Primate cell fusion disentangles gene regulatory divergence in neurodevelopment.

Among primates, humans display a unique trajectory of development that is responsible for the many traits specific to our species. However, the inaccessibility of primary human and chimpanzee tissues has limited our ability to study human evolution. Comparative in vitro approaches using primate-derived induced pluripotent stem cells have begun to reveal species differences on the cellular and molecular levels1,2. In particular, brain organoids have emerged as a promising platform to study primate neural development in vitro3-5, although cross-species comparisons of organoids are complicated by differences in developmental timing and variability of differentiation6,7. Here we develop a new platform to address these limitations by fusing human and chimpanzee induced pluripotent stem cells to generate a panel of tetraploid hybrid stem cells. We applied this approach to study species divergence in cerebral cortical development by differentiating these cells into neural organoids. We found that hybrid organoids provide a controlled system for disentangling cis- and trans-acting gene-expression divergence across cell types and developmental stages, revealing a signature of selection on astrocyte-related genes. In addition, we identified an upregulation of the human somatostatin receptor 2 gene (SSTR2), which regulates neuronal calcium signalling and is associated with neuropsychiatric disorders8,9. We reveal a human-specific response to modulation of SSTR2 function in cortical neurons, underscoring the potential of this platform for elucidating the molecular basis of human evolution.

Reliability of human cortical organoid generation.

The differentiation of pluripotent stem cells in three-dimensional cultures can recapitulate key aspects of brain development, but protocols are prone to variable results. Here we differentiated multiple human pluripotent stem cell lines for over 100 d using our previously developed approach to generate brain-region-specific organoids called cortical spheroids and, using several assays, found that spheroid generation was highly reliable and consistent. We anticipate the use of this approach for large-scale differentiation experiments and disease modeling.

HEB and E2A function as SMAD/FOXH1 cofactors.

Nodal signaling, mediated through SMAD transcription factors, is necessary for pluripotency maintenance and endoderm commitment. We identified a new motif, termed SMAD complex-associated (SCA), that is bound by SMAD2/3/4 and FOXH1 in human embryonic stem cells (hESCs) and derived endoderm. We demonstrate that two basic helix-loop-helix (bHLH) proteins-HEB and E2A-bind the SCA motif at regions overlapping SMAD2/3 and FOXH1. Furthermore, we show that HEB and E2A associate with SMAD2/3 and FOXH1, suggesting they form a complex at critical target regions. This association is biologically important, as E2A is critical for mesendoderm specification, gastrulation, and Nodal signal transduction in Xenopus tropicalis embryos. Taken together, E proteins are novel Nodal signaling cofactors that associate with SMAD2/3 and FOXH1 and are necessary for mesendoderm differentiation.

Single-cell transcriptomic landscape of the developing human spinal cord.

Understanding spinal cord assembly is essential to elucidate how motor behavior is controlled and how disorders arise. The human spinal cord is exquisitely organized, and this complex organization contributes to the diversity and intricacy of motor behavior and sensory processing. But how this complexity arises at the cellular level in the human spinal cord remains unknown. Here we transcriptomically profiled the midgestation human spinal cord with single-cell resolution and discovered remarkable heterogeneity across and within cell types. Glia displayed diversity related to positional identity along the dorso-ventral and rostro-caudal axes, while astrocytes with specialized transcriptional programs mapped into white and gray matter subtypes. Motor neurons clustered at this stage into groups suggestive of alpha and gamma neurons. We also integrated our data with multiple existing datasets of the developing human spinal cord spanning 22 weeks of gestation to investigate the cell diversity over time. Together with mapping of disease-related genes, this transcriptomic mapping of the developing human spinal cord opens new avenues for interrogating the cellular basis of motor control in humans and guides human stem cell-based models of disease.

Chromatin and transcriptional signatures for Nodal signaling during endoderm formation in hESCs.

