Fetal mesenchymal stem cells promote osteogenesis via Notch signaling

doi:10.21203/rs.3.rs-2880264/v1

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Fetal mesenchymal stem cells promote osteogenesis via Notch signaling

https://doi.org/10.21203/rs.3.rs-2880264/v1

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Background

Mesenchymal stem cells (MSCs) as a source of osteoblasts play a key role in bone repair and regeneration. Fetal mesenchymal stem cells (Fetal-MSCs) exhibit lower immunogenicity, higher potential for expansion, and higher differentiation potential into various cell lineages than adult MSCs. Herein, in vitro and in vivo evaluations were performed to study the role of human Fetal-MSCs in osteogenic differentiation and bone regeneration in comparison to that of adult bone marrow mesenchymal stem cells (BM-MSCs).

Methods

Fetal-MSCs were isolated from fetal tissues derived from ectopic pregnancy at eight and half and half weeks of gestation and studied for karyotypic stability, proliferation over successive passages, and expression of embryonal and mesenchymal markers using flow cytometry. The ability of BM-MSCs and Fetal-MSCs to differentiate into osteoblasts was assessed using alkaline phosphatase (ALP) and alizarin red staining(ARS). The expression of osteogenic markers such as ALP, osteopontin(OPN), RUNX2, and osteocalcin(OCN) was evaluated using quantitative reverse transcription polymerase chain reaction. mRNA sequencing was performed to identify the differences in gene expression between Fetal-MSCs and BM-MSCs. In addition, their effects were assessed in rat calvarial defect model using micro-computed tomography, immunohistochemistry, and histological examination.

Results

Fetal-MSCs were more efficient than BM-MSCs in promoting osteogenesis and had a greater bone regeneration capacity in vitro and in vivo. In particular, Hairy ears Y-linked (HEY) 1 and HEY2, which are involved in osteoblast development, showed a significantly high differential expression between Fetal-MSCs and BM-MSCs and were the key target genes in the Notch signaling pathway. Notch signaling plays a key role in the osteogenic differentiation of Fetal-MSCs.

Conclusions

We demonstrated for the first time that Notch signaling plays a key regulatory role in promoting the osteogenic differentiation of Fetal-MSCs. In addition, Fetal-MSCs with their strong bone formation ability may be a promising alternative to adult-derived MSCs for bone disease treatment.

Fetal mesenchymal stem cells

Bone marrow mesenchymal stem cells

Notch signaling

Osteogenic differentiation

Bone regeneration

Bone diseases are common conditions affecting human health worldwide, raising major challenges, including socioeconomic and healthcare issues [1]. Annually, more than 20 million individuals worldwide experience bone tissue loss due to trauma or pathological and physiological bone resorption [2, 3]. Approximately 10% of patients undergo an abnormal healing process, which results in insufficient bone formation [4]. Therefore, efficient and affordable bone repair strategies are required. Currently, several innovative strategies, including biophysical, local, and systemic cell-based therapies, are being used to stimulate the healing of bone defects and control skeletal development [2, 4].

Human mesenchymal stem cell (MSC) transplantation is a promising alternative for active bone healing and replacement of missing bone. An adequate supply of MSCs is important for efficient bone regeneration. The MSC characteristics, such as flexibility, self-renewal capacity, and multilineage potential, make them desirable transplantation options for bone-associated disorders. [5]. MSCs are multipotent progenitor cells that can be isolated from various tissues, such as adult bone marrow, adipose tissue, skin, umbilical cord, and placenta, and they can differentiate into several cell types, including osteoblasts, chondrocytes, and adipocytes [6].

