Marius Wernig Laboratory
Only those who attempt the absurd can achieve the impossible.
Research in the Wernig Lab
Our lab is generally interested in the mechanisms that determine cell fate identity. Our focus is on epigenetic reprogramming i.e. ways to induce cell fate changes by defined factors such as the reprogramming of somatic cells into pluripotent stem (or iPS) cells. More recently, we have demonstrated that mouse fibroblasts can directly be converted to functional neuronal cells that we termed induced neuronal (iN) cells (Vierbuchen et al., 2010, Nature). The iN cells were generated through expression of the three transcription factors Ascl1, Myt1l, and Brn2. This surprising discovery opened the door to a new area of investigation. We are currently working to apply our finding to human cells, explore the molecular mechanism of the action of the three transcription factors, and determine the neuronal subtype of resulting iN cells. A long term goal is to use this method to evaluate whether iN cells can be used to model neurological diseases. In addition, the emerging iPS cell technology provides new fascinating translational applications such as patient-specific stem cell therapy or disease phenocopy through differentiation into the neural lineage. Our lab has developed new methods to generate iPS cells from human fibroblasts with defined mutations and explores various technologies to improve gene targeting in human iPS cells with a long term goal to correct disease-causing mutations. This work is made possible through a very generous CIRM grant. Another interest of the laboratory is to study self-renewal and differentiation in neural stem/progenitor cells and apply these findings to the tumor precursor cells of glioblastoma. This will shed some light into glioma generation and potentially lead to alternative treatment strategies of this devastating brain disease.
Generation of pure GABAergic neurons by transcription factor programming.
2017; 14 (6): 621-628
Approaches to differentiating pluripotent stem cells (PSCs) into neurons currently face two major challenges-(i) generated cells are immature, with limited functional properties; and (ii) cultures exhibit heterogeneous neuronal subtypes and maturation stages. Using lineage-determining transcription factors, we previously developed a single-step method to generate glutamatergic neurons from human PSCs. Here, we show that transient expression of the transcription factors Ascl1 and Dlx2 (AD) induces the generation of exclusively GABAergic neurons from human PSCs with a high degree of synaptic maturation. These AD-induced neuronal (iN) cells represent largely nonoverlapping populations of GABAergic neurons that express various subtype-specific markers. We further used AD-iN cells to establish that human collybistin, the loss of gene function of which causes severe encephalopathy, is required for inhibitory synaptic function. The generation of defined populations of functionally mature human GABAergic neurons represents an important step toward enabling the study of diseases affecting inhibitory synaptic transmission.
View details for DOI 10.1038/nmeth.4291
View details for PubMedID 28504679
Induction of functional dopamine neurons from human astrocytes in vitro and mouse astrocytes in a Parkinson's disease model
2017; 35 (5): 444-?
Cell replacement therapies for neurodegenerative disease have focused on transplantation of the cell types affected by the pathological process. Here we describe an alternative strategy for Parkinson's disease in which dopamine neurons are generated by direct conversion of astrocytes. Using three transcription factors, NEUROD1, ASCL1 and LMX1A, and the microRNA miR218, collectively designated NeAL218, we reprogram human astrocytes in vitro, and mouse astrocytes in vivo, into induced dopamine neurons (iDANs). Reprogramming efficiency in vitro is improved by small molecules that promote chromatin remodeling and activate the TGFβ, Shh and Wnt signaling pathways. The reprogramming efficiency of human astrocytes reaches up to 16%, resulting in iDANs with appropriate midbrain markers and excitability. In a mouse model of Parkinson's disease, NeAL218 alone reprograms adult striatal astrocytes into iDANs that are excitable and correct some aspects of motor behavior in vivo, including gait impairments. With further optimization, this approach may enable clinical therapies for Parkinson's disease by delivery of genes rather than cells.
View details for DOI 10.1038/nbt.3835
View details for Web of Science ID 000400809800019
View details for PubMedID 28398344
Myt1l safeguards neuronal identity by actively repressing many non-neuronal fates
2017; 544 (7649): 245-?
