Hypoblast from human pluripotent stem cells regulates epiblast development.

Recently, several studies using cultures of human embryos together with single-cell RNA-seq analyses have revealed differences between humans and mice, necessitating the study of human embryos1-8. Despite the importance of human embryology, ethical and legal restrictions have limited post-implantation-stage studies. Thus, recent efforts have focused on developing in vitro self-organizing models using human stem cells9-17. Here, we report genetic and non-genetic approaches to generate authentic hypoblast cells (naive hPSC-derived hypoblast-like cells (nHyCs))-known to give rise to one of the two extraembryonic tissues essential for embryonic development-from naive human pluripotent stem cells (hPSCs). Our nHyCs spontaneously assemble with naive hPSCs to form a three-dimensional bilaminar structure (bilaminoids) with a pro-amniotic-like cavity. In the presence of additional naive hPSC-derived analogues of the second extraembryonic tissue, the trophectoderm, the efficiency of bilaminoid formation increases from 20% to 40%, and the epiblast within the bilaminoids continues to develop in response to trophectoderm-secreted IL-6. Furthermore, we show that bilaminoids robustly recapitulate the patterning of the anterior-posterior axis and the formation of cells reflecting the pregastrula stage, the emergence of which can be shaped by genetically manipulating the DKK1/OTX2 hypoblast-like domain. We have therefore successfully modelled and identified the mechanisms by which the two extraembryonic tissues efficiently guide the stage-specific growth and progression of the epiblast as it establishes the post-implantation landmarks of human embryogenesis.

Functional calcium-responsive parathyroid glands generated using single-step blastocyst complementation.

Patients with permanent hypoparathyroidism require lifelong replacement therapy to avoid life-threatening complications, The benefits of conventional treatment are limited, however. Transplanting a functional parathyroid gland (PTG) would yield better results. Parathyroid gland cells generated from pluripotent stem cells in vitro to date cannot mimic the physiological responses to extracellular calcium that are essential for calcium homeostasis. We thus hypothesized that blastocyst complementation (BC) could be a better strategy for generating functional PTG cells and compensating loss of parathyroid function. We here describe generation of fully functional PTGs from mouse embryonic stem cells (mESCs) with single-step BC. Using CRISPR-Cas9 knockout of Glial cells missing2 (Gcm2), we efficiently produced aparathyroid embryos for BC. In these embryos, mESCs differentiated into endocrinologically mature PTGs that rescued Gcm2-/- mice from neonatal death. The mESC-derived PTGs responded to extracellular calcium, restoring calcium homeostasis on transplantation into mice surgically rendered hypoparathyroid. We also successfully generated functional interspecies PTGs in Gcm2-/- rat neonates, an accomplishment with potential for future human PTG therapy using xenogeneic animal BC. Our results demonstrate that BC can produce functional endocrine organs and constitute a concept in treatment of hypoparathyroidism.

Pluripotent stem cells related to embryonic disc exhibit common self-renewal requirements in diverse livestock species.

Despite four decades of effort, robust propagation of pluripotent stem cells from livestock animals remains challenging. The requirements for self-renewal are unclear and the relationship of cultured stem cells to pluripotent cells resident in the embryo uncertain. Here, we avoided using feeder cells or serum factors to provide a defined culture microenvironment. We show that the combination of activin A, fibroblast growth factor and the Wnt inhibitor XAV939 (AFX) supports establishment and continuous expansion of pluripotent stem cell lines from porcine, ovine and bovine embryos. Germ layer differentiation was evident in teratomas and readily induced in vitro. Global transcriptome analyses highlighted commonality in transcription factor expression across the three species, while global comparison with porcine embryo stages showed proximity to bilaminar disc epiblast. Clonal genetic manipulation and gene targeting were exemplified in porcine stem cells. We further demonstrated that genetically modified AFX stem cells gave rise to cloned porcine foetuses by nuclear transfer. In summary, for major livestock mammals, pluripotent stem cells related to the formative embryonic disc are reliably established using a common and defined signalling environment. This article has an associated 'The people behind the papers' interview.

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.

Establishment of mouse expanded potential stem cells.

Mouse embryonic stem cells derived from the epiblast contribute to the somatic lineages and the germline but are excluded from the extra-embryonic tissues that are derived from the trophectoderm and the primitive endoderm upon reintroduction to the blastocyst. Here we report that cultures of expanded potential stem cells can be established from individual eight-cell blastomeres, and by direct conversion of mouse embryonic stem cells and induced pluripotent stem cells. Remarkably, a single expanded potential stem cell can contribute both to the embryo proper and to the trophectoderm lineages in a chimaera assay. Bona fide trophoblast stem cell lines and extra-embryonic endoderm stem cells can be directly derived from expanded potential stem cells in vitro. Molecular analyses of the epigenome and single-cell transcriptome reveal enrichment for blastomere-specific signature and a dynamic DNA methylome in expanded potential stem cells. The generation of mouse expanded potential stem cells highlights the feasibility of establishing expanded potential stem cells for other mammalian species.

Interspecies chimeras for human stem cell research.

