The neonatal Fc receptor (FcRn) is a pan-arterivirus receptor.

Arteriviruses infect a variety of mammalian hosts, but the receptors used by these viruses to enter cells are poorly understood. We identified the neonatal Fc receptor (FcRn) as an important pro-viral host factor via comparative genome-wide CRISPR-knockout screens with multiple arteriviruses. Using a panel of cell lines and divergent arteriviruses, we demonstrate that FcRn is required for the entry step of arterivirus infection and serves as a molecular barrier to arterivirus cross-species infection.

Isolation of Diverse Simian Arteriviruses Causing Hemorrhagic Disease.

Genetically diverse simian arteriviruses (simarteriviruses) naturally infect geographically and phylogenetically diverse monkeys, and cross-species transmission and emergence are of considerable concern. Characterization of most simarteriviruses beyond sequence analysis has not been possible because the viruses fail to propagate in the laboratory. We attempted to isolate 4 simarteriviruses, Kibale red colobus virus 1, Pebjah virus, simian hemorrhagic fever virus, and Southwest baboon virus 1, by inoculating an immortalized grivet cell line (known to replicate simian hemorrhagic fever virus), primary macaque cells, macrophages derived from macaque induced pluripotent stem cells, and mice engrafted with macaque CD34+-enriched hematopoietic stem cells.

PTP1B phosphatase dampens iPSC-derived neutrophil motility and antimicrobial function.

Neutrophils are rapidly recruited to sites of infection and are critical for pathogen clearance. Therapeutic use of primary neutrophils has been limited, as they have a short lifespan and are not amenable to genetic manipulation. Human induced pluripotent stem cells (iPSCs) can provide a robust source of neutrophils for infusion and are genetically tractable.

SOX18-enforced expression diverts hemogenic endothelium-derived progenitors from T towards NK lymphoid pathways.

Hemogenic endothelium (HE) is the main source of blood cells in the embryo. To improve blood manufacturing from human pluripotent stem cells (hPSCs), it is essential to define the molecular determinants that enhance HE specification and promote development of the desired blood lineage from HE. Here, using SOX18-inducible hPSCs, we revealed that SOX18 forced expression at the mesodermal stage, in contrast to its homolog SOX17, has minimal effects on arterial specification of HE, expression of HOXA genes and lymphoid differentiation.

Whole genome sequencing of CRISPR-Cas9-edited Mauritian cynomolgus macaque blastomeres reveals large-scale deletions and off-target edits.

Genome editing by CRISPR-Cas9 approaches offers promise for introducing or correcting disease-associated mutations for research and clinical applications. Nonhuman primates are physiologically closer to humans than other laboratory animal models, providing ideal candidates for introducing human disease-associated mutations to develop models of human disease. The incidence of large chromosomal anomalies in CRISPR-Cas9-edited human embryos and cells warrants comprehensive genotypic investigation of editing outcomes in primate embryos.

Generation of anti-GD2 CAR macrophages from human pluripotent stem cells for cancer immunotherapies.

Macrophages armed with chimeric antigen receptors (CARs) provide a potent new option for treating solid tumors. However, genetic engineering and scalable production of somatic macrophages remains significant challenges. Here, we used CRISPR-Cas9 gene editing methods to integrate an anti-GD2 CAR into the AAVS1 locus of human pluripotent stem cells (hPSCs).

CRISPR/Cas9 genome editing to create nonhuman primate models for studying stem cell therapies for HIV infection.

Nonhuman primates (NHPs) are well-established basic and translational research models for human immunodeficiency virus (HIV) infections and pathophysiology, hematopoietic stem cell (HSC) transplantation, and assisted reproductive technologies. Recent advances in CRISPR/Cas9 gene editing technologies present opportunities to refine NHP HIV models for investigating genetic factors that affect HIV replication and designing cellular therapies that exploit genetic barriers to HIV infections, including engineering mutations into CCR5 and conferring resistance to HIV/simian immunodeficiency virus (SIV) infections. In this report, we provide an overview of recent advances and challenges in gene editing NHP embryos and discuss the value of genetically engineered animal models for developing novel stem cell-based therapies for curing HIV.

