Induced Pluripotent Stem Cell Derivation and Ex Vivo Gene Correction Using a Mucopolysaccharidosis Type 1 Disease Mouse Model.

Mucopolysaccharidosis type 1 (MPS-1), also known as Hurler's disease, is a congenital metabolic disorder caused by a mutation in the alpha-L-iduronidase (IDUA) gene, which results in the loss of lysosomal enzyme function for the degradation of glycosaminoglycans. Here, we demonstrate the proof of concept of ex vivo gene editing therapy using induced pluripotent stem cell (iPSC) and CRISPR/Cas9 technologies with MPS-1 model mouse cell. Disease-affected iPSCs were generated from Idua knockout mouse embryonic fibroblasts, which carry a disrupting neomycin-resistant gene cassette (Neor) in exon VI of the Idua gene. Double guide RNAs were used to remove the Neor sequence, and various lengths of donor templates were used to reconstruct the exon VI sequence. A quantitative PCR-based screening method was used to identify Neor removal. The sequence restoration without any indel mutation was further confirmed by Sanger sequencing. After induced fibroblast differentiation, the gene-corrected iPSC-derived fibroblasts demonstrated Idua function equivalent to the wild-type iPSC-derived fibroblasts. The Idua-deficient cells were competent to be reprogrammed to iPSCs, and pluripotency was maintained through CRISPR/CAS9-mediated gene correction. These results support the concept of ex vivo gene editing therapy using iPSC and CRISPR/Cas9 technologies for MPS-1 patients.

Synergistic Cytotoxic Effect of Busulfan and the PARP Inhibitor Veliparib in Myeloproliferative Neoplasms.

Patients with high-risk myeloproliferative neoplasms (MPNs), and in particular myelofibrosis (MF), can be cured only with allogeneic hematopoietic stem cell transplantation (HSCT). Because MPNs and JAK2V617F-mutated cells show genomic instability, stalled replication forks, and baseline DNA double-strand breaks, DNA repair inhibition with poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors represents a potential novel therapy. Because the alkylating agent busulfan is integral in conditioning regimens for HSCT and leads to stalled replication forks through DNA strand cross-linking, we hypothesized that PARP inhibition with veliparib in combination with busulfan may lead to synergistic cytotoxicity in MPN cells. We first treated 2 MPN cell lines harboring the JAK2V617F mutation (SET2 and HEL) with veliparib at increasing concentrations and measured cell proliferation. SET2 and HEL cells were relatively sensitive to veliparib (IC50 of 11.3 µM and 74.2 µM, respectively). We next treated cells with increasing doses of busulfan in combination with 4 µM veliparib and found that the busulfan IC50 decreased from 27 µM to 4 µM in SET2 cells and from 45.1 µM to 28.1 µM in HEL cells. The mean combination index was .55 for SET2 cells and .40 for HEL cells. Combination treatment of SET2 cells caused G2M arrest in 53% of cells, compared with 30% with veliparib alone and 35% with busulfan alone. G2M arrest was associated with activation of the ATR-Chk1 pathway, as shown by an immunofluorescence assay for phosphorylated Chk1 (p-Chk1). We then tested in vivo the effect of combined low doses of busulfan and veliparib in a JAK2V617F MPN-AML xenotransplant model. Vehicle- and veliparib-treated mice had similar median survival of 39 and 40 days, respectively. Combination treatment increased median survival from 47 days (busulfan alone) to 50 days (P = .02). Finally, we tested the combined effect of busulfan and veliparib on CD34+ cells obtained from the bone marrow or peripheral blood of 5 patients with JAK2V617F-mutated and 2 patients with CALR-mutated MF. MF cells treated with the combination of veliparib and busulfan showed reduced colony formation compared with busulfan alone (87% versus 68%; P = .001). In contrast, treatment of normal CD34+ cells with veliparib did not affect colony growth. Here we show that in vivo confirmation that treatment with the PARP-1 inhibitor veliparib and busulfan results in synergistic cytotoxicity in MPN cells. Our data provide the rationale for testing novel pretransplantation conditioning regimens with combinations of PARP-1 inhibition and reduced doses of alkylators, such as busulfan and melphalan, for high-risk MPNs or MPN-derived acute myelogenous leukemia.

Depression, Loneliness, and Suicide Risk among Latino College Students: A Test of a Psychosocial Interaction Model.