MSCs have several beneficial immunomodulatory properties and secrete growth factors that facilitate repair processes [7, 8]. They exert paracrine effects and release growth factors, immunomodulatory cytokines, and chemokines to induce regeneration [5]. The ability of multipotent MSCs to regenerate bone, exhibit self-renewal properties, and maintain stemness during ectopic transplantation is demonstrated in several in vivo experiments [9, 10]. In this context, stem cells from fetuses, embryos, induced pluripotent stem cells (iPSCs), and other sources can be used as alternatives [11]. However, there is the possibility of tumorigenesis with the use of embryonic stem cells, and the use of iPSCs is still in the experimental stage with limited data; therefore, they are less attractive for practical applications [12]. However, fetal MSCs have certain advantages, especially when compared to adult-derived MSCs. They are less immunogenic because they do not express the human leukocyte antigen (HLA) type 2 surface antigens and respond slowly to γ-interferon stimulation [13]. In addition, they show greater potential for expansion, migration, and differentiation into various cell lineages [14], [15, 16]. For these reasons, fetal tissues (umbilical cord, cord blood, placenta, and amniotic fluid) derived from the perinatal period is commonly used [12, 17, 18].

The Notch signaling system controls signal transmission between cells. In mammals, canonical Notch signaling is mediated by five Notch ligands (Jagged-1 and 2, Delta-like (DLL)1, 3, and 4), and four Notch receptors (Notch 1–4) [19–21]. Notch signaling is triggered by the binding of Notch ligands to their receptors. Notch intracellular domain (NICD) is cleaved and released during activation, and then translocated from the cellular membrane to the nucleus. [22]. NICD binds transcriptional regulators of the CSL family in the nucleus to influence downstream targets.[23]. Notch signaling is crucial for determining cell fate, proliferation, apoptosis, migration [24, 25], and skeletal remodeling [26].

Currently, there is a paucity of information on the use of calvarial tissue and calvaria-derived stem cells from the fetus, especially that in the first trimester of gestation, for bone regeneration. In this study, we isolated and characterized Fetal stem cells and confirmed their osteogenic potential. We compared the osteogenic effect of human Fetal MSCs (Fetal-MSCs) to that of bone marrow MSCs (BM-MSCs) in vitro and in vivo and studied the involvement of Notch signaling in the osteogenic development of Fetal-MSCs.

Ethics

All experiments, including tissue collection, were approved by the Research Ethics Committee of CHA University and CHA Hospital, Korea. National guidelines and all state and federal guidelines were complied with in relation to the use of fetal tissues for research. Fetal-MSCs were isolated from the parts excluding the brain of an ectopic pregnancy at 8.5 weeks gestation, with donor consent for the use of tissue for research and commercialization.

Cell Isolation and Culture

Fetal tissues were examined for infections listed in Annex II of the EMEA Commission Directive 2006/17/EC, including human immunodeficiency virus types 1 and 2, hepatitis C and B, Treponema pallidum, cytomegalovirus, and Toxoplasma gondii. Human fetal MSC lines from fetuses without any diagnostic findings were used for long-term cultivation.

Fetal tissues other than the brain were isolated from mononuclear cells and digested for 30 min at 37°C with 0.1 mg/mL collagenase type I (Gibco) in phosphate buffered saline (PBS) supplemented with 10 µg/ml DNase (Roche); it was then washed thrice with Hanks balanced salt solution and incubated for 30 min at 37°C with 50 g/ml, followed by three washes. The samples were gently triturated 20 times. The cell suspension was propagated in Hanks balanced salt solution. The expansion media was based on a modified Eagle’s medium developed by Dulbecco (Welgen). Fetal-MSCs between passages 10 and 15 were used for osteoblast differentiation after the construction of the Research Cell Bank (passage 5). Human fetal MSCs were cultured in standard culture medium and osteogenic differentiation medium (OM, Gibco). High-glucose DMEM (Welgen) supplemented with 1% penicillin/streptomycin (Gibco) and 10% fetal bovine serum (Gibco) was used as the standard culture medium. The Fetal-MSCs were plated onto pre-coated cell culture dishes. After the cells reached 80–100% confluence, the medium was replaced with that from the StemPro™ osteogenesis differentiation kit (Gibco). The cells were then allowed to differentiate for 7–14 days.

Karyotype and DNA Repair Analysis

Karyotyping was performed at CHA Biotech Cytogenomic Services Facility. To determine the karyotype, cell division was inhibited in the metaphase, by treating the cells with 0.05 g/ml colcemid for 1–2 hours. G-band staining was used to examine chromosomes; and more than 100 metaphase cells were investigated [27].

