Hayato Fukusumi

Feeder-Free Generation and Long-Term Culture of Human Induced Pluripotent Stem Cells Using Pericellular Matrix of Decidua Derived Mesenchymal Cells

Human ES cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are usually generated and maintained on living feeder cells like mouse embryonic fibroblasts or on a cell-free substrate like Matrigel. For clinical applications, a quality-controlled, xenobiotic-free culture system is required to minimize risks from contaminating animal-derived pathogens and immunogens. We previously reported that the pericellular matrix of decidua-derived mesenchymal cells (PCM-DM) is an ideal human-derived substrate on which to maintain hiPSCs/hESCs. In this study, we examined whether PCM-DM could be used for the generation and long-term stable maintenance of hiPSCs. Decidua-derived mesenchymal cells (DMCs) were reprogrammed by the retroviral transduction of four factors (OCT4, SOX2, KLF4, c-MYC) and cultured on PCM-DM. The established hiPSC clones expressed alkaline phosphatase, hESC-specific genes and cell-surface markers, and differentiated into three germ layers in vitro and in vivo. At over 20 passages, the hiPSCs cultured on PCM-DM held the same cellular properties with genome integrity as those at early passages. Global gene expression analysis showed that the GDF3, FGF4, UTF1, and XIST expression levels varied during culture, and GATA6 was highly expressed under our culture conditions; however, these gene expressions did not affect the cells’ pluripotency. PCM-DM can be conveniently prepared from DMCs, which have a high proliferative potential. Our findings indicate that PCM-DM is a versatile and practical human-derived substrate that can be used for the feeder-cell-free generation and long-term stable maintenance of hiPSCs.

A method for efficiently generating neurospheres from human-induced pluripotent stem cells using microsphere arrays.

In vitro, human neural stem cells can be selectively expanded from fetal or adult neural tissues as neurospheres consisting of immature neural progenitor cells. Access to human neural tissues is limited, making it difficult to propagate and use primary neural stem or progenitor cells (NSPCs) from human neural tissues (hN-NSPCs). It was recently demonstrated that hN-NSPCs can be differentiated from either human embryonic stem cells (hESC-NSPCs) or human-induced pluripotent stem cells (hiPSC-NSPCs), and that hESC-NSPCs and hiPSC-NSPCs are adaptable, powerful substitutes for hN-NSPCs in both regenerative medicine and pharmacological or neurotoxicological assays. We here describe a new protocol to generate neurospheres consisting of hiPSC-NSPCs using microsphere arrays, the surface of which is modified with polyethylene glycol to render it nonadhesive to cells. Primary hiPSCs treated with noggin formed neurospheres on the microsphere arrays and could be stably propagated as free-floating spheroids. The hiPSC-NSPCs proliferating in these neurospheres were almost identical in phenotype to hN-NSPCs, in both cell-surface marker expression and their ability to differentiate into neuronal cells, although gene expression profiles showed that the hiPSC-NSPCs had higher neural and lower glial gene expression, along with mid-hindbrain-like regional specificity. This convenient propagation protocol can be used to evaluate the neurosphere-forming efficiency of hiPSC clones. This method will support the generation of neurospheres from hESCs and hiPSCs and contribute to the use of hESC-NSPCs and hiPSC-NSPCs in research.

Pathological classification of human iPSC-derived neural stem/progenitor cells towards safety assessment of transplantation therapy for CNS diseases

The risk of tumorigenicity is a hurdle for regenerative medicine using induced pluripotent stem cells (iPSCs). Although teratoma formation is readily distinguishable, the malignant transformation of iPSC derivatives has not been clearly defined due to insufficient analysis of histology and phenotype. In the present study, we evaluated the histology of neural stem/progenitor cells (NSPCs) generated from integration-free human peripheral blood mononuclear cell (PBMC)-derived iPSCs (iPSC-NSPCs) following transplantation into central nervous system (CNS) of immunodeficient mice. We found that transplanted iPSC-NSPCs produced differentiation patterns resembling those in embryonic CNS development, and that the microenvironment of the final site of migration affected their maturational stage. Genomic instability of iPSCs correlated with increased proliferation of transplants, although no carcinogenesis was evident. The histological classifications presented here may provide cues for addressing potential safety issues confronting regenerative medicine involving iPSCs.

Establishment of Human Neural Progenitor Cells from Human Induced Pluripotent Stem Cells with Diverse Tissue Origins

Human neural progenitor cells (hNPCs) have previously been generated from limited numbers of human induced pluripotent stem cell (hiPSC) clones. Here, 21 hiPSC clones derived from human dermal fibroblasts, cord blood cells, and peripheral blood mononuclear cells were differentiated using two neural induction methods, an embryoid body (EB) formation-based method and an EB formation method using dual SMAD inhibitors (dSMADi). Our results showed that expandable hNPCs could be generated from hiPSC clones with diverse somatic tissue origins. The established hNPCs exhibited a mid/hindbrain-type neural identity and uniform expression of neural progenitor genes.

Human Decidua-Derived Mesenchymal Cells Are a Promising Source for the Generation and Cell Banking of Human Induced Pluripotent Stem Cells.

