Prospect of Stem Cell Conditioned Medium in Regenerative Medicine

Background. Stem cell-derived conditioned medium has a promising prospect to be produced as pharmaceuticals for regenerative medicine. Objective. To investigate various methods to obtain stem cell-derived conditioned medium (CM) to get an insight into their prospect of application in various diseases. Methods. Systematic review using keywords “stem cell” and “conditioned medium” or “secretome” and “therapy.” Data concerning treated conditions/diseases, type of cell that was cultured, medium and supplements to culture the cells, culture condition, CM processing, growth factors and other secretions that were analyzed, method of application, and outcome were noted, grouped, tabulated, and analyzed. Results. Most of CM using studies showed good results. However, the various CM, even when they were derived from the same kind of cells, were produced by different condition, that is, from different passage, culture medium, and culture condition. The growth factor yields of the various types of cells were available in some studies, and the cell number that was needed to produce CM for one application could be computed. Conclusion. Various stem cell-derived conditioned media were tested on various diseases and mostly showed good results. However, standardized methods of production and validations of their use need to be conducted.


Introduction
Data of the use of stem cells in various diseases are accumulating. Some studies reported beneficial effects of stem cell therapy in degenerative diseases such as myocardial infarction and revealed that stem cells cause tissue repair due to their ability to secrete trophic factors that exert beneficial impact on the damaged tissue, rather than their capacity to differentiate into the needed cells [1]. Various studies on stem cell-derived secreted factors showed that the secreted factor alone without the stem cell itself may cause tissue repair in various conditions that involved tissue/organ damage. The secreted factors are referred to as secretome, microvesicles, or exosome and can be found in the medium where the stem cells are cultured; thus, the medium is called conditioned medium (CM) [2].
The use of secretome containing CM has several advantages compared to the use of stem cells, as CM can be manufactured, freeze-dried, packaged, and transported more easily. Moreover, as it is devoid of cells; there is no need to match the donor and the recipient to avoid rejection problems. Therefore, stem cell-derived conditioned medium have a promising prospect to be produced as pharmaceuticals for regenerative medicine.
To date, no clinical trial that used CM for a certain disease has been reported, except two pilot studies on the use of adipose derived mesenchymal stem cell CM for hair follicle regeneration [3] and fractional carbon dioxide resurfacing wound healing [4] in human, which showed good results. The use of CM for therapy is very appealing and may be booming in the near future, as studies on the use of CM for various diseases are accumulating [1,. Conditioned medium contains various growth factors and tissue regenerative agents, which were secreted by the stem cells. The fact that stem cells secrete various growth factors was also shown by various proteomic studies, which revealed the presence of various growth factors and other cytokines in the CM [5, 7-9, 13, 17, 20, 22, 28, 34, 35].
However, various studies reported the use of various kinds of stem cells and various methods to get the CM to cure various kinds of degenerative diseases in various animal models. Therefore, this systematic review aimed to investigate the various methods to get the CM and the various diseases that were treated, to get an insight into the various kinds of CM and their application benefit in various diseases.

Materials and Methods
We performed "all text" searches without time restriction on January 23, 2014, in Pubmed/Medline using keywords "stem cell" and "conditioned medium" or "secretome" and "therapy, " "all text" searches in Cochrane library (trials) using keywords "secretome" or "conditioned medium, " and "all text" searches in ClinicalTrials.gov using keywords "stem cell" and "conditioned medium" or "secretome" and "therapy. " In addition, relevant existing articles in our library were added.
Inclusion criteria are all studies that used CM for a certain disease. Exclusion criteria are studies that did not contain complete data concerning subject condition/disease model, source of CM, and outcome of treatment with CM.
Data collection is as follows: treated conditions/diseases, type of cell that was cultured, detailed composition of medium and supplements that was used to culture the cells, culture condition (hypoxia or normoxia) to get the CM, CM processing, growth factors, and other secretions that were analyzed; method (mode) of application and outcome of CM application were noted, grouped, and tabulated.
Data synthesis is as follows: data were grouped according to treated disease and cell types that were used to produce the CM. Further, to know the growth factor yields of the various types of cells, when available, growth factor levels were tabulated and grouped according to types of cells that yielded the growth factor containing conditioned medium, in relation to the number of cells, type and duration of culture, and processing of the conditioned medium. When the data was available, the number of cells that were needed to produce the CM for one application was computed.

