Stem cell therapy for faecal incontinence: Current state and future perspectives

Faecal continence is a complex function involving different organs and systems. Faecal incontinence is a common disorder with different pathogeneses, disabling consequences and high repercussions for quality of life. Current management modalities are not ideal, and the development of new treatments is needed. Since 2008, stem cell therapies have been validated, 36 publications have appeared(29 in preclinical models and seven in clinical settings), and six registered clinical trials are currently ongoing. Some publications have combined stem cells with bioengineering technologies. The aim of this review is to identify and summarise the existing published knowledge of stem cell utilization as a treatment for faecal incontinence. A narrative or descriptive review is presented. Preclinical studies have demonstrated that cellular therapy, mainly in the form of local injections of muscle-derived(muscle derived stem cells or myoblasts derived from them) or mesenchymal(bone-marrow-or adipose-derived) stem cells, is safe. Cellular therapy has also been shown to stimulate the repair of both acute and subacute anal sphincter injuries, and some encouraging functional results have been obtained. Stem cells combined with normal cells on bioengineered scaffolds have achieved the successful creation and implantation of intrinsically-innervated anal sphincter constructs. The clinical evidence, based on adipose-derived stem cells and myoblasts, is extremely limited yet has yielded some promising results, and appears to be safe. Further investigation in both animal models and clinical settings is necessary to drawing conclusions. Nevertheless, if the preliminary results are confirmed, stem cell therapy for faecal incontinence may well become a clinical reality in the near future.

Rat model of anal sphincter injury and two approaches for stem cell administration

AIM To establish a rat model of anal sphincter injury and test different systems to provide stem cells to injured area.METHODS Adipose-derived stem cells(ASCs) were isolated from BDIX rats and were transfected with green fluorescent protein(GFP) for cell tracking. Biosutures(sutures covered with ASCs) were prepared with 1.5 × 10~6 GFPASCs, and solutions of 10~6 GFP-ASCs in normal saline were prepared for injection. Anorectal normal anatomy was studied on Wistar and BDIX female rats. Then, we designed an anal sphincter injury model consisting of a 1-cm extra-mucosal miotomy beginning at the anal verge in the anterior middle line. The sphincter lesion was confirmed with conventional histology(hematoxylin and eosin) and immunofluorescence with 4', 6-diamidino-2-phenylindole(commonly known as DAPI), GFP and α-actin. Functional effect was assessed with basal anal manometry, prior to and after injury. After sphincter damage, 36 BDIX rats were randomized to three groups for:(1) Cell injection without repair;(2) biosuture repair; and(3) conventional suture repair and cell injection. Functional and safety studies were conducted on all the animals. Rats were sacrificed after 1, 4 or 7 d. Then, histological and immunofluorescence studies were performed on the surgical area.RESULTS With the described protocol, biosutures had been covered with at least 820000-860000 ASCs, with 100% viability. Our studies demonstrated that some ASCs remained adhered after suture passage through the muscle. Morphological assessment showed that the rat anal anatomy is comparable with human anatomy; two sphincters are present, but the external sphincter is poorly developed. Anal sphincter pressure data showed spontaneous, consistent, rhythmic anal contractions, taking the form of "plateaus" with multiple twitches(peaks) in each pressure wave. These basal contractions were very heterogeneous; their frequency was 0.91-4.17 per min(mean 1.6980, SD 0.57698), their mean duration was 26.67 s and mean number of peaks was 12.53. Our morphological assessment revealed that with the aforementioned surgical procedure, both sphincters were completely sectioned. In manometry, the described activity disappeared and was replaced by a gentle oscillation of basal line, without a recognizable pattern. Surprisingly, these findings appeared irrespective of injury repair or not. ASCs survived in this potentially septic area for 7 d, at least. We were able to identify them in 84% of animals, mainly in the muscular section area or in the tissue between the muscular endings. ASCs formed a kind of "conglomerate" in rats treated with injections, while in the biosuture group, they wrapped the suture. ASCs were also able to migrate to the damaged zone. No relevant adverse events or mortality could be related to the stem cells in our study. We also did not find unexpected tissue growths. CONCLUSION The proposed procedure produces a consistent sphincter lesion. Biosutures and injections are suitable for cell delivery. ASCs survive and are completely safe in this clinical setting.

