Chemically defined cytokine-free expansion of human haematopoietic stem cells.

Haematopoietic stem cells (HSCs) are a rare cell type that reconstitute the entire blood and immune systems after transplantation and can be used as a curative cell therapy for a variety of haematological diseases1,2. However, the low number of HSCs in the body makes both biological analyses and clinical application difficult, and the limited extent to which human HSCs can be expanded ex vivo remains a substantial barrier to the wider and safer therapeutic use of HSC transplantation3. Although various reagents have been tested in attempts to stimulate the expansion of human HSCs, cytokines have long been thought to be essential for supporting HSCs ex vivo4. Here we report the establishment of a culture system that allows the long-term ex vivo expansion of human HSCs, achieved through the complete replacement of exogenous cytokines and albumin with chemical agonists and a caprolactam-based polymer. A phosphoinositide 3-kinase activator, in combination with a thrombopoietin-receptor agonist and the pyrimidoindole derivative UM171, were sufficient to stimulate the expansion of umbilical cord blood HSCs that are capable of serial engraftment in xenotransplantation assays. Ex vivo HSC expansion was further supported by split-clone transplantation assays and single-cell RNA-sequencing analysis. Our chemically defined expansion culture system will help to advance clinical HSC therapies.

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.

Derivation of ground-state female ES cells maintaining gamete-derived DNA methylation.

Inhibitors of Mek1/2 and Gsk3ß, known as 2i, enhance the derivation of embryonic stem (ES) cells and promote ground-state pluripotency in rodents. Here we show that the derivation of female mouse ES cells in the presence of 2i and leukaemia inhibitory factor (2i/L ES cells) results in a widespread loss of DNA methylation, including a massive erasure of genomic imprints. Despite this global loss of DNA methylation, early-passage 2i/L ES cells efficiently differentiate into somatic cells, and this process requires genome-wide de novo DNA methylation. However, the majority of imprinting control regions (ICRs) remain unmethylated in 2i/L-ES-cell-derived differentiated cells. Consistently, 2i/L ES cells exhibit impaired autonomous embryonic and placental development by tetraploid embryo complementation or nuclear transplantation. We identified the derivation conditions of female ES cells that display 2i/L-ES-cell-like transcriptional signatures while preserving gamete-derived DNA methylation and autonomous developmental potential. Upon prolonged culture, however, female ES cells exhibited ICR demethylation regardless of culture conditions. Our results provide insights into the derivation of female ES cells reminiscent of the inner cell mass of preimplantation embryos.

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.

Improved development of mouse somatic cell nuclear transfer embryos by chlamydocin analogues, class I and IIa histone deacetylase inhibitors†.

In mammalian cloning by somatic cell nuclear transfer (SCNT), the treatment of reconstructed embryos with histone deacetylase (HDAC) inhibitors improves efficiency. So far, most of those used for SCNT are hydroxamic acid derivatives-such as trichostatin A-characterized by their broad inhibitory spectrum. Here, we examined whether mouse SCNT efficiency could be improved using chlamydocin analogues, a family of newly designed agents that specifically inhibit class I and IIa HDACs. Development of SCNT-derived embryos in vitro and in vivo revealed that four out of five chlamydocin analogues tested could promote the development of cloned embryos. The highest pup rates (7.1-7.2%) were obtained with Ky-9, similar to those achieved with trichostatin A (7.2-7.3%). Thus, inhibition of class I and/or IIa HDACs in SCNT-derived embryos is enough for significant improvements in full-term development. In mouse SCNT, the exposure of reconstructed oocytes to HDAC inhibitors is limited to 8-10 h because longer inhibition with class I inhibitors causes a two-cell developmental block. Therefore, we used Ky-29, with higher selectivity for class IIa than class I HDACs for longer treatment of SCNT-derived embryos. As expected, 24-h treatment with Ky-29 up to the two-cell stage did not induce a developmental block, but the pup rate was not improved. This suggests that the one-cell stage is a critical period for improving SCNT cloning using HDAC inhibitors. Thus, chlamydocin analogues appear promising for understanding and improving the epigenetic status of mammalian SCNT-derived embryos through their specific inhibitory effects on HDACs.

