BACKGROUND & AIMS:The devastation caused by necrotizing enterocolitis (NEC) has continued to claim the lives of infants despite advances in neonatal medicine. To address the acute, and often severe, intestinal epithelial damage caused by NEC, therapeutics that directly target epithelial recovery and cellular regeneration processes are needed. METHODS:We investigated the capacity of a decellularized human placental extract (HPE) to prevent and enhance recovery from NEC-like injury using in vitro and in vivo models. Healing responses in primary neonatal porcine ileal epithelial cells were analyzed following hypoxia or scratch-wound injury and HPE application. RESULTS:In vitro, HPE treatment accelerated scratch closure and increased proliferating cell number though did not enhance tight junction recovery following hypoxia. In vivo, NEC was induced in neonatal piglets through a combination of preterm delivery and formula feeding. Treated piglets received enteral HPE, while control animals had nothing by mouth prior to formula initiation. In piglets, HPE treatment increased weight gain, decreased macroscopic and histological damage, and increased ileal crypt epithelial cell proliferation. After observation of similar effects using in vitro and in vivo platforms, transcriptomic analysis of monolayer cultures treated with HPE was undertaken. Increased expression of pathways associated with epithelial wound healing, proliferation, and migration were identified, with key shared genes between those pathways. CONCLUSIONS:In sum, these findings suggest that HPE can enhance the reparative capacity of neonatal epithelium in the context of NEC.

Severe intestinal ischemia is a critical emergency diagnosis in both human and veterinary medicine, with mortality rates up to 80%. Its underlying pathophysiology is multifactorial but fundamentally linked to the preservation or loss of the intestine’s epithelial lining – a crucial barrier separating the gut contents from the sterile body. This barrier is maintained by proliferative intestinal stem cells (ISCs) that continuously replace the epithelial lining, renewing it entirely approximately every six days. Given ISCs’ essential role in maintaining intestinal barrier integrity, their function following injury merits deeper investigation. However, animal studies of ISCs have been limited to murine models, which have demonstrated poor translatability to human application. Thus, there is a pressing need for improved animal models to advance our understanding of the reparative processes driven by ISCs. Pigs closely approximate human gastrointestinal anatomy and physiology, offering a promising alternative as a large animal model. However, studies of porcine ISCs (pISCs) have been limited by an absence of known external markers for identification. Current methods rely on internal cellular markers, necessitating fixation and precluding in vitro analyses, or the use of an expensive and singularly available transgenic pig model. Altogether, these limitations have hindered progress in leveraging pISCs as a translational study tool. The objective of this study was to implement an alternative method for identifying pISCs using a flow cytometry (FCM)-based approach known as "side population" (SP) analysis. SP uses a cell-permeable dye that is actively effluxed by the membrane-bound transporters in cycling cells such as ISCs and has been effectively used in identifying both mouse and human ISCs. In contrast, non-cycling cells ( e.g., epithelial cells) retain the dye, creating a distinct FCM fluorescent signature. Thus, we hypothesized that SP analysis could be used to identify live pISCs in a wild-type pig model. To this end, we first optimized both chemical and mechanical dissociation protocols to produce a single-cell suspension that, upon FCM analysis, contained a significantly greater SP fluorescent signature compared to traditional dissociation techniques. We then validated the SP specificity by using a Ca 2+ channel blocker control (Verapamil) to inhibit transporter-mediated dye efflux, effectively preventing the SP. These findings are consistent with SP analysis identifying cycling cells, including ISCs. Ongoing analyses aim to exclude CD45+ leukocytes and CD166+ Paneth and goblet cells to further purify this SP. Additionally, 3D cell culture will be used to confirm ISC identity and their proliferative capacity. Furthermore, concurrent evaluations of novel surface markers such as CD24 and CD44 are pending, which may provide additional alternatives for histological pISC identification. Future directions will include fluorescence-activated cell sorting (FACS) for confirmatory downstream qPCR analysis of ISC markers such as LGR5, HopX, and Olfm4. These data represent the first implementation of SP analysis for identifying ISCs in a porcine model. Establishing