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. 2022 Oct 20;87(6):333–343. doi: 10.1159/000527514

Effect of Human Amniotic Epithelial Stem Cell Transplantation on Preterm Premature Rupture of Fetal Membrane Using the Amniotic Pore Culture Technique in vitro

Dahyeon Kang a, Min Jung Kang b, Deqi Kong a, Jae-eun Lee a, Ah-young Lee a, Dong Ho Geum a, Byung Soo Kim c, Yun Seop Kim a, Soon-Cheol Hong a,b,c,*
PMCID: PMC9909721  PMID: 36265471

Abstract

Objectives

The objective of this study was to evaluate the efficacy of cell therapy using human amniotic epithelial stem cells (hAESCs) for the treatment of premature rupture of membranes (PROM) in vitro.

Design

Using the amniotic pore culture technique (APCT), we mimicked the environment of PROM in vitro, thus enabling the observation of the healing process of hAESC-treated amniotic membranes.

Materials

Amniotic membrane samples were collected from placentas of pregnant women who underwent elective cesarean sections. APCT model and isolated hAESCs were used in this study. All patients who participated in this study provided their written informed consent prior to the commencement of the study.

Settings

To create the APCT model in vitro, isolated amniotic membranes were punched to create 5 mm diameter circles and re-punched to form a 1-mm pore at the center. Membranes were cultured in α-minimal essential medium, and the hAESCs were collected and cultured as well. Subsequently, the APCT models were divided into two groups: hAESC treated and control.

Methods

Within the culture period, pore sizes were calculated to evaluate the degree of tissue regeneration in both groups. We then evaluated the histology, cell density, and epithelial thickness of the regenerated tissues. Statistical analyses were performed using SPSS software ver. 20.0 (IBM, Armonk, NY, USA) with repeated-measures one-way analysis of variance or paired samples t test. The significance level was set at p < 0.05.

Results

As per the evaluation of the APCT model in vitro, the pore size in the hAESC-treated group reduced by 62.2% on day 6 (62.2 ± 0.19, n = 24), whereas in the control group, it shrank by only 36.8% (p < 0.05) (36.8 ± 0.19, n = 24). Furthermore, the epithelial thickness in the amniotic epithelial stem cell-treated group (10.08 ± 1.26 μm, n = 8) was significantly higher than that in the control group (5.87 ± 0.94 μm, n = 8). Cell density in the regenerated tissue in the amniotic epithelial stem cell-treated group (57 ± 2.77, n = 8) was significantly higher than that in the control group (49 ± 2.23, n = 8).

Limitations

In this study, we did not explore the molecular mechanisms by which hAESCs participate in membrane healing in the APCT model. Although our results showed a significant difference, this difference was not too obvious. Therefore, further research on the mechanisms of hAESCs is needed, with more amniotic tissues and APCT samples being tested.

Conclusions

We developed an APCT model to investigate the PROM conditions in vitro. By implanting donor hAESCs in the pores of the APCT model, we observed that hAESCs seeding accelerated pore healing in vitro. Thus, hAESCs may be a valuable source of cells for cell therapies in regenerative medicine.

Keywords: Amniotic membrane, Cell therapy, Premature rupture of membranes, Stem cells, Amniotic pore culture technique

Introduction

Over the past few decades, serious efforts have been made to improve the prognosis of preterm births [1]. Preterm births account for 75% of cases of perinatal mortality and more than half of long-term morbidity, and approximately 25–30% of preterm births occur after preterm premature rupture of membranes (PROM) [2, 3]. Preterm PROM is a condition that affects the prognosis of infants as the amniotic membrane ruptures before the completion of 37 gestational weeks and the onset of labor [4].

The risk of maternal infection and fetal prematurity depends on the gestational age of the membrane at the time of rupture, and the management decisions depend on this balance [4]. Therefore, the optimal approach for the evaluation and treatment of women with preterm PROM should be individualized depending on the gestational age and relative risks associated with prompt delivery versus risks of expectant management [4, 5, 6]. In cases where there is no indication for immediate delivery, particularly those in the case of amniotic membrane rupture before 34 gestational weeks, an expectant management method is recommended [7, 8, 9, 10, 11]. These strategies include the use of tocolytics to delay the delivery and administration of corticosteroids to promote fetal lung maturation [1, 4]. However, if the leakage site of the amniotic membrane is completely closed, it can be an effective treatment option to prevent the further deterioration of neonatal prognosis by avoiding intrauterine cord compression due to insufficient amniotic fluid and by preventing the ascending infection.

