Wharton's Jelly Mesenchymal Stem Cells Research Articles

BIOFUNCTIONALIZED 3D-BIOPRINTED VASCULAR-LIKE STRUCTURES: AN ADVANCED APPROACH TO PROMOTE ENDOTHELIAL TISSUE REGENERATION

Biofabrication is a popular technique to produce personalized constructs for tissue engineering. In this study we combined laponite (Lap), gellan gum (GG) with platelet-rich plasma (PRP) aiming to enhance the endothelial regeneration through the synergistic effects of their individual properties. Laponite has the ability to form porous three-dimensional networks mimicking the extracellular matrix structure, and PRP delivery of growth factors stimulates the endothelial cell proliferation and migration, offering a composite bioink for cell growth and support. The sustained release of these growth factors from the GG-laponite-PRP composite material over time provides a continuous source of stimulation for the cells, leading to more effective tissue engineering strategies for endothelial tissue regeneration. Four blend compositions comprising 1% w/v GG and 0.5 or 1% w/v Lap and 25% v/v PRP were combined with Wharton jelly mesenchymal stem cells (WJ-MSCs) and bioprinted into vessel-like structures with an inner diameter of 3 mm and a wall thickness of 1 mm. Stress/strain analysis revealed the elastomeric properties of the hydrogels with Young modulus values of 10 MPa. Increasing the Lap concentration led to a non-significant decrease of swelling ratio from 93 to 91%. Live/dead assay revealed cell viability of at least 76%, with the 0.5%Lap-GG viability exceeding 99% on day 21. Gradual increase of glycosaminoglycans accumulation and collagen production indicate promotion of ECM formation. The expression and membranous localization of PECAM-1 from day 7 and the granular intracellular localization of vWF after 2 weeks demonstrate in vitro endothelial functionality. In vivo subcutaneous implantation indicated the absence of any adverse immunological reactions. The results reveal the expression of both vWF and PECAM-1 by WJ-MSCs entrapped in all four construct compositions with significantly higher expression of vWF in the presence of PRP.

Early Detection of Functional Changes in an Intraocular Hypertension Rabbit Model Treated with Human Wharton Jelly Mesenchymal Stem Cells (hWJ-MSCs) Using Chromatic Pupillometry

Early Detection of Functional Changes in an Intraocular Hypertension Rabbit Model Treated with Human Wharton Jelly Mesenchymal Stem Cells (hWJ-MSCs) Using Chromatic Pupillometry

Advances in the Treatment of Autism Spectrum Disorder: A Comprehensive Case Report on Wharton Jelly Mesenchymal Stem Cell Transplantation

Advances in the Treatment of Autism Spectrum Disorder: A Comprehensive Case Report on Wharton Jelly Mesenchymal Stem Cell Transplantation

Potential antiaging activity of secretome gel of human Wharton's jelly mesenchymal stem cells (hWJ-MSCs) in UV-induced mice models.

Skin aging is a degenerative process that can be induced by UV irradiation. UV radiation can produce reactive oxidate stress which causes premature aging. This study aims to examine the antiaging potential of secretome gel (SC) from human Wharton Jelly Mesenchymal Stem Cells (hWJ-MSCs) in a UVB-induced mice model. The secretome was obtained from hWJ-MSCs and made in gel form. Male mice were radiated by UVB for 15 min twice daily for 14 days. The gel was topically applied to the mice's dorsal skin. Two treatments of secretome gel: secretome 1 is applied once and secretome 2 is applied twice daily after UVB radiation. TGF-β1, IL-10, and IL-18 gene expression was determined using RT-PCR. Hematoxylin Eosin staining was used to observe the inflammation and collagen density of skin tissue. An immunohistochemistry assay was used to analyze the protein expression of P53, COL4A1, MMP-2, and MMP-13. The data were statistically analyzed using the ANOVA test followed by the Tukey post hoc test (P<0.05). UVB induction caused loss of collagen, increasing inflammation and high expression of aging mediators. SC increased the gene expression of TGF-β1 and IL-10 and decreased IL-18 gene expression. Histopathological tests showed that SG increased collagen density, lowered inflammation, and repaired cell damage in skin tissue. Immunohistochemistry test showed that SC decreased MMP-2, MMP-13, and P53 expression, in contrast, increased COL4A1. The secretome gel of hWJ-MSCs showed antiaging activities with potential for preventing and curing skin aging.

