How Could We Slow or Reverse the Human Aging Process and Extend the Healthy Life Span with Heterochronous Autologous Hematopoietic Stem Cell Transplantation

Introduction

Heterochronous Transplantation of Autologous HSCs

HSCT has been used for the treatment of hematological and other malignancies, hereditary diseases, and some autoimmune diseases for almost 45 years. By 2014, more than 1 million HSCTs were performed worldwide. ³⁹ Currently, peripheral blood stem cells harvested by apheresis following mobilization with the granulocyte colony-stimulating factor (G-CSF) are the preferred source for HSCTs. ⁴⁰ For autologous transplantation, HSCs are collected in a disease free period and cryopreserved in liquid nitrogen where they retain complete engraftment potential after an extended period of cryostorage, ⁴¹ remaining viable for up to 100 or more years. ^42,43 The optimal age for collecting autologous HSCs is during an individual's healthy period of life, preferably between 20 and 40 years of age. This can be followed by the long-term cryopreservation and then reinfusion of the HSCs later in life.

The optimal time for reinfusion of haHSCs depends on the individual's age and health. The healthy life expectancy at birth in the United States in 2014 was 70 years. ⁴⁴ Based on this data, the optimum time for haHSCT would be somewhere between 50 and 70 years of age, when the preventive administration of young HSCs could postpone the deterioration of the immune system and delay the onset of age-related immune defects and accompanying morbidities (Fig. 1).

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FIG. 1.

Extension of healthy life span by using haHSCT. The life expectancy at birth of U.S. citizens in 2012 was 79 years (76 for men and 81 for women), whereas the healthy average life expectancy at birth— that is, the average number of years that a person can expect to live in “full health”—as 70 years. While the first 70 years of life is relatively disease-free, in the last 10 years of an average life span, elderly people suffer from one or more chronic and age-related diseases, many of them being due to the deterioration of their immune system. Serial haHSCT before the onset of the morbidity period (i.e., around the age of 60 years on average) could shift the life curve to the right, that is, postpone the advent of disease, prolong a healthy life span, and extend longevity. haHSCT, heterochronous autologous hematopoietic stem cell transplantation.

Since haHSCT can be performed in nonconditioned recipients, the transplantation of young autologous HSCs will not cause complete hematopoietic and immune chimerism. Instead, a partial displacement of the old population of bone marrow (BM) stem cells would take place, leading to an old/young HSC chimerism. Animal studies have shown that transplanted singeneic HSCs engraft effectively in nonirradiated and nonconditioned recipients, and they establish a stable long-term multilineage hematopoiesis. ^{45 –49} There is a linear correlation between the percentage of chimerism and the number of cells transplanted, finally reaching a plateau at over 50% of the highest percentage of chimerism reported in animals. ^{50 –53}

Many centers now perform unconditioned allogeneic stem cell transplants in patients with severe combined immunodeficiency disease (SCID). Dvorak et al. reported results of a multicenter study showing excellent donor T cell engraftment rates for SCID patients after receiving an unconditioned unrelated or matched sibling donor stem cell transplant. This demonstrates that stem cells transplanted into an unconditioned individual do not require the space generated in the BM compartment by conditioning to engraft, and is indirect evidence that the young stem cells will home and engraft successfully in human BM. ⁵⁴

The chimerism after an autologous HSCT is found not only in the BM and blood but also in organs such as the spleen, thymus, and lungs, and is enhanced after serial transplantations. ^48,55 Extrapolated to human medicine, serial infusions of autologous HSCs in the last quarter of the average life span could result in an effective hematopoietic chimerism in the recipient. We have recently shown in a nonconditioned autologous CD34⁽⁺⁾ hematopoietic stem and progenitor cell (HSPC) transplantation setting, that ∼44 million CD34⁽⁺⁾ autologous HSPCs would allow achievement of 20% young-in-old chimerism in a 70 kg human, which could also be used as a model for the use of HSCPs in gene and cell therapy in the future. ⁵³ Similarly, other animal studies indicate that 10%–20% chimerism would lead to positive effects of neo-hematopoiesis and neo-immunopoiesis, which will be discussed later.

