Cell therapy based on stem cells or their extracellular vesicles during kidney graft preservation: Current state of the art and novelties
1 ORGAN SHORTAGE AND EXTENDED CRITERIA DONORS
Transplantation often remains the best therapeutic option in terms of life quality and disease prognosis improvement to treat chronic or even acute organ failure. According to a report published by the World Health Organization in 2023,1 less than 10% of the world's organ needs are covered. Focusing on the kidney, which is now the most transplanted organ in the world, the latest report of the Global Observatory on Donation and Transplantation published in 2023 (based on 2022 data)2 pointed out the gap between supply and demand: there are currently more patients on the active waiting list than there are grafts available for them worldwide. Kidney grafts can come from two main different sources: living donors, which represent a minority of donations, and deceased donors. Nevertheless, the organ shortage, which has been worsening year after year, has led to extend donation criteria over the years. This means, for example, the use of deceased donors not only after brainstem death but also after unpredictable irreversible circulatory arrest with immediate cardiopulmonary resuscitation attempted by trained providers (according to the Maastricht classification, second category) and after circulatory arrest occurring based on a decision to withhold or withdraw life-sustaining treatment (according to the Maastricht classification, third category). These death circumstances are usually associated with intensive donor reanimation processes consisting in noradrenaline administration, in massive vascular filling to prevent reanimation complications such as inflammation, haemodynamic instability or acute kidney failure. Extended criteria donors also include older donors aged over 65 years and donors with comorbidities such as arterial hypertension, cardiopathy, diabetes and even chronic kidney failure. The growing need for organs may also result in organs being transported from more distant regions. In all these situations kidney grafts are more susceptible to be affected by ischaemia‒reperfusion (IR) injuries.
2 ISCHAEMIA‒REPERFUSION: A FAR-REACHING IMPACT AT EVERY SCALE
IR is a pathophysiological phenomenon taking place from the donor's reanimation to the recipient's transplantation. Ischaemia is induced by the sudden arrest of oxygen and nutrients supply during the organ retrieval step, which may be prolonged during organ preservation sequence depending on its modalities. Reperfusion occurs when anastomoses are performed between the graft and the recipient and refers to the massive oxygen supply in a medium, which was previously deprived of oxygen.3 On a microscopic scale, this phenomenon is associated with shifts in mitochondrial metabolism and function, by a release of reactive oxidative species causing cytoskeleton destruction, complement system activation and recruitment of innate and adaptative immune cells.4 Faced with these perturbations, the cell eventually dies by necrosis, phagocytosis or apoptosis. On a macroscopic scale considering renal grafts, IR mainly leads to graft endothelial injuries, which can be further translated clinically by acute tubular necrosis, primary non-function or delayed graft function usually characterised by the use of dialysis in the 7 days post-transplantation. Because of these injuries and immune system activation, IR can be responsible for shorter graft survival and even acute or chronic graft rejection.4
3 FROM KIDNEY GRAFT DYNAMIC PERFUSION …
Since 2000s, researchers have developed several techniques to improve kidney graft conditioning to counteract IR-induced kidney injuries. From cold storage, which has been the main preservation strategy for decades, dynamic hypothermic machine perfusion (HMP) has greatly improved graft conditioning.5 According to the Arrhenius's equation, hypothermia causes a 50% decrease of cell metabolic activity every 10°C. Placed in an iso-osmotic extracellular preservation liquid at a mean temperature of 4°C, the renal graft is connected to the preservation solution with its artery and a surgical aortic patch, then the blood is reabsorbed into the venous system, thus allowing continuous kidney perfusion. This technology has increased the microcirculation in the graft, reduced delayed graft function, chronic graft dysfunction and ensured a better graft survival at 1 year compared to standard static cold storage (SCS) in deceased donor kidney transplantation.5, 6
Normothermic machine perfusion (NMP) has also emerged as a new strategy to optimise graft preservation. It is defined by a warmed oxygenated circulation at a mean temperature between 34°C and 37°C through the kidney to recreate near-physiological conditions.7 Oxygen can be delivered by using either whole blood, pyridoxylated bovine haemoglobin or a combination of preservation solution and purified red blood cells.8 It has been shown to be feasible and safe in a randomised clinical trial.7 Furthermore, NMP has several advantages compared to SCS and HMP techniques. NMP can help cells to maintain an aerobic metabolism to minimise ischaemia-induced damage. Graft perfusion parameters, temperature and other items can also be monitored in real time through NMP. It is also a way to deliver therapeutics including those based on cell therapy.9
4 …TO CELL THERAPY DELIVERY
In the field of organ transplantation, cell therapy has emerged in recent years as a promising option for the future of graft preservation in dynamic perfusion.
