The Source of Eternal Youth (1)
The Fountain of Youth: A tale of parabiosis, stem cells, and rejuvenation
Massimo Conese et al., Open Medicine, 2017.
For links, see the original article.
A story about parabiosis, stem cells and rejuvenation
Translated by Evgenia Ryabtseva.
Introduction
The Spanish conquistador Juan Ponce de Leon became famous for discovering the Florida peninsula, although he had been looking for something completely different all his life – the legendary "source of eternal youth".
This is just one example of the continuous search that people have devoted to finding the opportunity to live forever. An interesting fact is that another frequently mentioned approach to rejuvenation consisted in attempts to transfer the heat and fluids of youth from young people to old. Examples of this approach include sexual acts with virgins – a method prescribed even by scientists in the 17th and 18th centuries, as well as bathing in blood or ingesting it.
Blood, blood plasma and their components are produced and used in modern medicine to improve the functioning of stem cells, as well as to stimulate tissue regeneration and repair their damage. Platelet-rich blood derivatives, such as platelet-rich plasma and platelet-rich fibrin, produce and release growth factors with anti-apoptotic and angiogenic effects. This stimulates the regenerative abilities of stem cells and progenitor cells both locally and when injected from the outside. Platelet-rich plasma has gained popularity as a means used in various orthopedic surgical interventions carried out to treat various diseases, including osteoarthritis, in plastic surgery to improve graft engraftment, as well as in the fight against skin necrosis. Thus, there is no doubt that blood and its derivatives can be successfully used not only in regenerative medicine strategies, but even as the "holy Grail" of rejuvenation – reversing the aging process.
History of parabiosis
The belief that blood can rejuvenate human organs was resurrected in 2005 and 2010 by a research team from Stanford University School of Medicine. These studies were based on observations according to which the regenerative ability of tissues fades with age. In tissues such as muscles, blood, liver, and brain, this extinction was attributed to reduced reactivity of tissue-specific stem cells and progenitor cells. However, aging muscle tissue successfully regenerates when implanted into the muscle of a young animal, whereas young muscle tissue demonstrates impaired regeneration when implanted into the muscle of an old animal. Both local and systemic factors may be responsible for these opposite effects. In order to test whether systemic factors can support tissue regeneration in young animals and/or inhibit regeneration in old animals, Conboy and colleagues in 2005 developed an experimental model in which, in contrast to transplantation, regenerating tissues of old animals were exposed exclusively to circulating factors of young animals and vice versa. Thus, they developed a method of parabiotic coupling of young and old mice (heterochronous parabiosis), for which the control formulation was the parabiotic coupling of two young or two old mice (isochronous parabiosis) (Figure 1).
In parabiosis, two mice are surgically connected to each other in such a way that they form a common circulatory system with a rapid and constant exchange of cells and soluble factors in physiological concentrations, occurring through their joint blood flow. Parabiosis was invented in 1864 by physiologist Paul Bert to study the formation of the circulatory system. Biochemist and gerontologist Clive McCay from Cornell University first used parabiosis to study aging, but this technique lost popularity after the 1970s, most likely because many rats died from a mysterious condition called parabiotic disease. It develops approximately 1-2 weeks after the animals are joined and may be a variant of immunological tissue rejection. It was only at the beginning of the 21st century that Irving Weissman and Thomas A. Rando of Stanford University brought parabiosis back to life to study the movement and fate of hematopoietic stem cells.
Figure 1. Parabiosis. Two mice are connected together and have a common blood flow. Heterochronous parabiosis is a surgical connection of young and old animals, whereas isochronous parabiosis is a paired connection of two young or two old animals.
The Stanford group studied the regeneration of muscle tissue and liver cells under parabiosis. After injury to muscle tissue, muscle regeneration was assessed by the formation of muscle tubules expressing a heavy chain of muscle myosin – a specific marker of regenerating muscle tubules in adult animals. Five days after the injury, the muscles of young animals showed marked regeneration in both isochronous and heterochronous parabiosis. At the same time, the damaged muscles of the old isochronous parabionts regenerated poorly, whereas parabiosis with young mice significantly improved the regeneration of the muscles of their old partners. The regeneration of aging muscles occurred almost exclusively due to the activation of resident old progenitor cells, and not due to the engraftment of circulating progenitor cells of their young partners (it was estimated by the presence of less than 0.1% of the cells expressing green fluorescent protein of their young transgenic partners). The age-related decline in the ability of muscle tissue to regenerate is partly due to an age-related violation of the activation of the Delta ligand of the Notch protein after muscle injury, therefore, Delta expression has also become an object of study. It should be noted that satellite muscle cells (satellite cells, myosatellites) The old partners of heterochronous parabiotes showed pronounced Delta activation comparable to that observed in their young partners and in young mice not exposed to parabiotic coupling (Figure 2).
In experiments on liver rejuvenation, as well as in the case of muscle tissue, the proliferation of albumin-positive cells in old isochronous parabionts was less pronounced than the proliferation observed in young isochronous parabionts, while parabiosis with a young partner significantly increased the proliferation activity of hepatocytes in old mice. As in the case of muscle tissue, the increased proliferation of hepatocytes in old mice was due to resident cells, and not to the engraftment of cells of young partners circulating in the bloodstream. The decrease in the proliferation activity of hepatocyte progenitor cells is due to the formation of a complex that includes CCAAT/enhancer binding protein-alpha (cEBP-α) and the brahma chromatin reconstruction factor (Brm), which inhibits gene expression triggered by the E2F transcription factor. In parallel with the effect on hepatocyte regeneration in the livers of old heterochronous parabionts, the formation of the cEBP-α-Brm complex was recorded, which was not observed in the liver of young isochronous parabionts, while in the former the formation of the complex was relatively weakly expressed (Figure 2).
