Tetsuro Kobayashi

Mitochondrial dysfunction associated with increased oxidative stress and α-synuclein accumulation in PARK2 iPSC-derived neurons and postmortem brain tissue

BackgroundParkinson’s disease (PD) is a neurodegenerative disease characterized by selective degeneration of dopaminergic neurons in the substantia nigra (SN). The familial form of PD, PARK2, is caused by mutations in the parkin gene. parkin-knockout mouse models show some abnormalities, but they do not fully recapitulate the pathophysiology of human PARK2.ResultsHere, we generated induced pluripotent stem cells (iPSCs) from two PARK2 patients. PARK2 iPSC-derived neurons showed increased oxidative stress and enhanced activity of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. iPSC-derived neurons, but not fibroblasts or iPSCs, exhibited abnormal mitochondrial morphology and impaired mitochondrial homeostasis. Although PARK2 patients rarely exhibit Lewy body (LB) formation with an accumulation of α-synuclein, α-synuclein accumulation was observed in the postmortem brain of one of the donor patients. This accumulation was also seen in the iPSC-derived neurons in the same patient.ConclusionsThus, pathogenic changes in the brain of a PARK2 patient were recapitulated using iPSC technology. These novel findings reveal mechanistic insights into the onset of PARK2 and identify novel targets for drug screening and potential modified therapies for PD.

Stress-induced production of chemokines by hair follicles regulates the trafficking of dendritic cells in skin

Langerhans cells (LCs) are epidermal dendritic cells with incompletely understood origins that associate with hair follicles for unknown reasons. Here we show that in response to external stress, mouse hair follicles recruited Gr-1hi monocyte-derived precursors of LCs whose epidermal entry was dependent on the chemokine receptors CCR2 and CCR6, whereas the chemokine receptor CCR8 inhibited the recruitment of LCs. Distinct hair-follicle regions had differences in their expression of ligands for CCR2 and CCR6. The isthmus expressed the chemokine CCL2; the infundibulum expressed the chemokine CCL20; and keratinocytes in the bulge produced the chemokine CCL8, which is the ligand for CCR8. Thus, distinct hair-follicle keratinocyte subpopulations promoted or inhibited repopulation with LCs via differences in chemokine production, a feature also noted in humans. Pre-LCs failed to enter hairless skin in mice or humans, which establishes hair follicles as portals for LCs.

Dysbiosis and Staphylococcus aureus Colonization Drives Inflammation in Atopic Dermatitis

Staphylococcus aureus skin colonization is universal in atopic dermatitis and common in cancer patients treated with epidermal growth factor receptor inhibitors. However, the causal relationship of dysbiosis and eczema has yet to be clarified. Herein, we demonstrate that Adam17(fl/fl)Sox9-(Cre) mice, generated to model ADAM17-deficiency in human, developed eczematous dermatitis with naturally occurring dysbiosis, similar to that observed in atopic dermatitis. Corynebacterium mastitidis, S. aureus, and Corynebacterium bovis sequentially emerged during the onset of eczematous dermatitis, and antibiotics specific for these bacterial species almost completely reversed dysbiosis and eliminated skin inflammation. Whereas S. aureus prominently drove eczema formation, C. bovis induced robust T helper 2 cell responses. Langerhans cells were required for eliciting immune responses against S. aureus inoculation. These results characterize differential contributions of dysbiotic flora during eczema formation, and highlight the microbiota-host immunity axis as a possible target for future therapeutics in eczematous dermatitis.

Hair follicle–derived IL-7 and IL-15 mediate skin-resident memory T cell homeostasis and lymphoma

The skin harbors a variety of resident leukocyte subsets that must be tightly regulated to maintain immune homeostasis. Hair follicles are unique structures in the skin that contribute to skin dendritic cell homeostasis through chemokine production. We demonstrate that CD4+ and CD8+ skin-resident memory T cells (TRM cells), which are responsible for long-term skin immunity, reside predominantly within the hair follicle epithelium of the unperturbed epidermis. TRM cell tropism for the epidermis and follicles is herein termed epidermotropism. Hair follicle expression of IL-15 was required for CD8+ TRM cells, and IL-7 for CD8+ and CD4+ TRM cells, to exert epidermotropism. A lack of either cytokine in the skin led to impaired hapten-induced contact hypersensitivity responses. In a model of cutaneous T cell lymphoma, epidermotropic CD4+ TRM lymphoma cell localization depended on the presence of hair follicle–derived IL-7. These findings implicate hair follicle–derived cytokines as regulators of malignant and non-malignant TRM cell tissue residence, and they suggest that the cytokines may be targeted therapeutically in inflammatory skin diseases and lymphoma.

