Design of a Multiparametric Perfusion Bioreactor System for Evaluating Sub-Normothermic Preservation of Rat Abdominal Wall Vascularized Composite Allografts.

Static cold storage (SCS), the current clinical gold standard for organ preservation, provides surgeons with a limited window of time between procurement and transplantation. In vascularized composite allotransplantation (VCA), this time limitation prevents many viable allografts from being designated to the best-matched recipients. Machine perfusion (MP) systems hold significant promise for extending and improving organ preservation. Most of the prior MP systems for VCA have been built and tested for large animal models. However, small animal models are beneficial for high-throughput biomolecular investigations. This study describes the design and development of a multiparametric bioreactor with a circuit customized to perfuse rat abdominal wall VCAs. To demonstrate its concept and functionality, this bioreactor system was employed in a small-scale demonstrative study in which biomolecular metrics pertaining to graft viability were evaluated non-invasively and in real time. We additionally report a low incidence of cell death from ischemic necrosis as well as minimal interstitial edema in machine perfused grafts. After up to 12 h of continuous perfusion, grafts were shown to survive transplantation and reperfusion, successfully integrating with recipient tissues and vasculature. Our multiparametric bioreactor system for rat abdominal wall VCA provides an advanced framework to test novel techniques to enhance normothermic and sub-normothermic VCA preservations in small animal models.

48 h Normothermic Machine Perfusion With Urine Recirculation for Discarded Human Kidney Grafts.

Normothermic machine perfusion (NMP) has reshaped organ preservation in recent years. In this preclinical study, prolonged normothermic perfusions of discarded human kidney grafts were performed in order to investigate perfusion dynamics and identify potential quality and assessment indicators. Five human discarded kidney grafts were perfused normothermically (37°C) for 48 h using the Kidney Assist device with a red-blood-cell based perfusate with urine recirculation. Perfusion dynamics, perfusate and urine composition as well as injury markers were measured and analyzed. Donor age ranged from 41 to 68 years. All but one kidney were from brain dead donors. Perfusions were performed successfully for 48 h with all discarded kidneys. Median arterial flow ranged from 405 to 841 mL/min. All kidneys excreted urine until the end of perfusion (median 0.43 mL/min at the end of perfusion). While sodium levels were consistently lower in urine compared to perfusate samples, this was only seen for chloride and potassium in kidney KTX 2. Lactate, AST, LDH as well as pro-inflammatory cytokines increased over time, especially in kidneys KTX 3 and 4. Ex vivo normothermic perfusion is able to identify patterns of perfusion, biological function, and changes in inflammatory markers in heterogenous discarded kidney grafts.

European Society for Organ Transplantation (ESOT) Consensus Statement on the Role of Pancreas Machine Perfusion to Increase the Donor Pool for Beta Cell Replacement Therapy.

The advent of Machine Perfusion (MP) as a superior form of preservation and assessment for cold storage of both high-risk kidney's and the liver presents opportunities in the field of beta-cell replacement. It is yet unknown whether such techniques, when applied to the pancreas, can increase the pool of suitable donor organs as well as ameliorating the effects of ischemia incurred during the retrieval process. Recent experimental models of pancreatic MP appear promising. Applications of MP to the pancreas, needs refinement regarding perfusion protocols and organ viability assessment criteria. To address the "Role of pancreas machine perfusion to increase the donor pool for beta cell replacement," the European Society for Organ Transplantation (ESOT) assembled a dedicated working group comprising of experts to review literature pertaining to the role of MP as a method of improving donor pancreas quality as well as quantity available for transplant, and to develop guidelines founded on evidence-based reviews in experimental and clinical settings. These were subsequently refined during the Consensus Conference when this took place in Prague.

The influence of liver transplantation on the interplay between gut microbiome and bile acid homeostasis in children with biliary atresia.

