Evaluation of inter-batch differences in stem-cell derived neurons

AbstractDifferentiated cells retain the genetic information of the donor but the extent to which phenotypic differences between donors or batches of differentiated cells are explained by variation introduced during the differentiation process is not fully understood. In this study, we evaluated four separate batches of commercially available neurons originating from the same iPSCs to investigate whether the differentiation process used in manufacturing iPSCs to neurons affected genome-wide gene expression and modified cytosines, or neuronal sensitivity to drugs. No significant changes in gene expression, as measured by RNA-Seq, or cytosine modification levels, as measured by the Illumina 450K arrays, were observed between batches relative to changes over time. As expected, neurotoxic chemotherapeutics affected neuronal outgrowth, but no inter-batch differences were observed in sensitivity to paclitaxel, vincristine and cisplatin. As a testament to the utility of the model for studies of neuropathy, we observed that genes involved in neuropathy had relatively higher expression levels in these samples across different time points. Our results suggest that the process used to differentiate iPSCs into neurons is consistent, resulting in minimal intra-individual variability across batches. Therefore, this model is reasonable for studies of human neuropathy, druggable targets to prevent neuropathy, and other neurological diseases

Application of Stem Cell Derived Neuronal Cells to Evaluate Neurotoxic Chemotherapy

The generation of induced pluripotent stem cells (iPSCs) and differentiation to cells composing major organs has opened up the possibility for a new model system to study adverse toxicities associated with chemotherapy. Therefore, we used human iPSC-derived neurons to study peripheral neuropathy, one of the most common adverse effects of chemotherapy and cause for dose reduction. To determine the utility of these neurons in investigating the effects of neurotoxic chemotherapy, we measured morphological differences in neurite outgrowth, cell viability as determined by ATP levels and apoptosis through measures of caspase 3/7 activation following treatment with clinically relevant concentrations of platinating agents (cisplatin, oxaliplatin and carboplatin), taxanes (paclitaxel, docetaxel and nab-paclitaxel), a targeted proteasome inhibitor (bortezomib), an antiangiogenic compound (thalidomide), and 5-fluorouracil, a chemotherapeutic that does not cause neuropathy. We demonstrate differential sensitivity of neurons to mechanistically distinct classes of chemotherapeutics. We also show a dose-dependent reduction of electrical activity as measured by mean firing rate of the neurons following treatment with paclitaxel. We compared neurite outgrowth and cell viability of iPSC-derived cortical (iCell® Neurons) and peripheral (Peri.4U) neurons to cisplatin, paclitaxel and vincristine. Goshajinkigan, a Japanese herbal neuroprotectant medicine, was protective against paclitaxel-induced neurotoxicity but not oxaliplatin as measured by morphological phenotypes. Thus, we have demonstrated the utility of human iPSC-derived neurons as a useful model to distinguish drug class differences and for studies of a potential neuroprotectant for the prevention of chemotherapy-induced peripheral neuropathy

The Association between Acceptance and Health for Individuals who are HIV-Positive

Given the increase in number of individuals infected with HIV and the prevalence of stressors and mental health problems among these persons, researchers continue to examine which appraisals and coping responses may be related to better mental health and physical health for HIV populations. Research shows that negative appraisals and avoidance coping are consistently associated with less adaptive functioning. As an alternative, acceptance may be a type of appraisal that precludes the need to engage in coping strategies, especially non-adaptive coping strategies (e.g., avoidance coping). Acceptance refers to the willingness to experience thoughts, feelings, and physical symptoms without engaging in efforts to avoid or control them. Furthermore, research demonstrates that higher levels of acceptance are associated with better functioning for persons living with chronic illnesses. However, the role of acceptance has yet to be empirically examined within HIV-positive populations. Therefore, the aim of the present study is to evaluate the relationships between acceptance, negative appraisals (i.e., threat appraisals), avoidance coping, psychological health (i.e., depression, quality of life, perceived functioning), medication adherence, and physical health (i.e., immune functioning). Analyses were conducted to examine both direct relationships and mediated relationships. Results indicated that levels of acceptance were related to better mental health. However, acceptance was not significantly associated with medication adherence, and medication adherence did not mediate the relationships between acceptance and mental health. Results also indicated that threat appraisals were associated with worse mental health, threat appraisals were related to some types of avoidance coping, and avoidance coping mediated the relationship between threat appraisals and depression. Overall, the present study suggests that acceptance and threat appraisals are important variables that impact mental health for those who are living with HIV and experiencing difficulties adjusting to the illness

