Isolation and culture of human primary keratinocytes-a methods review.

Keratinocyte culture is a necessary and widely used tool in a variety of experimental dermatological and biomedical studies. Literature search has revealed a variety of different protocols of human primary keratinocyte isolation and culture. Therefore, the aim of this paper was to review and summarize current trends in human primary keratinocyte culture techniques. We present data on the most popular and effective methods of human keratinocyte isolation and cultivation obtained from screening of 945 papers published during the last 10 years.

ß-escin activates ALDH and prevents cigarette smoke-induced cell death.

BACKGROUND: The tobacco use is one of the biggest public health threats worldwide. Cigarette smoke contains over 7000 chemicals among other aldehydes, regarded as priority toxicants. ß-escin (a mixture of triterpenoid saponins extracted from the Aesculus hippocastanum. L) is a potent activator of aldehyde dehydrogenase (ALDH) - an enzyme catalyzing oxidation of aldehydes to non-toxic carboxylic acids. PURPOSE: The aim of this study was to evaluate the effect of ß-escin on ALDH activity, ALDH isoforms mRNA expression and cytotoxicity in nasal epithelial cells exposed to cigarette smoke extract (CSE). METHODS: Nasal epithelial cells from healthy non-smokers were treated with ß-escin (1 µM) and exposed to 5% CSE. After 6- or 24-hours of stimulation cell viability, DNA damage, ALDH activity and mRNA expression of ALDH isoforms were examined. RESULTS: 24 h ß-escin stimulation revised CSE induced cytotoxicity and DNA damage. Cells cultured with ß-escin or exposed to CSE responded with strong increase in ALDH activity. This effect was more pronounced in cultures treated with combination of ß-escin and CSE. The strongest stimulatory effect on ALDH isoform mRNA expression was observed in cells cultured simultaneously with ß-escin and CSE: at 6 h for ALDH1A1 and ALDH3A1, and at 24 h for ALDH1A3, ALDH3A2, ALDH3B1, and ALDH18A1. Combined ß-escin and CSE treatment prevented the CSE-induced inhibition of ALDH2 expression at 24 h. CONCLUSIONS: ß-escin is an effective ALDH stimulatory and cytoprotective agent and might be useful in the prevention or supportive treatment of tobacco smoke-related diseases.

Safety and Efficacy of DT-DEC01 Therapy in Duchenne Muscular Dystrophy Patients: A 12 - Month Follow-Up Study After Systemic Intraosseous Administration.

Duchenne Muscular Dystrophy (DMD) is a progressive and fatal muscle-wasting disease with no known cure. We previously reported the preliminary safety and efficacy up to six months after the administration of DT-DEC01, a novel Dystrophin Expressing Chimeric (DEC) cell therapy created by fusion of myoblasts of DMD patient and the normal donor. In this 12-month follow-up study, we report on the safety and functional outcomes of three DMD patients after the systemic intraosseous administration of DT-DEC01. The safety of DT-DEC01 was confirmed by the absence of Adverse Events (AE) and Severe Adverse Events (SAE) up to 21 months after intraosseous DT-DEC01 administration. The lack of presence of anti-HLA antibodies and Donors Specific Antibodies (DSA) further confirmed DT-DEC01 therapy safety. Functional assessments in ambulatory patients revealed improvements in 6-Minute Walk Test (6MWT) and timed functions of North Star Ambulatory Assessment (NSAA). Additionally, improvements in PUL2.0 test and grip strength correlated with increased Motor Unit Potentials (MUP) duration recorded by Electromyography (EMG) in both ambulatory and non-ambulatory patients. DT-DEC01 systemic effect was confirmed by improved cardiac and pulmonary parameters and daily activity recordings. This follow-up study confirmed the safety and preliminary efficacy of DT-DEC01 therapy in DMD-affected patients up to 12 months after intraosseous administration. DT-DEC01 introduces a novel concept of personalized myoblast-based cellular therapy that is irrespective of the mutation type, does not require immunosuppression or the use of viral vectors, and carries no risk of off target mutations. This establishes DT-DEC01 as a promising and universally effective treatment option for all DMD patients.

Assessment of Motor Unit Potentials Duration as the Biomarker of DT-DEC01 Cell Therapy Efficacy in Duchenne Muscular Dystrophy Patients up to 12 Months After Systemic-Intraosseous Administration.

