Functional Properties of Human-Derived Mesenchymal Stem Cell Spheroids: A Meta-Analysis and Systematic Review

Mesenchymal stem cells (MSC) are adult multi-potent cells that can be isolated from many types of tissues including adipose tissue, bone marrow, and umbilical cord. They show great potential for cell therapy-based treatments, which is why they are being used in numerous clinical trials for a wide range of diseases. However, the success of placebo-controlled clinical trials has been limited, so new ways of improving the therapeutic effects of MSC are being developed, such as their assembly in a 3D conformation. In this meta-analysis, we review aggregate formation, in vitro functional properties and in vivo therapeutic potential displayed by adipose tissue, bone marrow, and umbilical cord-derived MSC, assembled as spheroids. The databases PubMed and SciELO were used to find eligible articles, using free-words and MeSH terms related to the subject, finding 28 published articles meeting all inclusion and exclusion criteria. Of the articles selected 15 corresponded to studies using MSC derived from bone marrow, 10 from adipose tissue and 3 from umbilical cord blood or tissue. The MSC spheroids properties analyzed that displayed enhancement in comparison with monolayer 2D culture, are stemness, angiogenesis, differentiation potential, cytokine secretion, paracrine and immunomodulatory effects. Overall studies reveal that the application of MSC spheroids in vivo enhanced therapeutic effects. For instance, research exhibited reduced inflammation, faster wound healing, and closure, functional recovery and tissue repair due to immunomodulatory effects, better MSC engraftment in damaged tissue, higher MSC survival and less apoptosis at the injury. Still, further research and clinical studies with controlled and consistent results are needed to see the real therapeutic efficacy of MSC spheroids.


Introduction
Mesenchymal stem cells (MSC) are plastic adherent, fibroblast-like, non-hematopoietic progenitor cells isolated from a variety of adult tissues. MSC have self-renewal capabilities, and they can differentiate into several tissue-specific lineages including osteoblasts, chondrocytes, adipocytes, hepatocytes and cardiomyocytes among others [1,2]. MSC can be harvested from several tissues, including bone-marrow (BM-MSC), adipose tissue (AT-MSC), umbilical cord (UC-MSC), Wharton's jelly (WJ-MSC), gingiva (G-MSC) and cartilage tissue (C-MSC), among others [3,4]. The International Society for Cellular Therapy (ISCT) establishes three key features to identify MSC: First, mesenchymal stem cells must display adherence to plastic when cultured under standard conditions. Second, MSC population must express CD105, CD73 and CD90 (≥95%) and not express CD45, CD34, HLA-DR, CD14 or CD11b, CD79a or CD19 (≤2%). Third, MSC cultured in vitro must show osteogenic, adipogenic and chondrogenic differentiation [5]. Besides their ability for multipotent differentiation, MSC are mainly considered as immune evasive cells and they secrete many key trophic factors contributing to tissue repair and regeneration [6][7][8]. Therefore, MSC appear to be an appealing alternative of treatment in regenerative medicine because it is possible to administrate allogenic populations of these cells or more differentiated lineages [9]. Based on MSC capacity to differentiate and mature into specific phenotypes, their immunomodulatory properties, and their distinct migratory and potent trophic effects during tissue regeneration, the potential for clinical applications is remarkable [10,11].
At the time of writing this review, there are currently 874 clinical studies reported using MSC to treat different diseases (www.clinicaltrials.gov). However, the beneficial effects of MSC based therapies in small clinical studies are often not substantiated by large randomized double blind, placebocontrolled clinical trials [12,13]. Several clinical studies, unable to meet the clinical goal, suggest that after prolonged ex vivo expansion of BM-MSC, their immune-suppressive properties change and display deficient survival rate posttransplantation [14,15]. This implies that it is necessary to comprehend the regenerative and immunomodulatory mechanisms by which MSC exert their action. New ways of enhancing the functional properties of MSC are also needed, which until now have only been cultivated in monolayer 2D culture for clinical applications. Although a simple procedure, some in vivo essential qualities and traits are compromised or lost.
Therefore, 3D cell culture emerges as a new therapeutic alternative, offering better preservation of those features, as allowing a better mimicking of in vivo conditions, particularly cells self-assembling, as observed during embryonic development, thus increasing cells interactions [16,17]. To promote this interaction, several spheroid formation techniques have been developed. Contrary to monolayer culture, three-dimensional culture of MSC as spheroids causes considerable changes in the gene expression pattern [18,19]. Diverse studies propose that the functionality of stem cells can be improved and unsuitable migration of cells, after injection in the target tissue, can be avoided, by aggregate formation [20][21][22]. However, the exact mechanism involved in 3D conformation is still unknown, although several signalling pathways have been proposed [23][24][25][26][27][28]. For example, it has been described that decreased expression of transcriptional co-activators yes-associated protein/transcriptional co-activator with PDZ-binding motif (YAP/TAZ) in MSC cultured in 3D conformation, was associated with a loss of the actin cytoskeleton [27,28]. In other study, Zhang et al., showed an increase in the expression of hypoxia-inducible factor (HIF)-1 and -2α in MSC spheroids which was related to increased resistance to apoptosis triggered by oxidative stress [23]. This meta-analysis was conducted to review spheroid formation, in vitro functional properties and in vivo therapeutic potential displayed by adipose tissue, bone marrow, and umbilical cord-derived MSC, assembled as spheroids.

