Mesenchymal stromal cells (MSCs) are emerging as an ideal candidate for regenerative medicine. It is known that the culture conditions impact the cellular properties of MSCs and their therapeutic ...behavior. Moreover, maintenance of MSCs in low oxygen tension for a short duration has shown to be beneficial for MSCs as it is similar to that of their physiological niche. However, the precise mechanism through which hypoxia pre-conditioning affects MSCs is not clear yet. Thus, in this study, we have investigated the effect of hypoxia exposure (1% O
2
) on tissue-specific MSCs over a period of time under serum-free culture conditions and evaluated the changes in expression of immuno-modulatory molecules and exosome biogenesis and secretion markers. It was observed that all MSCs responded differentially towards hypoxia exposure as indicated by the expression of HIF-1α. Moreover, this short-term exposure did not induce any changes in MSCs cellular morphology, proliferation rate, and surface marker profiling. In addition, we observed an enhancement in the expression of immunomodulatory factors (HLA-G, PGE-2, and IDO) after hypoxia exposure of 12 to 24 h in all tissue-specific MSCs. Interestingly, we have also observed the upregulation in exosome secretion that was further corelated to the upregulation of expression of exosome biogenesis and secretion markers (ALIX, TSG101, RAB27a, RAB27b). Though there was a differential response of MSCs where WJ-MSCs and BM-MSCs showed upregulation of these markers at 6–12 h of hypoxia pre-conditioning, while AD-MSCs showed similar changes beyond 24 h of hypoxia exposure.
Graphic Abstract
Exosomes are nanovesicles (30-120 nm) of endosomal origin. These exosomes contain various functional proteins and RNAs that could be used for therapeutic purposes. Currently, having a standard method ...for exosome isolation retaining its biological properties with increased yield and purity is a major challenge. The most commonly used method is differential ultracentrifugation but it has its own disadvantages, which include high time consumption, low yield due to disruption of exosome integrity, and high protein contaminants. In this study, we have identified an improved method addressing these problems for exosome isolation using ultracentrifugation since it is cost-effective and used worldwide.
We have compared differential ultracentrifugation with the modified method called one-step sucrose cushion ultracentrifugation for exosome isolation. The conditioned serum-free media from human mesenchymal stem cells cultured for 48 h was collected for exosome isolation. The cellular debris was removed by centrifugation at 300g for 10 min, followed by centrifugation at 10,000g for 30 min to remove microvesicles. Equal volumes of pre-processed conditioned media were used for exosome isolation by direct ultracentrifugation and one-step sucrose cushion ultracentrifugation. The exosomes isolated using these methods were characterized for their size, morphology, concentration, and surface marker protein expression.
It was observed that the recovery of exosomes with cup-shaped morphology from one-step sucrose cushion ultracentrifugation was comparatively high as estimated by nanoparticle tracking analysis and electron microscopy. These results were confirmed by Western blotting and flow cytometry.
We conclude that this one-step sucrose cushion ultracentrifugation method provides an effective and reproducible potential standard method which could be used for various starting materials for isolating exosomes. We believe that this method will have a wide application in the field of extracellular vesicle research where exosome isolation with high yield and purity is an imperative step. Schematic representation of comparison of UC and SUC exosome isolation methods for tissue-specific human mesenchymal stem cells. The SUC isolation method yields a greater number of cup-shaped exosomes with a relatively homogenous population for mass-scale production of exosomes for downstream analysis.
SUC One-step sucrose cushion ultracentrifugation, UC Direct ultracentrifugation.
Human Mesenchymal Stem Cells (hMSCs) represent a promising cell source for cell-based therapy in autoimmune diseases and other degenerative disorders due to their immunosuppressive, anti-inflammatory ...and regenerative potentials. Belonging to a glucocorticoid family, Dexamethasone (Dex) is a powerful anti-inflammatory compound that is widely used as therapy in autoimmune disease conditions or allogeneic transplantation. However, minimal immunomodulatory effect of hMSCs may limit their therapeutic uses. Moreover, the effect of glucocorticoids on the immunomodulatory molecules or other regenerative properties of tissue-specific hMSCs remains unknown.
Herein, we evaluated the in vitro effect of Dex at various dose concentrations and time intervals, 1000 ng/ml, 2000 ng/ml, 3000 ng/ml and 24 h, 48 h respectively, on the basic characteristics and immunomodulatory properties of Bone marrow derived MSC (BM-MSCs), Adipose tissue derived MSCs (AD-MSCs), Dental Pulp derived MSC (DP-MSCs) and Umbilical cord derived MSCs (UC-MSCs).
The present study indicated that the concentration of Dex did not ramify the cellular morphology nor showed cytotoxicity as well as conserved the basic characteristics of tissue specific hMSCs including cell proliferation and surface marker profiling. However, quite interestingly it was observed that the stemness markers (Oct-4, Sox-2, Nanog and Klf-4) showed a significant upregulation in DP-MSCs and AD-MSCs followed by UC-MSCs and BM-MSCs. Additionally, immunomodulatory molecules, Prostaglandin E-2 (PGE-2), Indoleamine- 2,3-dioxygenase (IDO) and Human Leukocyte Antigen-G (HLA-G) were seen to be upregulated in a dose-dependent manner. Moreover, there was a differential response of tissue specific hMSCs after pre-conditioning with Dex during mixed lymphocyte reaction, wherein UC-MSCs and DP-MSCs showed enhanced immunosuppression as compared to AD-MSCs and BM-MSCs, thereby proving to be a better candidate for therapeutic applications in immune-related diseases.
