Doctor of Philosophy, Stanford University, STMRM-PHD (2021)
BS, University of Wisconsin, Madison, Molecular Biology (2012)
Hiro Nakauchi, Postdoctoral Research Mentor
Removal of sperm tail using trypsin and pre-activation of oocyte facilitates intracytoplasmic sperm injection in mice and rats.
The Journal of reproduction and development
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.
View details for DOI 10.1262/jrd.2022-065
View details for PubMedID 36529517
Chimpanzee and pig-tailed macaque iPSCs: Improved culture and generation of primate cross-species embryos.
2022; 40 (9): 111264
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.
View details for DOI 10.1016/j.celrep.2022.111264
View details for PubMedID 36044843
Streamlined and quantitative detection of chimerism using digital PCR.
2022; 12 (1): 10223
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.
View details for DOI 10.1038/s41598-022-14467-5
View details for PubMedID 35715477
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.
View details for DOI 10.1016/j.cell.2022.05.024
View details for PubMedID 35738284
Generation of Functional Organs Using a Cell-Competitive Niche in Intra- and Inter-species Rodent Chimeras.
Cell stem cell
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.
View details for DOI 10.1016/j.stem.2020.11.019
View details for PubMedID 33373620
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.
View details for DOI 10.1038/s41434-020-0179-z
View details for PubMedID 32704085
Mosaicism diminishes the value of pre-implantation embryo biopsies for detecting CRISPR/Cas9 induced mutations in sheep
2018; 27 (6): 525-537
View details for DOI 10.1007/s11248-018-0094-x
View details for Web of Science ID 000451480100003
Intra-embryo Gene Cassette Knockin by CRISPR/Cas9-Mediated Genome Editing with Adeno-Associated Viral Vector
2018; 9: 286-+
View details for DOI 10.1016/j.isci.2018.10.030
View details for Web of Science ID 000454331400021
Intra-embryo Gene Cassette Knockin by CRISPR/Cas9-Mediated Genome Editing with Adeno-Associated Viral Vector.
2018; 9: 286–97
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.
View details for PubMedID 30447647
An interspecies barrier to tetraploid complementation and chimera formation
2018; 8: 15289
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.
View details for PubMedID 30327488
Generation of Vascular Endothelial Cells and Hematopoietic Cells by Blastocyst Complementation
STEM CELL REPORTS
2018; 11 (4): 988–97
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.
View details for PubMedID 30245211
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.
View details for PubMedID 30284144
Current opinion in genetics & development
2018; 52: 36–41
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.
View details for PubMedID 29859382
iPSC-Derived Organs In Vivo: Challenges and Promise
CELL STEM CELL
2018; 22 (1): 21–24
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.
View details for PubMedID 29304339
CRISPR/Cas9 microinjection in oocytes disables pancreas development in sheep
2017; 7: 17472
One of the ultimate goals of regenerative medicine is the generation of patient-specific organs from pluripotent stem cells (PSCs). Sheep are potential hosts for growing human organs through the technique of blastocyst complementation. We report here the creation of pancreatogenesis-disabled sheep by oocyte microinjection of CRISPR/Cas9 targeting PDX1, a critical gene for pancreas development. We compared the efficiency of target mutations after microinjecting the CRISPR/Cas9 system in metaphase II (MII) oocytes and zygote stage embryos. MII oocyte microinjection reduced lysis, improved blastocyst rate, increased the number of targeted bi-allelic mutations, and resulted in similar degree of mosaicism when compared to zygote microinjection. While the use of a single sgRNA was efficient at inducing mutated fetuses, the lack of complete gene inactivation resulted in animals with an intact pancreas. When using a dual sgRNA system, we achieved complete PDX1 disruption. This PDX1-/- fetus lacked a pancreas and provides the basis for the production of gene-edited sheep as a host for interspecies organ generation. In the future, combining gene editing with CRISPR/Cas9 and PSCs complementation could result in a powerful approach for human organ generation.
View details for PubMedID 29234093
Rapid Chromatin Switch in the Direct Reprogramming of Fibroblasts to Neurons
2017; 20 (13): 3236–47
How transcription factors (TFs) reprogram one cell lineage to another remains unclear. Here, we define chromatin accessibility changes induced by the proneural TF Ascl1 throughout conversion of fibroblasts into induced neuronal (iN) cells. Thousands of genomic loci are affected as early as 12 hr after Ascl1 induction. Surprisingly, over 80% of the accessibility changes occur between days 2 and 5 of the 3-week reprogramming process. This chromatin switch coincides with robust activation of endogenous neuronal TFs and nucleosome phasing of neuronal promoters and enhancers. Subsequent morphological and functional maturation of iN cells is accomplished with relatively little chromatin reconfiguration. By integrating chromatin accessibility and transcriptome changes, we built a network model of dynamic TF regulation during iN cell reprogramming and identified Zfp238, Sox8, and Dlx3 as key TFs downstream of Ascl1. These results reveal a singular, coordinated epigenomic switch during direct reprogramming, in contrast to stepwise cell fate transitions in development.
View details for PubMedID 28954238
Lessons from Interspecies Mammalian Chimeras
ANNUAL REVIEW OF CELL AND DEVELOPMENTAL BIOLOGY, VOL 33
2017; 33: 203–17
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.
View details for PubMedID 28806099
The Oligomeric States of the Purified Sigma-1 Receptor Are Stabilized by Ligands
JOURNAL OF BIOLOGICAL CHEMISTRY
2014; 289 (29): 20333-20344
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.
View details for DOI 10.1074/jbc.M113.537993
View details for Web of Science ID 000339395200044
View details for PubMedID 24847081
View details for PubMedCentralID PMC4106346
Solution Structure of the 2A Protease from a Common Cold Agent, Human Rhinovirus C2, Strain W12
2014; 9 (6)
Human rhinovirus strains differ greatly in their virulence, and this has been correlated with the differing substrate specificity of the respective 2A protease (2Apro). Rhinoviruses use their 2Apro to cleave a spectrum of cellular proteins important to virus replication and anti-host activities. These enzymes share a chymotrypsin-like fold stabilized by a tetra-coordinated zinc ion. The catalytic triad consists of conserved Cys (C105), His (H34), and Asp (D18) residues. We used a semi-automated NMR protocol developed at NMRFAM to determine the solution structure of 2Apro (C105A variant) from an isolate of the clinically important rhinovirus C species (RV-C). The backbone of C2 2Apro superimposed closely (1.41-1.81 Å rmsd) with those of orthologs from RV-A2, coxsackie B4 (CB4), and enterovirus 71 (EV71) having sequence identities between 40% and 60%. Comparison of the structures suggest that the differential functional properties of C2 2Apro stem from its unique surface charge, high proportion of surface aromatics, and sequence surrounding the di-tyrosine flap.
View details for DOI 10.1371/journal.pone.0097198
View details for Web of Science ID 000338503400005
View details for PubMedID 24937088
View details for PubMedCentralID PMC4061012
Improved expression and purification of sigma 1 receptor fused to maltose binding protein by alteration of linker sequence
PROTEIN EXPRESSION AND PURIFICATION
2013; 89 (2): 203-209
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.
View details for DOI 10.1016/j.pep.2013.03.013
View details for Web of Science ID 000319373300013
View details for PubMedID 23562661
View details for PubMedCentralID PMC3679933