HBO1 determines SMAD action in pluripotency and mesendoderm specification.

TGF-ß signaling family plays an essential role to regulate fate decisions in pluripotency and lineage specification. How the action of TGF-ß family signaling is intrinsically executed remains not fully elucidated. Here, we show that HBO1, a MYST histone acetyltransferase (HAT) is an essential cell intrinsic determinant for TGF-ß signaling in human embryonic stem cells (hESCs). HBO1-/- hESCs fail to response to TGF-ß signaling to maintain pluripotency and spontaneously differentiate into neuroectoderm. Moreover, HBO1 deficient hESCs show complete defect in mesendoderm specification in BMP4-triggered gastruloids or teratomas. Molecularly, HBO1 interacts with SMAD4 and co-binds the open chromatin labeled by H3K14ac and H3K4me3 in undifferentiated hESCs. Upon differentiation, HBO1/SMAD4 co-bind and maintain the mesoderm genes in BMP4-triggered mesoderm cells while lose chromatin occupancy in neural cells induced by dual-SMAD inhibition. Our data reveal an essential role of HBO1, a chromatin factor to determine the action of SMAD in both human pluripotency and mesendoderm specification.

YTHDF1 facilitates PRC1-mediated H2AK119ub in human ES cells.

Polycomb repressive complexes (PRCs) play critical roles in cell fate decisions during normal development as well as disease progression through mediating histone modifications such as H3K27me3 and H2AK119ub. How exactly PRCs recruited to chromatin remains to be fully illuminated. Here, we report that YTHDF1, the N6-methyladenine (m6 A) RNA reader that was previously known to be mainly cytoplasmic, associates with RNF2, a PRC1 protein that mediates H2AK119ub in human embryonic stem cells (hESCs). A portion of YTHDF1 localizes in the nuclei and associates with RNF2/H2AK119ub on a subset of gene loci related to neural development functions. Knock-down YTHDF1 attenuates H2AK119ub modification on these genes and promotes neural differentiation in hESCs. Our findings provide a noncanonical mechanism that YTHDF1 participates in PRC1 functions in hESCs.

Modulating Multiarticular Energy during Human Walking and Running with an Unpowered Exoskeleton.

Researchers have made advances in reducing the metabolic rate of both walking and running by modulating mono-articular energy with exoskeletons. However, how to modulate multiarticular energy with exoskeletons to improve the energy economy of both walking and running is still a challenging problem, due to the lack of understanding of energy transfer among human lower-limb joints. Based on the study of the energy recycling and energy transfer function of biarticular muscles, we proposed a hip-knee unpowered exoskeleton that emulates and reinforces the function of the hamstrings and rectus femoris in different gait phases. The biarticular exo-tendon of the exoskeleton assists hamstrings to recycle the kinetic energy of the leg swing while providing hip extension torque in the swing phase. In the following stance phase, the exo-tendon releases the stored energy to assist the co-contraction of gluteus maximus and rectus femoris for both hip extension and knee extension, thus realizing the phased modulation of hip and knee joint energy. The metabolic rate of both walking (1.5 m/s) and running (2.5 m/s) can be reduced by 6.2% and 4.0% with the multiarticular energy modulation of a hip-knee unpowered exoskeleton, compared to that of walking and running without an exoskeleton. The bio-inspired design method of this study may inspire people to develop devices that assist multiple gaits in the future.

Reducing the metabolic energy of walking and running using an unpowered hip exoskeleton.

