The Optimal Effective Dose of Adipose-Derived Stem Cell Exosomes in Wound Healing.

INTRODUCTION: Although the effect of adipose-derived mesenchymal stem cell exosomes (ADSC-exos) on wound healing with different doses are shown in various studies, efficient and sufficient doses of ADSC-exos are still unknown. The study aimed to determine the optimal dose of ADSC-exos in wound healing. METHODS: The 45 Sprague-Dawley rats were randomly divided into five groups, with seven animals in each. After dorsal circular defects were created, each wound was injected as follows: group 1: saline, group 2: 10 µg/mL of ADSC-exos, group 3: 100 µg/mL of ADSC-exos, group 4: 200 µg/mL of ADSC-exos, and group 5: 400 µg/mL of ADSC-exos. The effects of ADSC-exos on epithelization, angiogenesis, and collagen formation were analyzed macroscopically, histopathologically, and immunohistochemically on day 14. RESULTS: A total of 200 µg/mL and 400 µg/mL ADSC-exos groups had higher epithelial tongue length, epithelial tongue area, and angiogenesis scores than the other groups. Although there was no statistical difference in fibrosis scores among groups, collagen fibers were becoming well-organized as the ADSC-exos doses increased. While the wound area was clinically smaller in the 200 µg/mL ADSC-exos group, there was no statistically significant difference among groups on day 14. CONCLUSIONS: A total of 200 µg/mL of ADSC-exos was found to be the adequate and effective dose for re-epithelialization and angiogenesis in cutaneous wound healing. Moreover, the collagen density increased with a more regular pattern in the 200 µg/mL group, which can be important in scar regulation.

Dental Pulp-Derived Mesenchymal Stem Cells Increase Axon Numbers in Mental Nerve Repair.

Aim: The mental nerve, the extended part of the inferior alveolar nerve, is often injured during dentoalveolar, orthognathic, or tumor surgery. Numerous therapeutic interventions, including surgery and pharmacotherapy, have been used to enhance the recovery of nerve injuries. Dental pulp stem cells (DPSCs) represent an easily accessible source of adult stem cells that can be isolated from the pulp of extracted teeth. This study evaluated the effect of DPSCs on the regeneration of the mental nerve injury model of rabbits. Methods: In this presented study, DPSCs were cultured and cell characterizations were performed by using flow cytometry and immunostainings. Bilateral mental nerve injury models of rabbits were created. In the control group (n = 10), saline was applied, and in the study group (n = 10), 2 × 106 DPSCs were applied to the repaired nerve areas. After 3 weeks, animals were killed and histological examination was obtained by using Masson's trichrome staining. An unpaired Student's t test was used when comparing the groups. Differences were considered to be statistically significant at P values of less than 0.05. Results: The DPSCs demonstrated a homogeneous population of mesenchymal stromal cells which expressed cluster of differentiation CD44, CD73, CD90, and CD105 and lack of CD34, CD45, and HLA-DR. Our finding clearly demonstrated that a lower number of cross-sectioned axons were founded in the control group (60.18 ± 2.52) compared to the study group (72.96 ± 2.43) (p = 0.00). Conclusions: DPSCs promote mental nerve axonal regeneration. These results suggest that DPSCs provide an important accessible source of adult stem cells for mental nerve regeneration.

The efficacy of mesenchymal stem cell treatment and colistin-fosfomycin combination on colistin-resistant Acinetobacter baumannii sepsis model.