Bifunctional tissue-engineered composite construct for bone regeneration: The role of copper and fibrin.

Bifunctional tissue engineering constructs promoting osteogenesis and angiogenesis are essential for bone regeneration. Metal ion-incorporated scaffolds and fibrin encapsulation attract much attention due to low cost, nontoxicity, and tunable control over ion and growth factor release. Herein, we investigated the effect of Cu.nHA/Cs/Gel scaffold and fibrin encapsulation on osteogenic and angiogenic differentiation of Wharton's jelly mesenchymal stem cells (WJMSCs) in vitro and in vivo. Cu-laden scaffolds were synthesized using salt leaching/freeze drying and were characterized using standard techniques. WJMSCs were isolated from the human umbilical cord and characterized. WJMSCs with or without encapsulating in fibrin were seeded onto scaffolds, followed by differentiating into the osteogenic lineage for 7 and 21 days. Osteogenic and angiogenic differentiation were evaluated using real-time polymerase chain reaction, western blot, and Alizarin red staining. Then, scaffolds were implanted into critical-sized calvarial bone defects in rats and histological assessments were performed using hematoxylin/eosin, Masson's trichrome, and CD31 immunohistochemical staining at 4 and 12 weeks. The scaffolds had good physicochemical and biological characteristics suitable for cell attachment and growth. Cu and fibrin increased the expression of ALP, RUNX2, OCN, COLI, VEGF, and HIF1α in differentiated WJMSCs. Implanted scaffolds were also biocompatible and were integrated well with the host tissue. Increased collagen condensation, mineralization, and blood vessel formation were observed in Cu-laden scaffolds. The fibrin-encapsulated groups showed the highest collagen and cell densities, immune cell infiltration, and bone trabeculae. CD31-positive cell population increased with fibrin encapsulation and seeding onto Cu-laden scaffolds. Adding Cu to scaffolds and encapsulating cells in fibrin are promising methods that guide osteogenesis and angiogenesis cellular signaling, leading to better bone regeneration.

Chitosan microspheres loaded with WJMSCs facilitate the restoration of ovarian function in CTX‐induced premature ovarian failure mice

AbstractPremature ovarian failure (POF) is a complex disease affecting an increasing number of young women. Traditional treatments are limited in low efficacy and side effects. Wharton's jelly mesenchymal stem cells (WJMSCs) therapy holds promise in the treatment of POF. However, the clinical application of WJMSCs is hindered by challenges such as low cell viability, significant loss, and poor survival rates. In this study, we prepared three‐dimensional chitosan microspheres (CSM). CSM loading substantial improved the proliferative ability and cell viability of WJMSCs. Furthermore, transplantation of CSM loaded with WJMSCs into ovaries of POF mice increased serum levels of Estradiol (E2) and Anti‐Müllerian hormone (AMH), alongside decreased levels of follicle‐stimulating hormone (FSH). CSM+WJMSCs transplantation enhanced the number of growth follicles while reduced atresia follicles, and effective alleviated the ovarian fibrosis and apoptosis within the ovarian cortex. CSM also prolonged the residence time of WJMSCs in the ovarian capsule. Moreover, superovulation experiments illustrated that CSM+WJMSCs transplantation enhanced the total number of oocytes and reduced oocyte abnormality rates. Overall, CSM+WJMSCs transplantation promotes follicle development and regeneration, restoring ovarian function by inhibiting ovarian cell apoptosis and reducing levels of fibrosis. This study presents a novel therapeutic strategy that combines biomaterial and mesenchymal stem cells for the clinical treatment of premature ovarian failure.

