Direct generation of functional dopaminergic neurons from mouse and human fibroblasts

Stem Cells and Neurogenesis Unit, Division of Neuroscience, San Raffaele Scientific Institute, 20132 Milan, Italy.
Nature (Impact Factor: 41.46). 07/2011; 476(7359):224-7. DOI: 10.1038/nature10284
Source: PubMed


Transplantation of dopaminergic neurons can potentially improve the clinical outcome of Parkinson's disease, a neurological disorder resulting from degeneration of mesencephalic dopaminergic neurons. In particular, transplantation of embryonic-stem-cell-derived dopaminergic neurons has been shown to be efficient in restoring motor symptoms in conditions of dopamine deficiency. However, the use of pluripotent-derived cells might lead to the development of tumours if not properly controlled. Here we identified a minimal set of three transcription factors--Mash1 (also known as Ascl1), Nurr1 (also known as Nr4a2) and Lmx1a--that are able to generate directly functional dopaminergic neurons from mouse and human fibroblasts without reverting to a progenitor cell stage. Induced dopaminergic (iDA) cells release dopamine and show spontaneous electrical activity organized in regular spikes consistent with the pacemaker activity featured by brain dopaminergic neurons. The three factors were able to elicit dopaminergic neuronal conversion in prenatal and adult fibroblasts from healthy donors and Parkinson's disease patients. Direct generation of iDA cells from somatic cells might have significant implications for understanding critical processes for neuronal development, in vitro disease modelling and cell replacement therapies.

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    • "Furthermore, recent evidence demonstrates that the neuron-forming capacity of astrocytes is also active in vivo following neurodegeneration of the cortex and striatum (Guo et al., 2014; Magnusson et al., 2014; Niu et al., 2013; Torper et al., 2013), highlighting the existence of an endogenous cell source capable to restore connectivity and function following brain trauma. Similarly, it has been shown that not only astrocytes but also somatic cells more distant in lineage to the CNS, such as fibroblasts, can be reprogrammed either into NPCs or directly into various types of neurons, including glutamatergic , dopaminergic, and spinal motor neurons, by different cocktails of transcription factors and neuron-specific microRNAs (Caiazzo et al., 2011; Vierbuchen et al., 2010; Wernig et al., 2008; Yoo et al., 2011) and may thus be used as an exogenous cell source to restore damage following neuronal loss. The aim of this study is to explore the reprogramming potential of the neurogenic factor CEND1 in inducing the reprogramming of astrocytes and embryonic fibroblasts . "
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    ABSTRACT: Recent studies demonstrate that astroglia from non-neurogenic brain regions can be reprogrammed into functional neurons through forced expression of neurogenic factors. Here we explored the effect of CEND1 and NEUROG2 on reprogramming of mouse cortical astrocytes and embryonic fibroblasts. Forced expression of CEND1, NEUROG2, or both resulted in acquisition of induced neuronal cells expressing subtype-specific markers, while long-term live-cell imaging highlighted the existence of two different modes of neuronal trans-differentiation. Of note, a subpopulation of CEND1 and NEUROG2 double-transduced astrocytes formed spheres exhibiting neural stem cell properties. mRNA and protein expression studies revealed a reciprocal feedback loop existing between the two molecules, while knockdown of endogenous CEND1 demonstrated that it is a key mediator of NEUROG2-driven neuronal reprogramming. Our data suggest that common reprogramming mechanisms exist driving the conversion of lineage-distant somatic cell types to neurons and reveal a critical role for CEND1 in NEUROG2-driven astrocytic reprogramming. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    No preview · Article · Aug 2015 · Stem Cell Reports
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    • "In 2012, we have reported that mouse somatic cells—sertoli cells, can be directly converted to iNSCs by introduction of 8 transcription factors (Sheng et al., 2012a), which provides further evidence that somatic cells can be induced to not only pluripotent stem cells (PSCs) (Takahashi and Yamanaka, 2006; Takahashi et al., 2007; Yu et al., 2007), mature neurons (Vierbuchen et al., 2010; Pfisterer et al., 2011; Caiazzo et al., 2011; Kim et al., 2011), but also to adult stem cells. The above findings support the concept that the fates of any two different lineages are interchangeable, given sufficient and necessary conditions. "
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    ABSTRACT: Lmx1a plays a central role in the specification of dopaminergic (DA) neurons, which potentially could be employed as a key factor for trans-differentiation to DA neurons. In our previous study, we have converted somatic cells directly into neural stem cell-like cells, namely induced neural stem cells (iNSCs), which further can be differentiated into subtypes of neurons and glia in vitro. In the present study, we continued to test whether these iNSCs have therapeutic effects when transplanted into a mouse model of Parkinson's disease (PD), especially when Lmx1a was introduced into these iNSCs under a Nestin enhancer. iNSCs that over-expressed Lmx1a (iNSC-Lmx1a) gave rise to an increased yield of dopaminergic neurons and secreted a higher level of dopamine in vitro. When transplanted into mouse models of PD, both groups of mice showed decreased ipsilateral rotations; yet mice that received iNSC-Lmx1a vs. iNSC-GFP exhibited better recovery. Although few iNSCs survived 11weeks after transplantation, the improved motor performance in iNSC-Lmx1a group did correlate with a greater tyrosine hydroxylase (TH) signal abundance in the lesioned area of striatum, suggesting that iNSCs may have worked through a non-autonomous manner to enhance the functions of remaining endogenous dopaminergic neurons in brain. Copyright © 2014. Published by Elsevier B.V.
    Full-text · Article · Oct 2014 · Stem Cell Research
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    • "Here we report a highly efficient protocol for direct neural conversion that involves delayed transgene activation and improved culture conditions in combination with the addition of small molecules (SMs) that inhibit SMAD signalling and activate canonical WNT signalling14. This protocol results in at least a 10-fold improvement of conversion efficiency compared to previous reports5671516, and therefore provides a method for highly efficient generation of iNs from human fibroblasts without the need for a drug selection step. Furthermore, we provide the first evidence that hiNs survive and maintain their phenotype after transplantation into the adult rat brain, when transplanted 5 and 10 days after initiation of conversion. "
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    ABSTRACT: Induced neurons (iNs) offer a novel source of human neurons that can be explored for applications of disease modelling, diagnostics, drug screening and cell replacement therapy. Here we present a protocol for highly efficient generation of functional iNs from fetal human fibroblasts, and also demonstrate the ability of these converted human iNs (hiNs) to survive transplantation and maintain their phenotype in the adult rat brain. The protocol encompasses a delay in transgene activation after viral transduction that resulted in a significant increase in conversion efficiency. Combining this approach with treatment of small molecules that inhibit SMAD signalling and activate WNT signalling provides a further increase in the conversion efficiency and neuronal purity, resulting in a protocol that provides a highly efficient method for the generation of large numbers of functional and transplantable iNs from human fibroblasts without the use of a selection step. When transplanting the converted neurons from different stages of in vitro culture into the brain of adult rats, we observed robust survival and maintenance of neuronal identity four weeks post-transplantation. Interestingly, the positive effect of small molecule treatment observed in vitro did not result in a higher yield of iNs surviving transplantation.
    Full-text · Article · Sep 2014 · Scientific Reports
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