Optimization of the Decellularization Process and Evaluation of the Differentiation Potential of Wharton's Jelly Mesenchymal Stem Cells on a Decellularized 3D Scaffold of Testicular Tissue.

Umbilical cord Wharton's jelly-derived mesenchymal stem cell (WJMSC) is a new-found mesenchymal stem cell in recent years with multiple lineage potential. Due to its abundant resources, no damage procurement, and lower immunogenicity than other adult MSCs, WJMSC promises to be a good xenogenous cell candidate for tissue engineering. This in vivo pilot study explored the use of human umbilical cord Wharton's jelly mesenchymal stem cells (hWJMSCs) containing a tissue engineering construct xenotransplant in rabbits to repair full-thickness cartilage defects in the femoral patellar groove. We observed orderly spatial-temporal remodeling of hWJMSCs into cartilage tissues during repair over 16 months, with characteristic architectural features, including a hyaline-like neocartilage layer with good surface regularity, complete integration with adjacent host cartilage, and regenerated subchondral bone. No immune rejection was detected when xenograft hWJMSCs were implanted into rabbit cartilage defects. The repair results using hWJMSCs were superior to those of chondrogenically induced hWJMSCs after assessing gross appearance and histological grading scores. These preliminary results suggest that using novel undifferentiated hWJMSCs as seed cells might be a better approach than using transforming growth factor-β-induced differentiated hWJMSCs for in vivo tissue engineering treatment of cartilage defects. hWJMSC allografts may be promising for clinical applications.

Bone tissue engineering is a promising approach to overcome the limitations of traditional autograft bone transplantation. Graphene quantum dots (GQDs) have been suggested as an enhancement for osteogenic differentiation. This study aimed to investigate the ability of the fibrin hydrogel scaffold in the presence of graphene quantum dots to promote osteogenic differentiation of human Wharton's jelly-derived mesenchymal stem cells (hWJ-MSCs). The hWJ-MSCs were isolated from the Wharton's jelly of the human umbilical cord using a mechanical method. Fibrin hydrogel scaffolds were prepared by mixing 15 µl of thrombin solution with fibrinogen solution. GQDs were incorporated into the scaffolds at concentrations of 0, 5, and 10 µg/ml. Cell viability was determined through DAPI staining and the MTT assay. Osteogenic differentiation was assessed by measuring alkaline phosphatase (ALP) activity, quantifying calcium deposition using Alizarin Red S staining, and analyzing the gene expression of BGLAP, COL1A1, Runx-2 and ALP via qPCR. Scanning electron microscopy (SEM) was employed to analyze the scaffold architecture. SEM analysis revealed that the fibrin hydrogel exhibited a suitable architecture for tissue engineering, and DAPI staining confirmed cell viability. The MTT results indicated that the GQDs and fibrin hydrogel scaffold exhibited no cytotoxic effects. Furthermore, the incorporation of GQDs at a concentration of 10 µg/ml significantly enhanced ALP activity, calcium deposition, and the expression of osteogenesis-related genes compared to the control. The findings suggest that the combination of fibrin hydrogel and GQDs can effectively promote the osteogenic differentiation of hWJ-MSCs, contributing to the advancement of bone tissue engineering.

Current burn animal models have disadvantages, such as standardization issues. Many mesenchymal stem cell studies have been conducted on burn treatments; however, the efficacy of Wharton's jelly mesenchymal stem cells (WJ-MSCs) and the conditioned medium (CM) on partial- and full-thickness burns remains unclear. Therefore, we aimed to investigate the healing potential of WJ-MSCs and CM in treating partial- and full-thickness skin burns using a new carbon dioxide (CO2) laser model. Sixty-two male Wistar rats (200-250 g) were used, with 56 rats divided into 4 groups based on treatment after laser application. Furthermore, to create varying degrees of skin burns, a fractional CO 2 laser (10.2 mJ, 10,600 nm) was applied to 6 rats for 15-120 s. A 60-s and 120-s laser application was performed on the back of each animal to create partial-thickness and full-thickness burns, respectively. WJ-MSCs, CM, and minimal essential media (MEM) were applied to partial- and full-thickness skin burns determined in a pilot study 24 h later. However, no treatment was administered to the SHAM group. Treatment efficacy was evaluated semi-quantitatively through macroscopic, histopathological, and immunohistochemical analyses and quantitatively through real-time quantitative reverse transcription polymerase chain reaction on days 7 and 14. Macroscopic and histopathological analyses (epidermal thickness, dermal thickness, number of skin appendages, and collagen scoring) showed significant improvements in the CM, WJ-MSCs, and MEM groups compared with the controls. Immunohistochemical staining indicated increased levels of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) in the treated groups compared with those in the SHAM group. quantitative reverse transcription polymerase chain reaction revealed the upregulation of VEGF, VEGF receptor 1, FGF, and FGF receptor 1 in the CM and WJ-MSCs groups compared with that in the SHAM and MEM groups, highlighting their roles in angiogenesis and re-epithelialization. This study highlights the potential of WJ-MSCs and CM to enhance wound healing in CO 2 laser-induced partial and full-thickness skin burns. Additionally, a new noncontact CO 2 laser burn model that creates burns at varying degrees and is easy to standardize was developed.