In the clinical field, spontaneous healing of amniotic fluid is often observed after amniocentesis or the initial rupture of amniotic membranes [12]. Spontaneous resealing of amniotic membranes has been observed in up to 5% of preterm PROM [13], and in such cases, a small puncture in amniotic membranes caused by amniocentesis generally repairs itself spontaneously [14].

Based on these facts, we created a model of ruptured fetal membranes in vitro. We found that the amniotic membrane heals spontaneously [15]. In a previous natural healing study of the amnion, it was found that the degree of healing differs according to the pore size; that is, the smaller the pore size, the higher the healing rate and the larger the pore size, the more limited the healing rate of amniotic membranes [15, 16]. Amniotic epithelial stem cells derived from the amnion may be considered key factors in the spontaneous healing of damaged amniotic membranes [15, 16, 17, 18, 19]. Although the mechanism by which human amniotic epithelial stem cells (hAESCs) are involved in the healing of amniotic membranes is yet to be clarified, previous studies have suggested the possibility of using cell therapy for preterm PROM treatment, which led to the conception of this study. For the present study, we carried out a transplantation study of hAESCs from other donors' placenta to assess their potential in healing PROM by cell therapy, using the amniotic pore culture technique (APCT) model.

Materials and Methods

Isolation and Culture of hAESCs

Amniotic membranes were obtained from the placentas of pregnant women who underwent elective cesarean sections. The amniotic membranes were washed several times with phosphate-buffered saline (PBS; Biosesang, Seongnam, Korea) to remove all the blood [19]. To isolate hAESCs, amniotic membranes underwent digestion twice with 0.05% trypsin-ethylenediaminetetraacetic acid (Invitrogen, CA, USA) for 30 min at 37°C. Isolated hAESCs were cultured in 100 mm diameter cell culture dishes with α-minimal essential medium (α-MEM; Welgene, Daegu, Korea) supplemented with 10% fetal bovine serum (FBS; Welgene), 1% penicillin-streptomycin (P/S; Welgene), and 10 ng/mL epidermal growth factor (EGF; R&D Systems, Minneapolis, MN, USA) [16, 20]. All the procedures described below were performed under sterile conditions (Fig. 1a). This study was approved by the Institutional Review Board of the Korea University Anam Hospital, Seoul, Korea (no. 2009AN0036). All patients who participated in this study provided their written informed consent prior to the commencement of the study.

Fig. 1.

Fig. 1

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Isolation of hAESCs and the APCT model. a Human amniotic membranes were obtained from discarded placentas and hAESCs were isolated. b Explanation of the in vitro APCT model.

Flow Cytometry

For the evaluation of the hAESCs, these cells were cultured in basic media (α-MEM, 10% FBS, 1% P/S, and 10 ng/mL EGF) until 80% confluence was achieved. hAESCs were harvested using 0.05% trypsin-ethylenediaminetetraacetic acid (Life Technologies Corporation, NY, USA) and washed with 1× PBS. Isolated hAESCs were fixed with 4% paraformaldehyde for 10 min and washed three times with PBS. The experimental protocols were learned from the previous papers [20, 21, 22]. The cells were then incubated with antibodies against cluster of differentiation 19 (CD19), CD34, CD73, CD90 (Thy1), CD105 (endoglin), CD324 (E-cadherin), stage-specific embryonic antigen 4 (SSEA4), octamer-binding transcription factor 3/4 (Oct3/4), SRY-box transcription factor 2 (Sox2), Nanog (BD Biosciences, San Jose, CA, USA), and human leukocyte antigen (HLA) class I or II (Ancell, MN, USA) for 40 min on ice after a blocking procedure. Next, the cells were then analyzed with a BD FACS CaliburTM flow cytometer (BD FACSCalibur, BD Biosciences) using CellQuest Pro software (BD Biosciences).