Duchenne muscular dystrophy (DMD) is a lethal X-linked disease caused by mutations in the dystrophin gene, leading to muscle degeneration and wasting. Electromyography (EMG) is an objective electrophysiological biomarker of muscle fiber function in muscular dystrophies. A novel, DT-DEC01 therapy, consisting of Dystrophin Expressing Chimeric (DEC) cells created by fusing allogeneic myoblasts from normal donors with autologous myoblasts from DMD-affected patients, was assessed for safety and preliminary efficacy in boys of age 6-15 years old (n = 3). Assessments included EMG testing of selected muscles of upper (deltoideus, biceps brachii) and lower (rectus femoris and gastrocnemius) extremities at the screening visit and at 3, 6, and 12 months following systemic-intraosseous administration of a single low dose of DT-DEC01 therapy (Bioethics Committee approval no. 46/2019). No immunosuppression was administered. Safety of DT-DEC01 was confirmed by the lack of therapy-related Adverse Events or Serious Adverse Events up to 22 months following DT-DEC01 administration. EMG of selected muscles of both, ambulatory and non-ambulatory patients confirmed preliminary efficacy of DT-DEC01 therapy by an increase in motor unit potentials (MUP) duration, amplitudes, and polyphasic MUPs at 12 months. This study confirmed EMG as a reliable and objective biomarker of functional assessment in DMD patients after intraosseous administration of the novel DT-DEC01 therapy.

Dystrophin Expressing Chimeric (DEC) Cell Therapy for Duchenne Muscular Dystrophy: A First-in-Human Study with Minimum 6 Months Follow-up.

Duchenne Muscular Dystrophy (DMD) is a X-linked progressive lethal muscle wasting disease for which there is no cure. We present first-in-human study assessing safety and efficacy of novel Dystrophin Expressing Chimeric (DEC) cell therapy created by fusion of patient myoblasts with myoblasts of normal donor origin. We report here on safety and functional outcomes of the first 3 DMD patients. No study related adverse events (AE) and no serious adverse events (SAE) were observed up to 14 months after systemic-intraosseous administration of DEC01. Ambulatory patients showed improvements in functional tests (6-Minute Walk Test (6MWT), North Star Ambulatory Assessment (NSAA)) and both, ambulatory and non-ambulatory in PUL, strength and fatigue resistance which correlated with improvement of Electromyography (EMG) parameters. DEC01 therapy does not require immunosuppression, involves no risks of off target mutations, is not dependent upon the causative mutation and is therefore a universal therapy that does not use viral vectors and therefore can be readministered, if needed. This study was approved by the Bioethics Committee (approval No. 46/2019). Mechanism of action of the Dystrophin Expressing Chimeric Cell (DEC) cells created via ex vivo fusion of human myoblast from normal and DMD-affected donors. Following systemic-intraosseous administration, DEC engraft and fuse with the myoblasts of DMD patients, deliver dystrophin and improve muscle strength and function. (Created with BioRender.com).

Optimization of human myoblasts culture under different media conditions for application in the in vitro studies.

Human primary in vitro cell cultures are among the most challenging procedures in cellular biology laboratory practice. Myoblasts-progenitor of skeletal muscle origin represent a promising therapeutic cell source since the procedure of their isolation is not technically demanding, and the in vitro culture is relatively straightforward. Myoblasts could be considered as the candidates for clinical applications due to their regenerative potential, and as the carriers of therapeutic proteins introduced through genetic modifications. The main goal of this prospective study was to evaluate different myoblasts isolation strategies based on the pre-plating technique and cells density characteristics. Moreover, testing of different myoblast media formulations-both commercially available and in-house made was performed. Our goal was to establish the in vitro protocol of myoblasts culture allowing for preservation of the proliferative potential and desired phenotype. Our results revealed that in culture of myoblasts of human muscle origin, the pre-plate technique and cell density differences did not correlate with changes in the proliferative potential, however it was observed that low density cells maintained expression of the CD56 marker up to the higher passages. Assessment of different types of culture media confirmed the best performance for DMEM based media without Chicken Embryo Extract (CEE) addition. Cells cultured in DMEM+FBS medium revealed high expression of CD56 and CD90 antigens, absence of the hematopoietic markers and presented stable proliferation profile. This finding is in line with guidelines of regulatory agencies recommending removal of the xeno-derived reagents from the manufacturing process of Advanced Therapy Medicinal Products (ATMP). In this study, human myoblasts culture was optimized in vitro under different media conditions. The next approach in assessment of myoblasts propagation for potential clinical applications will be testing of the clinical grade human platelet lysate (hPL) instead of the FBS.