Healthy offspring from freeze-dried mouse spermatozoa held on the International Space Station for 9 months.

If humans ever start to live permanently in space, assisted reproductive technology using preserved spermatozoa will be important for producing offspring; however, radiation on the International Space Station (ISS) is more than 100 times stronger than that on Earth, and irradiation causes DNA damage in cells and gametes. Here we examined the effect of space radiation on freeze-dried mouse spermatozoa held on the ISS for 9 mo at -95 °C, with launch and recovery at room temperature. DNA damage to the spermatozoa and male pronuclei was slightly increased, but the fertilization and birth rates were similar to those of controls. Next-generation sequencing showed only minor genomic differences between offspring derived from space-preserved spermatozoa and controls, and all offspring grew to adulthood and had normal fertility. Thus, we demonstrate that although space radiation can damage sperm DNA, it does not affect the production of viable offspring after at least 9 mo of storage on the ISS.

The Number of Point Mutations in Induced Pluripotent Stem Cells and Nuclear Transfer Embryonic Stem Cells Depends on the Method and Somatic Cell Type Used for Their Generation.

Induced pluripotent stem cells hold great promise for regenerative medicine but point mutations have been identified in these cells and have raised serious concerns about their safe use. We generated nuclear transfer embryonic stem cells (ntESCs) from both mouse embryonic fibroblasts (MEFs) and tail-tip fibroblasts (TTFs) and by whole genome sequencing found fewer mutations compared with iPSCs generated by retroviral gene transduction. Furthermore, TTF-derived ntESCs showed only a very small number of point mutations, approximately 80% less than the number observed in iPSCs generated using retrovirus. Base substitution profile analysis confirmed this greatly reduced number of point mutations. The point mutations in iPSCs are therefore not a Yamanaka factor-specific phenomenon but are intrinsic to genome reprogramming. Moreover, the dramatic reduction in point mutations in ntESCs suggests that most are not essential for genome reprogramming. Our results suggest that it is feasible to reduce the point mutation frequency in iPSCs by optimizing various genome reprogramming conditions. We conducted whole genome sequencing of ntES cells derived from MEFs or TTFs. We thereby succeeded in establishing TTF-derived ntES cell lines with far fewer point mutations. Base substitution profile analysis of these clones also indicated a reduced point mutation frequency, moving from a transversion-predominance to a transition-predominance. Stem Cells 2017;35:1189-1196.

RNA sequencing-based identification of aberrant imprinting in cloned mice.

Animals cloned by somatic cell nuclear transfer (SCNT) provide a unique model for understanding the mechanisms of nuclear epigenetic reprogramming to a state of totipotency. Though many phenotypic abnormalities have been demonstrated in cloned animals, the underlying mechanisms are not well understood. In this study, we performed transcriptome-wide allelic expression analyses in brain and placental tissues of cloned mice. We found that Gab1, Sfmbt2 and Slc38a4 showed loss of imprinting in all cloned mice analyzed, which might be involved in placentomegaly of cloned mice. These three genes did not require de novo DNA methylation in growing oocytes for the establishment of imprinting, implying the involvement of a de novo DNA methylation-independent mechanism. Loss of Dlk1-Dio3 imprinting was also observed in nearly half of cloned mouse embryos and showed a strong correlation with embryonic lethality. Our findings are essential to understand the underlying mechanisms of developmental abnormalities of cloned animals. We also emphasize that particular attention should be paid to specific imprinted genes for therapeutic and agricultural applications of SCNT.

DNA Methylation Errors in Cloned Mouse Sperm by Germ Line Barrier Evasion.

The germ line reprogramming barrier resets parental epigenetic modifications according to sex, conferring totipotency to mammalian embryos upon fertilization. However, it is not known whether epigenetic errors are committed during germ line reprogramming that are then transmitted to germ cells, and consequently to offspring. We addressed this question in the present study by performing a genome-wide DNA methylation analysis using a target postbisulfite sequencing method in order to identify DNA methylation errors in cloned mouse sperm. The sperm genomes of two somatic cell-cloned mice (CL1 and CL7) contained significantly higher numbers of differentially methylated CpG sites (P = 0.0045 and P = 0.0116). As a result, they had higher numbers of differentially methylated CpG islands. However, there was no evidence that these sites were transmitted to the sperm genome of offspring. These results suggest that DNA methylation errors resulting from embryo cloning are transmitted to the sperm genome by evading the germ line reprogramming barrier.