The Comparative Effects of Schwann Cells and Wharton's Jelly Mesenchymal Stem Cells on the AIM2 Inflammasome Activity in an Experimental Model of Spinal Cord Injury

Inflammasome activation and the consequent release of pro-inflammatory cytokines play a crucial role in the development of sensory/motor deficits following spinal cord injury (SCI). Immunomodulatory activities are exhibited by Schwann cells (SCs) and Wharton's jelly mesenchymal stem cells (WJ-MSCs). In this study, we aimed to compare the effectiveness of these two cell sources in modulating the absent in melanoma 2 (AIM2) inflammasome complex in rats with SCI. The Basso, Beattie, Bresnahan (BBB) test, Nissl staining, and Luxol fast blue (LFB) staining were performed to evaluate locomotor function, neuronal survival, and myelination, respectively. Real-time polymerase chain reaction (RT-PCR), Western blotting, and enzyme-linked immunosorbent assay (ELISA) were employed to analyze the gene and protein expressions of inflammasome components, including AIM2, ASC, caspase-1, interleukin-1β (IL-1β), and IL-18. Both gene and protein expressions of all evaluated factors were decreased after SC or WJ-MSC treatment, with a more pronounced effect observed in the SCs group (P < 0.05). Additionally, SCs promoted neuronal survival and myelination. Moreover, the administration of 3 × 105 cells resulted in motor recovery improvement in both treatment groups (P < 0.05). Although not statistically significant, these effects were more prominent in the SC-treated animals. In conclusion, SC therapy demonstrated greater efficacy in targeting AIM2 inflammasome activation and the associated inflammatory pathway in SCI experiments compared to WJ-MSCs.

Effects of royal jelly on the antisenescence, mitochondrial viability and osteogenic differentiation capacity of umbilical cord-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are multipotent cells that have the ability to self-renew and regulate paracrine signalling and immune system processes. MSCs have extensive clinical applications in regeneration, functional reconstruction and cellular therapies. However, studies are needed to discover ways to improve the properties of MSCs, such as differentiation, and prevent senescence in culture, which are both very important for cell therapies. Royal jelly (RJ) is a nutritional substance produced by worker bees that contains a substantial amounts of proteins that are beneficial for cell growth and proliferation. RJ is widely used in traditional medicine today, and due to the specific components in its content, it has been reported to have antioxidant, antiproliferative, antimicrobial, neuroprotective, anti-inflammatory, immunomodulatory and anti-ageing properties. In our study, human Wharton's jelly mesenchymal stem cells (WJ-MSCs) derived from umbilical cord matrix were grown in culture medium supplemented with RJ. The control group comprised minimum essential medium (MEM) and 10% foetal bovine serum (FBS); RJ groups were formed using MEM, 10% FBS and 0.075mg/ml or 0.150mg/ml RJ. In our study, we evaluated the effect of RJ on WJ-MSC growth by MTT assay, proliferating cell nuclear antigen ELISA, β-galactosidase activity assay, MitoTracker Green staining and differentiation tests in adipogenic, osteogenic and chondrogenic cell lines. It was observed that the number of mitochondria increased, senescence decreased and osteogenic differentiation increased after differentiation induction after the addition of RJ to MSC culture. In general, the results of this study indicate that WJ-MSCs enhance mitochondrial numbers and important cellular activities, such as antisenescence and osteogenic differentiation, and with increasing evidence from further studies, RJ supplementation may be found beneficial for the use of MSCs in bone engineering regenerative medicine or cell therapy.

Wharton's Jelly mesenchymal stromal cell-derived extracellular vesicles promote nucleus pulposus cell anabolism in an in vitro 3D alginate-bead culture model.

Intradiscal transplantation of mesenchymal stromal cells (MSCs) has emerged as a promising therapy for intervertebral disc degeneration (IDD). However, the hostile microenvironment of the intervertebral disc (IVD) may compromise the survival of implanted cells. Interestingly, studies reported that paracrine factors, such as extracellular vesicles (EVs) released by MSCs, may regenerate the IVD. The aim of this study was to investigate the therapeutic effects of Wharton's Jelly MSC (WJ-MSC)-derived EVs on human nucleus pulposus cells (hNPCs) using an in vitro 3D alginate-bead culture model. After EV isolation and characterization, hNPCs isolated from surgical specimens were encapsulated in alginate beads and treated with 10, 50, and 100 μg/mL WJ-MSC-EVs. Cell proliferation and viability were assessed by flow cytometry and live/dead staining. Nitrite and glycosaminoglycan (GAG) content was evaluated through Griess and 1,9-dimethylmethylene blue assays. hNPCs in alginate beads were paraffin-embedded and stained for histological analysis (hematoxylin-eosin and Alcian blue) to assess extracellular matrix (ECM) composition. Gene expression levels of catabolic (MMP1, MMP13, ADAMTS5, IL6, NOS2), anabolic (ACAN), and hNPC marker (SOX9, KRT19) genes were analyzed through qPCR. Collagen type I and type II content was assessed with Western blot analysis. Treatment with WJ-MSC-EVs resulted in an increase in cell content and a decrease in cell death in degenerated hNPCs. Nitrite production was drastically reduced by EV treatment compared to the control. Furthermore, proteoglycan content was enhanced and confirmed by Alcian blue histological staining. EV stimulation attenuated ECM degradation and inflammation by suppressing catabolic and inflammatory gene expression levels. Additionally, NPC phenotypic marker genes were also maintained by the EV treatment. WJ-MSC-derived EVs ameliorated hNPC growth and viability, and attenuated ECM degradation and oxidative stress, offering new opportunities for IVD regeneration as an attractive alternative strategy to cell therapy, which may be jeopardized by the harsh microenvironment of the IVD.