Allogeneic MHC-matched T-cell receptor α/β-depleted bone marrow transplants in SHIV-infected, ART-suppressed Mauritian cynomolgus macaques.

Allogeneic hematopoietic stem cell transplants (allo-HSCTs) dramatically reduce HIV reservoirs in antiretroviral therapy (ART) suppressed individuals. However, the mechanism(s) responsible for these post-transplant viral reservoir declines are not fully understood. Therefore, we modeled allo-HSCT in ART-suppressed simian-human immunodeficiency virus (SHIV)-infected Mauritian cynomolgus macaques (MCMs) to illuminate factors contributing to transplant-induced viral reservoir decay.

Assessment of Endothelial-to-Hematopoietic Transition of Individual Hemogenic Endothelium and Bulk Populations in Defined Conditions.

Endothelial-to-hematopoietic transition (EHT) is a unique morphogenic event in which flat, adherent hemogenic endothelial (HE) cells acquire round, non-adherent blood cell morphology. Investigating the mechanisms of EHT is critical for understanding the development of hematopoietic stem cells (HSCs) and the entirety of the adult immune system, and advancing technologies for manufacturing blood cells from human pluripotent stem cells (hPSCs). Here we describe a protocol to (a) generate and isolate subsets of HE from hPSCs, (b) assess EHT and hematopoietic potential of HE subsets in bulk cultures and at the single-cell level, and (c) evaluate the role of NOTCH signaling during HE specification and EHT.

Assessment of safety and immunogenicity of MHC homozygous iPSC-derived CD34+ hematopoietic progenitors in an NHP model.

Administration of ex vivo expanded somatic myeloid progenitors has been explored as a way to facilitate a more rapid myeloid recovery and improve overall survival after myeloablation. Recent advances in induced pluripotent stem cell (iPSC) technologies have created alternative platforms for supplying off-the-shelf immunologically compatible myeloid progenitors, including cellular products derived from major histocompatibility complex (MHC) homozygous superdonors, potentially increasing the availability of MHC-matching cells and maximizing the utility of stem cell banking. However, the teratogenic and tumorigenic potential of iPSC-derived progenitor cells and whether they will induce alloreactive antibodies upon transfer remain unclear.

Generation of SIV-resistant T cells and macrophages from nonhuman primate induced pluripotent stem cells with edited CCR5 locus.

Adoptive therapies with genetically modified somatic T cells rendered HIV resistance have shown promise for AIDS therapy. A renewable source of HIV-resistant human T cells from induced pluripotent stem cells (iPSCs) would further facilitate and broaden the applicability of these therapies. Here, we report successful targeting of the CCR5 locus in iPSCs generated from T cells (T-iPSCs) or fibroblasts (fib-iPSCs) from Mauritian cynomolgus macaques (MCM), using CRISPR-Cas9 technology.

3D iPSC modeling of the retinal pigment epithelium-choriocapillaris complex identifies factors involved in the pathology of macular degeneration.

The retinal pigment epithelium (RPE)-choriocapillaris (CC) complex in the eye is compromised in age-related macular degeneration (AMD) and related macular dystrophies (MDs), yet in vitro models of RPE-CC complex that enable investigation of AMD/MD pathophysiology are lacking. By incorporating iPSC-derived cells into a hydrogel-based extracellular matrix, we developed a 3D RPE-CC model that recapitulates key features of both healthy and AMD/MD eyes and provides modular control over RPE and CC layers. Using this 3D RPE-CC model, we demonstrated that both RPE- and mesenchyme-secreted factors are necessary for the formation of fenestrated CC-like vasculature.

Generation of T cells from Human and Nonhuman Primate Pluripotent Stem Cells.

