This is the first study investigating the safety profile of stem cell therapy in patients with IPF. The clinical trial met its primary objective demonstrating an acceptable and tolerable safety profile of endobronchially administered autologous ADSCs-SVF, during the infusions and during short- and long-term follow-up. Although this study was not designed to delineate mechanisms of actions or authenticate efficacy, it provides indications that current therapeutic approach may be of some benefit. In particular, almost all studied participants (86%) followed-up for 12 months exhibited stable functional and exercise capacity status, evidence that needs to be further explored. Comparisons of available clinical and functional data in 5 subjects 3 months prior treatment initiation and during follow-up period also revealed no deterioration. Improvements in indicators of quality of life (SGRQ) were also reported indicating a potentially substantial psychological impact of cell-based therapies in patients with devastating chronic lung diseases with treatment, yet, ineffective.
The present results accelerate the rapidly expanded scientific knowledge and establish a rigid basis for future efficacy trials investigating the therapeutic use of cell-based therapies in patients with chronic lung diseases, including IPF overcoming fears, ethical issues and safety concerns. The latter mainly arise from the absence of a coordinated statement regarding the exact mechanisms of action and fate of MSCs, within an inflammatory, fibrotic and dysplastic microenvironment that severely hampered clinicians’ efforts to study stem cell therapeutic potential in adult injured lung [8, 12, 15]. This rather disappointing ascertainment currently dominates respiratory research field despite the fact that a continuing accumulation of data supports both safety and efficacy of stem cell therapy in animal models as assessed by attenuation of experimental lung fibrosis and inflammation potentially mediated through MSCs protective paracrine properties [15, 34–37].
In view of the significant lack of knowledge regarding the applicability of stem cell therapy in patients with IPF coupled with the current disappointing survival data arising from large multicentre clinical trials and the absence of a standard of care, we conducted a phase Ib, non-randomized, no placebo-controlled trial to study primarily the safety of the endobronchial infusion of autologous ADSCs-SVF in patients with IPF of mild to moderate disease severity (FVC>50%, DLCO>35%). As secondary exploratory endpoints we decided to include efficacy issues focusing on clinical (mMRC dyspnea scale), functional (FVC, DLCO), exercise capacity (6MWD) and quality of life indicators (SGRQ and CAT).
The most important finding of our study was the demonstration of an acceptable safety profile of adipose derived stem cell therapy locally administered within injured IPF lungs. Our approach exhibited a number of advantages including the following:
1) Firstly we used minimally manipulated autologous ADSCs-SVF lying in abundance within adipose tissue, thus eliminating the need for culturing over days to obtain a therapeutically viable number. In addition, cells were easily obtained by lipoaspiration, a procedure that is less painful than harvest of bone marrow. A growing body of evidence currently supports the notion that therapies involving minimally manipulated MSCs can overcome the fear and concern of undesirable alterations of allogeneic MSCs during ex vivo cellular expansion, including immunogenicity, contaminations, tumor, ectopic tissue formation and organ toxicity resulting from undesirable engraftment in the microvasculature [15, 16, 34, 56–59]. In our study, ADSCs-SVF, were neither cultured nor expanded and were directly infused within the patients’ lungs after implementing a two-step activation procedure using a cocktail of autologous growth factors (PRP)  and photobiostimulation , two novel approaches known to amplify paracrine beneficial effects of MSCs. Furthermore, recent preclinical and human studies have raised significant concerns with regards to possible dysfunctional migratory and paracrine properties of ADSCs-SVF derived from older individuals  and mice . In particular, Uji et al.  recently reported that stromal cells derived from the adipose tissue of relatively older mice exhibited reduced migratory capacity and failed to attenuate bleomycin-induced lung fibrosis. While the subject of an ongoing study, cultures of ADSCs-SVF derived from representative patients with IPF revealed a significant increase in the number of ADSCs-SVF colonies and an overexpression of anti-inflammatory (IL1-receptor antagonist) and angiogenic (vascular endothelial growth factor-VEGF) mediators, following activation with PRP and laser irradiation. Furthermore, no induction in the expression of mesenchymal markers (transforming growth factor-TGF-b, a-smooth muscle actin) or phenotypic characteristics compatible with fibroblast differentiation were observed (data not shown). In line with extended experimental [29, 34, 36, 37] and human data [40, 42, 43, 47, 56, 57] our patients did not experience any serious or clinically meaningful side effects, both during short-and long- term follow-up period, since no infusional toxicities, allergic reactions, disease acute exacerbations or ectopic tissue formation and tumor development were observed to date (24 months after the first infusion).