BACKGROUND: Biliary atresia (BA) causes neonatal cholestasis and rapidly progresses into cirrhosis if left untreated. Kasai portoenterostomy may delay cirrhosis. BA remains among the most common indications for liver transplantation (LT) during childhood. Liver function and gut microbiome are interconnected. Disturbed liver function and enterohepatic signaling influence microbial diversity. We, herein, investigate the impact of LT and reestablishment of bile flow on gut microbiome-bile acid homeostasis in children with BA before (pre, n = 10), 3 months (post3m, n = 12), 12 months (post12m, n = 9), and more than 24 months (post24 + m, n = 12) after LT. METHODS: We analyzed the intestinal microbiome of BA patients before and after LT by 16S-rRNA-sequencing and bioinformatics analyses, and serum primary and secondary bile acid levels. RESULTS: The gut microbiome in BA patients exhibits a markedly reduced alpha diversity in pre (p = 0.015) and post3m group (p = 0.044), and approximated healthy control groups at later timepoints post12m (p = 1.0) and post24 + m (p = 0.74). Beta diversity analysis showed overall community structure similarities of pre and post3m (p = 0.675), but both differed from the post24 + m (p < 0.001). Longitudinal analysis of the composition of the gut microbiome revealed the Klebsiella genus to show increased abundance in the post24 + m group compared with an age-matched control (p = 0.029). Secondary bile acid production increased 2+ years after LT (p = 0.03). Multivariable associations of microbial communities and clinical metadata reveal several significant associations of microbial genera with tacrolimus and mycophenolate mofetil-based immunosuppressive regimens. CONCLUSIONS: In children with BA, the gut microbiome shows strongly reduced diversity before and shortly after LT, and approximates healthy controls at later timepoints. Changes in diversity correlate with altered secondary bile acid synthesis at 2+ years and with the selection of different immunosuppressants.

Liver Transplantation in Recipients With a Positive Crossmatch: A Retrospective Single-Center Match-Pair Analysis.

A positive crossmatch (XM+) is considered a contraindication to solid abdominal organ transplantation except liver transplantation (LT). Conflicting reports exist regarding the effects of XM+ on post-transplant outcomes. The goal of this retrospective single-center analysis is to evaluate the influence of XM+ on relevant outcome parameters such as survival, graft rejection, biliary and arterial complications. Forty-nine adult patients undergoing LT with a XM+ between 2002 and 2017 were included. XM+ LT recipients were matched 1:2 with crossmatch negative (XM-) LT recipients based on the balance of risk (BAR) score. Patient and graft survival were compared using Kaplan-Meier survival analysis and the log-rank test. Comparative analysis of clinical outcomes in XM+ and XM- groups were conducted. Patient and graft survival were similar in XM+ and XM- patients. Rejection episodes did not differ either. Recipients with a strong XM+ were more likely to develop a PCR+ CMV infection. A XM+ was not associated with a higher incidence of biliary or arterial complications. Donor age, cold ischemia time, PCR+ CMV infection and a rejection episode were associated with the occurrence of ischemic type biliary lesions. A XM+ has no effects on patient and graft survival or other relevant outcome parameters following LT.

Internal Ileal Diversion as Treatment for Progressive Familial Intrahepatic Cholestasis Type 1-Associated Graft Inflammation and Steatosis after Liver Transplantation.

BACKGROUND: Progressive Familial Intrahepatic cholestasis type I (PFIC1) is a rare congenital hepatopathy causing cholestasis with progressive liver disease. Surgical interruption of the enterohepatic circulation, e.g., surgical biliary diversion (SBD) can slow down development of liver cirrhosis. Eventually, end stage liver disease necessitates liver transplantation (LT). PFIC1 patients might develop diarrhea, graft steatosis and inflammation after LT. SBD after LT was shown to be effective in the alleviation of liver steatosis and graft injury. CASE REPORT: Three PFIC1 patients received LT at the ages of two, two and a half and five years. Shortly after LT diarrhea and graft steatosis was recognized, SBD to the terminal ileum was opted to prevent risk for ascending cholangitis. After SBD, inflammation and steatosis was found to be reduced to resolved, as seen by liver biochemistry and ultrasounds. Diarrhea was reported unchanged. CONCLUSION: We present three PFIC1 cases for whom SBD to the terminal ileum successfully helped to resolve graft inflammation and steatosis.

