Phosphorylation mapping of laminin γ1-chain: Kinases, functional interaction sequences, and phosphorylationinterfering cancer mutations.

We computationally predicted all phosphorylation sites in the sequence of the human laminin γ1-chain (LAMC1), and computationally identified, for the first time, all kinases for experimentally observed phosphorylated residues of the LAMC1 and all missense deleterious LAMC1 mutations found in different cancer types that interfere with LAMC1 phosphorylation. Also, we mapped the above data to all the biologically functional interaction sequences of the LAMC1. Five kinases (CKII, GPCRK1, PKA, PKC, and CKI) are most enriched for LAMC1 phosphorylation, and the significance of ecto-kinases in this process was emphasized. PKA and PKC targeted more residues inside and close to functional interaction sequences compared with other kinases and in the functional interaction sequence RPESFAIYKRTR. Most phosphorylation-interfering mutations were found in cutaneous melanoma and uterine endometrioid carcinoma. The mutation R255H interfered with the experimentally observed phosphorylation of LAMC1 inside the functional interaction sequence TDIRVTLNRLNTF, while the mutations S181Y and S213Y interfered with the experimentally observed phosphorylation of LAMC1 outside the functional interaction sequences. Mutations R359C,H, R589H, R657C,H, R663I,G, and T1207 interfered with the predicted phosphorylation inside or close to the functional interaction sequences, whereas other mutations interfered outside. PKA- and PKC-predicted phosphorylation was mostly interfered with by mutations inside functional interaction sequences. Phosphorylation- interfering mutations and functional interaction sequences were suggested to promote specific cancer types or cancer progression in general.

Doxorubicin-related cardiotoxicity: review of fundamental pathways of cardiovascular system injury.

Over the years, advancements in the field of oncology have made remarkable strides in enhancing the efficacy of medical care for patients with cancer. These modernizations have resulted in prolonged survival and improved the quality of life for these patients. However, this progress has also been accompanied by escalation in mortality rates associated with anthracycline chemotherapy. Anthracyclines, which are known for their potent antitumor properties, are notorious for their substantial cardiotoxic potential. Remarkably, even after 6 decades of research, a conclusive solution to protect the cardiovascular system against doxorubicin-induced damage has not yet been established. A comprehensive understanding of the pathophysiological processes driving cardiotoxicity combined with targeted research is crucial for developing innovative cardioprotective strategies. This review seeks to explain the mechanisms responsible for structural and functional alterations in doxorubicin-induced cardiomyopathy.

Experimental models of high-risk bowel anastomosis in rats: A systematic review.

BACKGROUND: Anastomotic leaks remain one of the most dreaded complications in gastrointestinal surgery causing significant morbidity, that negatively affect the patients' quality of life. Experimental studies play an important role in understanding the pathophysiological background of anastomotic healing and there are still many fields that require further investigation. Knowledge drawn from these studies can lead to interventions or techniques that can reduce the risk of anastomotic leak in patients with high-risk features. Despite the advances in experimental protocols and techniques, designing a high-quality study is still challenging for the investigators as there is a plethora of different models used. AIM: To review current state of the art for experimental protocols in high-risk anastomosis in rats. METHODS: This systematic review was performed according to The Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. To identify eligible studies, a comprehensive literature search was performed in the electronic databases PubMed (MEDLINE) and Scopus, covering the period from conception until 18 October 2023. RESULTS: From our search strategy 102 studies were included and were categorized based on the mechanism used to create a high-risk anastomosis. Methods of assessing anastomotic healing were extracted and were individually appraised. CONCLUSION: Anastomotic healing studies have evolved over the last decades, but the findings are yet to be translated into human studies. There is a need for high-quality, well-designed studies that will help to the better understanding of the pathophysiology of anastomotic healing and the effects of various interventions.

A Simple and Effective Mechanical Method for Adipose-Derived Stromal Vascular Fraction Isolation.

