Tetsuhiro Kikuchi

Isolation of human induced pluripotent stem cell-derived dopaminergic progenitors by cell sorting for successful transplantation.

Summary Human induced pluripotent stem cells (iPSCs) can provide a promising source of midbrain dopaminergic (DA) neurons for cell replacement therapy for Parkinson’s disease. However, iPSC-derived donor cells inevitably contain tumorigenic or inappropriate cells. Here, we show that human iPSC-derived DA progenitor cells can be efficiently isolated by cell sorting using a floor plate marker, CORIN. We induced DA neurons using scalable culture conditions on human laminin fragment, and the sorted CORIN+ cells expressed the midbrain DA progenitor markers, FOXA2 and LMX1A. When transplanted into 6-OHDA-lesioned rats, the CORIN+ cells survived and differentiated into midbrain DA neurons in vivo, resulting in significant improvement of the motor behavior, without tumor formation. In particular, the CORIN+ cells in a NURR1+ cell-dominant stage exhibited the best survival and function as DA neurons. Our method is a favorable strategy in terms of scalability, safety, and efficiency and may be advantageous for clinical application.

Direct Comparison of Autologous and Allogeneic Transplantation of iPSC-Derived Neural Cells in the Brain of a Nonhuman Primate

Summary Induced pluripotent stem cells (iPSCs) provide the potential for autologous transplantation using cells derived from a patient’s own cells. However, the immunogenicity of iPSCs or their derivatives has been a matter of controversy, and up to now there has been no direct comparison of autologous and allogeneic transplantation in the brains of humans or nonhuman primates. Here, using nonhuman primates, we found that the autologous transplantation of iPSC-derived neurons elicited only a minimal immune response in the brain. In contrast, the allografts caused an acquired immune response with the activation of microglia (IBA-1+/MHC class II+) and the infiltration of leukocytes (CD45+/CD3+). Consequently, a higher number of dopaminergic neurons survived in the autografts. Our results suggest that the autologous transplantation of iPSC-derived neural cells is advantageous for minimizing the immune response in the brain compared with allogeneic grafts.

Small-molecule inhibitors of bone morphogenic protein and activin/nodal signals promote highly efficient neural induction from human pluripotent stem cells

The balance of bone morphogenic protein (BMP), transforming growth factor‐β (TGFβ)/activin/nodal, and Wnt signals regulates the early lineage segregation of human embryonic stem cells (ESCs). Here we demonstrate that a combination of small‐molecule inhibitors of BMP (Dorsomorphin) and TGFβ/activin/nodal (SB431542) signals promotes highly efficient neural induction from both human ESCs and induced pluripotent stem cells (iPSCs). The combination of small molecules had effects on both cell survival and purity of neural differentiation, under conditions of stromal (PA6) cell coculture and feeder‐free floating aggregation culture, for all seven pluripotent stem cell lines that we studied, including three ESC and four iPSC lines. Small molecule compounds are stable and cost effective, so our findings provide a promising strategy for controlled production of neurons in regenerative medicine.

Prolonged maturation culture favors a reduction in the tumorigenicity and the dopaminergic function of human ESC-derived neural cells in a primate model of Parkinson's disease

For the safe clinical application of embryonic stem cells (ESCs) for neurological diseases, it is critical to evaluate the tumorigenicity and function of human ESC (hESC)‐derived neural cells in primates. We have herein, for the first time, compared the growth and function of hESC‐derived cells with different stages of neural differentiation implanted in the brains of primate models of Parkinsons disease. We herein show that residual undifferentiated cells expressing ESC markers present in the cell preparation can induce tumor formation in the monkey brain. In contrast, a cell preparation matured by 42‐day culture with brain‐derived neurotrophic factor/glial cell line‐derived neurotrophic factor (BDNF/GDNF) treatment did not form tumors and survived as primarily dopaminergic (DA) neurons. In addition, the monkeys with such grafts showed behavioral improvement for at least 12 months. These results support the idea that hESCs, if appropriately matured, can serve as a source for DA neurons without forming any tumors in a primate brain. STEM CELLS 2012;30:935–945

Survival of human induced pluripotent stem cell-derived midbrain dopaminergic neurons in the brain of a primate model of Parkinson's disease.

Before induced pluripotent stem cells (iPSCs) can be used to treat neurologic diseases, human iPSC-derived neural cells must be analyzed in the primate brain. In fact, although mouse and human iPSCs have been used to generate dopaminergic (DA) neurons that are beneficial in rat models of Parkinsons disease (PD), human iPSC-derived neural progenitor cells (NPCs) have not been examined in primate brains. Here, we generated NPCs at different stages of predifferentiation using a feeder-free culture method, and grafted them into the brains of a monkey PD model and NOD-SCID mice. Magnetic resonance imaging (MRI), positron emission tomography (PET), immunocytochemistry, and behavioral analyses revealed that NPCs pretreated with Sonic hedgehog and fibroblast growth factor-8 followed by glial cell-derived neurotrophic factor, brain-derived neurotrophic factor, ascorbic acid, and dibutyryl cyclic AMP resulted in smaller grafts than those without these treatments, and survived as DA neurons in a monkey brain as long as six months. Thus, for the first time, we describe a feeder-free neural differentiation method from human iPSCs and an evaluation system that can be used to assess monkey PD models.