Both mature and stem cell-derived hepatocytes lost their phenotype and functionality under conventional culture conditions. However, the 3D scaffolds containing the main extracellular matrix constitutions, such as heparin, may provide appropriate microenvironment for hepatocytes to be functional. The current study aimed to investigate the efficacy of the differentiation capability of hepatocytes derived from human Wharton's jelly mesenchymal stem cells (WJ-MSCs) in 3D heparinized scaffold. In this case, the human WJ-MSCs were cultured on the heparinized and non-heparinized 2D collagen gels or within 3D scaffolds in the presence of hepatogenic medium. Immunostaining was performed for anti-alpha fetoprotein, cytokeratin-18 and -19 antibodies. RT-PCR was performed for detection of hepatic nuclear factor-4 (HNF-4), albumin, cytokeratin-18 and -19, glucose-6-phosphatase (G6P), c-met and Cyp2B. The results indicated that hepatogenic media induced the cells to express early liver-specific markers including HNF4, albumin, cytokeratin-18 and 19 in all conditions. The cells cultured on both heparinized culture conditions expressed late liver-specific markers such as G6P and Cyp2B as well. Besides, the hepatocytes differentiated in 3D heparinized scaffolds stored more glycogen that indicated they were more functional. Non-heparinized 2D gel was the superior condition for cholangiocyte differentiation as indicated by higher levels of cytokeratin 19 expression. In conclusion, the heparinized 3D scaffolds provided a microenvironment to mimic Disse space. Therefore, 3D heparinized collagen scaffold can be suggested as a good vehicle for hepatocyte differentiation.

Diprophylline drug as an efficient inducer loaded in chitosan-gelatin-umbilical cord serum hydrogel for neural differentiation of human Wharton's jelly-derived mesenchymal stem cells in vitro.

This review explores the potential of Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) in cartilage regeneration and osteoarthritis treatment. It covers key factors influencing chondrogenesis, including growth factors, cytokines, and hypoxia, focusing on precise timing. The effectiveness of three-dimensional cultures and scaffold-based strategies in chondrogenic differentiation is discussed. Specific biomaterials such as chitosan and hyaluronic acid are highlighted for tissue engineering. The document reviews clinical applications, incorporating evidence from animal research and early trials and molecular and histological assessments of chondrogenic differentiation processes. It addresses challenges and strategies for optimizing MSC-derived chondrocyte therapy, emphasizing the immunomodulatory properties of these cells. The review concludes as a comprehensive road map for future research and clinical applications in regenerative medicine.

To investigate the efficacy of Wharton's jelly mesenchymal stem cells (WJSCs) and their conditioned medium (CM) for corneal nerve regeneration in rats with diabetic keratopathy. Streptozotocin (STZ)-induced male diabetic (DM) rats (250–300 g) were divided into four groups (n=7/group): Control, DM, DM with WJSCs (DM+WJ), and DM with CM treatment (DM+CM). DM+WJ and DM+CM group received WJSCs or CM, respectively, topically with eye drops. Corneal sensibility, corneal epithelial layer integrity, histology, expression of GAP-43 and TUBB3 on mRNA level and their immunohistochemical expression were examined after two weeks of treatment. There were changes in corneal sensibility and corneal integrity between normal control and diabetic groups with/without WJSC or CM injection. Total central corneal thickness was significantly higher in DM+CM (249.81 ± 43.85 μm) than in control (174.72 ± 44.12 μm, P=0.004) and DM groups (190.15 ± 9.63 μm, P=0.03). GAP-43 mRNA expression levels of DM+WJ and DM+CM groups were higher compared with DM and control groups. TUBB3 mRNA level was increased after CM (P=0.047), but not after WJSCs treatment (P=1.00). GAP-43 and TUBB3 immunohistochemical expression of nerve fibers along the epithelial layer significantly increased in DM+WJ and DM+CM compared with DM group. Our findings showed that WJSCs and their CM improved corneal nerve regeneration in rats with diabetic keratopathy

