The global gene expression pattern between human fibroblasts and human neural stem cells
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ABSTRACT: Recent advances in direct reprogramming using cell type-specific transcription factors provide an unprecedented opportunity for rapid generation of desired human cell types from easily accessible tissues. However, due to the diversity of conversion factors that facilitate the process, an arduous screening step is inevitable to find the appropriate combination(s). Here, we show that under chemically defined conditions minimal pluripotency factors are sufficient to directly reprogram human fibroblasts into stably self-renewing neural progenitor/stem cells (NSCs), but without passing through a pluripotent intermediate stage. These NSCs can be expanded and propagated in vitro without losing their potential to differentiate into various neuronal subtypes and glia. Our direct reprogramming strategy represents a simple and advanced paradigm of direct conversion that will provide an unlimited source of human neural cells for cell therapy, disease modeling, and drug screening. We used microarray to compare the global gene expression pattern between human fibroblasts and human neural epitheliums from human ESCs or directly from fibroblasts. We cultured cells and harvested them and then extracted total RNA for microarray.
Project description:Recent advances in direct reprogramming using cell type-specific transcription factors provide an unprecedented opportunity for rapid generation of desired human cell types from easily accessible tissues. However, due to the diversity of conversion factors that facilitate the process, an arduous screening step is inevitable to find the appropriate combination(s). Here, we show that under chemically defined conditions minimal pluripotency factors are sufficient to directly reprogram human fibroblasts into stably self-renewing neural progenitor/stem cells (NSCs), but without passing through a pluripotent intermediate stage. These NSCs can be expanded and propagated in vitro without losing their potential to differentiate into various neuronal subtypes and glia. Our direct reprogramming strategy represents a simple and advanced paradigm of direct conversion that will provide an unlimited source of human neural cells for cell therapy, disease modeling, and drug screening. We used microarray to compare the global gene expression pattern between human fibroblasts and human neural epitheliums from human ESCs or directly from fibroblasts.
Project description:Direct conversion of somatic cells into neural stem cells (NSCs) by defined factors holds great promise for mechanistic studies, drug screening, and potential cell therapies for different neurodegenerative diseases. Here, we report that a single zinc-finger transcription factor, Zfp521, is sufficient for direct conversion of human fibroblasts into long-term self-renewable and multipotent NSCs. In vitro, Zfp521-induced NSCs maintained their characteristics in the absence of exogenous factor expression and exhibited morphological, molecular, developmental, and functional properties that were similar to control NSCs. Additionally, the single seeded induced NSCs were able to form NSC colonies with efficiency comparable to control NSCs and expressed NSC markers. The converted cells were capable of surviving, migrating and attaining neural phenotypes after transplantation into neonatal mouse- and adult rat brains, without forming tumors. Moreover, the Zfp521-induced NSCs predominantly expressed rostral genes. Our results suggest a facilitated approach for establishing human NSCs through Zfp521-driven conversion of fibroblasts.
Project description:Forced expression of pro-neural transcription factors was shown to mediate direct neuronal conversion of human fibroblasts. Since neurons are postmitotic, the conversion efficiency represents an important parameter. Here we present a minimalist approach combining two factor neuronal programming with small molecule-based inhibition of GSK3ß and SMAD signaling, which gives rise to functional neuron-like cells (iNs) of various neurotransmitter phenotypes with an overall yield of up to >200% and a final neuronal purity of up to >80%. Timcourse of reprogramming of fibroblasts towards an neuronal phenotype in two independent fibroblast lines
Project description:Direct cell reprogramming has enabled the direct conversion of skin fibroblasts into functional neurons and oligodendrocytes using a minimal set of cell lineage-specific transcription factors. This approach has substantial advantages since it is rapid and simple, generating the cell type of interest in a single step. However, it remains unknown whether this technology can be applied for directly reprogramming skin cells into astrocytes, the third neural lineage. Astrocytes play crucial roles in neuronal homeostasis and their dysfunctions contribute to the origin and progression of multiple human diseases. Herein, we carried out a screening using several transcription factors involved in defining the astroglial cell fate and identified NFIA, NFIB and SOX9 to be sufficient to convert with high efficiency embryonic and post-natal mouse fibroblasts into astrocytes (iAstrocytes). We proved both by gene expression profiling and functional tests that iAstrocytes are comparable to native brain astrocytes. This protocol can be then employed to generate functional iAstrocytes for a wide range of experimental applications. Induced astrocytes (iAstro) were compared to Fibroblasts (Fibro) as negative control and to primary astrocytes (astro) as positive control. Three biological replicates were analyzed for each experimental group.
