A transchromosomic rat model with human chromosome 21 shows robust Down syndrome features.

Progress in earlier detection and clinical management has increased life expectancy and quality of life in people with Down syndrome (DS). However, no drug has been approved to help individuals with DS live independently and fully. Although rat models could support more robust physiological, behavioral, and toxicology analysis than mouse models during preclinical validation, no DS rat model is available as a result of technical challenges. We developed a transchromosomic rat model of DS, TcHSA21rat, which contains a freely segregating, EGFP-inserted, human chromosome 21 (HSA21) with >93% of its protein-coding genes. RNA-seq of neonatal forebrains demonstrates that TcHSA21rat expresses HSA21 genes and has an imbalance in global gene expression. Using EGFP as a marker for trisomic cells, flow cytometry analyses of peripheral blood cells from 361 adult TcHSA21rat animals show that 81% of animals retain HSA21 in >80% of cells, the criterion for a "Down syndrome karyotype" in people. TcHSA21rat exhibits learning and memory deficits and shows increased anxiety and hyperactivity. TcHSA21rat recapitulates well-characterized DS brain morphology, including smaller brain volume and reduced cerebellar size. In addition, the rat model shows reduced cerebellar foliation, which is not observed in DS mouse models. Moreover, TcHSA21rat exhibits anomalies in craniofacial morphology, heart development, husbandry, and stature. TcHSA21rat is a robust DS animal model that can facilitate DS basic research and provide a unique tool for preclinical validation to accelerate DS drug development.

GHR-mutant pig derived from domestic pig and microminipig hybrid zygotes using CRISPR/Cas9 system.

BACKGROUND: Pigs are excellent large animal models with several similarities to humans. They provide valuable insights into biomedical research that are otherwise difficult to obtain from rodent models. However, even if miniature pig strains are used, their large stature compared with other experimental animals requires a specific maintenance facility which greatly limits their usage as animal models. Deficiency of growth hormone receptor (GHR) function causes small stature phenotypes. The establishment of miniature pig strains via GHR modification will enhance their usage as animal models. Microminipig is an incredibly small miniature pig strain developed in Japan. In this study, we generated a GHR mutant pig using electroporation-mediated introduction of the CRISPR/Cas9 system into porcine zygotes derived from domestic porcine oocytes and microminipig spermatozoa. METHODS AND RESULTS: First, we optimized the efficiency of five guide RNAs (gRNAs) designed to target GHR in zygotes. Embryos that had been electroporated with the optimized gRNAs and Cas9 were then transferred into recipient gilts. After embryo transfer, 10 piglets were delivered, and one carried a biallelic mutation in the GHR target region. The GHR biallelic mutant showed a remarkable growth-retardation phenotype. Furthermore, we obtained F1 pigs derived from the mating of GHR biallelic mutant with wild-type microminipig, and GHR biallelic mutant F2 pigs through sib-mating of F1 pigs. CONCLUSIONS: We have successfully demonstrated the generation of biallelic GHR-mutant small-stature pigs. Backcrossing of GHR-deficient pig with microminipig will establish the smallest pig strain which can contribute significantly to the field of biomedical research.

Interspecies organogenesis generates autologous functional islets.

Islet transplantation is an established therapy for diabetes. We have previously shown that rat pancreata can be created from rat pluripotent stem cells (PSCs) in mice through interspecies blastocyst complementation. Although they were functional and composed of rat-derived cells, the resulting pancreata were of mouse size, rendering them insufficient for isolating the numbers of islets required to treat diabetes in a rat model. Here, by performing the reverse experiment, injecting mouse PSCs into Pdx-1-deficient rat blastocysts, we generated rat-sized pancreata composed of mouse-PSC-derived cells. Islets subsequently prepared from these mouse-rat chimaeric pancreata were transplanted into mice with streptozotocin-induced diabetes. The transplanted islets successfully normalized and maintained host blood glucose levels for over 370 days in the absence of immunosuppression (excluding the first 5 days after transplant). These data provide proof-of-principle evidence for the therapeutic potential of PSC-derived islets generated by blastocyst complementation in a xenogeneic host.

Haploid embryonic stem cell lines derived from androgenetic and parthenogenetic rat blastocysts.