Over the last decades, there has been ongoing and evolving research concerning regenerative medicine, specifically, stem cells. The most common source of adult mesenchymal stem cells (MSCs) remains the adipose tissue and the easiest way to obtain such tissue is lipoaspirate. The fatty tissue obtained can be processed either in an enzymatic way, which is time-consuming and expensive and carries several dangers for the viability of the stem cells included, or with mechanical means which are fast, inexpensive, yield enough viable cells, and can be readily used for autologous transplantation in one-stage procedures. Herein, we demonstrate our non-enzymatic method for obtaining adipose-derived stromal vascular fraction comprising MSCs. The stromal vascular fraction was isolated via centrifugation, and the characteristics and numbers of the cells isolated have been tested with flow cytometry assay, cell culture, and differentiation. Over 91% of viable MSCs were isolated using the mechanical method. The cells retained the ability to differentiate into osteocytes, adipocytes, and chondrocytes. The method presented is simple, requiring no special equipment, and yields a viable population of stem cells in large numbers. These cells can be readily used in several operations (orthopedic, dentistry, fistulas, etc.) making feasible "one-stage" procedures, thus proving their benefits for the patient and the health care system.

Pegylated-polycaprolactone nano-sized drug delivery platforms loaded with biocompatible silver(i) complexes for anticancer therapeutics.

Cytotoxic potential of Ag(i) coordination compounds against cancer cells is widely recognized, but their frequently low water solubility and potential adverse interactions of Ag(i) ions in biological media require their incorporation into suitable platforms to ensure effective transport and delivery at target sites. Herein, we developed and evaluated the in vitro cytotoxic activity of a biodegradable copolymer-based nano-sized drug delivery system for three cytotoxically active and lipophillic Ag(i) compounds. In particular, polymer-based nanoparticles of the newly synthesized amphiphilic methoxy-poly(ethylene glycol)-poly(caprolactone) (mPEG-PCL) copolymer were prepared as carriers for [Ag(dmp2SH)(PPh3)2]NO3 (1), [Ag(dmp2SH)(xantphos)]NO3 (2) and [Ag(dmp2S)(xantphos)] (3) (dmP2SH = 4,6-dimethylpyrimidine-2-thiol, xantphos = 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene) which exhibit high cytotoxicity against HeLa cancer cells, while they maintain low toxicity against HDFa normal cells. Taking advantage of the favorable donor-acceptor Lewis acid-base and electrostatic interactions between functional groups of 1-3 and mPEG-PCL copolymer, the formation of [X]@mPEG-PCL (X = 1,2,3) nanoparticles with nearly spherical shape was achieved. Satisfactory loading capacities and encapsulation efficiencies were obtained (13-15% and 80-88%, respectively). Differences in their mean size diameters were observed, revealing a dependence on the individual structural characteristics of the Ag(i) compounds. In vitro release profiles of the nanoparticles showed an initial burst stage, followed by a prolonged release stage extending over 15 days, with their release rates being determined by the mean size of the nanoparticles, as well as the type and crystallinity of the encapsulated Ag(i) compounds. In vitro cytotoxicity studies revealed an increased cytotoxic activity of compounds 1-3 after their encapsulation in mPEG-PCL copolymer against HeLa cells, with the actual concentrations of the loaded compounds responsible for the inhibition of cell viability being reduced by 8 times compared to the compounds in free form. Therefore, the current drug delivery system improves the pharmacokinetic properties of the three cytotoxic and biocompatible Ag(i) compounds, and may be beneficial for future in vivo anticancer treatment.

Induced differentiation and molecular characterization of monocytes-derived multipotential cells generated from commonly discarded leukapheresis filters.

Monocyte-derived multipotential cells (MOMCs) are a subpopulation of monocytes that appear to be capable of differentiating into many cell populations, thus MOMCs can be an ideal autologous transplantable cell source for regenerative medicine. In this study, we generated MOMCs from leukapheresis filters, evaluated their ability to differentiate to endothelium and osteocytes and performed their molecular characterization. For this purpose, leukapheresis filters were collected from a hospital blood donation department and used for leukocytes isolation. The isolated leukocytes were cultured in a medium supplemented with SDF-1a for MOMCs generation. We evaluated the expression of the multipotency genes ZNF217, ZNF878, ESRRB, SALL4, KLF4, SOX2, NANOG, OCT4, GAPDH, CD34 and c- MYC in MOMCs with real-time reverse transcription PCR (qRT-PCR) and the differentiation capacity of MOMCs to osteocytes and endothelium with qRT-PCR. The results suggest that MOMCs can be generated using leukocytes isolated from leukapheresis filters in the presence of SDF-1a. Furthermore, MOMCs expressed all the tested factors responsible to activate the networks of pluripotency of cells and can differentiate into endothelium and osteocytes. Therefore, the blood donors could benefit and be rewarded with the potential use of their own immune system cells for future treatment in the frame of personalized regenerative medicine.

