Mesenchymal Stromal Cells Derived Conditioned Medium in Pulmonary Fibrosis: A Systematic Review and Meta-analysis

1College of Medicine, University of Sulaimani, Sulaimani, Iraq 2Department of Family and Community Medicine, College of Medicine, University of Sulaimani, Sulaimani, Iraq 3Physiology Research Center, Iran University of Medical Sciences, Tehran, Iran 4Pediatric Chronic Kidney Disease Research Center, Tehran University of Medical Sciences, Tehran, Iran 5Department of Epidemiology and Biostatistics, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran


Introduction
Idiopathic pulmonary fibrosis is a chronic and progressive disease with an unknown etiology. The prevalence and incidence of idiopathic pulmonary fibrosis have been increasing in recent decades 1 and it accounts for about 20% of all interstitial lung diseases. 2 About 3 million people have been reported worldwide to have idiopathic pulmonary fibrosis, with its prevalence increasing with age. 3 There is not a definitive treatment for pulmonary fibrosis. Initial studies have suggested the positive effects of corticosteroids and immunosuppressive agents in management of pulmonary fibrosis. However, systematic reviews have shown that there is insufficient evidence for the efficacy of these therapies. 4,5 The current guidelines approve pirfenidone and nintedanib for treatment of pulmonary fibrosis. However, the ideal therapeutic approach for management of pulmonary fibrosis is still under investigation. 6,7 Stem cells transplantation has been suggested as a therapeutic option for many conditions. 8-10 A systematic review in 2015 showed that administration of mesenchymal stromal cells (MSCs) in animal models of pulmonary fibrosis improved fibrosis, decreased collagen deposition in lung, and reduced the expression of profibrotic chemokines. 11 Although preclinical studies have shown the benefit of MSCs transplantation, this treatment option has some limitations, including the possibility of tumorigenesis. In addition, circulating MSCs play a critical role in the pathogenesis of pulmonary fibrosis. For example, bone marrow-derived mesenchymal progenitor cells are able to differentiate into fibroblasts. This conversion to myofibroblasts is intensified in the presence of tumor growth factor β (TGF-β), which is elevated in pulmonary fibrosis. 12 On the other hands, lung resident MSCs secretome has anti-fibrotic effects. 13 Therefore, caution should be exercised in the administration of MSCs to attenuate the progression of pulmonary fibrosis. The use of conditioned medium as an alternative option to stem cell therapy has been suggested in some studies. 14,15 Recent studies show that the paracrine effects of MSCs play a major role in treatment of acute and chronic conditions. [16][17][18] Therefore, administration of MSCsderived conditioned medium (MSCs-CM) may have all the effects of stem cell transplantation while omitting the side effects of stem cell administration. Preclinical studies have shown that administration of MSCs-CM could improve pulmonary fibrosis; [19][20][21] however, no consensus has yet been made in this field. Therefore, the present systematic review and meta-analysis intends to investigate the efficacy of MSCs-CM administration in improving pulmonary fibrosis.

Study Design and Search Strategy
An extensive search of the Medline, Embase, Scopus and Web of Science databases was conducted by the end of August 2019. PICO in the present study was defined as pulmonary fibrosis (problem), administration of MSCs-CM (intervention), comparison with non-treated animals (comparison), and pulmonary fibrosis score and fibrotic and profibrotic markers (outcome), respectively.
Keywords related to "pulmonary fibrosis" in combination with words related to "mesenchymal stem cell derived conditioned medium" were used in the search databases. Systematic searches were performed for each database using proper Boolean operators and standard tags. The search query in Medline (via PubMed) is presented in Appendix 1. In addition, a manual search was conducted on Google, Google Scholar and bibliography of relevant articles to find additional studies.

Eligibility Criteria
We investigated all original studies published/accepted in a peer-reviewed journal on the efficacy of MSCs-CM administration in managing pulmonary fibrosis in animal models. There was no time or language limitation for the search. Exclusion criteria included lack of an untreated control group, conditioned medium derived from non-MSCs, MSCs-derived secretome, genetically engineered MSCs-CM, in vitro studies and review articles.