INTRODUCTION: This study examines the role of mesenchymal stem cells (MSCs) in an experimental sepsis model developed with colistin-resistant Acinetobacter baumannii (CRAB). MATERIALS AND METHODS: BALB-c mice were divided into treatment groups (MSC, MSC + colistin (C)-fosfomycin (F), and C-F and control groups (positive and negative)). CRAB was administered to mice through intraperitoneal injection. Three hours later, C, F, and MSC were given intraperitoneally to the treatment groups. Colistin administration was repeated every 12 h, F administration was done every 4 h, and the second dose of MSC was administered after 48 h. Mice were sacrificed at 24 and 72 h. The bacterial load was determined as colony-forming units per gram (cfu/g). Histopathological examination was conducted on the left lung, liver, and both kidneys. IL-6 and C-reactive protein (CRP) levels in mouse sera were determined by enzyme-linked immunosorbent assay. RESULTS: Among the treatment groups, the C-F group had the lowest colony count in the lung (1.24 ± 1.66 cfu/g) and liver (1.03 ± 1.08 cfu/g). The highest bacterial clearance was observed at 72 h compared to 24 h in the MSC-treated groups (p = 0.008). The MSC + C-F group showed the lowest histopathological score in the liver and kidney (p = 0.009). In the negative control group, the IL-6 level at the 24th hour was the lowest (p < 0.001). Among the treatment groups, the CRP level was the lowest in the MSC + C-F group at 24 and 72 h. CONCLUSION: In a CRAB sepsis model, adding MSCs to a colistin-fosfomycin treatment may be beneficial in terms of reducing bacterial loads and preventing histopathological damage.

Stability of box genioplasty: a retrospective study.

In box genioplasty it is possible to advance, retrude, impact, and elongate, as well as to correct asymmetry. The aim of this study was to analyse the stability of box genioplasty as part of orthognathic correction. Twenty-five consecutive patients who had gone through the multidisciplinary pathway were selected. Menton and pogonion positions on radiographs taken just prior to surgery, and actual surgical movement on three-week and 12-month postoperative cephalograms, were compared. A one-sample Wilcoxon test was applied to assess whether the distributional change in advancement and vertical measurements was equal to zero. After treatment, anteroposterior changes in pogonion and vertical changes in menton were statistically insignificant (p>0.05). Our study demonstrated statistically significant stability of menton and pogonion positions after box genioplasty when surgical movement was only in the symphysis.

Local application of gingiva-derived mesenchymal stem cells on experimental periodontitis in rats.

BACKGROUND: Stem cell-based approaches in regenerative periodontal therapy have been used in different experimental models. In this study, the effect of local application of gingival mesenchymal stem cells (GMSC) in fibroin/chitosan oligosaccharide lactate hydrogel (F/COS) on periodontal regeneration was evaluated using experimental periodontitis model in rats. METHODS: Mesenchymal stem cells were isolated from the gingiva of rats and characterized. Viability tests and confocal imaging of GMSC in hydrogels were performed. Healthy control without periodontitis (Health; H; n=10), control with periodontitis but no application (Periodontitis; P; n=10), only hydrogel application (F/COS; n=10), and GMSC+F/COS (n=10) four groups were formed for in vivo studies. Experimental periodontitis was created with silk sutures around the maxillary second molars. GMSC labeled with green fluorescent protein (GFP) (250,000 cells/50 µL) in F/COS were applied to the defect. Animals were sacrificed at 2nd and 8th weeks and maxillae of the animals were evaluated by micro-computed tomography (micro-CT) and histologically. The presence of GFP-labeled GMSC was confirmed at the end of 8 weeks. RESULTS: Micro-CT analysis showed statistically significant new bone formation in the F/COS+GMSC treated group compared with the P group at the end of 8 weeks (p < 0.05). New bone formation was also observed in the F/COS group, but the statistical analysis revealed that this difference was not significant when compared with the P group (p > 0.05). Long junctional epithelium formation was less in the F/COS+GMSC group compared with the P group. Periodontal ligament and connective tissue were well-organized in F/COS+GMSC group. CONCLUSION: The results showed that local GMSC application in hydrogel contributed to the formation of new periodontal ligament and alveolar bone in rats with experimental periodontitis. Since gingiva is easly accessible tissue, it is promising for autologous cell-based treatments in clinical applications.

Relationship Between Temporomandibular Ankylosis And Maximum Mouth Opening In Children.

