Rejuvenation of Aged Progenitor Cells by Exposure to A Young Systemic Environment

Irina M. Conboy, Michael J. Conboy, Amy J. Wagers, Eric R. Girma, Irving L. Weissman & Thomas A. Rando

Nature 433, 760-764 (17 February 2005) | doi:10.1038/nature03260; Received 14 June 2004; Accepted 9

December 2004

Background:

Adult stem cells are multipotent, undifferentiated cells found in many organs of the body. They are capable of self renewal and differentiation into any cell type of their tissue of origin. Adult stem cells reside in small amounts in certain areas of tissues called niches. Adult stem cells are responsible for maintenance and renewal of damaged tissues. The stem cell theory of aging states that aging results from cumulative oxidative damage to tissues combined with diminished ability for adult stem cells to repair this damage.

Summary:

Reduced up-regulation of the Notch pathway (through reduction of the Delta ligand) correlates with age in mice. Past studies have shown that the Notch pathway is a key component in signaling satellite cells to repair muscle tissues (Conboy et. al
2003). In the mouse liver, age correlates with increased formation of complex involving cEBP-α and brahma (a chromatin remodeling factor), which inhibits transcription of the E2F gene and thus regeneration of the liver by hepatic stem cells (Iakova et. al 2003). This suggests that aging of organs and tissues result from environmental factors, and a modification of such factors could restore regenerative properties of older tissues.

In order to test this theory, two mouse strains were used. One strain was transfected with Green Fluorescent Protein (GFP), which was constitutively expressed while the other strain did not have GFP expressed. Three different types of pairs were made with theses mice: a young mouse from the transgenic strain paired with another young transgenic mouse, an old non-transgenic mouse paired with another old non-transgenic mouse (both of these pairings were isochronic), and a young transgenic mouse paired with an old non-transgenic mouse (heterochronic). Parabiosis (a single circulatory system) was established by suturing the mice together. After sufficient recuperation time, the hind limbs of all mice were damaged. The mice were injected with BrdU to measure the rate of proliferation. Five days after injury, the mice were harvested and stained for eMHC (a marker for myofiber genesis). Also, the number of muscle fibers expressing high levels of Delta was quantified by flow cytometry. The staining (fig. 1a) and quantification (fig. 1b and 1c) results are shown below.

As the results show, the older mice of heterochronic pairs showed significant increase in regenerative capabilities compared to old isochronic pairs, and this correlated with increased Delta ligand up-regulation. Also, younger mice of heterochronic pairs displayed a slight decrease in up-regulation of the Delta ligand compared to young isochronic pairs. Less than .01% of the tissues examined in old mice expressed GFP, verifying that increased growth is due to serum content rather than transplantation of younger mouse cells to the older mouse.

In order to further test this idea, satellite cells were harvested from old and young mice and were cultured in vitro. Cells were cultured with either serum from old mice or serum from young mice, and growth was quantified with BrdU staining. Young mouse cells cultured in old or young serum expressed higher levels of Delta than old mouse cells cultured in old serum. Delta up-regulation and Notch activation significantly increased when old mouse cells were cultured with young serum, and young mouse cells cultured with old serum showed decreased levels of Notch activation. These results are shown in Figures 2b and 2d. Culturing old mouse cells in young serum also significantly enhanced proliferation of satellite cells. Inhibition of Notch signaling reduced proliferation of both young and old mouse cells cultured with young serum. These in vivo and in vitro studies show that serum components alone are capable of increasing muscle satellite stem cell restorative capacity in mice.

Studies were done on the liver to generalize this concept to other tissues of the body. The liver is well chronicled for its deterioration with age. Similar experiments were conducted to reflect proliferation in the liver. Pairs of isochronic and heterochronic mice were produced, and they were stained with BrdU and Ki67 (a proliferation marker). The experiment yielded similar results, shown in Figure 3. Old mice parabiosed with young mice displayed significant increase in hepatocyte proliferation compared to old isochronic pairs. GFP detection was less than .01% in old mouse tissue, indicating proliferation of the older mice’s stem cells rather than transplantation of young mouse cells.

It has been shown that decline in hepatocyte proliferation with age is due to increased levels of the cEBP-α-Brm complex (Iakova et. al 2003). Brm is only found in older mice, and in this experiment, the cEBP-α-Brm complex was detected in old isochronic mice but not young isochronic mice. This detection was done by immunoprecipitation and quantification by western blotting. Levels of the complex were decreased in older mice of heterochronic pairs, and levels of the complex were increased in the younger mice of the pair (along with slightly decreased proliferation).

Both of these experiments show that it is possible to restore youthful characteristics of regeneration to tissues and organs by modifying the environmental signaling pathways present within the serum.

Analysis:

This paper was very interesting and used many different controls in order to show not that there were factors in the blood serum affecting regeneration but also that these factors were due to regulation of the Notch pathway, specifically of the Delta ligand. I found using a mouse strain transfected with GFP was an interesting and convincing method of distinguishing between transplantation of cells rather than regeneration. However, the paper lacks data at some points. The paper states that older mice in heterochronic pairs experienced significant increase in hepatocyte proliferation, yet there was no quantitative data provided. Also, I noticed that the transfected strain of mice (C57BL/Ka-Thy1.1) was a different strain than the non-transfected strain (C57BL/6). Therefore, depending on how similar these strains are, some of effects on proliferation could be due to using different strains. When analyzing the muscle tissue for the presence of GFP, the paper mentioned that there was a very small amount of GFP expressing cells in the older mouse tissue and that this was probably due to “infiltrating leukocytes.” In order to eliminate or possibly reduce this background signal, young nude mice could have been transfected with GFP and parabiosed with the older mice. Because nude mice lack a functional thymus, the number of infiltrating leukocytes could be reduced. Nude mice could also be used in place of the older C57BL/6 strain. It is possible that nude mice, due to their compromised immune system and therefore lack of immune response and rejection, would experience an increase in the effects of the young serum.