Microenvironmental modulation in tandem with human stem cell transplantation enhances functional recovery after chronic complete spinal cord injury.

While rapid advancements in regenerative medicine strategies for spinal cord injury (SCI) have been made, most research in this field has focused on the early stages of incomplete injury. However, the majority of patients experience chronic severe injury; therefore, treatments for these situations are fundamentally important. Here, we hypothesized that environmental modulation via a clinically relevant hepatocyte growth factor (HGF)-releasing scaffold and human iPS cell-derived neural stem/progenitor cells (hNS/PCs) transplantation contributes to functional recovery after chronic complete transection SCI. Effective release of HGF from a collagen scaffold induced progressive axonal elongation and increased grafted cell viability by activating microglia/macrophages and meningeal cells, inhibiting inflammation, reducing scar formation, and enhancing vascularization. Furthermore, hNS/PCs transplantation enhanced endogenous neuronal regrowth, the extension of graft axons, and the formation of circuits around the lesion and lumbar enlargement between host and graft neurons, resulting in the restoration of locomotor and urinary function. This study presents an effective therapeutic strategy for severe chronic SCI and provides evidence for the feasibility of regenerative medicine strategies using clinically relevant materials.

Single-cell transcriptomics of human gut T cells identifies cytotoxic CD4+CD8A+ T cells related to mouse CD4 cytotoxic T cells.

Cytotoxic CD4+ T cells (CD4-CTLs) show the presence of cytolytic granules, which include the enzymes granzyme and perforin. The cells have a pathogenic and protective role in various diseases, including cancer, viral infection, and autoimmune disease. In mice, cytotoxic CD4+ T cells express CD8αα+ and reside in the intestine (mouse CD4+CTLs; mCD4-CTLs). The population of cytotoxic CD4+ T cells in the human intestine is currently unknown. Moreover, it is unclear how cytotoxic CD4 T cells change in patients with inflammatory bowel disease (IBD). Here, we aimed to identify cytotoxic CD4+ T cells in the human intestine and analyze the characteristics of the population in patients with IBD using single-cell RNA-seq (scRNA-seq). In CD4+ T cells, granzyme and perforin expression was high in humanMAIT (hMAIT) cells and hCD4+CD8A+ T cell cluster. Both CD4 and CD8A were expressed in hTreg, hMAIT, and hCD4+CD8A+ T cell clusters. Next we performed fast gene set enrichment analysis to identify cell populations that showed homology to mCD4CTLs. The analysis identified the hCD4+CD8A+ T cell cluster (hCTL-like population; hCD4-CTL) similar to mouse CTLs. The percentage of CD4+CD8A+ T cells among the total CD4+ T cells in the inflamed intestine of the patients with Crohn's disease was significantly reduced compared with that in the noninflamed intestine of the patients. In summary, we identified cytotoxic CD4+CD8+ T cells in the small intestine of humans. The integration of the mouse and human sc-RNA-seq data analysis highlight an approach to identify human cell populations related to mouse cell populations, which may help determine the functional properties of several human cell populations in mice.

Critical roles of FGF, RA, and WNT signalling in the development of the human otic placode and subsequent lineages in a dish.

