Details of a Researcher - Okano, Hideyuki

Papers - Okano, Hideyuki

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Establishment of graded spinal cord injury model in a nonhuman primate

Iwanami A., Yamane J., Katoh H., Nakamura M., Momoshima S., Ishii H., Tanioka Y., Tamaoki N., Nomura T., Toyama Y., Okano Hideyuki

Journal of Neuroscience Research 80 ( 2 ) 172 - 181 2005.04

ISSN 0360-4012

Most previous studies on spinal cord injury (SCI) have used rodent models. Direct extrapolation of the results obtained in rodents to clinical cases is difficult, however, because of neurofunctional and anatomic differences between rodents and primates. In the present study, the development of histopathologic changes and functional deficits were assessed quantitatively after mild, moderate, and severe spinal cord contusive injuries in common marmosets. Contusive SCI was induced by dropping one of three different weights (15, 17, or 20 g) at the C5 level from a height of 50 mm. Serial magnetic resonance images showed significant differences in the intramedullary T1 low signal and T2 high signal areas among the three groups. Quantitative histologic analyses revealed that the number of motor neurons, the myelinated areas, and the amounts of corticospinal tract fibers decreased significantly as the injury increased in severity. Motor functions were evaluated using the following tests: original behavioral scoring scale, measurements of spontaneous motor activity, bar grip test, and cage-climbing test. Significant differences in all test results were observed among the three groups. Spontaneous motor activities at 10 weeks after injury were closely correlated with the residual myelinated area at the lesion epicenter. The establishment of a reliable nonhuman primate model for SCI with objective functional evaluation methods should become an essential tool for future SCI treatment studies. Quantitative behavioral and histopathologic analyses enabled three distinct grades of injury severity (15-g, 17-g, and 20-g groups) to be characterized with heavier weights producing more serious injuries, and relatively constant behavioral and histopathologic outcomes. © 2005 Wiley-Liss, Inc.

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Functional properties of dopaminergic neurones in the mouse olfactory bulb

Pignatelli Angela, Kobayashi Kazuto, Okano Hideyuki, Belluzzi Ottorino

Journal of Physiology 564 ( 2 ) 501 - 514 2005.04

ISSN 0022-3751

The olfactory bulb of mammals contains a large population of dopaminergic interneurones within the glomerular layer. Dopamine has been shown both in vivo and in vitro to modulate several aspects of olfactory information processing, but the functional properties of dopaminergic neurones have never been described due to the inability to recognize these cells in living preparations. To overcome this difficulty, we used a transgenic mouse strain harbouring an eGFP (enhanced green fluorescent protein) reporter construct under the promoter of tyrosine hydroxylase, the rate-limiting enzyme for cathecolamine synthesis. As a result, we were able to identify dopaminergic neurones (TH-GFP cells) in living preparations and, for the first time, we could study the functional properties of such neurones in the olfactory bulb, in both slices and dissociated cells. The most prominent feature of these cells was the autorhythmicity. In these cells we identified five main voltage-dependent conductances: the two having largest amplitude were a fast transient Na⁺ current and a delayed rectifier K⁺ current. In addition, we observed three smaller inward currents, sustained by Na⁺ ions (persistent type) and by Ca²⁺ ions (LVA and HVA). Using pharmacological tools and ion substitution methods we showed that the pacemaking process is supported by the interplay of the persistent Na⁺ current and of a T-type Ca²⁺ current. We carried out a complete kinetical analysis of the five conductances present in these cells, and developed a Hodgkin-Huxley model of TH-GFP cells, capable of reproducing accurately the properties of living cells, including autorhytmicity, and allowing a precise understanding of the process. © The Physiological Society 2005.

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Involvement of Hu and heterogeneous nuclear ribonucleoprotein K in neuronal differentiation through p21 mRNA post-transcriptional regulation