Generation of cloned mice from adult neurons by direct nuclear transfer.

Whereas cloning mammals by direct somatic cell nuclear transfer has been successful using a wide range of donor cell types, neurons from adult brain remain "unclonable" for unknown reasons. Here, using a combination of two epigenetic approaches, we examined whether neurons from adult mice could be cloned. First, we used a specific antibody to discover cell types with reduced amounts of a repressive histone mark-dimethylated histone H3 lysine 9 (H3K9me2)-and identified CA1 pyramidal cells in the hippocampus and Purkinje cells in the cerebellum as candidates. Second, reconstructed embryos were treated with trichostatin A (TSA), a potent histone deacetylase inhibitor. Using CA1 cells, cloned offspring were obtained at high rates, reaching 10.2% and 4.6% (of embryos transferred) for male and female donors, respectively. Cerebellar Purkinje cell nuclei were too large to maintain their genetic integrity during nuclear transfer, leading to developmental arrest of embryos. However, gene expression analysis using cloned blastocysts corroborated a high rate of genomic reprogrammability of CA1 pyramidal and Purkinje cells. Neurons from the hippocampal dentate gyrus and cerebral cortex, which had higher amounts of H3K9me2, could also be used for producing cloned offspring, but the efficiencies were low. A more thorough analysis revealed that TSA treatment was essential for cloning adult neuronal cells. This study demonstrates, to our knowledge for the first time, that adult neurons can be cloned by nuclear transfer. Furthermore, our data imply that reduced amounts of H3K9me2 and increased histone acetylation appear to act synergistically to improve the development of cloned embryos.

Establishment of paternal genomic imprinting in mouse prospermatogonia analyzed by nuclear transfer.

In mice, the establishment of paternal genomic imprinting in male germ cells starts at midgestation, as suggested by DNA methylation analyses of differentially methylated regions (DMRs). However, this information is based on averages from mixed populations of germ cells, and the DNA methylation pattern might not always provide a full representation of imprinting status. To obtain more detailed information on the establishment of paternal imprinting, single prospermatogonia at Embryonic Days 15.5 (E15.5), E16.5, and E17.5 and at Day 0.5 after birth were cloned using nuclear transfer; previous reports suggested that cloned embryos reflected the donor's genomic imprinting status. Then, the resultant fetuses (E9.5) were analyzed for the DNA methylation pattern of three paternal DMRs (IG-DMR, H19 DMR, and Rasgrf1 DMR) and the expression pattern of imprinted genes therein. The overall data indicated that establishment of genomic imprinting in all paternally imprinted regions was completed by E17.5, following a short intermediate period at E16.5. Furthermore, comparison between the methylation status of DMRs and the expression profiles of imprinted genes suggested that methylation of the IG-DMR, but not the H19 DMR, solely governed the control of its imprinted gene cluster. The Rasgrf1 DMR seemed to be imprinted later than the other two genes. We also found that the methylation status of the Gtl2 DMR, the secondary DMR that acquires DNA methylation after fertilization, was likely to follow the methylation status of the upstream IG-DMR. Thus, the systematic analyses of prospermatogonium-derived embryos provided additional important information on the establishment of paternal imprinting.

RNAi-mediated knockdown of Xist can rescue the impaired postimplantation development of cloned mouse embryos.

