Generating human artery and vein cells from pluripotent stem cells highlights the arterial tropism of Nipah and Hendra viruses.

Stem cell research endeavors to generate specific subtypes of classically defined "cell types." Here, we generate >90% pure human artery or vein endothelial cells from pluripotent stem cells within 3-4 days. We specified artery cells by inhibiting vein-specifying signals and vice versa. These cells modeled viral infection of human vasculature by Nipah and Hendra viruses, which are extraordinarily deadly (∼57%-59% fatality rate) and require biosafety-level-4 containment. Generating pure populations of artery and vein cells highlighted that Nipah and Hendra viruses preferentially infected arteries; arteries expressed higher levels of their viral-entry receptor. Virally infected artery cells fused into syncytia containing up to 23 nuclei, which rapidly died. Despite infecting arteries and occupying ∼6%-17% of their transcriptome, Nipah and Hendra largely eluded innate immune detection, minimally eliciting interferon signaling. We thus efficiently generate artery and vein cells, introduce stem-cell-based toolkits for biosafety-level-4 virology, and explore the arterial tropism and cellular effects of Nipah and Hendra viruses.

Lessons from Interspecies Mammalian Chimeras.

As chimeras transform from beasts of Greek mythology into tools of contemporary bioscience, secrets of developmental biology and evolutionary divergence are being revealed. Recent advances in stem cell biology and interspecies chimerism have generated new models with extensive basic and translational applications, including generation of transplantable, patient-specific organs.

Removal of sperm tail using trypsin and pre-activation of oocyte facilitates intracytoplasmic sperm injection in mice and rats.

We examined various methods to enhance the accessibility of intracytoplasmic sperm injection (ICSI) technology to more users by making the technique easier, more efficient, and practical. First, the methods for artificially removing the mouse sperm tail were evaluated. Trypsin treatment was found to efficiently remove the sperm tails. The resultant sperm cells had a lower oocyte activation capacity; however, the use of activated oocytes resulted in the same fecundity as that of fresh, untreated sperm. Pre-activated oocytes were more resistant to physical damage, showed higher survival rates, and required less time per injection. Testing this method in rats yielded similar results, although the oocyte activation method was different. Remarkably, this method resulted in higher birth rates of rat progeny than with conventional methods of rat ICSI. Our method thereby streamlines mouse and rat ICSI, making it more accessible to laboratories across many disciplines.

Sufficiency for inducible Caspase-9 safety switch in human pluripotent stem cells and disease cells.

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have promising potential for opening new avenues in regenerative medicine. However, since the tumorigenic potential of undifferentiated pluripotent stem cells (PSCs) is a major safety concern for clinical transplantation, inducible Caspase-9 (iC9) is under consideration for use as a fail-safe system. Here, we used targeted gene editing to introduce the iC9 system into human iPSCs, and then interrogated the efficiency of inducible apoptosis with normal iPSCs as well as diseased iPSCs derived from patients with acute myeloid leukemia (AML-iPSCs). The iC9 system induced quick and efficient apoptosis to iPSCs in vitro. More importantly, complete eradication of malignant cells without AML recurrence was shown in disease mouse models by using AML-iPSCs. In parallel, it shed light on several limitations of the iC9 system usage. Our results suggest that careful use of the iC9 system will serve as an important countermeasure against posttransplantation adverse events in stem cell transplantation therapies.

Mosaicism diminishes the value of pre-implantation embryo biopsies for detecting CRISPR/Cas9 induced mutations in sheep.

The production of knock-out (KO) livestock models is both expensive and time consuming due to their long gestational interval and low number of offspring. One alternative to increase efficiency is performing a genetic screening to select pre-implantation embryos that have incorporated the desired mutation. Here we report the use of sheep embryo biopsies for detecting CRISPR/Cas9-induced mutations targeting the gene PDX1 prior to embryo transfer. PDX1 is a critical gene for pancreas development and the target gene required for the creation of pancreatogenesis-disabled sheep. We evaluated the viability of biopsied embryos in vitro and in vivo, and we determined the mutation efficiency using PCR combined with gel electrophoresis and digital droplet PCR (ddPCR). Next, we determined the presence of mosaicism in ~ 50% of the recovered fetuses employing a clonal sequencing methodology. While the use of biopsies did not compromise embryo viability, the presence of mosaicism diminished the diagnostic value of the technique. If mosaicism could be overcome, pre-implantation embryo biopsies for mutation screening represents a powerful approach that will streamline the creation of KO animals.