Materials and Methods
2.1. Search Strategy. The following databases, PubMed and SciELO, were searched for eligible published articles until May 2019, using specific free-words and MeSH terms. Different combination of the following terms were used: type of cell: mesenchymal, stromal, stem cell, pluripotent, multipotent; cell organization: 3D, spheroid, cluster, organoid, aggregates; application: cell therapy, tissue regeneration, treatment, therapy, functional recovery; pathologies: skeletal muscle, muscle cartilage, tendon or joint pathologies; osteoarthritis, chronic injuries, immune diseases, neurodegenerative diseases, neurological pathology, bone pathology; culture conditions: hypoxia, low oxygen, xeno-free, serumfree, animal-free; properties: secretome, exosome, vesicles, immunoregulatory. Of the results obtained only articles published in English were included and, articles related to cancer and published earlier than 2008 were excluded. Other potential articles were identified from references within the selected articles or reviews related to the topic.

Selection
Criteria. Inclusion criteria are listed as: a) Human MSC; b) source of MSC either bone marrow, adipose tissue or umbilical cord; c) studies focused on cell therapy and regenerative medicine. Exclusion criteria are listed as a) article does not meet inclusion criteria; b) reviews and case reports; c) work focused on application and/or use of MSC spheroids in bioengineering.

Data Extraction.
The data extracted from included articles consisted of: authors, year, country, title, MSC tissue source, cell aggregation protocol, culture conditions, spheroid measurements, functional properties in vitro, altered markers for said properties, therapeutic effects in vivo and study model used.

Limitations.
Only articles published in English were included, which may leave out other eligible publications that were reported in other languages. Therefore, the results should be interpreted cautiously due to the limited data.

Results and Discussion
3.1. Search Results. After a screening of 254 articles identified by searching in PubMed (n =131) and SciELO (n =123) associated with MSC in 3D conformation, only 71 articles were assessed for eligibility according to the search strategy described in materials & methods. Of these 71 articles, only 28 articles were incorporated in this meta-analysis according to the inclusion criteria described in materials & methods (human MSC, MSC harvested from bone marrow, adipose tissue or umbilical cord, and studies focused on cell therapy and regenerative medicine ( Figure 1).
The list of the 28 articles chosen and their basic description (author(s), year, country, source of MSC, properties and parameters evaluated, and ref number) is shown in Table 1.
It can be noted that the earliest work included in this review is from May 2010 [29] and the latest being published in April 2019 [30]. Most articles are published in 2017 (n =5), succeeded by 2014, 2016 and 2018, each with 4 articles. The most articles collected are from work conducted in the USA with a total of 13 published articles comprising a 46.4% of all articles included, followed by South Korea with a total of 5 (17.9%) and China with 3 (10.7%) ( Table 1). Of the 28 articles selected 15 corresponded to studies using MSC derived 2 Stem Cells International from bone marrow, 10 from adipose tissue and 3 from umbilical cord blood or tissue. In all, the most evaluated characteristics of MSC spheroids are stemness, angiogenesis, differentiation, cytokine secretion, paracrine effect, metabolic function, and immunomodulatory effects (Table 1).

