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

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.

Human induced pluripotent stem cell line (ONHi001-A) generated from a patient with infantile neuroaxonal dystrophy having PLA2G6 c.517C > T (p.Q173X) and c.1634A > G (p.K545R) compound heterozygous mutations.

Infantile neuroaxonal dystrophy (INAD) is a rare neurodegenerative disease caused mainly by homozygous or compound heterozygous mutations in the PLA2G6 gene. We generated a human induced pluripotent stem cell (hiPSC) line (ONHi001-A) using fibroblasts derived from a patient with INAD. The patient exhibited c.517C > T (p.Q173X) and c.1634A > G (p.K545R) compound heterozygous mutations in the PLA2G6 gene. This hiPSC line may be useful for studying the pathogenic mechanism underlying INAD.

Human Genomic Safe Harbors and the Suicide Gene-Based Safeguard System for iPSC-Based Cell Therapy.

The use of human induced pluripotent stem cells (hiPSCs) and recent advances in cell engineering have opened new prospects for cell-based therapy. However, there are concerns that must be addressed prior to their broad clinical applications and a major concern is tumorigenicity. Suicide gene approaches could eliminate wayward tumor-initiating cells even after cell transplantation, but their efficacy remains controversial. Another concern is the safety of genome editing. Our knowledge of human genomic safe harbors (GSHs) is still insufficient, making it difficult to predict the influence of gene integration on nearby genes. Here, we showed the topological architecture of human GSH candidates, AAVS1, CCR5, human ROSA26, and an extragenic GSH locus on chromosome 1 (Chr1-eGSH). Chr1-eGSH permitted robust transgene expression, but a 2 Mb-distant gene within the same topologically associated domain showed aberrant expression. Although knockin iPSCs carrying the suicide gene, herpes simplex virus thymidine kinase (HSV-TK), were sufficiently sensitive to ganciclovir in vitro, the resulting teratomas showed varying degrees of resistance to the drug in vivo. Our findings suggest that the Chr1-eGSH is not suitable for therapeutic gene integration and highlight that topological analysis could facilitate exploration of human GSHs for regenerative medicine applications. Our data indicate that the HSV-TK/ganciclovir suicide gene approach alone may be not an adequate safeguard against the risk of teratoma, and suggest that the combination of several distinct approaches could reduce the risks associated with cell therapy. Stem Cells Translational Medicine 2019;8:627&638.

Systemic Intravenous Adoptive Transfer of Autologous Lymphokine-activated αß T-Cells Improves Temozolomide-induced Lymphopenia in Patients with Glioma.

In this clinical study, we investigated the safety and clinical usefulness of systemic adoptive immunotherapy using autologous lymphokine-activated αß T-cells (αß T-cells), combined with standard therapies, in patients with malignant brain tumors. Twenty-three patients with different malignant brain tumors, consisting of 14 treated with temozolomide (TMZ group) and 9 treated without temozolomide (non-TMZ group), received systemic intravenous injections of αß T-cells (mean=10.4 injections/patient for the TMZ group, and 4.78 for the non-TMZ group). No significant adverse effects associated with the αß T-cell injection were observed, and the total lymphocyte count (TLC) improved significantly in the TMZ group after five injections. Furthermore, CD8-positive or T-cell receptor V gamma -positive cells were increased with TLC in three patients with glioblastoma multiforme. These findings suggest that systemic αß T-cell immunotherapy is well tolerated, and may help restore an impaired and imbalanced T-cell immune status, and temozolomide- and/or radiotherapy-induced lymphopenia. Future prospective study is needed to clarify the clinical merits of this immunotherapy.

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.

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.

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.

Isolation and cellular properties of mesenchymal cells derived from the decidua of human term placenta.

