Rejuvenation of three germ layers tissues by exchanging old blood plasma with saline-albumin

Introduction

Heterochronic parabiosis has been used for decades in laboratory animals to investigate the effects of shared blood, organs and environmental enrichment, on the surgically connected partners [1, 2, 3–5]. The general conclusion of these studies was that the old partners had better health and/or repair of cartilage, muscle, liver, brain, spinal cord, kidneys, bone, skin, etc., and often the young animals experienced premature aging of their respective tissues [1, 3, 4, 6–8]. More recently, blood exchange studies demonstrated that blood heterochronicity alone, without the parabiotic sharing of organs or environment, quickly rejuvenates muscle and liver, but interestingly, not hippocampal neurogenesis or cognitive performance of old mice, and that young mice exhibit rapid and significant decline in hippocampal neurogenesis, agility, learning and liver regeneration after a single exchange with old blood [9].

Historically, the phenomena of heterochronic parabiosis and blood exchange remained unconfirmed with respect to the key assumption as to whether the addition of young factors is needed for rejuvenation, and if premature aging of young mice stemmed from the introduction of old blood factors or a simple dilution of young factors. To answer these questions in a well-controlled experimental set-up, we took advantage of our recently developed small animal blood exchange model [9].

Specifically, we performed a “neutral” blood exchange (NBE) by replacing the platelet-rich-plasma (PRP), fraction with physiological saline, supplemented with 5% purified commercial (fraction V) albumin (e.g. replenishing the depletion of blood albumin). Through a half-hour long series of small volume exchanges, 50% of the PRP of old and young mice was replaced with saline plus 5% mouse albumin while the circulating red and white blood cells were returned isochronically to the animal.

Young (2-4 months) and old (22-24 months) male C57/B6 mice underwent a single NBE; and, as a control for the procedure, we performed isochronic blood exchanges: young – young (YY) and old – old (OO) [9]. Subsequent analyses of muscle repair, liver adiposity and fibrosis, hippocampal neurogenesis and serum proteomics were all done at 6 days after a single NBE.

The procedure of small animal blood exchange is time consuming and labor intensive: blood vessels of mice are cannulated and checked for patency, the next day animals are checked for catheter patency again, and then undergo the exchange under general anesthesia. Of note, two mice are used for each NBE where one mouse provides freshly derived synchronic syngeneic blood cells, which are resuspended in saline + albumin, for the second animal that undergoes the NBE. We thus calculate the minimal number of experimental animals that is needed for statistically significant conclusions and allow for feasible experimental work.

A common error is to base the statistical significance of an observation on whether the sample size is large. The statistical significance for independence between samples or a measurement of statistical power (Power) is not solely dependent on the number of samples (N) but is measured through the effect size (ES) and Variance.

The metric used to determine the amount of statistical power in normal distributions is defined as:

$\begin{array}{c}X~N(0,1):\mu =0,{\sigma }^2=1(\text{normal,}\text{i}\text{.i}\text{.d}\text{.}\text{assumption})\\ n=\text{sample}\text{size},\widehat{\sigma }=\text{sample}\text{variance},\alpha =0.05\\ S_n^{(1)}=\left\{s_1,s_2,\dots ,s_n\right\},S_n^{(2)}=\left\{s_1,s_2,\dots ,s_n\right\}\\ \text{ES}(\text{effect}\text{size})=\frac{1}{n}\left({\displaystyle \sum _{i=1}^n{S}_n^{(1)}}-{\displaystyle \sum _{i=1}^n{S}_n^{(2)}}\right)\\ \text{critical}\text{valus}=\left|\text{X}\text{.ppf}(\frac{\alpha }{2})\right|=\left|{\Phi }^{-1}(\frac{\alpha }{2})\right|\\ \text{Power}=1-\text{X}\text{.cdf}\left(\text{critical}\text{value}-\frac{\text{ES}}{\widehat{\sigma }/\sqrt{n}}\right)\end{array}$

In Figure 1 below, we simulate power by modulating effect size and plotting these against the number of samples. A power of 0.8 is a high threshold standard of robust studies; lesser power is often used in published work and more than 0.8 power comes at a disproportionate cost of resources and time (diminishing returns of logistic function) [10, 11]. As shown in Figure 1, when the Variance of independent experiments is low and the difference in ES between the cohorts is high, e.g. as in our 2003-2020, including published and current studies on young, old and rejuvenated old cohorts [1–3, 4, 9] there is no difference in Power between N=4, 8, 19, or infinity. In contrast, in the absence of robust phenotypes, if two phenomena are detected as the same or have very modest effect, with high enough N the statistical power will strictly increase. Of note, this approach applies only to theoretical data obtained with no loss in precision, no detraction from statistical power and no preference. In the real-world this can permit not just detection of weak phenomena, but also overfitting and accumulation of artifacts that are misinterpreted as biological phenomena at some higher values of N.