Flow Cytometry Analysis

Flow cytometry analysis was performed on Fetal-MSCs at passage 5. Briefly, 2 × 10⁵ cells were incubated for 30 min with fluorescein isothiocyanate– or phycoerythrin-conjugated antibodies against SSEA4, SSEA3, TRA-1-81, CD29, CD34, CD44 CD45, CD90, and CD105 (BD Biosciences). Ten thousand events were recorded for each antibody using a Cytoflex Flow Cytometer and fluorescence-aided cell sorting (FACS). The results were analyzed using FlowJo software.

Alkaline Phosphatase (ALP) Staining

Fetal-MSCs (6×10⁴ cells/well) were cultured in an osteogenic (OM) medium for 3 and 7 days. The osteoblastic differentiation was commenced using 60 µg/mL ascorbic acid and 2 mM -glycerophosphate in complete cell culture media for the prescribed times, and the medium was replaced every 2 days. After seven days of osteogenic differentiation, the cells were fixed in 70% ethanol for 3 minutes, and ALP staining was conducted according to the manufacturer's instructions using an NBT/BCIP staining kit. Using reaction buffer and p-nitrophenyl phosphate as a substrate, the ALP activity was measured at 405 nm. After normalization to the total protein concentration, ALP activity was determined. Using the Pierce Protein Assay Kit and the bicinchoninic acid (BCA) technique, protein concentration was determined (Thermo Fisher Scientific, USA).

Alizarin Red Staining (ARS)

Fetal-MSCs (6×10⁴ cells/well) were cultured in OM for 7, 10 and 14 days. Following the osteogenic differentiation for the indicated number of days, cells were fixed in 70% ethanol for 3 min at room temperature, washed with distilled water, and stained with 40mM Alizarin Red (pH 4.2; Sigma-Aldrich MO, USA) for 30 min. For quantitative assessment of the degree of mineralization, the stain was dissolved in cetylpyridinium chloride (Sigma-Aldrich), and the absorbance at 570 nm was measured.

RNA Extraction and qRT-PCR

Total RNA was isolated from the BM-MSCs or Fetal-MSCs using the TRIzol® reagent (Invitrogen; Thermo Fisher Scientific Inc.), according to the manufacturer’s protocol. To quantify mRNA expression, total RNA (1 µg) was reverse-transcribed into cDNA using Maxime RT PreMix (iNtRON Biotechnology Inc.). The reaction mixtures were incubated at 45°C for 60 min and at 95°C for 5 min. qPCR was performed to quantify mRNA levels using the AMPIGENE qPCR Green Mix Lo-ROX (Enzo Life Sciences Inc.); the mRNA levels were normalized to that of glyceraldehyde 3-phosphate dehydrogenase. The thermocycling conditions were as follows: initial activation at 95°C for 2 min and 40 cycles at 95°C for 5 s and 60°C for 20 s.

mRNA Sequencing

Library preparation and sequencing of the total RNA from BM-MSCs or Fetal-MSCs and the library construction were performed using the QuantSeq 3’ mRNA-Seq Library Prep Kit (Lexogen, Inc., Austria), according to the manufacturer’s instructions. The library was amplified and the complete adapter sequences were added, for cluster generation. The final library was purified from PCR components. High-throughput sequencing was performed as single-end 75 sequencing using a NextSeq 500 (Illumina, Inc., USA). Data analysis of QuantSeq 3 mRNA-Seq reads was performed using Bowtie2 [28]. The Bowtie2 indices were generated either from the genome assembly sequence or from the representative transcript sequences for alignment to the genome and transcriptome. The alignment file was used to assemble the transcripts, estimate their abundance, and detect differential gene expression. Differentially expressed genes were determined based on counts from unique and multiple alignments using Bedtools [29]. The read count data were processed based on the quantile normalization method using EdgeR within R (R Development Core Team, 2016) with Bioconductor. Gene classification was based on searches of the DAVID and MEDLINE databases. Data mining and graphic visualization were performed using ExDEGA (Ebiogen Inc., Seoul, Korea).