a method for live pISC isolation is foundational for broader investigations into their regenerative potential. Thus, this work has the potential to broaden our understanding of ISCs, ultimately leveraging their reparative capabilities to advance therapeutic strategies in human and veterinary medicine alike. NIH T35OD011070 Interdisciplinary Biomedical Research Training Program; The Discovery Fund This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Intestinal ischemia and reperfusion injury (IRI) is a deadly and common condition. Death is associated with sepsis due to insufficient epithelial repair, requiring stem cell-driven regeneration, typically beginning 48 h after injury. Animal models are critical to advancing this field. To effectively study epithelial healing, models must survive clinically relevant intestinal ischemic injury extending to the crypt. Although mouse models are indispensable to intestinal research, their application for studying epithelial repair following severe IRI may be limited. Ischemic injury was induced in mouse and porcine jejunum for up to 3 h, with up to 72 h of reperfusion. Histologic damage was scored by Chiu-Park grade, and animal survival was assessed. Findings were compared between species. A mouse IRI literature review was performed to evaluate the purported degree of injury, duration of recovery, and reported survival rates. In mice and pigs, 3 h of ischemia induced severe, reliable injury extending into the crypt. However, at 48 h, mouse survival was only 23.5% compared with 100% survival in pigs. In literature, ischemia was induced for >1 h in only 4 of 102 mouse studies and none to 3 h. Recovery was attempted for 48 h in only six reports. Forty-seven studies reported intestinal crypt injury. Of those that featured histologic intestinal crypt damage, survival rates at 48 h ranged from 10 to 50% (median 30%). Mouse models are not ideal for studying intestinal stem cell-mediated recovery from severe IRI. Alternative large animal models, like pigs, are recommended. NEW & NOTEWORTHY Additional research is needed to improve recovery from severe intestinal ischemia. The selection of the ideal animal model is critical to facilitating this work. Based on our experimentation and literature review, porcine models, with increased translatability and an improved ability to survive both prolonged ischemia and the recovery period, appear to be the most appropriate choice for future studies.

Necrotizing enterocolitis (NEC) is a devastating condition with a ~30% mortality rate in pre-term, very low birth-weight infants. Premature infants lack vital in utero ingestion of amniotic fluid which accelerates intestinal maturation and reduces inflammation. Therefore, an acellular placental extract (PE) akin to amniotic fluid may be therapeutic for NEC. The objective of this study was to evaluate PE’s capacity to accelerate repair following or prevent the induction of NEC-like injury. We hypothesized that PE would enhance neonatal porcine intestinal epithelial cells’ capacity to repair in vitro and in vivo. NEC was induced in 12 piglets via pre-term cesarean delivery and hyperosmolar formula feeding. Prior to formula feeding, piglets were nil per os or enterally supplemented with PE. Tissue, collected at euthanasia, up to 72H after formula initiation, was evaluated grossly and histologically and with immunohistochemical staining for proliferation (Ki67), stem cell identity (Sox9), and apoptosis (CC3). Numerical outputs were assessed with Kolmogorov-Smirnov testing. PE’s effect on epithelial restitution was assessed with scratch assays performed on confluent monolayers derived from ~1-day old piglet ileum. Monolayers were pre-treated with either actin polymerization inhibitor, Latrunculin A, or a cell cycle inhibitor, hydroxyurea. After scratch, either media or PE in media was applied. Scratch-closure was monitored for 24H; percentage-closure was calculated and analyzed using 2-way ANOVA and Tukey multiple comparison. Scratch-wound leading-edge cells were examined for EdU and Ki67 immunofluorescent positivity which was evaluated using Kolmogorov-Smirnov analysis. Tight junction recovery was assessed using identically derived confluent monolayers grown on Transwell inserts and exposed to hypoxia (18H, 1%O2, 5%CO2). Following hypoxia, PE or media alone was added to the apical chamber. Transepithelial electrical resistance (TEER) was measured every 6-12H for 48H using a symmetrical disc electrode chamber. TEER rate of change was analyzed using a two-way ANOVA. For all analyses, significance was set at p<0.05. In vivo PE administration resulted in reduced gross and histological NEC damage in treated compared to untreated piglets (p=0.02). Ki67 and Sox9 expression increased (p=0.71; p=0.93) and CC3 expression decreased with PE treatment (p=0.65). Evaluation of these markers in additional animals is pending. In vitro, PE expedited scratch wound closure (p<0.05) and expanded proliferative cell number along scratch edges (EdU/DAPI, p<0.05; Ki67+EdU/DAPI, p<0.01). Other analyses are pending. PE application prevented gross NEC development in vivo and accelerated recovery in vitro. NIH #R44HD100243; #T32562967-39806. