Haeyoung Suh-Kim

Mesenchymal stem cells promote proliferation of endogenous neural stem cells and survival of newborn cells in a rat stroke model

Mesenchymal stem cells (MSCs) secrete bioactive factors that exert diverse responses in vivo. In the present study, we explored mechanism how MSCs may lead to higher functional recovery in the animal stroke model. Bone marrow-derived MSCs were transplanted into the brain parenchyma 3 days after induction of stroke by occluding middle cerebral artery for 2 h. Stoke induced proliferation of resident neural stem cells in subventricular zone. However, most of new born cells underwent cell death and had a limited impact on functional recovery after stroke. Transplantation of MSCs enhanced proliferation of endogenous neural stem cells while suppressing the cell death of newly generated cells. Thereby, newborn cells migrated toward ischemic territory and differentiated in ischemic boundaries into doublecortin+ neuroblasts at higher rates in animals with MSCs compared to control group. The present study indicates that therapeutic effects of MSCs are at least partly ascribed to dual functions of MSCs by enhancing endogenous neurogenesis and protecting newborn cells from deleterious environment. The results reinforce the prospects of clinical application using MSCs in the treatment of neurological disorders.

Expression of neuroD/BETA2 in mitotic and postmitotic neuronal cells during the development of nervous system†

NeuroD/BETA2, a basic helix‐loop‐helix transcription factor, has been shown to play a role in tissue‐specific differentiation of pancreatic and enteroendocrine cells. To gain further insight into the function of neuroD/BETA2 in the nervous system development, we examined the expression pattern of neuroD/BETA2 during embryonic and postnatal development by using in situ hybridization. Dynamic changes of neuroD/BETA2 expression in the central nervous system were observed during embryogenesis, especially in telencephalon, hippocampus, cerebellum, spinal cord, and olfactory epithelium. Moderate level of expression was also detected in developing pancreas in early embryogenesis. Although the neuroD/BETA2 expression in cerebellum and hippocampus increased over time, expression in cerebral cortex, spinal cord, as well as in fetal pancreas gradually decreased as embryogenesis proceeded. High level of the neuroD/BETA2 expression in developing cerebellum and hippocampus persisted throughout postnatal development and remained at a stable level in the adult brain. Interestingly, neuroD/BETA2 expression was detected not only in postmitotic but also in mitotic cells, as was evident in its expression in external granular layer of cerebellum and granule cells of the dentate gyrus during postnatal development. This observation suggests that neuroD/BETA2 may have a unique role in proliferation, differentiation, or both, of granule cells of cerebellum and dentate gyrus. Dev Den;217:361–367.

Neural Induction with Neurogenin1 Increases the Therapeutic Effects of Mesenchymal Stem Cells in the Ischemic Brain

Mesenchymal stem cells (MSCs) have been shown to ameliorate a variety of neurological dysfunctions. This effect is believed to be mediated by their paracrine functions, since these cells rarely differentiate into neuronal cells. It is of clinical interest whether neural induction of MSCs is beneficial for the replacement therapy of neurological diseases. Here we report that expression of Neurogenin1 (Ngn1), a proneural gene that directs neuronal differentiation of progenitor cells during development, is sufficient to convert the mesodermal cell fate of MSCs into a neuronal one. Ngn1‐expressing MSCs expressed neuron‐specific proteins, including NeuroD and voltage‐gated Ca2+ and Na+ channels that were absent in parental MSCs. Most importantly, transplantation of Ngn1‐expressing MSCs in the animal stroke model dramatically improved motor functions compared with the parental MSCs. MSCs with Ngn1 populated the ischemic brain, where they expressed mature neuronal markers, including microtubule associated protein 2, neurofilament 200, and vesicular glutamate transporter 2, and functionally connected to host neurons. MSCs with and without Ngn1 were indistinguishable in reducing the numbers of Iba1+, ED1+ inflammatory cells, and terminal deoxynucleotidyl transferase dUTP nick‐end labeling+ apoptotic cells and in increasing the numbers of proliferating Ki67+ cells. The data indicate that in addition to the intrinsic paracrine functions of MSCs, motor dysfunctions were remarkably improved by MSCs able to transdifferentiate into neuronal cells. Thus, neural induction of MSCs is advantageous for the treatment of neurological dysfunctions.

