Human-Induced Pluripotent Stem Cell-Derived Neural Progenitor Cells Showed Neuronal Differentiation, Neurite Extension, and Formation of Synaptic Structures in Rodent Ischemic Stroke Brains

Abstract

Ischemic stroke is a major cerebrovascular disease with high morbidity and mortality rates; however, effective treatments for ischemic stroke-related neurological dysfunction have yet to be developed. In this study, we generated neural progenitor cells from human leukocyte antigen major loci gene-homozygous-induced pluripotent stem cells (hiPSC-NPCs) and evaluated their therapeutic effects against ischemic stroke. hiPSC-NPCs were intracerebrally transplanted into rat ischemic brains produced by transient middle cerebral artery occlusion at either the subacute or acute stage, and their in vivo survival, differentiation, and efficacy for functional improvement in neurological dysfunction were evaluated. hiPSC-NPCs were histologically identified in host brain tissues and showed neuronal differentiation into vGLUT-positive glutamatergic neurons, extended neurites into both the ipsilateral infarct and contralateral healthy hemispheres, and synaptic structures formed 12 weeks after both acute and subacute stage transplantation. They also improved neurological function when transplanted at the subacute stage with γ-secretase inhibitor pretreatment. However, their effects were modest and not significant and showed a possible risk of cells remaining in their undifferentiated and immature status in acute-stage transplantation. These results suggest that hiPSC-NPCs show cell replacement effects in ischemic stroke-damaged neural tissues, but their efficacy is insufficient for neurological functional improvement after acute or subacute transplantation. Further optimization of cell preparation methods and the timing of transplantation is required to balance the efficacy and safety of hiPSC-NPC transplantation.

Keywords: cell replacement effect; induced pluripotent stem cells; intracerebral transplantation; ischemic stroke; neural progenitor cells; neurite extension; neuronal differentiation; synaptic structure formation.

Figure 1

Cellular properties of HLA-homo hiPSC-NPCs. (A) Representative phase contrast image. Bar = 500 μm. (B) Gene expression analysis using TaqMan assays; HLA-homo hiPSC-NPCs (HH-NPC: biological duplicate, technical duplicate; total n = 4), control hiPSC-NPCs (NPC: biological duplicate, technical duplicate; total n = 4), hf-NSPCs from fetal forebrain tissues (NSPC: biological triplicate, technical duplicate; total n = 6), and undifferentiated iPSCs (iPSC: biological duplicate, technical duplicate; total n = 4). Bar graphs are shown by mean ± SEM. (C) Immunocytochemical analysis of SOX1 and nestin (NES). Bar = 50 μm. (D) Flow cytometric analyses: red lines are the stained signal, and blue lines are negative controls. (E) Karyotype analysis carried out by G-banding. (F) In vitro differentiation assay. βIII tubulin (green), GFAP (red). Blue is nuclear staining using DAPI. Percentage of ELAVL3/4-positive cells was quantitatively determined independently. GFAP staining was evaluated by positive (+) or negative (−). Bar = 50 μm.

Figure 2

Infarct ratios in subacute and acute transplantation studies. The infarct ratio of each animal was measured using three hematoxylin and eosin-stained coronal sections. Target: the section centering on the point of transplant; +3 mm rostral, +3 mm rostral sections from the target; +3 mm caudal, +3 mm caudal sections from the target. The results are shown as box-and-whisker plots. Subacute: GSI (−) group (blue), GSI (+) group (red), and control group (green). Acute: GSI (−) group (blue), GSI (+) group (red), and control group (green).

Figure 3

Quantitative assessment of numbers of residual neurons in host brains. The number of residual neurons in the host brain was determined as the sum of NeuN immunopositive nuclei in the three sections. The results are shown as box-and-whisker plots. Subacute: GSI (−) group (blue), GSI (+) group (red), and control group (green). Acute: GSI (−) group (blue), GSI (+) group (red), and control group (green).