Based on the above analysis and preliminary YY, OO, YNBE and ONBE work with N=3 for each cohort, a Power of 0.8 and higher and robust significance of our conclusions was obtainable with N=4. We set up 4 of each isochronic YY and OO exchanges, which is a control repeat of the same studies (exchange process and analyses of muscle, liver, and hippocampal neurogenesis) that we previously published with 8 of YY and OO mice [9]. These were done in parallel with the new NBE studies. In work on repair of injured muscle, we performed four independent characterizations of adult myogenesis: regenerative index, fibrosis, minimum Feret diameter and myogenic proliferation with data agreement between all and between the mouse and human studies. In liver studies, we performed two characterizations of liver health: adiposity and fibrosis, latter was assayed by two independent methods; the data agreement was seen in 8 independent start-to-finish experiments with each cohort. In the studies of hippocampal neurogenesis, we performed 6-7 independent start-to-finish experiments with each cohort and these are in agreement with our in vitro studies with neural precursor cells. Each assay on each sample and tissue was done in multiple replicates. Additional tissues’ samples were used for work on other ideas and hypothesis (in progress). Of note, recent studies, comparable in scope and methods, published in high impact journals, have N =3-4 with even when the ES is lower, and Variance is larger than that of current work [12].

Muscle repair was assayed on the success in formation of new myofibers that replaced injury sites and the degree of fibrosis, both quantitatively assayed by hematoxylin and eosin (H&E) staining, and minimum Feret diameter of newly-formed eMyHC+ muscle fibers was quantified, as we previously published [1, 6, 7, 9, 13, 14]. YY muscles regenerated much better than OO, as expected [1, 9, 14, 15]. Interestingly, a single NBE improved regeneration, reduced fibrosis and increased minimum Feret diameter of de-novo myofibers in old mice to the point of no significant difference with the young, and NBE did not worsen these attributes of young muscle repair, Figure 2A, 2B. Muscle fibrosis was not different between YY and young control mice that did not experience blood exchange or OO and old control mice; regeneration was slightly better in YY than Y and in OO than O cohorts; and YY/Young cohorts had higher regeneration and lower fibrosis than OO/Old cohorts (Supplementary Figure 1).

To confirm these findings and to explore their evolutionary conservation, we took advantage of the fact that there is a procedure for human patients analogous to NBE, where most of the plasma is replaced by physiologic solution supplemented with commercial human albumin, called Therapeutic Plasma Exchange, TPE, which is FDA approved and routinely used in the clinic [16–18]. And so, we studied human blood samples, which were collected Pre and Post TPE, from four old individuals (65-70 years of age). We previously established that mouse primary myoblasts can be used for determining the effects of the proteins that are secreted by human cells on myogenic cell proliferation and differentiation [15, 19, 20]. Here we performed BrdU-based cell proliferation assay in C57.B6 primary myoblasts, which are over 95% pure as per Pax7/Myf5/MyoD markers ([13] and Supplementary Figure 1). These muscle progenitor cells were cultured in OPTI-MEM with 4% PreTPE versus PostTPE human serum for 20 hours in triplicate wells of two replicate 96 well plates (6 replicates total for each cohort, 15,000 cells per well). BrdU was added for the last 6 hours of culture, after which cells were fixed, immuno-detection of BrdU was performed, and high throughput automated microscopy was used for imaging 9 sites of each well and for subsequent data quantification.

As demonstrated in Figure 2, old human serum strongly reduced myogenic cell proliferation (which is consistent with the effects of the old mouse serum [1, 2, 6, 7, 13]). Interestingly, single TPE procedure removed this inhibition and allowed for robust proliferation of myogenic progenitors that were cultured with the PostTPE old serum, e.g. from the same individual(s) whose PreTPE serum was strongly inhibitory.