Background: Breast carcinoma (BC) is the most prevalent form of cancer in women and is classified into various subtypes based on the expression of ER, PR, and human epidermal growth factor 2 (HER2) receptors. Human epidermal growth factor 2-positive BC, in particular, presents significant challenges due to its aggressiveness and limited treatment options. Modulating apoptotic pathways is crucial for reducing tumor burden and enhancing treatment efficacy. Objectives: This study aimed to evaluate the inhibitory effects of secretomes derived from Wharton's jelly mesenchymal stem cells (WJ-MSC secretomes) on the proliferation of the SKBR3 cell line, as well as the subsequent changes in apoptosis-related gene expression after treatment. The research provides new insights into the therapeutic potential of WJ-MSC secretomes for HER2-positive BC. Methods: SKBR3 cells were exposed to WJ-MSC secretomes at doses of 10, 25, and 50 μg/mL for 24 and 48 hours. MTT assays and colony formation assays were used to evaluate cell viability, while annexin V/PI staining and mRNA expression analyses were conducted to assess apoptosis. Results: Treatment with WJ-MSC secretomes led to a significant, concentration- and time-dependent reduction in colony numbers and cell viability in the 25 and 50 μg/mL groups. Early and late apoptotic indices increased significantly in the 25 μg/mL group, with P-values of < 0.01 and < 0.05, respectively. The mRNA expression levels of Caspase-9 (P < 0.001), Caspase-3 (P < 0.05), and the Bax/Bcl-2 ratio (P < 0.01) significantly increased in the 25 μg/mL treatment group. Caspase-8 activity was unaffected by WJ-MSC secretomes. Conclusions: Wharton's jelly mesenchymal stem cells secretomes demonstrate potential as an anticancer agent for HER2-positive BC, acting through the intrinsic apoptotic pathway in a concentration- and time-dependent manner. This study is the first to detail the specific effects of WJ-MSC secretomes on HER2-positive SKBR3 cells, suggesting their novel application in targeted breast cancer therapy.

Articular cartilage exhibits a low regenerative capacity and limited potential for self-renewal. Recent research has demonstrated that exosomes and mesenchymal stem cells (MSCs) significantly enhance cartilage repair by promoting cellular proliferation, increasing extracellular matrix synthesis, and modulating the immune response. Additionally, hyaluronic acid (HA), a critical component of synovial fluid, plays a key role in facilitating cell migration. This study aims to compare the regenerative effects of Wharton's jelly-derived MSCs, MSC-derived exosomes, and their combination with hyaluronic acid in the treatment of cartilage defects. Additionally, we seek to evaluate the impact of hyaluronic acid when combined with MSCs and exosomes through histological analysis in a rat model. In this study, full-thickness cartilage defects were created in the trochlear grooves of both distal femurs in 48 adult rats. The knees were randomly assigned to six groups: Group I: Control-saline, Group II: Wharton's jelly mesenchymal stem cells (MSCs), Group III: Wharton's jelly MSC-derived exosomes (Exo), Group IV: Hyaluronic acid (HA), Group V: MSC and HA combination, and Group VI: Exo and HA combination. Each rat received a total of three intra-articular injections at weekly intervals, beginning two weeks post-surgery. Four weeks following the final injection, all rats were euthanized, and their femurs were dissected for analysis. All groups were assessed macroscopically using the International Cartilage Repair Society (ICRS) scoring system, following histological staining with hematoxylin-eosin (HE) and toluidine blue, and immunohistochemical staining with type II collagen antibodies. The quality of the repaired cartilage was subsequently evaluated according to the ICRS histological grading system by an independent, blinded observer. Macroscopic evaluations indicated that the ICRS scores of the MSC group (8.2 ± 0.7) were significantly higher (P < 0.05) than those of the control group (4.3 ± 0.7). The cartilage defects in the MSC group showed substantial repair, displaying the most effective cartilage regeneration among all groups. Furthermore, comparison between groups revealed that both the MSC and Exo groups demonstrated a higher rate of defect depth repair, a smaller demarcation border, and a smoother cartilage surface. This study demonstrates that exosomes are as effective as stem cell therapies in promoting cartilage repair, suggesting that exosomes may serve as a viable alternative to cell-based therapies for cartilage damage. However, the addition of hyaluronic acid to stem cells and exosomes showed no significant enhancement in cartilage repair. Our findings highlight a potentially effective therapeutic strategy for the treatment of osteochondral cartilage defects.