Differentiation of multipotent neural stem cells derived from Rett syndrome patients is biased toward the astrocytic lineage

BackgroundRett syndrome (RTT) is one of the most prevalent neurodevelopmental disorders in females, caused by de novo mutations in the X-linked methyl CpG-binding protein 2 gene, MECP2. Although abnormal regulation of neuronal genes due to mutant MeCP2 is thought to induce autistic behavior and impaired development in RTT patients, precise cellular mechanisms underlying the aberrant neural progression remain unclear.ResultsTwo sets of isogenic pairs of either wild-type or mutant MECP2-expressing human induced pluripotent stem cell (hiPSC) lines were generated from a single pair of 10-year-old RTT-monozygotic (MZ) female twins. Mutant MeCP2-expressing hiPSC lines did not express detectable MeCP2 protein during any stage of differentiation. The lack of MeCP2 reflected altered gene expression patterns in differentiated neural cells rather than in undifferentiated hiPSCs, as assessed by microarray analysis. Furthermore, MeCP2 deficiency in the neural cell lineage increased astrocyte-specific differentiation from multipotent neural stem cells. Additionally, chromatin immunoprecipitation (ChIP) and bisulfite sequencing assays indicated that anomalous glial fibrillary acidic protein gene (GFAP) expression in the MeCP2-negative, differentiated neural cells resulted from the absence of MeCP2 binding to the GFAP gene.ConclusionsAn isogenic RTT-hiPSC model demonstrated that MeCP2 participates in the differentiation of neural cells. Moreover, MeCP2 deficiency triggers perturbation of astrocytic gene expression, yielding accelerated astrocyte formation from RTT-hiPSC-derived neural stem cells. These findings are likely to shed new light on astrocytic abnormalities in RTT, and suggest that astrocytes, which are required for neuronal homeostasis and function, might be a new target of RTT therapy.

Restoration of the intrinsic properties of human dermal papilla in vitro.

Summary The dermal papilla (DP) plays pivotal roles in hair follicle morphogenesis and cycling. However, characterization and/or propagation of human DPs have been unsatisfactory because of the lack of efficient isolation methods and the loss of innate characteristics in vitro. We hypothesized that culture conditions sustaining the intrinsic molecular signature of the human DP could facilitate expansion of functional DP cells. To test this, we first characterized the global gene expression profile of microdissected, non-cultured human DPs. We performed a ‘two-step’ microarray analysis to exclude the influence of unwanted contaminants in isolated DPs and successfully identified 118 human DP signature genes, including 38 genes listed in the mouse DP signature. The bioinformatics analysis of the DP gene list revealed that WNT, BMP and FGF signaling pathways were upregulated in intact DPs and addition of 6-bromoindirubin-3′-oxime, recombinant BMP2 and basic FGF to stimulate these respective signaling pathways resulted in maintained expression of in situ DP signature genes in primarily cultured human DP cells. More importantly, the exposure to these stimulants restored normally reduced DP biomarker expression in conventionally cultured DP cells. Cell growth was moderate in the newly developed culture medium. However, rapid DP cell expansion by conventional culture followed by the restoration by defined activators provided a sufficient number of DP cells that demonstrated characteristic DP activities in functional assays. The study reported here revealed previously unreported molecular mechanisms contributing to human DP properties and describes a useful technique for the investigation of human DP biology and hair follicle bioengineering.

Human Induced Pluripotent Stem Cell–Derived Ectodermal Precursor Cells Contribute to Hair Follicle Morphogenesis In Vivo

Well-orchestrated epithelial-mesenchymal interactions are crucial for hair follicle (HF) morphogenesis. In this study, ectodermal precursor cells (EPCs) with the capacity to cross talk with hair-inductive dermal cells were generated from human induced pluripotent stem cells (hiPSCs) and assessed for HF-forming ability in vivo. EPCs derived from three hiPSC lines generated with 4 or 3 factors (POU5F1, SOX2, KLF4 +/- MYC) mostly expressed keratin 18, a marker of epithelial progenitors. When cocultured with human dermal papilla (DP) cells, a 4 factor 201B7 hiPSC-EPC line upregulated follicular keratinocyte (KC) markers more significantly than normal human adult KCs (NHKCs) and other hiPSC-EPC lines. DP cells preferentially increased DP biomarker expression in response to this line. Interestingly, 201B7 hiPSCs were shown to be ectodermal/epithelial prone, and the derived EPCs were putatively in a wingless-type MMTV integration site family (WNT)-activated state. Importantly, co-transplantation of 201B7 hiPSC-EPCs, but not NHKCs, with trichogenic mice dermal cells into immunodeficient mice resulted in HF formation. Human HF stem cell markers were detected in reconstituted HFs; however, a low frequency of human-derived cells implied that hiPSC-EPCs contributed to HF morphogenesis via direct repopulation and non-cell autonomous activities. The current study suggests a, to our knowledge, previously unrecognized advantage of using hiPSCs to enhance epithelial-mesenchymal interactions in HF bioengineering.