BACKGROUND: Pediatric dentists should have information regarding whether mouth opening is limited. In clinical practice, these professionals should collect and record oral area measurements at the pediatric patient's first medical examination. OBJECTIVES: The study's aim developed the standard mouth opening measurement in children by using ordinary least squares regression to develop a clinical prediction model in children with Temporomandibular Joint Ankylosis before preoperative surgery. METHODS: All participants completed their age, gender, and calculated height, weight, body mass index, and birth weight. Pediatric dentist performed all mouth-opening measurements. The oral-maxillofacial surgeon marked subnasal and pogonion points for the lower facial length of soft tissue. It was measured using the distance between the subnasal and pogonion with a digital vernier caliper. The widths of the three fingers (index, middle, and ring fingers) and four fingers (index, middle, ring, and little fingers) were also measured using a digital vernier caliper. RESULTS: Maximum mouth opening showed that three-finger width (R2 = 0.566, F = 185.479) and four-finger width (R2 = 0.462, F = 122.209) had a significant influence on the Maximum mouth opening (MMO) (p < 0.001). CONCLUSION: Pediatric dentists should collaborate with the treating maxillofacial surgeon to manage long-term treatment needs for individuals with Temporomandibular Joint Ankylosis.

Safety and efficacy of mesenchymal stromal cell therapy for multi-drug-resistant acute and late-acute graft-versus-host disease following allogeneic hematopoietic stem cell transplantation.

Graft versus host disease (GvHD) remains a significant risk for mortality and morbidity following allogeneic hematopoietic stem cell transplantation (HSCT). A growing literature supports successful applications of mesenchymal stromal cells (MSCs) for the treatment of steroid-refractory acute GvHD (aGvHD). However, there is limited knowledge about the effects of MSC treatment on late-acute GvHD (late aGvHD). In this article, we present our multicenter study on the safety and efficacy of MSC therapy for patients with steroid-refractory late aGvHD in comparison to those with aGvHD. The outcome measures include non-relapse mortality (NRM) and survival probability over a 2-year follow-up. The study includes a total of 76 patients with grades III-IV aGvHD (n = 46) or late aGvHD (n = 30), who had been treated with at least two lines of steroid-containing immunosuppressive therapy. Patients received weekly adipose or umbilical cord-derived MSC infusions at a dose of median 1.55 (ranging from 0.84 to 2.56) × 106/kg in the aGvHD group, and 1.64 (ranging from 0.85 to 2.58) × 106/kg in the late aGvHD group. This was an add-on treatment to ongoing conventional pharmaceutical management. In the aGvHD group, 23 patients received one or two infusions, 20 patients had 3-4, and three had ≥ 5. Likewise, in the late aGvHD group, 20 patients received one or two infusions, nine patients had 3-4, and one had ≥ 5. MSC was safe without acute or late adverse effects in 76 patients receiving over 190 infusions. In aGvHD group, 10.9% of the patients had a complete response (CR), 23.9% had a partial response (PR), and 65.2% had no response (NR). On the other hand, in the late aGvHD group, 23.3% of the patients had CR, 36.7% had PR, and the remaining 40% had NR. These findings were statistically significant (p = 0.031). Also, at the 2-year follow-up, the cumulative incidence of NRM was significantly lower in patients with late aGvHD than in patients with aGvHD at 40% (95% CI, 25-62%) versus 71% (95% CI, 59-86%), respectively (p = 0.032). In addition, the probability of survival at 2 years was significantly higher in patients with late aGvHD than in the aGvHD group at 59% (95% CI, 37-74%) versus 28% (95% CI, 13-40%), respectively (p = 0.002). To our knowledge, our study is the first to compare the safety and efficacy of MSC infusion(s) for the treatment of steroid-resistant late aGVHD and aGVHD. There were no infusion-related adverse effects in either group. The response rate to MSC therapy was significantly higher in the late aGvHD group than in the aGvHD group. In addition, at the 2-year follow-up, the survival and NRM rates were more favorable in patients with late aGVHD than in those with aGVHD. Thus, the results are encouraging and warrant further studies to optimize MSC-based treatment for late aGVHD.

The neuroprotective effect of mesenchymal stem cells in colistin-induced neurotoxicity.

