cell manufacturing. Current challenges impeding the translation of hPSC-HSPCs and immune cells as well as further directions to address these challenges are also discussed.

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.

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.

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.

Fabrication of Two-Dimensional and Three-Dimensional High-Resolution Binder-Free Graphene Circuits Using a Microfluidic Approach for Sensor Applications.

In this study, a simple microfluidic method, which can be universally applied to different rigid or flexible substrates, was developed to fabricate high-resolution, conductive, two-dimensional and three-dimensional microstructured graphene-based electronic circuits. The method involves controlled and selective filling of microchannels on substrate surfaces with a conductive binder-free graphene nanoplatelet (GNP) solution. The ethanol-thermal reaction of GNP solution at low temperatures (∼75 °C) prior to microchannel filling (preheating) can further reduce the GNP andprovide a homogeneous GNP solution, which in turn enhances conductivity, reduces sheet resistance (∼0.05 kΩ sq-1), enables room-temperature fabrication, and eliminates harsh postprocessing, which makes this fabrication technique compatible with degradable substrates. This method can also be used in combination with 3D printing to fabricate 3D circuits. The feature sizes of the graphene patterns can range from a few micrometers (down to ∼15 µm in width and ∼5 µm in depth) to a few millimeters and use very small amounts of GNP solution (∼2.5 mg of graphene to obtain ∼0.1 kΩ sq-1 of sheet resistance for 1 cm2). This microfluidic approach can also be implemented using other conductive liquids, such as conductive graphene-silver solutions. This technology has the potential to pave the way for low-cost, disposable, and biodegradable circuits for a range of electronic applications including near-field communication antennas and pressure or strain sensors.

Fabrication of High-resolution Graphene-based Flexible Electronics via Polymer Casting.

In this study, a novel method based on the transfer of graphene patterns from a rigid or flexible substrate onto a polymeric film surface via solvent casting was developed. The method involves the creation of predetermined graphene patterns on the substrate, casting a polymer solution, and directly transferring the graphene patterns from the substrate to the surface of the target polymer film via a peeling-off method. The feature sizes of the graphene patterns on the final film can vary from a few micrometers (as low as 5 µm) to few millimeters range. This process, applied at room temperature, eliminates the need for harsh post-processing techniques and enables creation of conductive graphene circuits (sheet resistance: ~0.2 kΩ/sq) with high stability (stable after 100 bending and 24 h washing cycles) on various polymeric flexible substrates. Moreover, this approach allows precise control of the substrate properties such as composition, biodegradability, 3D microstructure, pore size, porosity and mechanical properties using different film formation techniques. This approach can also be used to fabricate flexible biointerfaces to control stem cell behavior, such as differentiation and alignment. Overall, this promising approach provides a facile and low-cost method for the fabrication of flexible and stretchable electronic circuits.