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.

Couple similarity with respect to physical and mental disabilities.

AIMS: Existing research on relationships provides strong evidence of couple similarity on a wide range of variables including physical attributes, abilities, and attitudes. However, couple similarity with respect to disability has not been researched. This study investigated couple similarity with respect to both physical and mental disabilities, as well as associations with life satisfaction, among adult cohabiting couples in Denmark. METHODS: The study analysed data on self-reported mental and physical disabilities from a national survey involving 18,957 participants aged 16 to 65 years. RESULTS: The results showed that participants with a disability were more likely to have a partner with a disability. Further, results showed similarity by type and severity of disability as well as age of onset of disability. Having a partner with a disability was found to be associated with low life satisfaction among men with a disability. Results also showed an association among men with a disability between low life satisfaction and the onset of their disability after (as opposed to before) the start of their relationship. These associations were not found among women with a disability. CONCLUSIONS: The findings provide clear evidence for couple similarity with respect to disability. Findings on life satisfaction showed gender differences that might be explained by cultural gender norms that may play a particular role with respect to disability. Longitudinal research is required to research the factors that mediate how having or developing a disability affects relationships and wellbeing over time.

Interspecific in vitro assay for the chimera-forming ability of human pluripotent stem cells.

Functional assay limitations are an emerging issue in characterizing human pluripotent stem cells (PSCs). With rodent PSCs, chimera formation using pre-implantation embryos is the gold-standard assay of pluripotency (competence of progeny to differentiate into all three germ layers). In human PSCs (hPSCs), however, this can only be monitored via teratoma formation or in vitro differentiation, as ethical concerns preclude generation of human-human or human-animal chimeras. To circumvent this issue, we developed a functional assay utilizing interspecific blastocyst injection and in vitro culture (interspecies in vitro chimera assay) that enables the development and observation of embryos up to headfold stage. The assay uses mouse pre-implantation embryos and rat, monkey and human PSCs to create interspecies chimeras cultured in vitro to the early egg-cylinder stage. Intra- and interspecific chimera assays with rodent PSC lines were performed to confirm the consistency of results in vitro and in vivo. The behavior of chimeras developed in vitro appeared to recapitulate that of chimeras developed in vivo; that is, PSC-derived cells survived and were integrated into the epiblast of egg-cylinder-stage embryos. This indicates that the interspecific in vitro chimera assay is useful in evaluating the chimera-forming ability of rodent PSCs. However, when human induced PSCs (both conventional and naïve-like types) were injected into mouse embryos and cultured, some human cells survived but were segregated; unlike epiblast-stage rodent PSCs, they never integrated into the epiblast of egg-cylinder-stage embryos. These data suggest that the mouse-human interspecies in vitro chimera assay does not accurately reflect the early developmental potential/process of hPSCs. The use of evolutionarily more closely related species as host embryos might be necessary to evaluate the developmental potency of hPSCs.

Liver maturation deficiency in p57(Kip2)-/- mice occurs in a hepatocytic p57(Kip2) expression-independent manner.

Fetal hepatic stem/progenitor cells, hepatoblasts, are highly proliferative cells and the source of both hepatocytes and cholangiocytes. In contrast, mature hepatocytes have a low proliferative potency and high metabolic functions. Cell proliferation is regulated by cell cycle-related molecules. However, the correlation between cell cycle regulation and hepatic maturation are still unknown. To address this issue, we revealed that the cell cycle inhibitor p57(Kip2) was expressed in the hepatoblasts and mesenchymal cells of fetal liver in a spatiotemporal manner. In addition, we found that hepatoblasts in p57(Kip2)-/- mice were highly proliferative and had deficient maturation compared with those in wild-type (WT) mice. However, there were no remarkable differences in the expression levels of cell cycle- and bipotency-related genes except for Ccnd2. Furthermore, p57(Kip2)-/- hepatoblasts could differentiate into mature hepatocytes in p57(Kip2)-/- and WT chimeric mice, suggesting that the intrinsic activity of p57(Kip2) does not simply regulate hepatoblast maturation.

Practical selection methods for rat and mouse round spermatids without DNA staining by flow cytometric cell sorting.

Round spermatid injection (ROSI) into unfertilized oocytes enables a male with a severe spermatogenesis disorder to have children. One limitation of the application of this technique in the clinic is the identification and isolation of round spermatids from testis tissue. Here we developed an efficient and simple method to isolate rodent haploid round spermatids using flow cytometric cell sorting, based on DNA content (stained with Hoechst 33342 or Dye Cycle Violet) or by cell diameter and granularity (forward and side scatter). ROSI was performed with round spermatids selected by flow cytometry, and we obtained healthy offspring from unstained cells. This non-invasive method could therefore be an effective option for breeding domestic animals and human male infertility treatment. Mol. Reprod. Dev. 83: 488-496, 2016. © 2016 Wiley Periodicals, Inc.

