Hyun-Chul Koh

In vitro and in vivo analyses of human embryonic stem cell‐derived dopamine neurons

Human embryonic stem (hES) cells, due to their capacity of multipotency and self‐renewal, may serve as a valuable experimental tool for human developmental biology and may provide an unlimited cell source for cell replacement therapy. The purpose of this study was to assess the developmental potential of hES cells to replace the selectively lost midbrain dopamine (DA) neurons in Parkinsons disease. Here, we report the development of an in vitro differentiation protocol to derive an enriched population of midbrain DA neurons from hES cells. Neural induction of hES cells co‐cultured with stromal cells, followed by expansion of the resulting neural precursor cells, efficiently generated DA neurons with concomitant expression of transcriptional factors related to midbrain DA development, such as Pax2, En1 (Engrailed‐1), Nurr1, and Lmx1b. Using our procedure, the majority of differentiated hES cells (> 95%) contained neuronal or neural precursor markers and a high percentage (> 40%) of TuJ1+ neurons was tyrosine hydroxylase (TH)+, while none of them expressed the undifferentiated ES cell marker, Oct 3/4. Furthermore, hES cell‐derived DA neurons demonstrated functionality in vitro, releasing DA in response to KCl‐induced depolarization and reuptake of DA. Finally, transplantation of hES‐derived DA neurons into the striatum of hemi‐parkinsonian rats failed to result in improvement of their behavioral deficits as determined by amphetamine‐induced rotation and step‐adjustment. Immunohistochemical analyses of grafted brains revealed that abundant hES‐derived cells (human nuclei+ cells) survived in the grafts, but none of them were TH+. Therefore, unlike those from mouse ES cells, hES cell‐derived DA neurons either do not survive or their DA phenotype is unstable when grafted into rodent brains.

Protein-based human iPS cells efficiently generate functional dopamine neurons and can treat a rat model of Parkinson disease

Parkinson disease (PD) involves the selective loss of midbrain dopamine (mDA) neurons and is a possible target disease for stem cell-based therapy. Human induced pluripotent stem cells (hiPSCs) are a potentially unlimited source of patient-specific cells for transplantation. However, it is critical to evaluate the safety of hiPSCs generated by different reprogramming methods. Here, we compared multiple hiPSC lines derived by virus- and protein-based reprogramming to human ES cells (hESCs). Neuronal precursor cells (NPCs) and dopamine (DA) neurons delivered from lentivirus-based hiPSCs exhibited residual expression of exogenous reprogramming genes, but those cells derived from retrovirus- and protein-based hiPSCs did not. Furthermore, NPCs derived from virus-based hiPSCs exhibited early senescence and apoptotic cell death during passaging, which was preceded by abrupt induction of p53. In contrast, NPCs derived from hESCs and protein-based hiPSCs were highly expandable without senescence. DA neurons derived from protein-based hiPSCs exhibited gene expression, physiological, and electrophysiological properties similar to those of mDA neurons. Transplantation of these cells into rats with striatal lesions, a model of PD, significantly rescued motor deficits. These data support the clinical potential of protein-based hiPSCs for personalized cell therapy of PD.

Foxa2 and Nurr1 synergistically yield A9 nigral dopamine neurons exhibiting improved differentiation, function, and cell survival.

Effective dopamine (DA) neuron differentiation from neural precursor cells (NPCs) is prerequisite for precursor/stem cell‐based therapy of Parkinsons disease (PD). Nurr1, an orphan nuclear receptor, has been reported as a transcription factor that can drive DA neuron differentiation from non‐dopaminergic NPCs in vitro. However, Nurr1 alone neither induces full neuronal maturation nor expression of proteins found specifically in midbrain DA neurons. In addition, Nurr1 expression is inefficient in inducing DA phenotype expression in NPCs derived from certain species such as mouse and human. We show here that Foxa2, a forkhead transcription factor whose role in midbrain DA neuron development was recently revealed, synergistically cooperates with Nurr1 to induce DA phenotype acquisition, midbrain‐specific gene expression, and neuronal maturation. Thus, the combinatorial expression of Nurr1 and Foxa2 in NPCs efficiently yielded fully differentiated nigral (A9)‐type midbrain neurons with clearly detectable DA neuronal activities. The effects of Foxa2 in DA neuron generation were observed regardless of the brain regions or species from which NPCs were derived. Furthermore, DA neurons generated by ectopic Foxa2 expression were more resistant to toxins. Importantly, Foxa2 expression resulted in a rapid cell cycle exit and reduced cell proliferation. Consistently, transplantation of NPCs transduced with Nurr1 and Foxa2 generated grafts enriched with midbrain‐type DA neurons but reduced number of proliferating cells, and significantly reversed motor deficits in a rat PD model. Our findings can be applied to ongoing attempts to develop an efficient and safe precursor/stem cell‐based therapy for PD. STEM CELLS 2010;28:501–512

