Self-renewal of a purified Tie2+ hematopoietic stem cell population relies on mitochondrial clearance

Abstract

A single hematopoietic stem cell (HSC) is capable of reconstituting hematopoiesis and maintaining homeostasis by balancing self-renewal and cell differentiation. The mechanisms of HSC division balance, however, are not yet defined. Here we demonstrate, by characterizing at the single-cell level a purified and minimally heterogeneous murine Tie2⁺ HSC population, that these top hierarchical HSCs preferentially undergo symmetric divisions. The induction of mitophagy, a quality control process in mitochondria, plays an essential role in self-renewing expansion of Tie2⁺ HSCs. Activation of the PPAR (peroxisome proliferator-activated receptor)-fatty acid oxidation pathway promotes expansion of Tie2⁺ HSCs through enhanced Parkin recruitment in mitochondria. These metabolic pathways are conserved in human TIE2⁺ HSCs. Our data thus identify mitophagy as a key mechanism of HSC expansion and suggest potential methods of cell-fate manipulation through metabolic pathways.

Fig. 1. Tie2-GFP marks a distinct HSC subset

(A) LTC-IC frequencies of Tie2-GFP⁺ and Tie2-GFP⁻ HSCs were determined in a limiting-dilution assay (LDA). (B) Percentages of successful long-term reconstitution. (C) In vivo imaging of single Tie2-GFP⁺ HSCs immediately after local delivery in the calvarium. (Left) Top (upper) and side (lower) views of the delivery site. The arrow indicates the opening to the bone marrow. (Right) In vivo imaging of the same cell, 24 hours after local delivery (en face maximum intensity projection). (D) Single-cell gene expression analysis using the BioMark System array (Fluidigm) (see materials and methods).

Fig. 2. The PPAR-FAO pathway activates mitochondrial autophagy in HSCs

(A) Parkin and Pink1 levels in GW501516-treated (GW) Tie2-GFP⁺ HSCs. (B and C) PPARδ activators, GW501516 or L-165041 (L165), enhance colocalization of Tom20 with LAMP1. Treatment with carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used as a control for mitophagy induction [(B), left]. Mitochondrial net flux was calculated by Tom20-accumulation in the presence of leupeptin (Leup) [(B), right]. Representative immunofluorescence (IF) images of colocalized Tom20 with LAMP1 [4′,6-diamidino-2-phenylindole (DAPI), blue; Tom20, red; LAMP1, green; Merge, yellow] in PPARδ-agonist–treated HSCs. White dashed boxes represent the magnified images shown at right. Scale bars, 2 µm (C). (D) Representative images of GW501516-treated HSCs immunostained to label mtDNA (left) and quantified for mitophagy [12 hours oligomycin and antimycin A] (right top) and net mtDNA flux (right bottom). In (A), (B), and (D), error bars represent mean ± SEM (at least 10 cells analyzed per each condition per each experiment).

Fig. 3. Parkin is important for PPAR-FAO–induced mitophagy in HSCs

(A) Relative Tom20 colocalization (left) and mitochondria net flux (right) in GW501516-treated HSCs in the presence of siPark2. (B) Quantification of mitophagy based on the remaining mtDNA staining 12 hours after treatment with oligomycin and antimycin A. (C) Pink1-silencing reduces PPARδ-enhanced mitophagy. Relative Tom20 colocalization (top left), net mitochondria flux (top right), and representative IF images depicting LAMP1 and Tom20 colocalization in GW501516-treated HSCs in the presence of siPink1 (bottom). (D) GW501516 enhances colocalization of TOM20 with PARKIN (left) and increases mitochondrial net flux (right). Scale bars in (C) and (D), 2 µm.

Fig. 4. Self-renewing expansion of Tie2⁺ HSC through mitophagy

(A) Representative pictures of Tie2-GFP positivity, with bright-field images (BF) collected during symmetric division. (B) Representative repopulation kinetics of SD during the first and second bone marrow transplantation (BMT). PB-MNCs, peripheral blood mononuclear cells. (C) Division patterns of Tie2-GFP⁺ HSCs (left) and Tie2-GFP⁻ HSCs (right), in terms of reconstitution capacity. SD, symmetric division; AD, asymmetric division; SC, symmetric commitment. (D) Single Tie2-GFP⁺ HSC transplantation without supporting cells. Survival of recipient mice was examined by plotting Kaplan-Meier survival curves (also see fig. S13B). (E) PPARδ activation induces in vivo expansion of Tie2-GFP⁺ HSCs. The number of transplanted Tie2-GFP⁺ HSCs (donor cells) is denoted by the dashed line marked “Original” (also see fig. S13H). (F) Frequency of successful hematopoietic reconstitution of shParkin-silenced Tie2-GFP⁺ HSCs (also see fig. S13J).