Introduction: Efficient induction of the otic placode, the developmental origin of the inner ear from human pluripotent stem cells (hPSCs), provides a robust platform for otic development and sensorineural hearing loss modelling. Nevertheless, there remains a limited capacity of otic lineage specification from hPSCs by stepwise differentiation methods, since the critical factors for successful otic cell differentiation have not been thoroughly investigated. In this study, we developed a novel differentiation system involving the use of a three-dimensional (3D) floating culture with signalling factors for generating otic cell lineages via stepwise differentiation of hPSCs. Methods: We differentiated hPSCs into preplacodal cells under a two-dimensional (2D) monolayer culture. Then, we transferred the induced preplacodal cells into a 3D floating culture under the control of the fibroblast growth factor (FGF), bone morphogenetic protein (BMP), retinoic acid (RA) and WNT signalling pathways. We evaluated the characteristics of the induced cells using immunocytochemistry, quantitative PCR (qPCR), population averaging, and single-cell RNA-seq (RNA-seq) analysis. We further investigated the methods for differentiating otic progenitors towards hair cells by overexpression of defined transcription factors. Results: We demonstrated that hPSC-derived preplacodal cells acquired the potential to differentiate into posterior placodal cells in 3D floating culture with FGF2 and RA. Subsequent activation of WNT signalling induced otic placodal cell formation. By single-cell RNA-seq (scRNA-seq) analysis, we identified multiple clusters of otic placode- and otocyst marker-positive cells in the induced spheres. Moreover, the induced otic cells showed the potential to generate hair cell-like cells by overexpression of the transcription factors ATOH1, POU4F3 and GFI1. Conclusions: We demonstrated the critical role of FGF2, RA and WNT signalling in a 3D environment for the in vitro differentiation of otic lineage cells from hPSCs. The induced otic cells had the capacity to differentiate into inner ear hair cells with stereociliary bundles and tip link-like structures. The protocol will be useful for in vitro disease modelling of sensorineural hearing loss and human inner ear development and thus contribute to drug screening and stem cell-based regenerative medicine.

A robust culture system to generate neural progenitors with gliogenic competence from clinically relevant induced pluripotent stem cells for treatment of spinal cord injury.

Cell-based therapy targeting spinal cord injury (SCI) is an attractive approach to promote functional recovery by replacing damaged tissue. We and other groups have reported the effectiveness of transplanting neural stem/progenitor cells (NS/PCs) derived from human induced pluripotent stem cells (hiPSCs) in SCI animal models for neuronal replacement. Glial replacement is an additional approach for tissue repair; however, the lack of robust procedures to drive iPSCs into NS/PCs which can produce glial cells has hindered the development of glial cell transplantation for the restoration of neuronal functions after SCI. Here, we established a method to generate NS/PCs with gliogenic competence (gNS/PCs) optimized for clinical relevance and utilized them as a source of therapeutic NS/PCs for SCI. We could successfully generate gNS/PCs from clinically relevant hiPSCs, which efficiently produced astrocytes and oligodendrocytes in vitro. We also performed comparison between gNS/PCs and neurogenic NS/PCs based on single cell RNA-seq analysis and found that gNS/PCs were distinguished by expression of several transcription factors including HEY2 and NFIB. After gNS/PC transplantation, the graft did not exhibit tumor-like tissue formation, indicating the safety of them as a source of cell therapy. Importantly, the gNS/PCs triggered functional recovery in an SCI animal model, with remyelination of demyelinated axons and improved motor function. Given the inherent safety of gNS/PCs and favorable outcomes observed after their transplantation, cell-based medicine using the gNS/PCs-induction procedure described here together with clinically relevant iPSCs is realistic and would be beneficial for SCI patients.

Primed to Naive-Like Conversion of the Common Marmoset Embryonic Stem Cells.

Mammalian pluripotent stem cells are thought to exist in two states: naive and primed. Generally, unlike those in rodents, pluripotent stem cells in primates, including humans, are regarded as being in the primed pluripotent state. Recently, several groups reported the existence of naive pluripotent stem cells in humans. In this study, we report the conversion of primed state embryonic stem cells from common marmoset, a New World monkey, to the naive state using transgenes. The cells showed typical naive state features, including dome-like colony morphology, growth factor requirement, gene expression profile, X chromosome activation state, and energy metabolic status. Moreover, interspecies chimeric embryo formation ability with mouse embryos was increased in the naive state. This technique can be applied in basic medical research using nonhuman primates, such as preclinical use of naive pluripotent stem cells and generating genetically modified primates.

Loss of TET proteins in regulatory T cells promotes abnormal proliferation, Foxp3 destabilization and IL-17 expression.