Investigation of bipotent differentiation of hepatoblasts using inducible diphtheria toxin receptor-transgenic mice.

AIM: Hepatic progenitor cells, called hepatoblasts, are highly proliferative and exhibit bipotential differentiation into hepatocytes and cholangiocytes in the fetal liver. Thus, they are the ideal source for transplantation therapy. Although several studies have been performed in vitro, the molecular mechanisms regulating hepatoblast differentiation in vivo following transplantation remain poorly understood. The aim of this study was to investigate an in vivo model to analyze hepatoblast bipotency and proliferative ability. METHODS: Hepatic transplantation model using Cre-inducible diphtheria toxin receptor-transgenic mice (iDTR), and albafpCre mice expressing Cre under the control of albumin and α-fetoprotein (AFP) regulatory elements were established. Fresh hepatoblasts were transplanted into diphtheria toxin (DT)-injected iDTRalbafpCre mice and we analyzed their differentiation and proliferation abilities by immunostaining and gene expression profiles. RESULTS: Fresh hepatoblasts transplanted into DT-injected iDTRalbafpCre mice engrafted and differentiated into both hepatocytes and cholangiocytes. Additionally, the number of engrafted hepatoblast-derived hepatocytes increased following partial hepatectomy and serial DT injections. Expression levels of hepatic functional genes in transplanted hepatoblast-derived hepatocytes were similar to that of normal hepatocytes. CONCLUSION: In our iDTRalbafpCre transplantation model, fresh hepatoblasts could differentiate into hepatocytes and cholangiocytes. In addition, these donor cells were induced to proliferate by the following liver injury stimulation. This result suggests that this model is valuable for investigating hepatoblast differentiation pathways in vivo.

Generation of mouse functional oocytes in rat by xeno-ectopic transplantation of primordial germ cells.

Primordial germ cells (PGCs) are germ cell progenitors in the fetal genital ridge; female PGCs give rise to definitive oocytes that contribute to the next generation. Artificial PGCs have been induced in vitro from pluripotent stem cells and gonad-like tissue has been induced in vivo by cotransplantation of PGCs with PGC-free gonadal cells. To apply these technologies to human infertility treatment or conservation of rare species, PGC transplantation must be established in xenogenic animals. Here, we established a xenogeneic transplantation model by inducing ovary-like tissue from PGCs in xenogenic animals. We transplanted enzymatically dispersed PGCs with PGC-free gonadal cells under the kidney capsule of xenogenic immunodeficient animals. The transplanted cells formed ovary-like tissues under the kidney capsule. These tissues were histologically similar to the normal gonad and expressed the oocyte markers Vasa and Stella. In addition, mouse germinal vesicle-stage oocyte-like cells collected from ovary-like tissue in rats matured to metaphase II via in vitro maturation and gave rise to offspring by intracytoplasmic sperm injection. Our studies show that rat/mouse female PGCs and PGC-free gonadal cells can develop and reconstruct ovary-like tissue containing functional oocytes in an ectopic xenogenic microenvironment.

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.

Continuous cell supply from Krt7-expressing hematopoietic stem cells during native hematopoiesis revealed by targeted in vivo gene transfer method.

The nature of hematopoietic stem cells under normal hematopoiesis remained largely unknown due to the limited assays available to monitor their behavior in situ. Here, we develop a new mouse model to transfer genes specifically into the primitive hematopoietic stem cell compartment through the utilization of a modified Rcas/TVA system. We succeeded in transferring a GFP reporter gene into adult hematopoietic stem cells in vivo, which are predominantly quiescent, by generating pseudotyped-lentivirus. Furthermore, we demonstrate the utility of this system to study neonatal hematopoiesis, a developmental stage that has been difficult to analyze to date. Using the system developed in this study, we observed continuous multi-lineage hematopoietic cell supply in peripheral blood from Krt7-positive hematopoietic stem cells during unperturbed homeostatic condition. This powerful experimental system could provide a new standard tool to analyze hematopoiesis under physiological condition without transplantation.

