After HLA matching, donor age is generally the most critical factor determining survival after HSCT11. Grafts from older donors have been associated with increased graft failure even when controlling for cell number39,40, which may relate to declining inherent stem cell quality1–5, 41. Increased incidence of graft versus host disease (GVHD) in recipients of grafts from older donors has also been observed10,42, potentially related to an increase in antigen-experienced lymphocytes with age43,44 or the general increase in low-level inflammation that characterizes aging45. However, neither of these associations show consistent relationships with HSCT outcome10,46, and a combination of several age-related factors likely contribute. For these reasons, transplant physicians tend to favor younger donors, but finding the appropriately matched donor can sometimes be difficult or impossible. The probability of finding an unrelated matched donor varies with ethnicity, and can be especially problematic for some ethnic groups, e.g. African-Americans47. Elderly family members would have strong motivation to donate if suitably matched, and strategies to enhance aged grafts could help increase the possibility of success.
While hematopoietic malignancies treatable by autologous transplant increase in prevalence with age, elderly patients are often not eligible due to the rigors of the HSCT process as well as their own declining HSC quality. However, recent strategies employing reduced-intensity conditioning for elderly patients, as well as advances in transplant technique and supportive care, increasingly enable allogeneic and autologous HSCT in this population48–50. The ability to augment the function of aged grafts prior to infusion could facilitate successful, life-saving autologous transplants for older patients. Here we found that ex vivo pulse exposure to dmPGE2 can enhance the transplantation capacity of murine HSCs of advanced age. Previous work in young grafts has shown this effect to translate from zebrafish and mice18,19 to non-human primates51,52 and ultimately to enhancement of human cord blood transplantation21. Thus, the current findings have a high likelihood of translation.
PGE2 is an eicosanoid synthesized within most body tissues by many different cell types, acting in autocrine or paracrine fashion53. Activities mediated by PGE2 are highly pleiotropic, depending on the tissue/cell type and expression of its four G-protein coupled receptors EP1-435,54. EP1 signals primarily through PKC and Ca2+ mobilization, EP2 and EP4 induce cAMP production and subsequent cAMP response element-binding protein (CREB) activation as observed here, and EP3 inhibits cAMP production35,54. EP4 has been recognized as a key functional regulator for HSCs, including transplantation studies32,33. In the setting of radiation exposure, where dmPGE2 protects and enhances HSC function36, only dmPGE2 or EP4 agonism conferred survival from lethal irradiation55. In the current transcriptomic analysis, the predominance of CREB1-induced gene expression following dmPGE2 pulse in both young and old HSCs, along with strong EP4 expression but undetectable EP2 mRNA levels as observed here and previously by RNA-seq of purified murine pHSCs36, strongly supports EP4 as the relevant receptor mediating HSC enhancement regardless of age.
An interesting finding in this study was increased EP4 expression in HSCs with age. PGE2 production is known to increase with age in macrophages56 and decrease with age in gastrointestinal tissues57. In skeletal muscle, the capacity for PGE2 synthesis increases with age while receptor levels are downregulated58. It remains unclear if basal PGE2 levels change with age within the BM, and which factors would drive the increase in EP4 expression on HSCs. The intensity of CREB1-regulated genes was noticeably higher in old HSCs after dmPGE2 pulse and may be related to the number of EP4 receptors, though higher basal expression of these genes was also seen in control HSCs in a variable manner between aged mice (Fig. 4B). Thus, the change in CREB1-regulated genes was greater in some old mice but not in others, and the relevance of increased EP4 expression on aged HSCs remains uncertain. Ultimately, this investigation established that HSCs of advanced age do not lose expression or signaling through the pivotal EP4 receptor.
While the primary objective of these studies was not to compare old versus young HSC function, but rather to evaluate the effect of dmPGE2 on old grafts in parallel with young grafts, the transplantation experiments were performed simultaneously with the same cohorts of recipients and competitor cells. The studies indicated that the old and young grafts functioned similarly in regard to overall long-term and serial chimerism capacity. Since the old grafts contained approximately 24-fold higher pHSC frequency, and equivalent numbers of WBM cells were transplanted, these observations are in line with the reported substantial decrease in function of pHSCs with age2,3,6,7. We also observed myeloid-skewed reconstitution from aged HSCs as described2–4, 6,30. Interestingly, dmPGE2 pulse consistently increased the relative myeloid contribution of the young donor cells, bringing their lineage ratios closer to those of the aged. However, dmPGE2 also increased the overall frequencies of donor lymphoid cells in comparison to competitors, suggesting dmPGE2 has a positive effect on both major immune cell branches but augments myeloid reconstitution to a greater degree. DmPGE2 also augmented both branches for old HSCs compared to competitors without further affecting the inherent myeloid skew with age. Of interest, we have previously reported an increase in the proportion of myeloid cells in PB of mice transplanted with young HSCs pulsed with dmPGE2 following primary and secondary transplant, however this was not consistent across tertiary and quaternary transplants and was without overall effect on the enhancement of induced stem cell competitiveness59.