Ten-eleven translocation (TET) proteins regulate DNA methylation and gene expression by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Although Tet2/Tet3 deficiency has been reported to lead to myeloid cell, B-cell and invariant natural killer T (iNKT) cell malignancy, the effect of TET on regulatory T cells (Tregs) has not been elucidated. We found that Tet2/Tet3 deficiency in Tregs led to lethal hyperproliferation of CD4+Foxp3+ T cells in the spleen and mesenteric lymph nodes after 5 months of age. Additionally, in aged Treg-specific Tet2/Tet3-deficient mice, serum IgG1, IgG3, IgM and IgE levels were markedly elevated. High IL-17 expression was observed in both Foxp3+ and Fopx3- CD4+ T cells, and adoptive transfer of Tet2/Tet3-deficient Tregs into lymphopenic mice inhibited Foxp3 expression and caused conversion into IL-17-producing cells. However, the conserved non-coding DNA sequence-2 (CNS2) region of the Foxp3 gene locus, which has been shown to be particularly important for stable Foxp3 expression, was only partly methylated. We identified novel TET-dependent demethylation sites in the Foxp3 upstream enhancer, which may contribute to stable Foxp3 expression. Together, these data indicate that Tet2 and Tet3 are involved in Treg stability and immune homeostasis in mice.

HER2 Heterogeneity Is Associated with Poor Survival in HER2-Positive Breast Cancer.

Intratumoral human epidermal growth factor receptor 2 (HER2) heterogeneity has been reported in 16⁻36% of HER2-positive breast cancer and its clinical impact is under discussion. We examined the biological effects of HER2-heterogeneity on mouse models and analyzed metastatic brains by RNA sequence analysis. A metastatic mouse model was developed using 231-Luc (triple negative cells) and 2 HER2-positive cell lines, namely, HER2-60 and HER2-90 which showed heterogeneous and monotonous HER2 expressions, respectively. Metastatic lesions developed in 3 weeks in all the mice injected with HER2-60 cells, and in 69% of the mice injected with HER2-90 and 87.5% of the mice injected with 231-Luc. The median survival days of mice injected with 231-Luc, HER2-60, and HER2-90 cells were 29 (n = 24), 24 (n = 22) and 30 (n = 13) days, respectively. RNA sequence analysis showed that CASP-1 and its related genes were significantly downregulated in metastatic brain tumors with HER2-60 cells. The low expression of caspase-1 could be a new prognostic biomarker for early relapse in HER2-positive breast cancer.

Chromatin remodeler CHD7 regulates the stem cell identity of human neural progenitors.

Multiple congenital disorders often present complex phenotypes, but how the mutation of individual genetic factors can lead to multiple defects remains poorly understood. In the present study, we used human neuroepithelial (NE) cells and CHARGE patient-derived cells as an in vitro model system to identify the function of chromodomain helicase DNA-binding 7 (CHD7) in NE-neural crest bifurcation, thus revealing an etiological link between the central nervous system (CNS) and craniofacial anomalies observed in CHARGE syndrome. We found that CHD7 is required for epigenetic activation of superenhancers and CNS-specific enhancers, which support the maintenance of the NE and CNS lineage identities. Furthermore, we found that BRN2 and SOX21 are downstream effectors of CHD7, which shapes cellular identities by enhancing a CNS-specific cellular program and indirectly repressing non-CNS-specific cellular programs. Based on our results, CHD7, through its interactions with superenhancer elements, acts as a regulatory hub in the orchestration of the spatiotemporal dynamics of transcription factors to regulate NE and CNS lineage identities.

Naive-like ESRRB+ iPSCs with the Capacity for Rapid Neural Differentiation.