Notch Pathway Inhibition

Both BM-MSCs and Fetal-MSCs were cultured in OM. DAPT (N-[30]-S-phenylglycine t-butyl ester, LY-374973, Sigma-Aldrich S.r.l. Milano, Italy), which inhibits γ-secretase, was added to the culture medium at a final concentration of 20 µM. The inhibitor was dissolved in DMSO (Fisher Scientific, BP231-100) at 20 mM, stored at -80℃, and freshly diluted with the culture medium at each medium change during the entire differentiation period.

Calvarial Defect Rat Model

SD rats were housed in a specific pathogen-free animal laboratory for one week for acclimatization. The SD rats were handled in compliance with the guidelines for the care and use of animals at our institution. Animals gave their consent to the experimental techniques care and CHA laboratory animal research-center (IACUC210052) and conducted according to the ARRIVE guidelines .[30] Seven-week-old male SD rats were randomly assigned to three groups of seven each: (1) negative control group (CONT), (2) group treated with BM-MSCs, and (3) group treated with Fetal-MSCs. Animals were given oxygen and isoflurane(Hana Pharm. Co., Ltd.) at 4%, and rats were maintained at 2.5–3% through a nose cone during the procedure. A circular, full-thickness bone defect with a diameter of 4 mm was created on the lateral parietal bone using a bur. BM-MSCs, Fetal-MSCs (1×10⁴ cells/10 µl), and PBS (10 µl) were adsorbed onto collagen sponges (4 mm diameter; Advanced BioMatrix). Chemically adsorbed collagen sponges were placed in the calvarial region of the mice, and the defect was covered with pericranium. After eight weeks, rodents were subjected to euthanasia via CO₂ overdose in accordance with the guidelines for euthanasia established by AVMA.

µCT Analysis

CT images of rat calvaria fixed in a 10% neutral-buffered formalin solution were obtained using a high-resolution Skyscan 1173 micro-CT system (Bruker, Aartselaar, Belgium). Images were acquired at an effective pixel size of 21.9 µm, voltage of 80 kV, current of 125 µA, and exposure time of 1600 ms. Image reconstruction software (NRecon; Bruker) was used to reconstruct serial cross-sectional images with identical thresholds for global (0–0.08 in CS to image). The trabecular morphometry was evaluated by measuring percent bone volum (BV/TV), bone surface (BS), bone mineral density (BMD), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular number (Tb.N). 3D surface rendering images were created using a CT Analyzer (Bruker, Skyscan micro-CT).

Histological Analysis

Rat calvaria were decalcified using 10% ethylenediaminetetraacetic acid (EDTA; pH 7.4) for 1 month. The EDTA solution was replaced every two days. Decalcified femurs were washed, dehydrated, and embedded in paraffin. Using a ready-to-use IHC/ICC kit (BioVision, Inc.), we performed immunohistochemistry (IHC) on the calvarial bone, in accordance with the manufacturer’s protocol. Rat calvariae were fixed in 4% PFA and rinsed thrice with PBS. Subsequently, the calvariae were decalcified for 14 days with 12.5 mM EDTA (pH 8.0). The samples were paraffin-embedded for hematoxylin and eosin staining and Masson’s trichrome staining. The sections were incubated with anti- type 2 collagen alpha 1 (anti-COL2a1; 1:100), anti- RUNX2 (1:100), anti-osteocalcin (anti-OCN; 1:100), or anti-NOTCH1 (1:100) antibodies for 30 min at room temperature. This was followed by 20 min of incubation with HRP-anti-mouse or -rabbit IgG polymer at room temperature and washing with PBS. The sections were treated with 3,3'-diaminobenzidine for 10 min and counterstained with hematoxylin for 1 min. The EclipsTS2 microscope was used to obtain images (magnification, ×200).

Statistical Analysis

All data are presented as the mean ± SEM. All statistical analyses were performed using the GraphPad Prism software 8 (GraphPad Software Inc.). Student’s t-test was used to compare differences between two groups, and one-way ANOVA with post hoc Tukey's test was used to compare differences between more than two groups.