Yano Masato, Okano Hirotaka J., Okano Hideyuki

Journal of Biological Chemistry 280 ( 13 ) 12690 - 12699 2005.04

ISSN 0021-9258

The Hu family is a group of neuronal RNA-binding proteins required for neuronal differentiation in the developing nervous system. Previously, Hu proteins have been shown to enhance the stabilization and/or translation of target mRNAs, such as p21 (CIP1), by binding to AU-rich elements in untranslated regions (UTRs). In this study, we show that Hu induces p21 expression, cell cycle arrest, and neuronal differentiation in mouse neuroblastoma N1E-115 cells. p21 expression is also up-regulated during Me₂SO-induced differentiation in N1E-115 cells and is controlled by post-transcriptional mechanisms through its 3′-UTR. To investigate the molecular mechanisms of Hu functions, we used a proteomics strategy to isolate Hu-interacting proteins and identified heterogeneous nuclear ribonucleoprotein (hnRNP) K. hnRNP K also specifically binds to CU-rich sequences in p21 mRNA 3′-UTR and represses its translation in both nonneuronal and neuronal cells. Further, using RNA interference experiments, we show that the Hu-p21 pathway contributes to the regulation of neurite outgrowth and proliferation in N1E-115 cells, and this pathway is antagonized by hnRNP K. Our results suggest a model in which the mutually antagonistic action of two RNA-binding proteins, Hu and hnRNP K, control the timing of the switch from proliferation to neuronal differentiation through the post-transcriptional regulation of p21 mRNA. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

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An oncolytic HSV-1 mutant expressing ICP34.5 under control of a nestin promoter increases survival of animals even when symptomatic from a brain tumor

Kambara Hirokazu, Okano Hideyuki, Chiocca E. Antonio, Saeki Yoshinaga

Cancer Research 65 ( 7 ) 2832 - 2839 2005.04

ISSN 0008-5472

Oncolytic herpes simples virus-1 (HSV-1) mutants possessing mutations in the ICP34.5 and ICP6 genes have proven safe through clinical trials. However, ICP34.5-null viruses may grow poorly in cells due to their inability to prevent host-cell shut-off of protein synthesis caused by hyperphosphorylation of eukaryotic initiation factor 2α. To increase tumor selectivity, glioma-selective expression of ICP34.5 in the context of oncolysis may be useful. Malignant gliomas remain an incurable disease. One molecular marker of malignant gliomas is expression of the intermediate filament nestin. Expression of nestin mRNA was confirmed in 6 of 6 human glioma lines and in 3 of 4 primary glioma cells. Normal human astrocytes were negative. A novel glioma-selective HSV-1 mutant (rQNestin34.5) was thus engineered by expressing ICP34.5 under control of a synthetic nestin promoter. Replication, cellular propagation, and cytotoxicity of rQNestin34.5 were significantly enhanced in cultured and primary human glioma cell lines compared with control virus. However, replication, cellular propagation, and cytotoxicity of rQNestin34.5 in normal human astrocytes remained quantitatively similar to that of control virus. In glioma cell lines infected with rQNestin34.5, the level of phospho-eukaryotic initiation factor 2α was lower than that of cells infected by control rHsvQ1, confirming selective ICP34.5 expression in glioma cells. In vivo, rQNestin34.5 showed significantly more potent inhibition of tumor growth compared with control virus. Treatment in the brain tumor model was instituted on animal's display of neurologic symptoms, which usually led to rapid demise. rQNestin34.5 treatment doubled the life span of these animals. These results show that rQNestin34.5 could be a potent agent for the treatment of malignant glioma. ©2005 American Association for Cancer Research.

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

Nakamura Yasuhiro, Yamamoto Munehiko, Oda Eriko, Kanemura Yonehiro, Kodama Eri, Yamamoto Atsuyo, Yamamoto Hideyuki, Miyado Kenji, Okano Hirotaka James, Fukagawa Ryouji, Higaki Koichi, Yamasaki Mami, Okano Hideyuki

Journal of Histochemistry and Cytochemistry 53 ( 4 ) 423 - 430 2005.04

ISSN 0022-1554

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 humain 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.

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Components of the transcriptional mediator complex are required for asymmetric cell division in C. elegans

