Klebsiella oxytoca Complex: Update on Taxonomy, Antimicrobial Resistance, and Virulence.

Klebsiella oxytoca is actually a complex of nine species-Klebsiella grimontii, Klebsiella huaxiensis, Klebsiella michiganensis, K. oxytoca, Klebsiella pasteurii, Klebsiella spallanzanii, and three unnamed novel species. Phenotypic tests can assign isolates to the complex, but precise species identification requires genome-based analysis. The K. oxytoca complex is a human commensal but also an opportunistic pathogen causing various infections, such as antibiotic-associated hemorrhagic colitis (AAHC), urinary tract infection, and bacteremia, and has caused outbreaks. Production of the cytotoxins tilivalline and tilimycin lead to AAHC, while many virulence factors seen in Klebsiella pneumoniae, such as capsular polysaccharides and fimbriae, have been found in the complex; however, their association with pathogenicity remains unclear. Among the 5,724 K. oxytoca clinical isolates in the SENTRY surveillance system, the rates of nonsusceptibility to carbapenems, ceftriaxone, ciprofloxacin, colistin, and tigecycline were 1.8%, 12.5%, 7.1%, 0.8%, and 0.1%, respectively. Resistance to carbapenems is increasing alarmingly. In addition to the intrinsic blaOXY, many genes encoding ß-lactamases with varying spectra of hydrolysis, including extended-spectrum ß-lactamases, such as a few CTX-M variants and several TEM and SHV variants, have been found. blaKPC-2 is the most common carbapenemase gene found in the complex and is mainly seen on IncN or IncF plasmids. Due to the ability to acquire antimicrobial resistance and the carriage of multiple virulence genes, the K. oxytoca complex has the potential to become a major threat to human health.

Genome Analysis of Klebsiella oxytoca Complex for Antimicrobial Resistance and Virulence Genes.

Klebsiella oxytoca complex comprises nine closely related species causing human infections. We curated genomes labeled Klebsiella (n = 14,256) in GenBank and identified 588 belonging to the complex, which were examined for precise species, sequence types, K- and O-antigen types, and virulence and antimicrobial resistance genes. The complex and Klebsiella pneumoniae share many K- and O-antigen types. Of the complex, K. oxytoca and Klebsiella michiganensis appear to carry more virulence genes and be more commonly associated with human infections.

A Pandrug-Resistant Providencia Carrying Two blaIMP Carbapenemase-Encoding Genes Including blaIMP-69, a New blaIMP Variant, on a Newly Identified Worldwide-Distributed IncC Plasmid.

Imipenemase (IMP) is a metallo-ß-lactamase that confers resistance to almost all ß-lactams. Identification of IMP genes is essential for understanding and combatting antibiotic resistance. In this study, we report a pandrug-resistant Providencia strain from a human rectal swab. This strain carried 2 blaIMP carbapenemase genes, blaIMP-69 and blaIMP-4. IMP-69 is a novel IMP variant with an amino acid substitution at A21T compared with IMP-8. blaIMP-69 was found in a blaIMP-69-aacA4 array of an integron on a 165-kilobase (kb) IncC self-transmissible plasmid, whereas blaIMP-4 was located in a blaIMP-4-qacG-aacA4-catB3 array of an integron on a 19-kb nonself-transmissible plasmid. Such coexistence has the potential to allow the generation of new, hybrid blaIMP variants by homologous recombination. The blaIMP-69-carrying IncC plasmid belonged to the core-genome plasmid multilocus sequence typing (cgPMLST) 3.5 type. We found that cgPMLST 3.5 IncC plasmids have been circulating worldwide for decades and may represent a common vehicle mediating the spread of antimicrobial resistance.

Circular RNA circRHOT1 promotes hepatocellular carcinoma progression by initiation of NR2F6 expression.