The first stages of embryonic differentiation are driven by signaling pathways hardwired to induce particular fates. Endoderm commitment is controlled by the TGF-ß superfamily member, Nodal, which utilizes the transcription factors, SMAD2/3, SMAD4 and FOXH1, to drive target gene expression. While the role of Nodal is well defined within the context of endoderm commitment, mechanistically it is unknown how this signal interacts with chromatin on a genome wide scale to trigger downstream responses. To elucidate the Nodal transcriptional network that governs endoderm formation, we used ChIP-seq to identify genomic targets for SMAD2/3, SMAD3, SMAD4, FOXH1 and the active and repressive chromatin marks, H3K4me3 and H3K27me3, in human embryonic stem cells (hESCs) and derived endoderm. We demonstrate that while SMAD2/3, SMAD4 and FOXH1 associate with DNA in a highly dynamic fashion, there is an optimal bivalent signature at 32 gene loci for driving endoderm commitment. Initially, this signature is marked by both H3K4me3 and H3K27me3 as a very broad bivalent domain in hESCs. Within the first 24h, SMAD2/3 accumulation coincides with H3K27me3 reduction so that these loci become monovalent marked by H3K4me3. JMJD3, a histone demethylase, is simultaneously recruited to these promoters, suggesting a conservation of mechanism at multiple promoters genome-wide. The correlation between SMAD2/3 binding, monovalent formation and transcriptional activation suggests a mechanism by which SMAD proteins coordinate with chromatin at critical promoters to drive endoderm specification.

Engineering brain assembloids to interrogate human neural circuits.

The development of neural circuits involves wiring of neurons locally following their generation and migration, as well as establishing long-distance connections between brain regions. Studying these developmental processes in the human nervous system remains difficult because of limited access to tissue that can be maintained as functional over time in vitro. We have previously developed a method to convert human pluripotent stem cells into brain region-specific organoids that can be fused and integrated to form assembloids and study neuronal migration. In contrast to approaches that mix cell lineages in 2D cultures or engineer microchips, assembloids leverage self-organization to enable complex cell-cell interactions, circuit formation and maturation in long-term cultures. In this protocol, we describe approaches to model long-range neuronal connectivity in human brain assembloids. We present how to generate 3D spheroids resembling specific domains of the nervous system and then how to integrate them physically to allow axonal projections and synaptic assembly. In addition, we describe a series of assays including viral labeling and retrograde tracing, 3D live imaging of axon projection and optogenetics combined with calcium imaging and electrophysiological recordings to probe and manipulate the circuits in assembloids. The assays take 3-4 months to complete and require expertise in stem cell culture, imaging and electrophysiology. We anticipate that these approaches will be useful in deciphering human-specific aspects of neural circuit assembly and in modeling neurodevelopmental disorders with patient-derived cells.

The Effect of Botulinum Toxin on Hair Follicle Cell Regeneration Under Continuous Stress Conditions: a Pilot Animal Study.

We analyzed the effect of botulinum toxin (BTX) type A on the regeneration of hair follicle cells under continuous stress conditions. Thirty 6-week-old C57BL/6 mice were used, and hair loss was induced on their backs (10 control (CTL) mice, reared under normal conditions without stress; 10 mice, exposed to continuous stress (STRESS) by fixing in an enclosed space; 10 BTX + STRESS mice, injected subcutaneously with 1 IU of BTX (0.1 cc) where the hair follicles were removed under the same stress conditions). There was less hair growth in the STRESS and BTX + STRESS groups compared to that in the CTL group at 2 weeks. At 3 weeks, the telogen stage was mainly observed in the STRESS group whereas the anagen stage was observed in the CTL and BTX + STRESS groups. A substantial increase in terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells was observed in the STRESS group compared to that in the CTL and BTX + STRESS groups. Substance P (SP) immunoreactivity cell levels increased in the STRESS group at 2 and 3 weeks compared to those in the BTX + STRESS group. SP expression increased at 2 and 3 weeks in the STRESS group compared to that in the CTL and BTX + STRESS groups. A delay in the regeneration cycle of the hair follicle cells occurred when stress was applied, and an almost normal regeneration cycle occurred when BTX was injected subcutaneously. Therefore, BTX may be a positive indicator for hair loss treatment.

Long-term maturation of human cortical organoids matches key early postnatal transitions.