Αn optimized, simplified and clinically approved culture system to produce, in large scale, dendritic cells capable of priming specific T cells.

Cancer immunotherapy using dendritic cells (DCs) able to induce specific immune responses to naïve T lymphocytes raises great research interest. However, the extremely complex and expensive methods used to produce DCs, combined with the limited number of autologous DCs in the circulation make any application almost impossible. Aim of the study is the development of an optimized and simplified system to easily produce in large scale cord blood-derived DCs, loaded with common tumor antigens, capable of promoting controlled Th1 immunoresponses following clinically approved maturation with vaccines. CD34+cells cultured in the presence of a cytokine cocktail in miniPERM® bioreactors and the generated DCs were matured using anti-flu vaccines. Autologous T cells plated with DCs pulsed with overlapping peptides CEA and WT1 for multiple stimulations. 200 billion of myeloid DCs were produced and matured in just 8 h in bioreactors, presenting an increased expression of the co-stimulatory molecules and also high levels of Th1 related cytokines. Upon just the 2nd stimulation, the T cells exhibited specificity following stimulation with the CEA/WT1 peptides and strong cytotoxic capacity in co-culture with a colorectal cancer (CRC)-cell line. The high produced doses of DCs, easily maturated with clinically approved agents, and capable of priming specific T cells, could potentially strengthen the further progress in DCs-mediated cancer immunotherapy field.

Intravitreal Administration Effect of Adipose-Derived Mesenchymal Stromal Cells Combined with Anti-VEGF Nanocarriers, in a Pharmaceutically Induced Animal Model of Retinal Vein Occlusion.

Antiangiogenic therapeutic agents (anti-VEGF) have contributed to the treatment of retinal vein occlusion (RVO) while mesenchymal stromal cell- (MSCs-) mediated therapies limit eye degeneration. The aim of the present study is to determine the effect of adipose-derived MSCs (ASCs) combination with nanocarriers of anti-VEGF in a pharmaceutically induced animal model of RVO. Nanoparticles (NPs) of thiolated chitosan (ThioCHI) with encapsulated anti-VEGF antibody were prepared. ASCs were isolated and genetically modified to secrete the green fluorescence GFP. Twenty-four New Zealand rabbits were divided into the I-IV equal following groups: ASCs, ASCs + nanoThioCHI-anti-VEGF, RVO, and control. For the RVO induction, groups I-III received intravitreal (iv) injections of MEK kinase inhibitor, PD0325901. Twelve days later, therapeutic regiments were administered at groups I-II while groups III-IV received BSS. Two weeks later, the retinal damage evaluated via detailed ophthalmic examinations, histological analysis of fixed retinal sections, ELISA for secreted cytokines in peripheral blood or vitreous fluid, and Q-PCR for the expression of related to the occlusion and inflammatory genes. Mild retinal edema and hemorrhages, limited retinal detachment, and vasculature attenuation were observed in groups I and II compared with the pathological symptoms of group III which presented a totally disorganized retinal structure, following of positive immunostaining for neovascularization and related to RVO markers. Important reduction of the high secreted levels of inflammatory cytokines was quantified in groups I and II vitreous fluid, while the expression of the RVO-related and inflammatory genes has been significantly decreased especially in group II. GFP+ ASCs, capable of being differentiated towards neural progenitors, detected in dissociated retina tissues of group II presenting their attachment to damaged area. Conclusively, a stem cell-based therapy for RVO is proposed, accompanied by sustained release of anti-VEGF, in order to combine the paracrine action of ASCs and the progressive reduction of neovascularization.

Healing of colonic anastomosis in rats under obstructive ileus conditions.