Results and Discussion
We got 39 articles that met the inclusion criteria, and 7 were excluded due to incomplete data. Various conditions/diseases were treated by various cell-derived CM and mostly showed promising results ( Table 1).
The various conditioned media, even when they were derived from same kind of cells, were produced by different condition, that is, from different passage, number of cells, culture medium, and culture condition ( Table 2). The growth factor yields of the various types of cells can be seen in Table 3, and the cell number that is needed to produce CM for one application can be seen in Table 4.
In the two cases of kidney disease, it can be concluded that CM from hu-ESC-MSC can improve the condition, and the needed growth factor level is presumably enough as CM processing includes a 25-time concentration step [29]. However, for hu-UCB-USSC or mBM-MSC-CM, lack of data concerning CM processing and growth factor level of the CM [30] prevent further analysis to conclude whether the failure to improve the condition is due to the lack of certain growth factor or due to the level of growth factors that was too low to give an effect.
The conditioned medium can be harvested from various kinds of cells (Table 2). Moreover, there are various methods to get the conditioned medium, which may interfere with the growth factor types and levels that were harvested by the methods. Only some of the various studies using CM checked the growth factor levels ( Table 3) [5-10, 13, 17, 20, 22, 24, 25, 27, 28, 33, 36] and the same type of cells yielded different growth factor levels, when cell number, culture medium and condition, and CM processing were different [6,24]. Moreover, growth factor measures also differed, that is, pg/mL or ng/mL [6,8,10,13,17,24,27,28,33], pg/ g DNA [9,36], fg/cell [25], spot density [5], and positive/negative [20] (Table 3). The measure of pg/ g DNA and fg/cell can be computed into pg or ng/mL provided the DNA content/cell and cell number is known. However, in some studies, the exact cell number that was used to produce the CM was not mentioned [7,8,10,13,28,33,36]. In addition, most studies measured different sets of growth factors and other cytokines/factors (Table 3).

Culture Medium and Supplement.
Some studies used fetal bovine serum or other supplement containing complete medium, while other studies used serum-free media. Moreover, the basal media used were variable, for example, MEM, DMEM, DMEM/F12, M199, EBM2, EGM-2, in vivo 15, or chemically defined medium, and the same type of cell might be cultured in different kind of basal medium ( Table 2). Culture medium in in vitro culture represents microenvironment in in vivo condition and may determine cell fate and thus cell secretion [37]. Therefore, the same type of cells may secrete different level of growth factors when they were cultured in different medium, as can be seen in Table 3 [25, 27].

Culture Duration.
Production of CM varies in culture duration from sixteen hours to five days (Table 3). In case complete medium was used, short culture duration might leave certain serum derived growth factors that was not consumed by the cells and might add to the growth factor level, or, on the contrary, suppress growth factor secretion by the cells. Possibility of the presence of residual growth factor from the medium can be seen in a study, which showed that medium without cell contained a TGF-b1 level of 2.49 ± 2.39 pg/mL (Table 3) [24].
Most studies produced CM in monolayer culture, but several studies used spheroid cultures (Table 3). Spheroid cultures need a special handling and equipment (spinner flask) but yield more cells compared to conventional monolayer cultures, and thus more secreted factors [6,24] (Table 4). In addition, cells located at the center of the spheroid may be in relative hypoxic condition compared to cells on the surface, thus further increasing certain growth factor yield.

Secreted Factor's Role in Improvement of Diseases.
Various cytokines were secreted by stem cells into the CM, and they played a role in the improvement of various diseases/conditions. Those cytokines can be grouped into growth factors, proinflammatory and anti-inflammatory cytokines, and other cytokines. Various studies used various methods to assess various cytokines in the conditioned CM, from the conventional ELISA assays [6,10,24,25,27,33,36] to proteomic profiling methods [5, 7-9, 13, 17, 20, 22, 28, 34, 35].
Further, studies that analyzed various growth factors reported the presence of the various growth factors, which were secreted by various stem cells into their conditioned medium (Table 3), except for human MSC (Lonza) that did not secrete FGF-2, PDGFBB, BMP-2, and SDF-1 but secreted IGF-1, VEGF, TGF 1, and HGF [33]. Moreover, different culture condition and medium may yield different level of growth factor secretions [6].