Data Gathering and Synthesis
Records obtained from the systematic search were screened by two independent reviewers. Potential relevant studies were then studied in full details and relevant articles were saved. Data were recorded by two researchers independently in a pre-designed checklist. These data included first author's name, year of publication, number of animals, species, gender and age of animal, pulmonary fibrosis induction model, interval time between pulmonary fibrosis induction to treatment, source of MSCs, administration route, administered volume, type of graft (xenograft or allograft), follow-up duration and assessed outcome. Data were recorded as mean and standard deviation. Since in most animal studies, data are presented in graphs using plot digitizer software, mean and standard deviation were extracted. The use of this technique is a common method in animal meta-analysis studies. [22][23][24][25] Outcome First, we aimed to evaluate the effect of MSCs-CM on pulmonary fibrosis score, lung collagen deposition, collagen subtypes expression, TGF-β1 expression and other profibrotic chemokines. Since meta-analysis should be performed when the outcome has been reported in at least two studies, we had to assess pulmonary fibrosis score, lung collagen deposition, type I collagen, type III collagen, TGF-β1 expression and interleukin 6 (IL-6) expression.

Risk of Bias Assessment
Quality control of articles was performed based on SYRCLE's risk of bias tool -a 10-item risk assessment tool for pre-clinical studies. 26 Statistical Analysis Data were analyzed using STATA 14.0 statistical software. Mean and standard deviation of the outcomes were recorded and a pooled effect size with a 95% confidence interval (CI) was reported for each study based on standardized mean difference (SMD) calculation. I 2 was used for assessment of heterogeneity and Egger's test for publication bias. Finally, subgroup analysis and meta-regression were performed to find the source of heterogeneity and to find the factors affecting the efficacy of MSCs-CM on the improvement of pulmonary fibrosis. P < 0.05 was considered as the level of significance in all analyses.

Characteristics
Finally, 274 non-repetitive records were screened and data from 7 studies 19,21,27-31 were included in the meta-analysis ( Figure 1). Four studies were performed on rats while three were conducted on mice. Four studies used a bleomycininduced pulmonary fibrosis model while three studies used other models such as silica-induced and radiation-induced pulmonary fibrosis. The time interval between pulmonary fibrosis induction to MSCs-CM administration ranged from 0 to 28 days. Four studies performed MSCs-CM transplantation within the first 24 hours after pulmonary fibrosis induction. The type of MSCs used was bone marrow-derived mesenchymal stromal cells (BMMSCs) in three studies, adipose tissue-derived mesenchymal stromal cells (ADSCs) in two studies, and amnioticderived mesenchymal tissue cells (AMSCs) in two studies. MSCs-CM was injected intravenously in five studies and in situ in two studies. The transplanted volume of MSCs-CM ranged from 10 μL to 1000 μL. Follow-up duration varied between 11 and 28 days. Six studies examined fibrosis score, seven studies examined collagen deposition in lung tissue, four studies examined the expression of type I collagen, three studies type III collagen, three studies TGF-β1 expression and three studies IL-6 expression in lung tissue. Table 1 illustrates the characteristics of the included studies.
Risk of Bias Assessment SYRCLE's risk of bias tool showed random sequence generation, baseline characteristics of included animals, random housing, random outcome assessment, selective reporting, and other sources of bias in all eligible studies were of low risk. However, the risk of bias of allocation concealment and blinding of trial caregiver was unclear in six studies. Blinding of outcome assessment and incomplete outcome data in one study had high risk of bias (Table 2 and Figure 2A).

Meta-analysis: Efficacy of MSCs-CM on Pulmonary
Fibrosis Score Five studies reported the efficacy of MSCs-CM on pulmonary fibrosis score. Meta-analysis of this section showed that administration of MSCs-CM significantly reduced the score of pulmonary fibrosis. In other words, administration of MSCs-CM improves pulmonary fibrosis (SMD = -2.36; 95% CI: -3.21 to -1.51; P < 0.0001) ( Figure 3A). Subgroup analysis and meta-regression were performed despite mild heterogeneity between studies (I 2 = 52.0%; P = 0.064) ( Table 3 and Figure 4). Subgroup analysis indicated that the possible source of heterogeneity was animal species, type of MSCs and route of administration of MSCs-CM. It is worth noting that in all conditions, administration of MSCs-CM improved pulmonary fibrosis (P < 0.001) ( Table 3).
Meta-regression also showed that none of the time interval between pulmonary fibrosis induction to MSCs-CM administration (P = 0.722), administered MSCs-CM volume (P = 0.160), number of MSCs for conditioned medium derivation (P = 0.754) and follow-up duration (P = 0.920) had any effect on the efficacy of MSCs-CM on pulmonary fibrosis score (Figure 4).

