The novel Tenzing 7 delivery catheter designed to deliver intermediate catheters to the face of embolus without crossing: clinical performance predicted in anatomically challenging model.

BACKGROUND: In large vessel occlusionstroke, navigation of aspiration catheters (AC) can be impeded by vessel tortuosity and the ophthalmic artery origin. A novel tapered delivery catheter was designed to facilitate delivery without disturbing the embolus. We assessed AC deliverability in vitro and validated the observations in a first-in-human experience. METHODS: In a vascular model with three challenging craniocervical scenarios, two commercial AC were advanced from the carotid to the middle cerebral artery by four neurointerventionalists. Catheter deliverability with standard microwire and microcatheter (MC) combinations and the Tenzing 7 (T7) Delivery Catheter (Route 92 Medical, San Mateo, CA) were compared. Operators rated aspects of catheter deliverability on a 5-point scale. Results were compared with device delivery patterns at a neurovascular center before and after clinical introduction of T7. RESULTS: In vitro, success rate and speed were higher with T7 (96%; mean 30±10 s) than with MC (65%; 72±47 s, p<0.001 each), with fewer interactions with the occlusion site (T7: 54% vs MC: 77%, p=0.004). T7 received superior ratings regarding carotid artery deflection (T7: 2, IQR1-3 vs MC: 3, IQR2-3, p<0.001), guide catheter pushback (T7: 2, IQR1-3 vs MC: 3, IQR3-3, p<0.001) and ophthalmic artery passage (T7: 1.5, IQR1-2 vs MC: 4, IQR3-5, p<0.001). Before introduction of T7 at a single center, delivery of AC to a large vessel occlusion without crossing was achieved in 15/123 cases (12%). With T7, this rate was 28/31 patients (90.3%). CONCLUSION: Compared with microcatheter and microwire combinations, T7 improves aspiration catheter delivery in vitro, minimizing the need to cross the occlusion. Initial clinical experience appears to validate the model's observations.

Hematopoietic and endothelial differentiation of human induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) provide an unprecedented opportunity for modeling of human diseases in vitro, as well as for developing novel approaches for regenerative therapy based on immunologically compatible cells. In this study, we employed an OP9 differentiation system to characterize the hematopoietic and endothelial differentiation potential of seven human iPSC lines obtained from human fetal, neonatal, and adult fibroblasts through reprogramming with POU5F1, SOX2, NANOG, and LIN28 and compared it with the differentiation potential of five human embryonic stem cell lines (hESC, H1, H7, H9, H13, and H14). Similar to hESCs, all iPSCs generated CD34(+)CD43(+) hematopoietic progenitors and CD31(+)CD43(-) endothelial cells in coculture with OP9. When cultured in semisolid media in the presence of hematopoietic growth factors, iPSC-derived primitive blood cells formed all types of hematopoietic colonies, including GEMM colony-forming cells. Human induced pluripotent cells (hiPSCs)-derived CD43(+) cells could be separated into the following phenotypically defined subsets of primitive hematopoietic cells: CD43(+)CD235a(+)CD41a(+/-) (erythro-megakaryopoietic), lin(-)CD34(+)CD43(+)CD45(-) (multipotent), and lin(-)CD34(+)CD43(+)CD45(+) (myeloid-skewed) cells. Although we observed some variations in the efficiency of hematopoietic differentiation between different hiPSCs, the pattern of differentiation was very similar in all seven tested lines obtained through reprogramming of human fetal, neonatal, or adult fibroblasts with three or four genes. Although several issues remain to be resolved before iPSC-derived blood cells can be administered to humans for therapeutic purposes, patient-specific iPSCs can already be used for characterization of mechanisms of blood diseases and for identification of molecules that can correct affected genetic networks.

Induced Pluripotent Stem Cell-Derived Dopaminergic Neurons from Adult Common Marmoset Fibroblasts.

The common marmoset monkey (Callithrix jacchus; Cj) is an advantageous nonhuman primate species for modeling age-related disorders, including Parkinson's disease, due to their shorter life span compared to macaques. Cj-derived induced pluripotent stem cells (Cj-iPSCs) from somatic cells are needed for in vitro disease modeling and testing regenerative medicine approaches. Here we report the development of a novel Cj-iPSC line derived from adult marmoset fibroblasts. The Cj-iPSCs showed potent pluripotency properties, including the development of mesodermal lineages in tumors after injection to immunocompromised mice, as well as ectoderm and endoderm lineages after in vitro differentiation regimens, demonstrating differentiated derivatives of all three embryonic layers. In addition, expression of key pluripotency genes (ZFP42, PODXL, DNMT3B, C-MYC, LIN28, KLF4, NANOG, SOX2, and OCT4) was observed. We then tested the neural differentiation capacity and gene expression profiles of Cj-iPSCs and a marmoset embryonic stem cell line (Cj-ESC) after dual-SMAD inhibition. Exposure to CHIR99021 and sonic hedgehog (SHH) for 12 and 16 days, respectively, patterned the cells toward a ventralized midbrain dopaminergic phenotype, confirmed by expression of FOXA2, OTX2, EN-1, and tyrosine hydroxylase. These results demonstrate that common marmoset stem cells will be able to serve as a platform for investigating regenerative medicine approaches targeting the dopaminergic system.

Human induced pluripotent stem cells free of vector and transgene sequences.

Reprogramming differentiated human cells to induced pluripotent stem (iPS) cells has applications in basic biology, drug development, and transplantation. Human iPS cell derivation previously required vectors that integrate into the genome, which can create mutations and limit the utility of the cells in both research and clinical applications. We describe the derivation of human iPS cells with the use of nonintegrating episomal vectors. After removal of the episome, iPS cells completely free of vector and transgene sequences are derived that are similar to human embryonic stem (ES) cells in proliferative and developmental potential. These results demonstrate that reprogramming human somatic cells does not require genomic integration or the continued presence of exogenous reprogramming factors and removes one obstacle to the clinical application of human iPS cells.

Induced pluripotent stem cell lines derived from human somatic cells.

Somatic cell nuclear transfer allows trans-acting factors present in the mammalian oocyte to reprogram somatic cell nuclei to an undifferentiated state. We show that four factors (OCT4, SOX2, NANOG, and LIN28) are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem (ES) cells. These induced pluripotent human stem cells have normal karyotypes, express telomerase activity, express cell surface markers and genes that characterize human ES cells, and maintain the developmental potential to differentiate into advanced derivatives of all three primary germ layers. Such induced pluripotent human cell lines should be useful in the production of new disease models and in drug development, as well as for applications in transplantation medicine, once technical limitations (for example, mutation through viral integration) are eliminated.

A severe de novo methylation of episomal vectors by human ES cells.

Episomal vectors can allow efficient genetic modification of cells and have the potential advantage of avoiding chromosomal position of integration effects. Here we explore the use of an Epstein-Barr virus-based episomal vector with human embryonic stem (ES) cells, and find high initial transfection rates, but a rapid loss of reporter gene expression. Similar to mouse ES cells, human ES cells express high levels of the de novo DNA methyltransferases, and we detected dramatic CpG methylation and minor non-CpG methylation on the episomes recovered from the human ES cells 7 days after the transfection, which was not present on the same episome recovered from 293 cells. Interestingly, the oriP region of the episomes was relatively excluded from this methylation. These findings define some of the limitations of using episomal vectors with human ES cells and offer a unique platform for analyzing epigenetic gene silencing in human ES cells.