NEUROD1 reinforces endocrine cell fate acquisition in pancreatic development.

NEUROD1 is a transcription factor that helps maintain a mature phenotype of pancreatic ß cells. Disruption of Neurod1 during pancreatic development causes severe neonatal diabetes; however, the exact role of NEUROD1 in the differentiation programs of endocrine cells is unknown. Here, we report a crucial role of the NEUROD1 regulatory network in endocrine lineage commitment and differentiation. Mechanistically, transcriptome and chromatin landscape analyses demonstrate that Neurod1 inactivation triggers a downregulation of endocrine differentiation transcription factors and upregulation of non-endocrine genes within the Neurod1-deficient endocrine cell population, disturbing endocrine identity acquisition. Neurod1 deficiency altered the H3K27me3 histone modification pattern in promoter regions of differentially expressed genes, which resulted in gene regulatory network changes in the differentiation pathway of endocrine cells, compromising endocrine cell potential, differentiation, and functional properties.

IsletSwipe, a mobile platform for expert opinion exchange on islet graft images.

We previously developed a deep learning-based web service (IsletNet) for an automated counting of isolated pancreatic islets. The neural network training is limited by the absent consensus on the ground truth annotations. Here, we present a platform (IsletSwipe) for an exchange of graphical opinions among experts to facilitate the consensus formation. The platform consists of a web interface and a mobile application. In a small pilot study, we demonstrate the functionalities and the use case scenarios of the platform. Nine experts from three centers validated the drawing tools, tested precision and consistency of the expert contour drawing, and evaluated user experience. Eight experts from two centers proceeded to evaluate additional images to demonstrate the following two use case scenarios. The Validation scenario involves an automated selection of images and islets for the expert scrutiny. It is scalable (more experts, images, and islets may readily be added) and can be applied to independent validation of islet contours from various sources. The Inquiry scenario serves the ground truth generating expert in seeking assistance from peers to achieve consensus on challenging cases during the preparation for IsletNet training. This scenario is limited to a small number of manually selected images and islets. The experts gained an opportunity to influence IsletNet training and to compare other experts' opinions with their own. The ground truth-generating expert obtained feedback for future IsletNet training. IsletSwipe is a suitable tool for the consensus finding. Experts from additional centers are welcome to participate.

ISL1 controls pancreatic alpha cell fate and beta cell maturation.

BACKGROUND: Glucose homeostasis is dependent on functional pancreatic α and ß cells. The mechanisms underlying the generation and maturation of these endocrine cells remain unclear. RESULTS: We unravel the molecular mode of action of ISL1 in controlling α cell fate and the formation of functional ß cells in the pancreas. By combining transgenic mouse models, transcriptomic and epigenomic profiling, we uncover that elimination of Isl1 results in a diabetic phenotype with a complete loss of α cells, disrupted pancreatic islet architecture, downregulation of key ß-cell regulators and maturation markers of ß cells, and an enrichment in an intermediate endocrine progenitor transcriptomic profile. CONCLUSIONS: Mechanistically, apart from the altered transcriptome of pancreatic endocrine cells, Isl1 elimination results in altered silencing H3K27me3 histone modifications in the promoter regions of genes that are essential for endocrine cell differentiation. Our results thus show that ISL1 transcriptionally and epigenetically controls α cell fate competence, and ß cell maturation, suggesting that ISL1 is a critical component for generating functional α and ß cells.

Decellularized Pancreatic Tail as Matrix for Pancreatic Islet Transplantation into the Greater Omentum in Rats.

Infusing pancreatic islets into the portal vein currently represents the preferred approach for islet transplantation, despite considerable loss of islet mass almost immediately after implantation. Therefore, approaches that obviate direct intravascular placement are urgently needed. A promising candidate for extrahepatic placement is the omentum. We aimed to develop an extracellular matrix skeleton from the native pancreas that could provide a microenvironment for islet survival in an omental flap. To that end, we compared different decellularization approaches, including perfusion through the pancreatic duct, gastric artery, portal vein, and a novel method through the splenic vein. Decellularized skeletons were compared for size, residual DNA content, protein composition, histology, electron microscopy, and MR imaging after repopulation with isolated islets. Compared to the other approaches, pancreatic perfusion via the splenic vein provided smaller extracellular matrix skeletons, which facilitated transplantation into the omentum, without compromising other requirements, such as the complete depletion of cellular components and the preservation of pancreatic extracellular proteins. Repeated MR imaging of iron-oxide-labeled pancreatic islets showed that islets maintained their position in vivo for 49 days. Advanced environmental scanning electron microscopy demonstrated that islets remained integrated with the pancreatic skeleton. This novel approach represents a proof-of-concept for long-term transplantation experiments.