Pluripotent stem cells (PSCs) have the potential to provide homogeneous cell populations of T cells that can be grown at a clinical scale and genetically engineered to meet specific clinical needs. OP9-DLL4, a stromal line ectopically expressing the Notch ligand Delta-like 4 (DLL4) is used to support differentiation of PSCs to T-lymphocytes. This article outlines several protocols related to generation of T cells from human and non-human primate (NHP) PSCs, including initial hematopoietic differentiation of PSC on OP9 feeders or defined conditions, followed by coculture of the OP9-DLL4 cells with the PSC-derived hematopoietic progenitors (HPs), leading to efficient differentiation to T lymphocytes.

SOX17 integrates HOXA and arterial programs in hemogenic endothelium to drive definitive lympho-myeloid hematopoiesis.

SOX17 has been implicated in arterial specification and the maintenance of hematopoietic stem cells (HSCs) in the murine embryo. However, knowledge about molecular pathways and stage-specific effects of SOX17 in humans remains limited. Here, using SOX17-knockout and SOX17-inducible human pluripotent stem cells (hPSCs), paired with molecular profiling studies, we reveal that SOX17 is a master regulator of HOXA and arterial programs in hemogenic endothelium (HE) and is required for the specification of HE with robust lympho-myeloid potential and DLL4CXCR4 phenotype resembling arterial HE at the sites of HSC emergence.

Megakaryocytic Expansion in Gilteritinib-Treated Acute Myeloid Leukemia Patients Is Associated With AXL Inhibition.

Numerous recurrent genetic mutations are known to occur in acute myeloid leukemia (AML). Among these common mutations, Fms-like tyrosine kinase 3 remains as one of the most frequently mutated genes in AML. We observed apparent marrow expansion of megakaryocytes in three out of six patients with Flt3-mutated AML following treatment with a recently FDA-approved Flt3 inhibitor, gilteritinib which possesses activity against internal tandem duplication and tyrosine kinase domain Flt3 mutations and also inhibits tyrosine kinase AXL.

Transplantation of T-cell receptor α/β-depleted allogeneic bone marrow in nonhuman primates.

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a potentially curative treatment for hematologic cancers and chronic infections such as human immunodeficiency virus (HIV). Its success in these settings is attributed to the ability of engrafting immune cells to eliminate cancer cells or deplete the HIV reservoir (graft-versus-host effect [GvHE]). However, alloHSCT is commonly associated with graft-versus-host diseases (GvHDs) causing significant morbidity and mortality, thereby requiring development of novel allogeneic HSCT protocols and therapies promoting GvHE without GvHD using physiologically relevant preclinical models.

Genome editing of CCR5 by CRISPR-Cas9 in Mauritian cynomolgus macaque embryos.

The discovery that CCR5 serves as an R5-HIV-1 co-receptor, coupled with findings of protection from HIV infection in individuals lacking CCR5, led to the exploration of novel therapeutic strategies for HIV infection based on genome editing of CCR5. Advancing translation of CCR5-mutant-based cellular therapies for HIV requires development of novel physiologically relevant animal models. Mauritian cynomolgus macaques (MCMs), with high degree of MHC allele sharing, are valuable models for HIV-1 research and stem cell therapies.

Generation of Human Neutrophils from Induced Pluripotent Stem Cells in Chemically Defined Conditions Using Modified mRNA.

This protocol describes a rapid and efficient feeder-, serum-, and xeno-free method for neutrophil generation from hiPSCs using modified mRNA (mmRNA), which directs hematoendothelial programming of hiPSCs. Hematoendothelial progenitors were cultured with GM-CSF, FGF-2, and UM171 to expand myelomonocytic progenitors, followed by treatment with G-CSF and retinoic acid agonist Am580 to induce neutrophil maturation. This protocol is suitable for generating functional neutrophils from iPSCs to interrogate the role of genes in a neutrophil development and function.

