Long‐term mid‐onset dietary restriction rejuvenates hematopoietic stem cells and improves regeneration capacity of total bone marrow from aged mice

Abstract Currently, the world's aging population is expanding rapidly, leading to a rise in aged hematopoietic cell transplantation (HCT) recipients and aged donors. However, the age of donors is negatively related to the prognosis after transplantation due to functional decline in hematopoietic stem cells (HSCs) during aging. Previously, we showed that an early‐onset dietary restriction (DR) significantly retards early aging of HSCs. However, the effects of a mid‐onset DR on HSCs remain unknown. In the current study, we performed 30% DR in 15‐ to 18‐month‐old mice (equivalent to 50–60 human years) for short‐term (4 months) and long‐term (9 months). We show that DR reduces and rectifies the imbalance of the HSC pool in aged mice. Short‐term DR improves hematopoietic reconstitution in purified HSC transplantations, but not in bone marrow transplantations. Intriguingly, long‐term mid‐onset DR improves the hematopoietic regeneration of aging HSCs with a particular enhancement of lymphoid outputs even in total bone marrow transplantation settings. Mechanistically, long‐term DR rejuvenates the aberrantly regulated mitochondrial pathways in aging HSCs and is accompanied by increased quiescence and reduced DNA damage signaling in HSCs. Short‐term DR showed a similar trend of rescuing these aging hallmarks but to a much lesser extent. Together, the current study suggests that mid‐onset DR ameliorates the function of aging HSCs and long‐term DR even improved hematopoietic reconstitution in bone marrow transplantation, which could potentially have considerable implications in HCT of humans when only old donors are available.


| INTRODUC TI ON
Hematopoietic cell transplantation (HCT) is a key treatment for curing many hematopoietic malignancies. The world's aging population has been expanded remarkably, resulting in a rise in aged HCT recipients and donors (Visram et al., 2018). However, for every 10-year increment over 60 years in donor age, there is a 5.5% increase in the hazard ratio for overall mortality after HCT (Kollman et al., 2016).
Previously, we have shown that an early-onset dietary restriction (DR) improved HSC repopulating capacity in early aging of C57BL/6 J mice (Tang et al., 2016). However, the effects of a mid-onset DR on sorted HSCs and unpurified hematopoietic cells which are dominantly used in clinical HCT have not been reported so far.
In this study, we analyzed the effects of a 30% DR regimen applied for short-term (4 months) and long-term (9 months) in C57BL/6 J mice starting at 15-18 months of age (the equivalent change in humans would roughly be 50-60 years (Dutta & Sengupta, 2016)) on the function of HSCs and bone marrow cells in transplantations as well as in undisturbed conditions.

| Short-term DR rejuvenates aged HSCs but only long-term DR benefits in bone marrow transplantation
Short-term DR significantly reduced the size of HSC pool in the old mice, in particular, the myeloid-biased HSCs (CD150 high HSCs and CD41 + HSCs), whereas the lymphoid-biased HSCs (CD150 low HSCs and CD41 − HSCs) was not altered . DR also significantly rescued the aging-associated change in frequencies of LT-HSCs but failed to rescue the diminishment of LMPPs (Figure 1e).
To study the impact of DR on HSC function, purified HSCs from the above treated mice were transplanted along with competitor cells.
Interestingly, DR-derived HSCs led to higher chimerisms in whole white blood cells (WBC) and B lymphocytes, and the ratio of chimerism in B cells/chimerism in myeloid cells, with similar chimerisms in myeloid cells . However, clinical HCT dominantly use unpurified hematopoietic cells as graft sources. Although short-term DR improves regeneration of HSCs per se, it also reduces the frequency of HSCs which may have negative effect on HCT (Figure 1a). Of note, the short-term DR showed neutral effects in bone marrow transplantations (BMT), indicating that the functional improvement achieved by short-term DR could not compensate reduced frequency of HSCs (Figure 1j,k). We further performed another competitive BMT using bigger number of bone marrow cells from DR donors to reach same amount of HSCs to be transplanted as from AL donors. In this experiment, DR donor-derived bone marrow generated higher chimerisms in lymphoid and myeloid cells in competitive serial transplantations ( Figure S1).
We further studied the effect of a longer-term (9 months) DR on aged mice. Comparing to short-term DR, long-term DR showed similar effects on HSCs and progenitors and significant reduction of CD150 low HSCs (Figure 1l-p). Intriguingly, long-term DR increased the chimerisms in both B cells and myeloid cells in competitive BMT ( Figure 1q-x). Furthermore, DR donor generated higher ratio of chimerism in B cells/chimerism in myeloid cells (Figure 1t). Notably, the beneficial effect on lymphocytes starts as early as 1 month after BMT, which is essential in early anti-infection activities post-transplantation ( Figure 1q,u).

