Human UC samples were obtained from three healthy donors (n = 3) who delivered by cesarean section after full-term pregnancies of 38–40 weeks and provided informed consent. The use of human UC tissue was approved by the Medical Ethics Committee of the First People Hospital of Yunnan Province (KHLL2022-KY155). Three-centimeter segments from three different regions of human UCs toward the placenta (maternal segment), the center (middle segment) and toward the fetal region (fetal segment) were collected and stored in saline. MSCs isolated and cultured from 3 cm of UC tissue near the placenta are named maternal-MSCs. MSCs isolated and cultured from the central 3 cm of the entire UC length are named middle-MSCs.MSCs isolated and cultured from 3 cm of UC tissue near the fetal segment are named fetal-MSCs.Veins and arteries were removed from the UC segments, and Wharton’s jelly tissues were cut into 1–3 mm³ pieces and cultured as explants via the adherent method in 10-cm culture dishes containing complete medium consisting of MSC serum-free medium (MSC-T4; CSTI, Japan) and 5% UltraGRO™ (Helios, United States). After 10–15 days, the tissue explants were removed, and primary hUCMSCs (P0) were detached with CTS™ TrypLE™ Select (Gibco, United States) and cultured to passage 3 (P3). Then, hUCMSC marker identification was performed using a flow cytometer (Beckman Coulter, Chaska, MN, United States). P3 hUCMSCs were incubated with antibodies against CD44-FITC, CD90-PE, CD105-APC, CD73-APC, CD19-PerCP, CD45-FITC, CD34-FITC, CD11b-APC and HLA-DR-APC for 20 min at room temperature, and flow cytometry was performed according to the manufacturer’s instructions. All flow cytometry antibodies were obtained from BioLegend (BioLegend, United States). Finally, the adipogenic, osteogenic, and chondrogenic abilities of the hUCMSCs were assessed. P3 hUCMSCs were cultured in osteogenic, adipogenic or chondrogenic differentiation medium (Gibco, United States) according to the manufacturer’s instructions. Oil red O staining, Alizarin Red staining and Alcian Blue staining were used to evaluate adipogenesis, osteogenesis, and chondrogenesis, respectively.

Total RNA was extracted from UCMSCs using a TRIzol reagent kit (Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s protocol. After the total RNA was extracted, mRNA was then enriched using Oligo (dT) beads. The mRNA samples were fragmented into short fragments and reverse transcribed into cDNA with random primers. Second-strand cDNA was synthesized with DNA polymerase I, RNase H, dNTPs and buffer. Then, the cDNA fragments were purified with a QiaQuick PCR extraction kit (Qiagen, Venlo, Netherlands), the ends were repaired, poly(A) was added, and Illumina sequencing adapters were ligated. The ligation products were size selected by agarose gel electrophoresis, amplified by PCR, and sequenced using an Illumina NovaSeq 6,000 by Gene Denovo Biotechnology Co. (Guangzhou, China). RNA differential expression analysis was performed with DESeq2 (4) software. Genes with a false discovery rate (FDR) less than 0.05 and an absolute fold change ≥2 were considered differentially expressed genes (DEGs). Gene Ontology (GO) biological function enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed for all DEGs.

A transwell system (0.4-μm pore polycarbonate, Corning, United States) was used to coculture MSCs with AC16 cells. AC16 cells were purchased from the Beina Chuanglian Biotechnology Institute (Beijing, China). AC16 cells were seeded at a density of 10⁵ cells/well in 6-well plates, and MSCs were seeded at a density of 10⁵ cells/well in a transwell chamber and cultured overnight at 37°C. Then, the cells were subjected to OGD as previously described. After being replaced in glucose- and FBS-free DMEM, AC16 cells and MSCs were exposed to hypoxia (94% N₂, 5% CO₂, and 1% O₂) in an anaerobic incubator (Thermo Fisher Scientific, United States) at 37°C for 72 h. Flow cytometry was used to determine the apoptotic rates of AC16 cells using an Annexin V-FITC apoptosis detection kit (Beyotime, China).