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Intestinal transplantation (IT) is the only treatment for intestinal failure patients who cannot tolerate parenteral nutrition. Although cold storage (CS) is the gold standard for intestinal allograft preservation, normothermic machine perfusion (NMP) has improved transplantation success in other organs. Allograft immune modulation is thought to contribute to this success. The objective of this study was to assess the impact of NMP on intestinal immune cell populations as it relates to ultimate allograft and recipient health. We hypothesized that NMP would stabilize overall immune cell populations while reducing pro-inflammatory cohorts, thus improving IT success. Porcine intestines were stored for 6H at 4°C (CS-T6, n=6) or perfused at 34°C (NMP-T6, n=8) and transplanted. Samples were collected following intestinal procurement (T0), storage (T6), 1-hour post-transplant (T1PT), and euthanasia (T48). Flow cytometric profiling and quantification of intraepithelial (IEL) and lamina propria (LP) lymphocytes and NK cells were performed at T0, T6, and T48. Immunofluorescence (IF) identified CD3+ T-cells at T1PT. One-way ANOVA analyzed cell counts with significance set at p< 0.05. Flow cytometry revealed that NMP preserved CD3+ IELs in the jejunum at T6 (63.74% vs 32.79%, p< 0.0001), and in the ileum at T6 (51.31% vs 42.77%, p< 0.0001) and at T48 (57.73% vs 34.21%, p<0.0001). Immunofluorescent cell quantification confirmed this preservation of CD3+ IEL at T1PT in the crypts of the jejunum (4.9% vs 2.1 %, p< 0.05) and ileum (6.9% vs 2.8%, p< 0.001) compared to CS. However, upon further subpopulation analyses using flow cytometry, ileal IEL T-cells were reduced throughout NMP compared to CS (T6 28.7% vs 44.0%; T48 16.65% vs 22.37%; p< 0.0001). NK cells were also decreased in ileal and jejunal LP at NMP-T6 compared to T0 (3.6% vs 5.6%, p< 0.01; 2.4% vs 4.3% p< 0.05). NMP reduces NK cells, a cell type known to be associated with rejection. Interestingly, NMP preserved the overall CD3+ T cell populations in the jejunum and ileum at T6, T1PT, and T48. The subpopulation of IELs, however, were reduced in the ileum following NMP. The role of these populations, specifically in IT, has yet to be determined. Further characterization of T-cell subpopulations and dynamics is needed. Complimentary evaluations of the microbiome and proteomic responses to IT NMP are ongoing. Understanding immune cell dynamics will improve IT outcomes for patients requiring this critical, lifesaving surgery. U.S. Department of Defense PR181265; NIH K01OD010199 SERCA, T32OD011130-15. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Gastrointestinal disease is a leading cause of death in mature horses. A lack of in vitro modeling has impeded the development of novel therapeutics. The objectives of this study were to develop and further characterize a small intestinal monolayer cell culture derived from equine jejunum including establishing normal measurements of intestinal permeability and restitution. Three-dimensional enteroids, derived from postmortem sampling of equine jejunum, were utilized to develop confluent epithelial monolayers. The presence of differentiated intestinal epithelial cell types and tight junctions were confirmed using histology, reverse transcription PCR (RT-PCR), RNAscope, protein immunofluorescence and transmission electron microscopy. Transepithelial resistance (TER) and macromolecule flux were assessed as measurements of paracellular and transcellular permeability. Scratch assays were utilized to model and assess intestinal restitution. Monolayer cell cultures reached 100% confluency by ~5–7 days. Equine jejunum monolayers were confirmed as epithelial in origin, with identification of differentiated intestinal epithelial cell types and evidence of tight junction proteins. Function of the intestinal barrier was supported by acquisition of physiologically normal TER values (179.9 ± 33.7 ohms*cm2) and limited macromolecule flux (22 ± 8.8% at 60 min). Additionally, following a scratch wound, epithelial cell monolayers migrated to close gap defects within 24 h. In conclusion, this study describes the development of a novel intestinal epithelial monolayer cell culture for equine jejunum, and provides evidence of intestinal epithelial cell differentiation, formation of physiologically relevant barrier function and use as a model of intestinal restitution to test potential therapeutics for equine colic.