Multidisciplinary Treatment of Liver Metastases from Intracranial SFTs/HPCs: A Report of Three Consecutive Cases.

In the 2016 WHO classification of tumors of the central nervous system, hemangiopericytomas (HPCs) and solitary fibrous tumors (SFTs) were integrated into a new entity (SFT/HPC). Metastases to bone, liver, lung, and abdominal cavity are of concern. Only 37 cases of patients with liver metastases due to intracranial SFTs/HPCs have been reported. Herein, we present our experience in the management of patients with liver metastases from intracranial SFTs/HCPs. All consecutive patients who were treated for liver metastases from intracranial SFTs/HPCs from January 2014 to December 2020 were enrolled. Overall, three patients were treated for liver metastasis from SFTs/HPCs with curative intent. Two patients with bilobar metastases at presentation required surgical resection, transarterial embolization, stereotactic radiofrequency ablation (SRFA) and systemic therapy. One patient with a singular right liver lobe metastasis was treated with SRFA alone. This patient shows no evidence of liver metastases 39 months following diagnosis. Of the two patients with bilobar disease, one died 89 months following diagnosis, while one is still alive 73 months following diagnosis. Long-term survival can be achieved using a multimodal treatment concept, including surgery, loco-regional and systemic therapies. Referral to a specialized tertiary cancer center and comprehensive long-term follow-up examinations are essential.

Successful Pregnancy in a Kidney-Pancreas Transplanted Patient on LifeCycle Pharma Tacrolimus (LCPT)-Based Immunosuppression.

BACKGROUND There has been, to our knowledge, no reports on LifeCycle Pharma tacrolimus (LCPT) taken during pregnancy after simultaneous pancreas-kidney transplantation (SPK). Here, we report a 25-year-old female SPK recipient who gave birth to a healthy infant in posttransplant month 32. We analyzed the long-term graft function, obstetric/neonatal course, LCPT dosage, tacrolimus (TAC) levels, concomitant medication, and complications. CASE REPORT Her medical history consisted of type 1 diabetes with chronic nephropathy, arterial hypertension, and atypical haemolytic uremic syndrome with critical deterioration of her general condition requiring clinically indicated early termination of her first pregnancy prior to SPK. SPK was performed according to surgical standards. The immunosuppressive prophylaxis consisted of thymoglobulin, mycophenolate mofetil, standard TAC formulation, and steroids. Due to rapid TAC metabolism, the patient was converted from a standard TAC formulation to LCPT in the first month posttransplant. Her long-term immunosuppression, including the obstetric and peripartal course, consisted of LCPT, prednisolone, and azathioprine. She was normotensive without antihypertensive medication and maintained excellent function of both grafts during the observation period of 48 months posttransplant. All (mostly infectious) complications were reversible, especially temporary polyoma viremia within normal renal function, and 2 episodes of urosepsis. No relapse of her pretransplant episode of atypical haemolytic uremic syndrome occurred posttransplant. Her child is in good health at the age of 12 months without any malformations. CONCLUSIONS This case suggests that pregnancy after SPK under LCPT is feasible. Further studies are needed to expand the empirical knowledge surrounding tacrolimus.

Normothermic machine perfusion of kidneys: current strategies and future perspectives.

PURPOSE OF REVIEW: This review aims to summarize the latest original preclinical and clinical articles in the setting of normothermic machine perfusion (NMP) of kidney grafts. RECENT FINDINGS: Kidney NMP can be safely translated into the clinical routine and there is increasing evidence that NMP may be beneficial in graft preservation especially in marginal kidney grafts. Due to the near-physiological state during NMP, this technology may be used as an ex-vivo organ assessment and treatment platform. There are reports on the application of mesenchymal stromal/stem cells, multipotent adult progenitor cells and microRNA during kidney NMP, with first data indicating that these therapies indeed lead to a decrease in inflammatory response and kidney injury. Together with the demonstrated possibility of prolonged ex-vivo perfusion without significant graft damage, NMP could not only be used as a tool to perform preimplant graft assessment. Some evidence exists that it truly has the potential to be a platform to treat and repair injured kidney grafts, thereby significantly reducing the number of declined organs. SUMMARY: Kidney NMP is feasible and can potentially increase the donor pool not only by preimplant graft assessment, but also by ex-vivo graft treatment.