The positive control for myoblast proliferation (BrdU assay on cells in Growth medium, GM), no-serum control (OPTI-MEM) and the negative control for non-specific fluorescence (BrdU assay on myoblasts in GM that were not treated with BrdU) are shown in Supplementary Figure 1 that demonstrates robust myoblast proliferation in GM, good proliferation in OPTI-MEM and confirms that BrdU immunofluorescence was highly specific.

To start dissecting the possible mechanisms by which exchange of old mammals with saline plus albumin exerts these rejuvenative effects, we examined whether ectopic albumin (human serum albumin, HSA) might be a determinant. First, we performed HSA dose curve, which demonstrated that ectopic albumin does not promote myoblast proliferation (Figure 2F). Based on this dose-curve, we added 4% HSA to our myoblast proliferation assay (where cells were cultured with 4% Pre versus Post TPE human serum). Interestingly, while there was consistently better myoblast proliferation with PostTPE serum as compared to the PreTPE, HSA neither rescued the proliferation of PreTPE myoblast cultures, nor added to the proliferation of the Post-TPE cultures (Figure 2).

Western Blotting confirmed that serum albumin does not decrease with age (Supplementary Figure 2), and there is no net gain of albumin in NBE/TPE, but rather back-supplementation of the procedure depleted serum, albumin. In people, albumin levels correlate with disease, nutrition, and socio-economic status rather than chronological age; and even when health, etc. status are not considered, albumin diminishes only marginally, by 2-4% at 75 years of age from its 26 years of age levels [21–24].

We also looked at the antioxidant properties of PreTPE versus PostTPE serum. Albumin has antioxidant activity [16], and thus we tested if Post TPE serum might promote myogenic cell proliferation simply through improved antioxidant properties. Interestingly, we did not find antioxidative difference between Pre and PostTPE serum (Supplementary Figure 3).

These results establish that NBE and TPE have positive effects on adult myogenesis and that ectopic albumin does not rescue the old serum-imposed inhibition of myogenic proliferation.

In the original work on rejuvenation of old animals and aging of young through heterochronic parabiosis, the effects were shown not only for skeletal muscle, but also for the liver and brain-hippocampal neurogenesis [1, 7]. The hippocampal neurogenesis data was reviewed in Nature in 2004 [7] but it was not published in 2005 [1]; nevertheless, it was repeated and published in Nature in 2011 [25]. Similarly, the effects of heterochronic blood exchange were studied with all these three germ layers derived tissues [9]. Hence, we performed the analyses of hippocampal neurogenesis and liver health (degree of adiposity and fibrosis) to examine whether in addition to skeletal muscle, there might be rejuvenative effects in the absence of young blood.

Neurogenesis in the subgranular zone (SGZ) of the hippocampus continues throughout adult life, dramatically declines with advanced age and is influenced by blood heterochronicity [7, 9, 14, 26]. To assay the effects of NBE on SGZ neurogenesis, proliferation of the neural precursor cells in the SGZ of Dentate Gyrus was quantified throughout the thickness of hippocampi, using immuno-detection of the proliferation marker KI67 in serial brain cryosections (Figure 3A, 3B). YY mice had ~10-fold higher numbers of Ki67+ SGZ cells than old, as expected [9, 15, 26] and notably, there was a significant and large ~8-fold increase in hippocampal neurogenesis in old mice after a single NBE, with no statistical reduction of hippocampal neurogenesis in young mice (Figure 3A, 3B).

This increase was larger than what was observed after heterochronic parabiosis or through defined pharmacology of modulating the TGF-beta and oxytocin pathways [15]. SGZ localization of proliferating cells was used, as typically done, for determination of their neural precursor cell identity [27]; Ki67 was nuclear and non-specific immunofluorescence of isotype-matched IgG controls was negligible (Figure 3 and Supplementary Figure 6.

To continue the study with ectopic albumin, we performed BrdU proliferation assay with neural precursor cells, NPC, which provide a good in vitro correlation to the efficiency of hippocampal neurogenesis [15, 28]. Interestingly, in contrast to the lack of effects on myogenic cells, ectopic albumin enhanced NPC proliferation by itself – in the absence of serum and improved NPC proliferation when old serum was present in the cultures (Supplementary Figure 3B). These results agree with previously published enhancement of proliferation of retinal precursor cells by albumin [29] and with efficient proliferation of human iPSC-derived NPCs on electrospun serum albumin fibrous scaffolds [30].