Efficacy of Wharton Jelly Mesenchymal Stromal Cells infusions in moderate to severe SARS-Cov-2 related acute respiratory distress syndrome: a phase 2a double-blind randomized controlled trial.

The COVID-19 pandemic caused a wave of acute respiratory distress syndrome (ARDS) with a high in-hospital mortality, especially in patients requiring invasive mechanical ventilation. Wharton Jelly-derived Mesenchymal Stromal Cells (WJ-MSCs) may counteract the pulmonary damage induced by the SARS-CoV-2 infection through pro-angiogenic effects, lung epithelial cell protection, and immunomodulation. In this randomized, double-blind, placebo-controlled phase 2a trial, adult patients receiving invasive mechanical ventilation for SARS-CoV-2 induced moderate or severe ARDS were assigned to receive 1 intravenous infusion of 1 × 106 WJ-MSCs/kg or placebo within 48 h of invasive ventilation followed by 2 infusions of 0.5 × 106 WJ-MSCs/kg or placebo over 5 days. The primary endpoint was the percentage of patients with a PaO2/FiO2 > 200 on day 10. Thirty patients were included from November 2020 to May 2021, 15 in the WJ-MSC group and 15 in the placebo group. We did not find any significant difference in the PaO2/FiO2 ratio at day 10, with 18 and 15% of WJ-MSCs and placebo-treated patients reaching a ratio >200, respectively. Survival did not differ in the 2 groups with a 20% mortality rate at day 90. While we observed a higher number of ventilation-free days at 28 days in the WJ-MSC arm, this difference was not statistically significant (median of 11 (0-22) vs. 0 (0-18), p = 0.2). The infusions were well tolerated, with a low incidence of anti-HLA alloimmunization after 90 days. While treatment with WJ-MSCs appeared safe and feasible in patients with SARS-CoV2 moderate or severe ARDS in this phase 2a trial, the treatment was not associated with an increased percentage of patients with P/F > 200 at 10d, nor did 90 day mortality improve in the treated group. https://beta.clinicaltrials.gov/study/NCT04625738, identifier NCT04625738.

Human Wharton's jelly-derived mesenchymal stem cells prevent pregnancy loss in a rat by JAK/STAT-mediated immunomodulation.

Spontaneous abortion (SA) is a multiple-original syndrome with immune imbalance as one of its major risk factors. As Wharton's jelly-mesenchymal stem cells (WJ-MSCs) are considered to be able to prevent abortion, this study aims to explore the currently poorly understood underlining molecular signaling pathways and regulatory mechanisms of WJ-MSCs in pregnancy maintenance. Abortion mode is established by subcutaneous injection of bromocriptine in rat on day 9 and abortion prevention is achieved by WJ-MSCs injection via tail vein. WJ-MSCs were cultured with/without the inhibitors of JAK/STAT or NF-κB. The uterus was collected on the 14th day of gestation and the rate of embryo absorption was calculated. The expression of Th1/Th2/Th3 cytokines in decidual, placental tissue, and peripheral blood was analyzed. WJ-MSCs treatment significantly reduced the abortion rate in bromocriptine-treated pregnancy such that it was not significantly different from a normal pregnancy. JAK/STAT inhibition abolished pregnancy preserving effects of WJ-MSCs but NF-κB inhibition did not. The levels of Th1-related cytokines and mRNA levels in the bromocriptine abortion model were significantly higher than the normal pregnancy group and ethanol control group, while levels of the Th2-related cytokines and mRNA levels significantly decreased. WJ-MSCs transfusion into the abortion model restored cytokine profiles such that they were not significantly different from the normal pregnancy group and ethanol control group. JAK/STAT inhibition of WJ-MSCs prevented their effect on cytokine and mRNA levels, but NF-κB inhibition did not. WJ-MSCs significantly lower the rate of embryo resorption of spontaneous abortion by reducing Th1-related cytokines while increasing Th2 and Th3-related cytokines in JAK/STAT-dependent manner.