BACKGROUND: Walking and running are the most common means of locomotion in human daily life. People have made advances in developing separate exoskeletons to reduce the metabolic rate of walking or running. However, the combined requirements of overcoming the fundamental biomechanical differences between the two gaits and minimizing the metabolic penalty of the exoskeleton mass make it challenging to develop an exoskeleton that can reduce the metabolic energy during both gaits. Here we show that the metabolic energy of both walking and running can be reduced by regulating the metabolic energy of hip flexion during the common energy consumption period of the two gaits using an unpowered hip exoskeleton. METHODS: We analyzed the metabolic rates, muscle activities and spatiotemporal parameters of 9 healthy subjects (mean ± s.t.d; 24.9 ± 3.7 years, 66.9 ± 8.7 kg, 1.76 ± 0.05 m) walking on a treadmill at a speed of 1.5 m s-1 and running at a speed of 2.5 m s-1 with different spring stiffnesses. After obtaining the optimal spring stiffness, we recruited the participants to walk and run with the assistance from a spring with optimal stiffness at different speeds to demonstrate the generality of the proposed approach. RESULTS: We found that the common optimal exoskeleton spring stiffness for walking and running was 83 Nm Rad-1, corresponding to 7.2% ± 1.2% (mean ± s.e.m, paired t-test p < 0.01) and 6.8% ± 1.0% (p < 0.01) metabolic reductions compared to walking and running without exoskeleton. The metabolic energy within the tested speed range can be reduced with the assistance except for low-speed walking (1.0 m s-1). Participants showed different changes in muscle activities with the assistance of the proposed exoskeleton. CONCLUSIONS: This paper first demonstrates that the metabolic cost of walking and running can be reduced using an unpowered hip exoskeleton to regulate the metabolic energy of hip flexion. The design method based on analyzing the common energy consumption characteristics between gaits may inspire future exoskeletons that assist multiple gaits. The results of different changes in muscle activities provide new insight into human response to the same assistive principle for different gaits (walking and running).

Generation of H1 PAX6WT/EGFP reporter cells to purify PAX6 positive neural stem/progenitor cells.

Neural conversion from human pluripotent cells (hPSCs) is a potential therapy to neurological disease in the future. However, this is still limited by efficiency and stability of existed protocols used for neural induction from hPSCs. To overcome this obstacle, we developed a reporter system to screen PAX6+ neural progenitor/stem cells using transcription activator like effector nuclease (TALEN). We found that knock-in 2 A-EGFP cassette into PAX6 exon of human embryonic stem cells H1 with TALEN-based homology recombination could establish PAX6WT/EGFP H1 reporter cell line fast and efficiently. This reporter cell line could differentiate into PAX6 and EGFP double positive neural progenitor/stem cells (NPCs/NSCs) after neural induction. Those PAX6WT/EGFP NPCs could be purified, expanded and specified to post-mitotic neurons in vitro efficiently. With this reporter cell line, we also screened out 1 NPC-specific microRNA, hsa-miR-99a-5p, and 3 ESCs-enriched miRNAs, hsa-miR-302c-5p, hsa-miR-512-3p and hsa-miR-518 b. In conclusion, the TALEN-based neural stem cell screening system is safe and efficient and could help researcher to acquire adequate and pure neural progenitor cells for further application.

Magnesium sulphate can alleviate oxidative stress and reduce inflammatory cytokines in rat placenta of intrahepatic cholestasis of pregnancy model.

