Evgenia Verovskaya

Counting stem cells: methodological constraints

The number of stem cells contributing to hematopoiesis has been a matter of debate. Many studies use retroviral tagging of stem cells to measure clonal contribution. Here we argue that methodological factors can impact such clonal analyses. Whereas early studies had low resolution, leading to underestimation, recent methods may result in an overestimation of stem-cell counts. We discuss how restriction enzyme choice, PCR bias, high-throughput sequencing depth and tagging method could affect the conclusions of clonal studies.

Asymmetry in skeletal distribution of mouse hematopoietic stem cell clones and their equilibration by mobilizing cytokines

Upon transplant, functional HSC clones preferentially expand in certain skeletal locations, exhibiting only limited migration toward other niches.

Barcoded Vector Libraries and Retroviral or Lentiviral Barcoding of Hematopoietic Stem Cells

Cellular barcoding is a relatively recent technique aimed at clonal analysis of a proliferating cell population of any kind. The method was shown to be particularly successful in monitoring clonal contributions of hematopoietic stem cells (HSCs). An essential step of the method is retroviral or lentiviral labeling of the hematopoietic cells. The unique feature of the method is the generation of a vector library containing specific artificial DNA tags, generally known as barcodes. The library must satisfy multiple essential requirements. Importantly, considering the number of possible variations within the barcode sequence, the actual size of the barcoded vector library, and the number of clonogenic (stem) cells in the given experiment should be in ratios far from saturation. Excessive bias in barcodes frequencies must be avoided, and the library size must be assessed prior to the sequencing analysis. The final sequencing results must undergo statistical filtering. If all requirements are met, the method ensures profound sensitivity and accuracy for monitoring of the clonal fluctuations in a wide range of biological experiments.

Noncanonical Wnt Comes of Age in Hematopoietic Stem Cells

Understanding molecular mechanisms of aging is crucial in efforts to reverse it. In a recent issue of Nature, Florian et al. (2013) report that increasing levels of noncanonical Wnt signaling accompany hematopoietic stem cell (HSC) aging, which can be modulated to functionally rejuvenate HSCs.

Aging of Murine Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) are unique in their ability to self-renew and differentiate into all mature blood lineages. The equilibrium between these processes is crucial for tissue maintenance during the lifetime of the organism. However, with age the functionality of HSCs declines, resulting in development of anemias, deficiencies of immune response, and increased risk of hematopoietic malignancies. Aged HSCs are characterized by preferential differentiation toward myeloid lineage, impaired self-renewal, and engraftment. Recent evidence provides clues to the understanding of these processes on cellular and molecular levels. Key components contributing to stem cell aging are shifts in the transcriptome and epigenome, accompanied by dysfunction of DNA repair pathways. In this chapter we will focus on studies and conceptual models of murine HSC aging.