Understanding cell lineage relationships is fundamental to understanding development, and can shed light on disease etiology and progression. We present a method for automated tracking of lineages of ...proliferative, migrating cells from a sequence of images. The method is applicable to image sequences gathered either in vitro or in vivo. Currently, generating lineage trees from progenitor cells over time is a tedious, manual process, which limits the number of cell measurements that can be practically analyzed. In contrast, the automated method is rapid and easily applied, and produces a wealth of measurements including the precise position, shape, cell-cell contacts, motility and ancestry of each cell in every frame, and accurate timings of critical events, e.g. mitosis and cell death. Furthermore, it automatically produces graphical output that is immediately accessible. Application to clonal development of mouse neural progenitor cells growing in cell culture reveals complex changes in cell cycle rates during neuron and glia production. The method enables a level of quantitative analysis of cell behavior over time that was previously infeasible.
Neural stem cell activity in the ventricular-subventricular zone (V-SVZ) decreases with aging, thought to occur by a unidirectional decline. However, by analyzing the V-SVZ transcriptome of male mice ...at 2, 6, 18, and 22 months, we found that most of the genes that change significantly over time show a reversal of trend, with a maximum or minimum expression at 18 months. In vivo, MASH1+ progenitor cells decreased in number and proliferation between 2 and 18 months but increased between 18 and 22 months. Time-lapse lineage analysis of 944 V-SVZ cells showed that age-related declines in neurogenesis were recapitulated in vitro in clones. However, activated type B/type C cell clones divide slower at 2 to 18 months, then unexpectedly faster at 22 months, with impaired transition to type A neuroblasts. Our findings indicate that aging of the V-SVZ involves significant non-monotonic changes that are programmed within progenitor cells and are observable independent of the aging niche.
•RNA sequencing analysis of the adult V-SVZ NSC niche at 2, 6, 18, and 22 months•During aging, most V-SVZ niche genes show max/min expression at 18 months•In vivo MASH1+ cells cycle slowest at 18 months but at 22 months return to 2-month rate•Time-lapse analyses of isolated SVZ cells show that age-associated changes are programmed
Temple and colleagues show through a multi-time-point study that age-associated changes in gene expression and cell behavior in the adult V-SVZ are predominantly non-monotonic. While neurogenesis declines with aging, the number and cell division rate of transit-amplifying progenitor cells declines to 18 months and then surprisingly increases between 18 and 22 months. Furthermore, they demonstrate that these behaviors are recapitulated in single progenitor cells growing in clonal culture, indicating that age-associated changes are programmed and niche independent.
Tau protein aggregates are a defining neuropathological feature of "tauopathies," a group of neurodegenerative disorders that include Alzheimer's disease. In the current study, we develop a ...Drosophila split-luciferase-based sensor of tau-tau interaction. This model, which we term "tauLUM," allows investigators to quantify tau multimerization at individual time points or longitudinally in adult, living animals housed in a 96-well plate. TauLUM causes cell death in the adult Drosophila brain and responds to both pharmacological and genetic interventions. We find that transgenic expression of an anti-tau intrabody or pharmacological inhibition of HSP90 reduces tau multimerization and cell death in tauLUM flies, establishing the suitability of this system for future drug and genetic modifier screening. Overall, our studies position tauLUM as a powerful in vivo discovery platform that leverages the advantages of the Drosophila model organism to better understand tau multimerization.
During embryonic development, endothelial cells (ECs) undergo vasculogenesis to form a primitive plexus and assemble into networks comprised of mural cell-stabilized vessels with molecularly distinct ...artery and vein signatures. This organized vasculature is established prior to the initiation of blood flow and depends on a sequence of complex signaling events elucidated primarily in animal models, but less studied and understood in humans. Here, we have developed a simple vascular differentiation protocol for human pluripotent stem cells that generates ECs, pericytes, and smooth muscle cells simultaneously. When this protocol is applied in a 3D hydrogel, we demonstrate that it recapitulates the dynamic processes of early human vessel formation, including acquisition of distinct arterial and venous fates, resulting in a vasculogenesis angiogenesis model plexus (VAMP). The VAMP captures the major stages of vasculogenesis, angiogenesis, and vascular network formation and is a simple, rapid, scalable model system for studying early human vascular development in vitro.
Age-related macular degeneration (AMD) is a common cause of central visual loss in the elderly. Retinal pigment epithelial (RPE) cell loss occurs early in the course of AMD and RPE cell ...transplantation holds promise to slow disease progression. We report that subretinal transplantation of RPE stem cell (RPESC)-derived RPE cells (RPESC-RPE) preserved vision in a rat model of RPE cell dysfunction. Importantly, the stage of differentiation that RPESC-RPE acquired prior to transplantation influenced the efficacy of vision rescue. Whereas cells at all stages of differentiation tested rescued photoreceptor layer morphology, an intermediate stage of RPESC-RPE differentiation obtained after 4 weeks of culture was more consistent at vision rescue than progeny that were differentiated for 2 weeks or 8 weeks of culture. Our results indicate that the developmental stage of RPESC-RPE significantly influences the efficacy of RPE cell replacement, which affects the therapeutic application of these cells for AMD.
•Adult RPESC-derived RPE cell transplantation rescues vision in an animal model•Vision rescue depends on differentiation stage of RPESC-derived RPE transplant
Stern and colleagues report that the differentiation state of stem cell-derived retinal pigment epithelial (RPE) cells influences their ability to rescue vison. They transplanted RPE cells differentiated for 2 weeks (immature), 4 weeks (intermediate progenitor stage), or 8 weeks (mature RPE) and found that the intermediate RPE progenitor stage was more effective than immature or mature RPE at vison rescue.
