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

Abstract

We developed neural cell sheets being positive for mature motoneuron markers, such as βIII tubulin, islet1 and HB9, from human induced pluripotent stem (hiPS) cells after embryoid body formation. We transplanted the neural cell sheets into the complete transection sites of spinal cord injury (SCI) model mice. We assessed functional and histological analyses of the SCI mice and compared the data with those of hiPS cell derived neural stem/progenitor (NSP) cell transplanted SCI mice.Neurofilament medium (NFM), βIII tubulin, and HB9 positive cells were significantly increased in vitro in neural cell sheets compared with those in NSP cells. The neural cell sheets expressed remarkably high mRNA of NFM and islet 1 compared with NSP cells. After transplantation, the neural cell sheets elongated human neural cell adhesion molecule (hNCAM) positive axons over the L1 spinal level and carried a neuron tracer cholera toxin β subunit (CTβ) to downstream region. The sheet transplantation showed significantly better locomotor recovery of SCI mice until day 65 than NSP cell transplantation. The sheet transplantation tended to improve spinal cord atrophy in caudal part of injured/grafted site to the same extent as NSP cell transplantation. We suggest that the neural cell sheets derived from hiPS cells may be one of the most effective treatment for SCI, through the mature neural and extendable scaffold functions.

Transplantation of Human iPS Cell-Derived Neural Cells with an Artificial Nerve Conduit Leads to Cellular Retention in the Transplanted Area and Improves Motor Function in a Mouse Spinal Cord Injury Model

“…The model itself is described below, and timelines showing both NSPC devel-opment and transplantation into the model are shown in Figure 1. We developed NSPCs from hiPSCs, as reported previously 14,16) . The hiPSC line 253G1 was obtained from RIKEN BRC (Ibaraki, Japan, RRID: CVCL_B518, http://cellbank.brc.riken.jp/cell_bank/ CellInfo/?cellNo=HPS0002) and maintained according to the standard protocol 34) .…”

“…The cells were then cultured in fibronectin (FN, BD Biosciences, San Diego, CA, USA)-coated dishes for 4 days. We introduced 1 μM retinoic acid (RA, Sigma-Aldrich, St. Louis, MO, USA), 10 nM noggin (NOG)-Fc (R&D Systems, Minneapolis, MN, USA), and 10 nM Sonic hedgehog protein (SHH, R&D Systems) into the culture twice (on Days −3 and −1) 14,16,[35][36][37][38] . In preparation, the cells were disaggregated into single-cell suspensions and transplanted on Day 0.…”

“…Cells and cryostat sections were blocked for 2 hours in PBSTG (PBS containing 0.2% Tween 20 [Kanto Chemical, Tokyo, Japan] and 5% goat serum [ThermoFisher Scientific]). Immunofluorescence staining with antibody to βIII tubulin (G712A, Promega, Madison, WI, USA) was then performed, as previously reported [14][15][16][17] . Cells in the conduit were examined morphologically, and immunofluorescence was examined with a stereomicroscope and confocal microscope (LSM-510, Zeiss, Oberkochen, Germany).…”

“…We have succeeded in generating, from ES cells and human iPSCs (hiPSCs), NSPCs that can differen-tiate into cells with a spinal motoneuron phenotype [14][15][16][17][18][19] . Expression of neuron markers, neurofilament medium (NFM) and βIII tubulin, and of spinal motoneuron marker HB9 was increased by stimulation with retinoic acid (RA), noggin (NOG), and sonic hedgehog protein (SHH) 16,18,19) .…”

“…Implantation of a nerve conduit has the potential to connect the completely transected spinal cord by surrounding and protecting transplanted NSPCs. Tissue engineering involving development of two-or three-dimensional scaffolds made up of stem cells or biomaterials has been applied in animal models of various types of tissue damage and also clinically 14,17,[21][22][23][24][25][26][27] . Many types of artificial nerve conduits have been designed and used as alternatives to autologous nerve grafts for peripheral nerve injuries 23,28,29) .…”