Human Umbilical Cord Mesenchymal Stem Cells Encapsulated with Pluronic F-127 Enhance the Regeneration and Angiogenesis of Thin Endometrium in Rat via Local IL-1β Stimulation

Thin endometrium (< 7 mm) could cause low clinical pregnancy, reduced live birth, increased spontaneous abortion, and decreased birth weight. However, the treatments for thin endometrium have not been well developed. In this study, we aim to determine the role of Pluronic F-127 (PF-127) encapsulation of human umbilical cord mesenchymal stem cells (hUC-MSCs) in the regeneration of thin endometrium and its underlying mechanism. Thin endometrium rat model was created by infusion of 95% ethanol. Thin endometrium modeled rat uterus were treated with saline, hUC-MSCs, PF-127, or hUC-MSCs plus PF-127 separately. Regenerated rat uterus was measured for gene expression levels of angiogenesis factors and histological morphology. Angiogenesis capacity of interleukin-1 beta (IL-1β)-primed hUC-MSCs was monitored via quantitative polymerase chain reaction (q-PCR), Luminex assay, and tube formation assay. Decreased endometrium thickness and gland number and increased inflammatory factor IL-1β were achieved in the thin endometrium rat model. Embedding of hUC-MSCs with PF-127 could prolong the hUC-MSCs retaining, which could further enhance endometrium thickness and gland number in the thin endometrium rat model via increasing angiogenesis capacity. Conditional medium derived from IL-1β-primed hUC-MSCs increased the concentration of angiogenesis factors (basic fibroblast growth factor (bFGF), vascular endothelial growth factors (VEGF), and hepatocyte growth factor (HGF)). Improvement in the thickness, number of glands, and newly generated blood vessels could be achieved by uterus endometrium treatment with PF-127 and hUC-MSCs transplantation. Local IL-1β stimulation-primed hUC-MSCs promoted the release of angiogenesis factors and may play a vital role on thin endometrium regeneration.


Introduction
Thin endometrium is one of the main reasons for decreased clinical pregnancy, increased ectopic pregnancy rates, increased spontaneous abortion, and reduced live birth weight during application of assisted reproductive technol-ogy (ART) [1][2][3]. Thin endometrium is defined as endometrium thickness less than 7 mm [4]. The incidence of patients diagnosed as thin endometrium is from 2.4% to 8.5% [5,6]. Endometrium thickness was strongly associated with success of fertility. Many treatments such as "hormonal," "vascular," and "growth factor" have been attempted to increase the endometrium thickness [7]. However, until now none of these has been proven effective. Thus, improving endometrium thickness to increase clinical pregnancy and live birth for infertile couples is still a challenge for the clinicians.
Mesenchymal stem cells (MSCs) are widely used in repairing damaged tissues and achieving regeneration by promoting local capillary angiogenesis, inhibiting fibrosis, and executing immunomodulatory functions [8]. MSCs can be isolated from various tissues such as adipose tissues, bone marrow, umbilical cord, and placenta. Human umbilical cord is one of most economic and easily collectable sources to isolate MSCs, which is usually the medical waste after delivery [9]. HUC-MSCs are characterized by their highly proliferative capability, low immunogenicity, and in particular angiogenesis, which are clinically utilized for tissue repairing, and treatments of degenerative and inflammatory diseases with intravenous administration or local incubation [10]. More than 90% of MSCs were retained in the liver, lungs, and spleen rather than other organs through intravenous administration [11]. Limited numbers and poorly survived MSCs would reside in the uterus with direct cell infusion into uterus cavity. Therefore, biodegradable scaffolds could be applied for retention of MSCs in the injury endometrium [12].
PF-127 is one of the United States Food and Drug Administration (USFDA)-approved thermosensitive biodegradable hydrogels for clinical application [13]. PF-127 exhibits as liquid solution phase at low temperature (4°C) and is able to transform into solid gel solution at body temperature [14]. PF-127 characterizes as porous structure, which could further enhance the therapeutic effects by extending half-lives of drugs in serum [15]. These characteristics enable PF-127 to load the extracellular vesicles or cells in low temperature and form gel in the irregularly damaged space to promote regeneration [16][17][18][19]. PF-127 transplanted with hUC-MSCs was applied to regenerate intrauterine adhesion (IUA) rat uterus [20]. However, the effect about encapsulation of PF-127 with hUC-MSCs to regenerate thin endometrium remains to be elucidated.
It is known that underlying mechanisms of hUC-MSCsbased regeneration therapy is associated with paracrine function [21]. Local microenvironment is one of the issues that could modulate paracrine activities of MSCs [22]. Exposure MSCs on local titanium (Ti) particle could dysregulate MSCs population and alter vessel formation, which further induce local chronic Inflammation [23]. Incubation of MSCs with tumor necrosis factor-alpha (TNF-α) would promote the secretion of proangiogenic cytokines, which induce angiogenesis and tissue repair [24]. Interferon-γ (IFN-γ)treated MSCs was found to have enhanced prostaglandin E2 expression, which improves the antifibrotic ability of MSCs [25]. However, whether local microenvironment could enhance the expression of growth factors and angiogenesis factors of hUC-MSCs to regenerate thin endometrium is largely unknown. Moreover, the cellular and molecular mechanisms of regenerative effect on thin endometrium are yet to be discovered.
Thus, in the present study, we aim to investigate the therapeutic effects of PF-127 embedded hUC-MSCs on regeneration of thin endometrium. We found that PF-127-loaded hUC-MSCs could elevate angiogenesis with IL-1β stimulation, which further facilitated thin endometrium regeneration. Our findings may uncover a novel underlying mechanism for regeneration of thin endometrium.