Flow cytometry was used to determine the apoptotic rates of AC16 cells using an Annexin V-FITC apoptosis detection kit (Beyotime, China) according to the manufacturer’s instructions. The cells were harvested using trypsin and resuspended in 195 μL of binding buffer. The cells were stained with 5 μL of Annexin V-FITC and 10 μL of PI for 15 min at 25°C. The stained cells were examined by flow cytometry (NovoCyte D2060R, Agilent Technologies, United States).

Seven-week-old male Sprague–Dawley (SD) rats were purchased from Beijing Vital River Laboratory Animal Technology Co. The rats were fed under standard animal room conditions (humidity, 55%–60%; temperature, 25°C). Food and water were freely available throughout the experiments. The animal experiments were approved by the Animal Ethical and Welfare Committee of MDKN Biotech Co., Ltd (Approval No. MDKN- 2022-068). The MI model was established by ligating the LAD as described previously. Briefly, the rats were anesthetized by isoflurane inhalation (3%–5%), endotracheal intubation was performed for mechanical ventilation, and electrocardiographic changes were recorded. A left thoracotomy was performed between the third and fourth intercostal spaces, and the LAD artery was ligated with 8–0 nylon thread. Successful ligation was verified by observing a pale left ventricular wall below the ligation site and ST-segment elevation and QRS widening via electrocardiography. The sham-operated control rats also underwent the same surgery, except that the suture placed under the left coronary artery was not tied.

Immediately after MI induction, 1 × 10⁷ hUCMSCs suspended in 50 µL of normal saline [containing 5% human serum albumin (HAS)] were injected intramyocardially at 3 different sites into the infarct border zone. Rats with MI were randomized into the following groups: 1) MI + NS group: 50 µL of normal saline (containing 5% HAS); 2) MI + fetal-MSCs group: 1 × 10⁷ hUCMSCs from the fetal segment were injected; 3) MI + middle-MSCs group: 1 × 10⁷ hUCMSCs from the central segment were injected; and 4) MI + maternal-MSCs group: 1 × 10⁷ hUCMSCs from the placenta segment were injected.

The rats were anesthetized with 3%–5% isoflurane 3 weeks after surgery. M-mode echocardiography was performed with a high-resolution Vevo 3,100 microultrasound imaging system. The left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD) were measured on the parasternal LV long-axis view. The LVEF and LVFS were computed automatically by the echocardiography software to evaluate heart function.

Masson’s trichrome staining was used to evaluate myocardial infarct size. Briefly, the myocardium was fixed in 4% paraformaldehyde (PFA, pH 7.4) and then embedded in paraffin. The heart samples were cut into three 5 μm-thick sections from the bottom to the apex of each heart. The heart sections were stained with Masson’s trichrome according to the manufacturer’s instructions. Images were scanned with a Pannoramic MIDI scanner (3D HISTECH), and the area of the infarcted zone was analyzed using ImageJ software. The infarct area was calculated as the average percentage of the fibrosis area relative to the total area.

Paraffin blocks were prepared and cut into 5 μm-thick sections. The slides were deparaffinized, dehydrated and subjected to antigen retrieval in hot citric acid buffer. The slides were permeabilized with 0.2% Triton-100 and blocked with 3% bovine serum albumin (BSA) in PBS for 1 h. The slides were incubated with CD31 primary antibodies (1:300, GB113151, Servicebio) overnight at 4°C, followed by incubation with Cy3-labeled goat anti-rabbit IgG secondary antibodies (1:300, GB21303, Servicebio) for 2 h at room temperature. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Servicebio) for 10 min at room temperature. Images were scanned with a Pannoramic MIDI scanner (3D HISTECH), viewed with CaseViewer software (3D HISTECH), and analyzed using ImageJ software by a double-blinded investigator. For each rat, we analyzed 6 randomly selected regions of the infarcted area of each slide at 400 × magnification.