Yoda Akinori, Kouike Hiroko, Okano Hideyuki, Sawa Hitoshi

Development 132 ( 8 ) 1885 - 1893 2005.04

ISSN 0950-1991

Asymmetric cell division is a fundamental process that produces cellular diversity during development. In C. elegans, the Wnt signaling pathway regulates the asymmetric divisions of a number of cells including the T blast cell. We found that the let-19 and dpy-22 mutants have defects in their T-cell lineage, and lineage analyses showed that the defects were caused by disruption in the asymmetry of the T-cell division. We found that let-19 and dpy-22 encode homologs of the human proteins MEDD13/TRAP240 and MED12/TRAP230, respectively, which are components of the Mediator complex. Mediator is a multi-component complex that can regulate transcription by transducing the signals between activators and RNA polymerase in vitro. We also showed that LET-19 and DPY-22 form a complex in vivo with other components of Mediator, SUR-2/MED23 and LET-425/MED6. In the let-19 and dpy-22 mutants, tlp-1, which is normally expressed asymmetrically between the T-cell daughters through the function of the Wnt pathway, was expressed symmetrically in both daughter cells. Furthermore, we found that the let-19 and dpy-22 mutants were defective in the fusion of the Pn.p cell, a process that is regulated by bar-1/β-catenin. Ectopic cell fusion in bar-1 mutants was suppressed by the let-19 or dpy-22 mutations, while defective cell fusion in let-19 mutants was suppressed by lin-39/Hox mutations, suggesting that let-19 and dpy-22 repress the transcription of lin-39. These results suggest that LET-19 and DPY-22 in the Mediator complex repress the transcription of Wnt target genes.

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Lecithinized brain-derived neurotrophic factor promotes the differentiation of embryonic stem cells in vitro and in vivo

Kitagawa Aki, Nakayama Toshiaki, Takenaga Mitsuko, Matsumoto Kayo, Tokura Yukie, Ohta Yuki, Ichinohe Manabu, Yamaguchi Yoko, Suzuki Noboru, Okano Hideyuki, Igarashi Rie

Biochemical and Biophysical Research Communications 328 ( 4 ) 1051 - 1057 2005.03

ISSN 0006-291X

The addition of lecithin molecules to brain-derived neurotrophic factor (BDNF) has been reported to markedly enhance its pharmacological effect in vivo. In the current study, we show that lecithinized BDNF (PC-BDNF) has a higher affinity than BDNF for neural precursor cells. Although BDNF only slightly increased the expression of the genes for Mash-1, p35, 68 kDa neurofilament, and TrkB receptor, PC-BDNF caused a significant increase in their expression. PC-BDNF also increased the level of neurofilament protein and dramatically increased TrkB mRNA gene expression, which was followed by a sustained activation of the p42/p44 extracellular-regulated kinases. Finally, transplantation of PC-BDNF-treated cells was more effective than BDNF-treated cells at improving impaired motor function caused by spinal cord injury. These findings showed that PC-BDNF has a better potential than BDNF for promoting neural differentiation, partly due to a higher cellular affinity. Furthermore, PC-BDNF-treated cells could be useful for transplantation therapy for central nervous system injuries. © 2005 Elsevier Inc. All rights reserved.

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Loss of imprinting of Igf2 alters intestinal maturation and tumorigenesis in mice

Sakatani Takashi, Kaneda Atsushi, Iacobuzio-Donahue Christine A., Carter Mark G., De Boom Witzel Sten, Okano Hideyuki, Ko Minora S H, Ohlsson Rolf, Longo Dan L., Feinberg Andrew P.

Science 307 ( 5717 ) 1976 - 1978 2005.03

ISSN 0036-8075

Loss of imprinting (LOI) of the insulin-like growth factor II gene (IGF2) is an epigenetic alteration that results in a modest increase in IGF2 expression, and it is present in the normal colonic mucosa of about 30% of patients with colorectal cancer. To investigate its role in intestinal tumorigenesis, we created a mouse model of Igf2 LOI by crossing female H19 ^+/- mice with male Apc^+/Min mice. Mice with LOI developed twice as many intestinal tumors as did control littermates. Notably, these mice also showed a shift toward a less differentiated normal intestinal epithelium, reflected by an increase in crypt length and increased staining with progenitor cell markers. A similar shift in differentiation was seen in the normal colonic mucosa of humans with LOI. Thus, altered maturation of nonneoplastic tissue may be one mechanism by which epigenetic changes affect cancer risk.