Materials and Methods
2.1. Animals. Eight-week-old female Sprague-Dawley (SD) rats weighting 200-250 g were purchased from Chengdu Dossy Experimental Animals Co., Ltd. (Chengdu, Sichuan, China). After one-week adaption, the rats with consecutive 4-day estrous cycles were used in the experiment. All the animal experiments were carried out according to the guidelines indicated in the "Guide for the Care and Use of Laboratory Animals" [26]. The animal experiments were approved by the Ethics Review Board of Jinxin Research Institute for Reproductive Medicine and Genetics (approval ID: 2020YXLSD07).

Isolation and Culturing of hUC-MSCs.
With the permission of the Ethics Review Board of Chengdu Jinjiang Hospital for Maternal and Child Health Care (approval ID: 2020SZLSD02), 3 fresh human umbilical cords were collected from the Chengdu Jinjiang Hospital for Maternal and Child Health Care. The consent forms were signed by the three donors. Approximately 10-cm human umbilical cord was received from full-term birth after Caesarean section. Umbilical cord was collected in sterilized laboratory bottle with Dulbecco's Phosphate-Buffered Saline (DPBS) (Thermo Fisher Scientific, Grand Island, New York, USA) containing 100 U/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific) on ice. Umbilical cord was cut into 3 cm pieces and rinsed with ice-cold DPBS to remove blood clots. Blood vessels were completely removed from umbilical cord by using scissors and forceps. Afterwards, umbilical cord was cut into small pieces (1 to 2 mm 3 ) with scalpel. The small pieces of umbilical cord tissues were seeded into 10-cm dishes for 30-min incubation in 5% CO 2 at 37°C without medium. Half hour later, 7 ml of complete growth medium consisting of minimum essential medium α (α-MEM) (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (ExCell Bio, Taicang, Jiangsu, China) was added in the dishes to culture. After two weeks of culturing, small pieces of umbilical cord were removed. HUC-MSCs outgrowth from the explant was further passaged with TrypLE Express (Thermo Fisher Scientific) for characterization of cell transplantation.

Preparation and Characterization of PF-127 and C/GB
Hydrogel. PF-127 hydrogel was prepared with the following protocol as described previously [20]. 2.8. Determination of Biocompatibility of PF-127 and C/GP Hydrogel in Rat. Eight female SD rats (200-250 g) were used to evaluate the biocompatibility of PF-127 and C/GP hydrogel in vivo. All the rats were anesthetized with 4% of Nembutal (60 mg/kg). Three hundred microliter of PF-127 and C/GP hydrogel was subcutaneously injected into forelimbs and hindlimbs on both sides. Four weeks later, all the rats were sacrificed. Tissues around the hydrogel injection site were collected for q-PCR.