Transplantation of human neural stem cells transduced with Olig2 transcription factor improves locomotor recovery and enhances myelination in the white matter of rat spinal cord following contusive injury

BackgroundContusive spinal cord injury is complicated by a delayed loss of oligodendrocytes, resulting in chronic progressive demyelination. Therefore, transplantation strategies to provide oligodendrocyte lineage cells and to enhance the extent of myelination appear to be justified for spinal cord repair. The present study investigated whether transplantation of human neural stem cells (NSCs) genetically modified to express Olig2 transcription factor, an essential regulator of oligodendrocyte development, can improve locomotor recovery and enhance myelination in a rat contusive spinal cord injury model.ResultsHB1.F3 (F3) immortalized human NSC line was transduced with a retroviral vector encoding Olig2, an essential regulator of oligodendrocyte development. Overexpression of Olig2 in human NSCs (F3.Olig2) induced activation of NKX2.2 and directed differentiation of NSCs into oligodendrocyte lineage cells in vitro. Introduction of Olig2 conferred higher proliferative activity, and a much larger number of F3.Olig2 NSCs were detected by 7 weeks after transplantation into contused spinal cord than that of parental F3 NSCs. F3.Olig2 NSCs exhibited frequent migration towards the white matter, whereas F3 NSCs were mostly confined to the gray matter or around the lesion cavities. Most of F3.Olig2 NSCs occupying the spared white matter differentiated into mature oligodendrocytes. Transplantation of F3.Olig2 NSCs increased the volume of spared white matter and reduced the cavity volume. Moreover, F3.Olig2 grafts significantly increased the thickness of myelin sheath around the axons in the spared white matter. Finally, animals with F3.Olig2 grafts showed an improvement in the quality of hindlimbs locomotion.ConclusionTransplantation of NSCs genetically modified to differentiate into an oligodendrocytic lineage may be an effective strategy to improve functional outcomes following spinal cord trauma. The present study suggests that molecular factors governing cell fate decisions can be manipulated to enhance reparative potential of the cell-based therapy.

Immune following suppression mesenchymal stem cell transplantation in the ischemic brain is mediated by TGF-β.

Transplantation of mesenchymal stem cells (MSCs) has been shown to enhance the recovery of brain functions following ischemic injury. Although immune modulation has been suggested to be one of the mechanisms, the molecular mechanisms underlying improved recovery has not been clearly identified. Here, we report that MSCs secrete transforming growth factor-beta (TGF-β) to suppress immune propagation in the ischemic rat brain. Ischemic stroke caused global death of resident cells in the infarcted area, elevated the monocyte chemoattractant protein-1 (MCP-1) level, and evoked massive infiltration of circulating CD68+ immune cells through the impaired blood-brain barrier. Transplantation of MSCs at day 3 post-ischemia blocked the subsequent upregulation of MCP-1 in the ischemic area and the infiltration of additional CD68+ immune cells. MSC-conditioned media decreased the migration and MCP-1 production of freshly isolated immune cells in vitro, and this effect was blocked by an inhibitor of TGF-β signaling or an anti-TGF-β neutralizing antibody. Finally, transplantation of TGF-β1-silenced MSCs failed to attenuate the infiltration of CD68+ cells into the ischemic brain, and was associated with only minor improvements in motor function. These results indicate that TGF-β is key to the ability of MSCs to beneficially attenuate immune reactions in the ischemic brain. Our findings offer insight into the interactions between allogeneic MSCs and the host immune system, reinforcing the prospective clinical value of using MSCs in the treatment of neurological disorders involving inflammation-mediated secondary damage.

Effects of estrogen on lifespan and motor functions in female hSOD1 G93A transgenic mice

Amyotrophic lateral sclerosis (ALS) is a progressive disease which is caused by degeneration of motor neurons in the central nervous system. The incidence of ALS is higher in men than women, but the female advantage disappears with increased age. Here, we report evidence that the female advantage is due to the protective role of estrogen. In an ALS mouse model carrying the human Cu/Zn superoxide dismutase (hSOD1) G93A transgene, ovariectomy did not alter the onset age of the disease while reducing the female lifespan by 7 days and making it comparable to that of the male transgenic mice. Treatment of ovariectomized females with 17beta-estradiol (E2) did not delay the onset of disease, but prevented progression of ALS motor dysfunctions as shown by extension reflex test for a limited time window. Importantly, E2 treatment rescued the lifespans in overiectomized females. These findings will provide important new insights to interpretation of disease progression in post-menopausal female ALS patients.

Id proteins facilitate self-renewal and proliferation of neural stem cells.

Members of helix-loop-helix (HLH) protein family of Id (inhibitor of differentiation) dimerize with bHLH transcription factors and function as negative regulators of differentiation during development. Most of inhibitory roles of Id proteins have been demonstrated in non-neural tissues, and their roles in the developing nervous system are not clearly demonstrated. In this study, we show that Id1, Id2, and Id3 increase self-renewing and proliferation potential of cortical neural stem cells (NSCs) while inhibiting neuronal differentiation. In electrophoretic mobility gel shift and luciferase assays, Id proteins interfered with binding of NeuroD/E47 complexes to the E-box sequences and inhibited E-box-mediated gene expression. Overexpression of Id proteins in NSCs increased both the number and the size of neurospheres in colony-forming assays. Expression of Hes1 and Hes5 was not increased by overexpression of Id proteins under the condition in which Nestin expression was increased. In utero electroporation of Id yielded higher numbers of Ki67-positive and Sox2-positive cells in the mouse embryonic brain. The study suggests Id proteins play independent roles in the maintenance of neural stem properties.

