Anna Elizabeth Eastman

All Publications

• Resolving Cell Cycle Speed in One Snapshot with a Live-Cell Fluorescent Reporter. Cell reports Eastman, A. E., Chen, X., Hu, X., Hartman, A. A., Pearlman Morales, A. M., Yang, C., Lu, J., Kueh, H. Y., Guo, S. 2020; 31 (12): 107804

  Abstract

  Cell proliferation changes concomitantly with fate transitions during reprogramming, differentiation, regeneration, and oncogenesis. Methods to resolve cell cycle length heterogeneity in real time are currently lacking. Here, we describe a genetically encoded fluorescent reporter that captures live-cell cycle speed using a single measurement. This reporter is based on the color-changing fluorescent timer (FT) protein, which emits blue fluorescence when newly synthesized before maturing into a red fluorescent protein. We generated a mouse strain expressing an H2B-FT fusion reporter from a universally active locus and demonstrate that faster cycling cells can be distinguished from slower cycling ones on the basis of the intracellular fluorescence ratio between the FT's blue and red states. Using this reporter, we reveal the native cell cycle speed distributions of fresh hematopoietic cells and demonstrate its utility in analyzing cell proliferation in solid tissues. This system is broadly applicable for dissecting functional heterogeneity associated with cell cycle dynamics in complex tissues.

  View details for DOI 10.1016/j.celrep.2020.107804

  View details for PubMedID 32579930

  View details for PubMedCentralID PMC7418154

• The palette of techniques for cell cycle analysis. FEBS letters Eastman, A. E., Guo, S. 2020

  Abstract

  The cell division cycle is the generational period of cellular growth and propagation. Cell cycle progression needs to be highly regulated to preserve genomic fidelity while increasing cell number. In multicellular organisms, the cell cycle must also coordinate with cell fate specification during development and tissue homeostasis. Altered cell cycle dynamics play a central role also in a number of pathophysiological processes. Thus, extensive effort has been made to define the biochemical machineries that execute the cell cycle and their regulation, as well as implementing more sensitive and accurate cell cycle measurements. Here, we review the available techniques for cell cycle analysis, revisiting the assumptions behind conventional population-based measurements and discussing new tools to better address cell cycle heterogeneity in the single-cell era. We weigh the strengths, weaknesses, and trade-offs of methods designed to measure temporal aspects of the cell cycle. Finally, we discuss emerging techniques for capturing cell cycle speed at single-cell resolution in live animals.

  View details for DOI 10.1002/1873-3468.13842

  View details for PubMedID 32441778

• Modeling Glioma Intratumoral Heterogeneity with Primary Human Neural Stem and Progenitor Cells. bioRxiv : the preprint server for biology Gao, D., Liu, D. D., Eastman, A. E., Womack, N. L., Ohene-Gambill, B. F., Baez, M., Weissman, I. L. 2024

  Abstract

  Glioblastoma multiforme (GBM) is a deadly form of glioma notable for its significant intratumoral heterogeneity, which is believed to drive therapy resistance. GBM has been observed to mimic a neural stem cell hierarchy reminiscent of normal brain development. However, it is still unclear how cell-of-origin shapes intratumoral heterogeneity. Here, we develop a model of glioma initiation using neural stem and progenitor cells (NSPCs) purified from fetal human brain tissue. We previously described a method to prospectively isolate and culture tripotent neural stem cells (NSCs), bipotent glial progenitor cells (GPCs), and unipotent oligodendrocyte precursor cells (OPCs). We transduced these isogenic lines with dominant-negative TP53R175H and NF1 knockdown, a commonly-used genetic model of GBM in mice. These reprogrammed lines robustly engrafted when transplanted into the brains of immunodeficient mice, and showed significant expansion over time. Engrafted cells were reextracted from the mouse brain for single cell RNA sequencing (scRNA-seq), in order to quantify how the cell-of-origin modulates the cellular subtypes found in the resulting tumor. This result revealed the strong influence the cell-of-origin plays in glioma heterogeneity. Our platform is highly adaptable and allows for modular and systematic interrogation of how cell-of-origin shape the tumor landscape.