Transplantation of PF-127-Encapsulated hUC-MSCs.
Forty-eight rats were used to generate thin endometrium modeling by injection of 95% ethanol into the right side of uterine horns and rinsing twice with saline. To further investigate the efficiency of PF-127-encapsulated hUC-MSCs on reconstructing the function of destroyed endometrium, all the rats were randomly assigned into 4 groups. They consisted of a saline group (n = 12 uterine horns), PF-127 group (n = 12 uterine horns), hUC-MSCs group (n = 12 uterine horns), and PF-127-encapsulated hUC-MSCs group (n = 12 uterine horns). Nine days after thin endometrium modeling, 200 μl of saline, PF-127 hydrogel, hUC-MSCs (5 × 10 6 ), and PF-127 hydrogel-encapsulated hUC-MSCs (5 × 10 6 ) were injected into the right side of the uterine horns for treatment. Nine days after the treatment, all the rats were sacrificed. The right side uterine horns were collected for q-PCR, TaqMan-based quantitative real-time PCR (TaqMan qPCR), HE, and immunohistochemistry (IHC) analyses.
3 Stem Cells International 2.10. Preparation of IL-1β Conditioned Medium. Three lines of hUC-MSCs were seeded into 6-well plate and grown until approximately 80%-90% confluency. Cells were switched into a-MEM medium for overnight starving. To generate the IL-1β-conditioned medium, cells were incubated in a-MEM medium with different concentrations of IL-1β (PeproTech) (0, 20, 40, and 100 ng/ml) for 24 hours. Subsequently, the cells cultured in various concentrations of IL-1β induction were collected for q-PCR. Condition medium was harvested for Luminex analysis and tube formation assay test.
2.11. HE Examination. Collected tissues were fixed in 4% paraformaldehyde (PFA) for 4 hours. All the fixed samples were automatically dehydrated with Leica TP1020 with standard protocol (Leica, Buffalo Grove, Illinois, USA). The dehydrated samples were embedded in paraffin and cut into 4-μm sections (Leica). Afterwards, the sections were dewaxed and rehydrated in xylene and a graded series of ethanol. HE staining was applied to observe the structure of collected uterus. Fiji software was applied to evaluate the thickness of endometrium and gland number. The thickness of the uterine horn was determined by measuring the vertical distances from the lumen to the myometrium at 4 different directions (lateral and longitudinal side). 2.13. q-PCR. Total RNA was extracted by RNAiso Plus (Takara, Dalian, Liaoning, China) following the manufacturer's protocol. One microgram of total RNA was transcribed to cDNA by using the PrimeScript RT reagent Kit with gDNA Eraser (Takara) in accordance with the manufacturer's instructions. Q-PCR reactions were performed using KAPA SYBR® FAST Universal Kit (Sigma-Aldrich) on a 7500 Real-Time PCR Systems (Thermo Fisher Scientific). The mRNA expression levels were normalized to 18S and B2M in rat tissues and cultured cells, respectively. Data was analyzed based on 2 -ΔΔCt method [35].
The sequences of primers are summarized in Table 1: 2.14. TaqMan qPCR and DNA Calculation. Genomic DNA was extracted by GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific) following the manufacturer's

Stem Cells International
Ifng, and Il2 expressions were observed in PF-127 hydrogel in comparison to C/GB hydrogel (P < 0:01) (Figure 2(e)). Thus, we concluded that PF-127 hydrogel exhibited larger porosity and better in vivo biocompatibility, which fulfills the demands for an injectable biomaterial.

Establishment of Thin Endometrium Rat Model.
To characterize the thin endometrium rat model, HE staining and q-PCR were performed on the 95% ethanol and saline-treated uterus. Nine days after the modeling, thinner endometrium thickness was obtained from the ethanoltreated group (232:60 ± 51:62 μm) in comparison to the saline treated group (431:57 ± 22:76 μm) (P < 0:01) (Figures 3(a) and 3(b)). The total number of endometrial glands in the ethanol treated group (7:83 ± 1:40) was significantly lower than that in saline treated group (25:33 ± 3:33) (Figure 3(c)). We further performed q-PCR to evaluate inflammatory makers including Il1b, Tnfa, and Ifng in the uterus. Significantly higher expression of Il1b was shown in the modeling groups (P < 0:01) (Figure 3(d)). Hence, we concluded that we could generate thin endometrium with increased Il1b expression level in rat.  (Figures 4(a) and 4(b)). Moreover, the endometrial gland number in PF-127-encapsulated hUC-MSCs transplan-tation group (15:50 ± 2:15) was significantly higher than that in the saline group (6:75 ± 0:86) (Figure 4(c)). Increasing the trend of gland number was observed in the either hUC-MSCs or PF-127-treated group. Endometrial thickness was able to observe the increasing trend following transplantation with hUC-MSCs alone. However, neither of these two increasing trends achieved a statistically significant difference. Furthermore, we tested the retaining of hUC-MSCs in rat uterus by combining with or without PF-127 using TaqMan qPCR. Nine days after the treatment, only PF-127 and hUC-MSCs cotransplantation group could observe the residing of human DNA (Figure 4(d)). Human DNA was not able to detect in the group with direct hUC-MSCs injection. Thus, our results demonstrated that PF-127 could prolong the retention of hUC-MSCs, which enhanced the endometrium regeneration capacity of hUC-MSCs. Endometrial angiogenesis was one of the critical steps for regeneration. Therefore, IHC and q-PCR were performed to quantify the vascularization of endometrium. Significantly more VEGFA-positive blood vessels were observed in PF-127-encapsulated hUC-MSCs group (9:41 ± 0:77) in comparison to saline-treated group (7:34 ± 0:62) (P < 0:05) (Figures 5(a) and 5(b)). The same trend was also observed in VWF-positive blood vessels, in which strong VWFpositive blood vessels were found in hydrogel embedded hUC-MSCs group (8:143 ± 0:79) (Figures 5(a) and 5(c)). To further confirm the vascularization capacity, gene expressions of angiogenesis markers Vegfa and Nos3 were evaluated with q-PCR. Similarly, gene expressions of Vegfa and Nos3 were significantly increased in PF-127-encapsulated hUC-MSCs group compared to saline group (P < 0:05) (Figures 5(d) and 5(e)). These data indicated that hUC-MSCs embedded in thermosensitive hydrogel could regenerate thin endometrium with enhanced angiogenesis.

