Masahiko Abematsu

Neurons derived from transplanted neural stem cells restore disrupted neuronal circuitry in a mouse model of spinal cord injury

The bodys capacity to restore damaged neural networks in the injured CNS is severely limited. Although various treatment regimens can partially alleviate spinal cord injury (SCI), the mechanisms responsible for symptomatic improvement remain elusive. Here, using a mouse model of SCI, we have shown that transplantation of neural stem cells (NSCs) together with administration of valproic acid (VPA), a known antiepileptic and histone deacetylase inhibitor, dramatically enhanced the restoration of hind limb function. VPA treatment promoted the differentiation of transplanted NSCs into neurons rather than glial cells. Transsynaptic anterograde corticospinal tract tracing revealed that transplant-derived neurons reconstructed broken neuronal circuits, and electron microscopic analysis revealed that the transplant-derived neurons both received and sent synaptic connections to endogenous neurons. Ablation of the transplanted cells abolished the recovery of hind limb motor function, confirming that NSC transplantation directly contributed to restored motor function. These findings raise the possibility that epigenetic status in transplanted NSCs can be manipulated to provide effective treatment for SCI.

Treatment of a Mouse Model of Spinal Cord Injury by Transplantation of Human Induced Pluripotent Stem Cell-Derived Long-Term Self-Renewing Neuroepithelial-Like Stem Cells†‡§

Because of their ability to self‐renew, to differentiate into multiple lineages, and to migrate toward a damaged site, neural stem cells (NSCs), which can be derived from various sources such as fetal tissues and embryonic stem cells, are currently considered to be promising components of cell replacement strategies aimed at treating injuries of the central nervous system, including the spinal cord. Despite their efficiency in promoting functional recovery, these NSCs are not homogeneous and possess variable characteristics depending on their derivation protocols. The advent of induced pluripotent stem (iPS) cells has provided new prospects for regenerative medicine. We used a recently developed robust and stable protocol for the generation of long‐term, self‐renewing, neuroepithelial‐like stem cells from human iPS cells (hiPS‐lt‐NES cells), which can provide a homogeneous and well‐defined population of NSCs for standardized analysis. Here, we show that transplanted hiPS‐lt‐NES cells differentiate into neural lineages in the mouse model of spinal cord injury (SCI) and promote functional recovery of hind limb motor function. Furthermore, using two different neuronal tracers and ablation of the transplanted cells, we revealed that transplanted hiPS‐lt‐NES cell‐derived neurons, together with the surviving endogenous neurons, contributed to restored motor function. Both types of neurons reconstructed the corticospinal tract by forming synaptic connections and integrating neuronal circuits. Our findings indicate that hiPS‐lt‐NES transplantation represents a promising avenue for effective cell‐based treatment of SCI. STEM CELLS2012;30:1163–1173

Epigenetic regulation in neural stem cell differentiation

The central nervous system (CNS) is composed of three major cell types – neurons, astrocytes, and oligodendrocytes – which differentiate from common multipotent neural stem cells (NSCs). This differentiation process is regulated spatiotemporally during the course of mammalian development. It is becoming apparent that epigenetic regulation is an important cell‐intrinsic program, which can interact with transcription factors and environmental cues to modulate the differentiation of NSCs. This knowledge is important given the potential of NSCs to produce specific CNS cell types that will be beneficial for clinical applications. Here we review recent findings that address molecular mechanisms of epigenetic and transcription factor‐mediated regulation that specify NSC fate during CNS development, with a particular focus on the developing mammalian forebrain.

Chromatin remodeling in neural stem cell differentiation.

Chromatin remodeling is a dynamic alteration of chromatin structure that regulates several important biological processes. It is brought about by enzymatic activities that catalyze covalent modifications of histone tail or movement of nucleosomes along the DNA, and these activities often require multisubunit protein complexes for its proper functions. In concert with DNA methylation and noncoding RNA-mediated processes, histone modification such as acetylation and methylation regulates gene expression epigenetically, without affecting DNA sequence. Recent advances have revealed that this intrinsic regulation, together with protein complexes such as RE1 silencer of transcription/neuron-restrictive silencer factor (REST/NRSF) and switch/sucrose nonfermentable (SWI/SNF), play critical roles in neural stem cell fate determination.