Differential actions of the proneural genes encoding Mash1 and neurogenins in Nurr1-induced dopamine neuron differentiation

The steroid receptor-type transcription factor Nurr1 has a crucial role in the development of the mesencephalic dopamine (DA) neurons. Although ectopic expression of Nurr1 in cultured neural precursor cells is sufficient in establishing the DA phenotype, Nurr1-induced DA cells are morphologically and functionally immature, suggesting the necessity of additional factor(s) for full neuronal differentiation. In this study, we demonstrate that neurogenic basic helix-loop-helix (bHLH) factors Mash1, neurogenins (Ngns) and NeuroD play contrasting roles in Nurr1-induced DA neuronal differentiation. Mash1, but not Ngn2, spatially and temporally colocalized with aldehyde dehydrogenase 2 (AHD2), a specific midbrain DA neuronal progenitor marker, in the early embryonic ventral mesencephalon. Forced expression of Mash1 caused immature Nurr1-induced DA cells to differentiate into mature and functional DA neurons as judged by electrophysiological characteristics, release of DA, and expression of presynaptic DA neuronal markers. By contrast, atonal-related bHLHs, represented by Ngn1, Ngn2 and NeuroD, repressed Nurr1-induced expression of DA neuronal markers. Domain-swapping experiments with Mash1 and NeuroD indicated that the helix-loop-helix domain, responsible for mediating dimerization of bHLH transcription factors, imparts the distinct effect. Finally, transient co-transfection of the atonal-related bHLHs with Nurr1 resulted in an E-box-independent repression of Nurr1-induced transcriptional activation of a reporter containing Nurr1-binding element (NL3) as well as a reporter driven by the native tyrosine hydroxylase gene promoter. Taken together, these findings suggest that Mash1 contributes to the generation of DA neurons in cooperation with Nurr1 in the developing midbrain whereas atonal-related bHLH genes inhibit the process.

Neurite Outgrowth Induced by Cyclic AMP Can Be Modulated by the α Subunit of Go

Abstract: Although abundant Go has been found in nervous tissues and it has been implicated in neuronal differentiation, the mechanism of how Go modulates neuronal differentiation has not been defined. Here, we report that the α subunit of Go (αo) modulates neurite outgrowth by interfering with the signaling pathway initiated by cyclic AMP (cAMP). In F11 cells, cAMP induced neurite outgrowth and activated cAMP‐responsive element binding protein (CREB). Specific inhibition of cAMP‐dependent protein kinase reduced both CREB activity and neurite outgrowth (NOG). Interestingly, cAMP reduced phosphorylation of extracellular signal‐regulated kinase (Erk). Neither a dominant negative form nor an active form of Ras altered neurite outgrowth. Expression of αo (αowt) decreased the average length of neurites but increased the number of neurites per cell. An active mutant, αoQ205L, which lost GTPase activity and thus could not bind to Gβγ, gave similar results, suggesting that the effect of αo is not mediated through Gβγ. Expression of αowt or αoQ205L also prohibited CREB activation. Thus, activation of Erk may not be essential for neuronal differentiation in F11 cells and αo may cause changes in NOG by inhibiting CREB activation.

Hepatocyte growth factor reduces astrocytic scar formation and promotes axonal growth beyond glial scars after spinal cord injury

The formation of glial scars impedes growth of regenerating axons after CNS injuries such as spinal cord injury (SCI). Hepatocyte growth factor (HGF), originally identified as a mitogen for hepatocytes, exerts pleiotropic functions in the nervous system. HGF has been implicated in peripheral wound healing via regulation of the transforming growth factor beta (TGFβ), which is also a potent inducer of glial scar formation in CNS. In the present study, we found that HGF completely blocked secretion of TGFβ1 and β2 from activated astrocytes in culture. HGF also prevented expression of specific chondroitin sulfate proteoglycan (CSPG) species. To determine whether HGF inhibits glial scar formation in an in vivo SCI model, HGF overexpressing mesenchymal stem cells (HGF-MSCs) were transplanted into hemisection spinal cord lesions at C4. Transplantation of HGF-MSCs markedly diminished TGFβ isoform levels and reduced the extent of astrocytic activation. In addition, HGF-MSCs also significantly decreased neurocan expression and glycosaminoglycan chain deposition around hemisection lesions. Furthermore, animals treated with HGF-MSCs showed increased axonal growth beyond glial scars and improvement in recovery of forepaw function. Our results indicate that anti-glial scar effects of HGF, together with its known neurotrophic functions, could be utilized to ameliorate functional deficits following SCI.