Detection of KRAS G12D in colorectal cancer stool by droplet digital PCR

AIM To assess KRAS G12 D mutation detection by droplet digital PCR(dd PCR) in stool-derived DNA from colorectal cancer(CRC) patients.METHODS In this study, tumor tissue and stool samples were collected from 70 patients with stage Ⅰ-Ⅳ CRC diagnosed by preoperative biopsy. KRAS mutational status was determined by pyrosequencing analysis of DNA obtained from formalin-fixed paraffinembedded(FFPE) tumor tissues. The KRAS G12 D mutation was then analyzed by dd PCR in FFPE tumors and stool-derived DNA from patients with this point mutation. Wild-type(WT) tumors, as determined by pyrosequencing, were included as controls; analysis of FFPE tissue and stool-derived DNA by dd PCR was performed for these patients as well.RESULTS Among the total 70 patients included, KRAS mutations were detected by pyrosequencing in 32(45.71%), whereas 38(54.29%) had WT tumors. The frequency of KRAS mutations was higher in left-sided tumors(11 located in the right colon, 15 in the left, and 6 in the rectum). The predominant point mutation was KRAS G12 D(14.29%, n = 10), which was more frequent in early-stage tumors(I-IIA, n = 7). In agreement with pyrosequencing results, the KRAS G12 D mutation was detected by dd PCR in FFPE tumor-derived DNA, and only a residual number of mutated copies was found in WT controls. The KRAS G12 D mutation was also detected in stool-derived DNA in 80% of all fecal samples from CRC patients with this point mutation. CONCLUSION dd PCR is a reliable and sensitive method to analyze KRAS G12 D mutation in stool-derived DNA from CRC patients, especially at early stages. This non-invasive approach is potentially applicable to other relevant biomarkers for CRC management.

Stem cell therapy applied for digestive anastomosis: Current state and future perspectives

BACKGROUND Digestive tract resections are usually followed by an anastomosis.Anastomotic leakage,normally due to failed healing,is the most feared complication in digestive surgery because it is associated with high morbidity and mortality.Despite technical and technological advances and focused research,its rates have remained almost unchanged the last decades.In the last two decades,stem cells(SCs)have been shown to enhance healing in animal and human studies;hence,SCs have emerged since 2008 as an alternative to improve anastomoses outcomes.AIM To summarise the published knowledge of SC utilisation as a preventative tool for hollow digestive viscera anastomotic or suture leaks.METHODS PubMed,Science Direct,Scopus and Cochrane searches were performed using the key words“anastomosis”,“colorectal/colonic anastomoses”,“anastomotic leak”,“stem cells”,“progenitor cells”,“cellular therapy”and“cell therapy”in order to identify relevant articles published in English and Spanish during the years of 2000 to 2021.Studies employing SCs,performing digestive anastomoses in hollow viscera or digestive perforation sutures and monitoring healing were finally included.Reference lists from the selected articles were reviewed to identify additional pertinent articles.METHODS Given the great variability in the study designs,anastomotic models,interventions(SCs,doses and vehicles)and outcome measures,performing a reliable meta-analysis was considered impossible,so we present the studies,their results and limitations.RESULTS Eighteen preclinical studies and three review papers were identified;no clinical studies have been published and there are no registered clinical trials.Experimental studies,mainly in rat and porcine models and occasionally in very adverse conditions such as ischaemia or colitis,have been demonstrated SCs as safe and have shown some encouraging morphological,functional and even clinical results.Mesenchymal SCs are mostly employed,and delivery routes are mainly local injections and cell sheets followed by biosutures(sutures coated by SCs)or purely topical.As potential weaknesses,animal models need to be improved to make them more comparable and equivalent to clinical practice,and the SC isolation processes need to be standardised.There is notable heterogeneity in the studies,making them difficult to compare.Further investigations are needed to establish the indications,the administration system,potential adjuvants,the final efficacy and to confirm safety and exclude definitively oncological concerns.CONCLUSION The future role of SC therapy to induce healing processes in digestive anastomoses/sutures still needs to be determined and seems to be currently far from clinical use.