Generation of heterozygous (MCRIi030-A-1) and homozygous (MCRIi030-A-2) NR2F2/COUP-TFII knockout human iPSC lines.

The NR2F2 gene encodes the transcription factor COUP-TFII, which is upregulated in embryonic mesoderm. Heterozygous variants in NR2F2 cause a spectrum of congenital anomalies including cardiac and gonadal phenotypes. We generated heterozygous (MCRIi030-A-1) and homozygous (MCRIi030-A-2) NR2F2-knockout induced pluripotent stem cell (iPSC) lines from human fibroblasts using a one-step protocol for CRISPR/Cas9 gene-editing and episomal-based reprogramming. Both iPSC lines exhibited a normal karyotype, typical pluripotent cell morphology, pluripotency marker expression, and the capacity to differentiate into the three embryonic germ layers. These lines will allow us to explore the role of NR2F2 during development and disease.

The mRNA-based reprogramming of fibroblasts from a SOD1E101G familial amyotrophic lateral sclerosis patient to induced pluripotent stem cell line UOWi007.

Dermal fibroblasts were donated by a 43 year old male patient with clinically diagnosed familial amyotrophic lateral sclerosis (ALS), carrying the SOD1E101G mutation. The induced pluripotent stem cell (iPSC) line UOWi007-A was generated using repeated mRNA transfections for pluripotency transcription factors Oct4, Klf4, Sox2, c-Myc, Lin28 and Nanog. The iPSCs carried the SOD1E101G genotype and had a normal karyotype, expressed expected pluripotency markers and were capable of in vitro differentiation into endodermal, mesodermal and ectodermal lineages. This iPSC line may be useful for investigating familial ALS resulting from a SOD1E101G mutation.

Generation and characterization of a human induced pluripotent stem cell line UOWi005-A from dermal fibroblasts derived from a CCNFS621G familial amyotrophic lateral sclerosis patient using mRNA reprogramming.

Dermal fibroblasts from a 59 year old male patient with amyotrophic lateral sclerosis (symptomatic at the time of collection), attributed to a mutation in the cyclin F gene (CCNFS621G), were reprogrammed using mRNA and microRNA-delivered OSKM factors to induced pluripotent stem cells (iPSCs). The generated iPSCs were confirmed pluripotent, expressing typical pluripotency markers and were capable of three germ layer differentiation. This is the first reported reprogramming of cells with a mutation in the cyclin F gene, and represents a novel resource for the study of amyotrophic lateral sclerosis.

Application of a low cost array-based technique - TAB-Array - for quantifying and mapping both 5mC and 5hmC at single base resolution in human pluripotent stem cells.

5-hydroxymethylcytosine (5hmC), an oxidized derivative of 5-methylcytosine (5mC), has been implicated as an important epigenetic regulator of mammalian development. Current procedures use DNA sequencing methods to discriminate 5hmC from 5mC, limiting their accessibility to the scientific community. Here we report a method that combines TET-assisted bisulfite conversion with Illumina 450K DNA methylation arrays for a low-cost high-throughput approach that distinguishes 5hmC and 5mC signals at base resolution. Implementing this approach, termed "TAB-array", we assessed DNA methylation dynamics in the differentiation of human pluripotent stem cells into cardiovascular progenitors and neural precursor cells. With the ability to discriminate 5mC and 5hmC, we identified a large number of novel dynamically methylated genomic regions that are implicated in the development of these lineages. The increased resolution and accuracy afforded by this approach provides a powerful means to investigate the distinct contributions of 5mC and 5hmC in human development and disease.

Bone marrow mesenchymal stromal cells rescue cardiac function in streptozotocin-induced diabetic rats.