Immunocytochemistry

hAESCs cultured on gelatin-coated coverslips were rinsed thrice with PBS and once with washing buffer. The cells were then fixed in 4% paraformaldehyde for 10 min at room temperature. The samples were incubated with primary antibodies against SSEA4 (1:500; R&D), Oct3/4 (1:500; R&D), the mannose receptor (1:500; Abcam, San Francisco, CA, USA), microtubule-associated protein 2 (MAP2; 1:500; Invitrogen), nestin (Nes; 1:500; Millipore), alpha-fetoprotein (AFP; 1:500; Millipore), and perilipin A (Plin1; 1:300; Abcam) at 4°C for 1 day after blocking. The cells were rinsed twice with washing buffer and incubated with the appropriate secondary antibodies: mouse immunoglobulin G (IgG) (1:1,000; Invitrogen), rabbit IgG (1:1,000; Invitrogen), and rat IgG (1:1,000; Abcam) at room temperature for 1 h. Cells were then rinsed with washing buffer, and nuclei were stained with 4′,6-diamidino-2-phenylindole (1:1,000; Life Technologies) for 5 min and washed thrice. One drop of mounting medium was used to mount the coverslips onto the slides for permanent preservation.

In vitro Cell Differentiation

Cell differentiation was induced using the following culture conditions and methods: the isolated hAESCs (2 × 104 cells/cm2) were cultured on gelatin-coated coverslips in 24-well plates with basic medium supplemented with 10% FBS and 1% P/S for 24 h. Cultured cells were induced to differentiate into 3 cell types representing the three germ layer lineages: neurons (ectoderm), adipocytes (mesoderm), and hepatocytes (endoderm).

Ectodermal Lineage

To induce differentiation into neurons, hAESCs (2 × 104 cells/cm2) were cultured for 1 day in basic media and then switched to a neuronal differentiation medium (containing 50 μM trans-retinoic acid, 20 ng/mL basic fibroblast growth factor [bFGF], B-27 [1:100], N-2 [1:200], and 1% P/S in Dulbecco's minimal essential medium/Ham's F12). After 15–20 days, the presence of differentiated neuronal cells was visualized by immunocytochemical staining for nestin and MAP2.

Endodermal Lineage

To induce differentiation into hepatocytes, hAESCs (2 × 104 cells/cm2) were cultured in basic media and then switched to a hepatocyte differentiation medium (containing 10% FBS, 1% P/S, 10 ng/mL EGF, 10 ng/mL fibroblast growth factor 4 [FGF4], 1× isopropylthioxanthone, 1 μM dexamethasone, 20 ng/mL oncostatin M [OSM], and 20 ng/mL hepatocyte growth factor [HGF] in Iscove's modified Dulbecco's medium). After 15–20 days, differentiated hepatocytes were visualized by immunocytochemical staining for AFP.

Mesodermal Lineage

To induce differentiation into adipocytes, hAESCs (2 × 104 cells/cm2) were cultured for 1 day in basic media and then switched over to an adipocyte differentiation medium (0.5 mm 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, 1 μM IDMT, and 10 μm/mL insulin in α-MEM with 10% FBS and 1% P/S). After 15–20 days, differentiated adipocytes were visualized by immunocytochemical staining for perilipin A and the mannose receptor.

APCT Model

APCT Model in vitro Culture

Freshly isolated amniotic membranes were collected from 8 patients who provided written informed consent. The amniotic membranes were washed twice with cold PBS. The membranes were punched using a 5-mm disposable biopsy punch (Kai Medical, Japan) on a glass dish with PBS. Next, the punched amniotic tissues were re-punched with a 1-mm biopsy punch to form a pore. Amniotic tissues with 1-mm pores were attached to a 24-well plate and cultured in α-MEM, with 10% heat-inactivated FBS, 1% P/S, and 10 ng/mL EGF, as a standard culture medium.

Seeding of hAESCs in the APCT Model

Prior to seeding the hAESCs in the APCT model, the cultured amniotic membranes were stabilized for 24 h in a humidified incubator at 37°C, supplied with 5% CO2. APCT models were divided into two groups: hAESC-treated group and a control group without treatment with hAESCs. Each group consisted of three replicates of APCT models from 8 patients, which was 24 samples in total (n = 24).