Colistin is an effective antibiotic against multidrug-resistant gram-negative bacterial infections; however, neurotoxic effects are fundamental dose-limiting factors for this treatment. Stem cell therapy is a promising method for treating neuronal diseases. Multipotent mesenchymal stromal cells (MSC) represent a promising source for regenerative medicine. Identification of neuroprotective agents that can be co-administered with colistin has the potential to allow the clinical application of this essential drug. This study was conducted to assess the potential protective effects of MSC, against colistin-induced neurotoxicity, and the possible mechanisms underlying any effect. Forty adult female albino rats were randomly classified into four equal groups; the control group, the MSC-treated group (A single dose of 1 × 106/mL MSCs through the tail vein), the colistin-treated group (36 mg/kg/d colistin was given for 7 d) and the colistin and MSC treated group (36 mg/kg/d colistin was administered for 7 d, and 1 × 106/mL MSCs). Colistin administration significantly increased GFAP, NGF, Beclin-1, IL-6, and TNF-α immunreactivity intensity. MSC administration in colistin-treated rats partially restored each of these markers. Histopathological changes in brain tissues were also alleviated by MSC co-treatment. Our study reveals a critical role of inflammation, autophagy, and apoptosis in colistin-induced neurotoxicity and showed that they were markedly ameliorated by MSC co-administration. Therefore, MSC could represent a promising agent for prevention of colistin-induced neurotoxicity.

EVs vs. EVs: MSCs and Tregs as a source of invisible possibilities.

Extracellular vesicles (EVs) are produced by various cells and exist in most biological fluids. They play an important role in cell-cell signaling, immune response, and tumor metastasis, and also have theranostic potential. They deliver many functional biomolecules, including DNA, microRNAs (miRNA), messenger RNA (mRNA), long non-coding RNA (lncRNA), lipids, and proteins, thus affecting different physiological processes in target cells. Decreased immunogenicity compared to liposomes or viral vectors and the ability to cross through physiological barriers such as the blood-brain barrier make them an attractive and innovative option as diagnostic biomarkers and therapeutic carriers. Here, we highlighted two types of cells that can produce functional EVs, namely, mesenchymal stem/stromal cells (MSCs) and regulatory T cells (Tregs), discussing MSC/Treg-derived EV-based therapies for some specific diseases including acute respiratory distress syndrome (ARDS), autoimmune diseases, and cancer.

A pilot study for treatment of severe COVID-19 pneumonia by aerosolized formulation of convalescent human immune plasma exosomes (ChipEXO™).

This is a single-center prospective, open-label, single arm interventional study to test the safety and efficacy of recently described ChipEXO™ for severe COVID-19 pneumonia. The ChipEXO™ is a natural product derived from convalescent human immune plasma of patients recovered from moderate COVID-19 infection. In September 2021, 13 patients with pending respiratory failure were treated with ChipEXO™ adapted for aerosolized formulation delivered via jet nebulizer. Patients received 1-5x1010 nano vesicle/5 mL in distilled water twice daily for five days as an add-on to ongoing conventional COVID-19 treatment. The primary endpoint was patient safety and survival over a 28-day follow-up. The secondary endpoint was longitudinal assessment of clinical parameters following ChipEXO™ to evaluate treatment response and gain insights into the pharmacodynamics. ChipEXO™ was tolerated well without any allergic reaction or acute toxicity. The survival rate was 84.6% and 11 out of 13 recovered without any sequel to lungs or other organs. ChipEXO™ treatment was effective immediately as shown in arterial blood gas analyses before and two hours after exosome inhalation. During the 5 days of treatment, there was a sustainable and gradual improvement on oxygenation parameters: i.e. respiratory rate (RR) [20.8% (P < 0.05)], oxygen saturation (SpO2) [6,7% (P < 0.05)] and partial pressure of oxygen to the fraction of inspired oxygen (PaO2/FiO2) [127.9% (P < 0.05)] that correlated with steep decrease in the disease activity scores and inflammatory markers, i.e. the sequential organ failure assessment (SOFA) score (75%, p < 0.05), C-reactive protein (46% p < 0.05), ferritin (58% p = 0.53), D-dimer (28% p=0.46). In conclusion, aerosolized ChipEXO™ showed promising safety and efficacy for life-threatening COVID-19 pneumonia. Further studies on larger patient populations are required to confirm our findings and understand the pathophysiology of improvement toward a new therapeutic agent for the treatment of severe COVID-19 pneumonia.