IL-1β-Primed hUC-MSCs Improve Angiogenesis via
Releasing of Neovascularization Factors. Local microenvironmental cues could induce MSCs to release bioactive factors and signals to regenerate the damaged tissues. Our data indicated that high Il1b expression was observed in modeled uterus. Meanwhile, enhanced angiogenesis was found in PF-127-encapsulated hUC-MSCs group. Variable concentrations of IL-1β (0, 20, 40, and 100 ng/ml) were applied to prime hUC-MSCs for mimicking the hUC-MSCs in response to the local environment of modeled uterus. To investigate the angiogenesis role of IL-1β-primed hUC-MSCs, q-PCR was carried out to evaluate the expression of angiogenesis genes (bFGF, EGF, and HGF). Significantly upregulated bFGF was found in the hUC-MSCs treated with different concentrations of IL-1β (P < 0:01) (Figure 6(a)). EGF was increased significantly in hUC-MSCs incubation with 20 and 100 ng/ml of IL-1β (P < 0:01 ) (Figure 6(a)). Expression level of HGF was significantly upregulated in the hUC-MSCs primed with IL-1β (P < 0:05 ) (Figure 6(a)). To further confirm the paracrine functions of hUC-MSCs stimulated with IL-1β, Luminex was performed to evaluate the concentration of angiogenesis protein (bFGF, VEGF, and HGF). Luminex data showed significantly increased bFGF concentration in the supernatant with IL-1β stimulation in comparison to that without stimulation (IL − 1β = 20 ng/ml, P < 0:01; IL − 1β = 40 ng/ml and 100 ng/ml, P < 0:05) (Figure 6(b)). Significantly increased concentration of VEGF was observed in the medium derived from 100 ng/ml IL-1β-primed hUC-MSCs (P < 0:05) ( Figure 6(b)). Increased concentration of HGF was found in the medium originated from IL-1β-stimulated hUC-MSCs ( Figure 6(b)). Moreover, supernatant from 20 ng/ml IL-1β-primed hUC-MSCs was used to measure the angiogenesis potential with tube formation assays in vitro. Total tube length was significantly increased in HUVEC incubated in the medium derived from 20 ng/ml IL-1β-primed hUC-MSCs (P < 0:01) (Figures 6(c) and 6(d)). Our data indicated that hUC-MSCs in response to local highly expressed IL-1β promoted neovascularization potential.