PURPOSE: In our study, we try to investigate whether magnesium sulphate (MgSO4) could provide protection against oxidative damage and inflammatory response in rat placenta of intrahepatic cholestasis of pregnancy (ICP) model. METHODS: The rat model of ICP was established by injecting s.c. 17α-ethinyl estradiol (EE) daily for 5 days. MgSO4, as an therapeutic drug for ICP, was injected i.p. daily for 3 days. Age-matched pregnant rats served as controls. The level of serum total bile acid (TBA) was measured. The data including the number and weight of offsprings on day 20 of pregnancy were collected. We observed ultrastructural changes of mitochondria and endoplasmic reticulum (ER) in placenta by transmission electron microscope. The antioxidant proteins peroxiredoxin-6 (Prdx6) and nuclear factor erythroid 2-related factor-2 (Nrf2) were analyzed by Western Blot. The inflammatory cytokines including IL-1ß, TNF-α and IFN-γ were investigated by real-time PCR (RT-PCR) and enzyme-linked immune-sorbent assay (ELISA). RESULTS: The weight of offsprings on day 20 of pregnancy increased in ICP rats treated with MgSO4 (ICP + MG group) compared with that in ICP rats (ICP group). However, the level of TBA was not reduced. The damage of mitochondria and ER was observed in placenta, which was much more slighter in ICP + MgSO4 group as compared with that in ICP group. Prdx6 and Nrf2 were increased, while the inflammatory cytokines including IL-1ß, TNF-α and IFN-γ were decreased in ICP + MgSO4 group compared with that in ICP group. CONCLUSIONS: MgSO4 had beneficial effect on improving growth of offsprings in rat model of ICP. The protective effect of MgSO4 on alleviating oxidative damage and inflammatory response in placenta may play an important role in the process. MgSO4 may improve the function of placenta.

Generation of a PPM1A-deficient human induced pluripotent stem cell line using CRISPR-Cas9 technology.

PPM1A is a member of the serine/threonine protein phosphatase family. It can bind to a variety of proteins to dephosphorylate them, and extensively regulates many life activities such as cell growth, cell stress, immune response, and tumor formation. Here we constructed a human induced pluripotent stem cell (hiPSC) line with knockout of PPM1A using CRISPR/Cas9-mediated gene targeting. This cell line exhibits normal karyotype, pluripotency, and trilineage differentiation potential, which could provide a useful cellular resource for exploring the mechanism of PPM1A in regulating downstream signaling pathways and explore the application of PPM1A in anti-tumor and anti-infection.

STAMBP is Required for Long-Term Maintenance of Neural Progenitor Cells Derived from hESCs.

Mutations in STAMBP have been well-established to cause congenital human microcephaly-capillary malformation (MIC-CAP) syndrome, a rare genetic disorder characterized by global developmental delay, severe microcephaly, capillary malformations, etc. Previous biochemical investigations and loss-of-function studies in mice have provided insights into the mechanism of STAMBP, however, it remains controversial how STAMBP deficiency leads to malformation of those affected tissues in patients. In this study, we investigated the function and underlying mechanism of STAMBP during neural differentiation of human embryonic stem cells (hESCs). We found that STAMBP is dispensable for the pluripotency maintenance or neural differentiation of hESCs. However, neural progenitor cells (NPCs) derived from STAMBP-deficient hESCs fail to be long-term maintained/expanded in vitro. We identified the anti-apoptotic protein CFLAR is down-regulated in those affected NPCs and ectopic expression of CFLAR rescues NPC defects induced by STAMBP-deficiency. Our study not only provides novel insight into the mechanism of neural defects in STAMBP mutant patients, it also indicates that the death receptor mediated apoptosis is an obstacle for long-term maintenance/expansion of NPCs in vitro thus counteracting this cell death pathway could be beneficial to the generation of NPCs in vitro.

Generation of an induced pluripotent stem cell line from patient with atrial fibrillation with KCNQ1 p.Ser209Pro mutation.

Gain-of-function mutations in the KCNQ1 gene can cause atrial fibrillation. In this study, we generated an induced stem cell line (GRCHJUi001) from one member of an atrial fibrillation family line, whom had heterozygous mutation in the KCNQ1 gene c.625 T > C (p.Ser209Pro), and the cell line showed maintenance of stem cells characterized by morphology, normal karyotype, and pluripotency.

Characterization of gene regulatory networks underlying key properties in human hematopoietic stem cell ontogeny.