Transplantation of Pancreatic Islets Into the Omentum Using a Biocompatible Plasma-Thrombin Gel: First Experience at the Institute for Clinical and Experimental Medicine in Prague.

BACKGROUND: Islet transplantation represents an established therapeutic option for people with type 1 diabetes who have hypoglycemia unawareness syndrome and frequent problematic hypoglycemic episodes when other methods comprising diabetes education and use of technological support fail. Because the current standard method of islet infusion into the liver has some limitations, novel approaches are under investigation. METHODS: We report our first results with 2 cases of islet transplantation into an omental pouch using a biocompatible plasma-fibrin gel. The recipients received 12,350 and 5,350 islet equivalents per kilogram that were mixed with autologous plasma, seeded during a laparoscopic procedure on the omentum, overlaid with human thrombin solution, and fixed by flapping the omentum over. RESULTS: During a 9-month follow-up, neither patient experienced any moderate or severe hypoglycemia. Their glucose control significantly improved, insulin dose decreased by approximately 50%, and C-peptide at 1 year was 0.22 and 0.14 pmol/mL, respectively. The postoperative course was uneventful, but C-peptide production in the first patient progressively declined at 1 year and hypoglycemic episodes recurred. CONCLUSIONS: Though the results for these first 2 cases are not fully satisfactory, we have demonstrated the feasibility, safety, and ability of this novel method to restore insulin production. Further refinements to improve immediate islet survival seem necessary.

The Potential of Pancreatic Organoids for Diabetes Research and Therapy.

The success of clinical transplantation of pancreas or isolated pancreatic islets supports the concept of cell-based cure for diabetes. One limitation is the shortage of cadaver human pancreata. The demand-supply gap could potentially be bridged by harnessing the self-renewal capacity of stem cells. Pluripotent stem cells and adult pancreatic stem cells have been explored as possible cell sources. Recently, a system for long-term culture of proposed adult pancreatic stem cells in a form of organoids was developed. Generated organoids partially mimic the architecture and cell-type composition of pancreatic tissue. Here, we review the attempts over the past decade, to utilize the organoid cell culture principles in order to identify, expand, and differentiate the adult pancreatic stem cells from different compartments of mouse and human pancreata. The development of the culture conditions, effects of specific growth factors and small molecules is discussed. The potential utility of the adult pancreatic stem cells is considered in the context of other cell sources.

NEUROD1 Is Required for the Early α and ß Endocrine Differentiation in the Pancreas.

Diabetes is a metabolic disease that involves the death or dysfunction of the insulin-secreting ß cells in the pancreas. Consequently, most diabetes research is aimed at understanding the molecular and cellular bases of pancreatic development, islet formation, ß-cell survival, and insulin secretion. Complex interactions of signaling pathways and transcription factor networks regulate the specification, growth, and differentiation of cell types in the developing pancreas. Many of the same regulators continue to modulate gene expression and cell fate of the adult pancreas. The transcription factor NEUROD1 is essential for the maturation of ß cells and the expansion of the pancreatic islet cell mass. Mutations of the Neurod1 gene cause diabetes in humans and mice. However, the different aspects of the requirement of NEUROD1 for pancreas development are not fully understood. In this study, we investigated the role of NEUROD1 during the primary and secondary transitions of mouse pancreas development. We determined that the elimination of Neurod1 impairs the expression of key transcription factors for α- and ß-cell differentiation, ß-cell proliferation, insulin production, and islets of Langerhans formation. These findings demonstrate that the Neurod1 deletion altered the properties of α and ß endocrine cells, resulting in severe neonatal diabetes, and thus, NEUROD1 is required for proper activation of the transcriptional network and differentiation of functional α and ß cells.

Transplant Options for Patients With Diabetes and Advanced Kidney Disease: A Review.