Human iPS cell-derived dopaminergic neurons function in a primate Parkinson’s disease model

Induced pluripotent stem cells (iPS cells) are a promising source for a cell-based therapy to treat Parkinson’s disease (PD), in which midbrain dopaminergic neurons progressively degenerate. However, long-term analysis of human iPS cell-derived dopaminergic neurons in primate PD models has never been performed to our knowledge. Here we show that human iPS cell-derived dopaminergic progenitor cells survived and functioned as midbrain dopaminergic neurons in a primate model of PD (Macaca fascicularis) treated with the neurotoxin MPTP. Score-based and video-recording analyses revealed an increase in spontaneous movement of the monkeys after transplantation. Histological studies showed that the mature dopaminergic neurons extended dense neurites into the host striatum; this effect was consistent regardless of whether the cells were derived from patients with PD or from healthy individuals. Cells sorted by the floor plate marker CORIN did not form any tumours in the brains for at least two years. Finally, magnetic resonance imaging and positron emission tomography were used to monitor the survival, expansion and function of the grafted cells as well as the immune response in the host brain. Thus, this preclinical study using a primate model indicates that human iPS cell-derived dopaminergic progenitors are clinically applicable for the treatment of patients with PD.

Purification of functional human ES and iPSC-derived midbrain dopaminergic progenitors using LRTM1

Human induced pluripotent stem cells (iPSCs) can provide a promising source of midbrain dopaminergic (mDA) neurons for cell replacement therapy for Parkinsons disease (PD). However, iPSC-derived donor cells inevitably contain tumorigenic or inappropriate cells. To eliminate these unwanted cells, cell sorting using antibodies for specific markers such as CORIN or ALCAM has been developed, but neither marker is specific for ventral midbrain. Here we employ a double selection strategy for cells expressing both CORIN and LMX1A::GFP, and report a cell surface marker to enrich mDA progenitors, LRTM1. When transplanted into 6-OHDA-lesioned rats, human iPSC-derived LRTM1+ cells survive and differentiate into mDA neurons in vivo, resulting in a significant improvement in motor behaviour without tumour formation. In addition, there was marked survival of mDA neurons following transplantation of LRTM1+ cells into the brain of an MPTP-treated monkey. Thus, LRTM1 may provide a tool for efficient and safe cell therapy for PD patients.

MHC matching improves engraftment of iPSC-derived neurons in non-human primates

The banking of human leukocyte antigen (HLA)-homozygous-induced pluripotent stem cells (iPSCs) is considered a future clinical strategy for HLA-matched cell transplantation to reduce immunological graft rejection. Here we show the efficacy of major histocompatibility complex (MHC)-matched allogeneic neural cell grafting in the brain, which is considered a less immune-responsive tissue, using iPSCs derived from an MHC homozygous cynomolgus macaque. Positron emission tomography imaging reveals neuroinflammation associated with an immune response against MHC-mismatched grafted cells. Immunohistological analyses reveal that MHC-matching reduces the immune response by suppressing the accumulation of microglia (Iba-1+) and lymphocytes (CD45+) into the grafts. Consequently, MHC-matching increases the survival of grafted dopamine neurons (tyrosine hydroxylase: TH+). The effect of an immunosuppressant, Tacrolimus, is also confirmed in the same experimental setting. Our results demonstrate the rationale for MHC-matching in neural cell grafting to the brain and its feasibility in a clinical setting.Major histocompatibility complex (MHC) matching improves graft survival rates after organ transplantation. Here the authors show that in macaques, MHC-matched iPSC-derived neurons provide better engraftment in the brain, with a lower immune response and higher survival of the transplanted neurons.

Idiopathic Parkinson's disease patient‐derived induced pluripotent stem cells function as midbrain dopaminergic neurons in rodent brains

Patient‐specific induced pluripotent stem cells (iPSCs) are a promising source for cell transplantation therapy. In Parkinsons disease (PD) patients, however, their vulnerability and the transmission of pathological α‐Synuclein are possible drawbacks that may prevent PD‐specific iPSCs (PDiPSCs) from being used in clinical settings. In this study, we generated iPSCs from idiopathic PD patients and found that there was no significant vulnerability between dopaminergic (DA) neurons generated from healthy individuals and idiopathic PD patients. PDiPSC‐derived DA neurons survived and functioned in the brains of PD model rats. In addition, in the brains of α‐Synuclein transgenic mice, PDiPSC‐derived DA neurons did not cause pathological α‐Synuclein accumulation in the host brain or in the grafts. These results suggested that iPSCs derived from idiopathic PD patients are feasible as donor cells for autologous transplantation to treat PD.

X-linked severe combined immunodeficiency (X-SCID) rats for xeno-transplantation and behavioral evaluation

BACKGROUND To evaluate the in vivo function of human dopaminergic (DA) neurons, Parkinsons disease (PD) model rats made by the hemi-lateral injection of 6-hydroxydopamine (6-OHDA) are widely used as host animals. In the case of such xeno-transplantation, however, immunosuppression is needed for good survival of the grafted cells. NEW METHODS In order to determine whether human mature neurons can survive in X-linked severe combined immunodeficiency (X-SCID) rats without immunosuppression, we grafted human embryonic stem cell (ESC)-derived DA neurons into the striatum of X-SCID rats. We next treated the X-SCID rats with 6-OHDA and grafted mouse fetal DA neurons or human induced pluripotent stem cell (iPSC)-derived DA neurons to examine whether these rats can be used as PD model rats. RESULTS X-SCID rats did not elicit immune responses against human ESC-derived DA neurons and consequently resulted in good survival of the cells without immunosuppression. Furthermore, 6-OHDA-lesioned X-SCID rats exhibited rotational behavior, which was recovered by grafting mouse fetal DA neurons or human iPSC-derived DA neurons. COMPARISON WITH EXISTING METHODS Immunosuppression by drugs such as Cyclosporine A requires daily injection, which is stressful for rats and moreover may cause renal or hepatic failure. Furthermore, blood levels of the drug may not be stable, which weakens the reliability of the data. CONCLUSIONS Our results provide a more accessible and reliable method to evaluate the in vivo function of human DA neurons, potentially offering a pre-clinical study for the application of pluripotent stem cells.