Characterization of Fetal-MSCs

Fetal-MSCs were isolated from mononuclear cells, harvested, and cultured as described earlier. Analysis under the microscope revealed that the cells isolated from the primary tissue multiplied fast in the expanded culture medium. After the primary cells were passaged more than four times, their morphology became similar to that of fibroblast-like cells. We observed the morphology of Fetal-MSCs using phase-contrast microscopy; similar to adult MSCs, Fetal-MSCs grew as spindle-shaped adherent cells (Fig. 1A). These morphologies are consistent with those described earlier for MSCs [16]. Fetal-MSCs were passaged and maintained in an undifferentiated state with a spindle-shaped, fibroblastic morphology over 20 passages. Human karyotype (Fig. 1B) and cell growth curves (Fig. 1C) were normal. FACS was performed to confirm the expression of MSC surface markers. Fetal-MSCs were negative for embryonic stem markers (SSEA-3, and TRA-1-81) and hematopoietic markers (CD34 and CD45) except for SSEA4 (5.19% positive).however, they were positive for MSC-specific surface markers such as CD29, CD44, CD90, and CD105 (Fig. 1D) [2], [31]. This confirmed that the isolated cells were MSCs and demonstrated the characteristics of Fetal-MSCs, including cell morphology, karyotype, cell growth curves, and phenotypes.

Comparison of the Osteogenic Differentiation Potential of Stem Cells

ALP staining and ARS were performed, to compare the osteogenic differentiation potential of BM-MSCs and Fetal-MSCs. ALP staining and activity assays were conducted on day 3 and7 of differntiation cells. The optical density (OD) values in ALP staining and the activity increased significantly, indicating that Fetal-MSCs had a higher osteoblastic potential than BM-MSCs (Fig. 2A). ARS was utilized to identify calcium deposits inside the mineralized matrix. On days 7 and 10 of differentiation, Fetal-MSCs showed a high OD in ARS (Fig. 2B). qRT-PCR analysis results showed that the relative expression of mRNA encoding osteogenic markers such as ALP, OCN, osteopontin (OPN), and RUNX2 was significantly higher in Fetal-MSCs compared to that in BM-MSCs (Fig. 2C).

mRNA-seq Analysis of Human BM-MSCs and Fetal-MSCs

We sequenced mRNA to evaluate gene expression patterns and identify differentially expressed genes linked with the osteogenic effect of Fetal-MSCs relative to BM-MSCs. In addition, we aimed to identify specific signaling pathways closely associated with the osteogenic effect of Fetal-MSCs. We determined the global gene expression patterns, constructed a heatmap of differentially expressed genes identified via mRNA sequencing (Fig. 3A), and ranked the genes based on their mRNA expression levels. Among the genes associated with osteoblast development, Hairy ears Y-linked (HEY)1 was the most significantly enriched and was expressed at high levels (Fig. 3B). HEY1 is a key target gene of the Notch signaling pathway; therefore, we examined the correlation between the Notch signaling pathway and the osteogenic differentiation ability of Fetal-MSCs. We confirmed that HEY2 showed the greatest increase in expression among the Notch signaling–related genes (Fig. 3C). qRT-PCR results showed that HEY1 and HEY2 levels were significantly correlated with those of the bone differentiation markers, ALP and RUNX2, in differentiating Fetal-MSCs (Fig. 3D). These results show that bone differentiation in Fetal-MSCs acts as a positive regulator of Notch signaling.

Notch Signaling Promotes the Osteogenic Differentiation of Fetal-MSCs

Analyses of gene expression revealed the parricipation of Notch signaling in the osteogenic development of Fetal-MSCs. The Fetal-MSCs were treated with DAPT, a γ-secretase inhibitor, to block Notch signaling and observe the effects on osteogenesis. The presence of Notch inhibitors in the OM considerable influenced the expression of osteogenic biomarkers and genes in Fetal-MSCs. In particular, DAPT significantly decreased the OD in ALP staining and the activity at the time points examined, days 3 and 7, when compared to that seen in the OM-only group (Fig. 4A). Furthermore, DAPT induced a significant decrease in ARS uptake and quantitative staining on days 7 and 14 of differentiation (Fig. 4B). In the presence of DAPT, the expression of ALP, RUNX2, HEY1, HEY2, and HES4, which was upregulated during bone differentiation, was suppressed (Fig. 4C). These results show that the bone differentiation of Fetal-MSCs is dependent on Notch signaling.

