Coagulation activation and hepatic engraftment of human amnion-derived cells after intraportal transplantation in rats

ABSTRACT

Aims

Human amniotic epithelial cells (hAECs) have emerged as a promising cell source for regenerative medicine, with intraportal transplantation as a potential delivery route. However, the extent to which hAECs induce an immediate inflammatory response remains unclear. This study investigated tissue factor (TF) expression in isolated hAECs and assessed coagulation activation following intraportal transplantation in a rat model.

Materials and methods

TF expression was analyzed before and after cryopreservation, while thrombin – antithrombin (TAT) complex levels were measured to evaluate coagulation activation. To assess engraftment and hepatocyte-like function, hAECs were transplanted into non-albuminemic rats, followed by serial measurements of serum human albumin levels and histological liver analysis.

Results

The findings indicate that hAECs express TF pre- and post-cryopreservation. Intraportal transplantation resulted in a significant increase in plasma TAT levels, which was mitigated by heparin administration. Furthermore, human albumin levels increased post-transplantation, and albumin-positive cells were detected in the liver on day 21.

Conclusion

These results suggest that intraportal hAEC transplantation is a feasible approach for hepatic engraftment; however, TF expression may trigger coagulation activation, potentially leading to an instant blood-mediated inflammatory reaction. Further research is warranted to optimize anticoagulation strategies and evaluate long-term engraftment efficacy for clinical applications.

Plain Language Summary

This study explores the safety and effectiveness of transplanting special cells called human amnion-derived epithelial cells (hAECs) into the liver through the bloodstream. These cells are collected from the amniotic membrane, a part of the placenta that is usually discarded after childbirth. hAECs are promising for treating liver diseases because they can develop into liver-like cells and are less likely to be rejected by the immune system. However, when cells are injected into the bloodstream, they can sometimes trigger a rapid immune and clotting response, known as an instant blood-mediated inflammatory reaction (IBMIR), which may damage the transplanted cells. In this study, we tested whether hAECs cause such a response in rats. We found that the cells produce a protein called tissue factor, which can activate blood clotting. After transplantation, we observed increased levels of clotting markers in the blood. Despite this reaction, the cells were able to reach the liver, survive, and begin producing human albumin – a protein normally made by liver cells. These findings provide new insight into both the risks and the potential benefits of using hAECs in liver therapy. They also suggest possible strategies to improve the safety of this approach in future clinical applications.

1. Introduction

Hepatocyte transplantation has been explored as a minimally invasive alternative to whole organ liver transplantation, particularly for congenital metabolic disorders. Despite promising preclinical outcomes, clinical implementation remains limited due to issues related to cell supply, insufficient engraftment, and immune rejection [1,2]. Advances in regenerative medicine have led to investigations into alternative cell sources such as human amniotic epithelial cells (hAECs), which exhibit multipotent characteristics, including differentiation into hepatocytes [3–5], as well as immunomodulatory properties that could aid in overcoming immune rejection barriers [6]. In addition, hAECs are relatively easy to obtain as they are separated from the placenta (considered a medical waste product).

Intraportal transplantation of hepatocytes has been demonstrated as a feasible approach for targeted delivery to the liver. Previously, we reported that, compared to intravenous or trans-splenic transplantation, intraportal hAEC transplantation could be performed safely and efficiently [7]. However, previous studies have highlighted a major limitation associated with this route: the potential activation of coagulation and immune responses, leading to an instant blood-mediated inflammatory reaction (IBMIR), as demonstrated in pancreatic islet [8,9] and hepatocyte [10] transplantation. IBMIR is triggered by direct cell-blood contact and involves activation of both the coagulation cascade and complement system [11]. Tissue factor (TF) expression on transplanted cells has been identified as a key initiator of this reaction [12,13], and strategies to mitigate this effect are critical for optimizing engraftment outcomes.

Therefore, this study aimed to evaluate the role of TF expression in hAECs and its contribution to IBMIR following intraportal transplantation in a rat model. We assessed coagulation activation by monitoring thrombin – antithrombin (TAT) complex levels and examined hAEC engraftment and function in vivo through histological and biochemical analysis.