Optimal glycemic control in kidney transplant recipients with diabetes is associated with improved morbidity and better patient and allograft survival. Transplant options for patients with diabetes requiring insulin therapy and chronic kidney disease who are suitable candidates for kidney transplantation should include consideration of ß-cell replacement therapy: pancreas or islet transplantation. International variation related to national regulatory policies exists in offering one or both options to suitable candidates and is further affected by pancreas/islet allocation policies and transplant waiting list dynamics. The selection of appropriate candidates depends on patient age, coexistent morbidities, the timing of referral to the transplant center (predialysis versus on dialysis) and availability of living kidney donors. Therefore, early referral (estimated glomerular filtration rate < 30 mL/min/1.73 m2) is of the utmost importance to ensure adequate time for informed decision making and thorough pretransplant evaluation. Obesity, cardiovascular disease, peripheral vascular disease, smoking, and frailty are some of the conditions that need to be addressed before acceptance on the transplant list, and ideally before dialysis becoming imminent. This review offers insights into selection of pancreas/islet transplant candidates by transplant centers and an update on posttransplant outcomes, which may have practice implications for referring nephrologists.

US food and drug administration (FDA) panel endorses islet cell treatment for type 1 diabetes: A pyrrhic victory?

Allogeneic islet transplantation is a standard of care treatment for patients with labile type 1 diabetes in many countries around the world, including Japan, the United Kingdom, Australia, much of continental Europe, and parts of Canada. The United States is now endorsing islet cell treatment for type 1 diabetes, but the FDA has chosen to consider islets as a biologic that requires licensure, making the universal implementation of the procedure in the clinic very challenging and opening the manufacture of islet grafts to private companies. The commercialization of human tissues raises significant legal and ethical issues and ironically leads to a situation where treatments developed as a result of the scientific and economic efforts of academia over several decades become exploited exclusively by for-profit entities.

Bioluminescence Imaging In Vivo Confirms the Viability of Pancreatic Islets Transplanted into the Greater Omentum.

PURPOSE: The liver is the most widely used site for pancreatic islet transplantation. However, several site-specific limitations impair functional success, with instant blood-mediated inflammatory reaction being the most important. The aim of this study was to develop a preclinical model for placement of the islet graft into a highly vascularized omental flap using a fibrin gel. For this purpose, we tested islet viability by bioluminescence imaging (BLI). PROCEDURES: Pancreatic islets were isolated from luciferase-positive and luciferase-negative rats, mixed at a 1:1 ratio, placed into a plasma-thrombin bioscaffold, and transplanted in standard (10 pancreatic islets/g wt; n = 10) and marginal (4 pancreatic islets/g wt; n = 7) numbers into the omentums of syngeneic diabetic animals. For the control, 4 pancreatic islets/g were transplanted into the liver using the standard procedure (n = 7). Graft viability was tested by bioluminescence at days 14, 30, 60, and 90 post transplant. Glucose levels, intravenous glucose tolerance, and serum C-peptide were assessed regularly. RESULTS: Nonfasting glucose levels < 10 mmol/l were restored in all animals. While islet viability in the omentum was clearly detected by stable luminescence signals throughout the whole study period, no signals were detected from islets transplanted into the liver. The bioluminescence signals were highly correlated with stimulated C-peptide levels detected at 80 days post transplant. Glucose tolerance did not differ among the 3 groups. CONCLUSIONS: We successfully tested a preclinical model of islet transplantation into the greater omentum using a biocompatible scaffold made from autologous plasma and human thrombin. Both standard and marginal pancreatic islet numbers in a gel-form bioscaffold placed in the omentum restored glucose homeostasis in recipients with diabetes. Bioluminescence was shown promising as a direct proof of islet viability.

Distant diabetes care: what can the patient, nurse and patient do.

Due to the spread of new coronavirus disease, COVID-19, social interactions between people have been significantly reduced. In healthcare, outpatient care is a high-risk frontline of infection transmission in both patients and healthcare professionals. The presence of routine digital communication, remote data management and the availability of glucose monitoring and insulin delivery devices have given diabetology a certain advantage in this situation. However, the potential of these modalities has not been fully utilized so far. We provide an overview of practical methods of distance patient management, which can be used in most diabetes outpatient clinics without any difficult adjustments or additional investments. This approach can be used in different patients according to their treatment strategies and individual abilities.

Early worsening of diabetic retinopathy after simultaneous pancreas and kidney transplantation-Myth or reality?