Dex preconditioning improved the hMSCs immunomodulatory property and may have reduced the challenge associated with minimal potency and strengthen their therapeutic efficacy. Preconditioning of tissue specific hMSCs with dexamethasone biomanufacturers the enhanced potential hMSCs with better stemness and immunomodulatory properties for future therapeutics.
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•Novel method for scaffold preparation has been adopted.•Chitosan as a bio activators was coated on the pore wall of the polycaprolactone three-dimensional scaffolds.•Porosity and ...mechanical strength of the scaffolds shows the encouraging results.•Cell attachment & morphology, cytotoxicity, hemocompatibility shows the bio-compatibility of the fabricated scaffolds.•The osteoblast differentiation of chitosan 2.5 % proven to be superior in all the groups and makes it an adequate candidate for bone regeneration.
The study's purpose was to fabricate a 3-D porous scaffold, in which chitosan was coated onto the pore wall of polycaprolactone (PCL) scaffolds as a bioactive agent to maximize the cell recognition signals, to improve the osteoconductivity of the scaffolds. The pppporogen leaching technique has been modified and used in the fabrication process, comprising of the coating of chitosan over the porogen followed by transferring of coating to the pore wall of the PCL scaffold. The cytotoxicity and hemolysis results indicated chitosan's presence over the surface of the scaffold's pore walls has significantly enhanced its biocompatibility. Scaffolds coated with 2.5 %(w/v) chitosan shows 6.74 % increase in porosity and 207.96 % upsurge in mechanical strength, compared to PCL scaffolds. The Gene-expression also proves the study groups of scaffolds show the minimal osteogenic expression. Therefore, chitosan coating over the scaffold's pore wall's surface opens an unconventional approach for tissue engineering applications.
The journey into the field of stem cell biology has been an endeavor of paramount advancement in biomedicine, establishing new horizons in the avenue of materiobiology. The creative drive of the ...scientific community focuses on ameliorating the utilization of stem cells, which is currently untapped on a large scale. With similar motivation, we present a nascent strategy of maneuvering biogenic carbon quantum dots (CQDs) to eclipse the toxic hurdles of chemical synthesis of carbon allotropes to serve as a biocompatible trident in stem cell biology employing a three-prong action of stem cell differentiation, imaging, and migration. The derivation of CQDs from garlic peels as a biogenic precursor abets in realizing the optophysical features of CQDs to image mesenchymal stem cells without hampering the biological systems with cytotoxicity. We report the versatility of biogenic CQDs to generate reactive oxygen species (ROS) to robustly influence stem cell migration and concomitantly chondrocyte differentiation from human Wharton's jelly mesenchymal stem cells (hWJ-MSCs). This was orchestrated without the use of chondrogenic induction factors, which was confirmed from the expression of chondrogenic markers (Col II, Col X, ACAN). Even the collagen content of cells incubated with CQDs was quite comparable with that of chondrocyte-induced cells. Thus, we empirically propose garlic peel-derived CQDs as a tangible advancement in stem cell biology from a materiobiological frame of reference to hone significant development in this arena.
Argulus spp. are economically important fish ectoparasites. The development of antiparasitic drugs is thus important and real time PCR is an indispensable tool in drug development. The analytical ...potential of RT-PCR depends upon accurate normalisation by the use of stable reference genes. Here, we identified stable reference genes of Argulus siamensis for validation of efficacy of drugs and drug targets. Seven candidate genes were evaluated by evaluating their expression under different states of Argulus using the RefFinder tool. The four algorithms together generated a comprehensive ranking with elongation factor-1 alpha (EF-1α) being the most stable and 18S ribosomal protein (18S) the least stable gene. Taking EF-1α and 18S genes as references, the effectiveness of six anti-parasitic compounds against Argulus was evaluated by studying their effect on the expression pattern of few ion channel genes; this was to understand their mode of action, besides validating the reference genes. EF-1α was found to be the most stable gene in the validation. Collectively, this study is the first report to validate the optimal reference genes of A. siamensis for normalisation, and the potential of the ion channel genes for evaluating effective drug targets in parasite control.