2. Materials and methods

This study was conducted in accordance with the principles of the Declaration of Helsinki and approved by the Institutional Review Board of Tohoku University (approval number: 2018–1–203). Written informed consent was obtained in advance from all participating donors for the collection and use of amniotic membranes. All animals in this study were handled in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. Animal experiments were performed in compliance with the ARRIVE guidelines and approved by the Animal Care and Use Committee of Tohoku University (approval number: 2019–191). The experimental unit in all animal experiments was a single rat, and a total of 62 rats were used in this study.

All surgical procedures were performed under general anesthesia using inhaled isoflurane to minimize pain and distress. Animals were monitored until recovery and observed daily for signs of pain or abnormal behavior. No unexpected adverse events occurred during the study. A small number of animals experienced perioperative death during cell transplantation, which was anticipated and addressed by predefined exclusion criteria.

Humane endpoints were predefined. Animals exhibiting severe distress, such as sustained weight loss exceeding 10% for consecutive days or inability to feed orally due to postoperative complications, were promptly euthanized by overdose of inhaled isoflurane. Daily monitoring was conducted for clinical signs, including reduced activity, poor grooming, and abnormal behavior.

Animals that died during or immediately after cell transplantation were excluded from downstream analysis and not included in the reported n values. Perioperative mortality was an anticipated event due to the invasive nature of the procedure and was handled consistently across experimental groups in accordance with this criterion. While the exact number of excluded animals was not systematically recorded, all analyzed data reflect only animals that survived beyond the immediate post-transplantation period.

All rats, including Wistar/ST and Nagase analbuminemic (NAR)/Slc strains, were purchased from Kumagai-Shigeyasu Co., Ltd. (Sendai, Japan) and housed under specific pathogen-free (SPF) conditions. Animals were kept in individually ventilated cages with autoclaved bedding and provided autoclaved rodent chow and water ad libitum. Environmental enrichment, including nesting materials and chew sticks, was supplied throughout the study. NAR/Slc rats are a genetically modified strain lacking serum albumin. None of the animals had undergone any prior procedures before the current study.

2.1. hAEC isolation

We collected the amniotic membranes from the placentas of mothers with no infectious diseases who underwent cesarean delivery at the Department of Obstetrics at a university-affiliated hospital. We obtained written informed consent in advance based on the approval of the Institutional Review Board (2018–1–203). We isolated hAECs from the amniotic membranes as described previously [14]. We cryopreserved the isolated cells in a liquid-nitrogen storage tank until transplantation. Prior to using the cells, we thawed them quickly in a water bath at 37°C, sedimented by centrifugation at 300 × g for 5 min, and resuspended in 4°C Dulbecco’s phosphate-buffered saline (DPBS; Gibco® DPBS; Thermo Fisher Scientific, MA, USA). We counted the cells using an automated cell counter (TC20™; BIO-RAD, CA, USA) and assessed cell viability using the trypan blue exclusion assay (T8154; Sigma-Aldrich, MO, USA).

2.2. Flow cytometry analysis

We performed fluorescence-activated cell sorting (FACS) to assess TF expression in isolated hAECs before and after cryopreservation. We thawed the cryopreserved hAECs using a 37°C water bath. We divided the cells into three groups (n = 3; 1 × 10⁶ hAECs/200 μL of DPBS in each group) as follows: group 1, unstained (i.e., negative control); group 2, stained with anti-coagulation factor III antibody (Sino Biological, Beijing, China); and group 3, PE mouse IgG2a (Abcam, OR, USA) stained (i.e., isotype control). We analyzed the cells using a BD Accuri™ C6 Cytometer (Becton Dickinson, NJ, USA).

2.3. Transplantation procedure

We performed intraportal hAEC transplantation using a 27 G insulin syringe advanced to the hepatic hilum, injecting hAECs over 30 s. A schematic illustration of the intraportal cell injection procedure is provided in Figure 1 to aid reproducibility. This procedure follows established techniques and is not intended as a novel surgical method. We achieved hemostasis by directly pressing the puncture site for 2 min.

Figure 1.

Experimental model of intraportal transplantation.