Early worsening of diabetic retinopathy due to sudden glucose normalization is a feared complication of pancreas transplantation; however, its rate or severity has not been studied prospectively. We followed up 43 pancreas and kidney recipients for a composite endpoint comprising new need for laser therapy, newly diagnosed proliferation, macular edema, visual acuity worsening, and blindness over 12 months. Although 37% of patients met this primary endpoint, its severity was rather low. Mean central retinal thickness and proportion of patients with subclinical macular edema increased significantly, with spontaneous resolution in half of them. Visual acuity did not change. There was no significant difference in the absolute glycated hemoglobin (HbA1c) drop, age, and diabetes duration between the patients who met and those who did not meet the primary endpoint, but a higher proportion of patients with worsening had a recent history of laser treatment. Retinopathy remained stable in 62.8% of patients. In 26%, the visual acuity significantly improved. Although retinopathy worsening was documented in more than one-third of patients, its evolution was not related to the magnitude of metabolic change; rather, it corresponded to the expected natural course of retinopathy. Nonetheless, comprehensive ophthalmologic care should be a substantial component of the recipient management.

Targeting CXCR1/2 Does Not Improve Insulin Secretion After Pancreatic Islet Transplantation: A Phase 3, Double-Blind, Randomized, Placebo-Controlled Trial in Type 1 Diabetes.

OBJECTIVE: Reparixin is an inhibitor of CXCR1/2 chemokine receptor shown to be an effective anti-inflammatory adjuvant in a pilot clinical trial in allotransplant recipients. RESEARCH DESIGN AND METHODS: A phase 3, multicenter, randomized, double-blind, parallel-assignment study (NCT01817959) was conducted in recipients of islet allotransplants randomized (2:1) to reparixin or placebo in addition to immunosuppression. Primary outcome was the area under the curve (AUC) for C-peptide during the mixed-meal tolerance test at day 75 ± 5 after the first and day 365 ± 14 after the last transplant. Secondary end points included insulin independence and standard measures of glycemic control. RESULTS: The intention-to-treat analysis did not show a significant difference in C-peptide AUC at both day 75 (27 on reparixin vs. 18 on placebo, P = 0.99) and day 365 (24 on reparixin vs. 15 on placebo, P = 0.71). There was no statistically significant difference between treatment groups at any time point for any secondary variable. Analysis of patient subsets showed a trend for a higher percentage of subjects retaining insulin independence for 1 year after a single islet infusion in patients receiving reparixin as compared with patients receiving placebo (26.7% vs. 0%, P = 0.09) when antithymocyte globulin was used as induction immunosuppression. CONCLUSIONS: In this first double-blind randomized trial, islet transplantation data obtained with reparixin do not support a role of CXCR1/2 inhibition in preventing islet inflammation-mediated damage.

Heterogeneity of Human Pancreatic Islet Isolation Around Europe: Results of a Survey Study.

BACKGROUND: Europe is currently the most active region in the field of pancreatic islet transplantation, and many of the leading groups are actually achieving similar good outcomes. Further collaborative advances in the field require the standardization of islet cell product isolation processes, and this work aimed to identify differences in the human pancreatic islet isolation processes within European countries. METHODS: A web-based questionnaire about critical steps, including donor selection, pancreas processing, pancreas perfusion and digestion, islet counting and culture, islet quality evaluation, microbiological evaluation, and release criteria of the product, was completed by isolation facilities participating at the Ninth International European Pancreas and Islet Transplant Association (EPITA) Workshop on Islet-Beta Cell Replacement in Milan. RESULTS: Eleven islet isolation facilities completed the questionnaire. The facilities reported 445 and 53 islet isolations per year over the last 3 years from deceased organ donors and pancreatectomized patients, respectively. This activity resulted in 120 and 40 infusions per year in allograft and autograft recipients, respectively. Differences among facilities emerged in donor selection (age, cold ischemia time, intensive care unit length, amylase concentration), pancreas procurement, isolation procedures (brand and concentration of collagenase, additive, maximum acceptable digestion time), quality evaluation, and release criteria for transplantation (glucose-stimulated insulin secretion tests, islet numbers, and purity). Moreover, even when a high concordance about the relevance of one parameter was evident, thresholds for the acceptance were different among facilities. CONCLUSIONS: The result highlighted the presence of a heterogeneity in the islet cell product process and product release criteria.

Sirolimus vs mycophenolate mofetil (MMF) in primary combined pancreas and kidney transplantation. Results of a long-term prospective randomized study.