The clinical promise of cell-based therapies is generally recognized, and has driven an intense search for good cell sources. In this study, we isolated plastic-adherent cells from human term decidua vera, called decidua-derived-mesenchymal cells (DMCs), and compared their properties with those of bone marrow-derived-mesenchymal stem cells (BM-MSCs). The DMCs strongly expressed the mesenchymal cell marker vimentin, but not cytokeratin 19 or HLA-G, and had a high proliferative potential. That is, they exhibited a typical fibroblast-like morphology for over 30 population doublings. Cells phenotypically identical to the DMCs were identified in the decidua vera, and genotyping confirmed that the DMCs were derived from the maternal components of the fetal adnexa. Flow cytometry analysis showed that the expression pattern of CD antigens on the DMCs was almost identical to that on BM-MSCs, but some DMCs expressed the CD45 antigen, and over 50% of them also expressed anti-fibroblast antigen. In vitro, the DMCs showed good differentiation into chondrocytes and moderate differentiation into adipocytes, but scant evidence of osteogenesis, compared with the BM-MSCs. Gene expression analysis showed that, compared with BM-MSCs, the DMCs expressed higher levels of TWIST2 and RUNX2 (which are associated with early mesenchymal development and/or proliferative capacity), several matrix metalloproteinases (MMP1, 3, 10, and 12), and cytokines (BMP2 and TGFB2), and lower levels of MSX2, interleukin 26, and HGF. Although DMCs did not show the full multipotency of BM-MSCs, their higher proliferative ability indicates that their cultivation would require less maintenance. Furthermore, the use of DMCs avoids the ethical concerns associated with the use of embryonic tissues, because they are derived from the maternal portion of the placenta, which is otherwise discarded. Thus, the unique properties of DMCs give them several advantages for clinical use, making them an interesting and attractive alternative to MSCs for regenerative medicine.

ABCB1 is predominantly expressed in human fetal neural stem/progenitor cells at an early development stage.

ABCB1 is a human ABC transporter originally characterized by its ability to cause resistance to chemotherapy drugs in cancer cells, and later found to be functionally expressed in human neural stem/progenitor cells (NSPCs) in vitro. Here, we performed a detailed examination of ABCB1's expression on human NSPCs in vitro and in human fetal brain tissues, and analyzed the cellular properties of the human NSPCs expressing ABCB1. We confirmed that ABCB1 was expressed on the surface of human NSPCs, and its level correlated well with those of Nestin and CD133. The population of fluorescence-activated cell sorter-sorted human NSPCs expressing high levels of ABCB1 showed enrichment of proliferating cells, higher expression of 246 genes (e.g., RGS6, IGFBP7, GFAP, TNC, Hes1), and lower expression of 71 genes (e.g., STMN2, DLX5, BASP1, DCX, CD24) compared with human NSPCs expressing low or no ABCB1. In situ, ABCB1 was selectively expressed in cells in the ventricular or subventricular regions of lateral ventricles that expressed Nestin in human development. These findings suggest that ABCB1 is predominantly expressed in immature human fetal NSPCs in vitro and at early developmental stages in vivo, and that it may be a useful marker for human NSPCs.

Expression of the Neural RNA-binding protein Musashi1 in pediatric brain tumors.

Musashi1 (MSI1) is an evolutionarily conserved RNA-binding protein, selectively expressed in neural stem cells (NSCs) and considered a versatile marker for normal NSCs and tumor cell diagnosis. Here, we examined MSI1 expression in primary pediatric brain tumors, medulloblastomas and ependymomas, by double immunostaining with lineage phenotypic markers (Lin). These tumors highly express MSI1 and are heterogeneous, containing both MSI1+/Lin- tumor cells in regions of relatively high cellularity and proliferative activity and MSI1+/Lin+ tumor cells in regions of lower cellularity. These findings suggest that MSI1 may be a useful marker for characterizing tumor heterogeneity and for examining in situ the analogy between normal NSCs and MSI1+ cells in pediatric brain tumors. This test could be easily applied to routine clinical diagnosis.

A novel marker for Purkinje cells, KIAA0864 protein. An analysis based on a monoclonal antibody HFB-16 in developing human cerebellum.