BACKGROUND: The anastomosis leak in colon resections is a crucial post-operative complication with significant morbidity and mortality.  Methods: Forty (40) Wistar rats were allocated in two groups. In SHAM group only anastomosis was performed. In ILEUS group anastomosis was performed following one day of ileus. Animals in both groups were subdivided in two groups according to the day they were sacrificed, 4th or 8th post-operative day. A number of variables between the groups were estimated. RESULTS: Body weight loss was higher following obstructive ileus on both days. Adhesion score in 4th and 8th post-operative day was higher in ILEUS1, ILEUS2 groups compared to SHAM1, SHAM2 groups respectively (p<0.001 for both). Neovascularization decreased following obstructive ileus compared to control on the 4th day (ILEUS1 vs. SHAM1, p=0.038). Bursting pressure was lower in ILEUS2 group than SHAM2 group (p<0.001). The number of fibroblasts decreased following obstructive ileus compared to control on the 4th and 8th day (ILEUS1 vs. SHAM1, p=0.001, ILEUS2 vs SHAM2, p=0.016). Hydroxyproline concentration was decreased in ILEUS2 group compared to SHAM2 group (p<0.001). CONCLUSIONS: The balance of collagenolysis and collagenogenesis plays a decisive role in the healing of anastomoses following bowel obstruction. Under those circumstances, anastomosis' bursting pressure is reduced owning to decreased neovascularization, reduced fibroblast presence and lower hydroxyproline concertation. In our study, local inflammation, neocollagen concentration and collagenase activity were not associated with this adverse effect. However, further research should delineate the mechanisms of healing of colonic anastomoses and identify those factors that can improve our outcomes.

Dysregulation of Caveolin-1 Phosphorylation and Nuclear Translocation Is Associated with Senescence Onset.

We recently reported that the inability of osteoarthritic (OA) chondrocytes to repair oxidative stress (OS) induced DNA damage is linked to Cav-1 overexpression/improper localization. We speculated that the senescent status of OA cells was responsible for this Cav-1 dysregulation. Here, to further investigate this hypothesis, we used Wharton Jelly derived mesenchymal stem cells (WJ-MSCs) and investigated Cav-1 function as cells reached replicative senescence or upon stress induced senescence (SIPS). We showed that Cav-1 is upregulated, phosphorylated and translocated to the nucleus in young WJ-MSCs upon acute exogenous OS, and that it returns back to basal/nonphosphorylated levels and exports the nucleus in the recovery phase. However, as cells reach senescence, this regulation is lost. OS did not induce any Cav-1-mediated response, which is concomitant with the inability of older cells to restore DNA damage. Furthermore, downregulation of Cav-1 resulted in persistent OS-induced DNA damage and subsequent onset of senescence. We also report that the establishment of senescence is mediated by autophagy stimulation, since downregulation of autophagy key molecule Atg5, simultaneously with Cav-1 downregulation, was found to inhibit SIPS. Basically, we propose that Cav-1 involvement in DNA damage response can lead to senescence, either because the damage is extensive or because Cav-1 is absent/unable to perform its homeostatic role.

Methylene blue for treatment of hospitalized Covid-19 patients: a randomized, controlled, open-label clinical trial, phase 2

ABSTRACT Background: There is no pharmacological intervention on the treatment of hypoxemia and respiratory distress in COVID-19 patients. Objective: The objective of the study was to study the effect of the reduced form of methylene blue (MB) on the improvement of oxygen saturation (SpO2) and respiratory rate (RR). Methods: In an academic medical center, 80 hospitalized patients with severe COVID-19 were randomly assigned to receive either oral MB along with standard of care (SOC) (MB group, n = 40) or SOC only (SOC group, n=40). The primary outcomes were SpO2 and RR on the 3rd and 5th days. The secondary outcomes were hospital stay and mortality within 28 days. Results: In the MB group, a significant improvement in SpO2 and RR was observed on the 3rd day (for both, p < 0.0001) and also the 5th day (for both, p < 0.0001). In the SOC group, there was no significant improvement in SpO2 (p = 0.24) and RR (p = 0.20) on the 3rd day, although there was a significant improvement of SpO2 (p = 0.002) and RR (p = 0.01) on the 5th day. In the MB group in comparison to the SOC group, the rate ratio of increased SpO2 was 13.5 and 2.1 times on the 3rd and 5th days, respectively. In the MB group compared with the SOC group, the rate ratio of RR improvement was 10.1 and 3.7 times on the 3rd and 5th days, respectively. The hospital stay was significantly shortened in the MB group (p = 0.004), and the mortality was 12.5% and 22.5% in the MB and SOC groups, respectively. Conclusions: The addition of MB to the treatment protocols significantly improved SpO2 and respiratory distress in COVID-19 patients, which resulted in decreased hospital stay and mortality. ClinicalTrials.gov: NCT04370288

METHYLENE BLUE FOR TREATMENT OF HOSPITALIZED COVID-19 PATIENTS: A RANDOMIZED, CONTROLLED, OPEN-LABEL CLINICAL TRIAL, PHASE 2.

