Controlling Immune Rejection Is a Fail-Safe System against Potential Tumorigenicity after Human iPSC-Derived Neural Stem Cell Transplantation

<div><p>Our previous work reported functional recovery after transplantation of mouse and human induced pluripotent stem cell-derived neural stem/progenitor cells (hiPSC-NS/PCs) into rodent models of spinal cord injury (SCI). Although hiPSC-NS/PCs proved useful for the treatment of SCI, the tumorigenicity of the transplanted cells must be resolved before they can be used in clinical applications. The current study sought to determine the feasibility of ablation of the tumors formed after hiPSC-NS/PC transplantation through immunoregulation. Tumorigenic hiPSC-NS/PCs were transplanted into the intact spinal cords of immunocompetent BALB/cA mice with or without immunosuppressant treatment. <i>In vivo</i> bioluminescence imaging was used to evaluate the chronological survival and growth of the transplanted cells. The graft survival rate was 0% in the group without immunosuppressants versus 100% in the group with immunosuppressants. Most of the mice that received immunosuppressants exhibited hind-limb paralysis owing to tumor growth at 3 months after iPSC-NS/PC transplantation. Histological analysis showed that the tumors shared certain characteristics with low-grade gliomas rather than with teratomas. After confirming the progression of the tumors in immunosuppressed mice, the immunosuppressant agents were discontinued, resulting in the complete rejection of iPSC-NS/PC-derived masses within 42 days after drug cessation. In accordance with the tumor rejection, hind-limb motor function was recovered in all of the mice. Moreover, infiltration of microglia and lymphocytes was observed during the course of tumor rejection, along with apoptosis of iPSC-NS/PC-generated cells. Thus, immune rejection can be used as a fail-safe system against potential tumorigenicity after transplantation of iPSC-NS/PCs to treat SCI.</p></div

Representative <i>in vivo</i> images and quantitative analysis of photon counts derived from grafted hiPSC-NS/PCs.

<p>(A) Bioluminescence images of representative mice at 0, 21, 97, and 142 days after human induced pluripotent stem cell-derived neural stem/progenitor cell (hiPSC-NS/PC) transplantation. Upper panel: BALB/cA mouse with immunosuppressant treatment (FK506 plus anti-cluster of differentiation (CD) 4 monoclonal antibody (mAb); With-IS group); middle panel: BALB/cA mouse with immunosuppressant treatment, followed by discontinuation of immunosuppressants 100 days later (IS off group); lower panel: BALB/cA mouse without immunosuppressant treatment (Without-IS group). (B) Quantitative analysis of photon counts derived from grafted hiPSC-NS/PCs. Graft survival rate was 100% (n = 17/21, three animals died and one animal was sacrificed by day 21) in BALB/cA mice with immunosuppressant treatment (FK506 plus anti-CD4 mAb), versus 0% (n = 0/6) in BALB/cA mice without immunosuppressant treatment. After discontinuing the administration of FK506 and anti-CD4 mAb, all the grafted cells were rejected by day 164 and drastic reductions in signal intensity were observed. Data represent the mean value ± the standard error of the mean. (*p<0.05; Friedman’s test followed by Dunn’s post-hoc test.)</p

Lymphoid population in peripheral blood.

<p>Peripheral blood cells were analyzed using fluorescence-activated cell sorting-based flow cytometry. Data were gated on cluster of differentiation (CD) 4-positive or CD8-positive T-cell subsets. CD4-positive T-cells were depleted immediately after administration of FK506 plus anti-CD4 monoclonal antibody, but recovered after discontinuing immunosuppressant treatment. IS(+), with immunosuppressants; IS(−), without immunosuppressants.</p

Tumor formation by grafted hiPSC-NS/PCs in the mouse spinal cord.

<p>Representative hematoxylin-eosin (HE)-stained (A) and human nuclear antigen (HNA)-stained (B) images of sagittal sections of spinal cord at 79 days after cell transplantation. HE staining revealed a biphasic tumor pattern with high and low cell density areas. The high cell density area contained compact bipolar cells with rosenthal fibers, whereas the low cell density area contained loose-textured multipolar cells with microcysts. The low cell density area surrounded by the square box is shown at higher magnification to the left of the image. Immunostaining for glial fibrillary acidic protein (GFAP), HNA, GFP, nestin, β-III tubulin (C, D, E), and Oligo-1 (F). The human induced pluripotent stem cell-derived neural stem/progenitor cell (hiPSC-NSC)-derived tumors mainly consisted of undifferentiated cells that stained positively for nestin, with low numbers of differentiated cells (e.g., β-III tubulin-positive neurons, GFAP-positive astrocytes, and Oligo-1-positive oligodendrocyte precursor cells). Nestin-positive cells were located in the center of the tumor, whereas differentiated cells were localized to the tumor margin (D, E, F). The boxed area in (C) corresponds to the higher magnification images in (D). Tumors contained a paucity of octamer-binding transcription factor (Oct) 4-positive (G) and Ki-67-positive (H) cells. The Ki-67 index was 7.0%. Scale bars in A–C, 500 μm; D–H, 100 μm.</p

Fate of hiPSC-NS/PCs transplanted into the mouse spinal cord.

<p>Fate of hiPSC-NS/PCs transplanted into the mouse spinal cord.</p

Detection of bioluminescence and fluorescence signals in lentivirally transfected 253G1-NS/PCs <i>in vitro</i>.