Repair of Rat Calvarial Bone Defect by Using Exosomes of Umbilical Cord-Derived Mesenchymal Stromal Cells Embedded in Chitosan/Hydroxyapatite Scaffolds.

PURPOSE: To seek out the bone regeneration effect of human umbilical cord-mesenchymal stem cell (hUC-MSC)-derived exosomes of loaded chitosan/hydroxyapatite (CS/HA) scaffold in a rat calvarium bone regeneration model. MATERIALS AND METHODS: The hUC-MSC exosomes were purified and characterized. The scaffolds were prepared by a freeze-drying method. Animals were divided into five groups, and the CS/HA/exosome (CS/HA/Exo) scaffolds were transplanted to 5 × 2-mm critical-sized calvarial bone defects for repair in rats. All animals were sacrificed at the postoperative sixth week. Immunohistochemical and histologic analyses were performed. RESULTS: Scanning electron microscopy (SEM) images showed that the exosomes were round-shaped vesicles with bounded membrane, and the diameter of the exosomes was 83.728 ± 27.269 nm. Histologic analysis showed that mean new bone volumes were statistically significantly higher in the CS/HA/Exo group (1.83 ± 0.54, PCS/Exo-CS/HA/Exo = .000), and other new bone volumes in the other groups were statistically significant compared with the control (CS/Exo 1.50 ± 0.14 mm3; CS 1.20 ± 0.43 mm3; control 1.06 ± 0.10 mm3; and CS/HA 1.43 ± 0.66 mm3). CONCLUSION: The CS/HA/Exo combination is a novel treatment for bone defect repair to induce bone formation. The CS scaffold can significantly promote bone regeneration compared with the control. Moreover, the combination with HA and exosomes is promising for applications in bone tissue regeneration.

Assessment of clinical, biochemical, and radiological outcomes following intra-articular injection of Wharton jelly-derived mesenchymal stromal cells in patients with knee osteoarthritis: A prospective clinical study.

The aim of the present study was to perform clinical, biochemical, and radiological evaluation of the efficacy of mesenchymal stem cells derived from Wharton jelly (WJ) present within the human umbilical cord in the treatment of knee osteoarthritis. Between 2018 and 2019, 10 patients with knee osteoarthritis for whom the conservative treatment was not beneficial were included in the study. Patients were clinically, radiologically, and biochemically evaluated before treatment initiation. Thereafter, the patients were intra-articularly injected using a solution containing 1 × 108 WJ-derived MSCs. Evaluations were performed on day 21 (V1) and 42 (V2) and month 3 (V3), 6 (V4), and 12 (V5) after the procedure. At 1-year post-injection, visual analogue scale, Western Ontario and McMaster Universities Osteoarthritis Index, and Lequesne scores of patients were lower than those observed during the initial evaluation, whereas the mean 36-Item Short Form Health Survey score was higher. Cartilage thicknesses were found to be increased in all regions except in the medial femur, medial posterior femur, lateral posterior femur, and lateral posterior tibia regions in magnetic resonance imaging. A significant increase was observed in tumor necrosis factor-alpha, interleukin-1ß, adiponectin, resistin, and interleukin-6 levels compared with pre-injection values. The leptin levels at 6-month and 1-year controls were lower than the pre-injection levels, and the decrease observed at 6 months was significant. In patients with knee osteoarthritis, intra-articular WJ-derived MSC injection causes significant pain reduction, satisfactory functional improvement, and increased patient satisfaction following a 1-year follow-up. These clinical improvements were supported by magnetic resonance images, along with changes in adiponectin and leptin levels in synovial fluid. Level of evidence: IV.

The Effect of Mesenchymal Stromal Cells on the Mortality of Patients with Sepsis and Septic Shock: A Promising Therapy.