Wharton's jelly mesenchymal stem cells: a concise review of their secretome and prospective clinical applications.

Accumulating evidence indicates that most primary Wharton's jelly mesenchymal stem cells (WJ-MSCs) therapeutic potential is due to their paracrine activity, i.e., their ability to modulate their microenvironment by releasing bioactive molecules and factors collectively known as secretome. These bioactive molecules and factors can either be released directly into the surrounding microenvironment or can be embedded within the membrane-bound extracellular bioactive nano-sized (usually 30-150nm) messenger particles or vesicles of endosomal origin with specific route of biogenesis, known as exosomes or carried by relatively larger particles (100nm-1μm) formed by outward blebbing of plasma membrane called microvesicles (MVs); exosomes and MVs are collectively known as extracellular vesicles (EVs). The bioactive molecules and factors found in secretome are of various types, including cytokines, chemokines, cytoskeletal proteins, integrins, growth factors, angiogenic mediators, hormones, metabolites, and regulatory nucleic acid molecules. As expected, the secretome performs different biological functions, such as immunomodulation, tissue replenishment, cellular homeostasis, besides possessing anti-inflammatory and anti-fibrotic effects. This review highlights the current advances in research on the WJ-MSCs' secretome and its prospective clinical applications.

PSEN1 E280A Cholinergic-like Neurons and Cerebral Spheroids Derived from Mesenchymal Stromal Cells and from Induced Pluripotent Stem Cells Are Neuropathologically Equivalent.

Alzheimer's disease (AD) is a chronic neurological condition characterized by the severe loss of cholinergic neurons. Currently, the incomplete understanding of the loss of neurons has prevented curative treatments for familial AD (FAD). Therefore, modeling FAD in vitro is essential for studying cholinergic vulnerability. Moreover, to expedite the discovery of disease-modifying therapies that delay the onset and slow the progression of AD, we depend on trustworthy disease models. Although highly informative, induced pluripotent stem cell (iPSCs)-derived cholinergic neurons (ChNs) are time-consuming, not cost-effective, and labor-intensive. Other sources for AD modeling are urgently needed. Wild-type and presenilin (PSEN)1 p.E280A fibroblast-derived iPSCs, menstrual blood-derived menstrual stromal cells (MenSCs), and umbilical cord-derived Wharton Jelly's mesenchymal stromal cells (WJ-MSCs) were cultured in Cholinergic-N-Run and Fast-N-Spheres V2 medium to obtain WT and PSEN 1 E280A cholinergic-like neurons (ChLNs, 2D) and cerebroid spheroids (CSs, 3D), respectively, and to evaluate whether ChLNs/CSs can reproduce FAD pathology. We found that irrespective of tissue source, ChLNs/CSs successfully recapitulated the AD phenotype. PSEN 1 E280A ChLNs/CSs show accumulation of iAPPβ fragments, produce eAβ42, present TAU phosphorylation, display OS markers (e.g., oxDJ-1, p-JUN), show loss of ΔΨm, exhibit cell death markers (e.g., TP53, PUMA, CASP3), and demonstrate dysfunctional Ca2+ influx response to ACh stimuli. However, PSEN 1 E280A 2D and 3D cells derived from MenSCs and WJ-MSCs can reproduce FAD neuropathology more efficiently and faster (11 days) than ChLNs derived from mutant iPSCs (35 days). Mechanistically, MenSCs and WJ-MSCs are equivalent cell types to iPSCs for reproducing FAD in vitro.