Which Defects in an Aged Individual Could Be Alleviated by haHSCT?

Based on the experience related to HSCT in humans and animals, various defects of the immune system could be alleviated by haHSCT. After successful implantation into the BM, young HSCs are capable of completely rebuilding hematopoiesis as well as constituting the complete repertoire of immune cells that represent the innate and adaptive immune systems. Similarly, transplanted stem cells have the capacity to supplement the exhausted stem cell repertoire in various other somatic organs, including the spleen and thymus. ^56,57

Based on current knowledge, haHSCT could mitigate the long list of age-related defects of immunity detailed in Table 1. Regarding the innate immunity arm, haHSCT could, for instance, mitigate the impaired cytotoxic capacity of natural killer (NK) cells, the decreased production of cytokines and chemokines by activated NK cells, ⁵⁸ impaired migration and antigen presenting competence of the dendritic cells (DCs), ⁵⁹ chronic activation, the increased production of proinflammatory cytokines, decreased phagocytic capacity, the increased expression of adhesion molecules, and the decreased chemotactic activity of macrophages ^{60 –66} ; all of which are associated with increased morbidity and mortality in aged patients with infections. ⁶⁷

Similarly, haHSCT could contribute to the mitigation of a long list of impairments observed in the adaptive immunity arm, that is, in both B cell and T cell compartments in aged animals and humans, such as a decreased number of B-lymphocytes, ⁶⁸ a decreased production in long-term immunoglobulin-producing B lymphocytes and loss of immunoglobulin diversity and affinity, ⁶⁹ reduced responses to vaccination, ^{70 –73} shrinkage in the receptor repertoire and an increased amount of autoantibodies, ^74,75 a reduction in the naive T cell pool and a shift from naive to memory phenotype cells, ⁷⁶ a decrease in the diversity of the antigen-recognition repertoire, ^77,78 diminished proliferative response, ⁶⁰ as well as many other impairments. ^60,79 Similarly, one could expect a favorable effect on the deteriorated T cell subsets, such as on the decreased repertoire and number of γδ T cells, ⁸⁰ on their impaired expansion, ⁸¹ on their chronic stimulation, ⁸² and on a reduction in the numbers and proliferation capacity of the natural killer T cells (NKT) subsets. ^83,84

Other potential beneficial effects of haHSCTs could relate to enhancing an aged stem cell pool, which exhibits impaired repopulation potential, myeloid skewage, decreased homing efficiency, and clonal hematopoiesis. ^{25,85 –88}

Besides, various studies document that BM- and mobilized peripheral blood-derived HSCs can engraft into various organs, including the spleen, mesenteric lymph nodes, heart, kidney, liver, pancreas, central nervous system, lung epithelial cells, skin, eye, and gastrointestinal tract, where they regenerate tissues and restore their functions. It also seems that the donor stromal and stem cells can migrate to the thymus, where they participate in the positive selection of thymocytes, resulting in the rejuvenation of thymic function, preceding the improved functions of the adaptive arm of the immune system. ^{56,57,89 –91}

In addition, certain BM-derived subpopulations, such as the very small embryonic-like (VSEL) stem cells, seem to especially contribute to the rejuvenation of the tissues and to an extension of longevity. ^{92 –95} Although the issue of VSELs remains controversial to some authors, ⁹⁶ it seems that the evidence of their regenerative potential is accumulating. ⁹⁷

Based on these data, haHSCT should be seriously considered as a realistic option of prevention or therapy of age-related immune disorders.

Existing Studies

The heterochronous principle, that is, infusing young cells of an individual into the same individual when older, has already been used in the umbilical cord blood (UCB) banking. Similarly, rejuvenation of an immune system with autologous leukocytes collected and cryopreserved from a young subject was described by Charron in 2007. ⁹⁸ Aspinall et al. in 2013 managed to prove the validity of this approach by transplanting young naive T cells into old recipients, which led to their successful incorporation into the peripheral T cell pool. ⁹⁹ Transfusion of autologous blood-derived leukocytes from an individual taken at an earlier age therefore seems to be a valid replacement strategy for rejuvenating and restoring certain immune functions. ¹⁰⁰ However, with very few exceptions, haHSCT has never been explored as a realistic option of life span extension. ¹⁰¹