4.1 Mesenchymal stem cells or equivalent
Mesenchymal stem cells (MSCs) or multipotent adult progenitor cells are multipotent adult stem cells (which can be derived from various substrates including bone marrow) that can differentiate into three different cell types: osteocytes, chondrocytes and adipocytes. They are deficient in major histocompatibility complex (MHC) class II and costimulatory molecules (CD80, CD86 and CD40) and they set out a low expression of MHC class I.10 Thanks to their ability to release anti-inflammatory and pro-tolerogenic cytokines, they constitute a relevant source for cell therapy. Indeed these cells can promote tissue repair through paracrine factors and direct cell interactions.11 More specifically, the delivery of MSCs during NMP offers an opportunity to improve their biodistribution and avoid in this way the adverse effects potentially caused by a systemic administration. In 2019, Brasile et al.12 performed a titration of MSCs on five pairs of human kidney allografts from donation after circulatory death, conditioned in an exsanguinous warm perfusate. Treatment based on a MSC infusion from 25 × 106 to 100 × 106 MSCs revealed a reduced inflammatory response and an adenosine triphosphate (ATP) synthesis increase. Another study13 used a porcine kidney auto-transplantation procedure, exposed to oxygenated HMP followed by NMP, during which 10 × 106 porcine or human MSCs were injected; the procedure was safe and did not affect either urine or perfusate levels of neutrophil gelatinase-associated lipocalin (NGAL), a biomarker of acute kidney failure (tubular necrosis), or histological kidney injury for 14 days post-transplantation. Besides, creatininaemia on days 2–7 was significantly lower in the NMP + human MSCs group, than in NMP alone. Furthermore, in 2021, Thompson et al.14 carried out an experiment based on MAPCs delivery during NMP. Five pairs of human kidney grafts from deceased donors declined for transplantation were perfused in NMP for 7 h with an oxygenated red cell-based perfusate and the test group was treated with MAPCs isolated from the same human bone marrow following 60 min of NMP. They showed therefore that such a combined strategy was feasible and safe too.
4.2 Kidney progenitor cells or urine progenitor cells
Kidney/progenitor cells (KPCs) or urine progenitor cells (UPCs)15 are derived from the kidney and can be isolated from urine. Like MSCs, they are able to differentiate into adipocytes, chondrocytes and osteocytes but they also have the specific capacity to differentiate into endothelial and epithelial cells. In addition to their differentiation properties, they have also been described as inflammation modulators too.15 In 2022, Arcolino et al.16 carried out an innovative cell therapy strategy based on kidney stem progenitor cells (KSPCs) isolated from the urine of preterm neonates17 (nKSPCs) and injected into human kidneys discarded for transplantation during NMP. These cells expressed nephron and stromal progenitor markers.17 They found that administration of KPCs during NMP was also a safe and feasible procedure, inducing a de novo expression of SIX2 in the renal proximal tubular cells, known to be a transcription factor involved in cell self-renewal and survival,16 and upregulating regenerative markers such as SOX9 and VEGF, while significantly reducing levels of renal injury biomarkers and inflammatory cytokines.