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The RNA-binding protein HuD regulates neuronal cell identity and maturation

Akamatsu Wado, Fujihara Hiroaki, Mitsuhashi Takayuki, Yano Masato, Shibata Shinsuke, Hayakawa Yoshika, Okano Hirotaka James, Sakakibara Shin Ichi, Takano Hiroshi, Takano Toshiya, Takahashi Takao, Noda Tetsuo, Okano Hideyuki

Proceedings of the National Academy of Sciences of the United States of America 102 ( 12 ) 4625 - 4630 2005.03

ISSN 0027-8424

Neural Hu proteins (HuB/C/D) are RNA-binding proteins that have been shown to induce neuronal differentiation activity when overexpressed in immature neural progenitor cells or undifferentiated neuronal tumors. Newly generated HuD-deficient mice exhibited a transient impaired-cranial-nerve-development phenotype at an early embryonic stage. Adult HuD-deficient mice exhibited an abnormal hind-limb reflex and poor rotarod performance. Analysis of neurosphere formation revealed that the number and self-renewal capacity of the neural stem/progenitor cells were increased in HuD-deficient mice. HuD-deficient primary neurospheres also generated a smaller number of neurons. Cohort analysis of the cellular proliferative activity by using BrdUrd and iododeoxuridine labeling revealed that the number of differentiating quiescent cells in the embryonic cerebral wall was decreased. Long-term administration of BrdUrd revealed that the number of slowly dividing stem cells in the adult subventricular zone was increased in the HuD-deficient mice. Taken together, the results suggest that HuD is required at multiple points during neuronal development, including negative regulation of proliferative activity and neuronal cell-fate acquisition of neural stem/progenitor cells. © 2005 by The National Academy of Sciences of the USA.

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Hes1-deficient mice show precocious differentiation of Paneth cells in the small intestine

Suzuki Katsumasa, Fukui Hirokazu, Kayahara Takahisa, Sawada Mitsutaka, Seno Hiroshi, Hiai Hiroshi, Kageyama Ryoichiro, Okano Hideyuki, Chiba Tsutomu

Biochemical and Biophysical Research Communications 328 ( 1 ) 348 - 352 2005.03

ISSN 0006-291X

We have previously shown that Hes1 is expressed both in putative epithelial stem cells just above Paneth cells and in the crypt base columnar cells between Paneth cells, while Hes1 is completely absent in Paneth cells. This study was undertaken to clarify the role of Hes1 in Paneth cell differentiation, using Hes1-knockout (KO) newborn (P0) mice. Electron microscopy revealed premature appearance of distinct cells containing cytoplasmic granules in the intervillous region in Hes1-KO P0 mice, whereas those cells were absent in wild-type (WT) P0 mice. In Hes1-KO P0 mice, the gene expressions of cryptdins, exclusively present in Paneth cells, were all enhanced compared with WT P0 mice. Immunohistochemistry demonstrated increased number of both lysozyme-positive and cryptdin-4-positive cells in the small intestinal epithelium of Hes1-KO P0 mice as compared to WT P0 mice. Thus, Hes1 appears to have an inhibitory role in Paneth cell differentiation in the small intestine. © 2005 Elsevier Inc. All rights reserved.

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Leukemia inhibitory factor induces multi-lineage differentiation of adult stem-like cells in kidney via kidney-specific cadherin 16

Hishikawa Keiichi, Marumo Takeshi, Miura Shigeki, Nakanishi Asato, Matsuzaki Yumi, Shibata Katsunori, Kohike Hiroko, Komori Takuya, Hayashi Matsuhiko, Nakaki Toshio, Nakauchi Hiromitsu, Okano Hideyuki, Fujita Toshiro

Biochemical and Biophysical Research Communications 328 ( 1 ) 288 - 291 2005.03

ISSN 0006-291X

Side population (SP) is reported to be a stem cell-rich population. In the presence of leukemia inhibitory factor (LIF), cultured kidney SP cells differentiated into multi-lineage in collagen gel but not in synthesized polymer that has no cell adhesion factor. In cultured kidney SP cells, gene expression of kidney-specific cadherin 16 was specifically upregulated in collagen gel but not in synthesized polymer. Moreover, decreasing cadherin 16 expression using siRNA abolished LIF-induced multi-lineage differentiation of kidney SP in collagen gel. These results indicated that LIF induced multi-lineage differentiation of adult stem-like cells in kidney via cadherin 16. © 2005 Elsevier Inc. All rights reserved.