The present study was conducted to establish haploid embryonic stem (ES) cell lines using fluorescent marker-carrying rats. In the first series, 7 ES cell lines were established from 26 androgenetic haploid blastocysts. However, only 1 ES cell line (ahES-2) was found to contain haploid cells (1n = 20 + X) by fluorescence-activated cell sorting (FACS) and karyotypic analyses. No chimeras were detected among the 10 fetuses and 41 offspring derived from blastocyst injection with the FACS-purified haploid cells. In the second series, 2 ES cell lines containing haploid cells (13% in phES-1 and 1% in phES-2) were established from 2 parthenogenetic haploid blastocysts. Only the phES-2 cell population was purified by repeated FACS to obtain 33% haploid cells. Following blastocyst injection with the FACS-purified haploid cells, no chimera was observed among the 11 fetuses; however, 1 chimeric male was found among the 47 offspring. Although haploid rat ES cell lines can be established from both blastocyst sources, FACS purification may be necessary for maintenance and chimera production.

Hypomorphic phenotype of Foxn1 gene-modified rats by CRISPR/Cas9 system.

The Foxn1 gene is known as a critical factor for the differentiation of thymic and skin epithelial cells. This study was designed to examine the phenotype of Foxn1-modified rats generated by the CRISPR/Cas9 system. Guide-RNA designed for first exon of the Foxn1 and mRNA of Cas9 were co-injected into the pronucleus of Crlj:WI zygotes. Transfer of 158 injected zygotes resulted in the birth of 50 offspring (32 %), and PCR identified five (10 %) as Foxn1-edited. Genomic sequencing revealed the deletion of 44 or 60 bp from and/or insertion of 4 bp into the Foxn1 gene in a single allele. The number of T-cells in the peripheral blood lymphocytes of mutant rats decreased markedly. While homozygous deleted mutant rats had no thymus, the mutant rats were not completely hairless and showed normal performance in delivery and nursing. Splicing variants of the indel-mutation in the Foxn1 gene may cause hypomorphic allele, resulting in the phenotype of thymus deficiency and incomplete hairless. In conclusion, the mutant rats in Foxn1 gene edited by the CRISPR/Cas9 system showed the phenotype of thymus deficiency and incomplete hairless which was characterized by splicing variants.

High revivability of vitrified-warmed bovine mature oocytes after recovery culture with α-tocopherol.

The objective of this study was to investigate whether developmental competence of vitrified-warmed bovine oocytes can be improved by antioxidant treatment during recovery culture. In experiment 1, one of the two antioxidants (either l-ascorbic acid or α-tocopherol) was added as a supplement to the recovery culture medium to which postwarming oocytes were exposed for 2 h before IVF. The exposure to α-tocopherol had a positive effect on rescuing the oocytes as assessed by the blastocyst yield 8 days after the IVF (35.1-36.3% vs 19.2-25.8% in untreated postwarming oocytes). Quality of expanding blastocysts harvested on Day 8 was comparable between α-tocopherol-treated vitrification group and fresh control group in terms of total cell number and chromosomal ploidy. In experiment 2, level of reactive oxygen species, mitochondrial activity, and distribution of cortical granules in α-tocopherol-treated postwarming oocytes were assessed. No obvious differences from the control data were found in these parameters. However, the treatment with α-tocopherol increased the percentage of zygotes exhibiting normal single aster formation (90.3% vs 48.0% in untreated postwarming oocytes; 10 h post-IVF). It was concluded that α-tocopherol treatment of vitrified-warmed bovine mature oocytes during recovery culture can improve their revivability, as shown by the high blastocyst yield and the higher mean total cell number in the blastocysts.

Adverse effect of cake collapse on the functional integrity of freeze-dried bull spermatozoa.

Under optimal freeze-drying conditions, solutions exhibit a cake-like porous structure. However, if the solution temperature is higher than the glass transition temperature of the maximally freeze-concentrated phase (Tg') during drying phase, the glassy matrix undergoes viscous flow, resulting in cake collapse. The purpose of the present study was to investigate the effect of cake collapse on the integrity of freeze-dried bull spermatozoa. In a preliminary experiment, factors affecting the Tg' of conventional EGTA buffer (consisting of Tris-HCl, EGTA and NaCl) were investigated in order to establish the main experimental protocol because EGTA buffer Tg' was too low (-45.0°C) to suppress collapse. Modification of the EGTA buffer composition by complete removal of NaCl and addition of trehalose (mEGTA buffer) resulted in an increase of Tg' up to -27.7°C. In the main experiment, blastocyst yields after ooplasmic injection of freeze-dried sperm preserved in collapsed cakes (drying temperature: 0 or -15°C) were significantly lower than those of sperm preserved in non-collapsed cake (drying temperature: -30°C). In conclusion, freeze-dried cake collapse may be undesirable for maintaining sperm functions to support embryonic development, and can be inhibited by controlling both Tg' of freeze-drying buffer and temperature during the drying phase.