A 5-cm upper midline abdominal incision was made caudal to the xiphoid process. A 27-gauge insulin syringe was inserted peripherally at the confluence of the gastrosplenic vein (●), and the needle tip was advanced toward the porta hepatis (★) under direct visualization. The cell suspension was injected slowly over 30 seconds. This figure is provided to enhance reproducibility of the intraportal injection technique and is not intended to illustrate a novel surgical method.

2.4. Analysis of coagulation activation after intraportal hAEC transplantation

We analyzed TAT level changes after hAEC transplantation using enzyme-linked immunosorbent assay (ELISA). This assay was performed to evaluate coagulation activation as a marker of thromboinflammatory response. We intraportally administered 1 × 10⁷ hAECs in 200 μL DPBS into 10–12-week-old male Wistar/ST rats in the experimental group; rats in the control group were administered only 200 μL of DPBS (n = 10 in each group). In addition, to investigate how anticoagulants affect IBMIRs upon hAEC transplantation, we supplemented 0.1 units/weight (g) of heparin with 1 × 10⁷ hAECs in 200 μL of DPBS. We inserted a silicone tube (100-00N; Kaneka Medics, Osaka, Japan) into the left jugular vein before the abdominal opening operation. We drained the central venous (CV) catheter using saline solution. For the heparin group, we first administered heparin from the CV, boosted it with 100 μL of saline, and performed cell transplantation 2 min later. We set the CV blood collection time point before the abdominal opening operation as 0 min and collected 400 μL of blood from the CV catheter at 15, 60, 120, 180, and 240 min after hAEC infusion. We collected the blood in BD Microtainer® Lavender top tubes (Becton Dickinson, NJ, USA) and gently mixed the samples via inversion, and then immediately centrifuged them at 15,000 rpm for 2 min at 4°C to collect the plasma, which was stored at −80°C. We analyzed the TAT (NBP2–68132; Novus Biologicals, MA, USA) level after thawing.

2.5. Analysis of human albumin (hAlb) level after hAEC transplantation

We used 9–10-week-old Nagase analbuminemic (NAR)/Slc male rats (n = 10) as recipients for the intraportal transplantation of 1 × 10⁷ hAECs in 200 μL of DPBS. This measurement was intended to assess hepatocyte-like function of transplanted hAECs. Rats in the negative control group received only 200 μL of DPBS (n = 4). We defined the time point when we collected 200 μL of tail vein blood before transplantation as day 0. We collected 200-μL blood samples on days 3, 7, and 21 into BD Microtainer® yellow top tubes (Becton Dickinson, NJ, USA), mixed the samples via inversion, and then immediately centrifuged them at 15,000 rpm for 2 min at 4°C to collect the serum, which was stored at −80°C. We performed hAlb analysis after thawing the samples, using a commercial Human Albumin ELISA kit (ab108788; Abcam, OR, USA).

2.6. In vivo imaging assessment of hAEC engraftment

We directly labeled the cells with 1,1′-dio-ctadecyltetramethyl indotricarbocyanine iodide (XenoLight DiR®; Perkin Elmer, Waltham, MA, USA). This imaging approach was used to visualize the biodistribution and short-term retention of transplanted cells. Staining solution was prepared at a final concentration of 1 μg/mL in DPBS and hAECs were incubated in it at 37°C for 30 min. Next, we centrifuged the cells at 300 × g for 5 min and washed them thrice with DPBS at 4°C. We used 7 − 9-week-old male Wistar/ST rats, transplanting each animal with 1 × 10⁷ hAECs resuspended in 200 μL of DPBS and dividing them into three groups (n = 6 per group) as follows: group 1, 200 μL DPBS alone; group 2, 1 × 10⁷ hAECs in 200 μL of DPBS; and group 3, transplantation of 1 × 10⁷ hAECs in 200 μL DPBS performed 2 min after the administration of 0.1 units/g (target rat bodyweight) of heparin through the penile vein. After intraportal hAEC infusion, we placed the anesthetized rats in an in vivo imaging system (IVIS) for spectrum computed tomography (IVIS spectrum CT; Perkin Elmer) and captured images using IVIS® at 1, 3, and 24 h. For rats in the heparin group, we first injected heparin intravenously through the penile vein and transplanted the cells 2 min later. We acquired images of the stained 1 × 10⁶ hAECs in 200 μL of DPBS and 1 × 10⁶ hAECs in 200 μL of DPBS supplemented with three units of heparin in vitro using IVIS®.