BACKGROUND: There is no pharmacological intervention on the treatment of hypoxemia and respiratory distress in COVID-19 patients. OBJECTIVE: The objective of the study was to study the effect of the reduced form of methylene blue (MB) on the improvement of oxygen saturation (SpO2) and respiratory rate (RR). METHODS: In an academic medical center, 80 hospitalized patients with severe COVID-19 were randomly assigned to receive either oral MB along with standard of care (SOC) (MB group, n = 40) or SOC only (SOC group, n=40). The primary outcomes were SpO2 and RR on the 3rd and 5th days. The secondary outcomes were hospital stay and mortality within 28 days. RESULTS: In the MB group, a significant improvement in SpO2 and RR was observed on the 3rd day (for both, p < 0.0001) and also the 5th day (for both, p < 0.0001). In the SOC group, there was no significant improvement in SpO2 (p = 0.24) and RR (p = 0.20) on the 3rd day, although there was a significant improvement of SpO2 (p = 0.002) and RR (p = 0.01) on the 5th day. In the MB group in comparison to the SOC group, the rate ratio of increased SpO2 was 13.5 and 2.1 times on the 3rd and 5th days, respectively. In the MB group compared with the SOC group, the rate ratio of RR improvement was 10.1 and 3.7 times on the 3rd and 5th days, respectively. The hospital stay was significantly shortened in the MB group (p = 0.004), and the mortality was 12.5% and 22.5% in the MB and SOC groups, respectively. CONCLUSIONS: The addition of MB to the treatment protocols significantly improved SpO2 and respiratory distress in COVID-19 patients, which resulted in decreased hospital stay and mortality. ClinicalTrials.gov: NCT04370288.

Application of methylene blue -vitamin C -N-acetyl cysteine for treatment of critically ill COVID-19 patients, report of a phase-I clinical trial.

COVID-19 is a global catastrophic event that causes severe acute respiratory syndrome. The mechanism of the disease remains unclear, and hypoxia is one of the main complications. There is no currently approved protocol for treatment. The microbial threat as induced by COVID-19 causes the activation of macrophages to produce a huge amount of inflammatory molecules and nitric oxide (NO). Activation of macrophages population into a pro-inflammatory phenotype induces a self-reinforcing cycle. Oxidative stress and NO contribute to this cycle, establishing a cascade inflammatory state that can kill the patient. Interrupting this vicious cycle by a simple remedy may save critical patients' lives. Nitrite, nitrate (the metabolites of NO), methemoglobin, and prooxidant-antioxidant-balance levels were measured in 25 ICU COVID-19 patients and 25 healthy individuals. As the last therapeutic option, five patients were administered methylene blue-vitamin C-N-acetyl Cysteine (MCN). Nitrite, nitrate, methemoglobin, and oxidative stress were significantly increased in patients in comparison to healthy individuals. Four of the five patients responded well to treatment. In conclusion, NO, methemoglobin and oxidative stress may play a central role in the pathogenesis of critical COVID-19 disease. MCN treatment seems to increase the survival rate of these patients. Considering the vicious cycle of macrophage activation leading to deadly NO, oxidative stress, and cytokine cascade syndrome; the therapeutic effect of MCN seems to be reasonable. Accordingly, a wider clinical trial has been designed. It should be noted that the protocol is using the low-cost drugs which the FDA approved for other diseases. TRIAL REGISTRATION NUMBER: NCT04370288.

Autologous administration of adipose stromal cells improves skin flap survival through neovascularization: An experimental study.

One of the most severe complications in aesthetic and reconstructive surgeries is the partial or total necrosis of a skin flap. In our experimental study, we demonstrated the use of adipose-derived stem cells in the increase of skin flap survival rates. Stem cells were isolated from the fat of Wistar rats and genetically modified to permanently produce a green fluorescent protein (GFP). Two random-pattern skin flaps (2 cm × 8 cm) were elevated on the dorsal area of the spine, and after being separated from the surgical wounds with a thin silicone sheet, they were placed back onto their original location. Then, the autologous GFP-producing cells were injected intradermally into the dorsal area of the rats. At the seventh day, after the implantation of the stem cells, a clinical and immunohistochemical control was performed. The fluorescence microscopy revealed green vascular formations, suggesting that autologous GFP stromal cells were converted into endothelial cells through neovascularization. In the control skin flaps, where no stromal cells were used, no fluorescence was observed. The statistical analysis showed significantly lower necrosis rates in the right-sided flaps (i.e., the flaps where adipose-derived stromal cells were injected) compared with the left-sided ones. Findings from our study demonstrate that adipose-derived stem cells play an important role in the improvement of skin flap survival. Neovascularization is an effective way of achieving it.