Effect of leukemia inhibitory factor and forskolin on establishment of rat embryonic stem cell lines.

This study was designed to investigate whether supplementation of 2i medium with leukemia inhibitory factor (LIF) and/or forskolin would support establishment of germline-competent rat embryonic stem (ES) cell lines. Due to the higher likelihood of outgrowth rates, supplementation of forskolin with or without LIF contributed to the higher establishment efficiency of ES cell lines in the WDB strain. Germline transmission competency of the chimeric rats was not influenced by the profile of ES cell lines until their establishment. When the LIF/forskolin-supplemented 2i medium was used, the rat strain used as the blastocyst donor, such as the WI strain, was a possible factor negatively influencing the establishment efficiency of ES cell lines. Once ES cell lines were established, all lines were found to be germline-competent by a progeny test in chimeric rats. In conclusion, both LIF and forskolin are not essential but can play a beneficial role in the establishment of "genuine" rat ES cell lines.

Rescue of vitrified-warmed bovine oocytes with rho-associated coiled-coil kinase inhibitor.

Cryotolerance of matured bovine oocytes is not fully practical even though a promising vitrification procedure with a ultrarapid cooling rate was applied. The present study was conducted to investigate whether recovery culture of vitrified-warmed bovine oocytes with an inhibitor (Y-27632) of Rho-associated coiled-coil kinase (ROCK) can improve the developmental potential after in vitro fertilization (IVF) and in vitro culture. Immediately after warming, almost all oocytes appeared to be morphological normal. Treatment of the postwarming oocytes with 10 µM Y-27632 for 2 h resulted in the significantly higher oocyte survival rate before IVF as well as higher cleavage rate and blastocyst formation rate. Quality analysis of the resultant blastocysts in terms of total cell number and apoptotic cell ratio also showed the positive effect of the Y-27632 treatment. Time-dependent change in mitochondrial activity of the vitrified-warmed oocytes was not influenced by ROCK inhibition during the period of recovery culture. However, the ability of ooplasm to support single-aster formation was improved by the ROCK inhibition. Thus, inhibition of ROCK activity in vitrified-warmed bovine oocytes during a short-term recovery culture can lead to higher developmental competence, probably due to decreased apoptosis and normalized function of the microtubule-organizing center.

Nonviral Ex Vivo Genome Editing in Mouse Bona Fide Hematopoietic Stem Cells with CRISPR/Cas9.

Knock-in therapy, in which an insertion site can be controlled, would be more suitable for the treatment of genetic blood disorders as compared to conventional gene therapy with lentivirus vectors that introduce genes into the genome randomly. Recent advancements in genome editing technology have substantially improved the knock-in efficiency, making it a reality. We present the details of a virus-free CRISPR/Cas9-based genome editing method for bona fide mouse hematopoietic stem cells.

Secretory GFP reconstitution labeling of neighboring cells interrogates cell-cell interactions in metastatic niches.

Cancer cells inevitably interact with neighboring host tissue-resident cells during the process of metastatic colonization, establishing a metastatic niche to fuel their survival, growth, and invasion. However, the underlying mechanisms in the metastatic niche are yet to be fully elucidated owing to the lack of methodologies for comprehensively studying the mechanisms of cell-cell interactions in the niche. Here, we improve a split green fluorescent protein (GFP)-based genetically encoded system to develop secretory glycosylphosphatidylinositol-anchored reconstitution-activated proteins to highlight intercellular connections (sGRAPHIC) for efficient fluorescent labeling of tissue-resident cells that neighbor on and putatively interact with cancer cells in deep tissues. The sGRAPHIC system enables the isolation of metastatic niche-associated tissue-resident cells for their characterization using a single-cell RNA sequencing platform. We use this sGRAPHIC-leveraged transcriptomic platform to uncover gene expression patterns in metastatic niche-associated hepatocytes in a murine model of liver metastasis. Among the marker genes of metastatic niche-associated hepatocytes, we identify Lgals3, encoding galectin-3, as a potential pro-metastatic factor that accelerates metastatic growth and invasion.

Challenging endeavour for preservation of freeze-dried mammalian spermatozoa.