Purpose: Sepsis and septic shock are the major causes of death in intensive care units. This study aimed to evaluate the clinical safety and efficacy of mesenchymal stem cells (MSCs) in sepsis and septic shock patients. Methods: Ten patients were enrolled in the study. Adipose-derived MSC infusions were given (1 × 106/kg, on the 1st, 3rd, 5th, 7th, and 9th days of therapy) together with standard therapy. Before the MSC applications, blood samples were collected for cytokine assessment (TNF-α, IFN-γ, IL-2, IL-4, IL-6, IL-10). The clinical and laboratory improvements were recorded and compared with control groups selected retrospectively. The clinical trial was registered on 16.03.2022 with the registration number NCT05283317. Results: In the study group, the ages of patients ranged from 22 to 68 years, and APACHE II scores ranged from 14 to 42. In the control group, ages ranged from 22 to 80 years and their APACHE II scores were between 14-35. The survival rate in the study group was 100% on the 14th day whereas it was 70% on the 28th day. A significant decrease in the SOFA score (adjusted), clinical, and laboratory improvements were observed during the MSC administration. However, no significant cytokine level changes were observed. In the control group, the survival rate of 20 patients was 70% on the 14th day, whereas 60% was on the 28th day. While deaths were observed in the control group in the first week of treatment, deaths in the MSCs group were observed between the 15th and 28th days. Conclusion: MSCs treatment may have a positive impact on the survival rates of sepsis during the early phase. However, further randomized controlled studies with a large group of patients are needed. Trial Registration. This trial is registered with ClinicalTrials.gov Identifier: NCT05283317.

Efficacy of stem cell-based therapies for colistin-induced nephrotoxicity.

The increase in infections with multidrug resistant bacteria has forced to return to the use of colistin, antibiotic with known nephrotoxicity. Mesenchymal stem cells (MSCs) are being extensively investigated for their potential in regenerative medicine. This study aimed to investigate the possible protective mechanisms of the MSCs against kidney injury induced by colistin. Forty adult female albino rats were randomly classified into 4 equal groups; the control group, the MSC-treated group (a single dose of 1 ×106 /ml MSCs through the tail vein), the colistin-treated group (36 mg/kg/day colistin was given for 7 days), and the both colistin and MSC group (36 mg/kg/day colistin and 1 ×106 /ml MSCs). Main outcome measures were histopathological alterations, kidney malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and immunohistological autophagy evaluation. MSC repressed the progression of colistin-induced kidney injury as evidenced by the improvement of histopathological alterations and the substantial increase MDA, and decrease SOD and CAT in serum levels. Moreover, MSC resulted in a profound reduction in oxidative stress as manifested by decreased MDA and increased SOD in serum. Notably, MSC suppressed colistin-induced autophagy; it reduced renal levels of Beclin-1, P62 and LC3A/B. Furthermore, MSC decreased renal levels of eNOS. Lastly, MSC efficiently decreased expression of the TUNEL positive cell number. MSC confers protection against colistin-induced kidney injury by alleviating oxidative stress, nitric oxide synthase besides modulating reducing autophagy and apoptosis.

The evaluation of changes in maxillofacial bones using cone beam tomography in acromegaly

Background: The aim of the study was to evaluate the changes in craniofacial dimensions of newly diagnosed anduntreated acromegaly patients, patients with non-functional pituitary adenoma and healthy individuals on ConeBeam Computed Tomography (CBCT).Material and Methods: 50 newly diagnosed acromegaly patients who did not receive any treatment for acromegalywere included in the study (Group A). Twenty patients with nonfunctional pituitary adenoma (Group B) and 30healthy individuals were included (Group C). Linear, angular and volumetric measurements were performed.Results: Mandibular length showed significant difference in acromegaly patients, and maxillar length statisticallysignificant difference was found between the A-B and B-C (p> 0,05), no difference was found between the A-C(p<0,05). SNB and ANB angle was statistically different in all groups, while SNA angle was statistically different between group A-C and B-C. In volumetric measurements, a statistically significant difference was foundbetween groups a-c and groups A-B (p< 0,05), no difference was found between groups B-C (p>0,05).Conclusions: CBCT measurements showed that mandibular volume and length were increased in the acromegalygroup compared to the group B-C. Present study is the first research that compares acromegaly patients in respectto changes in maxillofacial dimensions. (AU)

Preclinical Studies on Convalescent Human Immune Plasma-Derived Exosome: Omics and Antiviral Properties to SARS-CoV-2.