This study tested a psychosocial model of suicide risk in a sample of 156 Latino college students. Specifically, depression and loneliness were hypothesized to be important predictors of suicide risk (namely, hopelessness and suicidal behaviors) in Latino students. Results of conducting regression analyses indicated that, independent of age and gender, depression and loneliness were significant predictors of both indices of suicide risk examined in the present study. It is noteworthy that within the psychosocial predictor set of depression and loneliness, depression was consistently found to be nearly twice as strong a predictor than was loneliness. Moreover, we found evidence for a significant depression-loneliness interaction effect in predicting suicide risk. That is, the highest level of suicide risk was found among dysphoric Latino students who were also socially isolated. Our findings indicate that depression and loneliness are important factors to consider in understanding suicide risk among Latino college students.

Liver-Directed Human Amniotic Epithelial Cell Transplantation Improves Systemic Disease Phenotype in Hurler Syndrome Mouse Model.

Mucopolysaccharidosis type 1 (MPS1) is an inherited lysosomal storage disorder caused by a deficiency in the glycosaminoglycan (GAG)-degrading enzyme α-l-iduronidase (IDUA). In affected patients, the systemic accumulation of GAGs results in skeletal dysplasia, neurological degeneration, multiple organ dysfunction, and early death. Current therapies, including enzyme replacement and bone marrow transplant, improve life expectancy but the benefits to skeletal and neurological phenotypes are limited. In this study, we tested the therapeutic efficacy of liver-directed transplantation of a placental stem cell, which possesses multilineage differentiation potential, low immunogenicity, and high lysosomal enzyme activity. Unfractionated human amniotic epithelial cells (hAECs) were transplanted directly into the liver of immunodeficient Idua knockout mouse neonates. The hAECs engraftment was immunohistochemically confirmed with anti-human mitochondria staining. Enzyme activity assays indicated that hAECs transplantation restored IDUA function in the liver and significantly decreased urinary GAG excretion. Histochemical and micro-computed tomography analyses revealed reduced GAG deposition in the phalanges joints and composition/morphology improvement of cranial and facial bones. Neurological assessment in the hAEC treated mice showed significant improvement of sensorimotor coordination in the hAEC treated mice compared to untreated mice. Results confirm that partial liver cell replacement with placental stem cells can provide long-term (>20 weeks) and systemic restoration of enzyme function, and lead to significant phenotypic improvement in the MPS1 mouse model. This preclinical data indicate that liver-directed placental stem cell transplantation may improve skeletal and neurological phenotypes of MPS1 patients. Stem Cells Translational Medicine 2017;6:1583-1594.

The effect of acute stress and long-term corticosteroid administration on plasma metabolites in an urban and desert songbird.

In response to stressful stimuli, animals activate the hypothalamic-pituitary-adrenal axis, which can result in transition to the "emergency life history stage." A key adaptive characteristic of this life history stage is the mobilization of energy stores. However, few data are available on the metabolic response to acute stress in wild-caught, free-ranging birds. We quantified the effect of acute capture and restraint stress on plasma glucose, free fatty acid, and uric acid in free-ranging Abert's towhees Melozone aberti. Furthermore, birds were caught from urban and desert localities of Phoenix, Arizona, to investigate potential effects of urban versus desert habitats on the corticosterone (CORT) and metabolic response to acute stress. Complementing work on free-ranging birds, captive towhees received CORT-filled Silastic capsules to investigate the response of urban and desert conspecifics to long-term CORT administration. We quantified the effect of CORT administration on baseline plasma glucose and uric acid, liver and pectoralis muscle glycogen stores, kidney phosphoenolpyruvate carboxykinase (PEPCK-C, a key gluconeogenic enzyme), and body mass. Acute stress increased plasma CORT and glucose and decreased plasma uric acid but had no effect on plasma free fatty acid. There was no difference between urban and desert localities in body mass, fat scores, and the response to acute stress. CORT administration decreased body mass but had no effect on glucose and uric acid, pectoral muscle glycogen, or kidney PEPCK-C. However, liver glycogen of CORT-treated urban birds increased compared with corresponding controls, whereas glycogen decreased in CORT-treated desert birds. This study suggests that Abert's towhees principally mobilize glucose during acute stress but urban and desert towhees do not differ in their CORT and metabolic response to acute stress or long-term CORT administration.