Purification and characterization of human neural stem and progenitor cells.

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.

EpoR stimulates rapid cycling and larger red cells during mouse and human erythropoiesis.

The erythroid terminal differentiation program couples sequential cell divisions with progressive reductions in cell size. The erythropoietin receptor (EpoR) is essential for erythroblast survival, but its other functions are not well characterized. Here we use Epor-/- mouse erythroblasts endowed with survival signaling to identify novel non-redundant EpoR functions. We find that, paradoxically, EpoR signaling increases red cell size while also increasing the number and speed of erythroblast cell cycles. EpoR-regulation of cell size is independent of established red cell size regulation by iron. High erythropoietin (Epo) increases red cell size in wild-type mice and in human volunteers. The increase in mean corpuscular volume (MCV) outlasts the duration of Epo treatment and is not the result of increased reticulocyte number. Our work shows that EpoR signaling alters the relationship between cycling and cell size. Further, diagnostic interpretations of increased MCV should now include high Epo levels and hypoxic stress.

Reprogramming progressive cells display low CAG promoter activity.

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.

Resolving Cell Cycle Speed in One Snapshot with a Live-Cell Fluorescent Reporter.

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.

The palette of techniques for cell cycle analysis.

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.

YAP Non-cell-autonomously Promotes Pluripotency Induction in Mouse Cells.

Yes-associated protein (YAP) is known to promote the stemness of multiple stem cell types, including pluripotent stem cells, while also antagonizing pluripotency during early embryogenesis. How YAP accomplishes these distinct functions remains unclear. Here, we report that, depending on the specific cells in which it is expressed, YAP could exhibit opposing effects on pluripotency induction from mouse somatic cells. Specifically, YAP inhibits pluripotency induction cell-autonomously but promotes it non-cell-autonomously. For its non-cell-autonomous role, YAP alters the expression of many secreted and matricellular proteins, including CYR61. YAP's non-cell-autonomous promoting effect could be recapitulated by recombinant CYR61 and abrogated by CYR61 depletion. Thus, we define a YAP-driven effect on enhancing pluripotency induction largely mediated by CYR61. Our work highlights the importance of considering the distinct contributions from heterologous cell types in deciphering cell fate control mechanisms and calls for careful re-examination of the co-existing bystander cells in complex cultures and tissues.

Publisher Correction: MLL-AF9 initiates transformation from fast-proliferating myeloid progenitors.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

MLL-AF9 initiates transformation from fast-proliferating myeloid progenitors.

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.

Cell cycle dynamics in the reprogramming of cellular identity.

Reprogramming of cellular identity is fundamentally at odds with replication of the genome: cell fate reprogramming requires complex multidimensional epigenomic changes, whereas genome replication demands fidelity. In this review, we discuss how the pace of the genome's replication and cell cycle influences the way daughter cells take on their identity. We highlight several biochemical processes that are pertinent to cell fate control, whose propagation into the daughter cells should be governed by more complex mechanisms than simple templated replication. With this mindset, we summarize multiple scenarios where rapid cell cycle could interfere with cell fate copying and promote cell fate reprogramming. Prominent examples of cell fate regulation by specific cell cycle phases are also discussed. Overall, there is much to be learned regarding the relationship between cell fate reprogramming and cell cycle control. Harnessing cell cycle dynamics could greatly facilitate the derivation of desired cell types.

MKL1-actin pathway restricts chromatin accessibility and prevents mature pluripotency activation.

Actin cytoskeleton is well-known for providing structural/mechanical support, but whether and how it regulates chromatin and cell fate reprogramming is far less clear. Here, we report that MKL1, the key transcriptional co-activator of many actin cytoskeletal genes, regulates genomic accessibility and cell fate reprogramming. The MKL1-actin pathway weakens during somatic cell reprogramming by pluripotency transcription factors. Cells that reprogram efficiently display low endogenous MKL1 and inhibition of actin polymerization promotes mature pluripotency activation. Sustained MKL1 expression at a level seen in typical fibroblasts yields excessive actin cytoskeleton, decreases nuclear volume and reduces global chromatin accessibility, stalling cells on their trajectory toward mature pluripotency. In addition, the MKL1-actin imposed block of pluripotency can be bypassed, at least partially, when the Sun2-containing linker of the nucleoskeleton and cytoskeleton (LINC) complex is inhibited. Thus, we unveil a previously unappreciated aspect of control on chromatin and cell fate reprogramming exerted by the MKL1-actin pathway.