The scale of the COVID-19 pandemic forced urgent measures for the development of new therapeutics. One of these strategies is the use of convalescent plasma (CP) as a conventional source for passive immunity. Recently, there has been interest in CP-derived exosomes. In this report, we present a structural, biochemical, and biological characterization of our proprietary product, convalescent human immune plasma-derived exosome (ChipEXO), following the guidelines set forth by the Turkish Ministry of Health and the Turkish Red Crescent, the Good Manufacturing Practice, the International Society for Extracellular Vesicles, and the Gene Ontology Consortium. The data support the safety and efficacy of this product against SARS-CoV-2 infections in preclinical models.

Pathogenic variants in RNPC3 are associated with hypopituitarism and primary ovarian insufficiency.

PURPOSE: We aimed to investigate the molecular basis underlying a novel phenotype including hypopituitarism associated with primary ovarian insufficiency. METHODS: We used next-generation sequencing to identify variants in all pedigrees. Expression of Rnpc3/RNPC3 was analyzed by in situ hybridization on murine/human embryonic sections. CRISPR/Cas9 was used to generate mice carrying the p.Leu483Phe pathogenic variant in the conserved murine Rnpc3 RRM2 domain. RESULTS: We described 15 patients from 9 pedigrees with biallelic pathogenic variants in RNPC3, encoding a specific protein component of the minor spliceosome, which is associated with a hypopituitary phenotype, including severe growth hormone (GH) deficiency, hypoprolactinemia, variable thyrotropin (also known as thyroid-stimulating hormone) deficiency, and anterior pituitary hypoplasia. Primary ovarian insufficiency was diagnosed in 8 of 9 affected females, whereas males had normal gonadal function. In addition, 2 affected males displayed normal growth when off GH treatment despite severe biochemical GH deficiency. In both mouse and human embryos, Rnpc3/RNPC3 was expressed in the developing forebrain, including the hypothalamus and Rathke's pouch. Female Rnpc3 mutant mice displayed a reduction in pituitary GH content but with no reproductive impairment in young mice. Male mice exhibited no obvious phenotype. CONCLUSION: Our findings suggest novel insights into the role of RNPC3 in female-specific gonadal function and emphasize a critical role for the minor spliceosome in pituitary and ovarian development and function.

TGF-B1-over-expressed adipose stem cells-derived secretome exhibits CD44 suppressor and anti-cancer properties via antagonistic effects against SMAD4 in breast cancer cells.

OBJECTIVES: This study aimed to investigate the effect of TGF-B1-transfected adipose-derived mesenchymal stem cell (AD-MSC) conditional medium (TGF-B1-CM) on CD44 expression and biological activities in MCF-7 and MDA-MB-231 cells. METHODS: In the study, the experimental groups were created as a standard medium, AD-MSC-CM, TGF-B1-CM, and TGF-B1 recombinant protein. The medium and proteins specified in these groups were applied to MCF-7 and MDA-MB-231 cells separately at 24, 48 and 72 hours. Western blot and immunofluorescent staining were performed with antibodies suitable for CD44 and canonical smad signaling pathway analyses between groups. Cellular proliferation in MCF-7 and MDA-MB-231 cells was measured by MTT. Biological activity analyses such as apoptosis, cell cycle, proliferation, DNA damage, and membrane depolarization between groups were tested on the Muse Cell Analyzer using appropriate kits. Cellular migration between groups was determined by showing cells that migrated to the scar area with in vitro scar formation. Statistics were performed with GraphPad Prism 8.02 software. RESULTS: It was determined that TGF-B1-CM activates the smad signaling pathway in MCF-7 and MDA-MB-231 cells. TGF-B1-CM increased pSMAD2/3 expression and decreased SMAD4 expression in breast cancer cells. A decrease in CD44 expression was found at points of increase in pSMAD2/3 expression. Decreased expression of SMAD4 in breast cancer cells with TGF-B1-CM was associated with decreased expression of CD44. In MCF-7 and MDA-MB-231 cells, TGF-B1-CM was found to increase apoptosis, decrease proliferation, disrupt membrane depolarization, and arrest cells at G0/G1 stage. TGF-B1-CM suppressed MCF-7 and MDA-MB-231 migrations. CONCLUSION: SMAD4-targeted therapeutic strategies may be considered to suppress CD44 expression in breast cancer cells. Both the anti-tumorigenic factors released by AD-MSCs and the secretomes obtained as a result of supporting these factors with the overexpression of TGF-B1, severely suppressed breast cancer cells. With this study, it was planned to obtain a targeted biological product that suppresses breast cancer cells in vitro.