Isolated hAESCs were sub-cultured in passage 2 and resuspended in α-MEM culture medium. Cell suspensions of 10 μL were applied to the 1-mm pore of the amniotic membrane (APCT model) at a concentration of 6 × 103 cells/cm2 in each well. In contrast, 10 μL of α-MEM culture medium without hAESCs was applied to the 1-mm pore of the amniotic membrane tissues in the control group. The medium was changed every 2 days during the culture period.

Pore Size Capture and Calculation

The pore sizes were calculated to evaluate the degree of tissue regeneration. The pore size was observed and measured using an optical microscope (Olympus) and TOMORO AcquCAM3 program. Pore images were analyzed using Image J.

Pre-Labeling of hAESCs

Isolated hAESCs used for cell therapy in the experimental group were labeled with the CFDA-SE fluorescent dye (Invitrogen). The hAESCs were stabilized in media (10% FBS and 1% P/S in α-MEM) before hAESC treatment.

Histological Analysis

The tissues used in the APCT model as mentioned previously were washed and fixed in 4% formaldehyde for 10 min at room temperature. After fixation, the tissues were washed with PBS. To prepare paraffin blocks, the tissues were incubated in xylene and a graded ethanol series (70%, 80%, 95%, and 100% EtOH) on a shaker at room temperature and then incubated in melted paraffin for 1 day. Tissue sections (2–3 μm thick) were incubated for 40 min in a 60°C dry oven and then deparaffinized and rehydrated. The sections were stained with hematoxylin and eosin, dehydrated, and mounted. The stained tissues were viewed at ×100 and ×200 magnification.

Statistics Analyses

All data are expressed as mean ± standard error. Statistical analyses were performed using SPSS software ver. 20.0 (IBM, Armonk) with repeated-measures one-way analysis of variance or paired samples t test. The significance level was set at p < 0.05.

Results

Characteristics and Morphology of hAESCs

The hAESCs are adherent cells that exhibit a round, spindle morphology with a large cytoplasm surrounding the nucleus. The cells maintained a single cell type and did not form colonies (Fig. 2a). Flow cytometry results showed that hAESCs partially expressed epithelial (CD324), somatic (CD90 and CD105), and embryonic (Oct3/4, Sox2, Nanog, and SSEA4) stem cell markers as well as MHC class I (HLA-ABC) antigens (Fig. 2c). However, hAESCs did not express hematopoietic stem cell markers (CD19 and CD34) or MHC class II (HLA-DR) antigens (Fig. 2b).

Fig. 2.

Fig. 2

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Characteristics of hAESCs. a Morphology of hAESCs at passage 2. b Flow cytometric analysis of hAESCs. Black color: control (unstained cells); orange color: marker (stained cells). c Percentage of expressed markers on hAESCs detected using flow cytometry. Data are shown as the mean ± SD from three independent experiments.

Flow cytometric analysis revealed the presence of pluripotency markers in the immunocytochemical staining of the isolated hAESCs. The hAESCs were incubated with antibodies against stem cell markers (Oct3/4 and SSEA4). As shown in Figure 3a, the pluripotency markers SSEA4 and OCT3/4 were expressed in the majority of the isolated hAESCs.

Fig. 3.

Fig. 3

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Pluripotency of hAESCs. a hAESCs showing positive staining for SSEA4 (green) and Oct3/4 (red) are indicated. Nuclei were counterstained with DAPI (blue). Scale bar, 25 μm. b Potential of hAESCs to be differentiated into cells of the three germ layers. Upper: potential of neuronal differentiation (nestin, MAP2, and GFAP staining), middle: potential of adipocyte differentiation (perilipin A and mannose receptor staining), and bottom: potential of hepatocyte differentiation (AFP, alpha-fetoprotein staining). Scale bar, 25 μm. DAPI, 4′,6-diamidino-2-phenylindole.

In addition, hAESCs were tested for their potential to differentiate into the representative cells of the three germ lineages. hAESCs were cultured in the differentiation medium for 20 days (Fig. 3b). Consequently, in hAESCs cultured in neuronal differentiation medium, we detected the expression of nestin, MAP2, and GFAP, indicating the potential of hAESCs to differentiate into neurons (ectoderm). Similarly, the expression of AFP protein indicated the potential of hAESCs to differentiate into hepatocytes (endoderm). We also observed the presence of small lipid vacuoles and the expression of perilipin A and mannose receptors in hAESCs after culture in adipocyte differentiation medium for 20 days. In summary, hAESCs have the potential to differentiate into representative cells of the three germ lineages.

hAESCs Accelerate Pore Healing in APCT Model in vitro

It is not only difficult to evaluate the potential of hAESCs as a source of cells for cell therapy in vivo, but there are also no appropriate models for evaluating their use in vitro. Thus, we used the APCT model established in our laboratory to reproduce the PROM in vitro (Fig. 1b). As per the results of our previous study, this APCT model has the potential advantage of enabling the examination of natural healing and effectiveness of hAESCs for cell therapy in vitro [15].