Cell Aggregation Protocol.
There are several methods and techniques to form MSC spheroids, here we have found that the most used procedure is the hanging drop method (n = 13) [19-21, 29, 31-39], followed by forced-aggregation (n = 4) [40][41][42][43], low attachment(n = 4) [44][45][46][47], spontaneous assembly (n = 4) [30,[48][49][50], then chitosan films (n = 2) [18,51], and finally hyaluronic acid gel (n = 1) [52] (Figure 2). The hanging drop technique consists of plating the MSC suspension in droplets of determined volume on the lid of a culture dish. The lid is turned over in a swift and careful move and placed on top of the culture plate which is filled with a solution to avoid drop evaporation. The spheroid is formed in the apex of the drop. This method can yield spheroids of controlled sizes, determined by the number of cells in each drop associated with the concentration of the cell suspension and volume of droplet, and there is no need for specialized and expensive equipment [32,38]. Even though this technique shows many advantages it still poses a problem for large scale production of MSC spheroids for therapeutic applications [53,54]. Other of the techniques used for the formation of MSC spheroids is the forced aggregation method which consists in applied centrifugal forces to induce MSC in vitro aggregation using micro-well plates in presence or not of biomaterials [40,42,43]. On the other hand, it has been showed that immunomodulatory activity of MSC does not seem to be spontaneous but requires MSC to be 'licensed' by inflammatory microenvironment to exert their effects [11,55]. In this line, Krampera and Ren demonstrated that MSC-mediated immunosuppression requires preliminary activation of the MSC by immune cells through the secretion of the proinflammatory cytokine IFN-g, alone or together with TNFa, IL-1a or IL-1b [56,57]. In this review, 4 of 28 articles selected, use cytokine priming to improve the functional properties of MSC ( Figure 2) [29,[42][43][44].

Culture Conditions.
For MSC spheroids to be eligible for clinical applications they need to be xeno-free (not contain-ing any animal-derived components). Serum containing media can carry unexpected agents risking viral or mycoplasma contamination [48]. Then, spheroid formation in media without FBS (fetal bovine serum) is critical. In this analysis, we identified that 8 of the 28 articles included, used serum-free media in their cell aggregation protocols ( Figure 2). The importance of FBS is highly noted for cell aggregation during spheroid formation, correlated with spheroids exhibiting faster assembly and more defined edges. Some of the strategies used to substitute FBS include using chemically defined media, in which all components are known, and composition can be controlled or supplementing media with patient-derived serum or human serum albumin [39].
Also controlling the oxygen level during spheroid assembly can have beneficial effects. Hypoxia as a priming method of MSC aggregates was used in three articles [30,33,46] ( Figure 2). Some research has pointed out that hypoxic conditions during cell aggregation can improve MSC properties. For example, the enhancement of the paracrine effect due to higher levels of IL-6, IL-8, and MCP-1 [46]. From a physiological point of view, MSC cultured under hypoxic conditions can better prepare cells for an ischemic environment typical of damaged tissue.

Spheroid Diameter
. Surprisingly, there does not seem to be homogeneity of spheroid size, 6 articles show a similar diameter range of 200-500 μm, 4 articles a diameter range between 100-200 μm, 3 a diameter between 0-50 μm, 1 has spheroids with diameter between 50-100 μm and finally 1 article with spheroid diameter> 500 μm ( Figure 2). Another measurement used to describe spheroid size is the number of cells within spheroids, and 5 articles displayed spheroids with 10,000-25,000 cells/spheroids, 3 with 200-1,000 cells/ spheroids and 1 with cell concentration greater than >25,000 cells/spheroids ( Figure 2). This is the initial number of cells at the beginning of the aggregation process. Some articles (n = 4) made no mention of MSC spheroid diameter or size ( Figure 2). Spheroid size is important because the diameter can determine nutrient and oxygen availability, as well as mechanical forces created by the cell-to-cell contacts between MSC, modulating gene expression [58].