A Click-iT 488 TUNEL Cell Apoptosis Detection Kit (Servicebio, China) was used to examine apoptosis in myocardial sections according to the manufacturer’s protocol. Images were scanned with a Pannoramic MIDI scanner (3D HISTECH) and viewed with CaseViewer software (3D HISTECH). Six randomly selected regions in the infarction border zone of each slide were analyzed at 200 × magnification.

Total RNA was extracted from hUCMSCs using TRIzol reagent (Takara, Japan) according to the manufacturer’s protocols. Subsequently, the RNA was reverse transcribed to cDNA with a PrimeScript™ RT reagent kit (Takara, Japan). RT‒qPCR was performed using TB Green Premix Ex TaqTM (Takara, Japan) and a StepOnePlus real-time PCR system (Azure Biosystems). The amplification reaction conditions consisted of 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 34 s. The sequences of the primers used were as follows: GATA4, forward: 5′-CGT​TCT​CAG​TCA​GTG​CGA​TGT​CTG-3′ and reverse: 5′-CCA​AGA​CCA​GGC​TGT​TCC​AAG​AG-3’; MYOCD, forward: 5′- CAA​CCC​TCA​CTT​TCT​GCC​CTC​ATC-3′ and reverse: 5′-GCC​ATC​GTG​TGC​TCC​TGA​GTT​C-3′; and GAPDH, forward: 5′-GCA​CCG​TCA​AGG​CTG​AGA​AC-3′, reverse: 5′- TGG​TGA​AGA​CGC​CAG​TGG​A-3′. Target gene mRNA levels were normalized to that of the reference gene GAPDH, and the relative mRNA levels were calculated using the 2^−ΔΔCT method.

Protein was extracted from hUCMSCs using RIPA lysis buffer (Beyotime, China) supplemented with 1% PMSF (Biosharp, China) and quantitated using a BCA protein assay kit (TargetMol, C0001). Protein samples were separated by SDS‒PAGE and transferred to PVDF membranes (Millipore). The membranes were blocked with 5% skim milk in Tris-buffered saline with Tween 20 (TBST) at room temperature for 2 h, incubated with primary antibodies against GATA-4 (1:2000, ab256782, Abcam), MYOCD (1:2000, PA5-100775, Thermo Fisher Scientific), and GAPDH (1:10,000, AF7021, Affinity) at 4°C overnight, and subsequently incubated with the corresponding HRP-conjugated secondary antibodies (1:10,000, Affinity) for 1 h at room temperature. The bands were visualized by enhanced chemiluminescence (ECL) and quantified using ImageJ software. The housekeeping protein GAPDH was quantified for normalization.

The data are presented as the means±SDs and were analyzed with GraphPad Prism 7 software (GraphPad Software, Inc.). Comparisons between 2 groups were performed by Student’s t-test, and multiple group comparisons were performed by one-way ANOVA. The Tukey correction was applied to account for multiple comparisons when performing post hoc tests. P < 0.05 was considered to indicate statistical significance.

MSCs were successfully isolated from three segments of human UCs and cultured for three passages. Flow cytometry confirmed that MSCs expressed specific mesenchymal cell markers, including CD44, CD73, CD90 and CD105 (≥95%), but lacked CD19, CD34, CD45, CD11b and HLA-DR (≤2%) (Figure 1C). Furthermore, the cells could differentiate into osteoblasts, adipocytes, and chondroblasts in vitro (Figure 1B). These results demonstrated that MSCs derived from different segments of the UC were successfully isolated and characterized.