Human stem-cell-derived models provide the promise of accelerating our understanding of brain disorders, but not knowing whether they possess the ability to mature beyond mid- to late-fetal stages potentially limits their utility. We leveraged a directed differentiation protocol to comprehensively assess maturation in vitro. Based on genome-wide analysis of the epigenetic clock and transcriptomics, as well as RNA editing, we observe that three-dimensional human cortical organoids reach postnatal stages between 250 and 300 days, a timeline paralleling in vivo development. We demonstrate the presence of several known developmental milestones, including switches in the histone deacetylase complex and NMDA receptor subunits, which we confirm at the protein and physiological levels. These results suggest that important components of an intrinsic in vivo developmental program persist in vitro. We further map neurodevelopmental and neurodegenerative disease risk genes onto in vitro gene expression trajectories to provide a resource and webtool (Gene Expression in Cortical Organoids, GECO) to guide disease modeling.

Chromatin accessibility dynamics in a model of human forebrain development.

Forebrain development is characterized by highly synchronized cellular processes, which, if perturbed, can cause disease. To chart the regulatory activity underlying these events, we generated a map of accessible chromatin in human three-dimensional forebrain organoids. To capture corticogenesis, we sampled glial and neuronal lineages from dorsal or ventral forebrain organoids over 20 months in vitro. Active chromatin regions identified in human primary brain tissue were observed in organoids at different developmental stages. We used this resource to map genetic risk for disease and to explore evolutionary conservation. Moreover, we integrated chromatin accessibility with transcriptomics to identify putative enhancer-gene linkages and transcription factors that regulate human corticogenesis. Overall, this platform brings insights into gene-regulatory dynamics at previously inaccessible stages of human forebrain development, including signatures of neuropsychiatric disorders.

Neuronal defects in a human cellular model of 22q11.2 deletion syndrome.

22q11.2 deletion syndrome (22q11DS) is a highly penetrant and common genetic cause of neuropsychiatric disease. Here we generated induced pluripotent stem cells from 15 individuals with 22q11DS and 15 control individuals and differentiated them into three-dimensional (3D) cerebral cortical organoids. Transcriptional profiling across 100 days showed high reliability of differentiation and revealed changes in neuronal excitability-related genes. Using electrophysiology and live imaging, we identified defects in spontaneous neuronal activity and calcium signaling in both organoid- and 2D-derived cortical neurons. The calcium deficit was related to resting membrane potential changes that led to abnormal inactivation of voltage-gated calcium channels. Heterozygous loss of DGCR8 recapitulated the excitability and calcium phenotypes and its overexpression rescued these defects. Moreover, the 22q11DS calcium abnormality could also be restored by application of antipsychotics. Taken together, our study illustrates how stem cell derived models can be used to uncover and rescue cellular phenotypes associated with genetic forms of neuropsychiatric disease.

Role of Bcl2-like 10 (Bcl2l10) in Regulating Mouse Oocyte Maturation.

Previously, we have shown that Bcl2l10 is highly expressed in metaphase II (MII)-stage oocytes. The objective of this study was to characterize Bcl2l10 expression in ovaries and to examine the function of Bcl2l10 in oocyte maturation using RNA interference. Bcl2l10 transcript expression was ovary and oocyte specific. Bcl2l10 was highly expressed in oocytes and pronuclear-stage embryos; however, its expression decreased at the two-cell stage and dramatically disappeared thereafter. Microinjection of Bcl2l10 double-stranded RNA into the cytoplasm of germinal vesicle oocytes resulted in a marked decrease in Bcl2l10 mRNA and protein and metaphase I (MI) arrest (78.9%). Most MI-arrested oocytes exhibited abnormalities in their spindles and chromosome configurations. Bcl2l10 RNA interference had an obvious effect on the activity of maturation-promoting factor but not on that of mitogen-activated protein kinase. We concluded that the role of Bcl2l10 is strongly associated with oocyte maturation, especially at the MI-MII transition.

Nonsurgical treatments for patients with radicular pain from lumbosacral disc herniation.