Immediate fat and nanofat-enriched fat grafting in breast reduction for scar management.

BACKGROUND: Reduction mammoplasty can be successful but surgical scars may continue to be a most undesirable and unavoidable outcome. Various medical and non-invasive methods are available to minimize scar formation but as yet no methods have been discovered to eliminate them. We hypothesize that immediate fat and nanofat-enriched fat graft transfer may improve the scar quality and optimize results. MATERIALS AND METHODS: This prospective study comprised 45 superomedial pedicle wise-pattern breast reduction patients divided into three groups of 15 in a randomized fashion. The control group had no additional injections whereas the other two groups received injections of fat and nanofat-enriched fat grafts immediately under their surgery scars, respectively. Surgical scar formation was evaluated at six months and scars were scored using the Vancouver scar scale and a visual analogue scale. RESULTS: Fat and nanofat-enriched fat graft-injected groups scored significantly better on all items of the Vancouver scar scale, except for scar height, compared to the control group (p < 0.05). Visual analogue scores were significantly lower in the fat and nanofat-enriched fat graft-injected groups compared to the control group (p < 0.05). CONCLUSIONS: In breast reduction patients, simultaneous fat and nanofat-enriched fat grafting appears to be a safe and promising strategy for scar management.

Effects of combination TGF-B1 transfection and platelet rich plasma (PRP) on three-dimension chondrogenic differentiation of rabbit dental pulp-derived mesenchymal stem cells.

Aim: The aim of this study was to evaluate the effects of standard culture medium and chondrogenic differentiation medium with PRP on chondrogenic differentiation of rabbit dental pulp-derived mesenchymal stem cells (rabbit DPSCs) that are transfected with transforming growth factor-beta 1 (TGF-B1) gene, based on the hypothesis of TGF- B1 and PRP can be effective on the chondrogenesis of stem cells. Materials and Methods: Rabbit DPSCs were characterized by using flow cytometry, immunofluorescent staining, quantitative Real Time Polymerase Chain Reaction (qRT-PCR) and differentiation tests. For the characterization, CD29, CD44 and CD45 mesenchymal cell markers were used. Rabbit DPSCs were transfected with TGF-B1 gene using electroporation technique in group 1; with PRP 10% in group 2; with chondrogenic medium in group 3; with both chondrogenic medium and PRP in group 4. DPSCs were cultured in medium with 10% inactive PRP in group 5, chondrogenic medium in group 6, chondrogenic medium with PRP 10% in group 7. SOX9, MMP13 and Aggrecan gene expression levels were evaluated in 3, 6, 12. and 24. days by qRT-PCR. Results: The expression levels of SOX9, MMP13 and Aggrecan were higher in group 2, 3 and group 7 in 3th day however in 24th day group 7 and group 2 were found higher. The expression levels changed by time-dependent. The extracellular matrix of the cells in experimental groups were positively stained with safranin O and toluidine blue. Conclusion: The combination in culture medium of TGF-B1 gene transfection and 10% PRP accelerates the chondrogenic differentiation of DPSCs.