Gluconate-Lactobionate-Dextran Perfusion Solutions Attenuate Ischemic Injury and Improve Function in a Murine Cardiac Transplant Model.

Static cold storage is the cheapest and easiest method and current gold standard to store and preserve donor organs. This study aimed to compare the preservative capacity of gluconate-lactobionate-dextran (Unisol) solutions to histidine-tryptophan-ketoglutarate (HTK) solution. Murine syngeneic heterotopic heart transplantations (Balb/c-Balb/c) were carried out after 18 h of static cold storage. Cardiac grafts were either flushed and stored with Unisol-based solutions with high-(UHK) and low-potassium (ULK) ± glutathione, or HTK. Cardiac grafts were assessed for rebeating and functionality, histomorphologic alterations, and cytokine expression. Unisol-based solutions demonstrated a faster rebeating time (UHK 56 s, UHK + Glut 44 s, ULK 45 s, ULK + Glut 47 s) compared to HTK (119.5 s) along with a better contractility early after reperfusion and at the endpoint on POD 3. Ischemic injury led to a significantly increased leukocyte recruitment, with similar degrees of tissue damage and inflammatory infiltrate in all groups, yet the number of apoptotic cells tended to be lower in ULK compared to HTK. In UHK- and ULK-treated animals, a trend toward decreased expression of proinflammatory markers was seen when compared to HTK. Unisol-based solutions showed an improved preservative capacity compared with the gold standard HTK early after cardiac transplantation. Supplemented glutathione did not further improve tissue-protective properties.

Forty-eight hours of normothermic kidney preservation applying urine recirculation.

Kidney transplantation is limited due to the organ scarcity and the large discrepancy between transplantable organs and patients on the waiting list. Ex-situ normothermic kidney preservation has been studied extensively as a tool to enlarge the donor pool and to enable viability assessment. Urine recirculation, for volume control, was applied to perfuse a discarded human kidney for 48 hours. Long-term kidney NMP was feasible under stable hemodynamic conditions with a physiological acid-base-balance and an intact histological morphology of the organ.

Perfusate Enzymes and Platelets Indicate Early Allograft Dysfunction After Transplantation of Normothermically Preserved Livers.

BACKGROUND: Normothermic machine perfusion (NMP) has become a clinically established tool to preserve livers in a near-physiological environment. However, little is known about the predictive value of perfusate parameters toward the outcomes after transplantation. METHODS: Fifty-five consecutive NMP livers between 2018 and 2019 were included. All of the livers were perfused on the OrganOx metra device according to an institutional protocol. Transplant and perfusion data were collected prospectively. RESULTS: Forty-five livers were transplanted after NMP. Five livers stem from donors after circulatory death and 31 (68.9%) from extended criteria donors. Mean (SD) cold ischemia time was 6.4 (2.3) h; mean (SD) total preservation time was 21.4 (7.1) h. Early allograft dysfunction (EAD) occurred in 13 of 45 (28.9%) patients. Perfusate aspartate aminotransferase (P = 0.008), alanine aminotransferase (P = 0.006), lactate dehydrogenase (P = 0.007) and their development over time, alkaline phosphatase (P = 0.013), and sodium (P = 0.016) correlated with EAD. Number of perfusate platelets correlated with cold ischemia time duration and were indicative for the occurrence of EAD. Moreover, von Willebrand Factor antigen was significantly higher in perfusates of EAD livers (P < 0.001), and Δ von Willebrand factor antigen correlated with EAD. Although perfusate lactate and glucose had no predictive value, EAD was more likely to occur in livers with lower perfusate pH (P = 0.008). ΔPerfusate alkaline phosphatase, Δperfusate aspartate aminotransferase, Δperfusate alanine aminotransferase, and Δperfusate lactate dehydrogenase correlated closely with model for early allograft function but not liver graft assessment following transplantation risk score. Bile parameters correlated with extended criteria donor and donor risk index. CONCLUSIONS: Biomarker assessment during NMP may help to predict EAD after liver transplantation. The increase of transaminases and lactate dehydrogenase over time as well as platelets and vWF antigen are important factors indicative for EAD.

