Single-cell analyses identify bioengineered niches for enhanced maintenance of hematopoietic stem cells

Abstract

The in vitro expansion of long-term hematopoietic stem cells (HSCs) remains a substantial challenge, largely because of our limited understanding of the mechanisms that control HSC fate choices. Using single-cell multigene expression analysis and time-lapse microscopy, here we define gene expression signatures and cell cycle hallmarks of murine HSCs and the earliest multipotent progenitors (MPPs), and analyze systematically single HSC fate choices in culture. Our analysis revealed twelve differentially expressed genes marking the quiescent HSC state, including four genes encoding cell–cell interaction signals in the niche. Under basal culture conditions, most HSCs rapidly commit to become early MPPs. In contrast, when we present ligands of the identified niche components such as JamC or Esam within artificial niches, HSC cycling is reduced and long-term multipotency in vivo is maintained. Our approach to bioengineer artificial niches should be useful in other stem cell systems.

Simultaneous tracking of division and differentiation from individual hematopoietic stem and progenitor cells reveals within-family homogeneity despite population heterogeneity

“…At the level of individual families, we observed substantial heterogeneity in division history ( Figure 2C), from families that did not proliferate to those with cells that had undergone 6 divisions. Consistent with earlier observations [27], after 24h most cells either remained undivided or were in generation one, with a few cells having undergone two or more divisions (Figure 2A). At 48h over 90% of the cells had undergone at least one division.…”

Long‐Term Perfusion Culture of Monoclonal Embryonic Stem Cells in 3D Hydrogel Beads for Continuous Optical Analysis of Differentiation

“…Deciphering the heterogeneity of a cell population plays an important role in understanding the mechanisms of cellular behavior by replacing ensemble measurements with insight into the dynamics of single cells or small subpopulations . Soluble factors, interactions between the extracellular matrix (ECM) and the cell, cell–cell interactions, as well as physical influences (e.g., shear stress, matrix elasticity) collectively determine cell signaling and behavior . For example, the 3D culture of cancer cell lines was shown to result in i) drastic changes in gene expression profiles when compared to 2D culture and ii) a change of phenotypic behavior upon stimulation with different drugs .…”

Cell division history determines hematopoietic stem cell potency

“…To investigate HSC division dynamics further we also sought to determine how the physical structure of the niche affects HSC behaviour. To do so we manufactured a bespoke polyethylene glycol (PEG) microwell culture system, which provides a softer environment with a high water content that more closely matches the physical characteristics of the in vivo HSC niche than 10/45 standard culture conditions (Gobaa et al 2011, Lutolf et al 2009, Roch et al 2017. Similar niche constructs have been shown to support stem cell self-renewal in other contexts (Gilbert et al 2010).…”

“…PEG-based hydrogel microwells were manufactured as previosuly described (Roch et al 2017). Briefly, arrays of hydrogel microwells were fabricated using standard photolithography to etch the microwell pattern into a silicon substrate onto which liquid thermocurable poly(dimethylsiloxane) (PDMS) was polymerized.…”