Intestinal epithelial stem cells (ISCs) are responsible for intestinal epithelial barrier renewal; thereby, ISCs play a critical role in intestinal pathophysiology research. While transgenic ISC reporter mice are available, advanced translational studies lack a large animal model. This study validates ISC isolation in a new porcine Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (LGR5) reporter line and demonstrates the use of these pigs as a novel colorectal cancer (CRC) model. We applied histology, immunofluorescence, fluorescence-activated cell sorting, flow cytometry, gene expression quantification, and 3D organoid cultures to whole tissue and single cells from the duodenum, jejunum, ileum, and colon of LGR5-H2B-GFP and wild-type pigs. Ileum and colon LGR5-H2B-GFP, healthy human, and murine biopsies were compared by mRNA fluorescent in situ hybridization (FISH). To model CRC, adenomatous polyposis coli (APC) mutation was induced by CRISPR/Cas9 editing in porcine LGR5-H2B-GFP colonoids. Crypt-base, green fluorescent protein (GFP) expressing cells co-localized with ISC biomarkers. LGR5-H2B-GFP(hi) cells had significantly higher LGR5 expression (p < .01) and enteroid forming efficiency (p < .0001) compared with LGR5-H2B-GFP(med/lo/neg) cells. Using FISH, similar LGR5, OLFM4, HOPX, LYZ, and SOX9 expression was identified between human and LGR5-H2B-GFP pig crypt-base cells. LGR5-H2B-GFP/APC(null) colonoids had cystic growth in WNT/R-spondin-depleted media and significantly upregulated WNT/beta-catenin target gene expression (p < .05). LGR5(+) ISCs are reproducibly isolated in LGR5-H2B-GFP pigs and used to model CRC in an organoid platform. The known anatomical and physiologic similarities between pig and human, and those shown by crypt-base FISH, underscore the significance of this novel LGR5-H2B-GFP pig to translational ISC research.

Intestinal transplantation (IT) is the final treatment option for intestinal failure. Static cold storage (CS) is the standard preservation method used for intestinal allografts. However, CS and subsequent transplantation induce ischemia-reperfusion injury (IRI). Severe IRI impairs epithelial barrier function, including loss of intestinal stem cells (ISC), critical to epithelial regeneration. Normothermic machine perfusion (NMP) preservation of kidney and liver allografts minimizes CS-associated IRI; however, it has not been used clinically for IT. We hypothesized that intestine NMP would induce less epithelial injury and better protect the intestine's regenerative ability when compared with CS. Full-length porcine jejunum and ileum were procured, stored at 4 °C, or perfused at 34 °C for 6 hours (T6), and transplanted. Histology was assessed following procurement (T0), T6, and 1 hour after reperfusion. Real-time quantitative reverse transcription polymerase chain reaction, immunofluorescence, and crypt culture measured ISC viability and proliferative potential. A greater number of NMP-preserved intestine recipients survived posttransplant, which correlated with significantly decreased tissue injury following 1-hour reperfusion in NMP compared with CS samples. Additionally, ISC gene expression, spheroid area, and cellular proliferation were significantly increased in NMP-T6 compared with CS-T6 intestine. NMP appears to reduce IRI and improve graft regeneration with improved ISC viability and proliferation.