BACKGROUND: Increasing evidence has revealed a close relationship between non-coding RNAs and cancer progression. Circular RNAs (circRNAs), a recently identified new member of non-coding RNAs, are demonstrated to participate in diverse biological processes, such as development, homeostatic maintenance and pathological responses. The functions of circRNAs in cancer have drawn wide attention recently. Until now, the expression patterns and roles of circRNAs in hepatocellular carcinoma (HCC) have remained largely unknown. METHODS: Bioinformatics method was used to screen differentially expressed novel circRNAs in HCC. Northern blotting, qRT-PCR, in situ hybridization (ISH) and RNA-FISH were utilized to analyzed the expression of circRHOT1 in HCC tisues.CCK8, colony formation, EdU assays were used to analyze proliferation of HCC cells. Transwell assay was utilized to analyze HCC cell migration and invasion. FACS was used for apoptosis analysis. Xenograft experiments were used to analyze tumor growth in vivo. Mass spectrum, RNA pulldown, RIP and EMSA was utilized to test the interaction between circRHOT1 and TIP60. RNA-sequencing method was used to analyze the downstream target gene of circRHOT1. RESULTS: We identified circRHOT1 (hsa_circRNA_102034) as a conserved and dramatically upregulated circRNA in HCC tissues. HCC patients displaying high circRHOT1 level possessed poor prognosis. Through in vitro and in vivo experiments, we demonstrated circRHOT1 significantly promoted HCC growth and metastasis. Regarding the mechanism, we conducted a RNA pulldown with a biotin-labeled circRHOT1-specific probe and found that circRHOT1 recruited TIP60 to the NR2F6 promoter and initiated NR2F6 transcription. Moreover, NR2F6 knockout inhibited growth, migration and invasion, whereas rescuing NR2F6 in circRHOT1-knockout HCC cells rescued the proliferation and metastasis abilities of HCC cells. CONCLUSION: Taken together, circRHOT1 inhibits HCC development and progression via recruiting TIP60 to initiate NR2F6 expression, indicating that circRHOT1 and NR2F6 may be potential biomarkers for HCC prognosis.

Sequence Type 273 Carbapenem-Resistant Klebsiella pneumoniae Carrying blaNDM-1 and blaIMP-4.

A carbapenem-resistant Klebsiella pneumoniae isolate was recovered from human blood. Its whole-genome sequence was obtained using Illumina and long-read MinION sequencing. The strain belongs to sequence type 273 (ST273), which was found recently and caused an outbreak in Southeast Asia. It has two carbapenemase genes, blaNDM-1 (carried by an ST7 IncN self-transmissible plasmid) and blaIMP-4 (located on a self-transmissible IncHI5 plasmid). Non-KPC-producing ST237 may represent a lineage of carbapenem-resistant K. pneumoniae, which warrants further monitoring.

Regulation of adipose-tissue-derived stromal cell orientation and motility in 2D- and 3D-cultures by direct-current electrical field.

Cell alignment and motility play a critical role in a variety of cell behaviors, including cytoskeleton reorganization, membrane-protein relocation, nuclear gene expression, and extracellular matrix remodeling. Direct current electric field (EF) in vitro can direct many types of cells to align vertically to EF vector. In this work, we investigated the effects of EF stimulation on rat adipose-tissue-derived stromal cells (ADSCs) in 2D-culture on plastic culture dishes and in 3D-culture on various scaffold materials, including collagen hydrogels, chitosan hydrogels and poly(L-lactic acid)/gelatin electrospinning fibers. Rat ADSCs were exposed to various physiological-strength EFs in a homemade EF-bioreactor. Changes of morphology and movements of cells affected by applied EFs were evaluated by time-lapse microphotography, and cell survival rates and intracellular calcium oscillations were also detected. Results showed that EF facilitated ADSC morphological changes, under 6 V/cm EF strength, and that ADSCs in 2D-culture aligned vertically to EF vector and kept a good cell survival rate. In 3D-culture, cell galvanotaxis responses were subject to the synergistic effect of applied EF and scaffold materials. Fast cell movement and intracellular calcium activities were observed in the cells of 3D-culture. We believe our research will provide some experimental references for the future study in cell galvanotaxis behaviors.

Endogenously produced itaconate negatively regulates innate-driven cytokine production and drives global ubiquitination in human macrophages.