However, the body of published work consistently demonstrates that albumin is a negative factor for brain health. With respect to reaching neural cells in vivo, Blood Brain Barrier becomes leaky with age [31, 32] and serum albumin crosses it and is found in cerebro-spinal-fluid of older individuals in a positive correlation with age and with certain types of dementias [33]. Moreover, in direct test, infusions of albumin into the brain were deleterious: causing neuro-inflammation, excessive TGF-beta1 and neuronal dysfunctions [34].

In our work the total levels of albumin were not altered, but rather replenished from those diminished by NBE/TPE, thus negative effects on the brain are not anticipated.

To expand our data in muscle (mesoderm) and brain (ectoderm) to an endodermal tissue lineage, and to compare all with the previous experiments on heterochronic parabiosis [1, 7] and heterochronic blood exchange [9], we studied liver adiposity and fibrosis in NBE vs. isochronically blood exchanged mice. Liver adiposity and fibrosis are known to increase with age and to be influenced by blood heterochronicity [1, 9, 14, 35–38].

Liver adiposity, or fat in liver, was assayed by conventionally used Oil Red staining Figure 4, [9, 14, 28, 39] and liver fibrosis was assayed by Albumin/Hoechst immunofluorescence, as published [2, 14] and shown in Supplementary Figure 6, and additionally, by Masson’s trichome (Figure 4). Adiposity and fibrosis were higher in the OO livers as compared to YY, as published [1, 9, 14]. Interestingly, after the NBE these parameters of liver health became much improved in the old mice, and did not worsen in the young mice (Figure 4).

These results show that NBE can supplant for the positive effects of young blood on old liver health, and that a dilution of young blood does not diminish the studied liver health attributes in young mice.

Summarily, these results establish broad tissues rejuvenation by a single replacement of old blood plasma with physiologic fluid: muscle repair was improved, fibrosis was attenuated, and inhibition of myogenic proliferation was switched to enhancement; liver adiposity and fibrosis were reduced; and hippocampal neurogenesis was increased. This rejuvenation is similar to (liver) or is stronger than (muscle and brain) that seen after heterochronic parabiosis or blood exchange.

These findings are most consistent with the conclusion that the age-altered systemic milieu inhibits the health and repair of multiple tissues in the old mice, and also exerts a dominant progeric effect on the young partners in parabiosis or blood exchange.

To address the molecular mechanism of this rapid rejuvenation of all three germ-layers derived tissues, we performed comparative antibody array proteomics on the mouse and human serum that were derived before versus after the NBE/TPE, as well as YY and OO mouse serum (Figure 5). Proteins of low, medium and high levels are detected with high sensitivity (high dynamic range) in this set-up, as demonstrated by Figure 5, Supplementary Figure 4, and Supplementary Table 1.

Multi-dimensional t-SNE analyses and Heatmapping of these data revealed that the ONBE proteome became significantly different from OO and regained some similarities to the YY proteome (Figure 5A–5C). Supplementary Figure 4 confirms the statistical significance of this comparative proteomics through Power Analysis, and shows the YY vs. OO Heatmap, where the age-specific differences are less pronounced than those between OO vs. ONBE, again emphasizing the robust effect of NBE on the molecular composition of the systemic milieu.

As true for the mouse data, human O Before TPE was also robustly different from O After TPE, establishing a significant change in the composition of human circulatory proteins one month after a single procedure (Figure 5D, 5E; and Supplementary Table 1). In an evolutionary-conserved fashion in both mice and humans, angiogenic regulators, immune regulators, and growth factors (including neurogenic ones), were changed in their systemic levels. A few NBE/TPE changed proteins were the same in both species. Very interestingly, even though we diluted blood serum, many of the NBE/TPE modulated proteins were up-regulated (Figure 5, Supplementary Figure 4 and Supplementary Table 1), suggesting that their production or secretion was inhibited by the age-elevated systemic factors in both mice and in people.

An age-associated increase in VCAM-1 was recently suggested to account for the negative circulatory effects on the brain [40]. We observed similar levels of VCAM-1 (p=0.953) between young and old serum which was confirmed by Western Blotting (Supplementary Figure 5), and an elevation (not decrease) in VCAM-1 after the NBE, e.g. when old tissues in mice were rejuvenated (Figure 5, Figures 2–4). Considering that there is little change in VCAM-1 in blood with age, it is unlikely that NBE acts through youthful normalization of this cell-surface protein, which might be shed into the circulation. TPE there was no significant change in VCAM-1 versus before the procedure (p= 0.072675), and similarly, we did not see a difference in CCL11 [25]. Of note, another cell-surface protein was implicated in brain aging, B2M [41], the invariant chain of the MHC class I molecule, which is indicative of broadly diminished inflammation [9, 42]. We have shown that systemic levels of B2M do not increase in the circulation of old mice as compared to young; and an exchange of old mice with young blood diminishes the tissue levels of B2M [9].