Post-Transplant Malignancies following Pancreas Transplantation: Incidence and Implications on Long-Term Outcome from a Single-Center Perspective.

Chronic immunosuppression is associated with an increased risk of malignancy. The main objective of this study is to evaluate the incidence and effect of post-transplant malignancies (PTMs) following pancreas transplantation. The 348 first pancreas transplants performed between 1985 and 2015 were retrospectively analyzed in this study. Incidences of PTMs, as well as patient and graft survival, were evaluated. Out of 348 patients, 71 (20.4%) developed a PTM. Median time to diagnosis was 130 months. Thirty-six patients (50.7%) developed skin cancers (four patients with melanoma, 32 with NMSCs). Solid organ malignancy occurred in 25 (35.2%), hematologic malignancy in ten patients (14.1%). Affected patients were transplanted earlier [2000 (IQR 1993-2004) vs. 2003 (IQR 1999-2008); p < 0.001]. No differences in induction therapy were seen, both groups demonstrated comparable patient and graft survival. Pancreas transplant recipients with solid organ and hematologic malignancies had a three- and six-fold increased hazard of death compared to those with skin cancers [aHR 3.04 (IQR 1.17-7.91); p = 0.023; aHR 6.07 (IQR 1.87-19.71); p = 0.003]. PTMs affect every fifth patient following pancreas transplantation. Skin cancers are the most common malignancies accounting for 50% of all PTMs. These results underscore the importance of close dermatologic follow-up.

Influence of early biliary complications on survival rates after pediatric liver transplantation-A positive outlook.

BACKGROUND: Early biliary complications (EBC) constitute a burden after pediatric liver transplantation frequently requiring immediate therapy. We aimed to assess the impact of EBC on short- and long-term patient and graft survival as well as post-transplant morbidity. METHODS: We analyzed 121 pediatric liver transplantations performed between 1984 and 2019 at the Medical University of Innsbruck for the occurrence of early (<90 days) biliary complications and investigated the influence of EBC on patient and graft survival. RESULTS: Early biliary complications occurred in 30 (24.8%) out of the 121 pediatric liver transplant recipients. Patient survival at 15 years (89.2% vs. 84.2%, p = .65) and all-cause (82.5% vs. 74.0%) and death-censored graft survival (82.5% vs. 75.1%, p = .71) at 10 years were similar between the EBC and the non-EBC group. The EBC group had a significantly longer ICU (25 vs. 16 days, p < .001) and initial hospital stay (64 vs. 42 days, p = .002). Livers of patients with EBC were characterized by multiple bile ducts (33.3% vs. 13.2%, p = .027), and patients with EBC had a higher risk to develop late biliary complications (OR 2.821 [95% CI 1.049-7.587], p = .044) and bowel obstruction/perforation (OR 4.388 [95% CI 1.503-12.812], p = .007). CONCLUSION: Early biliary complications after pediatric liver transplantation is frequent. The occurrence of EBC significantly increased post-transplant morbidity without affecting mortality. Multiple bile ducts were the only risk factor for the development of EBC in our cohort.

Ex Vivo Mesenchymal Stem Cell Therapy to Regenerate Machine Perfused Organs.