BackgroundIntestinal epithelial stem cells (ISC) are responsible for epithelial regeneration and are critical to the intestine's ability to regain barrier function following injury. Evaluating ISC biomarker expression in cases of small intestinal strangulation (SIS) may provide insight into clinical progression. ObjectivesIntestinal resection margins from cases of SIS were evaluated to determine if (1) evidence of injury could be identified using histomorphometry, (2) ISC biomarker expression was decreased in the proximal resection margin compared to control and distal resection margin, and (3) the ISC biomarker expression was associated with the number of preoperative risk factors negatively related to outcome, post-operative complications, or case outcome. Study designRetrospective cohort study. MethodsIntestinal samples were obtained intraoperatively from resection margins of adult horses with SIS and horses euthanised for reasons unrelated to colic. Preoperative risk factors negatively related to outcome, post-operative complications, and case outcome were obtained from medical records. Horses were grouped as euthanised intraoperatively, postoperatively, or survived to discharge. Histomorphometry and immunofluorescence were performed to evaluate tissue architecture and ISC and progenitor cell number. Groups were compared using one-way ANOVA. Associations between biomarker expression and the number of preoperative risk factors and post-operative complications negatively related to outcome were determined using linear regression modelling. ResultsThirty-six cases of SIS were evaluated. Ki67(+) cell counts were decreased in the proximal (mean = 15.45 cells; 95% CI = 10.27-20.63; SD = 4.17; p = 0.02) and distal resection margins (mean = 15.05; 95% CI = 8.46-21.64; SD = 4.141; p = 0.03) in horses euthanised postoperatively compared to control (mean = 23.62 cells; 95% CI = 19.42-27.83; SD = 5.883). In the distal resection margin, an increase in SOX9(+)Ki67(+) cells were associated with a decrease in the total number of preoperative risk factors negatively related to outcome (95% CI = 0.236-1.123; p = 0.008, SE = 0.1393). Main limitationsSmall population size. ConclusionsProliferating cell and ISC numbers may be associated with case outcome.

Background. Successful intestinal transplantation is currently hindered by graft injury that occurs during procurement and storage, which contributes to postoperative sepsis and allograft rejection. Improved graft preservation may expand transplantable graft numbers and enhance posttransplant outcomes. Superior transplant outcomes have recently been demonstrated in clinical trials using machine perfusion to preserve the liver. We hypothesized that machine perfusion preservation of intestinal allografts could be achieved and allow for transplantation in a porcine model. Methods. Using a translational porcine model, we developed a device for intestinal perfusion. Intestinal samples were collected at the time of organ procurement, and after 6 h of machine perfusion for gross and histologic evaluation, hourly chemistry panels were performed on the perfusate and were used for protocol optimization. Following transplantation, porcine recipient physical activity, systemic blood parameters, and vital signs were monitored for 2 d before sacrifice. Results. In initial protocol development (generation 1, n = 8 grafts), multiple metabolic, electrolyte, and acid-base derangements were measured. These factors coincided with graft and mesenteric edema and luminal hemorrhage and were addressed with the addition of dialysis. In the subsequent protocol (generation 2, n = 9 grafts), differential jejunum and ileum perfusion were observed resulting in gross evidence of ileal ischemia. Modifications in vasodilating medications enhanced ileal perfusion (generation 3, n = 4 grafts). We report successful transplantation of 2 porcine intestinal allografts after machine perfusion with postoperative clinical and gross evidence of normal gut function. Conclusions. This study reports development and optimization of machine perfusion preservation of small intestine and successful transplantation of intestinal allografts in a porcine model.