2010
USA Adipose tissue Gene expression and proteins levels determination, in-vivo therapeutic potential (diabetic wound model). [29] Bartosh et al.

South Korea
Adipose tissue Apoptotic markers and growth factors expression, in-vivo therapeutic potential (elastase-induced emphysema model). [49] Costa et al.

2017
China Bone marrow Cell preservation and survival analysis, transcriptomic analysis, immunomodulatory activity, in vivo therapeutic potential (colitis model). [34] Kim et al.

South Korea
Bone marrow Exosome production, MSC spheroid size, cell density and morphology evaluation. [35] Lawrence et al.

South Korea
Adipose tissue Hypoxia-induced angiogenic cytokines and extracellular matrix components expression, in-vivo proliferation potential (hindlimb ischemia model). [45] Li et al.

Germany
Adipose tissue Cytokine gene expression and protein levels, adipogenic potential, tissue healing-associated angiogenesis potential. [46] Park et al.

2017
USA Bone marrow IFN-g priming, IFN-g microparticle delivery, immune factors secretion, in-vitro immunomodulatory potential (T-cell activation and macrophage immune assays). [43] 5 Stem Cells International can be identified from the selected works. These traits can be studied by variations in gene expression, protein secretion, surface marker expression, culture, and differential staining. Stemness is usually evaluated by the expression of transcription factors Nanog, Oct-3/4, Sox-2, Klf4, c-Myc, and STAT3, but also the expression of surface markers like CD105, CD90, CD73, and CD34 among others [16,18,50]. The effect of MSC over angiogenesis, the development of new vessels, is assessed mainly by the expression of the factors VEGF and HGF, among others ( Table 2). Differentiation potential to several types of tissue is also studied as a characteristic trait of MSC, generally by different staining techniques like Alizarin Red, Alcian Blue, and Oil Red O staining, to determine osteogenic, chondrogenic and adipogenic differentiation, respectively. Cytokines and soluble factors secreted by MSC has been attributed to great therapeutic effects. Among the cytokines analyzed are growth factors, chemokines, interleukins, and more (Tables 1 and 2). There is also evidence of the paracrine anti-cancer, antiapoptotic and resistance to oxidative stress of MSC (Tables 1 and 2). There seems to be a great focus on immunomodulatory effects of MSC, so the expression of TSG-6, PGE2, LIF, IDO, STC1, IL-6, IL-8 and more, is normally evaluated, as well as the capacity of MSC to decrease levels of TNF-α (Table 2).

In Vivo Therapeutic Effects of MSC Spheroids.
Of the total pool of articles reviewed, only 13 assessed the therapeutic effects of MSC spheroids in vivo (Table 3), 2 articles used BM-MSC, 8 articles used AT-MSC and 3 articles used UC-MSC. Several study models are used to investigate in vivo effects, such as wound healing or pro-inflammatory disease models, but mostly ischemia animal models are used (Table 3). In vivo studies include spheroid transplantation or application of conditioned media derived from spheroid culture, into the target tissue. Overall studies reveal that the application of MSC spheroids in vivo has enhanced therapeutic effects compared to monolayer 2D culture. Studies showed better outcomes for MSC in 3D conformation including reduced inflammation, faster wound healing and closure, functional recovery and tissue repair due to immunomodulatory effects, better MSC engraftment in damaged tissue, higher MSC survival and less apoptosis at injury (Table 3).   40 Increased chemotactic potential induced by conditioned medium (3D, alginateencapsulated 3D versus 2D). Enhanced immunomodulatory potential. Enhanced angiogenic potential (alginate-encapsulated 3D versus 2D).
Higher migration of fibroblasts in the presence of alginate-encapsulated spheroids.
Higher expression of TSG-6 in encapsulated and non-encapsulated spheroids. Higher proangiogenic potential.    DSS and TNBS-induced mouse colitis model.
Wound healing nude mice model. 49 Enhanced therapeutic and regenerative effect (3D versus 2D). Greater regeneration of lung tissues and higher FGF2 and HGF expression.
Elastase-induced emphysema mouse model. 45 Higher cell survival and proliferation in ischemic tissue (3D versus 2D). Murine hindlimb ischemia model. 52 Enhanced therapeutic and regenerative effect (3D versus 2D). Spheroids promoted tissue repair and reduced the final atrophy.
Ischemia-reperfusion injury in SCID mice. 47 Enhanced survival and therapeutic effect (3D versus 2D). Higher survival, angiogenic efficacy and differentiation into epithelial cells. Greater effectiveness in functional recovery of ischemic skin flap.
Murine ischemic skin flap model.