Human hematopoiesis starts at early yolk sac and undergoes site- and stage-specific changes over development. The intrinsic mechanism underlying property changes in hematopoiesis ontogeny remains poorly understood. Here, we analyzed single-cell transcriptome of human primary hematopoietic stem/progenitor cells (HSPCs) at different developmental stages, including yolk-sac (YS), AGM, fetal liver (FL), umbilical cord blood (UCB) and adult peripheral blood (PB) mobilized HSPCs. These stage-specific HSPCs display differential intrinsic properties, such as metabolism, self-renewal, differentiating potentialities etc. We then generated highly co-related gene regulatory network (GRNs) modules underlying the differential HSC key properties. Particularly, we identified GRNs and key regulators controlling lymphoid potentiality, self-renewal as well as aerobic respiration in human HSCs. Introducing selected regulators promotes key HSC functions in HSPCs derived from human pluripotent stem cells. Therefore, GRNs underlying key intrinsic properties of human HSCs provide a valuable guide to generate fully functional HSCs in vitro.

Autophagy is essential for human myelopoiesis.

Emergency myelopoiesis (EM) is essential in immune defense against pathogens for rapid replenishing of mature myeloid cells. During the EM process, a rapid cell-cycle switch from the quiescent hematopoietic stem cells (HSCs) to highly proliferative myeloid progenitors (MPs) is critical. How the rapid proliferation of MPs during EM is regulated remains poorly understood. Here, we reveal that ATG7, a critical autophagy factor, is essential for the rapid proliferation of MPs during human myelopoiesis. Peripheral blood (PB)-mobilized hematopoietic stem/progenitor cells (HSPCs) with ATG7 knockdown or HSPCs derived from ATG7-/- human embryonic stem cells (hESCs) exhibit severe defect in proliferation during fate transition from HSPCs to MPs. Mechanistically, we show that ATG7 deficiency reduces p53 localization in lysosome for a potential autophagy-mediated degradation. Together, we reveal a previously unrecognized role of autophagy to regulate p53 for a rapid proliferation of MPs in human myelopoiesis.

Selecting Monoclonal Cell Lineages from Somatic Reprogramming Using Robotic-Based Spatial-Restricting Structured Flow.

Somatic cell reprogramming generates induced pluripotent stem cells (iPSCs), which serve as a crucial source of seed cells for personalized disease modeling and treatment in regenerative medicine. However, the process of reprogramming often causes substantial lineage manipulations, thereby increasing cellular heterogeneity. As a consequence, the process of harvesting monoclonal iPSCs is labor-intensive and leads to decreased reproducibility. Here, we report the first in-house developed robotic platform that uses a pin-tip-based micro-structure to manipulate radial shear flow for automated monoclonal iPSC colony selection (~1 s) in a non-invasive and label-free manner, which includes tasks for somatic cell reprogramming culturing, medium changes; time-lapse-based high-content imaging; and iPSCs monoclonal colony detection, selection, and expansion. Throughput-wise, this automated robotic system can perform approximately 24 somatic cell reprogramming tasks within 50 days in parallel via a scheduling program. Moreover, thanks to a dual flow-based iPSC selection process, the purity of iPSCs was enhanced, while simultaneously eliminating the need for single-cell subcloning. These iPSCs generated via the dual processing robotic approach demonstrated a purity 3.7 times greater than that of the conventional manual methods. In addition, the automatically produced human iPSCs exhibited typical pluripotent transcriptional profiles, differentiation potential, and karyotypes. In conclusion, this robotic method could offer a promising solution for the automated isolation or purification of lineage-specific cells derived from iPSCs, thereby accelerating the development of personalized medicines.

Generation of an NSD2-deficient human embryonic stem cell line using CRISPR/Cas9 technology.

NSD2 is a histone methyltransferase (HMT) and is involved in the epigenetic regulation of hematopoiesis and hematological cancers. To understand and illustrate the precise roles of NSD2 in hematopoietic development, here we constructed a human embryonic stem cell (hESC) line with knockout of NSD2 using CRISPR/Cas9-mediated gene targeting. The cell line maintained typical stem cell morphology and normal karyotype. Furthermore, the pluripotency of the cell line was evidenced by high expression level of pluripotency genes and differentiation potential into three germ layers. The cell line provides a good model for studying roles of NSD2 in embryonic development, especially hematopoiesis.