AK-5, which is a spontaneously regressing rat histiocytoma, is killed by necrosis (perforin mediated) and apoptosis. We have studied the induction of apoptosis in AK-5 tumor cells by each of the following: a factor from anti-AK-5 antiserum, dexamethasone, and natural killer cells. Partial inhibition in apoptosis was observed when AK-5 cells were transfected with Crm A gene, a specific inhibitor of ICE protease. Similarly peptide inhibitors Ac-YVAD-cmk and Ac-DEVD-CHO inhibited partially the formation of nuclear bodies and DNA fragmentation induced by each of the above-mentioned apoptotic inducers. Although NK cells were able to kill Crm A and bcl-2 transfected clones by cytotoxic action, they failed to induce DNA fragmentation in these clones, suggesting a dual mode of action by NK cells in the induction of target cell death. We were unable to detect ICE and YAMA/CPP32 transcripts in control AK-5 cells, but upon induction of the apoptotic process, there was significant expression of these transcripts in AK-5 cells. When bcl-2 gene was introduced into AK-5 cells there was complete inhibition of apoptosis, suggesting its affect to be upstream of ICE and YAMA proteases. These results suggest an important role for cysteine proteases in the execution of apoptosis, leading to tumor cell death and the regression of AK-5 tumor in syngeneic hosts.

Background. Successful intestinal transplantation is currently hindered by graft injury that occurs during procurement and storage, which contributes to postoperative sepsis and allograft rejection. Improved graft preservation may expand transplantable graft numbers and enhance posttransplant outcomes. Superior transplant outcomes have recently been demonstrated in clinical trials using machine perfusion to preserve the liver. We hypothesized that machine perfusion preservation of intestinal allografts could be achieved and allow for transplantation in a porcine model. Methods. Using a translational porcine model, we developed a device for intestinal perfusion. Intestinal samples were collected at the time of organ procurement, and after 6 h of machine perfusion for gross and histologic evaluation, hourly chemistry panels were performed on the perfusate and were used for protocol optimization. Following transplantation, porcine recipient physical activity, systemic blood parameters, and vital signs were monitored for 2 d before sacrifice. Results. In initial protocol development (generation 1, n = 8 grafts), multiple metabolic, electrolyte, and acid-base derangements were measured. These factors coincided with graft and mesenteric edema and luminal hemorrhage and were addressed with the addition of dialysis. In the subsequent protocol (generation 2, n = 9 grafts), differential jejunum and ileum perfusion were observed resulting in gross evidence of ileal ischemia. Modifications in vasodilating medications enhanced ileal perfusion (generation 3, n = 4 grafts). We report successful transplantation of 2 porcine intestinal allografts after machine perfusion with postoperative clinical and gross evidence of normal gut function. Conclusions. This study reports development and optimization of machine perfusion preservation of small intestine and successful transplantation of intestinal allografts in a porcine model.

Introduction: Cold storage (CS) preservation is the current gold standard for small bowel (SB) allografts, with post-transplantation allograft health monitored by histologic analysis of ileal biopsies. However, ileum may not represent overall graft health as CS has been shown to cause less histologic damage to the ileum compared to other SB segments. CS causes ischemia, which has been associated with epithelial loss and damage to intestinal stem cells (ISC). The extent to which ISCs are injured by CS and the potential differences between SB segments remains unknown. Given the role of ISCs in epithelial regeneration, we hypothesized that CS would decrease ISC viability and proliferative potential, and that this decrease would be greatest in the duodenum and jejunum. Methods: Nine porcine SB grafts were flushed with cold UW preservation solution and stored at 4°C for 6h. Crypts containing ISCs were isolated and placed in 3D culture from duodenum, jejunum, and ileum post-flushing (CO) or after 6h preservation to assess ISC viability and proliferation. Number and size of spheroids were quantified over 0-120h to test if ischemia affects the “stemness”, or spheroid/enteroid forming ability of crypts, a surrogate measure of stemness. Differences within and between groups were compared using a Kruskal-Wallis test and a Dunn’s multiple comparison post-test followed by a Mann-Whitney test. Significance was set at P<0.05. Results: No significant differences were found between plating efficiencies for CS and CO conditions for any segment. CS jejunal spheroid areas were significantly smaller at all time points compared to CO tissues. When CS segments were compared, duodenal or jejunal spheroids were significantly smaller than ileal spheroids at all time points (P<0.0001). Conclusions: CS injury does not appear to cause a difference in ISC viability between SB segments, but ISC proliferative potential is significantly diminished in both duodenum and jejunum compared to ileum. Our findings are clinically important as ileal biopsies and their histologic morphology serve as the marker of overall allograft health. However, ileum may not be the best marker of graft regenerative capacity. The addition of future porcine CS procedure data is warranted before a final conclusion can be made.