Meta-analysis: Efficacy of MSCs-CM on Lung Collagen Deposition
Six studies reported the efficacy of MSCs-CM on reduction of collagen deposition. Meta-analysis of this section  showed that administration of MSCs-CM significantly decreased collagen deposition in animals with pulmonary fibrosis (SMD = -1.70; 95% CI: -2.18 to -1.23; P < 0.0001) ( Figure 3B). Subgroup analysis showed that differences in animal species, pulmonary fibrosis induction model, type of MSCs, type of MSCs-CM administration, and type of graft (xenograft versus allograft) did not affect the efficacy of MSCs-CM in reducing collagen deposition in pulmonary fibrosis (Table 3).  Meta-regression also showed that none of the time interval factors between pulmonary fibrosis induction to MSCs-CM administration (P = 0.528), administered MSCs-CM volume (P = 0.814), and number of MSCs for conditioned medium derivation (P = 0.58) as well as follow-up duration (P = 0.867) had any effect on the efficacy of MSCs-CM on reducing collagen deposition in the lung ( Figure 5).

Meta-analysis: Efficacy of MSCs-CM on Type I Collagen and Type III Collagen Expression
Data from four and three experiments were entered to evaluate the efficacy of MSCs-CM on type I collagen and type III collagen expression, respectively. Analyses showed that administration of MSCs-CM decreased the expression of type I collagen (SMD = -6.27; 95% CI: -11.00 to -1.55; P = 0.009) and type III collagen (SMD = -5.16; 95% CI: -9.86 to -0.47; P = 0.031) after induction of pulmonary fibrosis (Figures 3C and D).

Discussion
The present meta-analysis for the first time collected and analyzed quantitative data on the effect of MSCs-CM on the pulmonary fibrosis in animal studies. Analyses showed that administration of MSCs-CM improved pulmonary fibrosis. The effect of MSCs-CM could be mediated by reducing collagen deposition and expression as well as inhibiting the production of inflammatory chemokines such as TGF-β1 and IL-6.
Risk of bias was low in most studies, which was one of the strengths of the present meta-analysis. However, blinding   of trial caregiver and allocation concealment details were not reported in six articles. Since the assessed outcome in the present study are histopathology assessment and protein expression assay, the blinding status seems to have little impact on the reported findings.
The reduction of fibrosis in the present study was evaluated by summarizing the data presented based on fibrosis score. This technique is a standard method for examining the rate of tissue fibrosis in the lung. 32,33 However, in interpreting the findings, the blinding status of outcome assessor is important. In the five studies included in this section, the risk of bias in the assessment of fibrosis score was low in four studies and unclear in one. Therefore, the findings are reliable.
Subgroup analysis showed that differences in the sources of MSCs did not affect the efficacy of MSCs-CM in improving fibrosis and collagen deposition. This finding is important because access to ADSCs cells is much easier than other sources such as AMSCs and BMMSCs. In addition, their preparation and autograft transplantation are much more practical. Therefore, since the differences in cellular source have no effect on the conditioned medium's performance, it is recommended to use ADSCs derived conditioned medium in future studies. In addition, a meta-regression analysis found that, unlike many chronic diseases, the interval between the occurrence of pulmonary fibrosis and the onset of treatment with MSCs-CM did not affect its efficacy. Therefore, it seems that even in the chronic phases of the disease, this treatment can be used to control the progression of pulmonary fibrosis. However, only one study identified a 4-week gap between the onset of pulmonary fibrosis and the onset of MSCs-CM administration. Therefore, the efficacy of MSCs-CM administration in the chronic phase of pulmonary fibrosis should be further investigated.
After MSCs-CM administration, type I and III collagen expression levels decreased significantly. Studies have shown that after pulmonary fibrosis, overexpression of type I and type III collagen is observed. 34,35 These studies show that expression of type I collagen is also increased by TGF-β1 expression. Therefore, administration of MSCs-CM seems to modulate collagen expression by reducing TGF-β1 expression in lung tissue.
Most of the studies included in the present metaanalysis used the intravenous route to administer MSCs-CM. Subgroup analyses also showed that there was no difference between intravenous and in situ administration of MSCs-CM. This is an advantage for MSCs-CM because intravenous administration is one of the simplest routes of drug administration, whereas in many cases, intravenous administration of MSCs is associated with concern such as tumorigenesis.
In conclusion, the present meta-analysis showed that administration of MSCs-CM improved pulmonary fibrosis. Since intravenous administration of this therapeutic option has fewer risks than MSCs and its intravenous administration has a similar efficacy to in situ administration, the conditioned medium can be used in future studies as an alternative to MSCs transplantation. It seems that the effect of MSCs-CM was mediated by reducing collagen deposition as well as inhibiting the production of inflammatory chemokines such as TGF-β1 and IL-6. In addition, it appears that ADSCs is the best source for preparation of conditioned medium.