37
Enhanced survival, paracrine secretion and therapeutic effect (3D versus 2D). Higher secretion of VEGF, HGF and TSG-6. Less apoptosis and tissue damage at injury site and higher vascularization.
Ischemia-reperfusion injury in rats kidneys.
Mouse hindlimb ischemia model. 50 Enhanced regenerative and therapeutic effect (3D versus 2D). Faster decreased of ALT, AST and total bilirubin. Spheroid treatment increases IFN-g and IL-6 serum levels and reduces TNF-a levels.

41
Higher therapeutic effect of conditioned medium (3Dversus 2D), spheroid-derived conditioned medium attenuated tissue destruction, reduced synovial inflammation and bone erosion.
Adjuvant-induced arthritis in wistar rats. 9 Stem Cells International 3.7. Limitations of This Study. There are some limitations to this study. First, there is a degree of heterogeneity in the sources of cells and techniques used to produce the MSC spheroids. Second, there are many publications excluded due to technical factors, but still holding conclusions that may be relevant to this research. Finally, there is still a lack of clinical research to test MSC real benefits on a large scale randomized controlled trials.

Conclusion
Based on the literature reviewed, we can conclude that the most used source of MSC to produce spheroids is bone marrow followed by adipose tissue and umbilical cord. Although MSC can be derived from many adult tissues such as bone marrow, adipose tissue, umbilical cord, and placenta, these sources present some limitations due to differences between donors, extensive in vitro cell culture expansion and clinical trials with inconsistent results [14,59]. In this line, MSC derived from human Induced pluripotent stem cells (iPSC) has emerged in the last decade as an excellent therapeutic strategy to overcome the limitations of MSC source, interdonor variability, senescence and culture [60][61][62]. Human-iPSC-derived MSC possess higher proliferative potential and telomerase activity as well as immunomodulatory and angiogenic properties which has been demonstrated in different preclinical models of disease and clinical trials [61,[63][64][65][66]. Interestingly, Ding et al., demonstrated that murine-iPSC-derived MSC cultured in an encapsulated 3D spheroid format display stronger immunomodulation in a murine heart transplantation model [67].
The cell aggregation protocol most used was the hanging drop technique followed by forced-aggregation, low attachment, spontaneous assembly, then chitosan films and hyaluronic acid gel.
The evidence here shows MSC traits are enhanced by 3D culture. Results from the 28 articles selected suggest that MSC display better stemness, angiogenesis, differentiation potential, cytokine secretion, paracrine and immunomodulatory effects when presented as spheroids. At the time of this review, no human trials using MSC spheroids were found at the National Institutes of Health (NIH) website.
In this context, genetically engineering MSC or threedimensional culture, express and secrete important paracrine and immunomodulatory factors such as IDO, PGE2 and TSG-6 suggesting that this might increase the in vivo therapeutic effect of MSC [23,31,42,43].
In comparison with monolayer culture, MSC in 3D conformation presents as an attractive alternative for therapeutic applications of MSC. However, more studies are needed to evaluate the real therapeutic efficacy of MSC spheroids as well as mechanisms and pathways involved.

Conflicts of Interest
The authors declare that they have no conflict of interests.