Interestingly, several animal studies have shown that heterochronous stem cell transplantation is associated with life span extension. Heterogeneous studies were reported, which were usually focused on different pathologies, so the life span extension was usually observed as a concomitant or secondary effect. Studying the biology of HSCs, Kamminga et al. in 2005 repeatedly transplanted 4–5 × 10⁶ unfractionated BM cells isolated from young (6- to 8-week old) mice to nonirradiated old (>16 months) mice. The transplanted recipients survived 10% longer than the nontransplanted animals, however, this lifespan extension did not reach statistical significance. ¹⁰² In 2011, Shen et al. conducted a study on age-related osteoporosis and found that transplanting young mesenchymal stem cells (MSCs) into old mice not only significantly slowed the loss of bone density but also prolonged their life span by 16.3%. ¹⁰³ Lavasani et al. administered muscle-derived stem cells from young wild-type mice to progeria mice, which conferred a lifespan extension of up to a third of what was usual, suggesting that there is a therapeutic potential in young cells to improve health and extend life spans. ¹⁰⁴ Then, in 2013, Kovina et al. focused for the first time on life span extension and transplanted 21.5-month-old C57BL/6 mice with young BM cells, which resulted in an extension of relative survival time by 39% ± 4% after haHSCT, compared to the control animals that received no treatment. Unfortunately, her study used limited numbers of animals. ¹⁰⁵ Kim et al. recently reported that young stem cells, when intravenously transplanted to old rats extended their life span by up to 31.3% and even improved their cognitive functions. Interestingly, this group used xenogeneic transplantation of human amniotic membrane-derived and adipose tissue-derived MSCs, and reported no side effects of tissue incompatibility. ¹⁰⁶

The central issue of the haHSCT principle is the postulated beneficial effect of young HSCs in an old environment. Interestingly, this has already been documented in humans, as Kollman et al. showed in a large retrospective study of more than 6000 cases of unrelated HSCTs between 2007 and 2011, an improved survival rate with younger donors compared with older donors. For every 10-year increment in donor age, there was a 5.5% increase in the hazard ratio for overall mortality. ¹⁰⁷ A similar study was conducted by McCurdy et al., who reported that in 928 adult transplantations of HSPCs used for patients with hematologic malignancy between 2008 and 2015, the mortality risks were significantly higher if the donor was over 30 years of age (hazard ratio, 1.39; p < 0.0001). ¹⁰⁸ This study indicates that younger stem cells are more immunologically efficient than older stem cells, which can be regarded as indirect evidence for the haHSCT principle of infusing young stem cells into older organism.

Discussion

The promises of the haHSCT concept seem very challenging to translate into clinical use. The immediate question, which could be asked based on the lack of clinical trials, is how such a clinical trial should be designed to support the haHSCT paradigm. Needless to say, a classical clinical trial does not seem to be feasible since several decades would be needed to statistically prove any extension of the human life span. There exists an option of using the autologous UCB HSCs units that are already available, but the first newborns whose UCB was cryopreserved in public and private UCB biobanks are now only in their 20s and they would need several more decades before being candidates for a clinical trial. Since every HSC collection is a medical procedure that can cause certain short-term adverse effects related to G-CSF mobilization and venipuncture, autologous UCB units collected at birth might represent a feasible source of HSCs for haHSCT. The evidence that collecting HSCs in one's youth could exhaust the stem cell reserve or cause leukemia has never been reported in trials so it seems that no serious long-term risk for the health of donors exists. ^109,110 This opinion is also supported by the fact that the International Donor Registry WMDA in the year 2019 contains more than 35 million volunteer BM donors. ¹¹¹

There are also certain limitations in translating animal longevity trials to clinical use, but due to the fact that mice have become a useful model for the study of transplantation, especially for the treatment and prevention strategies used in the patients undergoing HSCT, this has been used with success for translation to human medicine as reviewed recently. ^112,113

