Human Stem Cells for Modeling Amyotrophic Lateral Sclerosis Disease Mechanisms and Modifiers

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of the motor system. Although ALS has been extensively studied in post-mortem patient samples and animal models, there are currently no very effective treatments and there is no cure. One reason for the lack of treatment options in ALS may stem from the inaccessibility of living human motor neurons for use in disease research and subsequent therapeutic target validation. Recent developments in the field of stem cell biology can potentially provide access to living human motor neurons from individual ALS patients. It is now possible to derive induced pluripotent stem cells (iPS cells) from the somatic tissues of ALS patients and then to differentiate these iPS cells into motor neurons with the precise genetic makeup of the donor patient (iPS-MNs). Before iPS-MNs can be put to productive use, however, the iPS system as a whole must be validated as a reliable source of motor neurons with characteristics that closely resemble their endogenous or hES-derived counterparts. This thesis will first address a series of issues relating to the validation of iPS cells as a reliable source of motor neurons a then move on to expression profiling studies aimed at identifying a transcriptional signature of ALS in iPS-MNs. I will first describe a collaborative study aimed at determining whether or not iPS cells are as useful as ES cells for the production of motor neurons. By comparing motor neuron differentiation efficiency across a panel of 6 ES lines and 16 iPS lines, we demonstrated that iPS cells are equally capable of producing electrophysiologically active motor neurons as ES cells. Moreover, both ALS and control iPS lines produce motor neurons with equal efficiency, suggesting that iPS cells will be useful in the production of ALS iPS-MNs for disease research. In addition, our results identify some of the variables that contribute to differentiation efficiency, including donor identity and individual iPS/ES line identity. The following section will serve to provide a deeper molecular and electrophysiological understanding of human stem cell-derived motor neurons. I first generated expression profiles from purified hES-MNs to identify potential motor neuron-specific surface markers as well as maturational changes occurring in motor neurons in vitro. Using calcium imaging techniques, I then demonstrated that iPS-MNs behave functionally similarly to ES-MNs and described culture-wide rhythmic depolarizations that are likely influencing multiple properties of iPS-MNs. After characterizing the iPS-MN culture system, I made a first attempt at defining the transcriptional phenotypes of ALS in iPS-MNs. This work relied on the use of a motor neuron-specific lentiviral reporter that I developed to isolate and transcriptionally profile iPS-MNs from two control iPS lines and four ALS iPS lines. I show evidence of significant transcriptional differences between motor neurons isolated from ALS lines and those from control patients. These differences may in the future help to define ALS-specific phenotypes. Lastly, I conducted a meta-analysis comparing transcriptional changes in ALS iPS-MNs to those in existing models of ALS and identified some common stress-related features of ALS in iPS-MNs. In order to form new hypotheses about what sorts of individual patient-specific phenotypes may be present in iPS-MNs, I will then utilize published expression profiles from post-mortem ALS patient motor neurons to identify a previously-overlooked class of genes that exhibit expression levels highly correlated with individual age at ALS onset. This group of 43 onset-correlated genes contains many members with known or hypothesized relationships to neurodegenerative disease. I discuss how onset-correlated genes may function as disease-modifiers or biomarkers and design experiments to investigate these possibilities. Taken together, the work in this thesis will lay the foundations for developing a human iPS-based model of ALS and point toward numerous avenues of future investigation

A 44-year-old man with eye, kidney, and brain dysfunction

Retinal vasculopathy with cerebral leukodystrophy (RVCL) is a rare, autosomal dominant condition caused by mutations of the three-prime repair exonuclease-1 (TREX1). The phenotypic expressions range from isolated retinal involvement to varying degrees of retinopathy, cerebral infarction with calcium depositions, nephropathy, and hepatopathy. We report a case of RVCL caused by a novel TREX1 mutation. This patient’s multisystem presentation, retinal involvement interpreted as “retinal vasculitis”, and improvement of neuroimaging abnormalities with dexamethasone led to the accepted diagnosis of a rheumatologic disorder resembling Behçet’s disease. Clinicians should consider RVCL in any patient with retinal capillary obliterations associated with tumefactive brain lesions or nephropathy

Effect of Hydrocortisone on Mortality and Organ Support in Patients With Severe COVID-19: The REMAP-CAP COVID-19 Corticosteroid Domain Randomized Clinical Trial.