The study was intended to compare pancreas graft survival rates in two groups of pancreas and kidney transplant recipients prospectively randomized to treatment either with sirolimus or MMF. From 2002 to 2013, 238 type 1 diabetic recipients with end-stage kidney disease were randomized 1:1 to sirolimus or MMF treatment. Noncensored pancreas survival at 5 years was 76.4 and 71.6% for sirolimus and MMF groups, respectively (P > .05). Death-censored pancreas survival was better in the sirolimus group (P = .037). After removal of early graft losses pancreas survival did not differ between groups (MMF 83.1% vs sirolimus 91.6%, P = .11). Nonsignificantly more grafts were lost due to rejection in the MMF group (10 vs 5; P = .19). Cumulative patient 5-year survival was 96% in the MMF group and 91% in the sirolimus group (P > .05). Five-year cumulative noncensored kidney graft survival rates did not statistically differ (85.6% in the sirolimus group and 88.8% in MMF group). Recipients treated with MMF had significantly more episodes of gastrointestinal bleeding (7 vs 0, P = .007). More recipients in the sirolimus group required corrective surgery due to incisional hernias (21 vs 12, P = .019). ClinicalTrials No.: NCT03582878.

A novel model for in vivo quantification of immediate liver perfusion impairment after pancreatic islet transplantation.

Instant Blood-Mediated Inflammatory Reaction (IBMIR) is a major cause of graft loss during pancreatic islet transplantation, leading to a low efficiency of this treatment method and significantly limiting its broader clinical use. Within the procedure, transplanted islets obstruct intrahepatic portal vein branches and consequently restrict blood supply of downstream lying liver tissue, resulting typically in ischemic necrosis. The extent of ischemic lesions is influenced by mechanical obstruction and inflammation, as well as subsequent recanalization and regeneration capacity of recipient liver tissue. Monitoring of immediate liver perfusion impairment, which is directly related to the intensity of post-transplant inflammation and thrombosis (IBMIR), is essential for improving therapeutic and preventive strategies to improve overall islet graft survival. In this study, we present a new experimental model enabling direct quantification of liver perfusion impairment after pancreatic islet transplantation using ligation of hepatic arteries followed by contrast-enhanced magnetic resonance imaging (MRI). The ligation of hepatic arteries prevents the contrast agent from circumventing the portal vein obstruction and enables to discriminate between well-perfused and non-perfused liver tissue. Here we demonstrate that the extent of liver ischemia reliably reflects the number of transplanted islets. This model represents a useful tool for in vivo monitoring of biological effect of IBMIR-alleviating interventions as well as other experiments related to liver ischemia. This technical paper introduces a novel technique and its first application in experimental animals.

Retrospective Analysis of Bone Metabolism in Patients on Waiting List for Simultaneous Pancreas-Kidney Transplantation.

Posttransplant osteoporosis, which evolves from preexisting bone pathologies, represents a serious complication with deteriorating consequences. The aim of our study was to evaluate epidemiological data on bone mineral density (BMD) in subjects with type 1 diabetes (T1DM) in advanced stages of diabetic nephropathy indicated for simultaneous pancreas-kidney transplantation (SPK). We retrospectively compiled biochemical and densitometrical data from 177 patients with T1DM at CKD (chronic kidney disease) stages G4-G5 (115 men, 62 women, median age 40 yr, diabetes duration 23 yr) enrolled on waiting list for SPK for the first time between the years 2011 and 2016. Median Z-scores were as follows: lumbar spine (LS): -0.8 [interquartile range -1.75 to 0.1]; total hip (TH): -1.2 [-1.75 to -0.6]; femoral neck (FN): -1.2 [-1.9 to -0.7]; and distal radius (DR): -0.8 [-1.4 to -0.1]. We noted a gender difference in LS, with worse results for men (-1.1 vs. -0.3) even after adjusting for BMI (body mass index) and glomerular filtration (p < 0.001). Osteoporotic and osteopenic ranges (based on T-scores) for all major sites were 27.7% and 56.5%, respectively, with similar results across both genders. Women had a significantly higher proportion of normal BMD in LS than men (67.7 vs. 49.4%, p < 0.05). Patients with T1DM at CKD stages G4-G5 exhibited serious BMD impairment despite their young age. Men surprisingly displayed lower Z-scores and higher percentages of pathological BMD values in LS than women did. The introduction of adequate preventive measures during the advanced stages of diabetic nephropathy to prevent bone loss is recommended.

Preserved Expression of Skin Neurotrophic Factors in Advanced Diabetic Neuropathy Does Not Lead to Neural Regeneration despite Pancreas and Kidney Transplantation.