BRPF1 bridges H3K4me3 and H3K23ac in human embryonic stem cells and is essential to pluripotency.

Post-translational modifications (PTMs) on histones play essential roles in cell fate decisions during development. However, how these PTMs are recognized and coordinated remains to be fully illuminated. Here, we show that BRPF1, a multi-histone binding module protein, is essential for pluripotency in human embryonic stem cells (ESCs). BRPF1, H3K4me3, and H3K23ac substantially co-occupy the open chromatin and stemness genes in hESCs. BRPF1 deletion impairs H3K23ac in hESCs and leads to closed chromatin accessibility on stemness genes and hESC differentiation as well. Deletion of the N terminal or PHD-zinc knuckle-PHD (PZP) module in BRPF1 completely impairs its functions in hESCs while PWWP module deletion partially impacts the function. In sum, we reveal BRPF1, the multi-histone binding module protein that bridges the crosstalk between different histone modifications in hESCs to maintain pluripotency.

Establishing extended pluripotent stem cells from human urine cells.

BACKGROUND: Extended pluripotent stem cells (EPSCs) can contribute to both embryonic and trophectoderm-derived extraembryonic tissues. Therefore, EPSCs have great application significance for both research and industry. However, generating EPSCs from human somatic cells remains inefficient and cumbersome. RESULTS: In this study, we established a novel and robust EPSCs culture medium OCM175 with defined and optimized ingredients. Our OCM175 medium contains optimized concentration of L-selenium-methylcysteine as a source of selenium and ROCK inhibitors to maintain the single cell passaging ability of pluripotent stem cells. We also used Matrigel or the combination of laminin 511 and laminin 521(1:1) to bypass the requirement of feeder cells. With OCM175 medium, we successfully converted integration-free iPSCs from easily available human Urine-Derived Cells (hUC-iPSCs) into EPSCs (O-IPSCs). We showed that our O-IPSCs have the ability to form both intra- and extra- embryonic chimerism, and could contribute to the trophoblast ectoderm lineage and three germ layer cell lineages. CONCLUSIONS: In conclusion, our novel OCM175 culture medium has defined, optimized ingredients, which enables efficient generation of EPSCs in a feeder free manner. With the robust chimeric and differentiation potential, we believe that this system provides a solid basis to improve the application of EPSCs in regenerative medicine.

Generation of RYBP FLAG-HA knock-in human embryonic stem cell line through CRISPR/Cas9-mediated homologous recombination.

RYBP, a critical component of polycomb repressive complex1 (PRC1), is required for the pluripotency and differentiation of mouse embryonic stem cells(mESCs). However, its function and mechanism to regulate human embryonic stem cells(hESCs) remain unknown. Here, to investigate the role of RYBP in hESCs, we generate an hESC line with FLAG-HA tag knock-in to RYBP locus through CRISPR/Cas9-mediated homologous recombination. hESC with RYBP_FLAG-HA knock-in maintains normal morphology and karyotype, while it maintains pluripotency to differentiate into three germ layers.

Generation of a homozygous FIS1 knockout human embryonic stem cell line GIBHe015-B by CRISPR/Cas9 system.

The mitochondrial fission protein 1 (FIS1) is essential for mitochondrial division or fission and has been determined to mediate mitochondrial and peroxisomal fission. Other studies also found that FIS1 functions as an essential component of the mitophagy and apoptosis pathways in mammalian cells, suggesting that FIS1 has multiple important roles. Here, we generated homozygous FIS1 knockout human embryonic stem cells (hESCs) using the CRISPR/Cas9 system. This cell line exhibits normal karyotype, pluripotency, and trilineage differentiation potential, which could provide a useful cellular resource for exploring the functions of FIS1 and their implications in human health and diseases.