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Erratum

Sakuragawa Norio, Kakinuma Kenichi, Kikuchi Aiko, Okano Hideyuki, Uchida Saiko, Kamo Isao, Kobayashi Mamoru, Yokoyama Yasunobu

Journal of Neuroscience Research 79 ( 5 ) 725 2005.03

ISSN 0360-4012

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Epigenetics, I GF2 dosage and cancer risk

Kaneda [No Value], Feinberg [No Value], Iacobuzio-Donahue [No Value], Sakatani Takashi, Witzel Sten De Boom, Carter Mark, Ko Minoru, Longo Dan, Okano Hideyuki, Ohlsson [No Value]

Cancer Biology and Therapy 4 ( 3 ) 2005.03

ISSN 1538-4047

Comparative functional genomics revealed conservation and diversification of three enhancers of the isl1 gene for motor and sensory neuron-specific expression

Uemura Osamu, Okada Yohei, Ando Hideki, Guedj Mickael, Higashijima Shin Ichi, Shimazaki Takuya, Chino Naoichi, Okano Hideyuki, Okamoto Hitoshi

Developmental Biology 278 ( 2 ) 587 - 606 2005.02

ISSN 0012-1606

Islet-1 (Isl1) is a member of the Isl1 family of LIM-homeodomain transcription factors (LIM-HD) that is expressed in a defined subset of motor and sensory neurons during vertebrate embryogenesis. To investigate how this specific expression of isl1 is regulated, we searched for enhancers of the isl1 gene that are conserved in vertebrate evolution. Initially, two enhancer elements, CREST1 and CREST2, were identified downstream of the isl1 locus in the genomes of fugu, chick, mouse, and human by BLAST searching for highly similar elements to those originally identified as motor and sensory neuron-specific enhancers in the zebrafish genome. The combined action of these elements is sufficient for completely recapitulating the subtype-specific expression of the isl1 gene in motor neurons of the mouse spinal cord. Furthermore, by direct comparison of the upstream flanking regions of the zebrafish and human isl1 genes, we identified another highly conserved noncoding element, CREST3, and subsequently C3R, a similar element to CREST3 with two CDP CR1 recognition motifs, in the upstream regions of all other isl1 family members. In mouse and human, CRESTs are located as far as more than 300 kb away from the isl1 locus, while they are much closer to the isl1 locus in zebrafish. Although all of zebrafish CREST2, CREST3, and C3R activate gene expression in the sensory neurons of zebrafish, CREST2 of mouse and human does not have the sequence necessary for sensory neuron-specific expression. Our results revealed both a remarkable conservation of the regulatory elements regulating subtype-specific gene expression in motor and sensory neurons and the dynamic process of reorganization of these elements whereby each element increases the level of cell-type specificity by losing redundant functions with the other elements during vertebrate evolution. © 2004 Elsevier Inc. All rights reserved.

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Regenerative medicine for the central nervous system - Transplantation of neural stem cells into the injured spinal cord -

Kaneko Shinjiro, Nakamura Masaya, Ogawa Yuto, Watanabe Kota, Iwanami Akio, Toyama Yoshiaki, Okano Hideyuki

Japan Medical Association Journal 48 ( 2 ) 68 - 80 2005.02

ISSN 1346-8650

Advances in the stem cell biology of the central nervous system (CNS) and other studies enhance the possibility of regeneration of the damaged CNS, which has previously been considered to be almost impossible. The availability of signals to induce the appropriate differentiation of the transplanted and/or endogenous neural stem cells (NSCs) as well as the timing of the transplantation are vital factors for improving the functional recovery of the damaged CNS. Because the immediate post-traumatic microenvironment of the spinal cord is in an acute inflammatory stage, it does not favor the survival and differentiation of the transplants. Furthermore, in the chronic disease stage after injury, glial scars that inhibit the regeneration of neuronal axons form in the injured site. Thus, we consider that the optimal period of transplantation is 1-2 weeks after injury.

The transmembrane protein, Tincar, is involved in the development of the compound eye in Drosophila melanogaster

Hirota Yuki, Sawamoto Kazunobu, Takahashi Kuniaki, Ueda Ryu, Okano Hideyuki

Development Genes and Evolution 215 ( 2 ) 90 - 96 2005.02

ISSN 0949-944X

We previously cloned and characterized the Drosophila gene, tincar (tinc), which encodes a novel protein with eight putative transmembrane domains. Here, we have studied the expression pattern and functions of tinc during developmental processes. Tinc mRNA is expressed in the central and peripheral nervous systems, and midgut during embryogenesis. In the third-instar larval eye disc, tinc mRNA is strongly expressed in all the differentiating ommatidial cells within and in the vicinity of the morphogenetic furrow. Loss-of-function analysis using the RNA-interference method revealed severe defects of eye morphogenesis during the late developmental stages. Our results suggested that tinc may have an indispensable role in the normal differentiation of ommatidial cells. © Springer-Verlag 2005.