Vitronectin Mediates Survival of Human WJ-MSCs Under Inflammatory Temperature Stress Via Cell Cycle Arrest

BACKGROUND: Due to their anti-inflammatory and immunomodulatory capabilities, mesenchymal stem cells (MSCs) are being widely used in cell-based therapies for the treatment of a wide spectrum of inflammatory disorders. Despite their promises, substantial cell loss post transplantation leads to compromised therapeutic benefits in clinical trials, which remains a challenge to overcome. Inflammatory microenvironment comprises the presence of pro-inflammatory cytokines, elevated temperature, etc., which could hamper MSC viability following transplantation. Thus, identifying the underlying molecular factors controlling survival mechanism under such stress conditions becomes important. Also, since MSCs from different origins have significantly varied biology, choosing the appropriate MSC source could be critical in determining the fate of transplanted MSCs in a stressful milieu. OBJECTIVE: As extracellular matrix (ECM) components can mediate cell survival signals, in the present study, we have evaluated the role of ECM matricellular protein, vitronectin (VTN), in the survival of human umbilical cord-derived Wharton’s Jelly MSCs (WJ-MSCs) under the condition of inflammatory temperature stress. METHODS AND RESULTS: On exposure to 40°C, WJ-MSCs underwent cell cycle arrest with no significant change in viability status, along with an induction in VTN expression both at mRNA and protein levels. Interestingly, inhibition of pro-survival signalling pathways, ERK or PI3K, at 40°C led to further upregulation in VTN expression without any significant impact on viability or cell cycle arrest status. Interestingly, esiRNA mediated knockdown of VTN in WJ-MSCs at 40°C, with or without pro-survival pathway inhibitors, resulted in reduced cellular viability accompanied by a reversal in G0/G1 cell cycle arrest. CONCLUSIONS: These results established VTN as a protective factor that maintained the viability of WJ-MSCs at 40°C temperature stress. Finally, autophagy was indicated as a mechanism adopted by VTN in executing its pro-survival role.

WHARTON'S JELLY MSCS-DERIVED EXOSOMES AS CELL-FREE THERAPEUTIC STRATEGY FOR INTERVERTEBRAL DISC REGENERATION

The aim of this study was to investigate the regenerative effects of Wharton's Jelly Mesenchymal Stem Cells (WJ-MSCs) derived exosomes (WJ-Exos) on human nucleus pulposus cells (hNPCs) in an in vitro 3D model.WJ-Exos were isolated by tangent flow filtration of WJ-MSCs conditioned media and characterized by TEM, WB for markers expression and quantified with NTA. WJ-Exos PKH26-labeled uptake in hNPCs was detected by confocal microscopy. hNPCs, isolated from surgical specimens (n=4), culture expanded in vitro and encapsulated in alginate beads, were pre-treated with IL1β (10 ng/ml) for 24 hours, then with WJ-Exos at 10, 50 and 100 µg/ml. Cells with growth medium were used as control. We examined: i) cell proliferation and viability (flow cytometry), ii) nitrite production (Griess) iii) glycosaminoglycan (GAG) amount (DMBB), iv) histological staining for extracellular matrix (ECM) analysis and v) gene expression levels of catabolic and anabolic genes (qPCR). The investigations were performed in triplicate for each donor. One-way ANOVA analysis was used to compare the groups under exam and data were expressed as mean ± S.D.A dose dependent increase in hNPCs proliferation was noticed at all exos concentrations under study. Cell death decreased significantly in WJ-Exos 50 µg/ml samples (p ≤ 0,05) compared to IL1β treated hNPCs. Nitrite production was significantly attenuated at 10µg/ml of WJ-Exos (p ≤ 0,01). GAG content and histological analysis showed a difference in ECM synthesis between treated and untreated hNPCs (p ≤ 0,05). Catabolic and inflammatory markers were modulated by WJ-Exos at 100 µg/ml concentration (p ≤ 0,05) whereas 10 µg/ml group increased anabolic gene expression levels (p ≤ 0,05).These findings offer new opportunities for the potential use of exosomes as an attractive alternative cell-free strategy of IDD. WJ-MSC exosomes ameliorate hNPCs growth and viability, attenuate ECM degradation and oxidative stress-related IDD progression after IL1β stimulation.Financial support was received from the “iPSpine” and “RESPINE” Horizon 2020 projects.