  View details for DOI 10.1101/2024.10.20.619254

  View details for PubMedID 39484434

• Purification and characterization of human neural stem and progenitor cells. Cell Liu, D. D., He, J. Q., Sinha, R., Eastman, A. E., Toland, A. M., Morri, M., Neff, N. F., Vogel, H., Uchida, N., Weissman, I. L. 2023; 186 (6): 1179

  Abstract

  The human brain undergoes rapid development at mid-gestation from a pool of neural stem and progenitor cells (NSPCs) that give rise to the neurons, oligodendrocytes, and astrocytes of the mature brain. Functional study of these cell types has been hampered by a lack of precise purification methods. We describe a method for prospectively isolating ten distinct NSPC types from the developing human brain using cell-surface markers. CD24-THY1-/lo cells were enriched for radial glia, which robustly engrafted and differentiated into all three neural lineages in the mouse brain. THY1hi cells marked unipotent oligodendrocyte precursors committed to an oligodendroglial fate, and CD24+THY1-/lo cells marked committed excitatory and inhibitory neuronal lineages. Notably, we identify and functionally characterize a transcriptomically distinct THY1hiEGFRhiPDGFRA- bipotent glial progenitor cell (GPC), which is lineage-restricted to astrocytes and oligodendrocytes, but not to neurons. Our study provides a framework for the functional study of distinct cell types in human neurodevelopment.

  View details for DOI 10.1016/j.cell.2023.02.017

  View details for PubMedID 36931245

• Reprogramming progressive cells display low CAG promoter activity STEM CELLS Hu, X., Wu, Q., Zhang, J., Kim, J., Chen, X., Hartnnan, A. A., Eastnnan, A. E., Park, I., Guo, S. 2021; 39 (1): 43-54

  Abstract

  There is wide variability in the propensity of somatic cells to reprogram into pluripotency in response to the Yamanaka factors. How to segregate these variabilities to enrich for cells of specific traits that reprogram efficiently remains challenging. Here we report that the variability in reprogramming propensity is associated with the activity of the MKL1/SRF transcription factor and concurs with small cell size as well as rapid cell cycle. Reprogramming progressive cells can be prospectively identified by their low activity of a widely used synthetic promoter, CAG. CAGlow cells arise and expand during cell cycle acceleration in the early reprogramming culture of both mouse and human fibroblasts. Our work illustrates a molecular scenario underlying the distinct reprogramming propensities and demonstrates a convenient practical approach for their enrichment.

  View details for DOI 10.1002/stem.3295

  View details for Web of Science ID 000585046600001

  View details for PubMedID 33075202

  View details for PubMedCentralID PMC7821215

• MLL-AF9 initiates transformation from fast-proliferating myeloid progenitors NATURE COMMUNICATIONS Chen, X., Burkhardt, D. B., Hartman, A. A., Hu, X., Eastman, A. E., Sun, C., Wang, X., Zhong, M., Krishnaswamy, S., Guo, S. 2019; 10: 5767

  Abstract

  Cancer is a hyper-proliferative disease. Whether the proliferative state originates from the cell-of-origin or emerges later remains difficult to resolve. By tracking de novo transformation from normal hematopoietic progenitors expressing an acute myeloid leukemia (AML) oncogene MLL-AF9, we reveal that the cell cycle rate heterogeneity among granulocyte-macrophage progenitors (GMPs) determines their probability of transformation. A fast cell cycle intrinsic to these progenitors provide permissiveness for transformation, with the fastest cycling 3% GMPs acquiring malignancy with near certainty. Molecularly, we propose that MLL-AF9 preserves gene expression of the cellular states in which it is expressed. As such, when expressed in the naturally-existing, rapidly-cycling immature myeloid progenitors, this cell state becomes perpetuated, yielding malignancy. In humans, high CCND1 expression predicts worse prognosis for MLL fusion AMLs. Our work elucidates one of the earliest steps toward malignancy and suggests that modifying the cycling state of the cell-of-origin could be a preventative approach against malignancy.

  View details for DOI 10.1038/s41467-019-13666-5

  View details for Web of Science ID 000503222500003

  View details for PubMedID 31852898

  View details for PubMedCentralID PMC6920141

• Single-cell RNA sequencing reveals metallothionein heterogeneity during hESC differentiation to definitive endoderm STEM CELL RESEARCH Lu, J., Baccei, A., da Rocha, E., Guillermier, C., McManus, S., Finney, L. A., Zhang, C., Steinhauser, M. L., Li, H., Lerou, P. H. 2018; 28: 48-55

  Abstract

  Differentiation of human pluripotent stem cells towards definitive endoderm (DE) is the critical first step for generating cells comprising organs such as the gut, liver, pancreas and lung. This in-vitro differentiation process generates a heterogeneous population with a proportion of cells failing to differentiate properly and maintaining expression of pluripotency factors such as Oct4. RNA sequencing of single cells collected at four time points during a 4-day DE differentiation identified high expression of metallothionein genes in the residual Oct4-positive cells that failed to differentiate to DE. Using X-ray fluorescence microscopy and multi-isotope mass spectrometry, we discovered that high intracellular zinc level corresponds with persistent Oct4 expression and failure to differentiate. This study improves our understanding of the cellular heterogeneity during in-vitro directed differentiation and provides a valuable resource to improve DE differentiation efficiency.

