Diverse partial reprogramming strategies restore youthful gene expression and transiently suppress cell identity.

Partial pluripotent reprogramming can reverse features of aging in mammalian cells, but the impact on somatic identity and the necessity of individual reprogramming factors remain unknown. Here, we used single-cell genomics to map the identity trajectory induced by partial reprogramming in multiple murine cell types and dissected the influence of each factor by screening all Yamanaka Factor subsets with pooled single-cell screens. We found that partial reprogramming restored youthful expression in adipogenic and mesenchymal stem cells but also temporarily suppressed somatic identity programs. Our pooled screens revealed that many subsets of the Yamanaka Factors both restore youthful expression and suppress somatic identity, but these effects were not tightly entangled. We also found that a multipotent reprogramming strategy inspired by amphibian regeneration restored youthful expression in myogenic cells. Our results suggest that various sets of reprogramming factors can restore youthful expression with varying degrees of somatic identity suppression. A record of this paper's Transparent Peer Review process is included in the supplemental information.

Differentiation reveals latent features of aging and an energy barrier in murine myogenesis.

Skeletal muscle experiences a decline in lean mass and regenerative potential with age, in part due to intrinsic changes in progenitor cells. However, it remains unclear how age-related changes in progenitors manifest across a differentiation trajectory. Here, we perform single-cell RNA sequencing (RNA-seq) on muscle mononuclear cells from young and aged mice and profile muscle stem cells (MuSCs) and fibro-adipose progenitors (FAPs) after differentiation. Differentiation increases the magnitude of age-related change in MuSCs and FAPs, but it also masks a subset of age-related changes present in progenitors. Using a dynamical systems approach and RNA velocity, we find that aged MuSCs follow the same differentiation trajectory as young cells but stall in differentiation near a commitment decision. Our results suggest that differentiation reveals latent features of aging and that fate commitment decisions are delayed in aged myogenic cells in vitro.

Semisupervised adversarial neural networks for single-cell classification.

Annotating cell identities is a common bottleneck in the analysis of single-cell genomics experiments. Here, we present scNym, a semisupervised, adversarial neural network that learns to transfer cell identity annotations from one experiment to another. scNym takes advantage of information in both labeled data sets and new, unlabeled data sets to learn rich representations of cell identity that enable effective annotation transfer. We show that scNym effectively transfers annotations across experiments despite biological and technical differences, achieving performance superior to existing methods. We also show that scNym models can synthesize information from multiple training and target data sets to improve performance. We show that in addition to high accuracy, scNym models are well calibrated and interpretable with saliency methods.

Deep Convolutional and Recurrent Neural Networks for Cell Motility Discrimination and Prediction.

Cells in culture display diverse motility behaviors that may reflect differences in cell state and function, providing motivation to discriminate between different motility behaviors. Current methods to do so rely upon manual feature engineering. However, the types of features necessary to distinguish between motility behaviors can vary greatly depending on the biological context, and it is not always clear which features may be most predictive in each setting for distinguishing particular cell types or disease states. Convolutional neural networks (CNNs) are machine learning models allowing for relevant features to be learned directly from spatial data. Similarly, recurrent neural networks (RNNs) are a class of models capable of learning long term temporal dependencies. Given that cell motility is inherently spacio-temporal data, we present an approach utilizing both convolutional and long- short-term memory (LSTM) recurrent neural network units to analyze cell motility data. These RNN models provide accurate classification of simulated motility and experimentally measured motility from multiple cell types, comparable to results achieved with hand-engineered features. The variety of cell motility differences we can detect suggests that the algorithm is generally applicable to additional cell types not analyzed here. RNN autoencoders based on the same architecture are capable of learning motility features in an unsupervised manner and capturing variation between myogenic cells in the latent space. Adapting these RNN models to motility prediction, RNNs are capable of predicting muscle stem cell motility from past tracking data with performance superior to standard motion prediction models. This advance in cell motility prediction may be of practical utility in cell tracking applications.

Aging induces aberrant state transition kinetics in murine muscle stem cells.

Murine muscle stem cells (MuSCs) experience a transition from quiescence to activation that is required for regeneration, but it remains unknown if the trajectory and dynamics of activation change with age. Here, we use time-lapse imaging and single cell RNA-seq to measure activation trajectories and rates in young and aged MuSCs. We find that the activation trajectory is conserved in aged cells, and we develop effective machine-learning classifiers for cell age. Using cell-behavior analysis and RNA velocity, we find that activation kinetics are delayed in aged MuSCs, suggesting that changes in stem cell dynamics may contribute to impaired stem cell function with age. Intriguingly, we also find that stem cell activation appears to be a random walk-like process, with frequent reversals, rather than a continuous linear progression. These results support a view of the aged stem cell phenotype as a combination of differences in the location of stable cell states and differences in transition rates between them.

Murine single-cell RNA-seq reveals cell-identity- and tissue-specific trajectories of aging.

Aging is a pleiotropic process affecting many aspects of mammalian physiology. Mammals are composed of distinct cell type identities and tissue environments, but the influence of these cell identities and environments on the trajectory of aging in individual cells remains unclear. Here, we performed single-cell RNA-seq on >50,000 individual cells across three tissues in young and old mice to allow for direct comparison of aging phenotypes across cell types. We found transcriptional features of aging common across many cell types, as well as features of aging unique to each type. Leveraging matrix factorization and optimal transport methods, we found that both cell identities and tissue environments exert influence on the trajectory and magnitude of aging, with cell identity influence predominating. These results suggest that aging manifests with unique directionality and magnitude across the diverse cell identities in mammals.

Inferring cell state by quantitative motility analysis reveals a dynamic state system and broken detailed balance.

Cell populations display heterogeneous and dynamic phenotypic states at multiple scales. Similar to molecular features commonly used to explore cell heterogeneity, cell behavior is a rich phenotypic space that may allow for identification of relevant cell states. Inference of cell state from cell behavior across a time course may enable the investigation of dynamics of transitions between heterogeneous cell states, a task difficult to perform with destructive molecular observations. Cell motility is one such easily observed cell behavior with known biomedical relevance. To investigate heterogenous cell states and their dynamics through the lens of cell behavior, we developed Heteromotility, a software tool to extract quantitative motility features from timelapse cell images. In mouse embryonic fibroblasts (MEFs), myoblasts, and muscle stem cells (MuSCs), Heteromotility analysis identifies multiple motility phenotypes within the population. In all three systems, the motility state identity of individual cells is dynamic. Quantification of state transitions reveals that MuSCs undergoing activation transition through progressive motility states toward the myoblast phenotype. Transition rates during MuSC activation suggest non-linear kinetics. By probability flux analysis, we find that this MuSC motility state system breaks detailed balance, while the MEF and myoblast systems do not. Balanced behavior state transitions can be captured by equilibrium formalisms, while unbalanced switching between states violates equilibrium conditions and would require an external driving force. Our data indicate that the system regulating cell behavior can be decomposed into a set of attractor states which depend on the identity of the cell, together with a set of transitions between states. These results support a conceptual view of cell populations as dynamical systems, responding to inputs from signaling pathways and generating outputs in the form of state transitions and observable motile behaviors.