2.7. Histological analysis

We sampled the livers of Wistar/ST rats on days 1 and 3 after the transplantation of 1 × 10⁷ hAECs. This staining was performed to evaluate hepatic engraftment and differentiation of transplanted hAECs. We euthanized the NAR/Slc rats to remove their livers after blood collection on day 21 post-transplantation. Euthanasia was performed by overdose of inhaled isoflurane, in accordance with institutional guidelines for humane endpoints. We fixed the excised liver specimens with 4% formaldehyde, embedded them in paraffin, sectioned the samples, and stained them with antibodies specific to human epithelial cell adhesion molecule (hEpCAM; 1:500, ab223582; Abcam, OR, USA) and hAlb (1:200, #188835; R&B systems, MN, USA), using the EnVision^TM Kit (Dako, CA, USA) as secondary antibodies. We captured the images using an Olympus IX73 inverted microscope and analyzed them using the CellSens Dimension 1.12 software. We immunostained the cellblock specimens similarly.

2.8. Statistical analysis

We used the JMP® Pro 16.0.0 (SAS Inc., Cary, NC, USA) software for statistical analysis. We determined the statistical significance using Mann – Whitney U test, considering differences with P-values of p < 0.05 as statistically significant. Sample sizes were determined based on previous studies using similar transplantation models and endpoints, as well as feasibility and ethical considerations. No formal a priori sample size calculation was performed.

3. Results

3.1. TF expression in isolated hAecs

We assessed the expression of TF in isolated hAECs using flow cytometry before and after cryopreservation. As shown in Figure 2, freshly isolated hAECs exhibited a high positivity rate for anti-TF antibody staining compared to the isotype control. Similarly, cryopreserved hAECs that had been stored in liquid nitrogen demonstrated comparable TF expression levels, indicating that cryopreservation did not affect TF expression.

Figure 2.

TF expression in isolated hAECs.

Tissue factor (TF) expression was evaluated by flow cytometry in both freshly isolated and cryopreserved human amniotic epithelial cells (hAECs). TF was detected in both cell populations. (a) Representative histograms showing TF staining compared to isotype controls. (b) Quantification of TF-positive cells presented as mean ± SD. ** p< 0.01.

3.2. TAT level change after transplantation

Figure 3 illustrates the temporal changes in TAT levels following intraportal cell transplantation in rats, presented as fold changes relative to baseline (0 min). In the hAEC transplantation group, TAT levels increased approximately twofold by 60 minutes post-transplantation, indicating a rapid activation of the coagulation cascade. The elevated levels then plateaued and remained sustained up to 240 minutes. In contrast, no significant increase in TAT levels was observed in either the control group or the hAECs + heparin group, in which TAT levels remained near baseline throughout the observation period. These findings suggest that hAECs trigger an early procoagulant response, which is effectively suppressed by co-administration of heparin.

Figure 3.

Temporal changes in thrombin – antithrombin complex (TAT) levels following intraportal cell transplantation.

TAT levels were assessed in rat plasma at 0, 15, 60, and 240 minutes after transplantation and are expressed as fold changes relative to baseline (0 min). In the hAEC group, TAT levels increased approximately twofold by 60 minutes and remained elevated up to 240 minutes. In contrast, no significant increase in TAT levels was observed in the control or hAECs + heparin groups. Data are shown as mean ± SD (n = 10 per group).

Upper asterisks (*) indicate statistical significant differences between the hAEC group and the hAEC + heparin group; lower asterisks denote differences between the hAEC group and the control group. *p < 0.05, **p < 0.01.

3.3. Blood hAlb level increase after transplantation

Figure 4 shows the changes in serum hAlb levels following intraportal transplantation of hAECs into analbuminemic rats. hAlb levels peaked at day 3 post-transplantation (42.7 ± 34.2 µg/mL, p = 0.0047) and gradually declined through day 21, although the level on day 21 remained significantly higher compared to the control group (24.6 ± 21.0 vs 2.3 ± 0.1 µg/mL, p = 0.0046).