This work shifts the paradigm of blood heterochronicity away from dominance of young blood factors and establishes that replacement of a large volume of old blood with a neutral age physiological fluid (saline supplemented with 5% purified albumin), is sufficient for most if not all observed positive effects on muscle, brain and liver. Importantly, it shows that a currently approved FDA procedure promotes molecular and functional rejuvenation of the blood in older people, with improved proteomic profile and support for myogenic responses.

Promising work in progress follows an interesting observation that TPE anecdotally reduced incidents of viral diseases to zero in patients over the course of a year when for just flu-related hospitalizations, ~60% were from the same age group (https://www.cdc.gov/flu/about/season/flu-season-2017-2018.htm). TPE has the potential to improve recovery from viral illnesses and diseases, particularly for older people, through a number of ways, including changes in cytokine, interleukin, growth factor profiles and enhanced immunity, and restoration of virus-diminished oxytocin receptor [43] via productive attenuation of TGF-beta pathway [14]. Better myogenesis, angiogenesis and tissue vascularization in the old are also suggested by our data, hence better overall organ health and repair, better success of vaccination is predicted.

Our pilot longitudinal study with samples from several old individuals highlights the most robust effects of TPE that are observed even with this small sample size, providing insights for future work, in the classic tradition of case and pilot studies. Phase 2B and Phase 3 clinical trials are being actively developed; these will expand our findings and extend these to vaccination outcomes and improved recovery from viral illnesses.

This study does not exclude the possibility that factors that are present in young blood, as well as sharing of young organs and environmental enrichment, might also contribute to the rejuvenative effects of heterochronic parabiosis. It is yet to be determined whether and to what degree NBE/TPE also improve neuronal differentiation, neuroplasticity and cognition, metabolic health more generally than liver adiposity and fibrosis, as well as the health and maintenance of other tissues that have been reported rejuvenated in heterochronic parabioses.

Interestingly, a single NBE does not significantly worsen the examined parameters in young mice; albeit, a higher variation between YNBE mice is detected for all the studied tissues, as compared to the YY cohort. Thus, young animals might be able to offset, at variable individual degrees, one dilution of their platelet-rich-plasma (possibly, upregulating gene expression for a compensatory elevation of protein production and/or secretion). A higher myofiber diameter was detected in YNBE as compared to YY, which might reflect differences in myogenic proliferation and/or differentiation.

The functional phenotypes and comparative systemic proteomics suggest some evolutionary conservation and help to explain the rapid and profound resetting of the systemic milieu accompanied by tissue rejuvenation through exchange of old blood with saline-albumin. We simulate this in Figure 6, where many systemic age-elevated proteins, such as the TGF-beta family [7, 14, 1544–47], are known autoinducers, and where dilution will diminish their expression and feedback will decrease the levels for a prolonged time [48–56]. Consequentially, their secondary targets – other proteins whose expression was inhibited in the old (such as “young” factors needed for tissue maintenance and repair) become derepressed and increase (as we indeed observe for numerous proteins); and entire pathways (MAPK, for example) are no longer hijacked for non-canonical induction of TGF-beta, Smad1, and inflammation [57, 58], but instead engage in productive signaling by their cognate ligands, promoting tissue health, regeneration and homeostatic production of cytokines and growth factors [59–61] (with their own positive feed-backs), as we observe. When the levels of ligand(s) diminish, attenuators also diminish, and receptor(s) increase [62, 63]; signaling re-boot induces negative regulators, as we observed for follistatin and follistatin-like, secondarily diminishing the intensity of age-elevated pathways [64–66]. With variable feedbacks and protein half-lives, changes in every autoinductive protein, secondary target, receptor, etc. are not expected to be simultaneously detected at any given time point, but many are indeed observed at 6 days post NBE and 1 month after TPE.

