Fight Aging!

The thymus is a small but important organ; it is where thymocytes originally generated in the bone marrow mature to become T cells of the adaptive immune system. Unfortunately the active tissue of the thymus is slowly replaced by fat over the course of later life, and the supply of new T cells dwindles. This is a significant contributing cause of the age-related decline in immune function. Lacking reinforcements and replacements, the adaptive immune system becomes cluttered with senescent, exhausted, overspecialized, and just plain broken cells. It becomes overly active and inflammatory, but at the same time ineffective. It progressively becomes ever less competent when it comes to destroying cancerous and senescent cells, and defending against pathogens.

This is all well recognized, and over the years a range of efforts to regenerate the thymus have been undertaken. As of yet few have progressed much further than animal studies in mice. Recombinant KGF, which works quite well to enlarge the thymus in mice and non-human primates, failed utterly in a human trial, showing absolutely no effect. More recently, the staff at Intervene Immune have been combining some of the older and unreliable methods, such as use of growth hormone, into human tests of thymic regrowth. All of these approaches, and a few others, largely boil down to ways to upregulate FOXN1, the master controlling gene of thymic growth and T cell maturation activity. The most compelling studies in mice have been those in which FOXN1 expression was manipulated directly, and we might suspect that any therapy that grows a thymus, but fails to keep FOXN1 levels high going forward, will also fail to make a large and lasting impact on T cell generation. The thymus must be active, not just larger, and FOXN1 expression declines with age.

Tissue engineering offers an intriguing approach to the problem of the thymus, bypassing a lot of the hard work inherent in trying to manipulate expression of a given gene. (Of course replacing it with hard work of a different sort). Functional thymus tissue can be grown in small amounts, lacking a network of small-scale blood vessels, but able to be transplanted. Since thymocytes home to the thymus, thymic tissue located almost anywhere in the body will still be capable of doing its job, in principle. This has been demonstrated by implanting thymus organoids into lymph nodes, an approach being commercialized by Lygenesis. As the results here show, however, success still depends on building a suitably resilient tissue that will last for a long time following transplantation.

Gene Modification and Three-Dimensional Scaffolds as Novel Tools to Allow the Use of Postnatal Thymic Epithelial Cells for Thymus Regeneration Approaches

Many research groups have primarily focused on finding possible strategies to rejuvenate the thymus and have developed promising therapeutic approaches. However, few molecules and genes such as KGF, IL-22, IL-7, and Foxn1 have been identified as key players of the mechanistic pathway for endogenous thymic regeneration. Growth factors and hormone therapies were also explored in order to restore age-related or injury-related thymic degeneration, but, despite encouraging results, they have short-term effects and/or require a recurrent administration, which is complicated by their toxic effects on other tissues and organs.

Thymus transplantation represents another promising alternative to complement bone marrow transplantation or to treat congenital thymic anomalies, but T-cell reconstitution following thymus grafting is frequently incomplete and transient, complicated by a skewed T-cell receptor repertoire and an increased occurrence of autoimmunity.

The field of tissue engineering has put considerable efforts into the development of materials and techniques for the in vitro generation of tissues of clinical relevance. The major challenge for tissue engineering is to successfully recreate the complexity of the 3D structure of the thymic microenvironment and to fully rebuild the composition and organization of the thymic extracellular matrix (ECM). The use of thymic organoids formed by human thymic epithelial cells (TECs) and fibroblasts as well as seeding TECs into matrigel or other 3D biocompatible systems has been shown to promote a transient thymopoiesis in vivo. The use of de-cellularized thymic tissue has been suggested to overcome these limitations and has shown promising results in mouse models. However, the use of decellularized tissues, obtained from cadavers or patients undergoing cardiothoracic surgery, limits the applicability of such approaches to the availability of donors.

The use of postnatal TECs for thymic regeneration has revealed challenging because of the loss of thymopoietic function of TECs after in vitro culture. To avoid the use of embryonic TECs or induced pluripotent stem cell derived TECs, attempts have been made by combining mature TECs with different 3D systems for developing functional mini-thymus units. Several studies have been focused on the investigation of ideal biomaterials for human applicability, which need to be biocompatible, biodegradable, and easily detectable with imaging techniques regularly used in standard clinical practice. Collagen has been widely used in tissue engineering because it can be assembled in fibers closely reproducing the chemical and morphological characteristics of those present in soft tissues. Therefore, the production of collagen porous biomatrix could make this biomaterial suitable for the generation of thymic constructs.

Hence, we developed a potentially new therapeutic strategy that foresees transplantation of biomimetic scaffolds, mimicking the thymic ECM organization, obtained by seeding adult murine TECs and expanding them into 3D collagen type I scaffolds. In order to use postnatal TECs for the generation of transplantable thymic structure, we sought to induce a short-term expression of Oct4, a transcription factor involved in the maintenance or induction of pluripotency in embryonic cells, to obtain transient partial de-differentiation and promote their expansion. To create a physiologically relevant microenvironment to seed TECs, we tested 3D collagen type I scaffolds crosslinked with different amounts of 1,4-butanediol diglycidyl ether (BDDGE).

Here, we show that 3% BDDGE collagen-based scaffolds seeded with gene-modified TECs and transplanted subcutaneously in athymic nude mice were perfused and colonized by small new blood vessels and were able to sustain TEC survival in a 3D microenvironment. However, further improvement of the 3D scaffold composition is required to obtain long-term in vivo persistence of organoids that could allow the development of this approach for future clinical applications.