Discussion
Stem cell therapy has been used in restoring the function of damaged tissues [10]. However, limited MSCs could arrive or survive in the uterus by intravenous administration or local infusion [11]. Previous study indicated that biocompatible scaffolds encapsulated MSCs could significantly improve the treatment efficiency via prolonging cell retention and enhancing cell survival in vivo [38]. Therefore, we constructed thermosensitive hydrogel and hUC-MSCs for  Our results clearly demonstrated that thermosensitive PF-127encapsulated hUC-MSCs had long in vivo residing time, which could further alleviate thin endometrium with increased endometrium thickness, endometrial gland number, and vascularization capacity. Moreover, IL-1β microenvironment could induce hUC-MSCs to release angiogenesis factors, which played a key role during thin endometrium recovery. MSCs could be isolated from various tissues, such as bone marrow, adipose tissue, periapical cyst, dental pulp, placenta, and umbilical cord. However, the MSCs derived from different tissues would exhibit varying therapeutic effects with different diseases. Human periapical cystderived MSCs (hPCy-MSCs) could differentiate into dopaminergic neurons, which play an important role in brain regeneration and neurodegenerative disease modeling [39,40]. MSCs isolated from umbilical cord are able to differentiate into endometrial epithelial cell (EEC)-like and endometrial stromal cell (ESC)-like cells in vitro [41]. Previous study indicated that MSCs derived from various tissues were characterized with positive CD105, CD90, and CD73 and negative hematopoietic cells including HLA-DR, CD45, and CD34 [42]. In accordance with previous study, our data demonstrated that the MSCs isolated from umbilical cord expressed the typical MSCs surface makers and multilineage differentiation. In this light, hUC-MSCs characterized with extensive multipotency and high proliferative capacity would make it the most useful stem cell type in regeneration medicine in thin endometrium.
Thermosensitive hydrogels, characterized as liquid phase at low temperature and solid phase when temperature increasing, could develop equivalent hydrogel to cover the whole uterus [43]. Thermosensitive PF-127 and C/GP hydrogel were used to embed MSCs for tissues regeneration. Previous study indicated that constructing PF-127 with adipose-derived stem cell (ADSCs) was able to promote wound healing and cell proliferation [44]. PF-127 has been employed for in vitro differentiation scaffold for bone marrow-derived MSCs and dental-derived MSCs [45,46]. C/GP thermosensitive hydrogel was applied to encapsulate MSCs for in vitro proliferation and osteogenic differentiation [47,48]. Our finding showed that PF-127 takes shorter time to form solid hydrogel compared to C/GB hydrogel. These characters ensured that PF-127 could develop into solid hydrogel in the uterus within a short period of time without leaking from the uterus during clinic application in contrast to C/GB. In our study, SEM results demonstrated that 3D porous scaffold could be observed in both PF-127 and C/GB hydrogel. Three-dimensional porous structure scaffold promoted proliferation and differentiation of MSCs [34]. Our data indicated that significantly higher porosity  Stem Cells International was found in PF-127 compared to C/GB hydrogel. Larger porosity would be beneficial for biofactors releasing [34]. Four weeks after injection of PF-127 and C/GB hydrogel, significantly higher expression of inflammatory markers (Tnfa, Ifng, and Il2) was observed in the surrounding tissues with C/GB hydrogel injection in comparison to PF-127 injection. Our data indicated that high porosity and biocompatible PF-127 would be more suitable for encapsulation of hUC-MSCs for thin endometrium regeneration.
Thin endometrium was associated with lower clinical pregnancy and birth weight during the IVF-ET cycles [3,49,50]. Similar to previous studies [51,52], we modeled 10 Stem Cells International the thin endometrium by infusing the endometrium with 95% ethanol resulting in reduced endometrium thickness and gland number. Similar to the thin endometrium uterus environment, significantly higher expression of inflammatory marker Il1b was observed in the uterus after thin endometrium modeling [53,54]. Previous study indicated that increased endometrium thickness could be observed after 12-day uterine injection [55]. Similar to previous study, our data could observe the increasing trend of endometrium thickness, gland number, and newly generated blood vessels after 9 days of hUC-MSCs injection. However, this increasing trend was not significant. Our TaqMan qPCR results indicated that human-specific DNA was not able to detect after 9 days of only hUC-MSCs transplantation group.
Cotransplanted hUC-MSCs with PF-127 could retain in uterus for 9 days. Thus, the therapeutics effective of only hUC-MSCs injection would be very limited. Significantly increased endometrial thickness and gland number was only achieved by transplantation of PF-127/hUC-MSCs. Thin endometrium was highly characterized by poor vascular development, decreased VEGF expression, and poor epithelial growth [56]. Therefore, neovascularization is one of the critical steps for endometrium regeneration. Combination of scaffolds and MSCs could induce neovascularization potential of scared uterus via increasing expression of new blood vessel makers (VWF) [20,57]. Consistent with previous study, significantly increased vascular markers abundancy (Vegfa and Nos3) was observed in the thin endometrium modeled uterus with PF-127/ hUC-MSCs treatment. Moreover, significantly more VWFand VEGFA-positive blood vessels were confirmed by IHC staining after the modeled thin endometrium were treated with PF-127/hUC-MSCs. Thus, our results indicated that PF-127 encapsulation of hUC-MSCs could regenerate thin endometrium via promoting angiogenesis.