OBJECTIVES: In the present study, we investigated whether MSC-transplantation can revert cardiac dysfunction in streptozotocin-induced diabetic rats and the immunoregulatory effects of MSC were examined. BACKGROUND: Cardiac complications are one of the main causes of death in diabetes. Several studies have shown anti-diabetic effects of bone marrow mesenchymal stromal cells (MSC). METHODS/RESULTS: The rats were divided in three groups: Non-diabetic, Diabetic and Diabetic-Treated with 5 × 10(6) MSC 4 weeks after establishment of diabetes. Four weeks after MSC-therapy, systemic metabolic parameters, immunological profile and cardiac function were assessed. MSC-transplantation was able to revert the hyperglycemia and body weight loss of the animals. In addition, after MSC-transplantation a decrease in corticosterone and IFN-γ sera levels without restoration of insulin and leptin plasma levels was observed. Also, MSC-therapy improved electrical remodeling, shortening QT and QTc in the ECG and action potential duration of left ventricular myocytes. No arrhythmic events were observed after MSC-transplantation. MSC-therapy rescued the cardiac beta-adrenergic sensitivity by increasing beta-1 adrenergic receptor expression. Both alpha and beta cardiac AMPK and p-AMPK returned to baseline values after MSC-therapy. However, total ERK1 and p-ERK1/2 were not different among groups. CONCLUSION: The results indicate that MSC-therapy was able to rescue cardiac impairment induced by diabetes, normalize cardiac AMPK subunit expression and activity, decrease corticosterone and glycemia and exert systemic immunoregulation.

Insulin-like growth factor-1 receptor identifies a pool of human cardiac stem cells with superior therapeutic potential for myocardial regeneration.

RATIONALE: Age and coronary artery disease may negatively affect the function of human cardiac stem cells (hCSCs) and their potential therapeutic efficacy for autologous cell transplantation in the failing heart. OBJECTIVE: Insulin-like growth factor (IGF)-1, IGF-2, and angiotensin II (Ang II), as well as their receptors, IGF-1R, IGF-2R, and AT1R, were characterized in c-kit(+) hCSCs to establish whether these systems would allow us to separate hCSC classes with different growth reserve in the aging and diseased myocardium. METHODS AND RESULTS: C-kit(+) hCSCs were collected from myocardial samples obtained from 24 patients, 48 to 86 years of age, undergoing elective cardiac surgery for coronary artery disease. The expression of IGF-1R in hCSCs recognized a young cell phenotype defined by long telomeres, high telomerase activity, enhanced cell proliferation, and attenuated apoptosis. In addition to IGF-1, IGF-1R(+) hCSCs secreted IGF-2 that promoted myocyte differentiation. Conversely, the presence of IGF-2R and AT1R, in the absence of IGF-1R, identified senescent hCSCs with impaired growth reserve and increased susceptibility to apoptosis. The ability of IGF-1R(+) hCSCs to regenerate infarcted myocardium was then compared with that of unselected c-kit(+) hCSCs. IGF-1R(+) hCSCs improved cardiomyogenesis and vasculogenesis. Pretreatment of IGF-1R(+) hCSCs with IGF-2 resulted in the formation of more mature myocytes and superior recovery of ventricular structure. CONCLUSIONS: hCSCs expressing only IGF-1R synthesize both IGF-1 and IGF-2, which are potent modulators of stem cell replication, commitment to the myocyte lineage, and myocyte differentiation, which points to this hCSC subset as the ideal candidate cell for the management of human heart failure.

Sympathetic neuronal survival induced by retinal trophic factors.

Neuronal survival in the vertebrate peripheral nervous system depends on neurotrophic factors available from target tissues. In an attempt to identify novel survival factors, we have studied the effect of secreted factors from retinal cells on the survival of chick sympathetic ganglion neurons. Embryonic day 10 sympathetic neurons undergo programmed cell death after 48 h without appropriate levels of nerve growth factor (NGF). Retina Conditioned Media (RCM) from explants of embryonic day 11 retinas maintained for 4 days in vitro supported 90% of E10 chick sympathetic neurons after 48 h. Conditioned medium from purified chick retinal Muller glial cells supported nearly 100% of E10 chick sympathetic neurons. Anti-NGF (1 microg/mL) blocked the survival effect of NGF, but did not block the trophic effect of RCM. Neither BDNF nor NT4 (0.1-50 ng/mL) supported E10 sympathetic neuron survival. Incubation of chimeric immunoglobulin-receptors TrkA, TrkB, or TrkC had no effect on RCM-induced sympathetic neuron survival. The survival effects were not blocked by anti-GDNF, anti-TGFbeta, and anti-CNTF and were not mimicked by FGFb (0.1-10 nM). LY294002 at 50 microM, but not PD098059 blocked sympathetic survival induced by RCM. Further, the combination of RCM and NGF did not result in an increase in neuronal survival compared with NGF alone (82% survival after 48 h). The secreted factor in RCM is retained in subfractions with a molecular weight above 100 kDa, binds to heparin, and is unaffected by dialysis, but is heat sensitive. Our results indicate the presence of a high-molecular weight retinal secreted factor that supports sympathetic neurons in culture.