This study used a multiplex high‐throughput gene expression assay that simultaneously detects endogenous expression of multiple developmental, functional, and disease markers in induced pluripotent ...stem (iPS) cell‐derived retinal pigment epithelium (RPE). This assay provides the basis to screen for compounds that improve RPE function and maturation and target disease pathways, thus providing the basis for effective treatments of several retinal degenerative diseases.
There is continuing interest in the development of lineage‐specific cells from induced pluripotent stem (iPS) cells for use in cell therapies and drug discovery. Although in most cases differentiated cells show features of the desired lineage, they retain fetal gene expression and do not fully mature into “adult‐like” cells. Such cells may not serve as an effective therapy because, once implanted, immature cells pose the risk of uncontrolled growth. Therefore, there is a need to optimize lineage‐specific stem cell differentiation protocols to produce cells that no longer express fetal genes and have attained “adult‐like” phenotypes. Toward that goal, it is critical to develop assays that simultaneously measure cell function and disease markers in high‐throughput format. Here, we use a multiplex high‐throughput gene expression assay that simultaneously detects endogenous expression of multiple developmental, functional, and disease markers in iPS cell‐derived retinal pigment epithelium (RPE). We optimized protocols to differentiate iPS cell‐derived RPE that was then grown in 96‐ and 384‐well plates. As a proof of principle, we demonstrate differential expression of eight genes in iPS cells, iPS cell‐derived RPE at two different differentiation stages, and primary human RPE using this multiplex assay. The data obtained from the multiplex gene expression assay are significantly correlated with standard quantitative reverse transcription‐polymerase chain reaction‐based measurements, confirming the ability of this high‐throughput assay to measure relevant gene expression changes. This assay provides the basis to screen for compounds that improve RPE function and maturation and target disease pathways, thus providing the basis for effective treatments of several retinal degenerative diseases.
During CNS development, cell migrations play an important role, adding to the cellular complexity of different regions. Earlier studies have shown a robust migration of cells from basal forebrain ...into the overlying dorsal forebrain during the embryonic period. These immigrant cells include GABAergic neurons that populate the cerebral cortex and hippocampus. In this study we have examined the fate of other basal forebrain cells that migrate into the dorsal forebrain, identifying basal cells using an antibody that recognizes both early (dlx1/2) and late (dlx 5/6) members of the dlx homeobox gene family. We found that a subpopulation of cortical and hippocampal oligodendrocytes are also ventral-derived. We traced the origin of these cells to basal multipotent stem cells capable of generating both GABAergic neurons and oligodendrocytes. A clonal analysis showed that basal forebrain stem cells produce significantly more GABAergic neurons than dorsal forebrain stem cells from the same embryonic age. Moreover, stem cell clones from basal forebrain are significantly more likely to contain both GABAergic neurons and oligodendrocytes than those from dorsal. This indicates that forebrain stem cells are regionally specified. Whereas dlx expression was not detected within basal stem cells growing in culture, these cells produced dlx-positive products that are capable of migration. These data indicate that the developing cerebral cortex incorporates both neuronal and glial products of basal forebrain and suggest that these immigrant cells arise from a common progenitor, a dlx-negative basal forebrain stem cell.
Numb and Numblike, conserved homologs of
Drosophila Numb, have been implicated in cortical neurogenesis; however, analysis of their involvement in later stages of cortical development has been ...hampered by early lethality of double mutants in previous studies. Using Emx1
IREScre to induce more restricted inactivation of Numb in the dorsal forebrain of
numblike null mice beginning at E9.5, we have generated viable double mutants that displayed striking brain defects. It was thus possible to examine neurogenesis during the later peak phase (E12.5–E16.5). Loss of Numb and Numblike in dorsal forebrain resulted in neural progenitor hyperproliferation, delayed cell cycle exit, impaired neuronal differentiation, and concomitant defects in cortical morphogenesis. These findings reveal novel and essential function of Numb and Numblike during the peak period of cortical neurogenesis. Further, these double mutant mice provide an unprecedented viable animal model for severe brain malformations due to defects in neural progenitor cells.
More than 50 mutations in the
gene result in heterogeneous forms of frontotemporal lobar dementia with tau inclusions (FTLD-Tau). However, early pathogenic events that lead to disease and the degree ...to which they are common across
mutations remain poorly understood. The goal of this study is to determine whether there is a common molecular signature of FTLD-Tau.
We analyzed genes differentially expressed in induced pluripotent stem cell-derived neurons (iPSC-neurons) that represent the three major categories of
mutations: splicing (IVS10 + 16), exon 10 (p.P301L), and C-terminal (p.R406W) compared with isogenic controls. The genes that were commonly differentially expressed in
IVS10 + 16, p.P301L, and p.R406W neurons were enriched in trans-synaptic signaling, neuronal processes, and lysosomal function. Many of these pathways are sensitive to disruptions in calcium homeostasis. One gene,
, was significantly reduced across the three
mutant iPSC-neurons and in a mouse model of tau accumulation. We observed a significant reduction in calcium levels in
mutant neurons compared with isogenic controls, pointing to a functional consequence of this disrupted gene expression. Finally, a subset of genes commonly differentially expressed across
mutations were also dysregulated in brains from
mutation carriers and to a lesser extent in brains from sporadic Alzheimer disease and progressive supranuclear palsy, suggesting that molecular signatures relevant to genetic and sporadic forms of tauopathy are captured in a dish. The results from this study demonstrate that iPSC-neurons capture molecular processes that occur in human brains and can be used to pinpoint common molecular pathways involving synaptic and lysosomal function and neuronal development, which may be regulated by disruptions in calcium homeostasis.
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