A simple and efficient system for regulating gene expression in human pluripotent stem cells and derivatives.

Regulatable transgene expression in human pluripotent stem cells (hPSCs) and their progenies is often necessary to dissect gene function in a temporal and spatial manner. However, hPSC lines with inducible transgene expression, especially in differentiated progenies, have not been established due to silencing of randomly inserted genes during stem cell expansion and/or differentiation. Here, we report the use of transcription activator-like effector nucleases-mediated targeting to AAVS1 site to generate versatile conditional hPSC lines. Transgene (both green fluorescent protein and a functional gene) expression in hPSCs and their derivatives was not only sustained but also tightly regulated in response to doxycycline both in vitro and in vivo. We modified the donor construct so that any gene of interest can be readily inserted to produce hPSC lines with conditional transgene expression. This technology will substantially improve the way we study human stem cells.

Focal Seizures Induced by Paraneoplastic Syndrome Leading to a Diagnosis of Small Cell Lung Carcinoma.

Paraneoplastic neurological disorders are a rare complication of multiple neoplasms, such as lung, testis, and breast, and can be associated with positive antibody anti-Hu (anti-neuronal nuclear antibody type 1 or ANNA-1), anti-Ta, anti-Ma, and uncharacterized antibody, or be antibody-negative. Early treatment of the underlying tumor is the most likely modality that will lead to regression of the paraneoplastic neurological symptoms. Here, we present a case of a 73-year-old female with new-onset seizure activity from ANNA-1 encephalitis found to have undiagnosed small cell lung cancer to highlight the need for further workup for malignancy.

A case report of thrombocytopenic COVID-19 and Miller-Fisher syndrome on a concurrent chronic immune neuropathy.

RATIONALE: Miller-Fisher syndrome (MFS) is a rare subtype of Guillain-Barre syndrome with classic features of ataxia, areflexia, and ophthalmoplegia that can be caused by a preceding infection including COVID-19. We present a current, asymptomatic thrombocytopenic COVID-19 infection as a cause of MFS in a 60-year-old male with a concurrent chronic immune neuropathy. PATIENT CONCERNS: A 60-year-old male presenting with acute symptoms of MFS including ataxia, areflexia, and ophthalmoplegia on a chronic immune neuropathy for at least 1 year and concurrent asymptomatic COVID-19 positive infection. DIAGNOSIS: MFS suspected secondary to a current thrombocytopenic COVID-19 infection. INTERVENTIONS: Five days of intravenous immune globulin with continued monthly intravenous immune globulin as an outpatient, follow-up long-term in a neuromuscular clinic, electromyography as an outpatient, and continued physical therapy. OUTCOMES: The patient significantly improved after initial treatment. LESSONS: The full effect of COVID-19 on the various Guillain-Barre syndrome subtypes is unknown, although it clearly can be a cause of the various variants including being caused by a current, asymptomatic infection.

A Case Report and Literature Review of Nonketotic Hyperglycemic Hemichorea.

Nonketogenic hyperglycemic hemichorea, also recognized as diabetic striatopathy, is a rare manifestation of diabetes mellitus. The diagnosis of nonketotic hyperglycemic hemichorea is usually made through imaging along with a corresponding clinical picture. Early identification and treatment can lead to complete resolution of the symptoms and better patient outcomes. Here we present a 49-year-old female patient, with a past medical history of poorly controlled type 2 diabetes mellitus and prior left index finger amputation as a complication of her diabetes, who presented for evaluation of a two-week duration of sudden-onset left upper extremity choreiform movements.

Human Herpes Virus-6 (HHV-6) infectious encephalitis in an immunocompetent adult.

Human herpesvirus 6 (HHV-6) is one of the most prevalent childhood viruses. HHV-6 reactivation in allogeneic hematopoietic cell transplant recipients and solid organ transplant recipients is well described in medical literature. We present a case of HHV-6 reactivation causing encephalitis, which is rare in immunocompetent adults.

Systemic Lymphoma Masquerading as Multiple Sclerosis Relapse.

Multiple sclerosis (MS) is a chronic immune-mediated central nervous system disease that can affect both the brain and spinal cord. Given that MS can occur at any location in the brain or spinal cord and can lead to a variety of symptoms, this can lead to difficulty in diagnosing MS versus other conditions mimicking MS. Here we present a case of a 69-year-old female with a history of relapsing-remitting MS diagnosed in 2002 and melanoma status post-excision who exhibited progressive neurological decline over eight weeks characterized by right internuclear ophthalmoplegia, bilateral ataxia, and left hemiparesis sparing the face. Mimics of MS can include various inflammatory, neoplastic, infectious, metabolic, and genetic disorders. The diagnosis of MS-mimicking diseases can be especially challenging for someone with a known history of MS. A biopsy should be considered for new lesions seen on imaging if acute immunotherapies have no response to the clinical patient's symptoms. Given the wide variety of symptoms that can present with MS, it is important to keep a broad range of differential diagnoses when considering MS, even in those with a known history of MS.