Another intriguing question one might pose is whether transplanting autologous lymphocytes alone ^98,99 could have the same immune effect as does the presented haHSCT procedure. In recent years, it was confirmed by deep high-throughput sequencing of hypervariable regions of V-D-J gene segments that the CD4 and CD8 T cells in young individuals possess much more diverse T-cell receptor (TCR) repertoires than the old ones; for instance, an estimated TCRβ diversity in both CD4 and CD8 naive T cell repertoires was 60–120 million in the first two decades of life and only 8–57 million in individuals over 70 years old. ^{114 –116} Especially, the CD8 T cells are prone to these age-related defects. ¹¹⁷ As the reduced TCR repertoire diversity is associated with increased mortality in the elderly, ^118,119 beneficial effect of young autologous T cells is immanent. On the contrary, the latest research on the B cell dysfunction in senescence confirms that the aging B cells, which normally produce a diverse set of antibodies and participate in presentation of antigens and secretion of cytokines, contribute to immune senescence based on decline of B cell production in BM, myeloid skewing, and accumulation of old B cells in spleen and BM, termed age-associated B cells (ABCs). These ABCs have specific phenotype, they are quiescent and do not occupy germinal centers as the normal B cells but rather the T cell zones, and have altered B cell receptor repertoires compared to other B cell subpopulations. Their functions show skewing into autoimmune antibodies production, secretion of inflammatory cytokines, and an enhanced ability to present autoantigens. ¹²⁰ Based on these facts, supplementation with young autologous B cells seems a logical measure to counteract immune senescence.

However, there are several basic conceptual differences between the heterochronous infusion of isolated cryopreserved autologous lymphocytes and the haHSCT. ¹⁰¹ First, the former could only support the adaptive part of immunity by supplying a number of naive and differentiated lymphoid cells and boosting the immune system of an older “self” through effective incorporation into the T and B cell pool of the host. Second, this effect is time limited by the proliferative potential of lymphoid progenitors. Third, haHSCT has a broader impact on senescent immunity, starting with the homing, the formation of stable old/young chimerism, and an unlimited production of “fresh” cells that constantly substitute damaged parts of the innate and adaptive immune systems as well as hematopoietic progeny. This relates to NK cells, DCs, macrophages, and neutrophils, as well as naive and educated B and T cells with their complete subtype repertoire, and to the extended repertoire of hematopoietic progenitor clones. Homing of young stem cells into the thymus and other organs after HSCT can further improve the functionality of humoral and cellular immune response in the recipient. Obviously, this plethora of immune and hematopoietic effects is more abundant than when transplanting lymphocytes alone.

Finally, we agree with Fulop et al. that any interventions performed on the aging by targeting the rejuvenation of the immune system should aim to maintain its general homeostasis and functions, meaning that they will need to be personalized and the immune status and history of each individual would need to be taken into account to achieve and sustain a very complex immune equilibrium. ¹²¹

We expect that future research will answer some pending questions regarding the clinical translation of haHSCT, such as questions regarding the optimal time for haHSCT intervention, and several other technical issues. If haHSCT technology proves its potential, this will undoubtedly result in immense changes in medical practice and will affect society as a whole. Among other effects, haHSCT might lead to the rebirth of UCB cryostorage since the cryopreserved units of UCB could be considered not only valuable for allogeneic treatments but also valuable for autologous use in the long-term. However, such speculations are beyond the intent of this review.

Conclusion

Reliable methods and substances for life span extension have not yet been fully clinically proved. ^36,122,123 Obviously, the finding of a single substance that could systemically prevent the aging process such as agonists or antagonists of specific signaling pathways is unlikely, therefore, better strategies have to be explored. ¹²⁴ In this study, we have presented one of these strategies, that is, the transplantation of young healthy autologous HSCs, collected in youth and cryopreserved, into the same individual in need of immune reconstitution at a later date. This process, called haHSCT, seems to be a promising strategy for rejuvenating the senescent immune systems of the elderly, which may ultimately contribute to slowing or reversing the aging process, delay the onset of age-related diseases, and lead to an increase in the healthy life span. However, several scientific questions remain unresolved due to the current lack of scientific data and valid clinical trials in humans, which will only be performed at some time in the future. The lack of focused research indicates the necessity for further in vitro and in vivo animal model studies that would explain the mechanisms and complexity of proposed immune rejuvenation and life span extension.