Canine Follicle Stem Cell Candidates Reside in the Bulge and Share Characteristic Features with Human Bulge Cells

The hair follicle bulge has attracted great interest as a stem cell repository. Previous studies have focused on rodent or human bulge stem cells, and our understanding of those in other species is limited. In this study, we attempted to localize and characterize stem cell candidates in canine hair follicles. The canine skin xenografting study located label-retaining cells in the outer root sheath around the insertion point of the arrector pili muscle, where the immunoreactivity of human bulge markers, keratin 15 and follistatin, were detected. Canine bulge cell-enriched keratinocytes up-regulated human bulge biomarkers CD200 and DIO2, and conserved key cell regulators of bulge stem cells, such as SOX9 and LHX2. Importantly, canine bulge-derived keratinocytes were highly proliferative in vitro and, when combined with trichogenic dermal cells, reconstituted pilosebaceous structures as well as the epidermis in vivo. Successful detection of canine specific DNA sequences suggested that the regenerated tissue was of canine origin. In addition, canine specific bulge cell and sebocyte lineage markers were expressed in reconstituted pilosebaceous units, implying the multipotency of canine bulge cells. Our findings demonstrate a unique strategy utilizing canine bulge cells to investigate human stem cell biology and intractable hair disorders that involve the bulge region.

Canine hair-follicle keratinocytes enriched with bulge cells have the highly proliferative characteristic of stem cells

Homeostasis of the epidermis and skin appendages is maintained by tissue-specific stem cells. In mice and humans, two populations of epithelial stem cells have been identified: one in the basal layer of the interfollicular epidermis and another in the bulge area of hair follicles. However, our understanding of canine epithelial stem cells is extremely limited. In this study, in vitro colony-forming assays were performed to locate highly proliferative keratinocytes in canine skin. Their phenotypic resemblance to epithelial stem cells in other species was also assessed. When equal numbers of epidermal or hair-follicle keratinocytes were cultured, colonies derived from follicular keratinocytes were significantly larger both in total numbers and size, than those derived from epidermal keratinocytes. In addition, immunoreactivity for CD34, a putative bulge stem-cell marker in the mouse, was predominantly detected in follicular keratinocytes. Thus in dogs, follicular keratinocytes were distinct from epidermal keratinocytes in proliferative capacity and CD34 expression. Using microdissection, highly proliferative keratinocytes were located within the middle portion of hair follicles, including the bulge area. Immunohistochemical study revealed that keratin 15, an established marker of bulge stem cells in mice and humans, was also predominantly expressed in the canine bulge area. Flow cytometry analysis revealed high numbers of keratin-15-positive cells in the highly proliferative keratinocyte compartment. Of note, keratin 15(high) cells possessed the phenotypic characteristics of putative stem cells. This study represents the first in vitro identification and isolation of highly proliferative canine keratinocytes, which represent candidate epithelial stem cells.

Functional Neurons Generated from T Cell-Derived Induced Pluripotent Stem Cells for Neurological Disease Modeling

Summary Modeling of neurological diseases using induced pluripotent stem cells (iPSCs) derived from the somatic cells of patients has provided a means of elucidating pathogenic mechanisms and performing drug screening. T cells are an ideal source of patient-specific iPSCs because they can be easily obtained from samples. Recent studies indicated that iPSCs retain an epigenetic memory relating to their cell of origin that restricts their differentiation potential. The classical method of differentiation via embryoid body formation was not suitable for T cell-derived iPSCs (TiPSCs). We developed a neurosphere-based robust differentiation protocol, which enabled TiPSCs to differentiate into functional neurons, despite differences in global gene expression between TiPSCs and adult human dermal fibroblast-derived iPSCs. Furthermore, neurons derived from TiPSCs generated from a juvenile patient with Parkinsons disease exhibited several Parkinsons disease phenotypes. Therefore, we conclude that TiPSCs are a useful tool for modeling neurological diseases.