Figure 4.

Changes in the serum human albumin (hAlb) levels following hAEC transplantation.

Serum hAlb levels in NAR/Slc rats increased markedly by day 3 after intraportal hAEC transplantation, peaked at this time point, and then gradually declined through day 21. Values remained significantly higher than those in the control group throughout the observation period. Data are presented as mean ± SD. p < 0.05, p < 0.01.

3.4. Histological evaluation of transplanted cell

In the hAEC block specimens, hEpCAM strongly stained the hAEC cytoplasm. hEpCAM-positive cells were scattered in vivo throughout the hepatic lobules on days 1, 3, and 21. Although hEpCAM-positive cells could be similarly observed on days 1, 3, and 21, only the nucleoplasm was stained on day 21, and the cells were round. Moreover, we confirmed the presence of hAlb-positive cells on day 21. However, the number of such cells remained relatively low. These histological findings are shown in Figure 5. In addition to the histological assessment, we performed IVIS analysis to evaluate short-term hAEC engraftment. Our IVIS analysis demonstrated that almost all cells accumulated in the liver after intraportal transplantation and that the fluorescence intensity decayed over time. Finally, we evaluated the effect of heparin, which suppressed the increase in TAT level. This analysis revealed that heparin use did not affect the fluorescence intensity decay. These results are depicted in Figure 6.

Figure 5.

Immunohistochemical detection of transplanted hAecs in rat liver.

(a) hEpCAM staining of a hAEC cell block, showing strong cytoplasmic positivity. (b,c) hEpCAM staining of NAR/Slc rat liver tissue 21 days after intraportal transplantation, indicating widespread distribution of hEpCAM-positive cells. (d,e) Immunostaining for human albumin (hAlb) in liver sections 21 days post-transplantation, revealing hAlb-positive cells localized primarily around the portal vein.

Figure 6.

In vivo imaging of transplanted hAecs using IVIS.

hAECs labeled with 1,1′-dioctadecyltetramethyl indotricarbocyanine iodide (DiR) accumulated in the liver in both the hAEC-only and hAEC + heparin groups following intraportal transplantation. No significant differences in fluorescence intensity were observed between the two groups, suggesting comparable initial hepatic distribution.

4. Discussion

Our findings demonstrate that intraportal hAEC transplantation leads to significant coagulation activation, likely due to TF expression on the transplanted cells. Similar challenges have been reported in islet and hepatocyte transplantation [15,16], reinforcing the need for cross-disciplinary strategies. Future studies should explore alternative anticoagulant strategies beyond heparin to minimize bleeding risks while enhancing cell survival. Additionally, while hAECs demonstrated hepatocyte-like function in vivo, further work is required to optimize differentiation protocols and improve long-term engraftment. The potential for genetic modifications or preconditioning treatments to reduce TF expression prior to transplantation could represent a viable strategy for enhancing the safety profile of hAEC therapy.

hAECs can reportedly differentiate into cells of all three germ layers, including hepatocytes [4,17]. In addition, in a previous study, when hAECs were injected under the liver capsule of mice without inducing differentiation, the transplanted cells displayed hepatocyte-like morphological changes, and the expression of genes encoding cytochromes and hepatic enzymes was observed in mature hepatocytes 6 months after transplantation [18]. Furthermore, hAECs transplanted into mouse models of maple syrup urine disease and phenylketonuria reportedly improved the disease state and were viable in the livers of mice 18 months after transplantation [19,20]. In our study, hAECs injected into the portal vein displayed positive albumin staining, and the levels of the epithelial marker hEpCAM as well as hAlb in the liver increased significantly in the hAEC-transplantation group, indicating that transportal hAEC transplantation resulted in transplanted cell engraftment in the rat liver and hepatocyte-like functional expression. We confirmed strong hEpCAM expression in the hAECs following the cell block specimen staining. We identified neither hAlb-positive cells in pre-transplant hAECs nor hAlb-positive cells in the non-transplanted rat liver specimens.

