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.

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.

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. Thus, the purpose of this work was to examine and understand whether the release of exogenous signaling molecules from hydrogels can promote the survival of MSC spheroids. Our data show that more vascular endothelial growth factor (VEGF) but not platelet-derived growth factor BB (PDGF-BB) were released from MSC spheroids in comparison with 2D cultured MSCs. Aptamer-functionalized fibrin hydrogel (aFn) could release exogenous VEGF and PDGF-BB in a sustained manner. PDGF-BB-loaded aFn promoted MSC survival by ∼70% more than VEGF-loaded aFn under the hypoxic condition in vitro. Importantly, PDGF-BB-loaded aFn could double the survival rate of MSC spheroids in comparison with VEGF-loaded aFn during the one-week test in vivo. Therefore, this work demonstrated that defined exogenous signaling molecules (e.g., PDGF-BB) can function as biochemical cues for promoting the survival of MSC spheroids in vivo.

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. Here we recapitulated current advances in developing efficient protocols to generate hPSC-derived cardiovascular cell lineages, including cardiomyocytes, endothelial cells, and epicardial cells. We also discussed applications of hPSC-derived cells in combination with compatible bioactive materials, promising trials of cell transplantation in animal models of myocardial infarction, and potential hurdles to bring us closer to the ultimate goal of cell-based heart repair.

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. While male hPSCs efficiently differentiate into CD34 + CD31+ endothelial progenitors upon GSK3 inhibition, female hPSCs showed limited differentiation capacity using this protocol. Using VE-cadherin-GFP knockin reporter cells, female cells showed significantly increased differentiation efficiency when treated with VEGF during the second stage of endothelial progenitor differentiation. Interestingly, male cells showed no significant change in differentiation efficiency with VEGF treatment, but did show augmented early activation of VE-cadherin expression. A sex-based difference in endogenous expression of VEGF was identified that is likely the underlying cause of discrepancies in sex-dependent differentiation efficiency. These findings highlight the importance of sex differences in progenitor biology and the development of new stem cell differentiation protocols.