In Vivo Analysis and Micro-CT Evaluation

The role of Fetal-MSCs in bone reconstruction was investigated in a rat model of calvarial defects, in which holes were created using an electric drill. This was used to investigate the bone healing effect of Fetal-MSCs in vivo and to evaluate their capability in terms of ossification and bone formation, compared to that of BM-MSCs. BM-MSCs or Fetal-MSCs were loaded onto a collagen sponge and implanted into the defect in the rat calvarium. Micro-CT 3D images showed that at 8 weeks after BM-MSCs or Fetal-MSCs implantation, the defects were repaired. Moreover, the Fetal-MSCs group showed the smallest defect area with the highest closure range and healing percentage and demonstrated the highest healing effect and bone formation ability (Fig. 5A). The morphometric parameters evaluated using micro-CT analysis revealed that the bone volume fraction (BV/TV), bone surface (BS), mineral density (BMD), thickness (Tb.Th), and number (Tb.N) were significantly higher in the calvarial defects treated with Fetal-MSCs than in the defects treated with BM-MSCs (Fig. 5B).

Histological Analysis

Histological analysis was performed to assess the bone healing effect in the calvarial defect area and examine the molecular markers related to osteogenesis at the defect sites. Histological analysis showed that Fetal-MSCs induced bone regeneration and significantly enhanced bone formation in the calvarial defect sites. In the Fetal-MSCs group, the newly formed bone bridged defects more visibly and reinforced them with a thicker volume of bone tissue than that in the control and BM-MSCs groups. In addition, Fetal-MSCs showed enhanced cell proliferation and promoted osteogenic differentiation, as indicated by the increased expression of COL2a1, RUNX2, and OCN in the IHC analysis. The expression of NOTCH1 significantly increased in Fetal-MSCs, confirming the regeneration of bone tissue (Fig. 6A).

In this study, Fetal-MSCs were isolated from ectopic pregnancy fetuses at 8 weeks of gestation. Human Fetal and their characteristics, including cell morphology, growth curves, and MSC-specific surface markers, were identified. We examined the capacity of human Fetal-MSCs to develop into osteoblasts and stimulate bone regeneration in vitro and in vivo with that of adult BM-MSCs. mRNA sequencing indicated that HEY1 and HEY2, related to osteoblast development and the key target genes in the Notch signaling pathway, were significantly upregulated in Fetal-MSCs.

This study proposes a novel approach for studying bone regeneration and osteogenesis. We used stem cells derived from fetuses during the first trimester of gestation, as early as 8 weeks. Pathology classifies the embryonic stage as lasting up to nine weeks of gestation [2]. Human fetal tissues and stem cells have been studied worldwide since 1928 [32]; however, there is a paucity of knowledge regarding the characteristics, functional properties, and clinical applications of MSCs derived from fetuses during the first trimester of gestation. Owing to ethical controversies, most researchers use embryonic cells derived from voluntary interruption of pregnancy between 5 and 8 weeks [2].

The use of human Fetal-MSCs derived from calvarial bones is a new approach. In previous studies, human MSCs from first trimester fetal blood, liver, bone marrow, and limb buds were isolated, and the defining characteristics of MSCs, such as fibroblast-like morphology, expression of MSC markers, and the ability to differentiate into mesodermal lineages, were identified [12, 13, 33]. To the best of our knowledge, this is the first study to isolate human fetal stem cells derived from calvarial bones, and demonstrate their innate ability to regenerate bone. Calvarial sutures are a niche for stem cells during craniofacial regeneration. Throughout the embryonic and postnatal periods, sutures remain as active sites for bone formation [34]. Maruyama et al. identified, isolated, and characterized calvarial suture stem cells from a mouse model using Axin2. Axin2-expressing stem cells in the suture midline contributed cell-autonomously to damage healing and skeletal regeneration. [35]. In addition, they confirmed the effect of ectopic transplantation of calvarial suture stem cells at three weeks post-treatment, using an animal model. Axin2-expressing cells could self-differentiate into osteogenic lineage cells and multiply clonally in vivo. [35]. Kong et al. utilized human cranial suture stem cells derived from the residual skull tissues of patients aged 4–10 months who underwent craniotomy for unilateral coronal synostosis and analyzed their characteristics in vitro [36]. In this study, we identified, isolated, and characterized MSCs derived from the human Fetal bone. The findings would help advance cell-based therapies using stem cells derived from fetal tissues. The developmental stages and the tissue anatomical locations influence the differentiation potential of MSCs [37]; therefore, we hypothesized that the calvarial bone originates in early fetal tissues, which soon develop into skeletal tissue, and that it could help mediate bone regeneration therapy.