BACKGROUND CONTEXT: Lumbosacral disc herniation (LDH) is one of the most frequent musculoskeletal diseases causative of sick leave in the workplace and morbidity in daily activities. Nonsurgical managements are considered as first line treatment before surgical treatment. PURPOSE: This clinical practice guideline (CPG) is intended to provide physicians who treat patients diagnosed with LDH with a guideline supported by scientific evidence to assist in decision-making for appropriate and reasonable treatments. STUDY DESIGN/SETTING: A systematic review. PATIENT SAMPLE: Studies of human subjects written in Korean or English that met the following criteria were selected: patients aged ≥18 years, clinical presentation of low back and radicular leg pain, diagnosis of LDH on radiological evaluation including computed tomography or magnetic resonance imaging. OUTCOMES MEASURES: Pain and functional evaluation scales such as visual analogue scale, numeric rating scale, and Oswestry disability index METHODS: The MEDLINE (PubMed), EMBASE, Cochrane Review, and KoreaMed databases were searched for articles regarding non-surgical treatments for LDH published up to July 2017. Of the studies fulfilling these criteria, those investigating clinical results after non-surgical treatment including physical and behavioral therapy, medication, and interventional treatment in terms of pain control and functional improvements were chosen for this study. RESULTS: Nonsurgical treatments were determined to be clinically effective with regards to pain reduction and functional improvement in patients with LDH. Nevertheless, the evidence level was generally not evaluated as high degree, which might be attributed to the paucity of well-designed randomized controlled trials. Exercise and traction were strongly recommended despite moderate level of evidence. Epidural injection was strongly recommended with high degree of evidence and transforaminal approach was more strongly recommended than caudal approach. CONCLUSIONS: This CPG provides new and updated evidence-based recommendations for treatment of the patients with LDH, which suggested that, despite an absence of high degrees of evidence level, non-surgical treatments were clinically effective.

Low-dose botulinum toxin type A for the treatment of refractory piriformis syndrome.

STUDY OBJECTIVES: To evaluate the efficacy of a single, low-dose injection of botulinum toxin type A in relieving pain in Korean patients with piriformis syndrome resistant to conventional therapy, and to assess the drug's influence on these patients' quality of life. DESIGN: Prospective, single-site, open-label trial. SETTING: Rehabilitation medicine clinic in Seoul, Korea. PATIENTS: Twenty-nine patients with a confirmed diagnosis of chronic piriformis syndrome and 82 age- and sex-matched healthy subjects were enrolled from April 1, 2003-February 28, 2004. Intervention. In 20 of the patients, botulinum toxin type A 150 U was injected using computed tomographic guidance into the affected unilateral piriformis muscle. The other nine patients served as active controls and received an injection of dexamethasone 5 mg and 1% lidocaine. The healthy subjects did not receive any injection. MEASUREMENTS AND MAIN RESULTS: The patients' pain at baseline and at 4, 8, and 12 weeks after treatment was rated by using a numeric rating scale. Health-related quality of life was assessed by using the validated Korean version of the Medical Outcomes Study 36-Item Short Form Health Survey (SF-36) at baseline and at 4 weeks of treatment. Healthy subjects also completed the SF-36 at baseline. Pain intensity scores were significantly lower at 4, 8, and 12 weeks after treatment than at baseline (p<0.0001). Baseline scores from the SF-36 subscales, including those for physical functioning (p<0.0001), role physical (p<0.0001), bodily pain (p<0.0001), general health (p<0.0001), vitality (p<0.0001), and social functioning (p<0.002), were significantly lower in the patients than in the healthy subjects. Four weeks after treatment, physical functioning (p=0.003), role physical (p=0.021), bodily pain (p=0.016), general health (p=0.013), vitality (p=0.031) and social functioning (p=0.035) improved significantly from baseline in the patients. However, at 4 weeks, patients in the active control group were withdrawn from the study because their pain did not improve, and continuation without further medical care was considered unethical. CONCLUSION: A low dose of botulinum toxin type A relieved pain and improved quality of life in patients with refractory piriformis syndrome.

Leptin receptors are down-regulated in uterine implantation sites compared to interimplantation sites.