Cloning mammals by somatic cell nuclear transfer (SCNT) is highly inefficient. Most SCNT-generated embryos die after implantation because of unidentified, complex epigenetic errors in the process of postimplantation embryonic development. Here we identify the most upstream level of dysfunction leading to impaired development of clones by using RNAi against Xist, a gene responsible for X chromosome inactivation (XCI). A prior injection of Xist-specific siRNA into reconstructed oocytes efficiently corrected SCNT-specific aberrant Xist expression at the morula stage, but failed to do so thereafter at the blastocyst stage. However, we found that shortly after implantation, this aberrant XCI status in cloned embryos had been corrected autonomously in both embryonic and extraembryonic tissues, probably through a newly established XCI control for postimplantation embryos. Embryo transfer experiments revealed that siRNA-treated embryos showed 10 times higher survival than controls as early as embryonic day 5.5 and this high survival persisted until term, resulting in a remarkable improvement in cloning efficiency (12% vs. 1% in controls). Importantly, unlike control clones, these Xist-siRNA clones at birth showed only a limited dysregulation of their gene expression, indicating that correction of Xist expression in preimplantation embryos had a long-term effect on their postnatal normality. Thus, contrary to the general assumption, our results suggest that the fate of cloned embryos is determined almost exclusively before implantation by their XCI status. Furthermore, our strategy provides a promising breakthrough for mammalian SCNT cloning, because RNAi treatment of oocytes is readily applicable to most mammal species.

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.

Abnormal chromosome segregation at early cleavage is a major cause of the full-term developmental failure of mouse clones.

To clarify the causes of the poor success rate of somatic cell nuclear transfer (SCNT), we addressed the impact of abnormalities observed at early cleavage stages of development on further full-term development using 'less-damage' imaging technology. To visualize the cellular and nuclear division processes, SCNT embryos were injected with a mixture of mRNAs encoding enhanced green fluorescent protein coupled with α-tubulin (EGFP-α-tubulin) and monomeric red fluorescent protein 1 coupled with histone H2B (H2B-mRFP1) and monitored until the morula/blastocyst stage three-dimensionally. First, the rate of development of SCNT embryos and its effect on the full-term developmental ability were analyzed. The speed of development was retarded and varied in SCNT embryos. Despite the rate of development, SCNT morulae having more than eight cells at 70h after activation could develop to term. Next, chromosomal segregation was investigated in SCNT embryos during early embryogenesis. To our surprise, more than 90% of SCNT embryos showed abnormal chromosomal segregation (ACS) before they developed to morula stage. Importantly, ACS per se did not affect the rate of development, morphology or cellular differentiation in preimplantation development. However, ACS occurring before the 8-cell stage severely inhibited postimplantation development. Thus, the morphology and/or rate of development are not significant predictive markers for the full-term development of SCNT embryos. Moreover, the low efficiency of animal cloning may be caused primarily by genetic abnormalities such as ACS, in addition to the epigenetic errors described previously.

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.

Offspring from intracytoplasmic sperm injection of aged mouse oocytes treated with caffeine or MG132.

Postovulatory mammalian oocytes age significantly in culture. B6D2F1 or ICR strain mouse oocytes were collected 16 h after hCG injection and then cultured for up to 40 h post hCG at 37 °C under 5% CO(2) in air. After intracytoplasmic sperm injection (ICSI), B6D2F1 and ICR oocytes lost full-term developmental potential by 30 h and 26 h after hCG administration, respectively. However, using supplementation with 10 mM caffeine or 1-5 µM of MG132, we could obtain live offspring from oocytes at 34 h (BDF1, 5%-21%) or 28 h (ICR, 5%-18%), whereas none were obtained from untreated aged oocytes. Caffeine maintained normal meiotic spindle morphology, whereas MG132 maintained maturation-promoting factor activity. These treatments did not affect the potential of fresh oocytes for fertilization and subsequent development. Thus, it should be safe to use these chemicals in routine in vitro fertilization and offspring could be generated by ICSI of aged fertilization failed oocytes.

Survival and death of epiblast cells during embryonic stem cell derivation revealed by long-term live-cell imaging with an Oct4 reporter system.