A wide variety of electrophilic derivatives of itaconate, the Kreb's cycle-derived metabolite, are immunomodulatory, yet these derivatives have overlapping and sometimes contradictory activities. Therefore, we generated a genetic system to interrogate the immunomodulatory functions of endogenously produced itaconate in human macrophages. Endogenous itaconate is driven by multiple innate signals restraining inflammatory cytokine production. Endogenous itaconate directly targets cysteine 13 in IRAK4 (disrupting IRAK4 autophosphorylation and activation), drives the degradation of nuclear factor κB, and modulates global ubiquitination patterns. As a result, cells unable to make itaconate overproduce inflammatory cytokines such as tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6), and IL-1ß in response to these innate activators. In contrast, the production of interferon (IFN)ß, downstream of LPS, requires the production of itaconate. These data demonstrate that itaconate is a critical arbiter of inflammatory cytokine production downstream of multiple innate signaling pathways, laying the groundwork for the development of itaconate mimetics for the treatment of autoimmunity.

DPP4 Regulates the Th17/IL-17 Axis and Accelerates Epithelial Mesenchymal Transition to Promote Ovalbumin-Induced Asthma in Female C57BL/6J Mice.

BACKGROUND: Dipeptidyl peptidase-4 (DPP4) is a transmembrane glycoprotein, prevalent across a variety of tissues and cells and can be foundin a solubilised in peripheral blood. This paper aims at determining the role of sCD26/sDPP4 in Th17 cell polarization and airway epithelial cell to epithelial mesenchymal transition (EMT) in asthma. METHODS: Female C57BL/6J mice were treated with ovalbumin to constructed asthma mice. The CD4+ T cell, and bronchial epithelial cells (BECs) were purified from the spleens and bronchus of mice. The KRT8 expression in BECs were identified by immunofluorescence (IF). Th17 cells were differentiated from a CD4+ T cell. Flow cytometry was usewd to identify and calculate the Th17 and Treg cells. Mice woth asthma were treated by DPP4 overexpressing lentivirus or DPP4 inhibitor. Histopathological modifications were assessed by hematoxylin-eosin (HE), periodic acid Schiff (PAS), and Masson staining. The total number of leucocytes was detected using a hemocytometer. For detection, quantitative Real-time PCR (qRT-PCR), western blotting (WB), and IF were used to evaluate the expression of E-cadherin and alpha-smooth muscle actin (α-SMA). Enzyme-linked immunosorbent assay (ELISA) was performed to analyze the DPP4, IL-4, IL-5, IL-13 and IL-17 levels. RESULTS: The findings suggest that sCD26/sDPP4 promote CD4+ T cells differentiation into Th17 cells in a depending on the applied dose. sCD26/sDPP4 up-regulated the expression of α-SMA and down-regulated the expression of E-cadherin in TGF-ß1-induced mouse BECs, which was reversed by DPP4 inhibitor. Co-culture induced a synergic effect between Th17 cells and sCD26/sDPP4 on the formation of airway EMT in BECs. Furthermore, DPP4 inhibitor prevented lung-bronchial inflammatory infiltration, mucus secretion, goblet cell hyperplasia and collagen deposition in asthma mice. Meanwhile, DPP4 inhibitor decreased the levels of DPP4, IL-4, IL-5, IL-13, IL-17 and increased the total number of leukocytes in bronchoalveolar lavage fluid of asthma mice. In addition, DPP4 inhibitor also inhibited airway EMT and Th17 cell polarization in asthma mice. CONCLUSIONS: The results in this paper show that up-regulation of DPP4 enabled airway inflammation and airway remodeling in asthmatic mice by modulating the Th17/IL-17 axis and accelerating the airway EMT, which isa therapeutic target in asthma.

[Correlation among the levels of C-reactive protein and interleukin-18, quality of life, and lung function in patients with chronic obstructive pulmonary disease].