Generation of functional dopamine neurons from neural precursor cells isolated from the subventricular zone and white matter of the adult rat brain using nurr1 overexpression

Neural precursor (NP) cells from adult mammalian brains can be isolated, expanded in vitro, and potentially used as cell replacement source material for treatment of intractable brain disorders. Reduced ethical concerns, lack of teratoma formation, and possible ex vivo autologous transplantation are critical advantages to using adult NP donor cells over cells from fetal brain tissue or embryonic stem cells. However, the usage of adult NP cells is limited by the ability to induce specific neurochemical phenotypes in these cells. Here, we demonstrate induction of a dopaminergic phenotype in NP cells isolated from the subventricular zone (SVZ) and white matter of rodent adult brains using overexpression of the nuclear receptor Nurr1 in vitro. Forced expression of Nurr1, a transcriptional factor specific to midbrain dopamine (DA) neuron development, caused in the adult cells an acquisition of the DA neurotransmitter phenotype and sufficient differentiation toward morphologically, phenotypically, and ultrastructurally mature DA neurons. Co‐expression of neurogenic factor Mash1 and treatment with neurogenic cytokines brain‐derived neurotrophic factor and neurotrophin‐3 greatly enhanced Nurr1‐induced DA neuron yield. The Nurr1‐induced DA neurons demonstrated in vitro presynaptic DA neuronal functionality, releasing DA neurotransmitter in response to depolarization stimuli and specific DA reuptake. Furthermore, Nurr1‐engineered adult SVZ NP cells survived, integrated, and differentiated into DA neurons in vivo that can reverse the behavioral deficit in the host striatum of parkinsonian rats. These findings open the possibility for the use of precursor cells from adult brains as a cell source for neuronal replacement treatment of Parkinson disease.

Human embryonic stem cell‐derived neural precursors as a continuous, stable, and on‐demand source for human dopamine neurons

Human embryonic stem (hES) cells can be guided to differentiate into ventral midbrain‐type neural precursor (NP) cells that proliferate in vitro by specific mitogens. We investigated the potential of these NP cells derived from hES cells (hES‐NP) for the large‐scale generation of human dopamine (DA) neurons for functional analyses and therapeutic applications. To address this, hES‐NP cells were expanded in vitro for 1.5 months with six passages, and their proliferation and differentiation properties determined over the NP passages. Interestingly, the total hES‐NP cell number was increased by > 2 × 104‐folds over the in vitro period without alteration of phenotypic gene expression. They also sustained their differentiation capacity toward neuronal cells, exhibiting in vitro pre‐synaptic DA neuronal functionality. Furthermore, the hES‐NP cells can be cryopreserved without losing their proliferative and developmental potential. Upon transplantation into a Parkinson’s disease rat model, the multi‐passaged hES‐NP cells survived, integrated into the host striatum, and differentiated toward the neuronal cells expressing DA phenotypes. A significant reduction in the amphetamine‐induced rotation score of Parkinson’s disease rats was observed by the cell transplantation. Taken together, these findings indicate that hES‐NP cell expansion is exploitable for a large‐scale generation of experimental and transplantable DA neurons of human‐origin.

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.

Acquisition of in vitro and in vivo functionality of Nurr1-induced dopamine neurons

Neural precursor cells provide an expandable source of neurons and glia for basic and translational applications. However, little progress has been made in directing naive neural precursors toward specific neuronal fates such as midbrain dopamine (DA) neurons. We have recently demonstrated that transgenic expression of the nuclear orphan receptor Nurr1 is sufficient to drive dopaminergic differentiation of forebrain embryonic rat neural precursors in vitro. However, Nurr1‐induced DA neurons exhibit immature neuronal morphologies and functional properties and are unable to induce behavioral recovery in rodent models of Parkinsons disease (PD). Here, we report on the identification of key genetic factors that drive morphological and functional differentiation of Nurr1‐derived DA neurons. We show that coexpression of Nurr1, Bcl‐XL, and Sonic hedgehog (SHH) or Nurr1 and the proneural bHLH factor Mash1 is sufficient to drive naive rat forebrain precursors into neurons exhibiting the biochemical, electrophysiological, and functional properties of DA neuron in vitro. On transplantation into the striatum of Parkinsonian rats, precursor cells engineered with Nurr1/SHH/Bcl‐XL or Nurr1/Mash1 survived in vivo and differentiated into mature DA neurons that can reverse the behavioral deficits in the grafted animals.—Park, C.‐H., Kang, J. S., Shin, Y. H., Chang, M.‐Y., Chung, S., Koh, H.‐C., Zhu, M. H., Oh, S. B., Lee, Y.‐S., Panagiotakos, G., Tabar, V., Studer, L., and Lee, S.‐H. Acquisition of in vitro and in vivo functionality of Nurr1‐induced dopamine neurons. FASEB J. 20, E1910‐E1923 (2006)