Modulating Energy Among Foot-Ankle Complex With an Unpowered Exoskeleton Improves Human Walking Economy.

Over the course of both evolution and development, the human musculoskeletal system has been well shaped for the cushion function of the foot during foot-strike and the impulsive function of the ankle joint during push-off. Nevertheless, an efficient energy interaction between foot structure and ankle joint is still lacking in the human body itself, which may limit the further potential of economical walking. Here we showed the metabolic expenditure of walking can be lessened by an unpowered exoskeleton robot that modulates energy among the foot-ankle complex towards a more effective direction. The unpowered exoskeleton recycles negative mechanical energy of the foot that is normally dissipated in heel-strike, retains the stored energy before mid-stance, and then transfers the energy to the ankle joint to assist the push-off. The modulation process of the exoskeleton consumes no input energy, yet reduces the metabolic cost of walking by 8.19 ± 0.96 % (mean ± s.e.m) for healthy subjects. The electromyography measurements demonstrate the activities of target ankle plantarflexors decreased significantly without added effort for the antagonistic muscle, suggesting the exoskeleton enhanced the subjects' energy efficiency of the foot-ankle complex in a natural manner. Furthermore, the exoskeleton also provides cushion assistance for walking, which leads to significantly decreased activity of the quadriceps muscle during heel-strike. Rather than strengthening the functions of existing biological structures, developing the complementary energy loop that does not exist in the human body itself also shows its potential for gait assistance.

Generating functional cells through enhanced interspecies chimerism with human pluripotent stem cells.

Obtaining functional human cells through interspecies chimerism with human pluripotent stem cells (hPSCs) remains unsuccessful due to its extremely low efficiency. Here, we show that hPSCs failed to differentiate and contribute teratoma in the presence of mouse PSCs (mPSCs), while MYCN, a pro-growth factor, dramatically promotes hPSC contributions in teratoma co-formation by hPSCs/mPSCs. MYCN combined with BCL2 (M/B) greatly enhanced conventional hPSCs to integrate into pre-implantation embryos of different species, such as mice, rabbits, and pigs, and substantially contributed to mouse post-implantation chimera in embryonic and extra-embryonic tissues. Strikingly, M/B-hPSCs injected into pre-implantation Flk-1+/- mouse embryos show further enhanced chimerism that allows for obtaining live human CD34+ blood progenitor cells from chimeras through cell sorting. The chimera-derived human CD34+ cells further gave rise to various subtype blood cells in a typical colony-forming unit (CFU) assay. Thus, we provide proof of concept to obtain functional human cells through enhanced interspecies chimerism with hPSCs.

Regulating Metabolic Energy Among Joints During Human Walking Using a Multiarticular Unpowered Exoskeleton.

Researchers have found that the walking economy can be enhanced by recycling ankle metabolic energy using an unpowered ankle exoskeleton. However, how to regulate multiarticular energy to enhance the overall energy efficiency of humans during walking remains a challenging problem, as multiarticular passive assistance is more likely to interfere with the human body's natural biomechanics. Here we show that the metabolic energy of the hip and knee musculature can be regulated to a more energy-effective direction using a multiarticular unpowered exoskeleton that recycles negative mechanical energy of the knee joint in the late swing phase and transfers the stored energy to assist the hip extensors in performing positive mechanical work in the stance phase. The biarticular spring-clutch mechanism of the exoskeleton performs a complementary energy recycling and energy transfer function for hip and knee musculature. Through the phased regulation of the hip and knee metabolic energy, the target muscle activities decreased during the whole assistive period of the exoskeleton, which was the direct reason for 8.6 ± 1.5% (mean ± s.e.m) reduction in metabolic rate compared with that of walking without the exoskeleton. The proposed unpowered exoskeleton enhanced the user's multiarticular energy efficiency, which equals improving musculoskeletal structure by adding a complementary loop for efficient energy recycling and energy transfer.