HEY1 and HEY2, which are key target genes of the Notch signaling pathway, showed higher expression levels in the Fetal-MSC-treated group compared to that in the BM-MSC-treated group. The Notch signaling pathway plays an important role during the embryonic and postnatal periods by regulating cell fate decisions and coordinating cell proliferation, differentiation, and apoptosis, especially in skeletal tissues. Notch receptors regulate bone remodeling and regeneration, including osteoblast differentiation, matrix mineralization, osteoclast recruitment, cell fusion, and osteoblast/osteoclast progenitor cell proliferation [38]. The inhibition of NOTCH1 signaling decreased ALP activity and increased RUNX2 and COL1 expression [39]. Notch signaling interacts with the bone morphogenetic protein (BMP) and transforming growth factor (TGF)-β pathways, which are closely associated with the bone healing process; Notch receptor inhibition through pharmacological intervention impairs calvarial bone healing [38], [40]. Notch signaling influenced the osteogenic differentiation, osteoblast development, and bone healing effects of fetal MSCs. HEY are classical canonical Notch target genes that play pivotal roles in embryogenesis [38, 41].

Notch signaling influences bone healing positively. However, the exact mechanism by which Notch signaling regulates bone regeneration remains unclear, including whether Notch signaling accelerates or inhibits bone regeneration [42]. DAPT targets the release of NICD, which is derived from the interaction between the Jagged-1 ligand and Notch receptor. When Notch signaling is inhibited by the systemic application of DAPT, cartilage and bone callus formation increase via the promotion of MSC differentiation, resulting in accelerated fracture healing. DAPT use in fractures can enhance osteoclastogenesis and bone remodeling [43]. The effects of Notch signaling on osteoblasts and their progenitors may not be simply stimulatory or inhibitory, but rather cell context-dependent, depending on the developmental stage of bone formation and the differentiation status of each cell [44]. In addition, Notch signaling in the bone can regulate both osteoblastic and osteoclastic lineages through complicated pathways. Therefore, further studies investigating the relationship between Notch signaling and the regulation of osteogenesis and bone healing are required.

This study had some limitations. Ethical concerns and social issues regarding the medical use of fetus-derived stem cells have been raised, and the use of embryos and fetuses as sources for research and treatment remains controversial. Various guidelines have been formulated for the research and clinical use of human embryonic and fetal tissues. In the European Union, the “Ethical guidelines for the usage of human embryonic or fetal tissues for experimental and clinical neurotransplantation and research” was published by the Network for European CNS Transplantation and Restoration in 1994 [32]. Continuous research, efforts, and discussions on essential issues are required to develop more efficient stem cell therapies. Further research on animal models and human randomized controlled clinical trials are necessary for the clinical application of human Fetal stem cells in patients with bone-related conditions.

Future research should focus on the application of Fetal-MSCs in tissue engineering, combining the interdisciplinary study of stem cells, synthetic scaffolds, and bioactive molecules to support active bone healing or replace missing parts of the bone, which are actively investigated in preclinical and clinical trials [45, 46]. Scaffolds are porous structures with good biocompatibility, suitable porosity, and biodegradability; they serve as templates to guide cell attachment, differentiation, proliferation, and tissue regeneration [47]. Bioactive molecules, such as growth factors, including platelet-derived growth factor, BMPs, fibroblast growth factor 2, and TGF-β, are beneficial for bone regeneration and induction of bone formation [48]. Therefore, adequate use of early fetal stem cells in tissue engineering could help optimize bone regeneration strategies.

