Induction of neural cells with spinal motoneuron phenotype from human iPS cells and the transplantation to totally transected spinal cords in mice

Abstract

Transplantation of neural cells is a promising therapeutic strategy for spinal cord injury (SCI). Here we generated neurons including those with spinal motoneuron phenotype from human induced pluripotent stem (hiPS) cells by leading to formation of embryoid bodies (EB) and subsequent adherent culture for 4 days during which retinoic acid (RA), noggin (NOG) and sonic hedgehog (SHH) were introduced twice. Spinal motoneuron specific HB9 mRNA expression remarkably increased in the presence of RA, NOG and SHH. With this culture condition, the neurons expressed neurofilament middle chain (NFM), βIII tubulin and HB9 proteins in vitro. We then transplanted the hiPS derived human neurons into SCI mice with complete transection of Th11. Motor function of neuron transplanted SCI mice was significantly improved compared with those of vehicle injected SCI mice. Grafted cells survived and expressed βIII tubulin and HB9 diffusely 36 days after the transplantation. Galactocerebroside positive cells increased and glial fibrillary acidic protein (GFAP) expression reduced in the transection site of neuron transplanted SCI mice. The grafted motoneurons elongated human neural cell adhesion molecule (hNCAM) positive axons over the L1 spinal level. NCAM positive axons at L1 carried a neuron tracer cholera toxin β subunit (CTβ), which had been injected into Th10 of spinal cord, cranial region of the SCI/motoneuron grafted site (Th11). Collectively we succeeded in generating neurons which include those with HB9 positive spinal motoneuron phenotype from hiPS cells. The neurons brought about restoration of the motor function and histological improvement in an SCI. Rec.1/23/2015, Acc.4/15/2015, pp154-163

Human iPS cell derived neural cell sheets exhibit mature neural and extendable scaffold functions and promote recovery in injured mouse spinal cords

“…We have already tested a variety of culture conditions employing different culture media and growth factors, and found that medium containing the defined amounts of the three factors (RA, NOG and SHH) were most suitable for inducing the differentiation into neurons including those with spinal motoneuron phenotype from hiPS cells [7].…”

“…Female C57BL/6 mice (5-12 weeks old, Japan SLC) were used as transplant recipients because we wanted to use genetically normal mice [7,8]. Animals were housed less than 5/cage with mouse food and water adlib on a 12/12 hr light/dark cycle.…”

“…Neural cell sheets, NSP cells, and vehicle (PBS) were placed into the transection sites and covered with the temperature-responsive gelation polymer to retain the grafts [7,9,10]. The polymer was then covered with a cement protector before the suture of muscle layers.…”

“…Immunofluorescence staining was conducted as reported previously [6][7][8]. In brief, for all immunofluorescence procedures, consecutive tissue slices were made for immunohistochemistry of neuron associated proteins.…”

“…The neurons were functional in vivo even in the complete transection sites of SCI model mice and brought about motor functional recovery of hind limbs of the mice [7].…”