Translation of Conditioned Medium Usage in Patients.
In conditioned medium, various factors may be present as a cocktail and act in concert to promote regeneration. Therefore, it is important to analyze a complete set of growth factor and cytokine levels for every kind of stem cell-derived conditioned medium and to know the culture condition, conditioned medium processing, and diseases/conditions that are responsive to a certain conditioned medium treatment. When the content of the various cytokines in a certain conditioned medium is known, the result of the conditioned medium on a certain disease/condition can be determined, and the way to translation into patients is open. From studies that analyzed VEGF level we can conclude that most stem cells secrete VEGF. As VEGF plays a role on angiogenesis [36] that is important in regeneration of injured/damaged tissues/organs, various stem cell-derived conditioned media are able to cure various diseases and will have more impact on diseases with ischemia. In addition, VEGF may prevent apoptosis in hypoxic condition, thus preventing further damage [6].
Moreover, FGF2 is a more potent angiogenic factor compared to VEGF, with additional effect on proliferation of fibroblasts, preadipocytes, and endothelial, epithelial, and neural stem cells, on migration of neural crest derived glial and myogenic cells and on differentiation of neuroepithelial cells into mature neurons and glial cells [38].
Other growth factors contribute in the regeneration of injured/damaged tissue organs, with special emphasis on proliferation, that is, PDGF for connective tissue, glial, and other cells, EGF for mesenchymal, glial, and epithelial cells, and IGF-I and IGF-II for various kinds of cells [40]. In addition, PlGF that is a member of VEGF family increases the activity of VEGF in vitro and in vivo [41], KGF inhibits oxidative stress induced epithelial cell death [42], NGF promotes neurite outgrowth and neural cell survival [40], BDNF is neuroprotective, promotes cell survival, and reduces astroglial scar   formation [28], and some growth factors, including HEGF, FGF-7, EGF, and HGF promote liver regeneration [20]. Proinflammatory cytokines that play a role in regeneration are IL-1b due to its liver protective role [20], IL-8 due to its angiogenic activity [8,9,13], and IL-9 due to wound healing promotion activity [13,43]. In addition, antiinflammatory cytokines prevent inflammation and promote liver regeneration [20].
Moreover, one factor may contribute to more than one mode of regenerative action, such as MCP-1 that is involved in angiogenesis [9,13,17,20] and liver protection activity [20]. Further, for production of CM to be applied in various human diseases, data from animal studies that showed promising outcome are very valuable.

Production of CM for Translation into Various Human
Diseases. To use CM for various human diseases, production method of the CM needs to be standardized in terms of the type and number of cells that were needed to produce the CM, culture medium and condition, and conditioned medium processing. In addition, the volume and mode of delivery are also important. As various studies used various numbers and type of cells and various doses of CM, it is important to know the number of cells that yielded the CM for one application, which may be interpolated for human studies. Therefore, in Table 4 we summarized all data that may be needed for interpolation into human studies, that is, diseases that were treated, species and age or body weight of the animal, type of cell, culture medium and condition, number of cells to produce CM for one application, volume, and mode of application. Moreover, various possible applications of CM for various conditions are summarized in Figure 1.
In addition, for translation into patients, it is very important to analyze and to note the various cytokine contents of the various conditioned media. Further, for every conditioned medium with known cytokine content, validation of its use on various diseases needs to be conducted. Finally, the possibility of promotion of existing cancer should be tested for every CM, and caution should be taken before CM therapy to ensure that the recipient is free from cancer.
Advantages of production of various CM for patients lie in the possibility of mass production by pharmaceutical companies, when production methods have been standardized. Conditioned media are not like stem cells that need a good manufacturing practice (GMP) facility to be applied