Accumulation of p62 in degenerated spinal cord under chronic mechanical compression: Functional analysis of p62 and autophagy in hypoxic neuronal cells

Intracellular accumulation of altered proteins, including p62 and ubiquitinated proteins, is the basis of most neurodegenerative disorders. The relationship among the accumulation of altered proteins, autophagy, and spinal cord dysfunction by cervical spondylotic myelopathy has not been clarified. We examined the expression of p62 and autophagy markers in the chronically compressed spinal cord of tiptoe-walking Yoshimura mice. In addition, we examined the expression and roles of p62 and autophagy in hypoxic neuronal cells. Western blot analysis showed the accumulation of p62, ubiquitinated proteins, and microtubule-associated protein 1 light chain 3 (LC3), an autophagic marker, in the compressed spinal cord. Immunohistochemical examinations showed that p62 accumulated in neurons, axons, astrocytes, and oligodendrocytes. Electron microscopy showed the expression of autophagy markers, including autolysosomes and autophagic vesicles, in the compressed spinal cord. These findings suggest the presence of p62 and autophagy in the degenerated compressed spinal cord. Hypoxic stress increased the expression of p62, ubiquitinated proteins, and LC3-II in neuronal cells. In addition, LC3 turnover assay and GFP-LC3 cleavage assay showed that hypoxic stress increased autophagy flux in neuronal cells. These findings suggest that hypoxic stress induces accumulation of p62 and autophagy in neuronal cells. The forced expression of p62 decreased the number of neuronal cells under hypoxic stress. These findings suggest that p62 accumulation under hypoxic stress promotes neuronal cell death. Treatment with 3-methyladenine, an autophagy inhibitor decreased the number of neuronal cells, whereas lithium chloride, an autophagy inducer increased the number of cells under hypoxic stress. These findings suggest that autophagy promotes neuronal cell survival under hypoxic stress. Our findings suggest that pharmacological inducers of autophagy may be useful for treating cervical spondylotic myelopathy patients.

Arsenic Trioxide Prevents Osteosarcoma Growth by Inhibition of GLI Transcription via DNA Damage Accumulation

The Hedgehog pathway is activated in various types of malignancies. We previously reported that inhibition of SMO or GLI prevents osteosarcoma growth in vitro and in vivo. Recently, it has been reported that arsenic trioxide (ATO) inhibits cancer growth by blocking GLI transcription. In this study, we analyzed the function of ATO in the pathogenesis of osteosarcoma. Real-time PCR showed that ATO decreased the expression of Hedgehog target genes, including PTCH1, GLI1, and GLI2, in human osteosarcoma cell lines. WST-1 assay and colony formation assay revealed that ATO prevented osteosarcoma growth. These findings show that ATO prevents GLI transcription and osteosarcoma growth in vitro. Flow cytometric analysis showed that ATO promoted apoptotic cell death. Comet assay showed that ATO treatment increased accumulation of DNA damage. Western blot analysis showed that ATO treatment increased the expression of γH2AX, cleaved PARP, and cleaved caspase-3. In addition, ATO treatment decreased the expression of Bcl-2 and Bcl-xL. These findings suggest that ATO treatment promoted apoptotic cell death caused by accumulation of DNA damage. In contrast, Sonic Hedgehog treatment decreased the expression of γH2AX induced by cisplatin treatment. ATO re-induced the accumulation of DNA damage attenuated by Sonic Hedgehog treatment. These findings suggest that ATO inhibits the activation of Hedgehog signaling and promotes apoptotic cell death in osteosarcoma cells by accumulation of DNA damage. Finally, examination of mouse xenograft models showed that ATO administration prevented the growth of osteosarcoma in nude mice. Because ATO is an FDA-approved drug for treatment of leukemia, our findings suggest that ATO is a new therapeutic option for treatment of patients with osteosarcoma.

Basic fibroblast growth factor endows dorsal telencephalic neural progenitors with the ability to differentiate into oligodendrocytes but not γ-aminobutyric acidergic neurons