Among conventional fabrication techniques, freeze‐drying process has widely been investigated for polymeric implants. However, the understanding of the stem cell progenitor‐dependent cell ...functionality modulation and quantitative analysis of early osseointegration of highly porous scaffolds have not been explored. Here, we developed a novel, highly porous, multimaterial composite, chitosan/hydroxyapatite/polycaprolactone (CHT/HA/PCL). The in vitro studies have been performed using mesenchymal stem cells (MSCs) from three tissue sources: human bone marrow‐derived MSCs (BM‐MSCs), adipose‐derived MSCs (AD‐MSCs), and Wharton's jelly‐derived MSCs (WJ‐MSCs). Although cell attachment and metabolic activity 3‐4,5‐dimethylthiazol‐2yl‐(2,5 diphenyl‐2H‐tetrazoliumbromide) assay were ore enhanced in WJ‐MSC‐laden CHT/HA/PCL composites, scanning electron microscopy, real‐time gene expression (alkaline phosphatase ALP, collagen type I Col I, osteocalcin OCN, and bone morphogenetic protein 4 BMP‐4), and immunostaining (COL I, β‐CATENIN, OCN, and SCLEROSTIN SOST) demonstrated pronounced osteogenesis with terminal differentiation on BM‐MSC‐laden CHT/HA/PCL composites only. The enhanced cell functionality on CHT/HA/PCL composites was explained in terms of interplay among the surface properties and the optimal source of MSCs. In addition, osteogenesis in rat tibial model over 6 weeks confirmed a better ratio of bone volume to the total volume for BM‐MSC‐laden composites over scaffold‐only and defect‐only groups. The clinically conformant combination of 3D porous architecture with pore sizes varying in the range of 20 to 200 μm together with controlled in vitro degradation and early osseointegration establish the potential of CHT/HA/PCL composite as a potential cancellous bone analog.
A chitosan‐based, multimaterial was developed using freeze‐drying approach. The biocompatibility was assessed using three popular human stem cells of clinical relevance (bone marrow BM‐, adipose‐, fetal‐derived MSCs) was carried out for osseointegration. A better osseointegration was recorded with BM‐MSC‐laden composites in vitro and in vivo for reconstruction of preclinical rat tibial defects.
To study the incidence, clinical features and outcomes of multidrug-resistant (MDR) bacterial keratitis.
All cases of MDR-bacterial keratitis presenting to our institute over a period of 2 years were ...retrospectively analysed. Details of risk factors, size and depth of infiltrate, treatment, and outcome were noted. Antibiotic susceptibility tests were done on the ocular isolates from the culture of samples obtained from ocular infections, and resistance or sensitivity of the organisms to the commonly used antibiotics was studied.
Forty patients were diagnosed with MDR-bacterial keratitis in the study period. The mean age of patients was 50.9±25.4 years. Most common risk factors were vegetative trauma (n=12, 30.0%), followed by corneal transplantation (n=7, 17.5%) and systemic comorbidities (n=7, 17.5%). Infiltrate was small (<6 mm) in 22 (55%) and large (>6 mm) in 18 (45%) patients. It involved the superficial, mid and deep stroma in 11 (27.5%), 9 (22.5%) and 15 (37.5%) cases, respectively. Gram-negative bacilli (n=18, 45%) were the maximum, among which
(15%) was the most common. Resistance to 3 (n=17, 42.5%) and 4 (n=17, 42.5%) classes of antibiotics was the most commonly observed. One (2.5%) patient showed resistance to all seven classes of drugs tested. Complete resolution of infection was seen in 15 (37.5%) MDR patients on medical management alone. Five (12.5%) patients underwent therapeutic penetrating keratoplasty. Size of the infiltrate was found to have a significant correlation with the outcome (p=0.002).
MDR keratitis, despite being a challenge to treat, can be successfully managed by medical therapy alone, if appropriate therapy is started early in the clinical course.
Purpose
To study long-term visual and refractive outcomes and complications in eyes with anterior chamber intraocular lens (ACIOL) implantation.
Methods
Data of patients who underwent primary and ...secondary ACIOL implantation at L V Prasad Eye Institute, Bhubaneswar between 2011 and 2020 was collected, including details of post-operative visits. For analysis, sample was divided into: group Ia (primary ACIOL in cases without risk factors,
n
= 104); group Ib (primary ACIOL in cases with pre-existing risk factors,
n
= 49); and group II (secondary ACIOL,
n
= 40).
Results
A total of 193 eyes of 192 patients were included. Mean post-operative follow-up in groups I and II were 8.6 and 11.51 months, respectively. Mean pre-operative and last visit corrected distance visual acuity were 1.73 ± 0.11 and 0.42 ± 0.05 logMAR units in group Ia (
p
< 0.001), and 1.53 ± 0.14 and 0.49 ± 0.10 logMAR units in group Ib (
p
< 0.001). The mean spherical equivalent (MSE) for last refraction was −0.37 ± 0.18 diopters (D) and −0.15 ± 0.51 D in groups I and II, respectively. Of 76 eyes in which addition of 2.5 D (over the near emmetropic posterior chamber intraocular lens power) was taken for ACIOL, 40 (52.6%) had MSE within ± 0.5 D. Most common complications were transient corneal edema and anterior chamber reaction. Eyes on anti-glaucoma medications at last visit were eight (7.7%), 15 (30.6%), and two (5.0%) in groups Ia, Ib, and II, respectively.
Conclusion
We observed that ACIOLs have good visual and refractive outcomes. Raised IOP is a concern in eyes with pseudoexfoliation, but can be managed with close monitoring. Hence ACIOL can be a good option for managing aphakia after cataract surgery.
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