Mash1 and Neurogenin 2 Enhance Survival and Differentiation of Neural Precursor Cells After Transplantation to Rat Brains via Distinct Modes of Action

Neural precursor cells (NPCs) are regarded as a promising source of donor cells in transplantation-based therapies for neurodegenerative disorders. However, poor survival and limited neuronal differentiation of the transplanted NPCs remain critical limitations for developing therapeutic strategies. In this study, we investigated the effects of the proneural basic helix-loop-helix (bHLH) transcription factors Mash1 and Neurogenin 2 (Ngn2) in neuronal differentiation and survival of NPCs. Induction of Mash1 or Ngn2 expression strikingly enhanced neuronal differentiation of cultured NPCs in vitro. Ngn2-transduced NPCs underwent a rapid cell cycle arrest, which often accompanies differentiation. In contrast, cells continuously expanded upon Mash1 expression during NPC differentiation. Notably, sonic hedgehog (SHH) was upregulated by Mash1 and mediated the proliferative and survival effects of Mash1 on NPCs. Upon transplantation into adult rat brains, Mash1-expressing NPCs yielded large grafts enriched with neurons compared to control LacZ-transduced NPCs. Interestingly, enhancements in neuronal yield, as well as in donor cell survival, were also achieved by transplanting Ngn2-transduced NPCs. We show that a differentiation stage- and cell density-dependent survival effect of Ngn2 involves neurotrophin3 (NT3)/TrkC-mediated signaling. Together, these findings suggest potential benefits of bHLH gene manipulation to develop successful transplantation strategies for brain disorders.

Conditions for tumor-free and dopamine neuron-enriched grafts after transplanting human ES cell-derived neural precursor cells.

We have previously demonstrated derivation of neural precursor (NP) cells of a midbrain-type from human embryonic stem (hES) cells to yield an enriched population of dopamine (DA) neurons. These hES-derived NPs can be expanded in vitro through multiple passages without altering their DA neurogenic potential. Here, we studied two aspects of these hES-NP cells that are critical issues in cell therapeutic approaches for Parkinsons disease (PD): cell survival and tumorigenic potential. Neuroepithelial rosettes, a potentially tumorigenic structure, disappeared during hES-NP cell expansion in vitro. Although a minor population of cells positive for Oct3/4, a marker specific for undifferentiated hES cells, persisted in culture during hES-NP cell expansion, they could be completely eliminated by subculturing hES-NPs under differentiation-inducing conditions. Consistently, no tumors/teratomas are formed in rats grafted with multipassaged hES-NPs. However, extensively expanded hES-NP cells easily underwent cell death during differentiation in vitro and after transplantation in vivo. Transgenic expression of Bcl-XL and sonic hedgehog (SHH) completely overcame the cell survival problems without increasing tumor formation. These findings indicate that hES-NP cell expansion in conjunction with Bcl-XL+SHH transgene expression may provide a renewable and safe source of DA neurons for transplantation in PD.

Generation of Dopamine Neurons with Improved Cell Survival and Phenotype Maintenance Using a Degradation-Resistant Nurr1 Mutant†‡

Nurr1 is a transcription factor specific for the development and maintenance of the midbrain dopamine (DA) neurons. Exogenous Nurr1 in neural precursor (NP) cells induces the differentiation of DA neurons in vitro that are capable of reversing motor dysfunctions in a rodent model for Parkinson disease. The promise of this therapeutic approach, however, is unclear due to poor cell survival and phenotype loss of DA cells after transplantation. We herein demonstrate that Nurr1 proteins undergo ubiquitin‐proteasome‐system‐mediated degradation in differentiating NP cells. The degradation process is activated by a direct Akt‐mediated phosphorylation of Nurr1 proteins and can be prevented by abolishing the Akt‐target sequence in Nurr1 (Nurr1Akt). Overexpression of Nurr1Akt in NP cells yielded DA neurons in which Nurr1 protein levels were maintained for prolonged periods. The sustained Nurr1 expression endowed the Nurr1Akt‐induced DA neurons with resistance to toxic stimuli, enhanced survival, and sustained DA phenotypes in vitro and in vivo after transplantation. STEM CELLS 2009;27:2238–2246