Placental tissue is a biomaterial with remarkable potential for use in regenerative medicine. It has a three-layer structure derived from the fetus (amnion and chorion) and the mother (decidua), and it contains huge numbers of cells. Moreover, placental tissue can be collected without any physical danger to the donor and can be matched with a variety of HLA types. The decidua-derived mesenchymal cells (DMCs) are highly proliferative fibroblast-like cells that express a similar pattern of CD antigens as bone marrow-derived mesenchymal cells (BM-MSCs). Here we demonstrated that induced pluripotent stem (iPS) cells could be efficiently generated from DMCs by retroviral transfer of reprogramming factor genes. DMC-hiPS cells showed equivalent characteristics to human embryonic stem cells (hESCs) in colony morphology, global gene expression profile (including human pluripotent stem cell markers), DNA methylation status of the OCT3/4 and NANOG promoters, and ability to differentiate into components of the three germ layers in vitro and in vivo. The RNA expression of XIST and the methylation status of its promoter region suggested that DMC-iPSCs, when maintained undifferentiated and pluripotent, had three distinct states: (1) complete X-chromosome reactivation, (2) one inactive X-chromosome, or (3) an epigenetic aberration. Because DMCs are derived from the maternal portion of the placenta, they can be collected with the full consent of the adult donor and have considerable ethical advantages for cell banking and the subsequent generation of human iPS cells for regenerative applications.

Small-scale screening of anticancer drugs acting specifically on neural stem/progenitor cells derived from human-induced pluripotent stem cells using a time-course cytotoxicity test

Since the development of human-induced pluripotent stem cells (hiPSCs), various types of hiPSC-derived cells have been established for regenerative medicine and drug development. Neural stem/progenitor cells (NSPCs) derived from hiPSCs (hiPSC-NSPCs) have shown benefits for regenerative therapy of the central nervous system. However, owing to their intrinsic proliferative potential, therapies using transplanted hiPSC-NSPCs carry an inherent risk of undesired growth in vivo. Therefore, it is important to find cytotoxic drugs that can specifically target overproliferative transplanted hiPSC-NSPCs without damaging the intrinsic in vivo stem-cell system. Here, we examined the chemosensitivity of hiPSC-NSPCs and human neural tissue—derived NSPCs (hN-NSPCs) to the general anticancer drugs cisplatin, etoposide, mercaptopurine, and methotrexate. A time-course analysis of neurospheres in a microsphere array identified cisplatin and etoposide as fast-acting drugs, and mercaptopurine and methotrexate as slow-acting drugs. Notably, the slow-acting drugs were eventually cytotoxic to hiPSC-NSPCs but not to hN-NSPCs, a phenomenon not evident in the conventional endpoint assay on day 2 of treatment. Our results indicate that slow-acting drugs can distinguish hiPSC-NSPCs from hN-NSPCs and may provide an effective backup safety measure in stem-cell transplant therapies.

NTCT-07ANTI-GLIOMA ALKYLATING AGENTS SHOW DIFFERENT CHEMOSENSITIVITY AGAINST HUMAN NEURAL PROGENITOR CELLS FROM NEURAL TISSUES AND THOSE FROM iPS CELLS

Anti-glioma alkylating agents, like temozolomide (TMZ) or nimustine (ACNU), show in vitro and in vivo cytotoxicity against various glioma cells. Conventional neurotoxicity assays have been carried out using animal-derived primary neuronal cells or human-derived cell lines to examine cytotoxicity of anti-glioma agents against normal neural cells. However, these assays are not necessarily optimal evaluation system to predict neurotoxicity, and especially cytotoxicity against human-derived normal neuronal cells has not been fully examined. Human neural tissue-derived primary cells are ethically and practically less accessible materials, and those are also very difficult to obtain enough cells for high throughput in vitro screening assays. Recently, it was demonstrated that neural cells can be differentiated from human induced pluripotent stem cells (hiPSCs), and those are adaptable, powerful alternatives for tissue-derived primary cells in neurotoxicological assays. In this study, we generated hiPS-derived neural progenitor cells (hiPS-NPCs), expanded as free-floating spheroids (neurospheres) with self-renewal potential and differentiation capacity restricted to neural lineage (neurons and astrocytes), and examined cytotoxicity of anti-glioma alkylating agents against hiPS-NPCs in comparison with human neural tissue derived NPCs (hN-NPCs). The hiPS-NPCs were almost identical in phenotype to hN-NPCs, in both cell-surface marker expression and their ability to differentiate into neuronal cells, although gene expression profiles showed that the hiPS-NPCs had higher neural and lower glial gene expression, along with mid-hindbrain-like regional specificity. Cytotoxicity assays revealed both hN-NPCs and hiPS-NPCs showed different sensitivity against ACNU and TMZ, and gene expression pattern of O-6-methylguanine-DNA methyltransferase (MGMT) and mismatch repair (MMR)-related genes have been different between two human NPCs. These results suggest that human-derived normal neuronal cells show different chemosensitivity against anti-glioma alkylating agents, and MGMT and/or MMR-related genes might be involved with these properties. Our present findings will provide useful information for understanding of neurotoxicity of anti-glioma agents.