Reduced blood flow induced hypoxia in thin endometrium patient uterus [58]. Genome-wide single-cell RNA sequencing revealed decreased macrophages and natural killer cells in the human thin endometrium [59]. Highly expressed inflammatory genes including TNF-α, IFN-γ, and interleukin 1 receptor (IL1R1) were observed in thin endometrium through genomic mRNA sequencing analysis [53,54]. Previous study also indicated that local inflammatory environment was associated with occurrence of metabolic acidosis and shifting of extracellular pH [60]. MSCs could be stimulated by local injury microenvironment to release growth factors, exosomes, and complements to execute the therapeutic effects during regeneration [61]. Acidosis-primed MSCs could release the specified exosomes, which could increase the anti-inflammatory T cell subtypes in vitro [62]. Enhanced proliferation and neovascularization capacity were reported in the MSCs primed by hypoxia [63]. Inflammatory cytokine IL-1β-primed hUC-MSCs could release of growth factors to promote epidermal substitute engraftment and wound healing in vivo [64]. Significantly increased expression of Il1b in the modeled rat uterus might be the local stimulation molecules to promote hUC-MSCs therapeutic role. Our data indicated that upregulated expression levels of angiogenetic markers bFGF, EGF, and HGF were observed in the hUC-MSCs stimulated with IL-1β, which is in accordance with previous studies [65,66]. Moreover, enhanced secretion of bFGF, EGF, and VEGF was detected in medium derived from the IL-1β-primed hUC-MSCs, as previously reported [64]. Collagen-binding bFGF could regenerate the damaged uterus by promoting angiogenesis with increased blood vessels [67]. Intravenously administrated VEGF overexpressed MSCs could increase the endometrium thickness [52]. The expressions of EGF and HGF were required for the proliferation of endometrium of epithelial and stromal cells [68,69]. VEGF exhibited a vital role in endometrium regeneration, which was found highly expressed in human endometrium [70,71]. Since these angiogenic factors could promote proliferation, migration, and composition of capillary tubes, we further compared the neovascularization potential of medium derived from IL-1β-primed hUC-MSCs and nonprimed hUC-MSCs. Our results clearly proved that enhanced angiogenesis capacity was found in IL-1β-primed hUC-MSCs medium in tube formation assay. Taken together, our results demonstrated that the hUC-MSCs could respond to local IL-1β microenvironment, which promoted the release of angiogenic factors to enhance thin endometrium regeneration.
There are some limitations that need to be addressed in our study. Our data suggested that IL-1β environment could promote hUC-MSCs to secrete the angiogenesis cytokines to regenerate thin endometrium. The underlying molecular mechanisms of thin endometrium regeneration need to be further elucidated. Overexpressions of upregulated molecules including bFGF, EGF, HGF, and VEGF in hUC-MSCs should be tested in thin endometrium regeneration. Furthermore, to generalize environment stimulation on therapeutic role in MSCs, the mass spectrometric and transcriptome sequencing could be performed to compare the difference of secretome of hUC-MSCs with or without IL-1β stimulation. Moreover, the immune modulation and angiogenesis potential of stem cell would elevate the tumorigenesis risk during stem cell therapy. Previous studies indicated that local microenvironment could stimulate MSCs to secrete exosomes, which play a key role in stem cell therapy [22]. Therefore, in future studies, we plan to clarify that if application of exosomes released from IL-1β-primed hUC-MSCs on regeneration of thin endometrium to improve the safety and efficacy of our stem cell therapy.
Human umbilical cord tissue derived from different donors would produce hUC-MSCs with functional heterogeneity, which could bring therapeutic variations [72]. HUC-MSCs derived from different donors should be tested for their thin endometrium regeneration potential to make sure the therapeutic consistency. Moreover, hUC-MSCs have to be manufactured under GMP conditions for clinical application.

Conclusions
In this study, we found that PF-127 encapsulation of hUC-MSCs could restore the morphology of modeled thin endometrium, with increased endometrium thickness and gland number, and promoted neovascularization capacity. We 11 Stem Cells International further uncovered that local IL-1β microenvironment stimulated the hUC-MSCs to release angiogenic and endometrium regeneration factors to regenerate the structure of thin endometrium. Overall, we identify a novel underlying mechanism of combination of hUC-MSCs and biomaterials to regenerate thin endometrium.

Data Availability
The primary data presented in this study are available on request from the corresponding author.

Conflicts of Interest
The authors declare no conflict of interest.