Fetal-MSCs were more efficient than BM-MSCs in promoting bone regeneration because of the upregulation of Notch signaling in Fetal-MSCsthem. Fetal-MSCs could serve as an alternative source of adult stem cells for bone repair therapy. This study presents new perspectives on the clinical application of fetal stem cells.

ALP: alkaline phosphatase

BMD: bone mineral density

BM-MSCs: bone marrow mesenchymal stem cells

BMP: bone morphogenetic protein

DEG: differentially expressed gene

FACS: fluorescence-activated cell sorting

Fetal-MSCs: Fetal tissues-derived MSCs

HEY: hairy ears Y-linked

Fetal-MSCs: fetal mesenchymal stem cells

HLA: human leukocyte antigen

iPSCs: induced pluripotent stem cells

MSCs: mesenchymal stem cells

NICD: Notch intracellular domain

OM: osteogenic differentiation medium

Ethics approval and consent to participate

The mice used in the present study were maintained according to guidelines approved by the Institutional Animal Care and Use Committee of CHA medical university (Title of the approved project: Confirmation of bone regeneration efficacy using stem cells;, Name of the institutional approval committee: Institutional Animal Care and Use Committee of CHA Stem Cell Institute;, Approval number: IACUC210052;, Date of approval: 2021.04.20). and were conducted according to ILAR guide. Collection and use surgical human tissues were approved by the Institutional Review Board (IRB) of CHA medical university (Title of the approved project: Establishment of Neural Progenitor Cell Lines by utilizing Donated Fetus from Missed Abortion or Ecropic Pregnancy;, Name of the institutional approval committee: Cha Medical University, Gangnam Cha Hospital Research Ethics Review Committee;, Approval number: IRB No. GCI-18-34;, Date of approval: 2019.08.28 ). The study was conducted in accordance with the Declaration of Helsinki. Pericyte were collected from donors after obtaining their informed consent.

Consent for publication

Informed consent was obtained from all subjects involved in the study.

Availability of data and materials

All data generated or analyzed during current study are included in this published article. The full dateset is available in the National Center for Biotechnology 191 Information Gene Expression Omnibus database (NCBI GEO data base) with the accession 192 number GSE 229945. Raw data are accessible at 193 https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE229945. Secure token : wbgdyiqifbendmn

Competing interests

The authors declare that they have no competing interest.

Funding

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2022R1A2C2005916). This research was supported by the Korean Fund for Regenerative Medicine (KFRM) grant funded by the Korea government (the Ministry of Science and ICT, the Ministry of Health & Welfare). (22C0605L1). This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. RS-2023-00210067)

Authors' contributions:

J.L.: Project administration, data curation, formal analysis, methodology, writing original draft. H.-J.A.: Project administration, data curation, formal analysis, methodology, writing original draft. J.S.R.: Conceptualization, data curation, funding acquisition, manuscript review and editing T.K.: Data curation, formal analysis. J.W.: Data curation, formal analysis. J.K.: Data curation. S.H.S.: Human IRB and ectopic fetus isolation. S.L.: Conceptualization, funding acquisition, investigation, methodology, manuscript review and editing. All authors contributed to the article and approved the submitted version.

Acknowledgements

The authors thank the members in the lab for their helpful comments, suggestions, and assistance.

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Fetal mesenchymal stem cells promote osteogenesis via Notch signaling

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Abstract

Background

Methods

Results

Conclusions

Figures

Background

Methods

Ethics

Cell Isolation and Culture

Karyotype and DNA Repair Analysis

Flow Cytometry Analysis

Alkaline Phosphatase (ALP) Staining

Alizarin Red Staining (ARS)

RNA Extraction and qRT-PCR

mRNA Sequencing

Notch Pathway Inhibition

Calvarial Defect Rat Model

µCT Analysis

Histological Analysis

Statistical Analysis

Results

Characterization of Fetal-MSCs

Comparison of the Osteogenic Differentiation Potential of Stem Cells

mRNA-seq Analysis of Human BM-MSCs and Fetal-MSCs

Notch Signaling Promotes the Osteogenic Differentiation of Fetal-MSCs

In Vivo Analysis and Micro-CT Evaluation

Histological Analysis

Discussion

Conclusions

Abbreviations

Declarations

References

Supplementary Files

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