Despite the broad literature on embryonic stem cells (ESCs), their derivation process remains enigmatic. This may be because of the lack of experimental systems that can monitor this prolonged cellular process. Here we applied a live-cell imaging technique to monitor the process of ESC derivation over 10 days from morula to outgrowth phase using an Oct4/eGFP reporter system. Our imaging reflects the 'natural' state of ESC derivation, as the ESCs established after the imaging were both competent in chimeric mice formation and germ-line transmission. Using this technique, ESC derivation in conventional conditions was imaged. After the blastocoel was formed, the intensity of Oct4 signals attenuated in the trophoblast cells but was maintained in the inner cell mass (ICM). Thereafter, the Oct4-positive cells scattered and their number decreased along with apoptosis of the other Oct4-nagative cells likely corresponds to trophoblast and hypoblast cells, and then only the surviving Oct4-positive cells proliferated and formed the colony. All embryos without exception passed through this cell death phase. Importantly, the addition of caspase inhibitor Z-VAD-FMK to the medium dramatically suppressed the loss of Oct4-positive cells and also other embryo-derived cells, suggesting that the cell deaths was induced by a caspase-dependent apoptotic pathway. Next we imaged the ESC derivation in 3i medium, which consists of chemical compounds that can suppress differentiation. The most significant difference between the conventional and 3i methods was that there was no obvious cell death in 3i, so that the colony formation was rapid and all of the Oct4-positive cells contributed to the formation of the outgrown colony. These data indicate that the prevention of cell death in epiblast cells is one of the important events for the successful establishment of ESCs. Thus, our imaging technique can advance the understanding of the time-dependent cellular changes during ESC derivation.

Intracytoplasmic sperm injection with mouse spermatozoa preserved without freezing for six months can lead to full-term development.

Preservation of mammalian spermatozoa now plays an important role in fertility treatment, in generating hybrid animals, and in protecting endangered or extinct species. To date, the most common method of sperm preservation is freezing in liquid nitrogen (LN(2)). However, this method requires constant supplementation of the LN(2) and also involves some safety issues in transporting LN(2). Here we describe a new sperm preservation method that does not involve freezing. Mouse spermatozoa were cultured in four basic media (HEPES-CZB, potassium simplex optimization medium with amino acids [KSOMaa], K(+)-rich nuclear isolation medium [NIM], and PBS) with or without 10% bovine serum albumin (BSA) or 15% Ficoll as a protectant, and preserved in a refrigerator for up to 6 mo. These preserved sperm were then injected into fresh oocytes and cultured to the blastocyst stage in vitro or transferred into recipient females to demonstrate their genetic integrity. The results of sperm preservation for 1 mo suggested that NIM and PBS were better media than HEPES-CZB or KSOMaa and that BSA and Ficoll could improve either blastocyst or full-term development. Surprisingly, 18 pups were obtained using spermatozoa stored in these media for 6 mo. Moreover, this new method allowed easy production of healthy offspring even after transport of spermatozoa between two countries by aircraft at room temperature. In conclusion, this method allows for easy long-term preservation of mouse spermatozoa in a simple, modified medium at refrigerator temperature with very low cost and wide application.

Essential role of paternal chromatin in the regulation of transcriptional activity during mouse preimplantation development.

Several lines of evidence indicate that the formation of a transcriptionally repressive state during the two-cell stage in the preimplantation mouse embryo is superimposed on the activation of the embryonic genome. However, it is difficult to determine the profile of newly synthesized (nascent) RNA during this phase because large amounts of maternal RNA accumulate in maturing oocytes to support early development. Using 5-bromouridine-5'-triphosphate labeling of RNA, we have verified that nascent RNA synthesis was repressed between the two-cell and four-cell transition in normally fertilized but not in parthenogenetic embryos. Moreover, this repression was contributed by sperm (male) chromatin, which we confirmed by studying androgenetic embryos. The source of factors responsible for repressing nascent RNA production was investigated using different stages of sperm development. Fertilization with immature round spermatids resulted in a lower level of transcriptional activity than with ICSI at the two-cell stage, and this was consistent with further repression at the four-cell stage in the ICSI group. Finally, study on DNA replication and chromatin remodeling was performed using labeled histones H3 and H4 to differentiate between male and female pronuclei. The combination of male and female chromatin appeared to decrease nascent RNA production in the fertilized embryo. This study indicates that paternal chromatin is important in the regulation of transcriptional activity during mouse preimplantation development and that this capacity is acquired during spermiogenesis.