5'ValCAC tRNA fragment generated as part of a protective angiogenin response provides prognostic value in amyotrophic lateral sclerosis.

Loss-of-function mutations in the ribonuclease angiogenin are associated with amyotrophic lateral sclerosis. Angiogenin has been shown to cleave transfer RNAs during stress to produce 'transfer-derived stress-induced RNAs'. Stress-induced tRNA cleavage is preserved from single-celled organisms to humans indicating it represents part of a highly conserved stress response. However, to date, the role of tRNA cleavage in amyotrophic lateral sclerosis remains to be fully elucidated. To this end, we performed small RNA sequencing on a human astrocytoma cell line to identify the complete repertoire of tRNA fragments generated by angiogenin. We found that only a specific subset of tRNAs is cleaved by angiogenin and identified 5'ValCAC transfer-derived stress-induced RNA to be secreted from neural cells. 5'ValCAC was quantified in spinal cord and serum from SOD1G93A amyotrophic lateral sclerosis mouse models where we found it to be significantly elevated at symptom onset correlating with increased angiogenin expression, imbalanced protein translation initiation factors and slower disease progression. In amyotrophic lateral sclerosis patient serum samples, we found 5'ValCAC to be significantly higher in patients with slow disease progression, and interestingly, we find 5'ValCAC to hold prognostic value for amyotrophic lateral sclerosis patients. Here, we report that angiogenin cleaves a specific subset of tRNAs and provide evidence for 5'ValCAC as a prognostic biomarker in amyotrophic lateral sclerosis. We propose that increased serum 5'ValCAC levels indicate an enhanced angiogenin-mediated stress response within motor neurons that correlates with increased survival. These data suggest that the previously reported beneficial effects of angiogenin in SOD1G93A mice may result from elevated levels of 5'ValCAC transfer RNA fragment.

Human-derived neural progenitors functionally replace astrocytes in adult mice.

Astrocytes are integral components of the homeostatic neural network as well as active participants in pathogenesis of and recovery from nearly all neurological conditions. Evolutionarily, compared with lower vertebrates and nonhuman primates, humans have an increased astrocyte-to-neuron ratio; however, a lack of effective models has hindered the study of the complex roles of human astrocytes in intact adult animals. Here, we demonstrated that after transplantation into the cervical spinal cords of adult mice with severe combined immunodeficiency (SCID), human pluripotent stem cell-derived (PSC-derived) neural progenitors migrate a long distance and differentiate to astrocytes that nearly replace their mouse counterparts over a 9-month period. The human PSC-derived astrocytes formed networks through their processes, encircled endogenous neurons, and extended end feet that wrapped around blood vessels without altering locomotion behaviors, suggesting structural, and potentially functional, integration into the adult mouse spinal cord. Furthermore, in SCID mice transplanted with neural progenitors derived from induced PSCs from patients with ALS, astrocytes were generated and distributed to a similar degree as that seen in mice transplanted with healthy progenitors; however, these mice exhibited motor deficit, highlighting functional integration of the human-derived astrocytes. Together, these results indicate that this chimeric animal model has potential for further investigating the roles of human astrocytes in disease pathogenesis and repair.

Modeling ALS with iPSCs reveals that mutant SOD1 misregulates neurofilament balance in motor neurons.

Amyotrophic lateral sclerosis (ALS) presents motoneuron (MN)-selective protein inclusions and axonal degeneration but the underlying mechanisms of such are unknown. Using induced pluripotent cells (iPSCs) from patients with mutation in the Cu/Zn superoxide dismutase (SOD1) gene, we show that spinal MNs, but rarely non-MNs, exhibited neurofilament (NF) aggregation followed by neurite degeneration when glia were not present. These changes were associated with decreased stability of NF-L mRNA and binding of its 3' UTR by mutant SOD1 and thus altered protein proportion of NF subunits. Such MN-selective changes were mimicked by expression of a single copy of the mutant SOD1 in human embryonic stem cells and were prevented by genetic correction of the SOD1 mutation in patient's iPSCs. Importantly, conditional expression of NF-L in the SOD1 iPSC-derived MNs corrected the NF subunit proportion, mitigating NF aggregation and neurite degeneration. Thus, NF misregulation underlies mutant SOD1-mediated NF aggregation and axonal degeneration in ALS MNs.