The oligomeric states of the purified sigma-1 receptor are stabilized by ligands.

Sigma-1 receptor (S1R) is a mammalian member of the ERG2 and sigma-1 receptor-like protein family (pfam04622). It has been implicated in drug addiction and many human neurological disorders, including Alzheimer and Parkinson diseases and amyotrophic lateral sclerosis. A broad range of synthetic small molecules, including cocaine, (+)-pentazocine, haloperidol, and small endogenous molecules such as N,N-dimethyltryptamine, sphingosine, and steroids, have been identified as regulators of S1R. However, the mechanism of activation of S1R remains obscure. Here, we provide evidence in vitro that S1R has ligand binding activity only in an oligomeric state. The oligomeric state is prone to decay into an apparent monomeric form when exposed to elevated temperature, with loss of ligand binding activity. This decay is suppressed in the presence of the known S1R ligands such as haloperidol, BD-1047, and sphingosine. S1R has a GXXXG motif in its second transmembrane region, and these motifs are often involved in oligomerization of membrane proteins. Disrupting mutations within the GXXXG motif shifted the fraction of the higher oligomeric states toward smaller states and resulted in a significant decrease in specific (+)-[(3)H]pentazocine binding. Results presented here support the proposal that S1R function may be regulated by its oligomeric state. Possible mechanisms of molecular regulation of interacting protein partners by S1R in the presence of small molecule ligands are discussed.

Secreted Particle Information Transfer (SPIT) - A Cellular Platform for In Vivo Genetic Engineering.

A multitude of tools now exist that allow us to precisely manipulate the human genome in a myriad of different ways. However, successful delivery of these tools to the cells of human patients remains a major barrier to their clinical implementation. Here we introduce a new cellular approach for in vivo genetic engineering, Secreted Particle Information Transfer (SPIT) that utilizes human cells as delivery vectors for in vivo genetic engineering. We demonstrate the application of SPIT for cell-cell delivery of Cre recombinase and CRISPR-Cas9 enzymes, we show that genetic logic can be incorporated into SPIT and present the first demonstration of human cells as a delivery platform for in vivo genetic engineering in immunocompetent mice. We successfully applied SPIT to genetically modify multiple organs and tissue stem cells in vivo including the liver, spleen, intestines, peripheral blood, and bone marrow. We anticipate that by harnessing the large packaging capacity of a human cell's nucleus, the ability of human cells to engraft into patients' long term and the capacity of human cells for complex genetic programming, that SPIT will become a paradigm shifting approach for in vivo genetic engineering.

Skin graft with dermis and appendages generated in vivo by cell competition.

Autologous skin grafting is a standard treatment for skin defects such as burns. No artificial skin substitutes are functionally equivalent to autologous skin grafts. The cultured epidermis lacks the dermis and does not engraft deep wounds. Although reconstituted skin, which consists of cultured epidermal cells on a synthetic dermal substitute, can engraft deep wounds, it requires the wound bed to be well-vascularized and lacks skin appendages. In this study, we successfully generate complete skin grafts with pluripotent stem cell-derived epidermis with appendages on p63 knockout embryos' dermis. Donor pluripotent stem cell-derived keratinocytes encroach the embryos' dermis by eliminating p63 knockout keratinocytes based on cell-extracellular matrix adhesion mediated cell competition. Although the chimeric skin contains allogenic dermis, it is engraftable as long as autologous grafts. Furthermore, we could generate semi-humanized skin segments by human keratinocytes injection into the amnionic cavity of p63 knockout mice embryos. Niche encroachment opens the possibility of human skin graft production in livestock animals.

Secreted Particle Information Transfer (SPIT) - A Cellular Platform for In Vivo Genetic Engineering.

A multitude of tools now exist that allow us to precisely manipulate the human genome in a myriad of different ways. However, successful delivery of these tools to the cells of human patients remains a major barrier to their clinical implementation. Here we introduce a new cellular approach for in vivo genetic engineering, Secreted Particle Information Transfer (SPIT) that utilizes human cells as delivery vectors for in vivo genetic engineering. We demonstrate the application of SPIT for cell-cell delivery of Cre recombinase and CRISPR-Cas9 enzymes, we show that genetic logic can be incorporated into SPIT and present the first demonstration of human cells as a delivery platform for in vivo genetic engineering in immunocompetent mice. We successfully applied SPIT to genetically modify multiple organs and tissue stem cells in vivo including the liver, spleen, intestines, peripheral blood, and bone marrow. We anticipate that by harnessing the large packaging capacity of a human cell's nucleus, the ability of human cells to engraft into patients' long term and the capacity of human cells for complex genetic programming, that SPIT will become a paradigm shifting approach for in vivo genetic engineering.