Importance: Evidence regarding corticosteroid use for severe coronavirus disease 2019 (COVID-19) is limited. Objective: To determine whether hydrocortisone improves outcome for patients with severe COVID-19. Design, Setting, and Participants: An ongoing adaptive platform trial testing multiple interventions within multiple therapeutic domains, for example, antiviral agents, corticosteroids, or immunoglobulin. Between March 9 and June 17, 2020, 614 adult patients with suspected or confirmed COVID-19 were enrolled and randomized within at least 1 domain following admission to an intensive care unit (ICU) for respiratory or cardiovascular organ support at 121 sites in 8 countries. Of these, 403 were randomized to open-label interventions within the corticosteroid domain. The domain was halted after results from another trial were released. Follow-up ended August 12, 2020. Interventions: The corticosteroid domain randomized participants to a fixed 7-day course of intravenous hydrocortisone (50 mg or 100 mg every 6 hours) (n = 143), a shock-dependent course (50 mg every 6 hours when shock was clinically evident) (n = 152), or no hydrocortisone (n = 108). Main Outcomes and Measures: The primary end point was organ support-free days (days alive and free of ICU-based respiratory or cardiovascular support) within 21 days, where patients who died were assigned -1 day. The primary analysis was a bayesian cumulative logistic model that included all patients enrolled with severe COVID-19, adjusting for age, sex, site, region, time, assignment to interventions within other domains, and domain and intervention eligibility. Superiority was defined as the posterior probability of an odds ratio greater than 1 (threshold for trial conclusion of superiority >99%). Results: After excluding 19 participants who withdrew consent, there were 384 patients (mean age, 60 years; 29% female) randomized to the fixed-dose (n = 137), shock-dependent (n = 146), and no (n = 101) hydrocortisone groups; 379 (99%) completed the study and were included in the analysis. The mean age for the 3 groups ranged between 59.5 and 60.4 years; most patients were male (range, 70.6%-71.5%); mean body mass index ranged between 29.7 and 30.9; and patients receiving mechanical ventilation ranged between 50.0% and 63.5%. For the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively, the median organ support-free days were 0 (IQR, -1 to 15), 0 (IQR, -1 to 13), and 0 (-1 to 11) days (composed of 30%, 26%, and 33% mortality rates and 11.5, 9.5, and 6 median organ support-free days among survivors). The median adjusted odds ratio and bayesian probability of superiority were 1.43 (95% credible interval, 0.91-2.27) and 93% for fixed-dose hydrocortisone, respectively, and were 1.22 (95% credible interval, 0.76-1.94) and 80% for shock-dependent hydrocortisone compared with no hydrocortisone. Serious adverse events were reported in 4 (3%), 5 (3%), and 1 (1%) patients in the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively. Conclusions and Relevance: Among patients with severe COVID-19, treatment with a 7-day fixed-dose course of hydrocortisone or shock-dependent dosing of hydrocortisone, compared with no hydrocortisone, resulted in 93% and 80% probabilities of superiority with regard to the odds of improvement in organ support-free days within 21 days. However, the trial was stopped early and no treatment strategy met prespecified criteria for statistical superiority, precluding definitive conclusions. Trial Registration: ClinicalTrials.gov Identifier: NCT02735707

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Pathways Disrupted in Human ALS Motor Neurons Identified through Genetic Correction of Mutant SOD1

Direct electrical recording and stimulation of neural activity using micro-fabricated silicon and metal micro-wire probes have contributed extensively to basic neuroscience and therapeutic applications; however, the dimensional and mechanical mismatch of these probes with the brain tissue limits their stability in chronic implants and decreases the neuron–device contact. Here, we demonstrate the realization of a three-dimensional macroporous nanoelectronic brain probe that combines ultra-flexibility and subcellular feature sizes to overcome these limitations. Built-in strains controlling the local geometry of the macroporous devices are designed to optimize the neuron/probe interface and to promote integration with the brain tissue while introducing minimal mechanical perturbation. The ultra-flexible probes were implanted frozen into rodent brains and used to record multiplexed local field potentials and single-unit action potentials from the somatosensory cortex. Significantly, histology analysis revealed filling-in of neural tissue through the macroporous network and attractive neuron–probe interactions, consistent with long-term biocompatibility of the device.Stem Cell and Regenerative Biolog

The NF-?B Subunit c-Rel Stimulates Cardiac Hypertrophy and Fibrosis

Cardiac remodeling and hypertrophy are the pathological consequences of cardiovascular disease and are correlated with its associated mortality. Activity of the transcription factor NF-κB is increased in the diseased heart; however, our present understanding of how the individual subunits contribute to cardiovascular disease is limited. We assign a new role for the c-Rel subunit as a stimulator of cardiac hypertrophy and fibrosis. We discovered that c-Rel-deficient mice have smaller hearts at birth, as well as during adulthood, and are protected from developing cardiac hypertrophy and fibrosis after chronic angiotensin infusion. Results of both gene expression and cross-linked chromatin immunoprecipitation assay analyses identified transcriptional activators of hypertrophy, myocyte enhancer family, Gata4, and Tbx proteins as Rel gene targets. We suggest that the p50 subunit could limit the prohypertrophic actions of c-Rel in the normal heart, because p50 overexpression in H9c2 cells repressed c-Rel levels and the absence of cardiac p50 was associated with increases in both c-Rel levels and cardiac hypertrophy. We report for the first time that c-Rel is highly expressed and confined to the nuclei of diseased adult human hearts but is restricted to the cytoplasm of normal cardiac tissues. We conclude that c-Rel-dependent signaling is critical for both cardiac remodeling and hypertrophy. Targeting its activities could offer a novel therapeutic strategy to limit the effects of cardiac disease

Effect of Antiplatelet Therapy on Survival and Organ Support–Free Days in Critically Ill Patients With COVID-19

International audienc