Freshly isolated amniotic membranes were collected from 8 patients who provided written informed consent. These amniotic membranes were used to create APCT models with 1-mm pore in the center. We then divided the APCT models into two groups: the hAESC-treated group and the untreated control group. Each group consisted of three replicates, with eight samples in each replicate (n = 24).

Prior to employing the hAESCs in the APCT in vitro culture model, the cultured tissues were stabilized for 24 h at 37°C with 5% CO2. Isolated hAESCs were sub-cultured in passage 2 and resuspended in α-MEM culture medium. Then, 10 μL cell suspensions were applied to the 1-mm pore of the amniotic membrane (APCT model) at a concentration of 6 × 103 cells/cm2 in each well. In contrast, 10 μL of α-MEM culture medium without hAESCs was applied to the 1-mm pore of the amniotic membrane tissues in the control group. The medium was changed every 2 days during the 6-day culture period. The pore sizes of the two groups were measured and calculated to evaluate the degree of tissue regeneration.

As shown in Figure 4a, the pores in the hAESC-treated group recovered faster than those in the control group. As shown in Figure 4b, after 6 days of culture, the average pore size of the control group reduced by 36.8% (36.8 ± 0.19, n = 24). However, the average pore size of the hAESC-treated group reduced by 62.2% (62.2 ± 0.19, n = 24). The differences in healing between the two groups on day 6 were statistically significant (p < 0.05).

Fig. 4.

Fig. 4

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Tissue culture of hAESC-treated and control (hAESC untreated) tissues in the APCT model. Pore size-dependent regeneration of amniotic tissue. Pore size was measured for 6 days using ImageJ. a The pore sizes in the APCT model were compared between the hAESC-treated and control groups. Left: day 0 (after cell treatment); middle: day 4; and right: day 6. Scale bar, 250 μm. b The APCT model pore size reduced 62.2% (62.2 ± 0.19, n = 24) in the hAESC-treated group, which is more than that in the control group 36.8% (36.8 ± 0.19, n = 24). The result is reported as the percentage of pore size compared to that on day 0 and was analyzed by repeated-measures ANOVA. * denotes p < 0.05 (n = 24). ANOVA, analysis of variance.

Characteristics of Regeneration Tissues in the Pores of the APCT Model

In addition, we observed that the pores recovered within an average of 10 days, and most of the samples in both groups completely recovered. To investigate whether hAESCs were localized to the pores during recovery, hAESCs were pre-labeled with CFDA-SE (Fig. 5a) and seeded in the APCT model as described previously. At 10 days post-culture, the applied hAESCs were detected in the fully recovered tissues (Fig. 5b). These results showed that seeded hAESCs could localize to the pore area and participate in the healing process and regeneration of new tissues.

Fig. 5.

Fig. 5

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Pre-labeling of hAESCs by CFDA-SE in the hAESC-treated group in the APCT model. a hAESCs were detected and dispersed on fully recovered tissues. Scale bar, 100 μm. b At 10 days post-culture, the cells were detected mainly at the healed pore site and dispersed on fully recovered tissues. These findings indicate that CFDA-SE-labeled hAESCs can be localized to the injured area and can be quantified by their viability and proliferation in the regenerated tissue. Scale bar, 100 μm.

To evaluate the effectiveness of hAESCs in promoting tissue regeneration, we first examined the histology of the recovered tissues after 10 days of culture. As shown in Figure 6a, the hAESC-treated group exhibited better epithelialization than the control group.

Fig. 6.

