Stem cells have the remarkable potential to divide into different cell types and differentiate into various lineages in the body. Serving as a sort of repair system for the body, they can subdivide incessantly and can replenish lost functions of the body. Stem cells are derived from different tissues in the body such as the bone marrow, adipose tissue, umbilical cord, amniotic fluid and dental tissues.

When stem cells are administered, only a small percentage are integrated into the host. Tissue repair occurs via indirect effect called as the paracrine effect. This mechanism is achieved through the secretion of specific bioactive factors which results in tissue regeneration. Stem cells secrete a composite set of cytokines, chemokines and growth/trophic factors possessing unique biological activities. This cocktail of biological entities secreted from cells is called Secretome. Studies have demonstrated that the administration of stem cell conditioned medium containing bioactive factors exert the same regenerative effect as obtained with cell transplantation. The use of cell-free therapies in regenerative medicine holds several advantages over more conventional stem-cell based applications like:
n    Use of secretome for tissue repair rather than cell transplantation can prevent issues related to immune compatibility, tumorigenicity, and the transmission of infections associated with cell therapies.
n    Secretome use greatly reduces the time and cost associated with the expansion and maintenance of clonal cell lines since secretome therapies are prepared in advance in large quantities and stored in off-the-shelf fashion. They can be immediately available for treating acute conditions such as myocardial infarction, cerebral ischemia, or military trauma.
n    Furthermore, the proteins of secretomes could be tailored to enhance or reduce certain cell-specific effects to produce different therapeutic outcomes.
Stem cells produce a variety of secretomes in response to various stimuli and each of these molecules have specific actions. They are as follows:
n    Hepatocyte Growth Factor- accelerates wound healing
n    Transforming Growth Factor Beta- immunomodulatory effect
n    Vascular Endothelial Growth Factor- induces angiogenesis
n    Tumor necrosis factor-stimulated gene-6 counteracts inflammatory responses
n    Prostaglandin E2 has immunosuppressive activities
n    Galactin 1 and 9 and micro-vesicles and exosomes

Secretomes are delivered in the form the medium in which the stem cells are grown which is known as ‘Conditioned medium’. The stem cells are allowed to grow in specific culture mediums, and these mediums are administered parenterally or topically. The growth medium can be modified to enhance the secretion of specific factors. Secretome is highly dependent on the local micro environment. Pre-conditioning of Mesenchymal Stem Cells (MSCs) and the modification of their secreted contents has been achieved with alterations in the micro environment. This includes physiologic preconditioning by exposure to hypoxic conditions; molecular preconditioning by exposure to specific cytokines, chemokines or growth factors; pharmacological preconditioning via exposure to small molecules available in large stores; and preconditioning through cell-cell interactions.

Therapeutic activity
Hypoxic preconditioning and addition of an inflammatory stimulus have been shown to modulate the production and excretion of secretomes i.e. different potential therapeutic factors by the stem cells because the therapeutic activity of MSCs is stimulated by physiological need. One of the most common elements of tissue injury is the presence of hypoxia. Once MSC migrate to areas of hypoxia, production of various therapeutic paracrine mediators is increased. It was demonstrated that exposure of bone marrow (BM)-MSCs to 24 hours of hypoxia (1% oxygen) resulted in induction of Vascular endothelial growth factor (VEGF), Fibroblast growth factor 2 (FGF-2), Hepatocyte growth factor (HGF), and Insulin like growth factor 1 (IGF-1) production. This biological relevance of MSC-secreted growth factors stimulated by hypoxia can be seen in studies showing that conditioned media from MSCs grown under hypoxic but not normal conditions show therapeutic benefit in animal models. It was demonstrated that conditioned medium from hypoxia treated BM-MSC was capable of restoring neurological function in a rat model of traumatic brain injury significantly better than conditioned medium from normal BM-MSCs. Furthermore, it was demonstrated that efficacy was associated with production of HGF and VEGF which induced endogenous neurogenesis. Hypoxia not only triggers production of growth factors from MSC, but also allows the MSC to retain in their undifferentiated state, allowing their self-renewal without differentiation.

In addition to response to hypoxia, MSCs produce immune modulatory and regenerative factors in response to inflammatory stimuli also. One of the most studied mechanisms by which inflammation triggers MSCs activity to produce secretomes is treatment with interferon gamma (IFN-?). This cytokine is typically produced during inflammatory immune responses that are associated with autoimmunity mediated by cellular means, such as CD8 T cells and Natural Killer cells. Exposure to this inflammatory mediator induces production of other inhibitors of inflammation, including the complement inhibitor Factor H, as well as the immune modulatory molecules TGF-ß and HGF.

Another inflammatory mediator known to induce regenerative activities in MSCs is the macrophage-derived cytokine TNF-a. TNF-a pretreatment of MSCs imparted superior angiogenic activity in vitro and in-vivo in the cells, as assessed by expression of VEGF and in an animal model of critical limb ischemia as compared to untreated MSCs.

Conditioned medium
Obtaining a pure cell population from a donor is a hurdle. Subsequently, obtaining a secreted protein sample that is free of the serum typically present in culture media is difficult. However, it is ideal to obtain a serum-free sample as the presence of serum interferes with protein collection and analysis. To solve this issue, investigators incubate cells in serum-free media for several hours prior to secretome collection and study. The time of incubation is a factor that needs to be carefully optimized to avoid leakage. Additionally the washing of cells and the flasks in which they are cultured during the switch from serum-containing to serum-free media is another key consideration to prevent contamination. In cell populations that rely on the presence of serum, the minimum quantity of serum necessary for normal cell function needs to be optimized and further studies should be controlled for the presence of these serum proteins in the secretome. A final consideration in obtaining conditioned medium is the low quantities in which secreted proteins are produced and the degree of dilution of these proteins into the culture medium. There is a vast array of methodologies available to study cell secretomes of stem cells like the antibody arrays, bead based array, mass spectroscopy, electrophoresis and computer algorithms.

Conclusion
Secretomes have found application in many fields like wound healing, cardiac injury, brain diseases, lung injury as well as dental regeneration and retinal tissue injury. It is reasonable to conclude that the protective and restorative action of stem cells is associated with its paracrine effect via release of specific cytokines, chemokines and growth factors. Although stem-cell derived paracrine therapy may represent an extremely good and novel therapy; several aspects are yet to be addressed before clinical use of the secretomes. The large scale production of specific paracrine molecules obtained from cultured stem cells needs to be defined. Also, their incorporation into an appropriate dosage form is a big challenge. Thus, employing secretomes for regenerative medicine in clinical use will form a new generation breakthrough to repair and cure diseases. The next age of medicine will revolve around stem cells and will be marked as a driving force for revolution in medicine.

(Vandana B Patravale is Professor of Pharmaceutics & Devanshi Shah is M.Pharm- student, Institute of Chemical Technology, Mumbai)