Figure 4

In vivo survival of transplanted HLA-homo hiPSC-NPCs in the ischemic brain. (A) HNA- or Ki-67-positive cells in host brain tissues after 12 weeks from subacute transplantation of GSI-untreated or treated HLA-homo hiPSC-NPCs, and the number of positive cells are shown in box-and-whisker plots. GSI (−) group (blue), GSI (+) group (red). Bar = 50 μm. (B) HNA- or Ki-67-positive cells in host brain tissues after 12 weeks from acute transplantation of HLA-homo hiPSC-NPCs, and the number of positive cells are shown in box-and-whisker plots. GSI (−) group (blue), GSI (+) group (red). Bar = 50 μm.

Figure 5

In vivo differentiation of transplanted HLA-homo hiPSC-NPCs in the ischemic brain. (A) HNA-positive cells expressing SOX1 or ELAVL3/4 (open triangle) and expression of hSYP in host brain tissues after 12 weeks from subacute transplantation of GSI-untreated or treated HLA-homo hiPSC-NPCs. The results are shown in box-and-whisker plots. GSI (−) group (blue), GSI (+) group (red). Bar = 50 μm. (B) HNA-positive cells expressing SOX1 or ELAVL3/4 (open triangle) and expression of hSYP in host brain tissues after 12 weeks from acute transplantation of GSI-untreated or treated HLA-homo hiPSC-NPCs. The results are shown in box-and-whisker plots. GSI (−) group (blue), GSI (+) group (red). Bar = 50 μm.

Figure 5

In vivo differentiation of transplanted HLA-homo hiPSC-NPCs in the ischemic brain. (A) HNA-positive cells expressing SOX1 or ELAVL3/4 (open triangle) and expression of hSYP in host brain tissues after 12 weeks from subacute transplantation of GSI-untreated or treated HLA-homo hiPSC-NPCs. The results are shown in box-and-whisker plots. GSI (−) group (blue), GSI (+) group (red). Bar = 50 μm. (B) HNA-positive cells expressing SOX1 or ELAVL3/4 (open triangle) and expression of hSYP in host brain tissues after 12 weeks from acute transplantation of GSI-untreated or treated HLA-homo hiPSC-NPCs. The results are shown in box-and-whisker plots. GSI (−) group (blue), GSI (+) group (red). Bar = 50 μm.

Figure 6

Neurites growth from transplanted HLA-homo hiPSC-NPCs. (A) STEM121-positive fibers structures in both the ipsilateral (Ipsi) infarct and contralateral (Contra) healthy hemispheres. Bar = 20 μm. (B) Expression of HLA-ABC and growth-associated protein of 43-kDa (GAP-43) phosphorylated at Thr172 site [pGAP43(Thr172)] in both Ipsi and Contra hemispheres. Bar = 20 μm. (C) Distribution patterns of STEM121-positive fibers structures. Total amount of STEM121-positive fiber structures calculated by immunopositive pixel count analyses are shown in box-and-whisker plots. GSI (−) group (blue), GSI (+) group (red).

Figure 7

Maturation of the transplanted HLA-homo hiPSC-NPCs. (A) Expression of hSYP (green) and synaptic structures (yellow) formed by contact with drebrin A-expressing structures (red). Bar = 10 μm. (B) Expression of vGLUT1 or vGLUT2 (red) co-expressed with hSYP (green). Bar = 10 μm. Blue is nuclear staining using DAPI.

Figure 7

Maturation of the transplanted HLA-homo hiPSC-NPCs. (A) Expression of hSYP (green) and synaptic structures (yellow) formed by contact with drebrin A-expressing structures (red). Bar = 10 μm. (B) Expression of vGLUT1 or vGLUT2 (red) co-expressed with hSYP (green). Bar = 10 μm. Blue is nuclear staining using DAPI.

Figure 8

Neurological functional assessment. Results of neurological functional assessments were calculated as a relative ratio to baseline results [Neurological deficit score (NDS): post-IS (ischemic) 1-day; Step test, post-IS 2 days (acute transplantation) or 7 days (subacute transplantation)], and shown by mean ± SEM. Subacute: GSI (−) group (n = 10; blue), GSI (+) group (n = 10; red), and control group (n = 10; green). Acute: GSI (−) group (n = 10; blue), GSI (+) group (n = 9; red), and control group (n = 9; green).