Generation of an induced pluripotent stem cell line from a Huntington's disease patient with a long HTT-PolyQ sequence.

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an abnormal length of CAG repeats in the gene HTT, leading to an elongated poly-glutamine (poly-Q) sequence in huntingtin (HTT). We used non-integrative Sendai virus to reprogram fibroblasts from a patient with juvenile onset HD to induced pluripotent stem cells (iPSCs). Reprogrammed iPSCs expressed pluripotency-associated markers, exhibited a normal karyotype, and following directed differentiation generated cell types belonging to the three germ layers. PCR analysis and sequencing confirmed the HD patient-derived iPSC line had one normal HTT allele and one with elongated CAG repeats, equivalent to ≥180Q.

Generation of induced pluripotent stem cells from three individuals with Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disorder caused by abnormal glutamine (Q) expansion in the huntingtin protein due to elongated CAG repeats in the gene HTT. We used non-integrative episomal plasmids to generate induced pluripotent stem cells (iPSCs) from three individuals affected by HD: CH1 (58Q), and two twin brothers CH3 (44Q) and CH4 (44Q). The iPSC lines exhibited one healthy HTT allele and one with elongated CAG repeats, as confirmed by PCR and sequencing. All iPSC lines expressed pluripotency markers, exhibited a normal karyotype, and generated cells of the three germ layers in vitro.

Simple Workflow and Comparison of Media for hPSC-Cardiomyocyte Cryopreservation and Recovery.

Great progress has been made with protocols for the differentiation and functional application of hPSC-cardiomyocytes (hPSC-CMs) in recent years; however, the cryopreservation and recovery of hPSC-CMs still presents challenges and few reports describe in detail the protocols and general workflow. In order to facilitate cryopreservation and recovery of hPSC-CMs for a wide range of applications, we provide detailed information and step-by-step protocols. The protocols are simple and use common reagents. They are comprised of a fast dissociation, cryopreservation using standard equipment, and gentle recovery following thawing. We discuss various features of the protocols, as well as their utilization in the context of common hPSC-CM differentiation and application workflows. Finally, we compare two proprietary and two common in-house formulations of cryopreservation media used for hPSC-CMs, and despite differences in their price and composition find broadly similar recovery rates and cellular function after thawing. © 2019 The Authors. Basic Protocol 1: Dissociation and cryopreservation of hPSC-CMs Basic Protocol 2: Thawing and recovery of cryogenically frozen hPSC-CMs.