In the search for immunohistochemical markers of the developing human brain, a monoclonal antibody, HFB-16, was raised against homogenates from the cerebrum of a 15-gestational-week-old (GW) human fetus and screened on paraffin-embedded human embryonic brain specimens. This antibody was particularly useful as a marker for Purkinje cells in the developing human cerebellum. Positive immunoreactivities with HFB-16 first appeared in the Purkinje cell layer at 17 GW. From 20 to 24 GW, positive immunoreactivities were found above the lamina dissecans. After 25 GW, dendrites of Purkinje cells were found with the HFB-16 antibody, and the nerve fibers of the Purkinje cells became positive after 35 GW. Neurons in the dentate nucleus and external and internal granular layers reacted negatively to this antibody. After 1 year, when the external granular layer faded out, the dendrites of the Purkinje cells reached the pial surface of the cerebellum, and nerve fibers began to develop in the white matter. This antibody was also useful for characterization of components in heterotopic neurons found in various anomaly syndromes such as trisomy 13. Expressional cloning indicated the antigen against HFB-16 to be human KIAA0864 protein, which is supposed to be an alternative splicing product of p116Rip, whose function has not yet been elucidated. The antigenicity of the KIAA0864 protein was confirmed using human cDNA of the KIAA0864 protein, a protein expression vector, and an HFB-16 antibody.

In Vitro Screening of Exogenous Factors for Human Neural Stem/Progenitor Cell Proliferation Using Measurement of Total ATP Content in Viable Cells.

One of the newest and most promising methods for treating intractable neuronal diseases and injures is the transplantation of ex vivo-expanded human neural stem/progenitor cells (NSPCs). Human NSPCs are selectively expanded as free-floating neurospheres in serum-free culture medium containing fibroblast growth factor 2 (FGF2) and/or epidermal growth factor (EGF); however, the culture conditions still need to be optimized for performance and cost before the method is used clinically. Here, to improve the NSPC culture method for clinical use, we used an ATP assay to screen the effects of various reagents on human NSPC proliferation. Human NSPCs responded to EGF, FGF2, and leukemia inhibitory factor (LIF) in a dose-dependent manner, and the minimum concentrations eliciting maximum effects were 10 ng/ml EGF, 10 ng/ml FGF2, and 5 ng/ml LIF. EGF and LIF were stable in culture medium without NSPCs, although FGF2 was degraded. In the presence of human NSPCs, however, FGF2 and LIF were both degraded very rapidly, to below the estimated minimum concentration on day 3, but EGF remained above the minimum concentration for 5 days. Adding supplemental doses of each growth factor during the incubation promoted human NSPC proliferation. Among other supplements, insulin and transferrin promoted human NSPC growth, but progesterone, putrescine, selenite, D-glucose, and lactate were not effective and were cytotoxic at higher concentrations. Supplementing with conditioned medium from human NSPCs significantly increased human NSPC proliferation, but using a high percentage of the medium had a negative effect. These findings suggest that human NSPC culture is regulated by a balance in the culture medium between decreasing growth factor levels and increasing positive or negative factors derived from the NSPCs. Thus, in designing culture conditions for human NSPCs, it is useful to take the individual properties of each factor into consideration.

In vitro screening of exogenous factors for human neural stem/progenitor cell proliferation using measurement of total ATP content in viable cells.