Beneficial effects of ß-escin on muscle regeneration in rat model of skeletal muscle injury.

BACKGROUND: Recent advancements in understanding ß-escin action provide basis for new therapeutic claims for the drug. ß-escin-evoked attenuation of NF-κB-dependent signaling, increase in MMP-14 and decrease in COUP-TFII content and a rise in cholesterol biosynthesis could be beneficial in alleviating muscle-damaging processes. PURPOSE: The aim of this study was to investigate the effect of ß-escin on skeletal muscle regeneration. METHODS: Rat model of cardiotoxin-induced injury of fast-twich extensor digitorum longus (EDL) and slow-twich soleus (SOL) muscles and C2C12 myoblast cells were used in the study. We evaluated muscles obtained on day 3 and 14 post-injury by histological analyses of muscle fibers, connective tissue, and mononuclear infiltrate, by immunolocalization of macrophages and by qPCR to quantify the expression of muscle regeneration-related genes. Mechanism of drug action was investigated in vitro by assessing cell viability, NF-κB activation, MMP-2 and MMP-9 secretion, and ALDH activity. RESULTS: In rat model, ß-escin rescues regenerating muscles from atrophy. The drug reduces inflammatory infiltration, increases the number of muscle fibers and decreases fibrosis. ß-escin reduces macrophage infiltration into injured muscles and promotes their M2 polarization. It also alters transcription of muscle regeneration-related genes: Myf5, Myh2, Myh3, Myh8, Myod1, Pax3 and Pax7, and Pcna. In C2C12 myoblasts in vitro, ß-escin inhibits TNF-α-induced activation of NF-κB, reduces secretion of MMP-9 and increases ALDH activity. CONCLUSIONS: The data reveal beneficial role of ß-escin in muscle regeneration, particularly in poorly regenerating slow-twitch muscles. The findings provide rationale for further studies on ß-escin repositioning into conditions associated with muscle damage such as strenuous exercise, drug-induced myotoxicity or age-related disuse atrophy.

Polyphenolic compounds from Abutilon grandiflorum leaves.

The flavonol glycosides: kaempferol 3-O-beta-(6"-p-coumaroyl)-glucopyranoside, kaempferol and quercetin 3-O-beta-glucopyranosides and 3-O-beta-rutinosides were isolated and identified from the leaves of Abutilon grandiflorum. Their structures were established by chemical and spectroscopic methods. The presence of phenolic acids: p-hydroxybenzoic, p-coumaric, syringic, and vanillic was confirmed by TLC.

Stem cells migration during skeletal muscle regeneration - the role of Sdf-1/Cxcr4 and Sdf-1/Cxcr7 axis.

The skeletal muscle regeneration occurs due to the presence of tissue specific stem cells - satellite cells. These cells, localized between sarcolemma and basal lamina, are bound to muscle fibers and remain quiescent until their activation upon muscle injury. Due to pathological conditions, such as extensive injury or dystrophy, skeletal muscle regeneration is diminished. Among the therapies aiming to ameliorate skeletal muscle diseases are transplantations of the stem cells. In our previous studies we showed that Sdf-1 (stromal derived factor -1) increased migration of stem cells and their fusion with myoblasts in vitro. Importantly, we identified that Sdf-1 caused an increase in the expression of tetraspanin CD9 - adhesion protein involved in myoblasts fusion. In the current study we aimed to uncover the details of molecular mechanism of Sdf-1 action. We focused at the Sdf-1 receptors - Cxcr4 and Cxcr7, as well as signaling pathways induced by these molecules in primary myoblasts, as well as various stem cells - mesenchymal stem cells and embryonic stem cells, i.e. the cells of different migration and myogenic potential. We showed that Sdf-1 altered actin organization via FAK (focal adhesion kinase), Cdc42 (cell division control protein 42), and Rac-1 (Ras-Related C3 Botulinum Toxin Substrate 1). Moreover, we showed that Sdf-1 modified the transcription profile of genes encoding factors engaged in cells adhesion and migration. As the result, cells such as primary myoblasts or embryonic stem cells, became characterized by more effective migration when transplanted into regenerating muscle.

Flavonoids from Abutilon theophrasti flowers.