Freeze-drying (lyophilization) has been proposed as an alternative method for sperm preservation to overcome the disadvantages of the current cryopreservation method such as the high maintenance cost of frozen stocks, the problems associated with transportation of frozen materials and the potential risk of total loss of the frozen stock. Since freeze-dried spermatozoa after rehydration lose their motility, which is an essential requirement to complete physiological fertilization, a relatively difficult microinsemination technique must be applied to rehydrated spermatozoa. Theoretically, it has been supposed that freeze-dried spermatozoa could maintain their functions and abilities to interact with the oocyte cytoplasm after prolonged storage at refrigerator temperature. However, sufficient yield of transferable blastocysts and production of live offspring derived from freeze-dried sperm samples are still subjects to be challenged and overcome in large domestic species.

Procedure for bovine ICSI, not sperm freeze-drying, impairs the function of the microtubule-organizing center.

This study was designed to investigate whether freeze-dried (FD) bull spermatozoa maintained the function of the microtubule-organizing center (MTOC) after rehydration and intracytoplasmic sperm injection (ICSI). In a preliminary attempt, the cleavage and blastocyst formation rates in FD-ICSI zygotes (36 and 1%, respectively) were found to be considerably lower than those in control ICSI zygotes (67 and 21%, respectively) or in IVF zygotes (78 and 43%, respectively). An alkaline comet assay indicated that the DNA fragmentation index (length of comet tail % DNA liberated) was not significantly different between fresh and FD spermatozoa. In the main experiment, formation of sperm-asters in the FD-ICSI oocytes 7 h postinsemination occurred at a similar rate when compared with the control ICSI oocytes (41 vs. 49%). Among the oocytes exhibiting sperm aster formation, the extent of microtubule network assembly was comparable between the FD-ICSI and control ICSI groups. However, the MTOC of the ICSI oocytes was not as functional as that of IVF oocytes in terms of the aster formation rate (97%) and the fluorescent intensity of the microtubule network (2.0 folds). These results suggest that the freeze-drying process per se had no adverse effect on maintaining the MTOC function in bull spermatozoa.

Fetal sheep support the development of hematopoietic cells in vivo from human induced pluripotent stem cells.

We report that a sheep fetal liver provides a microenvironment for generating hematopoietic cells with long-term engrafting capacity and multilineage differentiation potential from human induced pluripotent stem cell (iPSC)-derived hemogenic endothelial cells (HEs). Despite the promise of iPSCs for making any cell types, generating hematopoietic stem and progenitor cells (HSPCs) is still a challenge. We hypothesized that the hematopoietic microenvironment, which exists in fetal liver but is lacking in vitro, turns iPSC-HEs into HSPCs. To test this, we transplanted CD45-negative iPSC-HEs into fetal sheep liver, in which HSPCs first grow. Within 2 months, the transplanted cells became CD45 positive and differentiated into multilineage blood cells in the fetal liver. Then, CD45-positive cells translocated to the bone marrow and were maintained there for 3 years with the capability of multilineage differentiation, indicating that hematopoietic cells with long-term engraftment potential were generated. Moreover, human hematopoietic cells were temporally enriched by xenogeneic donor-lymphocyte infusion into the sheep. This study could serve as a foundation to generate HSPCs from iPSCs.

Non-viral ex vivo genome-editing in mouse bona fide hematopoietic stem cells with CRISPR/Cas9.

We conducted two lines of genome-editing experiments of mouse hematopoietic stem cells (HSCs) with the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9). First, to evaluate the genome-editing efficiency in mouse bona fide HSCs, we knocked out integrin alpha 2b (Itga2b) with Cas9 ribonucleoprotein (Cas9/RNP) and performed serial transplantation in mice. The knockout efficiency was estimated at approximately 15%. Second, giving an example of X-linked severe combined immunodeficiency (X-SCID) as a target genetic disease, we showed a proof-of-concept of universal gene correction, allowing rescue of most of X-SCID mutations, in a completely non-viral setting. We inserted partial cDNA of interleukin-2 receptor gamma chain (Il2rg) into intron 1 of Il2rg via non-homologous end-joining (NHEJ) with Cas9/RNP and a homology-independent targeted integration (HITI)-based construct. Repaired HSCs reconstituted T lymphocytes and thymuses in SCID mice. Our results show that a non-viral genome-editing of HSCs with CRISPR/Cas9 will help cure genetic diseases.

Generation of Efficient Knock-in Mouse and Human Pluripotent Stem Cells Using CRISPR-Cas9.