For clinical applications, determining the optimal administration route for the transplantation of each cell type is essential [2,21]. In current clinical cell transplantation therapy, hematopoietic and mesenchymal stem cells are administered intravenously [22], while islet cells and hepatocytes are administered intraportally [23,24]. In plastic and orthopedic surgery, mesenchymal stem cells are administered subcutaneously or directly into the joints [25,26]. The optimal administration route for cell transplantation is determined by the location of the transplanted cells and the risk of complications associated with each administration route. For hAEC transplant therapy, Malhotra et al. reported that hAECs could be safely administered intravenously to infants with respiratory disease in clinical settings [27]. In contrast, the preferred hAEC transplantation route for liver diseases should allow safe and efficient engraftment in the liver. In a previous study, we examined hAEC distribution after intraportal transplantation using IVIS spectrum CT and revealed that the cells accumulated only in the liver and did not migrate to the lungs or other organs after intraportal administration [7]. The ability of transplanted cells to pass through blood vessels is diameter- and size-dependent [28,29]. Although this study was conducted only on small animals, its results could be helpful for future studies on larger experimental animals and in clinical settings.

TF expression in transplanted cells reportedly triggers the activation of the coagulation system, initiating IBMIRs [13]. IBMIRs could be attenuated by islet transplantation of freshly isolated cells with low-level TF expression [30]. However, in this study, we demonstrated that TF expression levels did not differ between freshly isolated and cryopreserved hAECs, consistent with the results of a previous study in which cryopreservation did not alter antigen expression in hAECs [31]. While these findings suggest that cryopreservation preserves TF expression, the broader effects of cryopreservation on hAEC viability, proliferation, and differentiation capacity were not evaluated in this study. Future investigations should include assessments of these parameters to ensure the therapeutic functionality of cryopreserved hAECs is maintained after thawing. TF is homeostatically expressed in various organs such as the placenta, brain, heart, and kidneys. hAECs might also homeostatically express TF during healthy pregnancy [32]. After determining that the isolated hAECs expressed TF, we assessed whether IBMIRs were induced by intraportal hAEC transplantation and confirmed that the TAT level increased immediately after cell administration. IBMIRs are assumably induced upon intraportal hAEC transplantation by coagulation system activation. Nevertheless, it should be noted that the TAT level change differed from that described in previous studies. The TAT evaluation after intraportal islet transplantation demonstrated that its level increased quickly, peaking at 60 min, then gradually decreased and converged to the pre-transplantation level [9]. In contrast, in this study, the TAT level increased immediately after intraportal transplantation and remained constant, potentially affected by the xenotransplantation model beyond the transplanted cell-related differences.

This study also demonstrated that heparin, with potent anticoagulant activity, effectively suppressed the TAT level increase to nearly that of the control group, indicating attenuation of coagulation activation. However, the clinical application of heparin during intraportal cell administration raises concerns related to the risk of intraperitoneal bleeding. To address this concern and enhance clinical applicability, future studies should explore alternative anticoagulant strategies. These may include low molecular weight heparin (LMWH), which offers more predictable pharmacodynamics, or direct thrombin inhibitors such as argatroban or dabigatran, which may reduce bleeding risk while maintaining antithrombotic efficacy. Although heparin administration effectively suppressed the elevation of TAT levels, indicating inhibition of coagulation activation, in vivo imaging using IVIS did not show a marked difference in fluorescence decay between the heparin-treated and untreated groups. This discrepancy suggests that while heparin may reduce thrombo inflammatory activation, it does not necessarily enhance short-term cell engraftment detectable by IVIS. Alternatively, this may reflect limitations in IVIS sensitivity or the presence of other factors influencing early cell viability, such as immune responses or hepatic microenvironmental conditions. Regarding islet transplantation, the simultaneous suppression of multiple cascades, such as that of complement system activation and anti-cytokine therapy, has already been proposed to be necessary for controlling IBMIRs [9,33]. As hAECs express high levels of the complement regulator CD59 [6], IBMIRs in hAEC transplantation might be regulated only by suppressing coagulation system activation. Future studies should evaluate further strategies to control IBMIRs in hAEC transplantation.