The aim of this research was to find a way to differentiate germ cells from umbilical cord Wharton's jelly mesenchymal stem cells (MSCs) to support in vitro spermatogenesis. A small piece of Wharton's jelly was cultured in high-glucose Dulbecco's modified Eagle's medium in present of 10% foetal calf serum. After the fourth passage, the cells were isolated and cultured in Sertoli cell-conditioned medium under induction of two different doses of retinoic acid (10-5 , 10-6 m). The differentiation of MSC to germ-like cells was evaluated by expression of Oct4, Nanog, Plzf, Stra8 and Prm1 genes during different days of culture through qPCR. The results showed that there were downregulation of Oct4 and Nanog and upregulation of pre-meiotic germ cell marker (stra8) and haploid cell marker (Prm1) when MSCs are differentiated over time. The expression of Bax gene (an apoptotic marker) was significantly observed in high dosage of retinoic acid (RA). As a result, RA has positive effects on proliferation and differentiation of MSCs, but its effects are related to dosage. The success of this method can introduce umbilical cord MSC as a source of germ cells for treatment of infertility in future.

IntroductionIn large-animal acute myocardial infarction (AMI) models, Wharton's jelly (umbilical cord matrix) mesenchymal stem cells (WJMSCs) effectively promote angiogenesis and drive functional myocardial regeneration. Human data are lacking.AimTo evaluate the feasibility and safety of a novel myocardial regeneration strategy using human WJMSCs as a unique, allogenic but immuno-privileged, off-the-shelf cellular therapeutic agent.Material and methodsThe inclusion criterion was first, large (LVEF ≤ 45%, CK-MB > 100 U/l) AMI with successful infarct-related artery primary percutaneous coronary intervention reperfusion (TIMI ≥ 2). Ten consecutive patients (age 32–65 years, peak hs-troponin T 17.3 ±9.1 ng/ml and peak CK-MB 533 ±89 U/l, sustained echo LVEF reduction to 37.6 ±2.6%, cMRI LVEF 40.3 ±2.7% and infarct size 20.1 ±2.8%) were enrolled.Results30 × 106 WJMSCs were administered (LAD/Cx/RCA in 6/3/1) per protocol at ≈ 5–7 days using a cell delivery-dedicated, coronary-non-occlusive method. No clinical symptoms or ECG signs of myocardial ischemia occurred. There was no epicardial flow or myocardial perfusion impairment (TIMI-3 in all; cTFC 45 ±8 vs. 44 ±9, p = 0.51), and no patient showed hs-troponin T elevation (0.92 ±0.29 ≤ 24 h before vs. 0.89 ±0.28 ≤ 24 h after; decrease, p = 0.04). One subject experienced, 2 days after cell transfer, a transient temperature rise (38.9°C); this was reactive to paracetamol with no sequel. No other adverse events and no significant arrhythmias (ECG Holter) occurred. Up to 12 months there was one new, non-index territory lethal AMI but no adverse events that might be attributable to WJMSC treatment.ConclusionsThis study demonstrated the feasibility and procedural safety of WJMSC use as off-the-shelf cellular therapy in human AMI and suggested further clinical safety of WJMSC cardiac transfer, providing a basis for randomized placebo-controlled endpoint-powered evaluation.

Designing convenient substrates is a pertinent parameter that can guide stem cell differentiation. Current research is directed toward differentiating mesenchymal stem cells (MSCs) into endothelial cells (ECs). It is generally accepted that MSCs cannot be easily differentiated into ECs without high concentrations of proangiogenic factors. To guide either bone marrow-derived mesenchymal stem cells (BM-MSCs) and Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) into ECs-like phenotype, poly(allylamine-hydrochloride)/poly(styrene-sulfonate) multilayers film (PAH/PSS) was used as culture coating and compared to type I collagen (as control coating). After 2 weeks of culture and in absence of angiogenic growth factors, PAH/PSS upregulated KDR, PECAM-1, and CDH5 genes, whereas combining PAH/PSS with endothelial growth media (EGM-2® ) led to the production of respective proteins by WJ-MSCs. In contrast, not fully EC-like phenotype is obtained from the differentiation of BM- or WJ-MSCs cultured on type I collagen. Thus, using PAH/PSS coating in synergy with EGM-2® appears as an ideal condition promoting WJ-MSCs differentiation into ECs-like. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 292-300, 2017.