Interspecies chimeric assays are a valuable tool for investigating the potential of human stem and progenitor cells, as well as their differentiated progeny. This Spotlight article discusses the different factors that affect interspecies chimera generation, such as evolutionary distance, developmental timing, and apoptosis of the transplanted cells, and suggests some possible strategies to address them. A refined approach to generating interspecies chimeras could contribute not only to a better understanding of cellular potential, but also to understanding the nature of xenogeneic barriers and mechanisms of heterochronicity, to modeling human development, and to the creation of human transplantable organs.

Bazedoxifene improves renal function and increases renal phosphate excretion in patients with postmenopausal osteoporosis.

INTRODUCTION: Because aging is a predictor of renal insufficiency in the general population, renal function is a concern in postmenopausal patients undergoing treatment for osteoporosis. Although high serum phosphate concentration is a predictor of renal insufficiency, the effect of selective estrogen receptor modulator (SERM) on renal function and phosphate homeostasis remains to be established. MATERIALS AND METHODS: We administered 20 mg/day bazedoxifene to 48 postmenopausal osteoporotic women who had been taking alfacalcidol for ≥ 6 months, and assessed lumbar spine bone mineral density (LS-BMD), renal function (by calculating estimated glomerular filtration rate using serum cystatin-C levels [eGFRcys] [range 38.0-98.2 mL/min/1.73 m2]), and phosphate homeostasis. RESULTS: LS-BMD was significantly higher 6 months after the initiation of bazedoxifene administration. eGFRcys had increased by 3 months after initiation and was stable until 12 months. Serum phosphate gradually decreased after initiation, reaching statistical significance at 6 months. The changes in serum phosphate were also significant when the maximum tubular reabsorption rate of phosphate was normalized to glomerular filtration rate (TmP/GFR), indicating that bazedoxifene treatment reduces serum phosphate by increasing the urinary excretion of phosphate. The change in eGFRcys after the initiation of bazedoxifene was significantly negatively correlated with the change in serum phosphate, suggesting that a reduction in serum phosphate improves renal function. CONCLUSION: Bazedoxifene improves renal function, possibly by increasing renal phosphate excretion, in postmenopausal osteoporotic women without severe renal insufficiency.

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.

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.

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 engraftable hematopoietic stem cells from induced pluripotent stem cells by way of teratoma formation.

In vitro generation of hematopoietic stem cells (HSCs) from induced pluripotent stem cells (iPSCs) has the potential to provide novel therapeutic approaches for replacing bone marrow (BM) transplantation without rejection or graft versus host disease. Hitherto, however, it has proved difficult to generate truly functional HSCs transplantable to adult host mice. Here, we demonstrate a unique in vivo differentiation system yielding engraftable HSCs from mouse and human iPSCs in teratoma-bearing animals in combination with a maneuver to facilitate hematopoiesis. In mice, we found that iPSC-derived HSCs migrate from teratomas into the BM and their intravenous injection into irradiated recipients resulted in multilineage and long-term reconstitution of the hematolymphopoietic system in serial transfers. Using this in vivo generation system, we could demonstrate that X-linked severe combined immunodeficiency (X-SCID) mice can be treated by HSCs derived from gene-corrected clonal iPSCs. It should also be noted that neither leukemia nor tumors were observed in recipients after transplantation of iPSC-derived HSCs. Taken our findings together, our system presented in this report should provide a useful tool not only for the study of HSCs, but also for practical application of iPSCs in the treatment of hematologic and immunologic diseases.

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.

Inter-cellular mRNA Transfer Alters Human Pluripotent Stem Cell State.

Inter-cellular transmission of mRNA is being explored in mammalian species using immortal cell lines (1-3). Here, we uncover an inter-cellular mRNA transfer phenomenon that allows for the adaptation and reprogramming of human primed pluripotent stem cells (hPSCs). This process is induced by the direct cell contact-mediated coculture with mouse embryonic stem cells (mESCs) under the condition impermissible for human primed PSC culture. Mouse-derived mRNA contents are transmitted into adapted hPSCs only in the coculture. Transfer-specific mRNA analysis show the enrichment for divergent biological pathways involving transcription/translational machinery and stress-coping mechanisms, wherein such transfer is diminished when direct cell contacts are lost. After 5 days of mESC culture, surface marker analysis, and global gene profiling confirmed that mRNA transfer-prone hPSC efficiently gains a naïve-like state. Furthermore, transfer-specific knockdown experiments targeting mouse-specific transcription factor-coding mRNAs in hPSC show that mouse-derived Tfcp2l1, Tfap2c, and Klf4 are indispensable for human naïve-like conversion. Thus, inter-species mRNA transfer triggers cellular reprogramming in mammalian cells. Our results support that episodic mRNA transfer can occur in cell cooperative and competitive processes(4), which provides a fresh perspective on understanding the roles of mRNA mobility for intra- and inter-species cellular communications.

Two cases of dementias with motor neuron disease evaluated by Pittsburgh compound B-positron emission tomography.

