Human induced pluripotent stem cells (hiPSCs) represent a potentially useful tool for studying the molecular mechanisms of disease thanks to their ability to generate patient-specific hiPSC clones. However, previous studies have reported that DNA methylation profiles, including those for imprinted genes, may change during passaging of hiPSCs. This is particularly problematic for hiPSC models of X-linked disease, because unstable X chromosome inactivation status may affect the detection of phenotypes. In the present study, we examined the epigenetic status of hiPSCs derived from patients with Rett syndrome, an X-linked disease, during long-term culture. To analyze X chromosome inactivation, we used a methylation-specific polymerase chain reaction (MSP) to assay the human androgen receptor locus (HUMARA). We found that single cell-derived hiPSC clones exhibit various states of X chromosome inactivation immediately after clonal isolation, even when established simultaneously from a single donor. X chromosome inactivation states remain variable in hiPSC clones at early passages, and this variability may affect cellular phenotypes characteristic of X-linked diseases. Careful evaluation of X chromosome inactivation in hiPSC clones, particularly in early passages, by methods such as HUMARA-MSP, is therefore important when using patient-specific hiPSCs to model X-linked disease.
Cutaneous metaplastic synovial cysts (CMSCs) are rare tumors typically comprising a solitary, well-circumscribed cystic mass that is not connected to the joint. Synovial cysts have been reported predominantly by orthopedists or pathologists; however, the presence of CMSC is not generally well recognized by dermatologists. Herein, we report a CMSC in a 68-year-old woman receiving systemic corticosteroid therapy for the treatment of eosinophilic granulomatosis with polyangiitis (EGPA). We attempt to delineate the clinical characteristics of this unusual neoplasm by reviewing the literature, focusing especially on dermatological descriptions. Histologic examination of the surgical specimen in the current case revealed that the cystic wall was lined with layers of flattened synovial cell-like cells and connective tissues, mimicking the synovial membrane. Positive immunoreactivity of the lining cells against vimentin was detected, but no immunoreactivity against cytokeratin, carcinoembryonic antigen (CEA), CD68, or S-100 was detected. The pathogenesis of CMSC remains unclear, but it has been tightly linked to direct traumatic stimuli or relative tissue fragility, which potentially accounts for CMSC development in our case. Most CMSCs reported by dermatologists are located on the extremities, whereas those described by other specialists tend to be distributed more globally. Preoperative diagnoses are often either epidermal cyst or suture/foreign body granuloma. Incomplete surgical excision of usual synovial cysts may lead to local recurrence, which has been reported in oral and maxillofacial surgery, but not in dermatologic surgery. This fact could be explained by the technical difficulties of surgical excision related to anatomical location. Dermatologists need to be aware of CMSC, and CMSC should be included in the differential diagnosis of subcutaneous cysts.
The mechanisms that allow enteric pathogens to colonize the intestine and host immunity as well as the indigenous microbiota to inhibit pathogen colonization remain poorly understood. Our laboratory is using Citrobacter rodentium, a mouse pathogen that models human infections by enteropathogenic E. coli, to understand the mechanisms that regulate the colonization and clearance of the pathogen in the gut. These studies have revealed how the pathogen colonizes and replicates successfully early during infection and how host immunity and the indigenous microbiota cooperate to eradicate the pathogen in the later stage of the infection. The impairment of the immune system to control the barrier function of the intestine leads to pathogen invasion and the induction of a second layer of host protective immunity to limit the systemic spread of the pathogen.
(Presented at the 1931st Meeting, January 17, 2017)
The generation of induced pluripotent stem cells (iPSCs) achieved by overexpression of Oct4, Sox2, Klf4 and c-Myc, transformed our classical views of the cellular epigenetic landscape and delivered a new concept for cell and tissue engineering. In addition to iPSCs, several other cell types have also been generated by master transcription factor (TF)-mediated transdifferentiation. However, the critical molecular mechanisms amongst diverse cellular identity changes are not well understood. Through the investigation of reprogramming mechanisms, we recently revealed that over-expression of constitutive active Smad3 boosted not only iPSC generation, but also 3 other master TF-mediated conversions, from B cells to macrophages, myoblasts to adipocytes, and human fibroblasts to neurons. This demonstrated that there were common mechanisms underlying different master TF-mediated cell conversions. To illuminate such mechanisms further, we have recently performed CRISPR/Cas9-mediated genome-wide knockout screening during reprogramming with a lentiviral gRNA library containing 90,000 gRNAs. This screening provided us with ~15 novel reprogramming roadblock genes as well as ~20 candidate genes essential for the reprogramming process but not for ES cell self-renewal. This data set will be a valuable resource to further understand how overexpression of master TFs alters cellular identity, and to achieve more faithful, efficient cell conversions for regenerative medicine.
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