In vitro evaluation of genipin-crosslinked gelatin hydrogels for vocal fold injection

Glottic insufficiency is one of the voice disorders affecting all demographics. Due to the incomplete closure of the vocal fold, there is a risk of aspiration and ineffective phonation. Current treatments for glottic insufficiency include nerve repair, reinnervation, implantation and injection laryngoplasty. Injection laryngoplasty is favored among these techniques due to its cost-effectiveness and efficiency. However, research into developing an effective injectable for the treatment of glottic insufficiency is currently lacking. Therefore, this study aims to develop an injectable gelatin (G) hydrogel crosslinked with either 1-ethyl-3-(3-dimethylaminpropyl)carbodiimide hydrochloride) (EDC) or genipin (gn). The gelation time, biodegradability and swelling ratio of hydrogels with varying concentrations of gelatin (6–10% G) and genipin (0.1–0.5% gn) were investigated. Some selected formulations were proceeded with rheology, pore size, chemical analysis and in vitro cellular activity of Wharton's Jelly Mesenchymal Stem Cells (WJMSCs), to determine the safety application of the selected hydrogels, for future cell delivery prospect. 6G 0.4gn and 8G 0.4gn were the only hydrogel groups capable of achieving complete gelation within 20 min, exhibiting an elastic modulus between 2 and 10 kPa and a pore size between 100 and 400 μm. Moreover, these hydrogels were biodegradable and biocompatible with WJMSCs, as > 70% viability were observed after 7 days of in vitro culture. Our results suggested 6G 0.4gn and 8G 0.4gn hydrogels as potential cell encapsulation injectates. In light of these findings, future research should focus on characterizing their encapsulation efficiency and exploring the possibility of using these hydrogels as a drug delivery system for vocal fold treatment.

Three-Dimensional Bioprinting of an In Vitro Lung Model.

In December 2019, COVID-19 emerged in China, and in January 2020, the World Health Organization declared a state of international emergency. Within this context, there is a significant search for new drugs to fight the disease and a need for in vitro models for preclinical drug tests. This study aims to develop a 3D lung model. For the execution, Wharton's jelly mesenchymal stem cells (WJ-MSC) were isolated and characterized through flow cytometry and trilineage differentiation. For pulmonary differentiation, the cells were seeded in plates coated with natural functional biopolymer matrix as membrane until spheroid formation, and then the spheroids were cultured with differentiation inductors. The differentiated cells were characterized using immunocytochemistry and RT-PCR, confirming the presence of alveolar type I and II, ciliated, and goblet cells. Then, 3D bioprinting was performed with a sodium alginate and gelatin bioink in an extrusion-based 3D printer. The 3D structure was analyzed, confirming cell viability with a live/dead assay and the expression of lung markers with immunocytochemistry. The results showed that the differentiation of WJ-MSC into lung cells was successful, as well as the bioprinting of these cells in a 3D structure, a promising alternative for in vitro drug testing.

AMPK Regulates DNA Methylation of PGC-1α and Myogenic Differentiation in Human Mesenchymal Stem Cells.

Adverse intrauterine environments can cause persistent changes in epigenetic profiles of stem cells, increasing susceptibility of the offspring to developing metabolic diseases later in life. Effective approaches to restore the epigenetic landscape and function of stem cells remain to be determined. In this study, we investigated the effects of pharmaceutical activation of AMP-activated protein kinase (AMPK), an essential regulator of energy metabolism, on mitochondrial programming of Wharton's Jelly mesenchymal stem cells (WJ-MSCs) from women with diabetes during pregnancy. Induction of myogenic differentiation of WJ-MSCs was associated with increased proliferator-activated receptor-γ coactivator-1α (PGC-1α) expression and mitochondrial DNA (mtDNA) abundance. Inhibition of DNA methylation by 5 Azacytidine significantly increased PGC-1α expression and mtDNA abundance in WJ-MSCs, which were abolished by AMPK inhibitor Compound C (CC), suggesting an AMPK-dependent role of DNA demethylation in regulating mitochondrial biogenesis in WJ-MSCs. Furthermore, activation of AMPK in diabetic WJ-MSCs by AICAR or metformin decreased the level of PGC-1α promoter methylation and increased PGC-1α expression. Notably, decreased PGC-1α promoter methylation by transient treatment of AMPK activators persisted after myogenic differentiation. This was associated with enhanced myogenic differentiation capacity of human WJ-MSCs and increased mitochondrial function. Taken together, our findings revealed an important role for AMPK activators in epigenetic regulation of mitochondrial biogenesis and myogenesis in WJ-MSCs, which could lead to potential therapeutics for preventing fetal mitochondrial programming and long-term adverse outcome in offspring of women with diabetes during pregnancy.