2) Secondly, in order to better characterize our isolated cell population and given significant controversies regarding isolation and characterization procedures between different laboratories we applied the most recent statement of IFATS/ISCT  and used a complete panel of surface antigens (n=10). Based on the recent definition of IFATS/ISCT our isolated cells, after being activated, developed characteristics of an immunophenotypic profile that lies between crude SVF cells and ADSCs for the following reasons: a) Consistently undetectable levels of CD45+ cells (characteristic of initially isolated SVF cells), b) levels of stromal markers (CD13, 29, 73, 90, 105) higher than those described in initially isolated SVF cells that did not reach peak levels seen in ADSCs which represent a more homogeneous cell population. c) Expression levels of stem cell associated marker CD34 were consistently high; however they were neither at peak values as observed in crude SVF population nor absent as noted in ADSCs. It seems that activation procedures may account for the temporal loss of expression markers that are present in the crude SVF cellular mixture rendering activated cells to exhibit a surface protein profile tending to resemble that of MSCs. Therefore, we applied the term ADSCs-SVF that better characterizes our isolated and activated cell population.
3) Thirdly, given that IPF pathogenesis is mainly restricted to the lungs and experimental data reports that intravenous stem cell administration is characterized by minimal lung uptake , we decided to deliver cells endobronchially in order to achieve maximum accumulation into sites of ongoing injury and thus, maximize their therapeutic potential. A semi-invasive technique of administration was chosen, namely bronchoscopy that was proven both well tolerable and accurate as estimated by scintigraphic analysis demonstrating prolonged strong signal intensity exhibited by radiolabeled stem cells and sustained even 24 hours after the infusion. Whether local administration of ADSCs-SVF will be proven more efficacious than systemic delivery remains to be elucidated.
4) Finally and most importantly we deliberately selected patients with mild to moderate disease severity since our study was designed to provide safety data and therefore events of disease acute exacerbation or progression that are more likely to occur in patients with end-stage lung disease could have masked our results. In addition, current experimental data demonstrates maximum beneficial effects during the early inflammatory stages of modeled disease that disappear later when established fibrosis has developed [36, 63]. Nevertheless, the last parallelism is arbitrary and should be treated cautiously. Future studies are warranted to support this notion.
Despite relative enthusiasm arising from our safety data, our trial is underpowered and exhibits a number of caveats that should be addressed cautiously before rigid conclusions can be drawn. This trial has not been designed neither to investigate efficacy nor to elucidate mechanisms of stem cells’ actions. Therefore exploratory efficacy data presented here should be interpreted cautiously and rigid conclusions cannot be drawn safely. At this point it is worth reporting that statistically significant improvements in indicators of quality of life that were observed in almost all our patients (86%) may reflect a placebo-effect and deserve additional verification. Furthermore, any statements that arise from functional and exercise capacity outcomes indicating disease stabilization would be too speculative and therefore should be avoided for the moment. It is also debatable whether a trend towards increase in sPAP at 6 and 12 months post-first endobronchial infusion could be attributed to therapeutic interventions or simply reflects an epiphenomenon due to unreliable methods of sPAP assessment such as cardiac echo. Additionally available efficacy data 24 months after first infusion were excluded from current analysis since all enrolled patients after completing the 12 month follow-up period were switched to pirfenidone treatment based on the recently published CAPACITY trials  and therefore it was impossible to attribute any functional alterations solely to cell-based therapy.
Furthermore the use of a heterogeneous mixture of cell population such as adipose tissue SVF raises significant methodological limitations since the exact contribution of each one of them could not be delineated based on our study design. To substantiate our findings and better define our isolated cell population, we utilized a complete panel of mesenchymal, hematopoietic and endothelial markers. As shown in Table 1 and in line with previous reports [21, 27, 54], the majority of our non-cultured, non-expanded ADSCs-SVF were of mesenchymal origin, meaning that they were positive for the minimally required markers such as CD29, 73, 90, 105 as well as for CD44, CD13 and CD116. In addition, ADSCs-SVF after activation did not express surface antigens CD31, 45, as expected. Finally, almost a third of our cells stained positive for CD34 indicating the presence of hematopoietic and endothelial progenitors. One alternative approach was to purify MSCs by subtracting CD45-, CD31- CD34- cells using immunomagnetic beads. Nevertheless, we decided to avoid manipulation of isolated cells since by infusing unpurified ADSCs-SVF we exploited the beneficial stemness of the entire cell population including MSCs, lymphocytes, endothelial progenitors and hematopoietic stem cells.
While our results indicate some reassurance regarding endobronchial infusion of stem cells in patients with IPF, significant work is sorely needed to understand complex stem cell properties as well as their behavior and fate within a fibrotic microenvironment. Currently, whether MSCs could differentiate into fibroblasts  given their common mesodermal origin and accelerate fibrotic cascade or even promote tumorigenesis on a longitudinal basis, is under debate. Alternatively, we may speculate that ADSCs-SVF may exert their beneficial effects through their unique paracrine activities (anti-inflammatory, anti-fibrotic, anti-apoptotic and immunomodulatory) and less by acting as cells with regenerative capacity. In line with this premise and given a potential association between global immune impairment [65–67] and fibrogenesis we may speculate that a proportion of ADSCs-SVF may act as T regulatory cells  restoring immune deregulation and attenuating inflammatory and fibrotic cascade. The above statement is currently only speculative and needs further exploration. Studies are underway to shed further light into stem cells’ behaviour and resolve mechanistic issues surrounding their use.