Production, safety and efficacy of iPSC-derived mesenchymal stromal cells in acute steroid-resistant graft versus host disease: a phase I, multicenter, open-label, dose-escalation study.

The therapeutic potential of donor-derived mesenchymal stromal cells (MSCs) has been investigated in diverse diseases, including steroid-resistant acute graft versus host disease (SR-aGvHD). However, conventional manufacturing approaches are hampered by challenges with scalability and interdonor variability, and clinical trials have shown inconsistent outcomes. Induced pluripotent stem cells (iPSCs) have the potential to overcome these challenges, due to their capacity for multilineage differentiation and indefinite proliferation.

Cryopreservation of Mauritian Cynomolgus Macaque () Sperm in Chemically Defined Medium.

The objective of this study was to optimize cryopreservation of sperm from Mauritian cynomolgus macaques (MCM) in defined conditions. Sperm viability and motility were compared between sperm cryopreserved in chemically-defined freezing media with variable osmolarity and the presence of either ethylene glycol or glycerol. The highest percentage viability (after freeze-thaw) was seen in sperm samples that were cryopreserved in medium with an osmolarity of 310 mOsm, while higher osmolarities markedly decreased sperm viability.

Effective and Rapid Generation of Functional Neutrophils from Induced Pluripotent Stem Cells Using ETV2-Modified mRNA.

Human induced pluripotent stem cells (hiPSCs) can serve as a versatile and scalable source of neutrophils for biomedical research and transfusion therapies. Here we describe a rapid efficient serum- and xenogen-free protocol for neutrophil generation, which is based on direct hematoendothelial programming of hiPSCs using ETV2-modified mRNA. Culture of ETV2-induced hematoendothelial progenitors in the presence of GM-CSF, FGF2, and UM171 led to continuous production of generous amounts of CD34CD33 myeloid progenitors which could be harvested every 8-10 days for up to 30 days of culture.

Resveratrol trimer enhances gene delivery to hematopoietic stem cells by reducing antiviral restriction at endosomes.

Therapeutic gene delivery to hematopoietic stem cells (HSCs) holds great potential as a life-saving treatment of monogenic, oncologic, and infectious diseases. However, clinical gene therapy is severely limited by intrinsic HSC resistance to modification with lentiviral vectors (LVs), thus requiring high doses or repeat LV administration to achieve therapeutic gene correction. Here we show that temporary coapplication of the cyclic resveratrol trimer caraphenol A enhances LV gene delivery efficiency to human and nonhuman primate hematopoietic stem and progenitor cells with integrating and nonintegrating LVs.

UM171 expands distinct types of myeloid and NK progenitors from human pluripotent stem cells.

Scaling up blood cell production from hPSCs is critical to advancing hPSC technologies for blood transfusion, immunotherapy, and transplantation. Here we explored the potential of the HSC agonist pyrimido-indole derivative UM171, to expand hematopoietic progenitors (HPs) derived from hPSCs in chemically defined conditions. We revealed that culture of hPSC-HPs in HSC expansion conditions (SFEM with added TPO, SCF, FLT3L, IL3 and IL6) in the presence of UM171 predominantly expanded HPs with a unique CD34CD41aCD45 phenotype that were enriched in granulocytic progenitors (G-CFCs).

Cymerus™ iPSC-MSCs significantly prolong survival in a pre-clinical, humanized mouse model of Graft-vs-host disease.

The immune-mediated tissue destruction of graft-vs-host disease (GvHD) remains a major barrier to greater use of hematopoietic stem cell transplantation (HSCT). Mesenchymal stem cells (MSCs) have intrinsic immunosuppressive qualities and are being actively investigated as a therapeutic strategy for treating GvHD. We characterized Cymerus™ MSCs, which are derived from adult, induced pluripotent stem cells (iPSCs), and show they display surface markers and tri-lineage differentiation consistent with MSCs isolated from bone marrow (BM).