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Substrate-Dependent Galvanotaxis of Directly Reprogrammed Human Neural Precursor Cells.

ABSTRACT: Background: Neural precursor cells (NPCs) hold great promise for neural repair. Endogenous NPCs, found in the subventricular zone of the adult brain, proliferate and migrate toward lesion sites; however, it is not sufficient for neural repair. NPCs are electrosensitive cells that undergo directed migration in an electric field (EF). Here, we examined the EF-induced migration of a clinically relevant human NPC population. Materials & Methods: We examined the effects of different substrates and microenvironments on human NPC galvanotaxis. Results: Human NPCs increased their migration speed in the presence of an EF, and the direction of migration (anodal vs. cathodal) varied between substrates. The secretome and extracellular pH were not significant factors in EF-induced migration; however, our results are consistent with substrate stiffness playing a role in the direction of cell migration. Conclusion: These findings provide insight into the importance of the microenvironment on modulating human NPC migration and highlight substrate-dependent considerations for neurorepair.

SUBMITTER: Ahmed U

PROVIDER: S-EPMC8370329 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundCell reprogramming is a promising avenue for cell-based therapies as it allows for the generation of multipotent, unipotent, or mature somatic cells without going through a pluripotent state. While the use of autologous cells is considered ideal, key challenges for their clinical translation include the ability to reproducibly generate sufficient quantities of cells within a therapeutically relevant time window.MethodsWe performed transfection of three distinct human somatic starting populations of cells with a non-integrating synthetic plasmid expressing Musashi 1 (MSI1), Neurogenin 2 (NGN2), and Methyl-CpG-Binding Domain 2 (MBD2). The resulting directly reprogrammed neural precursor cells (drNPCs) were examined in vitro using RT-qPCR, karyotype analysis, immunohistochemistry, and FACS at early and late time post-transfection. Electrophysiology (patch clamp) was performed on drNPC-derived neurons to determine their capacity to generate action potentials. In vivo characterization was performed following transplantation of drNPCs into two animal models (Shiverer and SCID/Beige mice), and the numbers, location, and differentiation profile of the transplanted cells were examined using immunohistochemistry.ResultsHuman somatic cells can be directly reprogrammed within two weeks to neural precursor cells (drNPCs) by transient exposure to Msi1, Ngn2, and MBD2 using non-viral constructs. The drNPCs generate all three neural cell types (astrocytes, oligodendrocytes, and neurons) and can be passaged in vitro to generate large numbers of cells within four weeks. drNPCs can respond to in vivo differentiation and migration cues as demonstrated by their migration to the olfactory bulb and contribution to neurogenesis in vivo. Differentiation profiles of transplanted cells onto the corpus callosum of myelin-deficient mice reveal the production of oligodendrocytes and astrocytes.ConclusionsHuman drNPCs can be efficiently and rapidly produced from donor somatic cells and possess all the important characteristics of native neural multipotent cells including differentiation into neurons, astrocytes, and oligodendrocytes, and in vivo neurogenesis and myelination.

Project description:IntroductionThe neurodevelopmental disorder fragile X syndrome (FXS) is the most common monogenic cause of intellectual disability associated with autism spectrum disorder. Inaccessibility to developing human brain cells is a major barrier to studying FXS. Direct-to-neural precursor reprogramming provides a unique platform to investigate the developmental profile of FXS-associated phenotypes throughout neural precursor and neuron generation, at a temporal resolution not afforded by post-mortem tissue and in a patient-specific context not represented in rodent models. Direct reprogramming also circumvents the protracted culture times and low efficiency of current induced pluripotent stem cell strategies.MethodsWe have developed a chemically modified mRNA (cmRNA) -based direct reprogramming protocol to generate dorsal forebrain precursors (hiDFPs) from FXS patient-derived fibroblasts, with subsequent differentiation to glutamatergic cortical neurons and astrocytes.ResultsWe observed differential expression of mature neuronal markers suggesting impaired neuronal development and maturation in FXS- hiDFP-derived neurons compared to controls. FXS- hiDFP-derived cortical neurons exhibited dendritic growth and arborization deficits characterized by reduced neurite length and branching consistent with impaired neuronal maturation. Furthermore, FXS- hiDFP-derived neurons exhibited a significant decrease in the density of pre- and post- synaptic proteins and reduced glutamate-induced calcium activity, suggesting impaired excitatory synapse development and functional maturation. We also observed a reduced yield of FXS- hiDFP-derived neurons with a significant increase in FXS-affected astrocytes.DiscussionThis study represents the first reported derivation of FXS-affected cortical neurons following direct reprogramming of patient fibroblasts to dorsal forebrain precursors and subsequently neurons that recapitulate the key molecular hallmarks of FXS as it occurs in human tissue. We propose that direct to hiDFP reprogramming provides a unique platform for further study into the pathogenesis of FXS as well as the identification and screening of new drug targets for the treatment of FXS.

Project description:Adipose tissue is the primary tissue affected in most single gene forms of severe insulin resistance, and growing evidence has implicated it as a site at which many risk alleles for insulin resistance identified in population-wide studies might exert their effect. There is thus increasing need for human adipocyte models in which to interrogate the function of known and emerging genetic risk variants. However, primary adipocyte cultures, existing immortalised cell lines and stem-cell based models all have significant biological or practical limitations. In an attempt to widen the repertoire of human cell models in which to study adipocyte-autonomous effects of relevant human genetic variants, we have undertaken direct reprogramming of skin fibroblasts to adipocyte-like cells by employing an inducible recombinant lentivirus overexpressing the master adipogenic transcription factor PPAR?2. Doxycycline-driven expression of PPAR?2 and adipogenic culture conditions converted dermal fibroblasts into triglyceride-laden cells within days. The resulting cells recapitulated most of the crucial aspects of adipocyte biology in vivo, including the expression of mature adipocyte markers, secreted high levels of the adipokine adiponectin, and underwent lipolysis when treated with isoproterenol/3-isobutyl-1-methylxanthine (IBMX). They did not, however, exhibit insulin-inducible glucose uptake, and withdrawal of doxycycline produced rapid delipidation and loss of adipogenic markers. This protocol was applied successfully to a panel of skin cells from individuals with monogenic severe insulin resistance; however, surprisingly, even cell lines harbouring mutations causing severe, generalised lipodystrophy accumulated large lipid droplets and induced adipocyte-specific genes. The direct reprogramming protocol of human dermal fibroblasts to adipocyte-like cells we established is simple, fast and efficient, and has the potential to generate cells which can serve as a tool to address some, though not all, aspects of adipocyte function in the presence of endogenous disease-causing mutations.

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Substrate-Dependent Galvanotaxis of Directly Reprogrammed Human Neural Precursor Cells.

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