Transplantation represents the treatment of choice for many end-stage diseases but is limited by the shortage of healthy donor organs. Ex situ normothermic machine perfusion (NMP) has the potential to extend the donor pool by facilitating the use of marginal quality organs such as those from donors after cardiac death (DCD) and extended criteria donors (ECD). NMP provides a platform for organ quality assessment but also offers the opportunity to treat and eventually regenerate organs during the perfusion process prior to transplantation. Due to their anti-inflammatory, immunomodulatory and regenerative capacity, mesenchymal stem cells (MSCs) are considered as an interesting tool in this model system. Only a limited number of studies have reported on the use of MSCs during ex situ machine perfusion so far with a focus on feasibility and safety aspects. At this point, no clinical benefits have been conclusively demonstrated, and studies with controlled transplantation set-ups are urgently warranted to elucidate favorable effects of MSCs in order to improve organs during ex situ machine perfusion.

The impact of donor pancreas extraction time on graft survival and postoperative complications in pancreas transplant recipients.

BACKGROUND: Simultaneous pancreas kidney transplantation (SPK) is the best therapeutic option for patients with diabetes mellitus type 1 and end-stage renal disease. Recently, donor organ extraction time has been shown to affect kidney and liver graft survival. This study aimed to assess the effect of pancreas donor extraction time on graft survival and postoperative complications. METHODS: We retrospectively analyzed all pancreas transplants performed in two Eurotransplant centers. The association of pancreas extraction time with pancreas graft survival was analyzed by a Cox proportional hazards regression analysis after 3 months, 1 and 5 year. Besides, the effect of pancreas extraction time on the incidence of severe postoperative complications was analyzed. RESULTS: A total of 317 pancreas transplants were included in this study. Death-censored pancreas graft survival was 85.7% after one year and 76.7% after five years. Median pancreas donor extraction time was 64 min [IQR: 52-79 min]. After adjustment for potential confounders, death censored graft survival after 30 days (HR 1.01, 95% CI 0.9-1.03 (p = 0.23), 1 year (HR 1.01, 95% CI 0.99-1.03 (p = 0.22) and 5 years (HR 1.00, 95% CI 0.99-1.02 (p = 0.57) was not associated with pancreas donor extraction time. However, extraction time was significantly associated with a higher incidence of Clavien-Dindo ≥3 complications compared to Clavien-Dindo 1 + 2 complications: OR 1.012, 95% CI 1.00-1.02 (p = 0.039). CONCLUSIONS: Our findings suggest that although no effect on graft survival was found, limiting pancreas extraction time can have a significant impact on lowering postoperative complications.

A Large-Scale Bank of Organ Donor Bone Marrow and Matched Mesenchymal Stem Cells for Promoting Immunomodulation and Transplant Tolerance.

Induction of immune tolerance for solid organ and vascular composite allografts is the Holy Grail for transplantation medicine. This would obviate the need for life-long immunosuppression which is associated with serious adverse outcomes, such as infections, cancers, and renal failure. Currently the most promising means of tolerance induction is through establishing a mixed chimeric state by transplantation of donor hematopoietic stem cells; however, with the exception of living donor renal transplantation, the mixed chimerism approach has not achieved durable immune tolerance on a large scale in preclinical or clinical trials with other solid organs or vascular composite allotransplants (VCA). Ossium Health has established a bank of cryopreserved bone marrow (BM), termed "hematopoietic progenitor cell (HPC), Marrow," recovered from deceased organ donor vertebral bodies. This new source for hematopoietic cell transplant will be a valuable resource for treating hematological malignancies as well as for inducing transplant tolerance. In addition, we have discovered and developed a large source of mesenchymal stem (stromal) cells (MSC) tightly associated with the vertebral body bone fragment byproduct of the HPC, Marrow recovery process. Thus, these vertebral bone adherent MSC (vBA-MSC) are matched to the banked BM obtained from each donor, as opposed to third-party MSC, which enhances safety and potentially efficacy. Isolation and characterization of vBA-MSC from over 30 donors has demonstrated that the cells are no different than traditional BM-MSC; however, their abundance is >1,000-fold higher than obtainable from living donor BM aspirates. Based on our own unpublished data as well as reports published by others, MSC facilitate chimerism, especially at limiting hematopoietic stem and progenitor cell (HSPC) numbers and increase safety by controlling and/or preventing graft-vs.-host-disease (GvHD). Thus, vBA-MSC have the potential to facilitate mixed chimerism, promote complementary peripheral immunomodulatory functions and increase safety of BM infusions. Both HPC, Marrow and vBA-MSC have potential use in current VCA and solid organ transplant (SOT) tolerance clinical protocols that are amenable to "delayed tolerance." Current trials with HPC, Marrow are planned with subsequent phases to include vBA-MSC for tolerance of both VCA and SOT.