Leptin is a circulating hormone that plays an important role in the regulation of metabolism, obesity, and reproduction. Leptin binds to its receptors on the cell membrane and is involved in the activation of STAT3. Recently, endometrium was suggested to be a novel target for leptin recently. We, therefore, examined the expression of leptin, leptin receptors, and STAT3 in the mouse uterus (implantation and interimplantation sites) to investigate the role of the leptin system during the early implantation period. Leptin mRNA was not detected in mouse uterine tissues or blastocysts, although adipose tissue, the positive control, showed a strong signal. Both of the receptor splice variants were expressed in the uterus and blastocysts, but the mRNA level was much lower in implantation sites compared to interimplantation sites. The mRNA expression of leptin receptors was determined to be higher in stromal cells than in the luminal epithelium using laser capture microdissection (LCM) analysis. Using immunohistochemistry, leptin was detected as a strong signal in the luminal epithelium and embryo, whereas the receptor was detected in subepithelial stromal cells rather than the luminal epithelium. As leptin itself was not detected by RT-PCR, the immunohistologically detected leptin may originate elsewhere, such as in adipose tissue. The differential expression of leptin receptors in implantation sites compared to interimplantation sites suggests that the leptin/leptin receptor system may be a delicate regulator of the implantation process.

Identification of differential gene expression in germinal vesicle vs. metaphase II mouse oocytes by using annealing control primers.

By using a new innovative technology, annealing control primer-polymerase chain reaction (ACP-PCR), we identified genes that are differentially expressed in immature GV vs. in mature MII mouse oocytes. The results of the present study will be valuable in understanding the components of oocyte maturation and provide a basis for studies of human oocyte maturation.

HEB associates with PRC2 and SMAD2/3 to regulate developmental fates.

In embryonic stem cells, extracellular signals are required to derepress developmental promoters to drive lineage specification, but the proteins involved in connecting extrinsic cues to relaxation of chromatin remain unknown. We demonstrate that the helix-loop-helix (HLH) protein, HEB, directly associates with the Polycomb repressive complex 2 (PRC2) at a subset of developmental promoters, including at genes involved in mesoderm and endoderm specification and at the Hox and Fox gene families. While we show that depletion of HEB does not affect mouse ESCs, it does cause premature differentiation after exposure to Activin. Further, we find that HEB deposition at developmental promoters is dependent upon PRC2 and independent of Nodal, whereas HEB association with SMAD2/3 elements is dependent of Nodal, but independent of PRC2. We suggest that HEB is a fundamental link between Nodal signalling, the derepression of a specific class of poised promoters during differentiation, and lineage specification in mouse ESCs.

A molecular basis for embryo apposition at the luminal epithelium.

To obtain a gene expression profile during embryo apposition to the luminal epithelium, we isolated mouse luminal epithelium from implantation (IM) and interimplantation (INTER) sites using laser capture microdissection (LCM), and analyzed their gene expression by microarray analysis. IM and INTER sites were sampled on day 4.5 after mating of female mice with fertile males (day 0.5 = vaginal plug). RNA was extracted, amplified, labeled, and hybridized to microarrays and results were analyzed using the significance analysis of microarrays (SAM) method. Comparison of IM and INTER sites by SAM identified 73 genes most highly ranked at IM, while 13 genes most highly expressed at the INTER sites, within the estimated false discovery rate (FDR) of 0.163. Among 73 genes at IM, 20 were ESTs or were of unknown function, and the remain 53 genes had known functions mainly relating to cellular structuring and others such as cell cycling, gene/protein expression, immune responses, invasion, metabolism, oxidative stress, or signal transduction. Specifically, of the 24 structural genes, 14 were implicated in extracellular matrix and tissue remodeling. Meanwhile, of the 13 genes that were highly expressed at INTER, eight were ESTs or of unknown function, and the remaining five were implicated in metabolism, signal transduction, and gene/protein expression. Among these 58 (53 + 5) genes with known functions, 13 genes (22.4%) were associated with Ca2+ for their function. Results of the present study suggest that (1) at IM sites, active tissue remodeling is occurring for embryo invasion while the INTER sites are relatively quiescent and (2) Ca2+ may be a vital regulatory factor in the apposition process. Investigations of human homologues of those genes expressed in the mouse luminal epithelium during apposition may help to understand the implantation process and/or implantation failure in humans.

Anti-inflammatory effects of botulinum toxin type a in a complete Freund's adjuvant-induced arthritic knee joint of hind leg on rat model.