A Novel Mouse Model of iNKT Cell-deficiency Generated by CRISPR/Cas9 Reveals a Pathogenic Role of iNKT Cells in Metabolic Disease.

iNKT cells play important roles in immune regulation by bridging the innate and acquired immune systems. The functions of iNKT cells have been investigated in mice lacking the Traj18 gene segment that were generated by traditional embryonic stem cell technology, but these animals contain a biased T cell receptor (TCR) repertoire that might affect immune responses. To circumvent this confounding factor, we have generated a new strain of iNKT cell-deficient mice by deleting the Traj18 locus using CRISPR/Cas9 technology, and these animals contain an unbiased TCR repertoire. We employed these mice to investigate the contribution of iNKT cells to metabolic disease and found a pathogenic role of these cells in obesity-associated insulin-resistance. The new Traj18-deficient mouse strain will assist in studies of iNKT cell biology.

Inhibition of Apoptosis Overcomes Stage-Related Compatibility Barriers to Chimera Formation in Mouse Embryos.

Cell types more advanced in development than embryonic stem cells, such as EpiSCs, fail to contribute to chimeras when injected into pre-implantation-stage blastocysts, apparently because the injected cells undergo apoptosis. Here we show that transient promotion of cell survival through expression of the anti-apoptotic gene BCL2 enables EpiSCs and Sox17+ endoderm progenitors to integrate into blastocysts and contribute to chimeric embryos. Upon injection into blastocyst, BCL2-expressing EpiSCs contributed to all bodily tissues in chimeric animals while Sox17+ endoderm progenitors specifically contributed in a region-specific fashion to endodermal tissues. In addition, BCL2 expression enabled rat EpiSCs to contribute to mouse embryonic chimeras, thereby forming interspecies chimeras that could survive to adulthood. Our system therefore provides a method to overcome cellular compatibility issues that typically restrict chimera formation. Application of this type of approach could broaden the use of embryonic chimeras, including region-specific chimeras, for basic developmental biology research and regenerative medicine.

Development of an all-in-one inducible lentiviral vector for gene specific analysis of reprogramming.

Fair comparison of reprogramming efficiencies and in vitro differentiation capabilities among induced pluripotent stem cell (iPSC) lines has been hampered by the cellular and genetic heterogeneity of de novo infected somatic cells. In order to address this problem, we constructed a single cassette all-in-one inducible lentiviral vector (Ai-LV) for the expression of three reprogramming factors (Oct3/4, Klf4 and Sox2). To obtain multiple types of somatic cells having the same genetic background, we generated reprogrammable chimeric mice using iPSCs derived from Ai-LV infected somatic cells. Then, hepatic cells, hematopoietic cells and fibroblasts were isolated at different developmental stages from the chimeric mice, and reprogrammed again to generate 2nd iPSCs. The results revealed that somatic cells, especially fetal hepatoblasts were reprogrammed 1200 times more efficiently than adult hepatocytes with maximum reprogramming efficiency reaching 12.5%. However, we found that forced expression of c-Myc compensated for the reduced reprogramming efficiency in aged somatic cells without affecting cell proliferation. All these findings suggest that the Ai-LV system enables us to generate a panel of iPSC clones derived from various tissues with the same genetic background, and thus provides an invaluable tool for iPSC research.

Generation of germline-competent rat induced pluripotent stem cells.

BACKGROUND: Recent progress in rat pluripotent stem cell technology has been remarkable. Particularly salient is the demonstration that embryonic stem cells (ESCs) in the rat (rESCs) can contribute to germline transmission, permitting generation of gene-modified rats as is now done using mouse ESCs (mESCs) or mouse induced pluripotent stem cells (iPSCs; miPSCs). However, determinations of whether rat iPSCs (riPSCs) can contribute to germ cells are not published. Here we report the germline competency of riPSCs. METHODOLOGY/PRINCIPAL FINDINGS: We generated riPSCs by transducing three mouse reprogramming factors (Oct3/4, Klf4, and Sox2) into rat somatic cells, followed by culture in the presence of exogenous rat leukemia inhibitory factor (rLIF) and small molecules that specifically inhibit GSK3, MEK, and FGF receptor tyrosine kinases. We found that, like rESCs, our riPSCs can contribute to germline transmission. Furthermore we found, by immunostaining of testis from mouse-rat interspecific chimeras with antibody against mouse vasa homolog, that riPSCs can contribute to embryonic development with chimera formation in mice (rat-mouse interspecific chimeras) and to interspecific germlines. CONCLUSIONS/SIGNIFICANCE: Our data clearly demonstrate that using only three reprogramming factors (Oct3/4, Klf4, and Sox2) rat somatic cells can be reprogrammed into a ground state. Our generated riPSCs exhibited germline transmission in either rat-rat intraspecific or mouse-rat interspecific chimeras.