Normal differentiation and induced reprogramming require the activation of target cell programs and silencing of donor cell programs. In reprogramming, the same factors are often used to reprogram many different donor cell types. As most developmental repressors, such as RE1-silencing transcription factor (REST) and Groucho (also known as TLE), are considered lineage-specific repressors, it remains unclear how identical combinations of transcription factors can silence so many different donor programs. Distinct lineage repressors would have to be induced in different donor cell types. Here, by studying the reprogramming of mouse fibroblasts to neurons, we found that the pan neuron-specific transcription factor Myt1-like (Myt1l) exerts its pro-neuronal function by direct repression of many different somatic lineage programs except the neuronal program. The repressive function of Myt1l is mediated via recruitment of a complex containing Sin3b by binding to a previously uncharacterized N-terminal domain. In agreement with its repressive function, the genomic binding sites of Myt1l are similar in neurons and fibroblasts and are preferentially in an open chromatin configuration. The Notch signalling pathway is repressed by Myt1l through silencing of several members, including Hes1. Acute knockdown of Myt1l in the developing mouse brain mimicked a Notch gain-of-function phenotype, suggesting that Myt1l allows newborn neurons to escape Notch activation during normal development. Depletion of Myt1l in primary postmitotic neurons de-repressed non-neuronal programs and impaired neuronal gene expression and function, indicating that many somatic lineage programs are actively and persistently repressed by Myt1l to maintain neuronal identity. It is now tempting to speculate that similar 'many-but-one' lineage repressors exist for other cell fates; such repressors, in combination with lineage-specific activators, would be prime candidates for use in reprogramming additional cell types.
View details for DOI 10.1038/nature21722
View details for Web of Science ID 000398897900040
View details for PubMedID 28379941
Partial Reprogramming of Pluripotent Stem Cell-Derived Cardiomyocytes into Neurons
Direct reprogramming of somatic cells has been demonstrated, however, it is unknown whether electrophysiologically-active somatic cells derived from separate germ layers can be interconverted. We demonstrate that partial direct reprogramming of mesoderm-derived cardiomyocytes into neurons is feasible, generating cells exhibiting structural and electrophysiological properties of both cardiomyocytes and neurons. Human and mouse pluripotent stem cell-derived CMs (PSC-CMs) were transduced with the neurogenic transcription factors Brn2, Ascl1, Myt1l and NeuroD. We found that CMs adopted neuronal morphologies as early as day 3 post-transduction while still retaining a CM gene expression profile. At week 1 post-transduction, we found that reprogrammed CMs expressed neuronal markers such as Tuj1, Map2, and NCAM. At week 3 post-transduction, mature neuronal markers such as vGlut and synapsin were observed. With single-cell qPCR, we temporally examined CM gene expression and observed increased expression of neuronal markers Dcx, Map2, and Tubb3. Patch-clamp analysis confirmed the neuron-like electrophysiological profile of reprogrammed CMs. This study demonstrates that PSC-CMs are amenable to partial neuronal conversion, yielding a population of cells exhibiting features of both neurons and CMs.
View details for DOI 10.1038/srep44840
View details for Web of Science ID 000396983300001
View details for PubMedID 28327614
View details for PubMedCentralID PMC5361100
Human AML-iPSCs Reacquire Leukemic Properties after Differentiation and Model Clonal Variation of Disease.
Cell stem cell
2017; 20 (3): 329-344 e7
Understanding the relative contributions of genetic and epigenetic abnormalities to acute myeloid leukemia (AML) should assist integrated design of targeted therapies. In this study, we generated induced pluripotent stem cells (iPSCs) from AML patient samples harboring MLL rearrangements and found that they retained leukemic mutations but reset leukemic DNA methylation/gene expression patterns. AML-iPSCs lacked leukemic potential, but when differentiated into hematopoietic cells, they reacquired the ability to give rise to leukemia in vivo and reestablished leukemic DNA methylation/gene expression patterns, including an aberrant MLL signature. Epigenetic reprogramming was therefore not sufficient to eliminate leukemic behavior. This approach also allowed us to study the properties of distinct AML subclones, including differential drug susceptibilities of KRAS mutant and wild-type cells, and predict relapse based on increased cytarabine resistance of a KRAS wild-type subclone. Overall, our findings illustrate the value of AML-iPSCs for investigating the mechanistic basis and clonal properties of human AML.
View details for DOI 10.1016/j.stem.2016.11.018
View details for PubMedID 28089908