The objective of the study is to verify histopathologically the anti-inflammatory effect of botulinum toxin type A (BoNT-A) in a Complete Freund's Adjuvant (CFA)-induced arthritic knee joint of hind leg on rat model using immunofluorescent staining of anti-ionized calcium-binding adaptor molecule 1 (Iba-1) and interleukin-1ß (IL-1ß) antibody. Twenty-eight experimental rats were injected with 0.1 ml of CFA solution in the knee joint of the hind leg bilaterally. Three weeks after CFA injection, the BoNT-A group (N = 14) was injected with 20 IU (0.1 ml) of BoNT-A bilaterally while the saline group (N = 14) was injected with 0.1 ml of saline in the knee joint of the hind leg bilaterally. One and two weeks after BoNT-A or saline injection, joint inflammation was investigated in seven rats from each group using histopathological and immune-fluorescent staining of Iba-1 and IL-1ß antibody. The number of Iba-1 and IL-1ß immune-reactive (IR) cells was counted in the BoNT-A and saline groups for comparison. There was a significant reduction in joint inflammation and destruction in the BoNT-A group at 1 and 2 weeks after BoNT-A injection compared with the saline group. The binding of Iba-1 and IL-1ß antibody was significantly lower in the BoNT-A group than the saline group at 1 and 2 weeks after BoNT-A injection. The number of Iba-1 and IL-1ß-IR cells at 1 and 2 weeks after the injection of BoNT-A were significantly different from the corresponding number of Iba-1 and IL-1ß-IR cells in the saline group. To conclude, BoNT-A had an anti-inflammatory effect in a CFA-induced arthritic rat model, indicating that BoNT-A could potentially be used to treat inflammatory joint pain.

A comparison of the short-term effects of a botulinum toxin type a and triamcinolone acetate injection on adhesive capsulitis of the shoulder.

OBJECTIVE: To evaluate the short-term clinical effects of the intra-articular injection of botulinum toxin type A (BoNT-A) for the treatment of adhesive capsulitis. METHODS: A prospective, controlled trial compared the effects of intra-articular BoNT-A (Dysport; 200 IU, n=15) with the steroid triamcinolone acetate (TA; 20 mg, n=13) in patients suffering from adhesive capsulitis of the shoulder. All patients were evaluated using a Numeric Rating Scale (NRS) of the pain intensity and a measurement of the range of motion (ROM) at baseline (before treatment) and at 2, 4, and 8 weeks post-treatment. RESULTS: The NRS at 2 weeks (BoNT-A vs. TA; 5.0 vs. 5.2), 4 weeks (4.1 vs. 4.9) and 8 weeks (3.8 vs. 4.6) of both treatment groups were significantly lower than that measured at baseline (7.4 vs. 7.6). The ROM of patients' shoulders increased significantly from baseline in both treatment groups. There was no significant difference in the NRS of pain intensity or the ROM between the two groups. Reduction in the pain intensity score was maintained for 8 weeks post-injection in both groups. There were no significant adverse events in either treatment group. CONCLUSION: The results suggest that there are no significant short-term differences between the intra-articular injections of BoNT-A and TA. Although BoNT-A has a high cost, it may be used as a safe alternative of TA to avoid the steroid-induced side effects or as a second-line agent, for patients who have failed to respond to the current treatments.

Function of COP9 signalosome in regulation of mouse oocytes meiosis by regulating MPF activity and securing degradation.

The COP9 (constitutive photomorphogenic) signalosome (CSN), composed of eight subunits, is a highly conserved protein complex that regulates processes such as cell cycle progression and kinase signalling. Previously, we found the expression of the COP9 constitutive photomorphogenic homolog subunit 3 (CSN3) and subunit 5 (CSN5) changes as oocytes mature for the first time, and there is no report regarding roles of COP9 in the mammalian oocytes. Therefore, in the present study, we examined the effects of RNA interference (RNAi)-mediated transient knockdown of each subunit on the meiotic cell cycle in mice oocytes. Following knockdown of either CSN3 or CSN5, oocytes failed to complete meiosis I. These arrested oocytes exhibited a disrupted meiotic spindle and misarranged chromosomes. Moreover, down-regulation of each subunit disrupted the activity of maturation-promoting factor (MPF) and concurrently reduced degradation of the anaphase-promoting complex/cyclosome (APC/C) substrates Cyclin B1 and Securin. Our data suggest that the CSN3 and CSN5 are involved in oocyte meiosis by regulating degradation of Cyclin B1 and Securin via APC/C.