We described the cases of two patients with dementia associated with motor neuron disease, the former with frontotemporal dementia (FTD) and the latter with Alzheimer's disease (AD), studied by the Pittsburgh compound B-positron emission tomography (PIB-PET). In the FTD patient, the PIB-PET revealed no amyloid accumulation in the cortex, whilst in the AD patient showed amyloid accumulation mainly in the frontal, parietal and lateral temporal lobes, besides the posterior cingulate gyrus and the precuneus. Thus, PIB-PET might facilitate the discrimination of different proteinopathies that cause neurodegenerative diseases, as dementia associated with ALS.

[New approved markers of bone turnover for osteoporosis in Japan].

Various markers of bone turnover are already under clinical use in Japan, and mostly for clinical investigation in some countries. Standard values including ranges and variations are summarized in the previous edition of the guideline. The information of additional new markers adapted by government is summarized including clinical features in the new edition 2012. Among the new markers, the methods for measurement for TRACP-5b and ucOC are developed in Japan. As P1NP and TRACP-5b levels are not affected by meals, biological variations are smaller compared with other markers. ucOC is unique because it is to evaluate vitamin K insufficiency for bone. New bone markers adapted in the Japanese guideline 2012 will facilitate clinicians to utilize of metabolic markers of bone for osteoporosis treatment.

Xenotransplantation and interspecies organogenesis: current status and issues.

Pancreas (and islet) transplantation is the only curative treatment for type 1 diabetes patients whose ß-cell functions have been abolished. However, the lack of donor organs has been the major hurdle to save a large number of patients. Therefore, transplantation of animal organs is expected to be an alternative method to solve the serious shortage of donor organs. More recently, a method to generate organs from pluripotent stem cells inside the body of other species has been developed. This interspecies organ generation using blastocyst complementation (BC) is expected to be the next-generation regenerative medicine. Here, we describe the recent advances and future prospects for these two approaches.

In vitro and in vivo functions of T cells produced in complemented thymi of chimeric mice generated by blastocyst complementation.

Blastocyst complementation is an intriguing way of generating humanized animals for organ preparation in regenerative medicine and establishing novel models for drug development. Confirming that complemented organs and cells work normally in chimeric animals is critical to demonstrating the feasibility of blastocyst complementation. Here, we generated thymus-complemented chimeric mice, assessed the efficacy of anti-PD-L1 antibody in tumor-bearing chimeric mice, and then investigated T-cell function. Thymus-complemented chimeric mice were generated by injecting C57BL/6 (B6) embryonic stem cells into Foxn1nu/nu morulae or blastocysts. Flow cytometry data showed that the chimeric mouse thymic epithelial cells (TECs) were derived from the B6 cells. T cells appeared outside the thymi. Single-cell RNA-sequencing analysis revealed that the TEC gene-expression profile was comparable to that in B6 mice. Splenic T cells of chimeric mice responded very well to anti-CD3 stimulation in vitro; CD4+ and CD8+ T cells proliferated and produced IFNγ, IL-2, and granzyme B, as in B6 mice. Anti-PD-L1 antibody treatment inhibited MC38 tumor growth in chimeric mice. Moreover, in the chimeras, anti-PD-L1 antibody restored T-cell activation by significantly decreasing PD-1 expression on T cells and increasing IFNγ-producing T cells in the draining lymph nodes and tumors. T cells produced by complemented thymi thus functioned normally in vitro and in vivo. To successfully generate humanized animals by blastocyst complementation, both verification of the function and gene expression profiling of complemented organs/cells in interspecific chimeras will be important in the near future.

[Bone and calcium metabolism in elderly women].

Bone mass of elderly women reduces after menopause, but the rate of reduction differ individually especially in the elderly, because bone metabolism of the elderly is influenced by many factors such as hormones, fractures, drugs, ability of exercise and/or nutritional level. Therefore, it is quite difficult to determine the normal ranges of the metabolism. As a whole, aging accelerates bone turnover slowly with wide range of individual variation. Subnormal renal function may effect on the excretion or metabolism of bone markers, such as osteocalcin. Urinary markers such as NTX corrected by urinary creatinine may be higher than the real level, because of low creatinine production in the elderly. These factors may influence on the elevated bone metabolism resulting in the discrepancy with morphometric evaluation.

Hedgehog Activation Regulates Human Osteoblastogenesis.

Two genetic diseases, Gorlin syndrome and McCune-Albright syndrome (MAS), show completely opposite symptoms in terms of bone mineral density and hedgehog (Hh) activity. In this study, we utilized human induced pluripotent stem cell (iPSC)-based models of the two diseases to understand the roles of Hh signaling in osteogenesis. Gorlin syndrome-derived iPSCs showed increased osteoblastogenesis and mineralization with Hh signaling activation and upregulation of a set of transcription factors in an osteogenic culture, compared with the isogenic control. MAS-specific iPSCs showed poor mineralization with low Hh signaling activity in the osteogenic culture; impaired osteoblastogenesis was restored to the normal level by treatment with an Hh signaling-activating small molecule. These data suggest that Hh signaling is a key controller for differentiation of osteoblasts from precursors. This study may pave a path to new drug therapies for genetic abnormalities in calcification caused by dysregulation of Hh signaling.