<p>Phase-contrast (A) and fluorescence (B) images of a neurosphere derived from tumorigenic 253G1 induced pluripotent stem cells. Neural stem/progenitor cells (NS/PCs) differentiated into β-III tubulin-positive neurons and glial fibrillary acidic protein (GFAP)-positive astrocytes <i>in vitro</i> (C). Bioluminescence imaging was used to detect bioluminescence signals in various numbers of 253G1-NS/PCs (0, 1.5 × 10⁵, 3 × 10⁵, 6 × 10⁵, and 1.2 × 10⁶ cells per well) (D). A direct linear correlation was found between cell numbers and photon counts <i>in vitro</i> (E). Scale bars in A–C, 1,000 μm.</p

Analysis of inflammatory cell infiltration and apoptotic cells during the immune rejection of hiPSC-NS/PC-derived tumors.

<p>Immunohistochemical (A) and quantitative (B) analyses of human nuclear antigen (HNA)-, GFP-, cluster of differentiation (CD) 11b-, CD3-, NKp46-, and terminal deoxynucleotidyl transferase (TdT)-positive cells after discontinuing immunosuppressant treatment on day 100. After discontinuing the immunosuppressants, HNA-positive cells were gradually rejected. Inflammatory cells, including CD11b-, CD3-, and NKp46-positive cells, increased up to day 122 and then gradually decreased thereafter. TdT-positive cells also became prominent by day 122 and then gradually disappeared (C). Scale bars in A, 200 μm. hiPSC-NS/PC, human induced pluripotent stem cell-derived neural stem/progenitor cell.</p

Grafted hiPSC-NS/PCs differentiate into three neural lineages without tumor formation in the injured spinal cord.

<p>(<b>A</b>) Representative H-E stained images of axial sections at the lesion epicenter at 12 weeks (84 days) after cell transplantation. No tumor formation was observed in the hiPSC-NS/PC-transplanted group during this time. H-E staining showed that cystic cavity formation at the lesion epicenter was prominent in the vehicle control group compared with the transplantation group, whereas there was no clear difference in the transverse area of the injured spinal cord. (<b>B</b>) Quantification of the cystic cavity area. Data represent the mean ± the SEM (n = 5 for each group, *p<0.05). (<b>C</b>) Immunostaining for Oct4 and Ki-67. (<b>C-1</b>) All colonies of undifferentiated iPSCs were positive for Oct4, a marker of undifferentiated ESCs and iPSCs. (<b>C-2</b>) Grafted hiPSC- NS/PCs yielded no Oct4-positive following transplantation into the injured spinal cord. (<b>C-3</b>) The percentage of HNu-positive cells that were also Ki-67-positive was 0.55±0.08%. (<b>D</b>) Differentiation of grafted hiPSC-NS/PCs. (<b>D-1-3</b>) Representative images of Venus-positive grafted hiPSC-NS/PCs immunostained with antibodies against NeuN to detect mature neurons, GFAP to detect astrocytes, and Olig1 to detect oligodendrocyte progenitor cells. (<b>D-4</b>) Percentages of cell type-specific, marker-positive cells among the Venus-positive grafted hiPSC-NS/PCs at 12 weeks post-engraftment. (<b>E</b>) In vitro differentiation of hiPSC-NS/PCs. (<b>E-1, 2</b>) Representative images of hiPSC-NS/PCs immunostained with antibodies against βIII-tubulin to detect neurons, and GFAP to detect astrocytes. (<b>E-3</b>) Percentages of cell type-specific marker-positive cells among the hiPSC-NS/PCs 10 days after the initiation of cell differentiation in vitro.</p

Grafted hiPSC-NS/PCs promote functional recovery after SCI.

<p>(<b>A-1</b>) Original open field rating scale (maximum 30 points). Immediately after injury, the score decreased to approximately zero for both groups. By 12 weeks after injury, the score had increased to a plateau of ∼21 in the transplantation group and ∼13 in the vehicle control group. Data represent the mean ± SEM (n = 5 animals for the transplantation group and n = 4 for the control group, *p<0.05). (<b>A-2</b>, <b>A-3</b>) Open field locomotor rating scale for the upper limbs (maximum 20 points) and the lower limbs and trunk (maximum 10 points). Data represent the mean ± SEM (n = 5 for the transplantation group and 4 for the control group, *p<0.05). (<b>B</b>) Bar grip test showing the time course of recovery of bar grip strength in the transplantation and the control groups. Each value indicates the percentage of pre-injury bar grip strength. Bar grip strength recovered gradually, reaching a plateau of ∼57% versus ∼40% of the pre-injury value in the transplantation group and the control group, respectively, by 12 weeks after injury. Data represent the mean ± SEM (n = 5 for the transplantation group and n = 4 for the control group, *p<0.05, **p<0.01). (<b>C</b>) Cage climbing test. The average test score was zero for both groups immediately after injury. By 12 weeks after injury, the mean score of the animals in the transplantation group had improved to 4, whereas the mean score of the animals in the control group had improved to 2.8. Data represent the mean ± the SEM (n = 5 for the transplantation group and n = 4 for the control group, *p<0.05, **p<0.01).</p

Grafted hiPSC-NS/PCs secrete neurotrophic factors and enhance angiogenesis after SCI.

<p>(<b>A-1</b>) Representative images of PECAM-1-positive blood vessels at the lesion epicenter. Scale bar: 50 µm. (<b>A-2</b>) Quantification of PECAM-1-positive blood vessels. The number of PECAM-1-positive blood vessels per unit area was higher in the transplantation group than in the control group. Data represent the mean ± SEM (n = 5 for the transplantation group and n = 4 for the control group, *p<0.05). (<b>B</b>) Quantitative RT-PCR and mRNA expression levels of VEGF and human neurotrophic factors. The expression levels of VEGF, NT3, NT4, and CNTF were all significantly higher in hiPSC-NS/PCs than in hDFs.</p