| DR ameliorates development of aging hallmarks in HSCs
Quiescence is an essential mechanism to protect HSCs against proliferation-induced damage and deteriorations in their functionality (Hu & Wang, 2019;Walter et al., 2015;Wilson et al., 2008). Exit from quiescent stage leads to accumulation of DNA damage and functional decline in HSCs (Walter et al., 2015). HSCs from DR mice showed a significant increase in the percentage at G0 phase and a decrease in the G1 phase and, correlatively, reduced formation of γ-H2AX foci and expression of p21 (Figure 2a-g). HSC quiescence is mediated by mitochondrial unfolded protein response (UPR mt ) which is increased upon elevated mitochondrial protein folding stress (PFS mt ) during aging (Mohrin et al., 2015;Mohrin, Widjaja, Liu, Luo, & Chen, 2018). Furthermore, with age, reduced Sirt2 expression and increased mitochondrial stress lead to aberrant activation of NLRP3 and caspase 1, which impairs HSC regenerative capacity (Luo et al., 2019). Notably, long-term DR remarkably rejuvenated expression of UPR mt genes (C1pP, HSP10 and HSP60), the UPR mt upstream suppressor SIRT7, and the SIRT2-NLRP3-caspase 1 axis in aging HSCs to levels approaching that of young HSCs; short-term DR also showed a similar trend of rescuing these aging hallmarks but to a much lesser extent (Figure 2h-n). In line with that, aging-associated increase of caspase 1-induced-pyroptosis F I G U R E 1 Short-term DR rejuvenates aged HSCs but only long-term DR benefits in bone marrow transplantation. Mice were fed with DR or AL diet for 4 months (OAL 4 m/ODR 4 m, a-k) or 9 months (OAL 9 m/ODR 9 m, l-x). (a-e and l-p) Frequencies of indicated populations in 4 months (a-e) or 9 months (l-p) treated mice determined by FACS (n = 4-7). (f-k and q-x) Competitive transplantation analysis. Donor-derived chimerisms and representative FACS plots in peripheral blood and bone marrow in recipients after HSC transplantation (f,g,i, n = 7-8 recipient mice per group) or bone marrow transplantation (j,k, short-term treatment, n = 5-11 recipient mice per group; (q-s and u-x) long-term treatment, n = 6-11 recipient mice per group). (h and t) The ratio of donor-derived chimerism in B cells versus myeloid cells in short-term HSCT (h) and long-term BMT (t). Results were displayed as mean ± SD. ns, not significant; *p < 0.05; **p < 0.01;***p < 0.001;****p < 0.0001. (f-h,j,k,q-t) Unpaired two-tailed Student's t test; (a,c-e,l,n-p) one-way ANOVA. Data show results from 2 independent experiments. YAL, young control was decreased under DR (Figure 2o,q). The rate of DAPI + cells was not changed by DR (Figure 2p).