Fig. 6

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Histological features after wound repair. a Regenerated amniotic tissues were stained with hematoxylin and eosin. Scale bar, 25 μm. b Analysis of cell density and epithelium thickness after recovery. * denotes p < 0.05, ** denotes p < 0.01, paired t test. Cell counts (number): normal (63 ± 2.32, n = 8), control (49 ± 2.23, n = 8), and hAESC-treated (57 ± 2.77, n = 8). Epithelium thickness (μm): normal (17.3 ± 0.51, n = 8), control (5.87 ± 0.94, n = 8), and hAESC treated (10.08 ± 1.26, n = 8).

Second, we measured the cell density and epithelium thickness in the tissues on day 10. During sectioning, the recovered amniotic membrane was cut vertically into 1-mm sections. We then counted the number of cells in the recovered tissue as the cell density. Normal amniotic tissue membranes without punching were used as positive controls.

The number of cells per mm length of regenerated tissue was 63 ± 2.32 (n = 8), 49 ± 2.23 (n = 8), and 57 ± 2.77 (n = 8) in the normal tissue (no punching), control group, and hAESC-treated group, respectively. As shown in Figure 6b, the hAESC-treated group showed a significantly higher cell density (57 ± 2.77, n = 8) than the control group (49 ± 2.32, n = 8). There was no statistically significant difference between the normal tissue and hAESC-treated groups. However, the epithelial thickness in the normal tissue group was 17.3 ± 0.51 μm (n = 8), which is significantly higher than that in the control group (5.87 ± 0.94 μm, n = 8) and the hAESC-treated group (10.08 ± 1.26 μm, n = 8) (Fig. 6b). In addition, we observed a higher epithelial thickness in the hAESC-treated group than in the control group.

Discussion

More than half of women with preterm PROM give birth within 1 week after the rupture of membranes [12]. Preterm PROM complicates pregnancy in 1–2% of women and is associated with preterm births in 30–40% of women [3, 7]. Infants born after preterm PROM are premature and face many problems, including respiratory distress, sepsis, intraventricular hemorrhage, necrotizing enterocolitis, and neurodevelopmental impairment [23, 24, 25].

The American College of Obstetricians and Gynecologists (2016) recommended expectant management without indication for immediate delivery at 24 0/7 to 33 6/7 weeks of gestation [26, 27]. As per the latest data, there may still be controversy over immediate delivery compared with expectant management before 34 weeks of gestation, but continuing the pregnancy without contraindication, a policy of expectant management with careful monitoring was associated with better outcomes for neonates [7, 11, 26].

Accordingly, there have been various discussions on ways to prolong pregnancy. Several studies have been conducted on implants or various sealants, such as dry collagen and gelatin plugs or liquid blood-derived sealants and intra-amniotic injection of maternal platelets mixed with fibrin frozen precipitates, to minimize or stop amniotic fluid leakage at the puncture site [26, 27, 28, 29, 30].

Apart from these sealants, stem cell potentiality has also been explored in PROM treatment. MacNeil et al. [31] suggested the possibility of minimizing amniotic fluid leakage by transplanting implantable membranes containing polyurethane and adipose-derived stem cells. In our previous study, we demonstrated the possibility of regeneration of hAESCs in the process of spontaneous healing of amniotic membranes in vitro [16]. Based on these possibilities, we illustrated the effect of donor hAESCs transplantation as a cell therapy in the preterm PROM using the APCT model.

The hAESCs originate from the amniotic membrane of the human placenta, which has immune neutrality, making it a relatively safe source of cells for cell therapy. Under specific culture conditions, hAESCs can differentiate into cells of all the three germ layers, and they do not form teratocarcinomas [32]. hAESCs can also regulate local immune responses [33]. In addition, hAESCs exhibit anti-inflammatory, antibacterial, and antiviral activities; participate in anti-angiogenic functions; promote epithelialization; and are non-tumorigenic [34]. In this study, hAESCs from donors were transplanted into the recipient APCT model and presented with low immunogenicity. We also found that hAESCs grew rapidly and had stem cell properties with the capability to differentiate into endodermal, mesodermal, and ectodermal lineages in vitro, which is consistent with the findings of previous studies [35, 36]. Based on these cell characterizations, hAESCs may have pluripotent properties and low immunogenicity. These properties make hAESCs an attractive option for use in cell therapy.