OBJECTIVE: To determine the correlation among the levels of C-reactive protein (CRP) and interleukin-18 (IL-18), quality of life, and pulmonary function in patients with chronic obstructive pulmonary disease (COPD),and to evaluate George's respiratory questionnaire (SGRQ). METHODS: From October 2008 to March 2009, 39 patients with an acute exacerbation COPD (the AECOPD group), 21 patients with stable COPD (the COPD group), and 22 normal people (the control group) were enrolled in our study. The serum CRP level was measured with Beckman Coulter Immage and the IL-18 level was measured by ELISA. All subjects completed the SGRQ assessment and all patients with COPD accepted pulmonary function test. RESULTS: The serum levels of CRP and IL-18, and SGRQ scores in the AECOPD group were significant1y higher than those in the COPD group and the control group (P<0.01). The serum levels of CRP and IL-18 and SGRQ scores in the COPD group were significant1y higher than those in the control group (P<0.01). The serum levels of CRP and IL-18 in the AECOPD group and the COPD group were negatively correlated with FEV1% and forced expiratory volume in one second (FEV1)/forced vital capacity (FVC). SGRQ scores in the AECOPD group and the COPD group were negatively correlated with FEV1% and FEV1/FVC. The serum levels of CRP, IL-18 in the AECOPD group and the COPD group were positively correlated with SGRQ scores. CONCLUSION: CRP and IL-18 are involved in the process of inflammatory reaction of COPD, which could be markers of the acute exacerbation period of COPD. The serum levels of CRP and IL-18 might be related to the lung dysfunction and the lower quality of life in the patients with COPD. SGRQ is an effective, susceptible, and feasible method to evaluate the quality of life in patients with COPD.

AI based colorectal disease detection using real-time screening colonoscopy.

Colonoscopy is an effective tool for early screening of colorectal diseases. However, the application of colonoscopy in distinguishing different intestinal diseases still faces great challenges of efficiency and accuracy. Here we constructed and evaluated a deep convolution neural network (CNN) model based on 117 055 images from 16 004 individuals, which achieved a high accuracy of 0.933 in the validation dataset in identifying patients with polyp, colitis, colorectal cancer (CRC) from normal. The proposed approach was further validated on multi-center real-time colonoscopy videos and images, which achieved accurate diagnostic performance on detecting colorectal diseases with high accuracy and precision to generalize across external validation datasets. The diagnostic performance of the model was further compared to the skilled endoscopists and the novices. In addition, our model has potential in diagnosis of adenomatous polyp and hyperplastic polyp with an area under the receiver operating characteristic curve of 0.975. Our proposed CNN models have potential in assisting clinicians in making clinical decisions with efficiency during application.

Contamination of SARS-CoV-2 in patient surroundings and on personal protective equipment in a non-ICU isolation ward for COVID-19 patients with prolonged PCR positive status.

OBJECTIVES: We performed an environmental sampling study to investigate the environmental contamination of SARS-CoV-2 by COVID-19 patients with prolonged PCR positive status of clinical samples. METHODS: We sampled the air from rooms for nine COVID-19 patients with illness or positive PCR > 30 days, before and after nasopharyngeal/oropharyngeal swabbing and before and after nebulization treatment. We also sampled patients' surroundings and healthcare workers' personal protection equipment (PPE) in a non-ICU ward. SARS-CoV-2 was detected by PCR. RESULTS: Eighty-eight samples were collected from high-touch surfaces and floors in patient rooms and toilets, with only the bedsheets of two patients and one toilet positive for SARS-CoV-2. All air samples (n = 34) were negative for SARS-CoV-2. Fifty-five samples collected from PPE were all negative. CONCLUSION: Contamination of near-patient surroundings was uncommon for COVID-19 patients with prolonged PCR positive status if environmental cleaning/disinfection were performed rigorously. Airborne transmission of SARS-CoV-2 was unlikely in these non-ICU settings.

The co-transfer of plasmid-borne colistin-resistant genes mcr-1 and mcr-3.5, the carbapenemase gene blaNDM-5 and the 16S methylase gene rmtB from Escherichia coli.