Although this study combined xenotransplantation from humans to rodents, we used no immunosuppressive drugs due to the potential adverse effects and because we aimed to verify the results early after transplantation. hAECs are cells of fetal origin, characterized by weak human leukocyte antigen (HLA)-Ia/II and strong HLA-G expression to escape maternal immunity [34]. These properties make hAECs ideal candidates for immunosuppressive drug-free transplantation to avoid strong adverse effects. Subcutaneous immunosuppressive agent-free transplantation was reportedly performed without adverse effects, rejection, or tumor formation in healthy human volunteers and children with lysosomal disease [35,36]. Nevertheless, the possibility that rejection around the cells might have occurred in this study cannot be ruled out. Further verification of this point in immunodeficient model animals or models using immunosuppressive agents would be necessary.

This study has some limitations. Firstly, although hAECs engrafted efficiently in the liver, our functional evaluation relied solely on serum hAlb levels, which only partially reflect hepatocyte-like function. We also did not assess the extent of differentiation into mature hepatocyte-like cells. To more comprehensively evaluate hepatic differentiation and function, future studies should incorporate additional hepatic markers, such as cytochrome P450 enzyme expression or metabolic function assays, including urea synthesis or drug metabolism capacity. Secondly, immunohistological analysis revealed that the number of hAlb-positive cells was significantly lower than that of hEpCAM-positive cells. This discrepancy may be due to differences in staining sensitivity, which warrants further validation through comparative immunostaining studies. In addition, it may reflect that a subset of transplanted hAECs had not fully differentiated into hepatocyte-like cells capable of albumin production. The number of engrafted cells that express and retain hepatocyte-like functions is directly related to the therapeutic efficacy of cell transplantation and thus represents an important topic for future studies. Thirdly, this study involved xenogeneic transplantation of human cells into rats without the use of immunosuppressive agents or immunodeficient models. Xenogeneic antigens might have affected cell viability or contributed to immune rejection. The observed TAT level profile also differed from previous reports, potentially reflecting the influence of xenogeneic immune responses. Likewise, the serum albumin level, which typically increases steadily after hepatocyte transplantation, instead rose and then declined slowly in our model, suggesting the possibility of prolonged IBMIR and xenograft rejection. Fourthly, while we used TAT levels as a surrogate marker for coagulation activation during IBMIR, we did not evaluate other key components of the IBMIR cascade, such as complement activation or inflammatory cytokine production. A more comprehensive assessment in future studies should include markers like C3a, C5a, IL-6, TNF-α, and IFN-γ, as well as histological analysis of immune cell infiltration at the graft site. Finally, although previous studies have reported favorable outcomes of hAEC transplantation without immunodeficient models or immunosuppressive agents [37], it remains necessary to confirm long-term engraftment and immune tolerance. Future research should consider using humanized mouse models or appropriate immunosuppressive regimens to evaluate cell viability, function, and immune interaction in a more clinically relevant setting. To that end, incorporating immunohistochemical analyses (e.g., CD3, F4/80), cytokine profiling, and assessment of MHC class II and cell death markers will be essential for understanding immune-mediated graft injury.

This study is the first to demonstrate that hAECs, when transplanted intraportally into a rat model, induce a significant coagulation response via TF expression, contributing to IBMIR. While TF-mediated IBMIR has been previously characterized in islet and hepatocyte transplantation, its impact in hAEC-based therapies has not been systematically investigated. These findings highlight a critical barrier to clinical application and suggest that safety strategies – such as anticoagulant modulation – must be optimized before hAEC therapies can be translated into hepatic indications. Importantly, our results also have broader implications for the translational advancement of hAEC-based therapies. Specifically, they underscore the unique advantages of hAECs as readily available, immunomodulatory cell sources for regenerative medicine, and emphasize the need for approaches that enhance engraftment while mitigating thrombo inflammatory responses [38]. By addressing these challenges, future research can help accelerate the clinical translation of hAEC transplantation toward effective and safe treatments for liver diseases.