| DR suppresses B lymphopoiesis in donor mice in undisturbed conditions
We further analyzed the effect of DR on downstream lineages in donor mice in undisturbed conditions. DR showed minor effects on bone marrow cells, peripheral blood WBCs, and myeloid lineages but the B lymphoid lineage and proliferation of lymphoid progenitors were severely suppressed, similar to our previous findings in an early-onset DR ( Figure S2a-x) (Tang et al., 2016). The ratio of lymphoid versus myeloid cells (L/M) in peripheral blood was decreased during aging and was not rescued by DR ( Figure S2y).  libitum, as what we have shown previously (Tang et al., 2016).
Mechanistically, we show that long-term DR rejuvenates the aberrantly regulated mitochondrial pathways in aging HSCs and is accompanied by increased quiescence and reduced DNA damage signaling in HSCs. Short-term DR showed a similar trend of rescuing these aging hallmarks but to a much lesser extent.

| E XPERIMENTAL PROCEDURE S
Experimental procedures are available online as supporting information.

| Mice and dietary restriction
C57BL/6 J mice were obtained from Hunan SJA Laboratory Animal Co. Ltd. and maintained in the animal facilities of Nanchang Royo Biotech under pathogen-free conditions on a 12-h light/12-h dark cycle at 23°C-25°C. DR was performed as previously described (Tang et al., 2016). Briefly, mice were housed individually and fed with a timed daily supply of food-70% of the normal food amount that the body weight-and gender-matched ad libitum (AL) mice consumed. For the AL group, mice were fed with an unlimited amount of food. All mouse experiments were approved by the Animal Experimental Ethical Inspection of Nanchang Royo Biotech Co. Ltd (RYEI20170513-1). Three-month-old mice were used as young control, and DR was initiated from 15-18 months of age (the equivalent change in humans would roughly be 50-60 years (Dutta & Sengupta, 2016)).

| Flow cytometry and HSC sorting
The following combinations were used for indicated populations: HSCs ( For HSC sorting, BM cells were pre-enriched for c-Kit + cells as described previously (Tang et al., 2016). All FACS gating strategies are shown in Figure S3.

| Immunostaining
Cells were sorted and stained as previously described (Ema et al., 2006). Briefly, cells were FACS-purified and dropped on poly-L-lysine-coated glass slides (Shanghai JingAn Biological) and fixed with 4% paraformaldehyde for 10 min at room temperature (RT), followed by permeabilization in 0.25% Triton/PBS for 10 min at RT and a blocking with 1% BSA/PBS for 1 h at RT. Then, cells were incubated with primary antibody anti-phospho-Histone H2AX (Ser139) Antibody (Merck) at 1:500 dilution overnight at 4°C followed by an incubation with secondary antibody anti-mouse Alexa Fluor488 (Invitrogen) for 1 h at RT. To visualize the nuclei, the cells were counterstained by DAPI. Images were acquired on a Leica SP5 fluorescent microscope and processed by LAS-AF-Lite_2.6.0. One hundred and fifty HSCs from 3 samples per group were scored blindly, and foci were counted manually according to previously published protocols (Gutierrez-Martinez et al., 2018).

| RNA Isolation, cDNA Synthesis, and Quantitative Real-Time PCR
Total RNA was isolated from freshly sorted cells by using RNAsimple Total RNA Kit (TianGen Biotech). TransScript-Uni One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech) was used for reverse transcriptions. qRT-PCR was performed with an ABI 7900 Real-Time PCR System (Applied Biosystems) and TransStart Tip Green qPCR SuperMix (TransGen Biotech). Relative expression of genes was normalized to β-actin in each sample and was calculated using the ΔCt method. Primer sets are listed in Table S1.

| Peripheral blood cell counting
Peripheral blood was collected from the orbital venous plexus containing 5 μl EDTA (0.5 M). PB cell count was performed on automated hematology analyzer (Sysmex, XS-500i) according to the manufacturer's instructions.

| Statistics
GraphPad Prism 7.0 software was used for statistical analysis. The unpaired two-tailed Student's t test was used for two-group datasets, and one-way ANOVA or two-way ANOVA was used for multi-group datasets to calculate p-values. All results were displayed as mean ±SD. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

CO N FLI C T S O F I NTE R E S T
The authors declare no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analyzed in this study.