To investigate the heterogeneity of hUCMSCs isolated from different sections of the UC, we used RNA-seq technology to analyze the transcriptome and gene expression of 9 types of MSCs derived from three independent UCs. Compared to fetal-MSCs and middle-MSCs, maternal-MSCs exhibited 10 upregulated genes and 3 downregulated genes (|log2(FC)| > 1, FDR< 0.05) (Figure 2A). Maternal-MSCs highly expressed MYOCD and GATA4 (Figure 2A), which are critical transcription factors for cardiomyocyte development. These results were validated by quantitative real-time PCR analysis (Figure 2B). We further confirmed the protein expression levels of MYOCD and GATA4 by western blot analysis and showed that maternal-MSCs expressed higher protein levels of MYOCD and GATA4 than fetal-MSCs and middle-MSCs (Figure 2C). In addition, we also quantitatively analyzed the differences in GATA4 and MYOCD gene expression in hUCMSCs isolated from different segments of 7 independent UCs. The qPCR results showed that the expression levels of MYOCD and GATA4 were increased in maternal-MSCs compared to fetal-MSCs and middle-MSCs (Supplementary Figure S1). Taken together, these results demonstrated the heterogeneity of hUCMSCs isolated from different segments of the UC and the maternal-MSCs might have greater potential in cardiac protection due to higher expression of the genes associated cardiomyocte development.

To test the capability of MSCs derived from different segments of the UC to protect cardiomyocytes in vitro, AC16 cells were cocultured with fetal-MSCs, middle-MSCs and maternal-MSCs in a transwell system. Subsequently, we investigated AC16 cell apoptosis under OGD conditions. OGD resulted in significant apoptosis compared to that in the control group (Figures 3A, B). Notably, the apoptosis rate of AC16 cells was significantly lower in the maternal-MSCs group than in the fetal-MSCs and middle-MSCs groups, and there was no significant difference between the fetal-MSCs group and the middle-MSCs group (Figure 3B). The results showed that maternal-MSCs could effectively protect AC16 cells from OGD-induced cell injury in vitro.

To investigate the therapeutic effects of MSCs derived from different segments of the UC, we established an MI model in rats by permanent left anterior descending (LAD) artery ligation, after which MSCs were immediately injected intramyocardially. At 21 days after-MI, transthoracic echocardiography was performed to evaluate the in vivo therapeutic effects of MSCs in the rat MI model. The results showed that the left ventricular ejection fraction (LVEF) and fractional shortening (FS) of infarcted hearts were significantly lower in the MI group than in the sham group, and MSC treatment significantly rescued the MI-induced decreases in LVEF and FS (Figures 4A–C). More importantly, the maternal-MSCs group exhibited significantly higher increases in LVEF and FS than the fetal-MSCs and middle-MSCs groups (Figures 4B, C). The results indicate that maternal-MSCs exert superior protective effects on cardiac function in the rat MI model.

To determine the extent of MI-related fibrosis, Masson trichrome staining was performed 21 days after the transplantation of MSCs (Figure 4D). ImageJ analysis demonstrated that the area of fibrosis was significantly decreased in the maternal-MSCs group compared with the fetal-MSCs and middle-MSCs groups (Figure 4E). The results showed that maternal-MSCs could effectively alleviate cardiac fibrosis.

To evaluate the therapeutic effects of MSCs derived from different segments of the UC on angiogenesis and the inhibition of apoptosis in vivo, cardiomyocyte apoptosis was assessed by TUNEL staining (Figure 5A), and capillaries were stained with an antibody against CD31 and analyzed by immunofluorescence (Figure 5B). TUNEL staining revealed that the percentage of apoptotic cells in the border zone of the cardiac infarction area was significantly lower in the MSC treatment group than in the MI group (Figure 5C). Furthermore, compared with the fetal-MSCs and middle-MSCs groups, the maternal-MSCs group had significantly fewer apoptotic cells (Figure 5C). Immunofluorescence staining revealed that the capillary density in the cardiac infarction area was significantly increased in the MSC treatment groups compared with the MI group (Figure 5D). Importantly, the capillary density in the maternal-MSCs group was significantly increased compared with that in the fetal-MSCs and middle-MSCs groups (Figure 5D). These results indicated that maternal-MSCs could improve cardiac function by promoting angiogenesis and cardiomyocyte survival.