Several groups have reported the existence of a form of pluripotency that resembles that of mouse embryonic stem cells (mESCs), i.e., a naive state, in human pluripotent stem cells; however, the characteristics vary between reports. The nuclear receptor ESRRB is expressed in mESCs and plays a significant role in their self-renewal, but its expression has not been observed in most naive-like human induced pluripotent stem cells (hiPSCs). In this study, we modified several methods for converting hiPSCs into a naive state through the transgenic expression of several reprogramming factors. The resulting cells express the components of the core transcriptional network of mESCs, including ESRRB, at high levels, which suggests the existence of naive-state hiPSCs that are similar to mESCs. We also demonstrate that these cells differentiate more readily into neural cells than do conventional hiPSCs. These features may be beneficial for their use in disease modeling and regenerative medicine.

An RNA-binding protein, Qki5, regulates embryonic neural stem cells through pre-mRNA processing in cell adhesion signaling.

Cell type-specific transcriptomes are enabled by the action of multiple regulators, which are frequently expressed within restricted tissue regions. In the present study, we identify one such regulator, Quaking 5 (Qki5), as an RNA-binding protein (RNABP) that is expressed in early embryonic neural stem cells and subsequently down-regulated during neurogenesis. mRNA sequencing analysis in neural stem cell culture indicates that Qki proteins play supporting roles in the neural stem cell transcriptome and various forms of mRNA processing that may result from regionally restricted expression and subcellular localization. Also, our in utero electroporation gain-of-function study suggests that the nuclear-type Qki isoform Qki5 supports the neural stem cell state. We next performed in vivo transcriptome-wide protein-RNA interaction mapping to search for direct targets of Qki5 and elucidate how Qki5 regulates neural stem cell function. Combined with our transcriptome analysis, this mapping analysis yielded a bona fide map of Qki5-RNA interaction at single-nucleotide resolution, the identification of 892 Qki5 direct target genes, and an accurate Qki5-dependent alternative splicing rule in the developing brain. Last, our target gene list provides the first compelling evidence that Qki5 is associated with specific biological events; namely, cell-cell adhesion. This prediction was confirmed by histological analysis of mice in which Qki proteins were genetically ablated, which revealed disruption of the apical surface of the lateral wall in the developing brain. These data collectively indicate that Qki5 regulates communication between neural stem cells by mediating numerous RNA processing events and suggest new links between splicing regulation and neural stem cell states.

Improvement of Foxp3 stability through CNS2 demethylation by TET enzyme induction and activation.

Since induced regulatory T cells (iTregs) can be produced in a large quantity in vitro, these cells are expected to be clinically useful to induce immunological tolerance in various immunological diseases. Foxp3 (Forkhead box P3) expression in iTregs is, however, unstable due to the lack of demethylation of the CpG island in the conserved non-coding sequence 2 (CNS2) of the Foxp3 locus. To facilitate the demethylation of CNS2, we over-expressed the catalytic domain (CD) of the ten-eleven translocation (TET) protein, which catalyzes the steps of the iterative demethylation of 5-methylcytosine. TET-CD over-expression in iTregs resulted in partial demethylation of CNS2 and stable Foxp3 expression. We also discovered that TET expression was enhanced under low oxygen (5%) culture conditions, which facilitated CNS2 DNA demethylation and stabilization of Foxp3 expression in a TET2- and TET3-dependent manner. In combination with vitamin C treatment, which has been reported to enhance TET catalytic activity, iTregs generated under low oxygen conditions retained more stable Foxp3 expression in vitro and in vivo and exhibited stronger suppression activity in a colitis model compared with untreated iTregs. Our data indicate that the induction and activation of TET enzymes in iTregs would be an effective method for Treg-mediated adoptive immunotherapy.

RNA Sequencing Analysis Reveals Interactions between Breast Cancer or Melanoma Cells and the Tissue Microenvironment during Brain Metastasis.