A knock-in can generate fluorescent or Cre-reporter under the control of an endogenous promoter. It also generates knock-out or tagged-protein with fluorescent protein and short tags for tracking and purification. Recent advances in genome editing with clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) significantly increased the efficiencies of making knock-in cells. Here we describe the detailed protocols of generating knock-in mouse and human pluripotent stem cells (PSCs) by electroporation and lipofection, respectively.

Generation of novel Il2rg-knockout mice with clustered regularly interspaced short palindromic repeats (CRISPR) and Cas9.

X-linked severe combined immunodeficiency (X-SCID) is an inherited genetic disorder. A majority of X-SCID subjects carries point mutations in the Interleukin-2 receptor gamma chain (IL2RG) gene. In contrast, Il2rg-knockout mice recapitulating X-SCID phenotype lack a large part of Il2rg instead of point mutations. In this study, we generated novel X-SCID mouse strains with small insertion and deletion (InDel) mutations in Il2rg by using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9. To this end, we injected Streptococcus pyogenes Cas9 (SpCas9) mRNA and single guide RNA targeting the exon 2, 3 or 4 of Il2rg into mouse zygotes. In the F0 generation, we obtained 35 pups and 25 out of them were positive for Surveyor assay, and most of mutants displayed dramatic reductions of T and B lymphocytes in the peripheral blood. By amplicon sequencing, 15 out of 31 founder mice were determined as monoallelic mutants with possible minor mosaicisms while 10 mice were mosaic. Finally, we established new strains with 7-nucleotide deletion and 1-nucleotide insertions in the exon 2 and the exons 3 and 4, respectively. Although no IL2RG protein was detected on T cells of exons 3 and 4 mutants, IL2RG protein was unexpectedly detected in the exon 2 mutants. These data indicated that CRISPR/Cas9 targeting Il2rg causes InDel mutations effectively and generates genetically X-SCID mice. Genetic mutations, however, did not necessarily grant phenotypical alteration, which requires an intensive analysis after establishing a strain to confirm their phenotypes.

Generation of pluripotent stem cell-derived mouse kidneys in Sall1-targeted anephric rats.

Regeneration of human kidneys in animal models would help combat the severe shortage of donors in transplantation therapy. Previously, we demonstrated by interspecific blastocyst complementation between mouse and rats, generation of pluripotent stem cell (PSC)-derived functional pancreas, in apancreatic Pdx1 mutant mice. We, however, were unable to obtain rat PSC-derived kidneys in anephric Sall1 mutant mice, likely due to the poor contribution of rat PSCs to the mouse metanephric mesenchyme, a nephron progenitor. Here, conversely, we show that mouse PSCs can efficiently differentiate into the metanephric mesenchyme in rat, allowing the generation of mouse PSC-derived kidney in anephric Sall1 mutant rat. Glomerular epithelium and renal tubules in the kidneys are entirely composed of mouse PSC-derived cells expressing key functional markers. Importantly, the ureter-bladder junction is normally formed. These data provide proof-of-principle for interspecific blastocyst complementation as a viable approach for kidney generation.

Production and rearing of germ-free X-SCID pigs.

Pigs with X-linked severe combined immunodeficiency (X-SCID) caused by a mutation of the interleukin-2 receptor gamma chain gene (IL2RG) are of value for a wide range of studies. However, they do not survive longer than 8 weeks because of their susceptibility to infections. To allow longer survival of X-SCID pigs, the animals must be born and reared under germ-free conditions. Here, we established an efficient system for piglet derivation by hysterectomy and used it to obtain and maintain a germ-free X-SCID pig. In four trials using pregnant wild-type pigs, 66% of piglets after hysterectomy started spontaneous breathing (range of 20-100% per litter). The resuscitation rate was found to negatively correlate with elapsed time from the uterus excision to piglet derivation (r=-0.97, P<0.05). Therefore, it is critical to deliver piglets within 5 min to achieve a high resuscitation rate (82% estimated from regression analysis). In a fifth trial with an IL2RG+/- pig, four piglets were delivered within 4.2 min of uterus excision and three were alive (75%). One of the live born piglets was genotypically and phenotypically determined to be X-SCID and was reared for 12 weeks. The X-SCID piglet was free from both bacteria and fungi at all time points tested by microbial culture and grew without any abnormal signs or symptoms. This study showed successful production and rearing of germ-free pigs, enabling experiments involving long-term follow-up of X-SCID pigs.