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A putative human breast stem cell population is enriched for steroid receptor-positive cells

Clarke Robert B., Spence Katherine, Anderson Elizabeth, Howell Anthony, Okano Hideyuki, Potten Christopher S.

Developmental Biology 277 ( 2 ) 443 - 456 2005.01

ISSN 0012-1606

Breast epithelial stem cells are thought to be the primary targets in the etiology of breast cancer. Since breast cancers mostly express estrogen and progesterone receptor (ERα and PR), we examined the biology of these ERα/PR-positive cells and their relationship to stem cells in normal human breast epithelium. We employed several complementary approaches to identify putative stem cell markers, to characterise an isolated stem cell population and to relate these to cells expressing the steroid receptors ERα and PR. Using DNA radiolabelling in human tissue implanted into athymic nude mice, a population of label-retaining cells were shown to be enriched for the putative stem cell markers p21 ^CIP1 and Msi-1, the human homolog of Drosophila Musashi. Steroid receptor-positive cells were found to co-express these stem cell markers together with cytokeratin 19, another putative stem cell marker in the breast. Human breast epithelial cells with Hoechst dye-effluxing "side population" (SP) properties characteristic of mammary stem cells in mice were demonstrated to be undifferentiated "intermediate" cells by lack of expression of myoepithelial and luminal apical membrane markers. These SP cells were 6-fold enriched for ERα-positive cells and expressed several fold higher levels of the ERα, p21 ^CIP1 and Msi1 genes than non-SP cells. In contrast to non-SP cells, SP cells formed branching structures in matrigel which included cells of both luminal and myoepithelial lineages. The data suggest a model where scattered steroid receptor-positive cells are stem cells that self-renew through asymmetric cell division and generate patches of transit amplifying and differentiated cells. © 2004 Elsevier Inc. All rights reserved.

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Transient inhibition of BMP signaling by Noggin induces cardiomyocyte differentiation of mouse embryonic stem cells

Yuasa Shinsuke, Itabashi Yuji, Koshimizu Uichi, Tanaka Tomofumi, Sugimura Keijiro, Kinoshita Masayoshi, Hattori Fumiyuki, Fukami Shin Ichi, Shimazaki Takuya, Okano Hideyuki, Ogawa Satoshi, Fukuda Keiichi

Nature Biotechnology 23 ( 5 ) 607 - 611 2005

ISSN 1087-0156

Embryonic stem (ES) cells are a promising source of cardiomyocytes, but clinical application of ES cells has been hindered by the lack of reliable selective differentiation methods. Differentiation into any lineage is partly dependent on the regulatory mechanisms of normal early development. Although several signals, including bone morphogenetic protein (BMP), Wnt and FGF, are involved in heart development, scarce evidence is available about the exact signals that mediate cardiomyocyte differentiation. While investigating the involvement of BMP signaling in early heart formation in the mouse, we found that the BMP antagonist Noggin is transiently but strongly expressed in the heart-forming region during gastrulation and acts at the level of induction of mesendoderm to establish conditions conducive to cardiogenesis. We applied this finding to develop an effective protocol for obtaining cardiomyocytes from mouse ES cells by inhibition of BMP signaling.

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Erratum

Yuasa Shinsuke, Itabashi Yuji, Koshimizu Uichi, Tanaka Tomofumi, Sugimura Keijiro, Kinoshita Masayoshi, Hattori Fumiyuki, Fukami Shin Ichi, Shimazaki Takuya, Ogawa Satoshi, Okano Hideyuki, Fukuda Keiichi

Nature Biotechnology 23 ( 7 ) 897 2005

ISSN 1087-0156

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

Kanemura Yonehiro, Mori Hideki, Nakagawa Atsuyo, Islam Mohammed Omedul, Kodama Eri, Yamamoto Atsuyo, Shofuda Tomoko, Kobayashi Satoshi, Miyake Jun, Yamazaki Tomohiko, Hirano Shun Ichiro, Yamasaki Mami, Okano Hideyuki

Cell Transplantation 14 ( 9 ) 673 - 682 2005

ISSN 0963-6897

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. Copyright © 2005 Cognizant Comm. Corp.

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