Genome engineering with Cas9 and AAV repair templates generates frequent concatemeric insertions of viral vectors.

CRISPR-Cas9 paired with adeno-associated virus serotype 6 (AAV6) is among the most efficient tools for producing targeted gene knockins. Here, we report that this system can lead to frequent concatemeric insertions of the viral vector genome at the target site that are difficult to detect. Such errors can cause adverse and unreliable phenotypes that are antithetical to the goal of precision genome engineering. The concatemeric knockins occurred regardless of locus, vector concentration, cell line or cell type, including human pluripotent and hematopoietic stem cells. Although these highly abundant errors were found in more than half of the edited cells, they could not be readily detected by common analytical methods. We describe strategies to detect and thoroughly characterize the concatemeric viral vector insertions, and we highlight analytical pitfalls that mask their prevalence. We then describe strategies to prevent the concatemeric inserts by cutting the vector genome after transduction. This approach is compatible with established gene editing pipelines, enabling robust genetic knockins that are safer, more reliable and more reproducible.

Improved expression and purification of sigma 1 receptor fused to maltose binding protein by alteration of linker sequence.

Sigma 1 receptor (S1R) is a eukaryotic membrane protein that functions as an inter-organelle signaling modulator and chaperone. Here we report an improved expression of S1R in Escherichia coli as a fusion to maltose binding protein (MBP) and a high-yield purification. Variants with linking amino acid sequences consisting of 0-5 alanine residues between MBP and S1R were created and tested in several E. coli expression strains in order to determine the best combination of construct and host for production of active MBP-S1R. Among the linker variations, the protein containing a 4-Ala linker exhibited superior expression characteristics (MBP-4A-S1R); this construct was most productively paired with E. coli B834-pRARE2 and a chemically defined growth and expression medium. A 3-step purification was developed, including extraction from the E. coli membrane fraction using a mixture of Triton X-100 and n-dodecyl-beta-D-maltopyranoside identified by screening constrainted by retention of binding function, and purification by amylose affinity and gel filtration chromatographies. This procedure yields ∼3.5mg of purified fusion protein per L of bacterial culture medium. Purified MBP-4A-S1R showed a 175-fold purification from the starting cellular lysate with respect to specific ligand binding activity, and is stable during concentration and freeze-thaw cycling.

Streamlined and quantitative detection of chimerism using digital PCR.

Animal chimeras are widely used for biomedical discoveries, from developmental biology to cancer research. However, the accurate quantitation of mixed cell types in chimeric and mosaic tissues is complicated by sample preparation bias, transgenic silencing, phenotypic similarity, and low-throughput analytical pipelines. Here, we have developed and characterized a droplet digital PCR single-nucleotide discrimination assay to detect chimerism among common albino and non-albino mouse strains. In addition, we validated that this assay is compatible with crude lysate from all solid organs, drastically streamlining sample preparation. This chimerism detection assay has many additional advantages over existing methods including its robust nature, minimal technical bias, and ability to report the total number of cells in a prepared sample. Moreover, the concepts discussed here are readily adapted to other genomic loci to accurately measure mixed cell populations in any tissue.

Chimpanzee and pig-tailed macaque iPSCs: Improved culture and generation of primate cross-species embryos.

As our closest living relatives, non-human primates uniquely enable explorations of human health, disease, development, and evolution. Considerable effort has thus been devoted to generating induced pluripotent stem cells (iPSCs) from multiple non-human primate species. Here, we establish improved culture methods for chimpanzee (Pan troglodytes) and pig-tailed macaque (Macaca nemestrina) iPSCs. Such iPSCs spontaneously differentiate in conventional culture conditions, but can be readily propagated by inhibiting endogenous WNT signaling. As a unique functional test of these iPSCs, we injected them into the pre-implantation embryos of another non-human species, rhesus macaques (Macaca mulatta). Ectopic expression of gene BCL2 enhances the survival and proliferation of chimpanzee and pig-tailed macaque iPSCs within the pre-implantation embryo, although the identity and long-term contribution of the transplanted cells warrants further investigation. In summary, we disclose transcriptomic and proteomic data, cell lines, and cell culture resources that may be broadly enabling for non-human primate iPSCs research.