Phosphorylation mapping of laminin α1-chain: Kinases in association with active sites.

Laminin-111 is a trimeric glycoprotein of the extracellular matrix (ECM) that holds a significant role in cell adhesion, migration and differentiation. Laminin-111 is the most studied laminin isoform, composed of three chains; α1, ß1 and γ1. Phosphorylation is the most common eukaryotic post - translational modification and has regulatory effect on protein function. Using bioinformatic tools we computationally predicted all the possible phosphorylation sites on human laminin α1-chain sequence (LAMA1) according to kinases binding motifs. Thus, we predicted, for the first time, the possibly responsible kinases for fifteen of the nineteen already published experimentally observed phosphorylated residues in LAMA1. Searching the literature extensively, we recorded all the known functional sites (active sites) in LAMA1. We combined the experimentally observed and predicted phosphorylated residues as well as the active sites in LAMA1, generating an analytic phosphorylation map of human laminin α1-chain, which is useful for further analysis. Our results indicated fourteen kinases that might be important for the phosphorylation of human laminin α1-chain, out of which three kinases with reported ecto-phosphorylation activity (PKA, PKC and CKII) were suggested to have a more significant role. Six cancer associated-active sites were correlated with kinases, three out which were correlated with only the above ecto - kinases.

Differentiation Capacity of Monocyte-Derived Multipotential Cells on Nanocomposite Poly(e-caprolactone)-Based Thin Films.

Background: Μonocyte-derived multipotential cells (MOMCs) include progenitors capable of differentiation into multiple cell lineages and thus represent an ideal autologous transplantable cell source for regenerative medicine. In this study, we cultured MOMCs, generated from mononuclear cells of peripheral blood, on the surface of nanocomposite thin films. Methods: For this purpose, nanocomposite Poly(e-caprolactone) (PCL)-based thin films containing either 2.5 wt% silica nanotubes (SiO2ntbs) or strontium hydroxyapatite nanorods (SrHAnrds), were prepared using the spin-coating method. The induced differentiation capacity of MOMCs, towards bone and endothelium, was estimated using flow cytometry, real-time polymerase chain reaction, scanning electron microscopy and fluorescence microscopy after cells' genetic modification using the Sleeping Beauty Transposon System aiming their observation onto the scaffolds. Moreover, Wharton's Jelly Mesenchymal Stromal Cells were cultivated as a control cell line, while Human Umbilical Vein Endothelial Cells were used to strengthen and accelerate the differentiation procedure in semi-permeable culture systems. Finally, the cytotoxicity of the studied materials was checked with MTT assay. Results: The highest differentiation capacity of MOMCs was observed on PCL/SiO2ntbs 2.5 wt% nanocomposite film, as they progressively lost their native markers and gained endothelial lineage, in both protein and transcriptional level. In addition, the presence of SrHAnrds in the PCL matrix triggered processes related to osteoblast bone formation. Conclusion: To conclude, the differentiation of MOMCs was selectively guided by incorporating SiO2ntbs or SrHAnrds into a polymeric matrix, for the first time.

Isolation of a novel embryonic stem cell cord blood-derived population with in vitro hematopoietic capacity in the presence of Wharton's jelly-derived mesenchymal stromal cells.

