Project description:Bulk RNA was extracted via trizol at Fibroblasts stage, 5 days, 10 days, 15 days, and 20 days of fibroblast to neuron conversion using traditional NC media, or NC media supplemented with ZM336372, pyrintegrin, AZ960, and KC7F2
Project description:Astrocyte-to-neuron conversion has developed into a promising avenue for neuronal replacement therapy. Neurons depend critically on mitochondria function and often die by ferroptosis during the conversion process. Here we examined the extent of adequate mitochondrial reprogramming by morphology and proteome analysis. While mitochondria profoundly changed their morphology during Neurogenin2 (Neurog2) – or Achaete-scute homolog 1 (Ascl1)-mediated astrocyte-to-neuron reprogramming, we found neuron-specific mitochondrial proteins, here identified in a comprehensive proteome analysis of isolated mitochondria from primary neurons and astrocytes, to be only partially and at late stages regulated during the process. To improve this, we used dCas9 technology to induce neuron-specific mitochondrial proteins early during reprogramming. This resulted not only in increased conversion efficiency, but also in faster neuronal generation. Taken together, reprogramming mitochondria in a cell type-specific manner has powerful effects on astrocyte-to-neuron conversion, suggesting mitochondria to be a driving force in this process.
Project description:The direct conversion of human skin fibroblasts to neurons has a low efficiency and unclear mechanism. Here, we show that the knockdown of PTBP2 (nPTB) significantly enhanced the transdifferentiation induced by ASCL1, MiR124-9/9* and p53 shRNA to generate mostly GABAergic neurons. Longitudinal RNAseq analyses identified the continuous induction of many RNA Splicing Regulators (RSRs). Among these, the knockdown of RBFOX3, which encodes the mature neuronal marker NeuN, significantly abrogated the transdifferentiation. Overexpression of RBFOX3 significantly enhanced the conversion induced by AMp; the enhancement was occluded by PTBP2 knockdown. We found that PTBP2 attenuation significantly favored neuron-specific alternative splicing (AS) of many genes involved in synaptic transmission, signal transduction, and axon formation. RBFOX3 knockdown significantly reversed the effect, while RBFOX3 overexpression enhanced it. The study reveals the critical role of neuron-specific AS in the direct conversion of human skin fibroblasts to neurons by showing that PTBP2 attenuation enhances this mechanism in concert with RBFOX3.
Project description:Transmission of Plasmodium spp. from a human host to a mosquito vector requires that some parasites abandon the asexual replicative cycle in the blood, associated with all clinical symptoms of malaria, and convert into non-replicative sexual precursors called gametocytes. The sexual conversion rate (i.e., the proportion of parasites that convert at each cycle) is variable, which enables parasites to adjust the balance between sexual and asexual development to the conditions of the human blood environment. Under some specific types of stress, parasites enhance sexual conversion rates to increase their relative investment in transmission. Sexual conversion is orchestrated by the master regulator AP2-G, a transcription factor of the ApiAP2 family. In Plasmodium falciparum, the species that produces the vast majority of human malaria cases and deaths, activation of ap2-g expression requires the GDV1 protein, which displaces heterochromatin from the ap2-g promoter. An antisense long noncoding RNA (lncRNA) encoded in the same locus as GDV1, named gdv1-as, is a negative regulator of GDV1 expression, but how the expression of this lncRNA is regulated or how specific types of stress enhance sexual conversion is not known. Here we show that environmental induction of sexual conversion is initiated by activation of gdv1 expression in a process dependent on the AP2-HS transcription factor, previously described as the activator of the malarial protective HS response. Next, GDV1 activates the expression of its own repressor, gdv1-as, by removing heterochromatin from its putative promoter. This positive-negative regulatory feedback loop limits GDV1 activation to a short burst of expression. This same mechanism operates for stimulation of sexual conversion by different environmental conditions, and AP2-HS is also needed for the activation of metabolic pathways that compensate depletion of phospholipid precursors. These findings provide new insight on the mechanisms underlying the fundamental developmental decision of malaria parasites and explain their plasticity to increase the investment in transmission when the conditions of the environment are unfavourable.
Project description:Engineering clinically relevant cells in vitro holds promise for regenerative medicine, but most protocols fail to faithfully recapitulate target cell properties. To address this, we developed CellNet, a network biology platform that determines whether engineered cells are equivalent to their target tissues, diagnoses aberrant gene regulatory networks, and prioritizes candidate transcriptional regulators to enhance engineered conversions. Using CellNet, we improved B cell to macrophage conversion, transcriptionally and functionally, by knocking down predicted B cell regulators. Analyzing conversion of fibroblasts to induced hepatocytes (iHeps), CellNet revealed an unexpected intestinal program regulated by the master regulator Cdx2. We observed functional engraftment of mouse colon by iHeps, thereby establishing their broader potential as endoderm progenitors and demonstrating direct conversion of fibroblasts into intestinal epithelium. Our studies illustrate how CellNet can be employed to improve direct conversion and to uncover unappreciated properties of engineered cells. 15 samples
Project description:Direct conversion of reactive glial cells to neurons is promising avenue for the replacement therapies after brain injury or neurodegeneration. The overexpression of developmental neurogenic fate determinants in glial cells converts them to neurons. For the repair purposes the conversion is confined to the pathology-induced neuroinflammatory environment. However, very little is known about the influence of injury-induced neuroinflammatory environment on the direct conversion process. We established the new in vitro culture system of postnatal astrocytes that reflects the direct conversion rate in the injured, neuroinflammatory environment in vivo. We could show that the growth factor combination corresponding to the injured environment defines the capacity of the glia to be directly converted to neurons. Using this culture, we showed that the chromatin structural protein high mobility group b2 (Hmgb2) regulate the direct conversion rate downstream of the growth factor combination. We could further show that Hmgb2 in cooperation with neurogenic fate determinants such as Neurog2 opens the chromatin containing neuronal maturation and synapse formation genes, leading to early chromatin re-arrangements during the direct fate conversion that are necessary for the full fate conversion. Our data demonstrate the novel, environmental cues controlled level of gene regulation during direct fate conversion necessary for the proper maturation of induced neurons that could be targeted to improve the repair process.
Project description:Methylation array was used to quantify CpG methylation to determine if NC+ZPAK media was affecting the age of the donor after neuron conversion. Beta values were determined using the ChAMP methylation analysis pipeline.