5. Conclusions

Our findings provide key insights into the early thromboinflammatory responses triggered by hAECs during intraportal transplantation, which constitute major barriers to safe and effective hepatic cell therapy. By identifying tissue factor expression as a major contributor to coagulation activation, and demonstrating that heparin administration effectively suppresses this response, this study provides a rationale for developing safer hAEC-based therapies.

These results contribute to the preclinical foundation required for advancing hAEC transplantation toward clinical translation. Future studies should further evaluate immune responses, long-term engraftment, and functional integration in immunocompetent models. Additionally, the development of modified hAECs with reduced thrombogenic potential or the use of alternative anticoagulants may offer safer transplantation protocols. Collectively, these findings support the further development of hAECs as a promising and clinically translatable cell therapy for congenital liver disorders and inherited metabolic diseases.

Funding Statement

This work was supported by the Clinical Research Promotion Program for Young Investigators of Tohoku University Hospital, Kawano Masanori Memorial Public Interest Incorporated Foundation for Promotion of Pediatrics, The Japan Foundation for Pediatric Research, Public Trust Surgery Research Fund, The Japan Society for Organ Preservation and Medical Biology, TERUMO LIFE SCIENCE FOUNDATION, and Takeda Science Foundation. The funders had no role in the design of the study; in the collection, analysis, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Article highlights

• Human amniotic epithelial cells (hAECs) were evaluated for intraportal transplantation in a rat model, focusing on engraftment, functional performance, and innate immune response.

• hAECs expressed tissue factor (TF) both before and after cryopreservation, which was associated with significant activation of the coagulation cascade following transplantation.

• Post-transplantation analysis showed a marked increase in thrombin – antithrombin (TAT) complexes, suggesting the induction of an instant blood-mediated inflammatory reaction (IBMIR).

• Heparin administration attenuated TAT elevation but did not enhance long-term cell persistence, indicating the need for alternative strategies to mitigate IBMIR while preserving engraftment.

• Immunohistochemical staining confirmed engraftment of hAECs in the rat liver and partial hepatocyte-like function, demonstrated by human albumin production and hEpCAM expression.

• Human albumin levels increased transiently after transplantation but declined over time, raising the possibility of immune-mediated clearance and highlighting the importance of evaluating immune responses.

• The use of Nagase analbuminemic rats enabled detection of human albumin without background interference, enhancing functional assessment sensitivity.

• The study proposes future directions including the use of humanized animal models, alternative anticoagulants, and further analysis of complement activation and immune infiltration.

• These findings identify a key translational barrier – coagulation and innate immune activation – relevant to the clinical application of hAEC-based therapies for liver diseases.

• Overall, the study contributes valuable insight toward developing safer and more effective protocols for hAEC transplantation in regenerative medicine.

Author contributions

Miyako Tanaka: Investigation, Data curation, Formal analysis, Writing – original draft.

Kazuaki Tokodai: Conceptualization, Methodology, Supervision, Project administration, Writing – review & editing, Corresponding author.

Kaoru Okada: Investigation, Formal analysis.

Masato Sato: Investigation, Resources.

Hitomi Okita: Investigation, Validation.

Takako Ito: Investigation, Data curation.

Tetsuro Hoshiai: Data interpretation, Writing – review & editing.

Masatoshi Saito: Data interpretation, Writing – review & editing.

Toshio Miki: Conceptualization, Supervision, Writing – review & editing.

Michiaki Unno: Conceptualization, Supervision, Writing – review & editing.

Takashi Kamei: Conceptualization, Writing – review & editing.

Masafumi Goto: Supervision, Writing – review & editing.

All authors reviewed and approved the final version of the manuscript and meet the ICMJE criteria for authorship.

Disclosure statement

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Reviewer disclosure

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Ethical declaration

This study was conducted in accordance with the principles of the Declaration of Helsinki and approved by the Institutional Review Board of Tohoku University (approval number: 2018–1–203). Written informed consent was obtained in advance from all participating donors for the collection and use of amniotic membranes. Animal experiments were performed in compliance with the ARRIVE guidelines and approved by the Animal Care and Use Committee of Tohoku University (approval number: 2019–191).

Data availability statement

The datasets generated and analyzed in the current study are available from the corresponding author on reasonable request.