4.3 Stem cell-derived extracellular vesicles, advances and barriers
To go further, some of the beneficial effects of stem cells still seem yet to be related to their secretome which is composed, among others, of extracellular vesicles (EVs).18 In this review, we have chosen to follow the International Society of Extracellular Vesicles 2023 guidelines, preferring the generic term EVs since the distinction between subtypes requires a rigorous characterisation.19 EVs represent a broad class of nanometric membrane-enclosed protein and ribonucleic acid (RNA)-containing particles, reflecting the original nature of the cell. They can be divided into exosomes, produced by the endosomal pathway, microvesicles produced from the plasma membrane or multivesicular bodies.20 EVs are involved in tissue repair and immunomodulation by moving and communicating with other cells through a paracrine and autocrine mode.21 Isolated EVs could therefore contribute to a cell-free tissue regenerative approach, bypassing some potential side effects linked with the use of living cells such as immunogenicity, oncogenicity or thrombosis/embolism.20 To expand on this point, in 2024 a deep RNA sequencing carried out on urine stem cell-derived EVs (USC-EVs) injected intravenously in rats with IR injuries showed that USC-EVs enriched with miR-216a-5p may represent a promising therapeutic option for acute kidney injury by reducing apoptosis22 and even pyroptosis.23
Considering this new perspective based on porcine UPC (pUPC)-derived EVs and the benefits brought by dynamic perfusion, our IRMETIST team decided to focus on the impact of pUPC-derived EVs on injured kidney grafts during HMP and NMP.24 On one hand, in an ex vivo preclinical porcine kidney preservation model, we exposed 17 porcine kidney grafts to 32 min of warm ischaemia, then to 24 h of hypothermic perfusion (Lifeport, Organ Recovery System) followed by 5 h of normothermic perfusion. Kidney grafts were randomly allocated in three different groups: HMP and NMP without EVs, EVs during HMP only and EVs during both HMP and NMP. This model showed that UPC-derived EVs in dynamic perfusion was feasible, safe without perfusion parameters disturbance. It contributed to reduce IR biomarkers when EVs were injected during HMP + NMP (group 3). Indeed, normothermic perfusates metabolic analyses associated to group 3 revealed a stimulation of antioxidant actors such as Haeme-oxygenase 1 and Nuclear factor erythroid-2-Related factor 2 (NRF2), as well as a decrease of sulphonylureas-analogue signalling pathway involved in K+/ATP inhibition, a decrease of pro-apoptotic and pro-fibrotic factors such as ecdysone analogue. Histological analyses of this group also showed less tubular dilatation and a significant reduction of a few pro-inflammatory cytokines such as IL-18, IL-1α and IL-1β. There was no effect on renal function neither on the creatinine clearance nor on NGAL perfusate's level. On the other hand, we developed an in vitro approach based on human glomerular endothelial renal cells in culture, exposed to 24 h of cold anoxia (4°C) followed by either 5 or 24 h of normothermia‒reoxygenation with 20% of oxygen, treated with UPC-derived EVs either during only anoxia or in both anoxia and normoxia, mimicking thus the ex vivo model. This in vitro experiment underlined an increased metabolic activity in cells treated with pUPC-EVs in both cold anoxia and normothermia‒reoxygenation, but only 24 h post-reoxygenation. This study was all in all the first to describe the proof of concept of combining kidney graft dynamic perfusion with cell therapy based on pUPC-derived EVs.
In terms of methods and results, some strengths of this innovative approach need to be highlighted. Indeed, it may be more appropriate to administer cell therapy at an earlier stage than during anastomosis or after transplantation to act as early as possible in the IR sequence. Dynamic perfusion may thus promote EVs renal biodistribution and limit potential risks associated to an intravenous administration. We were also able to independently assess the effect of pUPC-derived EVs during both HMP and NMP procedures, which has not been described before. Compared to other types of stem cells, UPCs have several advantages including first their renal origin, their easier isolation technique from a readily available substrate. Second, the use of a porcine source for UPCs and grafts may be clinically relevant because of the anatomical, physiological and functional similarities shared with their human counterparts.25 That is why precisely more and more scientists are trying to make progress on xenotransplantation; this preclinical porcine model and porcine cell therapy product may help to increase the number of clinical xenotransplantation trials, following in the footsteps of NYU Langone surgeons.26 Concerning EVs themselves, compared to whole cells, they represent an appropriate therapeutic source thanks to their lack of immunogenicity despite MHC class I expression,27 their nanometric size, which allows them to circulate more easily through the perfusion machine. They can also be cryopreserved at ‒80°C without additives and engineered to be loaded with a drug. For example, IL-10, an anti-inflammatory cytokine, loaded on macrophage-derived EVs has already been used to prevent IR injuries.28 Regarding the current public health issue, we hope that this cell therapy strategy will help to alleviate the graft shortage by improving the conditioning of marginal grafts. We can even dare to imagine that in a near future this approach could be used in organ repair centres to rehabilitate such marginal human organs or genetically modified porcine grafts prior to transplantation.
