Psa ncam microbeads

Primary β-cells were isolated by magnetic bead purification using the AutoMACS cell sorter (Miltenyi Biotech Inc., San Diego, CA) as previously described [35 (link)]. In brief, human islets were washed once, subjected to trypsin disassociation, and filtered using a 70 μm nylon mesh. Dispersed islets were stained using anti-human CA19-9 antibodies (Miltenyi Biotech Inc., San Diego, CA) and ran through the AutoMACS. The negative fraction was stained using PSA-NCAM microbeads (Miltenyi Biotech Inc., San Diego, CA) and the positive fraction was sorted using the AutoMACS. CA19-9^-PSA-NCAM⁺ cells were enriched for β-cells and were used for further analysis. Fraction enrichment was verified by measuring the signal of demethylated insulin DNA as previously described [8 (link)] (S1 Fig).

Both rat iPSCs, i.e., o-riPSCs and l-riPSCs, were expanded on feeder cell layers in basic-DMEM. For the expansion of rat iPSCs, 1000 U/mL LIF was added in basic-DMEM. For neurogenesis, riPSCs were incubated for 2 days without LIF and compacted to form embryoid bodies (EBs) in hanging drops. After that, EBs were exposed to 5×10^-5 M all-trans retinoic acid (RA, Sigma) in basic DMEM for 5 days and were placed in a magnetic cell separation system (MACS; Miltenyi Biotech; Auburn, CA; www.miltenyibiotec.com) by using PSA-NCAM micro beads (130-092-966 Miltenyi Biotech). The sample preparation, magnetic labeling, and magnetic separation with LS columns were conducted according to the manufacturer's instructions. Sorted neuron progenitors were attached to dishes coated with poly-d-lysine hydrobromide (P7280, Sigma)-laminin (L2020, Sigma), and then they were differentiated into motor neurons by addition of 25 ng/mL sonic hedgehog (recombinant mouse sonic hedgehog N-terminal 461-SH; R&D Systems; Minneapolis, MN; www.RnDSystems.com) and 2×10^-5 M RA in basic-DMEM. The culture medium was changed every day.