  View details for DOI 10.1016/j.scr.2018.01.015

  View details for Web of Science ID 000428799700011

  View details for PubMedID 29427839

• Influence of <i>ATM</i>-Mediated DNA Damage Response on Genomic Variation in Human Induced Pluripotent Stem Cells STEM CELLS AND DEVELOPMENT Lu, J., Li, H., Baccei, A., Sasaki, T., Gilbert, D. M., Lerou, P. H. 2016; 25 (9): 740-747

  Abstract

  Genome instability is a potential limitation to the research and therapeutic application of induced pluripotent stem cells (iPSCs). Observed genomic variations reflect the combined activities of DNA damage, cellular DNA damage response (DDR), and selection pressure in culture. To understand the contribution of DDR on the distribution of copy number variations (CNVs) in iPSCs, we mapped CNVs of iPSCs with mutations in the central DDR gene ATM onto genome organization landscapes defined by genome-wide replication timing profiles. We show that following reprogramming the early and late replicating genome is differentially affected by CNVs in ATM-deficient iPSCs relative to wild-type iPSCs. Specifically, the early replicating regions had increased CNV losses during retroviral (RV) reprogramming. This differential CNV distribution was not present after later passage or after episomal reprogramming. Comparison of different reprogramming methods in the setting of defective DDR reveals unique vulnerability of early replicating open chromatin to RV vectors.

  View details for DOI 10.1089/scd.2015.0393

  View details for Web of Science ID 000374854000007

  View details for PubMedID 26935587

  View details for PubMedCentralID PMC4854209

• Multi-Scale Imaging and Informatics Pipeline for In Situ Pluripotent Stem Cell Analysis PLOS ONE Gorman, B. R., Lu, J., Baccei, A., Lowry, N. C., Purvis, J. E., Mangoubi, R. S., Lerou, P. H. 2014; 9 (12): e116037

  Abstract

  Human pluripotent stem (hPS) cells are a potential source of cells for medical therapy and an ideal system to study fate decisions in early development. However, hPS cells cultured in vitro exhibit a high degree of heterogeneity, presenting an obstacle to clinical translation. hPS cells grow in spatially patterned colony structures, necessitating quantitative single-cell image analysis. We offer a tool for analyzing the spatial population context of hPS cells that integrates automated fluorescent microscopy with an analysis pipeline. It enables high-throughput detection of colonies at low resolution, with single-cellular and sub-cellular analysis at high resolutions, generating seamless in situ maps of single-cellular data organized by colony. We demonstrate the tool's utility by analyzing inter- and intra-colony heterogeneity of hPS cell cycle regulation and pluripotency marker expression. We measured the heterogeneity within individual colonies by analyzing cell cycle as a function of distance. Cells loosely associated with the outside of the colony are more likely to be in G1, reflecting a less pluripotent state, while cells within the first pluripotent layer are more likely to be in G2, possibly reflecting a G2/M block. Our multi-scale analysis tool groups colony regions into density classes, and cells belonging to those classes have distinct distributions of pluripotency markers and respond differently to DNA damage induction. Lastly, we demonstrate that our pipeline can robustly handle high-content, high-resolution single molecular mRNA FISH data by using novel image processing techniques. Overall, the imaging informatics pipeline presented offers a novel approach to the analysis of hPS cells that includes not only single cell features but also colony wide, and more generally, multi-scale spatial configuration.

  View details for DOI 10.1371/journal.pone.0116037

  View details for Web of Science ID 000347119100090

  View details for PubMedID 25551762

  View details for PubMedCentralID PMC4281228

• The Distribution of Genomic Variations in Human iPSCs Is Related to Replication-Timing Reorganization during Reprogramming CELL REPORTS Lu, J., Li, H., Hu, M., Sasaki, T., Baccei, A., Gilbert, D. M., Liu, J. S., Collins, J. J., Lerou, P. H. 2014; 7 (1): 70-78

  Abstract

  Cell-fate change involves significant genome reorganization, including changes in replication timing, but how these changes are related to genetic variation has not been examined. To study how a change in replication timing that occurs during reprogramming impacts the copy-number variation (CNV) landscape, we generated genome-wide replication-timing profiles of induced pluripotent stem cells (iPSCs) and their parental fibroblasts. A significant portion of the genome changes replication timing as a result of reprogramming, indicative of overall genome reorganization. We found that early- and late-replicating domains in iPSCs are differentially affected by copy-number gains and losses and that in particular, CNV gains accumulate in regions of the genome that change to earlier replication during the reprogramming process. This differential relationship was present irrespective of reprogramming method. Overall, our findings reveal a functional association between reorganization of replication timing and the CNV landscape that emerges during reprogramming.

  View details for DOI 10.1016/j.celrep.2014.03.007

  View details for Web of Science ID 000334298200009

  View details for PubMedID 24685138

  View details for PubMedCentralID PMC4133748