Metastasis is the main cause of treatment failure and death in cancer patients. Metastasis of tumor cells to the brain occurs frequently in individuals with breast cancer, non-small cell lung cancer, or melanoma. Despite recent advances in our understanding of the causes and in the treatment of primary tumors, the biological and molecular mechanisms underlying the metastasis of cancer cells to the brain have remained unclear. Metastasizing cancer cells interact with their microenvironment in the brain to establish metastases. We have now developed mouse models of brain metastasis based on intracardiac injection of human breast cancer or melanoma cell lines, and we have performed RNA sequencing analysis to identify genes in mouse brain tissue and the human cancer cells whose expression is associated specifically with metastasis. We found that the expressions of the mouse genes Tph2, Sspo, Ptprq, and Pole as well as those of the human genes CXCR4, PLLP, TNFSF4, VCAM1, SLC8A2, and SLC7A11 were upregulated in brain tissue harboring metastases. Further characterization of such genes that contribute to the establishment of brain metastases may provide a basis for the development of new therapeutic strategies and consequent improvement in the prognosis of cancer patients.

Tumour resistance in induced pluripotent stem cells derived from naked mole-rats.

The naked mole-rat (NMR, Heterocephalus glaber), which is the longest-lived rodent species, exhibits extraordinary resistance to cancer. Here we report that NMR somatic cells exhibit a unique tumour-suppressor response to reprogramming induction. In this study, we generate NMR-induced pluripotent stem cells (NMR-iPSCs) and find that NMR-iPSCs do not exhibit teratoma-forming tumorigenicity due to the species-specific activation of tumour-suppressor alternative reading frame (ARF) and a disruption mutation of the oncogene ES cell-expressed Ras (ERAS). The forced expression of Arf in mouse iPSCs markedly reduces tumorigenicity. Furthermore, we identify an NMR-specific tumour-suppression phenotype-ARF suppression-induced senescence (ASIS)-that may protect iPSCs and somatic cells from ARF suppression and, as a consequence, tumorigenicity. Thus, NMR-specific ARF regulation and the disruption of ERAS regulate tumour resistance in NMR-iPSCs. Our findings obtained from studies of NMR-iPSCs provide new insight into the mechanisms of tumorigenicity in iPSCs and cancer resistance in the NMR.

Long-term safety issues of iPSC-based cell therapy in a spinal cord injury model: oncogenic transformation with epithelial-mesenchymal transition.

Previously, we described the safety and therapeutic potential of neurospheres (NSs) derived from a human induced pluripotent stem cell (iPSC) clone, 201B7, in a spinal cord injury (SCI) mouse model. However, several safety issues concerning iPSC-based cell therapy remain unresolved. Here, we investigated another iPSC clone, 253G1, that we established by transducing OCT4, SOX2, and KLF4 into adult human dermal fibroblasts collected from the same donor who provided the 201B7 clone. The grafted 253G1-NSs survived, differentiated into three neural lineages, and promoted functional recovery accompanied by stimulated synapse formation 47 days after transplantation. However, long-term observation (for up to 103 days) revealed deteriorated motor function accompanied by tumor formation. The tumors consisted of Nestin(+) undifferentiated neural cells and exhibited activation of the OCT4 transgene. Transcriptome analysis revealed that a heightened mesenchymal transition may have contributed to the progression of tumors derived from grafted cells.

Global gene expression analysis following spinal cord injury in non-human primates.

Spinal cord injury (SCI) is a devastating condition with no established treatment. To better understand the pathology and develop a treatment modality for SCI, an understanding of the physiological changes following SCI at the molecular level is essential. However, studies on SCI have primarily used rodent models, and few studies have examined SCI in non-human primates. In this study, we analyzed the temporal changes in gene expression patterns following SCI in common marmosets (Callithrix jacchus) using microarray analysis and mRNA deep sequencing. This analysis revealed that, although the sequence of events is comparable between primates and rodents, the inflammatory response following SCI is significantly prolonged and the onset of glial scar formation is temporally delayed in primates compared with rodents. These observations indicate that the optimal time window to treat SCI significantly differs among different species. This study provides the first extensive analysis of gene expression following SCI in non-human primates and will serve as a valuable resource in understanding the pathology of SCI.