BACKGROUND: Recent studies highlight the existence of a population of cord blood (CB)-derived stem cells that bare embryonic features (very small embryonic-like stem cells [VSELs]) as the most primitive CB-stem cell population. In the present study, we present for the first time a novel and high purity isolation method of VSELs with in vitro hematopoietic capacity in the presence of Wharton's jelly-derived mesenchymal stromal cells (WJ-MSCs). METHODS: The experimental procedure includes isolation upon gradually increased centrifugation spins and chemotaxis to Stromal cell-derived factor 1a (SDF-1a). Τhis cell population is characterized with flow cytometry, alkaline phosphatase (ALP) staining and qRT-PCR. The functional role of the isolated VSELs is assayed following co-culture with WJ-MSCs or bone marrow-derived mesenchymal stromal cells (BM-MSCs), whereas the stimulation of the quiescent VSEL population is verified via cell cycle analysis. The in vitro hematopoietic capacity is evaluated in methylcellulose cultures and also through induction of erythroid differentiation. RESULTS: The final isolated subpopulation is characterized as a small-sized CD45/Lineage-/CXCR4+/CD133+/SSEA-4+cell population, positive in ALP staining and overexpressing the Oct3/4, Nanog and Sox-2 transcription factors. Upon the co-culture with MSCs, a stimulation of the quiescent VSEL population is observed. An impressive increase in the co-expression of the CD34+/CD45+ markers is observed following the co-culture with the WJ-MSCs, which is confirmed by the intense clonogenic ability suggesting in vitro differentiation toward all of the hematopoietic cell lineages and successful differentiation toward erythrocytes. DISCUSSION: Conclusively, we propose a novel, rapid and rather simplified isolation method of CB-VSELs, capable of in vitro hematopoiesis.

Acetylcholinesterase activity and bone biochemical markers in premature and full-term neonates.

Background Almost 30% of the premature infants have low body weight and bone mineral density due to prematurity. There is no consensus of screening premature neonates for metabolic bone disease; therefore, it is important to investigate the use of bone biochemical parameters. Latest studies involved the activity of acetylcholinesterase as a mediator in bone remodeling. It is hypothesized that there is a possible correlation of bone biochemical biomarkers and acetylcholinesterase (AChE) activity in premature infants. Methods We studied 50 neonates (26 preterm with gestational age <32 weeks, 24 full-term). Clinical data (sex, gestational week) and anthropometric parameters (body weight) were recorded. We directly measured the bone biochemical markers in serum such as alkaline phosphatase (ALP), calcium (Ca), phosphorus (P), magnesium (Mg) and parathyroid hormone (PTH). In addition, we measured the AChE activity. Results ALP and parathyroid hormone levels were higher, but Ca, P and AChE were lower in premature neonates group compared with full-term ones. There is a significant positive correlation of gestational age with body weight, Ca and AChE. A significant negative correlation was observed for ALP and PTH with gestational age. Conclusions We found a gestational age-related increase of AChE activity. There were significant relationships between AChE activity with P and PTH.

Accelerated healing of a diabetic foot ulcer using autologous stromal vascular fraction suspended in platelet-rich plasma.

We describe the case of a Type I diabetic patient with a refractory foot ulcer that remained unhealed for 2 years despite conventional therapy. Autologous adipose-derived stromal vascular fraction suspended in autologous platelet-rich plasma was applied to the wound, which completely healed within 1 month. The wound remained closed with no complications for a 2-year follow-up. Reporting of this and similar cases may lead to larger clinical trials that will prove the efficacy of this therapy that may offer accelerated healing and lessen the financial burden of more expensive therapeutic modalities.

Generation of an immortalized mesenchymal stem cell line producing a secreted biosensor protein for glucose monitoring.

Diabetes is a chronic disease characterized by high levels of blood glucose. Diabetic patients should normalize these levels in order to avoid short and long term clinical complications. Presently, blood glucose monitoring is dependent on frequent finger pricking and enzyme based systems that analyze the drawn blood. Continuous blood glucose monitors are already on market but suffer from technical problems, inaccuracy and short operation time. A novel approach for continuous glucose monitoring is the development of implantable cell-based biosensors that emit light signals corresponding to glucose concentrations. Such devices use genetically modified cells expressing chimeric genes with glucose binding properties. MSCs are good candidates as carrier cells, as they can be genetically engineered and expanded into large numbers. They also possess immunomodulatory properties that, by reducing local inflammation, may assist long operation time. Here, we generated a novel immortalized human MSC line co-expressing hTERT and a secreted glucose biosensor transgene using the Sleeping Beauty transposon technology. Genetically modified hMSCs retained their mesenchymal characteristics. Stable transgene expression was validated biochemically. Increased activity of hTERT was accompanied by elevated and constant level of stem cell pluripotency markers and subsequently, by MSC immortalization. Furthermore, these cells efficiently suppressed PBMC proliferation in MLR transwell assays, indicating that they possess immunomodulatory properties. Finally, biosensor protein produced by MSCs was used to quantify glucose in cell-free assays. Our results indicate that our immortalized MSCs are suitable for measuring glucose concentrations in a physiological range. Thus, they are appropriate for incorporation into a cell-based, immune-privileged, glucose-monitoring medical device.