Generation of Functional Organs Using a Cell-Competitive Niche in Intra- and Inter-species Rodent Chimeras.

Interspecies organ generation via blastocyst complementation has succeeded in rodents, but not yet in evolutionally more distant species. Early developmental arrest hinders the formation of highly chimeric fetuses. We demonstrate that the deletion of insulin-like growth factor 1 receptor (Igf1r) in mouse embryos creates a permissive "cell-competitive niche" in several organs, significantly augmenting both mouse intraspecies and mouse/rat interspecies donor chimerism that continuously increases from embryonic day 11 onward, sometimes even taking over entire organs within intraspecies chimeras. Since Igf1r deletion allows the evasion of early developmental arrest, interspecies fetuses with high levels of organ chimerism can be generated via blastocyst complementation. This observation should facilitate donor cell contribution to host tissues, resulting in whole-organ generation via blastocyst complementation across wide evolutionary distances.

Author Correction: CRISPR/Cas9 microinjection in oocytes disables pancreas development in sheep.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Interspecies chimeras.

By probing early embryogenesis and regeneration, interspecies chimeras provide a unique platform for discovery and clinical use. Although efficient generation of human:animal chimeric embryos remains elusive, recent advancements attempt to overcome incompatibilities in xenogeneic development and transplantation.

iPSC-Derived Organs In Vivo: Challenges and Promise.

Transplanting iPSCs into the embryos of another species can generate functional organs for basic research and translational applications. We discuss forward-looking approaches and address key remaining challenges of generating iPSC-derived human organs in vivo.

Intra-embryo Gene Cassette Knockin by CRISPR/Cas9-Mediated Genome Editing with Adeno-Associated Viral Vector.

Intra-embryo genome editing by CRISPR/Cas9 enables easy generation of gene-modified animals by non-homologous end joining (NHEJ)-mediated frameshift mutations or homology-directed repair (HDR)-mediated point mutations. However, large modifications, such as gene replacement or gene fusions, are still difficult to introduce in embryos without costly micromanipulators. Moreover, micromanipulation techniques for intra-embryo genome editing have been established in only a small set of animals. To overcome these issues, we developed a method of large-fragment DNA knockin without micromanipulation. In this study, we successfully delivered the knockin donor DNA into zygotes by adeno-associated virus (AAV) without removing the zona pellucida, and we succeeded in both large-DNA fragment knockin and whole exon exchange with electroporation of CRISPR/Cas9 ribonucleoprotein. By this method, we can exchange large DNA fragments conveniently in various animal species without micromanipulation.

Generation of Vascular Endothelial Cells and Hematopoietic Cells by Blastocyst Complementation.

In the case of organ transplantation accompanied by vascular anastomosis, major histocompatibility complex mismatched vascular endothelial cells become a target for graft rejection. Production of a rejection-free, transplantable organ, therefore, requires simultaneous generation of vascular endothelial cells within the organ. To generate pluripotent stem cell (PSC)-derived vascular endothelial cells, we performed blastocyst complementation with a vascular endothelial growth factor receptor-2 homozygous mutant blastocyst. This mutation is embryonic lethal at embryonic (E) day 8.5-9.5 due to an early defect in endothelial and hematopoietic cells. The Flk-1 homozygous knockout chimeric mice survived to adulthood for over 1 year without any abnormality, and all vascular endothelial cells and hematopoietic cells were derived from the injected PSCs. This approach could be used in conjunction with other gene knockouts which induce organ deficiency to produce a rejection-free, transplantable organ in which all the organ's cells and vasculature are PSC derived.

An interspecies barrier to tetraploid complementation and chimera formation.

To study development of the conceptus in xenogeneic environments, we assessed interspecies chimera formation as well as tetraploid complementation between mouse and rat. Overall contribution of donor PSC-derived cells was lower in interspecies chimeras than in intraspecies chimeras, and high donor chimerism was associated with anomalies or embryonic death. Organ to organ variation in donor chimerism was greater in interspecies chimeras than in intraspecies chimeras, suggesting species-specific affinity differences among interacting molecules necessary for organogenesis. In interspecies tetraploid complementation, embryo development was near normal until the stage of placental formation, after which no embryos survived.