A Three-Dimensional Xeno-Free Culture Condition for Wharton's Jelly-Mesenchymal Stem Cells: The Pros and Cons.

Xeno-free three-dimensional cultures are gaining attention for mesenchymal stem cell (MSCs) expansion in clinical applications. We investigated the potential of xeno-free serum alternatives, human serum and human platelet lysate, to replace the current conventional use of foetal bovine serum for subsequent MSCs microcarrier cultures. In this study, Wharton's Jelly MSCs were cultured in nine different media combinations to identify the best xeno-free culture media for MSCs culture. Cell proliferation and viability were identified, and the cultured MSCs were characterised in accordance with the minimal criteria for defining multipotent mesenchymal stromal cells by the International Society for Cellular Therapy (ISCT). The selected culture media was then used in the microcarrier culture of MSCs to determine the potential of a three-dimensional culture system in the expansion of MSCs for future clinical applications, and to identify the immunomodulatory potential of cultured MSCs. Low Glucose DMEM (LG) + Human Platelet (HPL) lysate media appeared to be good candidates for replacing conventional MSCs culture media in our monolayer culture system. MSCs cultured in LG-HPL achieved high cell yield, with characteristics that remained as described by ISCT, although the overall mitochondrial activity of the cells was lower than the control and the subsequent effects remained unknown. MSC microcarrier culture, on the other hand, showed comparable cell characteristics with monolayer culture, yet had stagnated cell proliferation, which is potentially due to the inactivation of FAK. Nonetheless, both the MSCs monolayer culture and the microcarrier culture showed high suppressive activity on TNF-α, and only the MSC microcarrier culture has a better suppression of IL-1 secretion. In conclusion, LG-HPL was identified as a good xeno-free media for WJMSCs culture, and although further mechanistic research is needed, the results show that the xeno-free three-dimensional culture maintained MSC characteristics and improved immunomodulatory activities, suggesting the potential of translating the monolayer culture into this culture system in MSC expansion for future clinical application.

Additive manufacturing of barium-doped calcium silicate/poly-ε-caprolactone scaffolds to activate CaSR and AKT signalling and osteogenic differentiation of mesenchymal stem cells.

3D-printed scaffolds are suitable for patient-specific implant preparation for bone regeneration in large-scale critical bone defects. In addition, these scaffolds should have mechanical and biological properties similar to those of natural bone tissue. In this study, 3D-printed barium-doped calcium silicate (BaCS)/poly-ε-caprolactone (PCL) composite scaffolds were fabricated as an alternative strategy for bone tissue engineering to achieve appropriate physicochemical characteristics and stimulate osteogenesis. Scaffolds containing 10% Ba (Ba10) showed optimal mechanical properties, preventing premature scaffold degradation during immersion while enabling ion release in a sustained manner to achieve the desired therapeutic goals. In addition, Wharton's jelly mesenchymal stem cells (WJMSCs) were used to assess biocompatibility and osteogenic differentiation behaviour. WJMSCs were cultured on the scaffold and permeabilised via ICP to analyse the presence of Si and Ba ions in the medium and cell lysates, suggesting that the ions released by the scaffold could effectively enter the cells. The protein expression of CaSR, PI3K, Akt, and JNK confirmed that CaSR could activate cells cultured in Ba10, thereby affecting the subsequent PI3k/Akt and JNK pathways and further promoting osteogenic differentiation. The in vivo performance of the proposed scaffolds was assessed using micro-CT and histological slices, which revealed that the BaCS scaffolds could further enhance bone regeneration, compared with bare scaffolds. These results suggest the potential use of 3D-printed BaCS/PCL scaffolds as next-generation substitutes for bone regeneration.