Basic fibroblast growth factor (bFGF) is commonly used to enrich and maintain neural stem cells in vitro. Olig2 is an essential transcription factor for oligodendrocyte lineage specification and is expressed predominantly in ventral neuroepithelial cells in the medial and lateral ganglionic eminence (GE), where oligodendrocyte progenitors originate. Here we report significant induction of Olig2 expression in dorsal neuroepithelium‐derived cells cultured in the presence of bFGF, in which Olig2‐expressing cells were initially negligible. Among Olig2‐expressing cells appearing after a 5‐day treatment with bFGF, 99.8% coexpressed nestin. There was no significant difference in proliferation or apoptosis in dorsal and ventral neuroepithelial cultures in the presence of bFGF, suggesting that bFGF induces ectopic expression of Olig2 in dorsal “cortical” neuroepithelial cells. Similarly, expression of Mash1, another ventral neuroepithelial cell marker gene, was also induced in cultured dorsal neuroepithelial cells in the presence of bFGF. Conversely, in this culture, expression of dorsal neuroepithelial cell markers, such as Neurogenin1, Neurogenin2, Pax6, and Emx2, was down‐regulated. These results suggested a possible ventralizing activity of bFGF. In fact, bFGF‐treated dorsal neuroepithelial cells acquired the potential to generate O4‐positive oligodendrocytes with efficacy comparable to that observed with GE‐derived cells. In marked contrast, bFGF did not enable dorsal neuroepithelial cells to generate γ‐aminobutyric acid (GABA) neurons, which normally develop only from GE in vivo. Thus, bFGF endows dorsal telencephalic neural progenitors with the ability to differentiate into oligodendrocytes but not GABAergic neurons, suggesting the presence of different mechanisms governing specification of dorsoventral cell identities of neuronal and glial cell lineages.

GLI2 is a novel therapeutic target for metastasis of osteosarcoma.

Aberrant activation of the Hedgehog (Hh) pathway has been reported in several malignancies. We previously demonstrated that knockdown of GLI2 inhibited proliferation of osteosarcoma cells through regulation of the cell cycle. In this study, we analyzed the function of GLI2 in the pathogenesis of osteosarcoma metastasis. Immunohistochemical studies showed that GLI2 was overexpressed in patient osteosarcoma specimens. Knockdown of GLI2 inhibited migration and invasion of osteosarcoma cells. In contrast, the forced expression of constitutively active GLI2 in mesenchymal stem cells promoted invasion. In addition, xenograft models showed that knockdown of GLI2 decreased lung metastasis of osteosarcomas. To examine clinical applications, we evaluated the efficacy of arsenic trioxide (ATO), which is a Food and Drug Administration‐approved antitumor drug, on osteosarcoma cells. ATO treatment suppressed the invasiveness of osteosarcoma cells by inhibiting the transcriptional activity of GLI2. In addition, the combination of Hh inhibitors including ATO, vismodegib and GANT61 prevented migration and metastasis of osteosarcoma cells. Consequently, our findings suggested that GLI2 regulated metastasis as well as the progression of osteosarcomas. Inhibition of the GLI2 transcription may be an effective therapeutic method for preventing osteosarcoma metastasis.

Induction of superficial cortical layer neurons from mouse embryonic stem cells by valproic acid

Within the developing mammalian cortex, neural progenitors first generate deep-layer neurons and subsequently more superficial-layer neurons, in an inside-out manner. It has been reported recently that mouse embryonic stem cells (mESCs) can, to some extent, recapitulate cortical development in vitro, with the sequential appearance of neurogenesis markers resembling that in the developing cortex. However, mESCs can only recapitulate early corticogenesis; superficial-layer neurons, which are normally produced in later developmental periods in vivo, are under-represented. This failure of mESCs to reproduce later corticogenesis in vitro implies the existence of crucial factor(s) that are absent or uninduced in existing culture systems. Here we show that mESCs can give rise to superficial-layer neurons efficiently when treated with valproic acid (VPA), a histone deacetylase inhibitor. VPA treatment increased the production of Cux1-positive superficial-layer neurons, and decreased that of Ctip2-positive deep-layer neurons. These results shed new light on the mechanisms of later corticogenesis.

Mechanisms of Neural Stem Cell Fate Determination: Extracellular Cues and Intracellular Programs

Multipotent neural stem cells (NSC) possess the ability to self-renew and to generate the three major central nervous system (CNS) cell types: neurons, astrocytes and oligodendrocytes. However, the molecular mechanisms that control NSC fate specification are not yet fully understood. Recent studies have provided evidence that soluble protein mediators such as cytokines and transcriptional factors play critical roles in cell fate determination. Furthermore, it has become apparent that epigenetic gene regulation plays an important intracellular role as cell-intrinsic programs in the specification of cell lineages. In this review, we focus on recent progress that addresses the mechanisms of NSC fate specification and their possible contribution in the field of regenerative medicine.