In this study, the hAESC-treated group recovered more quickly and completely than the control group, and it appeared that the hAESCs themselves supported this enhanced tissue regeneration. We also tested whether hAESCs could be detected in the recovered tissue using hAESCs pre-labeled with CFDA-SE. Importantly, labeled hAESCs were detected at the site of injury in the recovered tissue. Thus, the localization of hAESCs in recovered tissues strongly suggests that hAESCs survive in damaged tissues and contribute to healing. It can be assumed that the process by which hAESCs repair damaged tissues is owing to two mechanisms reported in cell-based therapeutic approaches: cell-to-cell and paracrine interactions [37, 38].

In response to tissue damage, amniotic stem cells can release paracrine factors that enhance cell survival and activate the endogenous system of recovery [39, 40]. Several previous studies have shown that hAESCs are capable of releasing cytokines, such as IL-6 and IL-10, with anti-inflammatory and immunomodulatory effects [37, 38], and they are also capable of releasing growth factors, such as epidermal growth factor, basic fibroblast growth factor, and transforming growth factors, to promote wound healing [41, 42, 43]. In addition, paracrine mediators can affect the biology of the adjacent parenchymal and stromal cells under different conditions. In addition to paracrine interactions, cell-to-cell interactions have also been reported to be involved in healing of damaged tissue: Qi showed that exosomal miR-135a, which was derived from human amniotic mesenchymal stem cells, promotes cutaneous fibroblast migration by directly inhibiting LAST2 (large tumor suppressor 2) expression [44].

Although many innovative studies have been carried out in stem cell research, it remains difficult to demonstrate amniotic membrane recovery in vivo and in vitro. The APCT model enabled a more precise and quantitative evaluation of the efficacy of hEASC therapy in PROM in vitro.

Altogether, the results of the present study suggest that hAESCs could be used as a promising source for cell therapy, as suggested by our research team. Therefore, the application of hAESCs in an in vivo animal model, to evaluate their potential as a source of cell therapy, needs to be conducted in the future.

This study has certain limitations. First, amniotic membranes from 8 patients with multiple replicates were collected to build the APCT model. Using these samples, we discovered that the pore healing size, cell density, and epithelial thickness in regenerated amniotic tissue changed significantly. However, the small sample size may be the reason that the observations of this study did not vary dramatically. To better verify the conclusion of this study, experiments using a larger number of APCT samples should be conducted in the future. Second, we did not explore the molecular mechanism by which hAESCs participate in membrane healing in the APCT model. Therefore, further research on the mechanisms of hAESCs is needed. Third, preceding this study, we conducted experiments at various concentrations to determine the concentration of hAESCs. When the concentration increased, the regeneration rate was extremely high. The experiment was performed at a median concentration that allowed good observation of regeneration over time. Further research is needed to determine the optimal concentration for cell therapy in vivo and in vitro. Fourth, it is necessary to compare the tensile strength between the undamaged natural amniotic membrane and the tissue repaired by hAESC treatment.

Statement of Ethics

This study was approved by the Institutional Review Board of the Korea University Anam Hospital, Seoul, Korea (No. 2009AN0036). Written informed consent was obtained from all subjects involved in the study.

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

This work was supported by the Korea University Future Research Grant (K1619411) from Korea University Anam Hospital, Seoul, Republic of Korea (K2014101) and by the Institute of Information and Communications Technology, Planning and Evaluation (IITP) grant funded by the Korean Government (MSIT) (2020-0-00423).

Author Contributions

Conceptualization: Soon-Choel Hong, Dahyeon Kang, Min Jung Kang, and Byung-Soo Kim; experimental performance: Dahyeon Kang, Min Jung Kang, and Dong Ho Geum; data analysis: Jar-Eun Lee, Deqi Kong, Ah-Young Lee, and Yun Seop Kim; original draft preparation: Dahyeon Kang and Min Jung Kang; manuscript revision: Min Jung Kang, Deqi Kong, and Soon-Cheol Hong; and funding acquisition: Soon-Cheol Hong. All the authors have read and agreed to the final version of the manuscript.

Data Availability Statement

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.

Funding Statement

This work was supported by the Korea University Future Research Grant (K1619411) from Korea University Anam Hospital, Seoul, Republic of Korea (K2014101) and by the Institute of Information and Communications Technology, Planning and Evaluation (IITP) grant funded by the Korean Government (MSIT) (2020-0-00423).

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