Diabetic peripheral neuropathy (DPN) is a common complication of diabetes with potential severe consequences. Its pathogenesis involves hyperglycemia-linked mechanisms, which may include changes in the expression of neurotrophic growth factors. We analyzed the expression of 29 factors potentially related to nerve degeneration and regeneration in skin biopsies from 13 type 1 diabetic pancreas and kidney recipients with severe DPN including severe depletion of intraepidermal nerve fibers (IENF) in lower limb skin biopsies (group Tx1 1st examination). The investigation was repeated after a median 28-month period of normoglycemia achieved by pancreas transplantation (group Tx1 2nd examination). The same tests were performed in 13 stable normoglycemic pancreas and kidney recipients 6-12 years posttransplantation (group Tx2), in 12 matched healthy controls (group HC), and in 12 type 1 diabetic subjects without severe DPN (group DM). Compared to DM and HC groups, we found a significantly higher (p < 0.05-0.001) expression of NGF (nerve growth factor), NGFR (NGF receptor), NTRK1 (neurotrophic receptor tyrosine kinase 1), GDNF (glial cell-derived neurotrophic factor), GFRA1 (GDNF family receptor alpha 1), and GFAP (glial fibrillary acidic protein) in both transplant groups (Tx1 and Tx2). Enhanced expression of these factors was not normalized following the median 28-month period of normoglycemia (Tx1 2nd examination) and negatively correlated with IENF density and with electrophysiological indices of DPN (vibration perception threshold, electromyography, and autonomic tests). In contrast to our expectation, the expression of most of 29 selected factors related to neural regeneration was comparable in subjects with severe peripheral nerve fiber depletion and healthy controls and the expression of six factors was significantly upregulated. These findings may be important for better understanding the pathophysiology of nerve regeneration and for the development of intervention strategies.

Nkx6.1 decline accompanies mitochondrial DNA reduction but subtle nucleoid size decrease in pancreatic islet ß-cells of diabetic Goto Kakizaki rats.

Hypertrophic pancreatic islets (PI) of Goto Kakizaki (GK) diabetic rats contain a lower number of ß-cells vs. non-diabetic Wistar rat PI. Remaining ß-cells contain reduced mitochondrial (mt) DNA per nucleus (copy number), probably due to declining mtDNA replication machinery, decreased mt biogenesis or enhanced mitophagy. We confirmed mtDNA copy number decrease down to <30% in PI of one-year-old GK rats. Studying relations to mt nucleoids sizes, we employed 3D superresolution fluorescent photoactivable localization microscopy (FPALM) with lentivirally transduced Eos conjugate of mt single-stranded-DNA-binding protein (mtSSB) or transcription factor TFAM; or by 3D immunocytochemistry. mtSSB (binding transcription or replication nucleoids) contoured "nucleoids" which were smaller by 25% (less diameters >150 nm) in GK ß-cells. Eos-TFAM-visualized nucleoids, composed of 72% localized TFAM, were smaller by 10% (immunochemically by 3%). A theoretical ~70% decrease in cell nucleoid number (spatial density) was not observed, rejecting model of single mtDNA per nucleoid. The ß-cell maintenance factor Nkx6.1 mRNA and protein were declining with age (>12-fold, 10 months) and decreasing with fasting hyperglycemia in GK rats, probably predetermining the impaired mtDNA replication (copy number decrease), while spatial expansion of mtDNA kept nucleoids with only smaller sizes than those containing much higher mtDNA in non-diabetic ß-cells.

Synthetic mRNA is a more reliable tool for the delivery of DNA-targeting proteins into the cell nucleus than fusion with a protein transduction domain.

Cell reprogramming requires efficient delivery of reprogramming transcription factors into the cell nucleus. Here, we compared the robustness and workload of two protein delivery methods that avoid the risk of genomic integration. The first method is based on fusion of the protein of interest to a protein transduction domain (PTD) for delivery across the membranes of target cells. The second method relies on de novo synthesis of the protein of interest inside the target cells utilizing synthetic mRNA (syn-mRNA) as a template. We established a Cre/lox reporter system in three different cell types derived from human (PANC-1, HEK293) and rat (BRIN-BD11) tissues and used Cre recombinase to model a protein of interest. The system allowed constitutive expression of red fluorescence protein (RFP), while green fluorescence protein (GFP) was expressed only after the genomic action of Cre recombinase. The efficiency of protein delivery into cell nuclei was quantified as the frequency of GFP+ cells in the total cell number. The PTD method showed good efficiency only in BRIN-BD11 cells (68%), whereas it failed in PANC-1 and HEK293 cells. By contrast, the syn-mRNA method was highly effective in all three cell types (29-71%). We conclude that using synthetic mRNA is a more robust and less labor-intensive approach than using the PTD-fusion alternative.