Nevertheless, we have to point out a few limitations. First of all, this study does not assess the long-term effect of our therapy due to the lack of transplantation; this important step should be carried out in a future experiment. From a pharmacokinetic point of view, many aspects of this strategy still need to be optimised. Indeed, it is required to elaborate on the dose‒response effect of pUPC-derived EVs injection and on the most appropriate injection time. It could also be considered to repeat the injection to emphasise its effects. Another experimental step should also be added to compare the impact of EVs + pUPCs to EVs alone. A study by Lin et al. in 201629 demonstrated that combined therapy with adipose-derived MSCs and adipose MSC-derived EVs could have a better effect on acute IR injury than either one, considering creatininaemia and levels of inflammatory, apoptotic and fibrotic biomarkers; we can therefore wonder whether there is a different impact by injecting pUPCs + pUPC-derived EVs than pUPC-derived EVs alone. From a pharmacodynamic point of view, the specific molecular mechanism of EVs recognition and uptake by the recipient remains unclear.30, 31 It can be suggested that the membranous ligands of EVs allow them to be captured by target cell receptors and that they can be internalised by the recipient cell through endocytosis or direct fusion with the plasma membrane.32 Several studies have shown that cells at 4°C significantly reduce their ability to capture EVs,33 suggesting that EVs uptake is an energy-dependent process. To expand on this point, an additional experiment could involve pre-labelled UPC-derived EVs to track their distribution within kidney grafts under dynamic perfusion. Indeed Durak-Kozica et al. in 201834 succeeded in visualising EVs uptake by human endothelial cells using a confocal laser microscope after staining of EVs with PKH26 marker. The composition of EVs in the study by Burdeyron et al. is not only undetermined, but also represents an obstacle to understand their mechanism. We may suggest further transcriptomic analyses through RNA sequencing as currently performed by Zhang et al. in 202022 to elaborate on the EVs components and to know if specific RNA transcripts contained into EVs are involved in this mechanism and are able to inhibit apoptosis and thus prevent IR.35 For example, it has already been demonstrated that miR-125b-5p, contained in human umbilical cord MSC-derived EVs inhibited miR-125b-5p/p53 signalling pathway in tubular epithelial cells and mediated kidney repair in acute kidney injury36; in addition, human urine-derived stem cells have been demonstrated to protect against renal IR injury in a rat model through exosomal miR-146a-5p, which targets IRAK1 and further inhibits the nuclear factor-κB pathway and thus, pro-inflammatory cytokine IL-1β maturation.37 Furthermore, the microenvironment has emerged as a key feature in EVs secretome and composition modulation. Indeed, a hypoxic preconditioning performed on adipose-derived MSCs has been described38 to improve the production of EVs and their protection ability against IR injuries in a rat kidney model, with antiapoptotic, pro-angiogenic and antioxidative effects.39 In addition, MSC-derived EVs could also mitigate renal IR injuries by reducing NK cells number.40 Finally, if we want to move towards clinical applications and avoid a maximum of immunological and infectious interspecies obstacles, it seems necessary to test the effect of human UPC-EVs in a kidney transplantation model.
5 CONCLUSION
Although stem cell-derived EVs have emerged as a promising new strategy in the field of organ conditioning in transplantation, pharmacokinetic and pharmacodynamic challenges still remain to fill the knowledge gap in terms of mechanistic understanding, as well as isolation and characterisation techniques need to be further developed to promote a clinical use of EVs.
However, such an approach first requires serious thinking about precise criteria to define the organs at risk of IR injury and delayed graft function. Furthermore, this entails a better harmonisation of reanimation, surgical and clinical practice between the different centres. Therefore, marginal grafts could be selected for the most optimal preservation protocol regarding the donor/recipient's clinical context and be repaired prior transplantation. In this way, in a future ageing society affected by a more severe organ shortage, the cell therapy approach combined with the improvement of graft dynamic preservation technologies may be a supplemental potential mean to optimise the transplantation procedure from extended criteria donors or long-distance grafts.
AUTHOR CONTRIBUTIONS
NCB wrote the initial draft of this manuscript; TH and CS reviewed the manuscript. All authors approved the final version of this review.
ACKNOWLEDGMENTS
We thank Pr. Luc Pellerin for reviewing the final version of the manuscript.
ETHICS STATEMENT
Not applicable (review).