Mesenchymal stem cells (MSCs), obtained from several anatomical sites, have already been described, characterized and used in therapeutic models for tissue repair. The umbilical cord mesenchymal stem cells, represented by cells from arteries and veins walls, as well as Wharton's jelly are easy to be obtained, highly available, require no invasive procedure, do not present risk to donors and do not present ethical limitation. The aim of this research was to analyze the plasticity of Wharton's jelly mesenchymal stem cells (WJ-MSCs) of goat, evaluating their behavior in vitro and characterizing them immunophenotypically. Thus, tests were performed on colony forming units, viability and cell growth curve, flow cytometry analysis and plasticity potential. Goat umbilical cord matrix cells exhibited fibroblastoid morphology with colony formation and self-renewal ability, always maintaining their undifferentiated state up to the eighth passage (P8). The growth curve kinetics exhibited the LAG, LOG, and DECAY phases, without displaying a PLATEAU phase. The plasticity assay demonstrated positive differentiation for osteogenic, adipogenic and chondrogenic lines, characterized by the synthesis of intracytoplasmic granules or extracellular matrix with the presence of calcium, lipids and proteoglycans. Flow cytometry demonstrated the expression of CD90 and CD105; absence of CD14 expression. It is concluded that the cell population isolated from the Wharton's jelly of goat constitutes a representative sample of mesenchymal stem cells, with great possibilities in the field of regenerative and reproductive medicine.

Following myocardial infarction (MI), progressive death of cardiomyocytes and subsequent loss of the extracellular matrix leads to drastic alterations in the structure and mechanical performance of the heart, thereby leading to infarct expansion and cardiac dysfunction. To compensate for the lack of reparative potency in infarcted hearts and to inhibit negative remodeling in the myocardium after MI, stem cell-based therapy in combination with hydrogels has emerged as a promising strategy to improve cardiac function recovery. In this study, a novel injectable hydrogel derived from decellularized Wharton's jelly extracellular matrix (DWJM) is prepared and examined the therapeutic performance of a combination of bioactive DWJM hydrogels and Wharton's jelly mesenchymal stem cells (WJMSCs) for myocardial repair in ischemic rats. In vitro examinations indicated that the DWJM hydrogel exhibited appropriate rheological performance and is capable of undergoing sol-gel transition at 37°C. After intramyocardial injection in MI rats, DWJM-trapped WJMSCs significantly improved cardiac function recovery, reduced scar formation, and promoted cardiomyogenesis and microvascular renewal compared to WJMSCs or DWJM hydrogels alone. The results demonstrated that the DWJM hydrogel and WJMSCs synergistically promoted myocardial repair, which further confirmed the promising stem cell therapy using the bioactive ECM hydrogel.

The persistence of hepatotoxicity induced by N-acetyl-para-aminophenol (Acetaminophen or Paracetamol, abbreviated as APAP) as the most common cause of acute liver failure in the United States, despite the availability of N-acetylcysteine, illustrates the clinical relevance of additional therapeutic approaches. While human mesenchymal stem cells (MSCs) have shown protection in mouse models of liver injury, the MSCs used are generally not cleared for human use and it is unclear whether these effects are due to xenotransplantation. Here we evaluated GMP manufactured clinical grade human Wharton's Jelly mesenchymal stem cells (WJMSCs), which are currently being investigated in human clinical trials, in a mouse model of APAP hepatotoxicity in comparison to human dermal fibroblasts (HDFs) to address these issues. C57BL6J mice were treated with a moderate APAP overdose (300mg/kg) and WJMSCs were administered 90min later. Liver injury was evaluated at 6 and 24h after APAP. WJMSCs treatment reduced APAP-induced liver injury at both time points unlike HDFs, which showed no protection. APAP-induced JNK activation as well as AIF and Smac release from mitochondria were prevented by WJMSCs treatment without influencing APAP bioactivation. Mechanistically, WJMSCs treatment upregulated expression of Gclc and Gclm to enhance recovery of liver GSH levels to attenuate mitochondrial dysfunction and accelerated recovery of pericentral hepatocytes to re-establish liver zonation and promote liver homeostasis. Notably, preventing GSH resynthesis with buthionine sulfoximine prevented the protective effects of WJMSCs. These data indicate that these GMP-manufactured WJMCs could be a clinically relevant therapeutic approach in the management of APAP hepatotoxicity in humans.