The above concept fits well with the age-imposed increase in systemic TGF-beta family ligands (GDF11 and TGF-beta 1, for example), which contributes to pro-geronic phenotypes [7, 14, 1544–47] and the fact that attenuation of TGF-beta signaling in old animals has effects that are similar to those of NBE [7, 14, 15, 28, 44]. NBE is also predicted to promote stronger rejuvenation than an Alk5 inhibitor, as that attenuates just one branch of one pathway, and because proteins other than the TGF-beta family that are elevated with age will be re-set to their younger levels of gene expression and/or signaling intensities by NBE/TPE (to be profiled in the future). Fitting the model that is shown in Figure 6 with experimental data on multiple time points after NBE/TPE, for multiple proteins and multiple levels of regulation (mRNA, protein, signaling intensities), is a focus of our long-term work.

It is also quite possible that multiple mechanisms contribute to the rejuvenation of the three germ layer tissues by NBE, with the above described model being just one. For example, while we did not see an effect in myogenesis, ectopic albumin might promote enhanced immunity in NBE/TPE, particularly after multiple in vivo procedures. There was a positive effect of albumin on NPC proliferation, which agrees with published results; and yet delivery of ectopic albumin worsened brain health, and CSF albumin is a marker of brain aging and disease. Overall, it does not seem that albumin is the only determinant of NBE/TPE, but it might have a contribution particularly when the age-elevated factors become diluted.

Of note, we grouped the proteins loosely, as many of them are pleiotropic and play a role in more than one functional group. For instance, lipocalins are homeostatic transporters of lipophilic molecules but also play a role in productive innate immunity [67], and IL-8 receptor beta, also known as CXCR2, plays a role in immune responses, angiogenesis and has a cross-talk with PI3K, p38/ERK, and JAK/Stat signaling pathways [68].

With respect to the proteins that are the same between mice and people, and were modulated in the same direction by the NBE and TPE, an increase in erythropoietin is likely to improve the numbers and health of erythrocytes, attenuating age-imposed anemia [69]. MIP2 controls migration of neutrophils to sites of inflammation, and an increase could help to resolve inflammaging [70]. PF4 promotes platelet aggregation, it is broadly chemotactic, plays a role in wound repair and has anti-microbial activity [71]. IL-8 receptor signaling promotes cell survival, migration, chemotaxis, angiogenesis, oligodendrocyte positioning, and might attenuate neuropathy [72].

Looking at the general protein categories, an increase of angiogenic regulators is expected to broadly contribute to improved vascularization [73, 74] and through this increased perfusion of tissues, to tissue repair. Immune regulators promote better surveillance and wound clearance in multiple tissues [75, 76]. Increases in neurotrophic growth factors BDNF and CNTF, and of GDF5 which broadly and positively regulates formation of cartilage, brown fat, and neuronal axons and dendrites, have implications for better maintenance and repair of damage in the CNS and periphery.

Modulation of multiple key cell-fate regulatory pathways, including attenuation of pSmad2,3 signaling by follistatin, is important for productive cell proliferation and regeneration in skeletal muscle, heart and generally, throughout the body [77–79]. And, follistatin-like has broad anti-inflammatory properties [80], which are also expected to promote better health broadly in a mammal. Similarly, a general positive effects are expected from the elevation by TPE of Tomoreguilin-1, which antagonizes Nodal and Junk signaling, was shown to combat cardiac hypertrophy [81], to be needed for CNS formation and maintenance [82], and to be neuroprotective, by binding toxic Alzheimer’s Disease amyloid beta [83].

Finally, improved tissue maintenance is typically accompanied by a healthy remodeling [77, 84, 85], which makes it of significance that MMP9, VCAM-1, TIMP2, and thrombospondin are being modulated by the NBE and/or TPE. Of note, while we observed an increase of TIPM2 after an NBE (Figure 4), e.g., in agreement with more active tissue remodeling, at the baseline there was no age-specific change in TIMP-2 (p=0.217 YY vs OO).

Summarily, the focus on the before versus after systemic proteomics defined the key changes that correlate and logically fit with the rapid and robust rejuvenation of multiple tissues. It is possible that many of the observed molecular systemic changes are simultaneously required for the enhancement of maintenance and health of old tissues, or alternatively, smaller groups of these proteins might suffice for multi-tissue or tissue-specific rejuvenation.

The theoretical significance of this study is in a better understanding of how blood heterochronicity acts to quickly and profoundly rejuvenate old mammals, and the clinical significance of this work is in developing TPE as a new modality to broadly improve organ health and repair in older individuals preventing illnesses that develop or become more severe in later decades of life.

Materials and Methods

Supplementary Materials

Supplementary Figures

Supplementary Table 1

Supplementary Dataset 1

Supplementary Dataset 2