Intestinal ischemia is a life-threatening emergency with mortality rates of 50%-80% due to epithelial cell death and resultant barrier loss. Loss of the epithelial barrier occurs in conditions including intestinal volvulus and neonatal necrotizing enterocolitis. Survival depends on effective epithelial repair; crypt-based intestinal epithelial stem cells (ISCs) are the source of epithelial renewal in homeostasis and after injury. Two ISC populations have been described: 1) active ISC [aISC; highly proliferative; leucine-rich-repeat-containing G protein-coupled receptor 5 (LGR5(+))-positive or sex-determining region Y-box 9 -antigen Ki67-positive (SOX9(+)Ki67(+))] and 2) reserve ISC [rISC; less proliferative; homeodomain-only protein X positive (HOPX+)]. The contributions of these ISCs have been evaluated both in vivo and in vitro using a porcine model of mesenteric vascular occlusion to understand mechanisms that modulate ISC recovery responses following ischemic injury. In our previously published work, we observed that rISC conversion to an activated state was associated with decreased HOPX expression during in vitro recovery. In the present study, we wanted to evaluate the direct role of HOPX on cellular proliferation during recovery after injury. Our data demonstrated that during early in vivo recovery, injury-resistant HOPX+ cells maintain quiescence. Subsequent early regeneration within the intestinal crypt occurs around 2 days after injury, a period in which HOPX expression decreased. When HOPX was silenced in vitro, cellular proliferation of injured cells was promoted during recovery. This suggests that HOPX may serve a functional role in ISC-mediated regeneration after injury and could be a target to control ISC proliferation. NEW & NOTEWORTHY This paper supports that rISCs are resistant to ischemic injury and likely an important source of cellular renewal following near-complete epithelial loss. Furthermore, we have evidence that HOPX controls ISC activity state and may be a critical signaling pathway during ISC-mediated repair. Finally, we use multiple novel methods to evaluate ISCs in a translationally relevant large animal model of severe intestinal injury and provide evidence for the potential role of rISCs as therapeutic targets.

Introduction: Previously published attempts at mitigating the effects of ischemia-reperfusion injury of intestinal grafts via machine perfusion are deemed not clinically translatable due to either the short length of the graft, or the increased risk of contamination due to the need for luminal access. This study provides the first clinically translatable protocol for the normothermic machine perfusion (NMP) of the full-length porcine intestine. Methods: Full-length, heart-beating donor, porcine intestinal grafts (N=3) were perfused ex-vivo at 34C via the superior mesenteric artery for 6 hours (h) using the donor’s whole blood, at a mean arterial pressure of 45-50mmHg. The machine consisted of perfusion and hemodiafiltration circuits. The perfusate was sampled for analysis and supplemented hourly with a mixture of nutrients and medication. Tissue biopsy samples were obtained before and after NMP. Results: Vascular resistance in all 3 grafts stabilized <0.4 at 2h. In grafts #2 and #3, which received an optimized hematocrit level and dialysis dose, lactate levels stabilized <3mmol/L by 4h. The higher lactate levels in graft #1 were associated with mild to moderate villus epithelial loss compared to minimal to none in grafts #2 and #3.Conclusions: Clinically translatable NMP of a full-length intestinal graft can be performed using our proprietary setup and FDA-approved, commercially available medication. Vascular resistance and lactate levels are real-time point-of-care tests that may prove valuable markers in the decision process of whether to transplant or discard an intestinal graft.