Nine flavonoid compounds: kaempferol 3-0-beta-(6"-p-coumaroyl)-glucopyranoside, myricetin 3-O-beta-glucopyranoside, quercetin 3-0-beta-glucopyranoside, quercetin 3-0-alpha-rhamnopyranosyl (1-->6)-beta-glucopyranoside, kaempferol 3-0-beta-glucopyranoside, kaempferol 3-0-alpha-rhamnopyranosyl (1-->6)-beta-glucopyranoside, quercetin 7-0-beta-glucoside, quercetin 7-0-beta-diglucoside, kaempferol 7-0-beta-diglucoside were isolated and identified from the flowers of Abutilon theophrasti.

Differentiation of interglycosidic linkages in permethylated flavonoid glycosides from linked-scan mass spectra (B/E).

A series of per-O-methylated flavonoid di- and tri-glycosides, linked with 1-2 and/or 1-6 glycosidic bonds between sugar rings that were isolated from different plant materials were analyzed. It was demonstrated that the fragmentation behavior of permethylated flavonoid glycosides is dependent on the glycosidic bond placement between sugars. Y(n) type fragment ions, created after glycosidic bond cleavage with oxygen retention on sugar at the reducing end for permethylated compounds, were observed in the normal and linked-scan mass spectra recorded for alpha(1-2) bonded conjugates of flavonoid di- and tri-glycosides. Moreover, for alpha (1-6) linked glycosides, Y fragments created after rearrangement and elimination of internal sugar residues were observed in addition to Y(n) type ions, but these fragment ions were not registered in normal desorption ionization spectra. This second type of fragmentation was also reported previously in collision-induced dissociation tandem mass spectrometry (CID MS/MS) spectra of some oligosaccharides and flavonoid glycosides, but their presence was independent of the glycosidic bonds placement between sugar rings.

Flavonoids in the leaves of Asclepias incarnata L.

Seven flavonoid compounds: quercelin 3-O-beta-galactopyranoside, 3-O-beta-glucopyranoside, 3-O-arabinoside, 3-O-beta-glucopyranosyl (1-->2)-beta-galactopyranoside, 3-O-beta-xylopyranosyl (1-->2)-beta-galactopyranoside, 3-O-alpha-rhamnopyranosyl (1-->2)-beta-galactopyranoside and kaempferol 3-beta-glucopyranoside were isolated and identified from the leaves of Asclepias incarnata, L. (Asclepiadaceae).

Flavonoids from flowers of Malva crispa L. (Malvaceae).

The following flavonoids were isolated and identified from the flowers of Malva crispa L.: kaempferol 3-O-beta-glucopyranoside, 3-O-(6"-tran-p-coumaroyl)-beta-D-glucopyranoside, 7-O-beta-D-glucopyranoside, 3-O-alpha-L-rhamnopyranosyl (1-->6)-beta-D-glucopyranoside and 3,7-O-diglucoside as well as quercetin 3-O-beta-D-glucopyranoside, 3-O-alpha-L-rhamnopyranosyl (1-->6) -beta-D-glucopyranoside and apigenin 7-O-beta-D-glucopyranoside. Their structures were established by chemical analysis, UV, 1H and 13C NMR spectrometry.

Flavonoid compounds in the flowers of Abutilon indicum (L.) Sweet (Malvaceae).

Seven flavonoid compounds: luteolin, chrysoeriol, luteolin 7-O-beta-glucopyranoside, chrysoeriol 7-O-beta-glucopyranoside, apigenin 7-O-beta-glucopyranoside, quercetin 3-O-beta-glucopyranoside, quercetin 3-O-alpha-rhamnopyranosyl (1 --> 6)-beta-glucopyranoside, were isolated and identified from the flowers of Abutilon indicum (L.) Sweet (Malvaceae).

Electrospray mass spectrometric decomposition of some glucuronic acid-containing flavonoid diglycosides.

Mass spectrometric fragmentation patterns of herbacetin 3-O-glucopyranoside-8-O-glucuronopyranoside (1), gossypetin 3-O-glucopyranoside-8-O-glucuronopyranoside (2) and takakin 7-O-glucopyranoside-8-O-glucuronopyranoside (3) were elucidated from mass spectra obtained with electrospray ionisation. The usefulness of the fragmentation patterns observed in the positive and negative mode for structural elucidation of the studied compounds is discussed. The fragmentation of 3 was substantially different from 1 and 2, especially in the negative mode. In order to explain these differences, theoretical calculations were performed.