Figure 2. Aging of muscles, liver and brain of old mice and rejuvenation through heterochronous parabiosis. Skeletal muscle regeneration after injury is associated with the activation of the Delta ligand of the Notch protein, which is lost with age (upper cells). Proliferation of hepatocytes in young animals correlates with a decrease in the cEBP-α-brahma complex (cEBP-α-Brm) compared to old mice (medium cells). Whereas in young animals, neurogenesis and angiogenesis may increase in the subventricular zone of the brain, where stem cells are present, this is not possible for old animals (lower cells). In general, heterochronous parabiosis reverses all phenotypic and molecular manifestations of aging through the influence of soluble factors and cells.
Finally, a decrease in the proliferation of progenitor cells was recorded in the muscles and liver of young mice after parabiotic coupling with old animals. This indicates that the blood of old mice is enriched with inhibitory factors, the concentration of which decreases with parabiosis due to dilution. The data obtained indicate that systemic factors can modulate molecular signaling pathways critical for the activation or inhibition of tissue-specific progenitor cells, as well as the fact that the systemic environment of organisms of young animals promotes successful regeneration, whereas in old animals it does not stimulate or even actively inhibits successful tissue regeneration. Ultimately, this work also demonstrated that tissue-specific stem cells and progenitor cells retain most of their inherent proliferative potential until old age, but age-related changes in the systemic environment and/or the niche in which the progenitor cells live prevent the full activation of these cells necessary for productive tissue regeneration.
In a 2010 paper, Wagers and co-authors attempted to understand the role of local, microenvironment/niche-driven, and systemic factors in the aging of hematopoietic stem cells and progenitor cells using an in vivo parabiotic mouse system, as well as to study the frequency of hematopoietic stem cells and the number of "long-term" hematopoietic stem cells. In order to distinguish the cells of young and old animals, congenic markers (differing by one chromosomal locus) were used. At the bone marrow level, aging is accompanied by a significant expansion of hematopoietic stem cells and progenitor cells, paradoxically associated with a decrease in the ability to renew blood and impaired differentiation potential after transplantation. The old heterochronous parabionts showed a significant decrease in the number of long-term hematopoietic stem cells to almost normal (typical for a young organism) values. It should be noted that this effect is the result of changes within the population of aging hematopoietic stem cells themselves, and not the movement of "young" cells into the bone marrow of an old partner. Moreover, heterochronous parabiosis also induced the restoration of the functions of long-term hematopoietic stem cells in old age. This was evidenced by the restoration of the graft's engraftment potential and the restoration of the normal ("young") ratio of B-lymphoid and myeloid cells. As in the case of hematopoietic stem cells, both the frequency of occurrence and the total number of osteoblastic niche cells isolated in old mice increased compared to young mice of the control group. In vitro experiments to study the interaction between young bone marrow cells and cells of an aging osteoblastic niche also demonstrated the expansion of hematopoietic stem cells. This indicated that the rejuvenating effect of parabiosis on these cells was mediated by the reversal of age-related changes in osteoblastic niche cells. In fact, the frequency of occurrence and the number of osteoblasts in old animals were restored to normal levels under the influence of the "young" systemic environment formed by heterochronous parabiosis. Moreover, niche cells isolated from old heterochromic parabionts showed a significantly reduced ability to cause the accumulation of hematopoietic stem cells and progenitor cells, unlike niche cells of old isochronous parabionts. An interesting fact is that osteoblastic niche cells isolated from young heterochronous parabionts induced a weak expansion of young hematopoietic stem cells and progenitor cells, compared with cells of young isochronous parabionts. These in vitro studies indicate the possibility of mutual influence of the aging circulatory environment on niche activity in young heterochronic partners and indicate the ability of system signals to restore aging niches. The aim of the subsequent experiments was to evaluate the ability of young hematopoietic stem cells and progenitor cells to restore hematopoiesis. The results obtained showed that, similar to the impairment of the ability of naturally aged hematopoietic cells to restore hematopoiesis after transplantation, young hematopoietic cells exposed in vitro to the cells of old isochronous parabionts showed a reduced ability to restore hematopoiesis. Apparently, interaction with aging cells of the osteoblastic niche is sufficient to induce functional disorders of hematopoietic stem cells. However, the cells of old heterochronous parabionts did not change the regenerative ability of young hematopoietic stem cells. Taken together, all these data indicate that functional changes in the cells of the regulatory niche of hematopoietic stem cells induced by aging can be reversed by the influence of circulating factors of a young organism. This indicates that the rejuvenating effect of the young circulatory environment on hematopoietic stem cells is mediated by signaling mechanisms triggered by rejuvenated osteoblastic niche cells.
In order to find out which factor is involved in the regulation of cell niche functions, the authors decided to study the possible role of insulin-like growth factor-1. This factor is known as a regulator of the aging process and longevity preserved in the course of evolution. In vitro and in vivo experiments have demonstrated that local, but not systemic, exposure to insulin-like growth factor-1 induces aging of niche cells regulating the activity of hematopoietic stem cells. At the same time, the neutralization of the signaling mechanism mediated by insulin-like growth factor-1 in the bone marrow microenvironment reverses the age-related changes in osteoblastic niche cells that distort the regulation of hematopoietic stem cells carried out by them.
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