Noninvasive evaluation of intragraft immune responses in upper extremity transplantation.

In vascularized composite allotransplantation (VCA), invasive tissue biopsies remain the gold standard in diagnosing rejection carrying significant morbidity. We aimed to show feasibility of tape-stripping for noninvasive immune monitoring in VCA. Tape-stripping was performed on allografts and native skin of upper extremity transplant recipients. Healthy nontransplanted individuals served as controls. The technique was also used in swine on naïve skin in nontransplanted animals, native skin of treated, transplanted swine, nonrejecting VCAs, and rejecting VCAs. Extracted protein was analyzed for differences in cytokine expression using Luminex technology. Significantly decreased levels of INFγ and IL-1Ra were seen between human allograft samples and native skin. In swine, rejecting grafts had increased IL-1Ra compared to naïve and native skin, decreased levels of GM-CSF compared to native skin, and decreased IL-10 compared to nonrejecting grafts. Unsupervised hierarchical clustering revealed rejecting grafts separated from the nonrejecting (P = 0.021). Variable importance in projection scores identified GM-CSF, IL-1Ra, and IL-2 as the most important profiles for group discrimination. Differences in cytokine expression are detectable in human VCA patient native skin and VCA graft skin using a noninvasive tape-stripping method. Swine studies suggest that differences in cytokines between rejecting and nonrejecting grafts are discernable.

Myogenic progenitor cell transplantation for muscle regeneration following hindlimb ischemia and reperfusion.

BACKGROUND: Muscle is severely affected by ischemia/reperfusion injury (IRI). Quiescent satellite cells differentiating into myogenic progenitor cells (MPC) possess a remarkable regenerative potential. We herein established a model of local application of MPC in murine hindlimb ischemia/reperfusion to study cell engraftment and differentiation required for muscle regeneration. METHODS: A clamping model of murine (C57b/6 J) hindlimb ischemia was established to induce IRI in skeletal muscle. After 2 h (h) warm ischemic time (WIT) and reperfusion, reporter protein expressing MPC (TdTomato or Luci-GFP, 1 × 106 cells) obtained from isolated satellite cells were injected intramuscularly. Surface marker expression and differentiation potential of MPC were analyzed in vitro by flow cytometry and differentiation assay. In vivo bioluminescence imaging and histopathologic evaluation of biopsies were performed to quantify cell fate, engraftment and regeneration. RESULTS: 2h WIT induced severe IRI on muscle, and muscle fiber regeneration as per histopathology within 14 days after injury. Bioluminescence in vivo imaging demonstrated reporter protein signals of MPC in 2h WIT animals and controls over the study period (75 days). Bioluminescence signals were detected at the injection site and increased over time. TdTomato expressing MPC and myofibers were visible in host tissue on postoperative days 2 and 14, respectively, suggesting that injected MPC differentiated into muscle fibers. Higher reporter protein signals were found after 2h WIT compared to controls without ischemia, indicative for enhanced growth and/or engraftment of MPC injected into IRI-affected muscle antagonizing muscle damage caused by IRI. CONCLUSION: WIT-induced IRI in muscle requests increased numbers of injected MPC to engraft and persist, suggesting a possible rational for cell therapy to antagonize IRI. Further investigations are needed to evaluate the regenerative capacity and therapeutic advantage of MPC in the setting of ischemic limb injury.