Improved methods to recover HIV-1 integrated proviruses and integration sites

Pathology: Medical Virolog

Rosai-Dorfman Disease of the Breast

Rosai-Dorfman disease (also known as sinus histiocytosis with massive lymphadenopathy) is a rare benign proliferative disorder of histiocytes that typically involves the lymph nodes and can also involve extranodal sites. Rosai-Dorfman disease confined to the breast is extremely rare, but important to recognize as it can mimic malignancy. We present the case of a 63-year-old woman who presented with a palpable breast lump that was highly suspicious for malignancy based on mammogram and ultrasound appearance. Biopsy revealed inflammatory tissue with lymphoplasmacytic and histiocytic predominance. The diagnosis of Rosai-Dorfman was made based on characteristic staining of histiocytes with S-100 and the presence of emperipolesis. Early recognition of this benign disease entity spared the patient further investigation and surgical intervention

Modeling Chemotherapeutic Neurotoxicity with Human Induced Pluripotent Stem Cell-Derived Neuronal Cells

There are no effective agents to prevent or treat chemotherapy-induced peripheral neuropathy (CIPN), the most common non-hematologic toxicity of chemotherapy. Therefore, we sought to evaluate the utility of human neuron-like cells derived from induced pluripotent stem cells (iPSCs) as a means to study CIPN. We used high content imaging measurements of neurite outgrowth phenotypes to compare the changes that occur to iPSC-derived neuronal cells among drugs and among individuals in response to several classes of chemotherapeutics. Upon treatment of these neuronal cells with the neurotoxic drug paclitaxel, vincristine or cisplatin, we identified significant differences in five morphological phenotypes among drugs, including total outgrowth, mean/median/maximum process length, and mean outgrowth intensity (P TUBB2A results in significantly increased sensitivity of neurons to paclitaxel (0.23 ± 0.06 decrease in total neurite outgrowth, P = 0.011). The variance in several neurite outgrowth and apoptotic phenotypes upon treatment with one of the neurotoxic drugs is significantly greater between than within neurons derived from four different individuals (P < 0.05), demonstrating the potential of iPSC-derived neurons as a genetically diverse model for CIPN. The human neuron model will allow both for mechanistic studies of specific genes and genetic variants discovered in clinical studies and for screening of new drugs to prevent or treat CIPN.</p

Modeling chemotherapeutic neurotoxicity with human induced pluripotent stem cell-derived neuronal cells.

There are no effective agents to prevent or treat chemotherapy-induced peripheral neuropathy (CIPN), the most common non-hematologic toxicity of chemotherapy. Therefore, we sought to evaluate the utility of human neuron-like cells derived from induced pluripotent stem cells (iPSCs) as a means to study CIPN. We used high content imaging measurements of neurite outgrowth phenotypes to compare the changes that occur to iPSC-derived neuronal cells among drugs and among individuals in response to several classes of chemotherapeutics. Upon treatment of these neuronal cells with the neurotoxic drug paclitaxel, vincristine or cisplatin, we identified significant differences in five morphological phenotypes among drugs, including total outgrowth, mean/median/maximum process length, and mean outgrowth intensity (P < 0.05). The differences in damage among drugs reflect differences in their mechanisms of action and clinical CIPN manifestations. We show the potential of the model for gene perturbation studies by demonstrating decreased expression of TUBB2A results in significantly increased sensitivity of neurons to paclitaxel (0.23 ± 0.06 decrease in total neurite outgrowth, P = 0.011). The variance in several neurite outgrowth and apoptotic phenotypes upon treatment with one of the neurotoxic drugs is significantly greater between than within neurons derived from four different individuals (P < 0.05), demonstrating the potential of iPSC-derived neurons as a genetically diverse model for CIPN. The human neuron model will allow both for mechanistic studies of specific genes and genetic variants discovered in clinical studies and for screening of new drugs to prevent or treat CIPN

ANOVA results comparing the AUCs of relative neurite outgrowth phenotypes among 4 genetically different LCL-iPSC-derived Neuron lines after 72 h drug treatment.

<p>ANOVA = one-way analysis of variance (not assuming equal variances), AUC = area under the concentration curve calculated from 0.001–100 μM.</p><p>ANOVA results comparing the AUCs of relative neurite outgrowth phenotypes among 4 genetically different LCL-iPSC-derived Neuron lines after 72 h drug treatment.</p