One of the newest and most promising methods for treating intractable neuronal diseases and injures is the transplantation of ex vivo-expanded human neural stem/progenitor cells (NSPCs). Human NSPCs are selectively expanded as free-floating neurospheres in serum-free culture medium containing fibroblast growth factor 2 (FGF2) and/or epidermal growth factor (EGF); however, the culture conditions still need to be optimized for performance and cost before the method is used clinically. Here, to improve the NSPC culture method for clinical use, we used an ATP assay to screen the effects of various reagents on human NSPC proliferation. Human NSPCs responded to EGF, FGF2, and leukemia inhibitory factor (LIF) in a dose-dependent manner, and the minimum concentrations eliciting maximum effects were 10 ng/ml EGF, 10 ng/ ml FGF2, and 5 ng/ml LIF. EGF and LIF were stable in culture medium without NSPCs, although FGF2 was degraded. In the presence of human NSPCs, however, FGF2 and LIF were both degraded very rapidly, to below the estimated minimum concentration on day 3, but EGF remained above the minimum concentration for 5 days. Adding supplemental doses of each growth factor during the incubation promoted human NSPC proliferation. Among other supplements, insulin and transferrin promoted human NSPC growth, but progesterone, putrescine, selenite, D-glucose, and lactate were not effective and were cytotoxic at higher concentrations. Supplementing with conditioned medium from human NSPCs significantly increased human NSPC proliferation, but using a high percentage of the medium had a negative effect. These findings suggest that human NSPC culture is regulated by a balance in the culture medium between decreasing growth factor levels and increasing positive or negative factors derived from the NSPCs. Thus, in designing culture conditions for human NSPCs, it is useful to take the individual properties of each factor into consideration.

Expression of tubulin beta II in neural stem/progenitor cells and radial fibers during human fetal brain development.

Recent studies revealed that the "radial glia" in fetal rodent brains are dividing neuronal precursor cells. However, in fetal primate brains, this issue remains unclear, with previous reports indicating that radial glia are a specialized form of astroglia. To investigate the relationship between radial fibers (RFs) and neural stem/progenitor cells in the fetal human brain, we generated polyclonal antibodies to human nestin protein and developed a new mAb, KNY-379, by screening for antibodies that immunostained RFs on paraffin-embedded human fetal brain specimens (12 gestational weeks). The immunostaining for KNY-379 antigen and nestin was seen over the RFs in brains at 8 gestational weeks. Furthermore, KNY-379 antigen and nestin were also detected in human neural stem/progenitor cells in neurosphere cultures. At 12 to 15 gestational weeks, the KNY-379 immunostaining of RFs remained in the periventricular zone and the deep part of the intermediate zone, but it also appeared in outgrowing axons in the cortical plate, in the superficial portion of the intermediate zone, and in apical dendrites in the molecular layer. In the later stages of fetal development (18-40 gestational weeks), this antigen remained in the outgrowing axons and dendrites, but was no longer associated with RFs. Expression cloning and immunoblot analysis demonstrated the antigen to be tubulin beta II, which would thus be a good marker for studying RFs and neural stem/progenitor cells in the early developing human brain.

Evaluation of in vitro proliferative activity of human fetal neural stem/progenitor cells using indirect measurements of viable cells based on cellular metabolic activity.

To scale up human neural stem/progenitor cell (NSPC) cultures for clinical use, we need to know how long these cells can live ex vivo without losing their ability to proliferate and differentiate; thus, a convenient method is needed to estimate the proliferative activity of human NSPCs grown in neurosphere cultures, as direct cell counting is laborious and potentially inaccurate. Here, we isolated NSPCs from human fetal forebrain and prepared neurosphere cultures. We determined the number of viable cells and estimated their proliferative activity in long-term culture using two methods that measure viable cell numbers indirectly, based on their metabolic activity: the WST-8 assay, in which a formazan dye is produced upon reduction of the water-soluble tetrazolium salt WST-8 by dehydrogenase activity, and the ATP assay, which measures the ATP content of the total cell plasma. We compared the results of these assays with the proliferative activity estimated by DNA synthesis using the 5-bromo-2'-deoxyuridine incorporation assay. We found the numbers of viable human NSPCs to be directly proportional to the metabolic reaction products obtained in the WST-8 and ATP assays. Both methods yielded identical cell growth curves, showing an exponentially proliferative phase and a change in the population doubling time in long-term culture. They also showed that human NSPCs could be expanded for up to 200 days ex vivo without losing their ability to proliferate and differentiate. Our findings indicated that indirect measurements of viable cells based on metabolic activity, especially the ATP assay, are very effective and reproducible ways to determine the numbers of viable human NSPCs in intact neurospheres.