Project description:Neural stem cells (NSCs) are valuable for both basic research and clinical application. We previously reported a chemical cocktail that could reprogram somatic cells into neural progenitor cells. However, the process of chemical induction is complex and the underlying mechanism remains largely elusive. Here, we identified a new culture condition that greatly promotes the efficiency of NSC generation directly from mouse fibroblasts based on our reported chemical cocktail. Transcriptome and epigenome analyses during the reprogramming process demonstrated that growth factors including IL6, FGF5 and LIF were dynamically activated and contributed to the chemical cocktail induced cell fate changes. Interestingly, fibroblast-to-NSC conversion requires the synergistic action of these growth factors. Moreover, the reprogramming capacity of both chemical cocktail and growth factors require nucleoporin Nup210 to activate endogenous neural transcription factors SoxB1 family to initiate NSC fate. Our study not only provides a novel protocol to generate NSC directly from mouse fibroblasts, but also reveals an important role of growth factors in chemical-induced cellular reprogramming.
Project description:Direct conversion from fibroblast to neuron has recently been successfully induced bypassing the pluripotent state. However, the conversion takes a few months with low percentages of success. Here we found that depletion of p53, which can converted fibroblasts into three major neural lineages: neurons, astrocytes and oligodendrocytes. Furthermore, our method provided a high efficiency of conversion in aging fibroblasts, where published methods failed. This finding may help developing a prototype for neuron replacement therapy, including foraging people vulnerable to neurological disorders. p53 has been shown to inhibit reprogramming of fibroblasts to iPS cells, by depletion of p53 in human fibroblasts, we study the function of p53 in induced neuron process. By induction of p53 knockdown fibroblasts with special neuron medium, we can get mature neurons directly. In the induction process, many neurogenic transcription factors were up-regulated, and we prove that p21 is not involved in this process.
Project description:Direct cell reprogramming has enabled the direct conversion of skin fibroblasts into functional neurons and oligodendrocytes using a minimal set of cell lineage-specific transcription factors. This approach has substantial advantages since it is rapid and simple, generating the cell type of interest in a single step. However, it remains unknown whether this technology can be applied for directly reprogramming skin cells into astrocytes, the third neural lineage. Astrocytes play crucial roles in neuronal homeostasis and their dysfunctions contribute to the origin and progression of multiple human diseases. Herein, we carried out a screening using several transcription factors involved in defining the astroglial cell fate and identified NFIA, NFIB and SOX9 to be sufficient to convert with high efficiency embryonic and post-natal mouse fibroblasts into astrocytes (iAstrocytes). We proved both by gene expression profiling and functional tests that iAstrocytes are comparable to native brain astrocytes. This protocol can be then employed to generate functional iAstrocytes for a wide range of experimental applications.
Project description:The four transcription factors Oct4, Sox2, Klf4, and c-Myc can induce pluripotency in mouse and human fibroblasts. We previously described direct reprogramming of adult mouse neural stem cells (NSCs) by Oct4 and either Klf4 or c-Myc. NSCs endogenously express Sox2, c-Myc, and Klf4 as well as several intermediate reprogramming markers. Here we report that exogenous expression of the germline-specific transcription factor Oct4 is sufficient to generate pluripotent stem cells from adult mouse NSCs. These one-factor induced pluripotent stem (1F iPS) cells are similar to embryonic stem cells in vitro and in vivo. Not only can these cells be efficiently differentiated into NSCs, cardiomyocytes and germ cells in vitro, but they are also capable of teratoma formation and germline transmission in vivo. Our results demonstrate that Oct4 is required and sufficient to directly reprogram NSCs to pluripotency.
Project description:Direct cell fate conversion allows the generation of somatic cells that are otherwise difficult to obtain directly from patients. The clinical applicability of this approach depends on obtaining an initial source of somatic cells from adult patients that is easy to harvest, store, and manipulate for reprogramming. Here we have generated induced neural progenitor cells (iNPCs) from neonatal as well as peripheral blood from human adults using single factor OCT4 based reprogramming. Unlike fibroblasts that share molecular hallmarks of neural crest, direct OCT4 reprogramming of human blood could be facilitated by SMAD+GSK-3 inhibition to overcome restrictions on neural fate conversion. Blood derived (BD)-iNPCs functionally differentiate in vivo, and respond to guided differentiation in vitro to produce both glia (astrocytes and oligodendrocytes) and multiple neuronal subtypes including dopamine releasing DA neurons (CNS related) and nociceptive neurons (PNS). Furthermore, BD nociceptive neurons phenocopy chemotherapy induced neurotoxicity in a system suitable for high throughput drug screening. Our findings provide an easily accessible approach to generate human NPCs that harbor extensive developmental potential, enabling the study of clinically relevant neural diseases directly from patient cohorts.