Bioinformatic analysis of dmPGE2 signaling in young versus old HSCs revealed that the core response pathways remained largely unchanged with age. Several age-independent genes were identified as both increased by CREB1 signaling and involved in the significantly predicted functions of ‘Cell survival’ and ‘Cellular homeostasis’. Many of these genes additionally have described roles in hematopoiesis, supporting their involvement in HSC modulation by dmPGE2. Vegfa encodes for vascular endothelial growth factor A (VEGF-A) which, while first discovered for its primary role in angiogenesis60, enhances human HPC formation61, promotes hematopoietic cell generation from embryonic stem cells of both mouse62 and human63, and regulates HSC survival64. Cebpb is the gene for CCAAT enhancer binding protein beta (C/EBPβ), a transcription factor that promotes lymphopoiesis65 and emergency myelopoiesis66,67 at the level of stem and progenitor cell regulation68,69. Gadd45b, encoding growth arrest and DNA-damage-inducible beta (GADD45β), appears essential for DNA damage protection and survival of HSCs/HPCs and induced pluripotent stem cells (iPSCs) under stress70. Cdkn1a encodes the cyclin-dependent kinase inhibitor P21, which preserves HSC quiescence under stress and promotes HSC self-renewal in serial transplantation71, while Nr4a2 encoding the transcription factor nuclear receptor subfamily 4 group A member 2, also known as NURR1, also attenuates HSC cycling72 and may contribute to the maintenance of stem cell quiescence during the stress of transplantation.
In addition to transplantation, steady-state hematopoiesis in older humans is subject to increased bone marrow failure and decreased hematologic tolerance of cytotoxic injury, as well as the increased propensity for myeloproliferative disorders and cancerous transformation8,9. A broader understanding of aged HSC function is essential to development of novel treatments for hematopoietic compromise in the elderly. The high-throughput genomic comparison of old and young HSC responses to dmPGE2 provided a unique modality for investigating changes in HSC stimulation response pathways with age. Several signaling alterations identified here may have relevance for targeting in treatment of age-induced HSC defects.
Genes differentially affected by dmPGE2 in young and old HSCs included numerous phosphoproteins induced in young but not in old, many of which were already elevated with age. IPA did not return any significant predictions for a common upstream regulator controlling these genes (no more than 5 had shared association with any given regulator), but functional categories including ‘Alternative splicing’ exhibited significant enrichment (Table 1). Abnormalities in alternative splicing have been implicated in the development of myeloproliferative disorders that increase in prevalence with age, with over 50% of myelodysplastic syndromes harboring spliceosome factor mutations in the dominant clone37,38. The current analysis suggests dmPGE2-responsive genes that become less responsive with age tend to be those with alternative splice variants, and tend to have higher mRNA levels detectable in old HSCs pre-stimulation (Table 2). However, the current experimental design did not distinguish between splice variants, and further investigation is needed to determine whether these transcripts could be affected by dysregulated splicing in HSCs of advanced age.
Several specific genes were identified as significantly oppositely affected by dmPGE2 stimulation in old versus young HSCs, revealing divergent molecular responses potentially related to aging defects. Ccbe1 and Rorb were particularly elevated with age and strongly decreased by dmPGE2 only in old HSCs. Ccbe1, encoding for collagen and calcium binding EGF domains 1 (CCBE1), is a secreted protein thought to function in remodeling of extracellular matrix and cell migration, and is an important factor in lymphangiogenesis73–75. It is also an essential mediator of erythroblastic island formation for erythropoiesis in fetal liver, though it is not required for postnatal erythropoiesis76. This gene has otherwise not been associated with hematopoiesis, and gene expression levels found here in young HSCs were near-zero at baseline with a very slight elevation by dmPGE2. However, the Ccbe1 transcript was much more detectable in aged HSCs and was strongly and consistently downregulated by dmPGE2 stimulation. Thus, transcription of Ccbe1 appears to be ‘turned on’ in HSCs by an unknown aging factor that may be sensitive to ‘turning back off’ by dmPGE2 signaling. However, a potential role for this protein in aged HSCs remains to be explored.
A more substantial target may be Rorb, which encodes for RAR related orphan receptor B (RORβ), a member of the highly conserved ROR family of receptor tyrosine kinases77. These kinases, including RORβ, are known to negatively regulate WNT/B-catenin signaling78,79, an important facilitator of HSC fate decisions80. In the context of dmPGE2 stimulation, dmPGE2 enhanced WNT signaling during zebrafish embryogenesis and was required for WNT-mediated regulation of HSC development81. In addition, RORβ is elevated with age in marrow-derived osteoprogenitor cells, contributing to development of osteoporosis79,82. Our study reveals that RORβ is elevated with age in HSCs, and decreased in response to dmPGE2 in an age-dependent manner. Rora and Rorc also exhibited unique expression patterns affected by both age and dmPGE2 treatment (Fig. 5B). Together, these findings may indicate a novel mechanism of age-associated dysregulation of HSC fate decisions through increased ROR expression, and reveal an intriguing avenue for downregulation of RORβ in aged HSCs through the PGE2 signaling pathway.
In conclusion, this study has identified that aged HSCs primarily retain the molecular capacity to respond to dmPGE2 pulse exposure and initiate transcriptional programs enhancing survival and long-term repopulating function, which has potential importance toward the goal of enhancing aged human grafts for transplantation. Moreover, age-related alterations in HSC signaling in response to PGE2 were identified as potential targets for treatment of age-related defects.