Hematopoietic stem cells (HSCs) are responsible for long-term maintenance and regeneration of the hematopoietic system. Loss of long-term (LT)-HSC function is a major contributor to decline in hematopoietic function with aging, leading to increased risk of infection, poor vaccination response, and increased susceptibility to hematologic malignancies. A number of LT-HSC-intrinsic alterations and LT-HSC-extrinsic changes in the bone marrow (BM) microenvironment have been associated with functional decline in aged LT-HSCs. Outstanding questions in the field are which of these mechanisms cause, rather than are a consequence of, physiological aging and what mechanism(s) represent key therapeutic targets to extend healthy LT-HSC function in older age?
Previously, we took the novel approach of examining LT-HSC frequency and function in mice at a wide array of ages with the rationale that interventions to extend LT-HSC function will be most effective starting at or before the age of onset of functional hematopoietic decline. We found that canonical markers of LT-HSC aging significantly accumulate by middle age (9-12mo) in C57BL/6 mice, including increased phenotypic LT-HSC frequency, reduced regenerative capacity, myeloid lineage bias at both transcriptional and functional levels, increased gH2.AX staining, and loss of polarity of CDC42 and tubulin. Furthermore, we found by reciprocal transplantation studies of young LT-HSCs into middle-aged recipient mice and middle-aged LT-HSCs into young recipient mice that LT-HSC-extrinsic changes in the middle-aged BM microenvironment were necessary and sufficient to cause LT-HSC aging. By transcriptome analysis, we identified decreased IGF1 signaling in LT-HSCs as a candidate mechanism causing LT-HSC aging.
Here, we systematically identify mesenchymal stromal cells (MSCs) as the major local producer of IGF1 in the BM of young mice and determine that this production is diminished by middle age. To evaluate the specific effect of MSC-produced IGF1 on LT-HSCs, we co-cultured LT-HSCs with Igf1conditional knockout MSCs, which was found to cause increased differentiation of LT-HSCs toward myeloid progenitor cells. In vivo, reduced IGF signaling in the BM microenvironment was modeled by transplantation of wild-type BM cells into Igf1 conditional knockout recipients, which phenocopied myeloid-biased hematopoiesis as observed in middle-aged mice. A similar myeloid-biased hematopoiesis phenotype was observed upon transplant of Igf1r conditional knockout LT-HSCs into wild-type recipients, supporting a model of direct communication between MSCs and LT-HSCs via IGF1 signaling.
To determine whether restoration of IGF1 signaling had the capacity to rejuvenate middle-aged LT-HSCs, we applied short-term (24hr) in vitro treatment of recombinant IGF1. IGF1 treatment restored polarity of CDC42 and tubulin, decreased gH2.AX staining, increased in vivo regenerative capacity, and increased lymphoid differentiation potential of middle-aged LT-HSCs at the transcriptional and functional levels. Furthermore, increasing local IGF1 levels in the middle-aged BM microenvironment by transient intrafemoral injection (once per week for 4 wks) restored mature B cell frequency and decreased mature myeloid cell frequency in the BM and peripheral blood, rebalancing the composition of hematopoietic cells to that observed in young mice.
At a mechanistic level, short-term IGF1 stimulation of middle-aged LT-HSCs was found to induce phosphorylation of IGF1R and the downstream signaling mediator AKT, and transcriptionally activate PI3K/AKT target genes. Open chromatin profiling by ATAC-seq revealed altered chromatin organization in middle-aged LT-HSCs in response to IGF1 stimulation, altering chromatin accessibility at promoters of genes enriched in intracellular and protein localization, regulation of transport and sequence-specific DNA binding, and enrichment of regulatory regions containing E2F2 binding sites.
In summary, our data strongly support that decreased local production of IGF1 in the BM microenvironment causes many hallmarks of LT-HSC aging. We propose that restoration of local IGF1 signaling, or its downstream target pathways, represents an attractive prophylactic strategy for extending hematopoietic healthspan into older age.
Trowbridge:Fate Therapeutics: Patents & Royalties: patent license.
Asterisk with author names denotes non-ASH members.