We found an unusual Escherichia coli strain with resistance to colistin, carbapenem and amikacin from sewage. We therefore characterized the strain and determined the co-transfer of the resistance determinants. Whole genome sequencing was performed using both Illumina HiSeq X10 and MinION sequencers. Short and long reads were subjected to de novo hybrid assembly. Sequence type, antimicrobial resistance genes and plasmid replicons were identified from the genome sequences. Phylogenetic analysis of all IncHI2 plasmids carrying mcr-1 available in GenBank was performed based on core genes. Conjugation experiments were performed. mcr-3.5 was cloned into E. coli DH5α. The strain belonged to ST410, a type with a global distribution. Two colistin-resistant genes, mcr-1.1 and mcr-3.5, a carbapenemase gene blaNDM-5, and a 16S methylase gene rmtB were identified on different plasmids of IncHI2(ST3)/IncN, IncP, IncX3 and IncFII, respectively. All of the four plasmids were self-transmissible and mcr-1.1, mcr-3.5, blaNDM-5 and rmtB were transferred together. mcr-1-carrying IncHI2 plasmids belonged to several sequence types with ST3 and ST4 being predominant. MIC of colistin (4 µg/ml) for DH5α containing mcr-3.5 was identical to that containing the original mcr-3 variant. In conclusion, carbapenem resistance, colistin resistance and high-level aminoglycoside resistance can be transferred together even when their encoding genes are not located on the same plasmid. The co-transfer of multiple clinically-important antimicrobial resistance represents a particular challenge for clinical treatment and infection control in healthcare settings. Isolates with resistance to both carbapenem and colistin are not restricted to a given sequence type but rather are diverse in clonal background, which warrants further surveillance. The amino acid substitutions of MCR-3.5 have not altered its activity against colistin.

Key evolutionary events in the emergence of a globally disseminated, carbapenem resistant clone in the Escherichia coli ST410 lineage.

There is an urgent need to understand the global epidemiological landscape of carbapenem-resistant Escherichia coli (CREC). Here we provide combined genomic and phenotypic characterization of the emergence of a CREC clone from the ST410 lineage. We show that the clone expands with a single plasmid, within which there is frequent switching of the carbapenemase gene type between blaNDM and blaOXA-181 with no impact on plasmid stability or fitness. A search for clone-specific traits identified unique alleles of genes involved in adhesion and iron acquisition, which have been imported via recombination. These recombination-derived allelic switches had no impact on virulence in a simple infection model, but decreased efficiency in binding to abiotic surfaces and greatly enhanced fitness in iron limited conditions. Together our data show a footprint for evolution of a CREC clone, whereby recombination drives new alleles into the clone which provide a competitive advantage in colonizing mammalian hosts.

Voltammetric Behavior of o-Nitrophenol and Damage to DNA.

The electrochemical behavior of o-nitrophenol was studied in detail with a glassy carbon electrode (GCE). The dependence of peak potential on pH indicated that equivalent electrons and protons were involved in the process of o-nitrophenol reduction. The interaction of o-nitrophenol with calf thymus DNA was investigated by adding DNA to the o-nitrophenol solution and by immobilizing DNA on GCE, respectively. The peak current decrement and peak potential shift in presence of DNA indicated that o-nitrophenol could interact with DNA. The result was demonstrated that the in situ DNA damage was detected by differential pulse voltammetry after the o-nitrophenol was electrochemically reduced.

Assessment of the characteristics and biocompatibility of gelatin sponge scaffolds prepared by various crosslinking methods.

This comparative study aims to identify a biocompatible and effective crosslinker for preparing gelatin sponges. Glutaraldehyde (GTA), genipin (GP), 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC), and microbial transglutaminase (mTG) were used as crosslinking agents. The physical properties of the prepared samples were characterized, and material degradation was studied in vitro with various proteases and in vivo through subcutaneous implantation of the sponges in rats. Adipose-derived stromal stem cells (ADSCs) were cultured and inoculated onto the scaffolds to compare the cellular biocompatibility of the sponges. Cellular seeding efficiency and digestion time of the sponges were also evaluated. Cellular viability and proliferation in scaffolds were analyzed by fluorescence staining and MTT assay. All the samples exhibited high porosity, good swelling ratio, and hydrolysis properties; however, material strength, hydrolysis, and enzymolytic properties varied among the samples. GTA-sponge and GP-sponge possessed high compressive moduli, and EDC-sponge exhibited fast degradation performance. GTA and GP sponge implants exerted strong in vivo rejections, and the former showed poor cell growth. mTG-sponge exhibited the optimal comprehensive performance, with good porosity, compressive modulus, anti-degradation ability, and good biocompatibility. Hence, mTG-sponge can be used as a scaffold material for tissue engineering applications.

Preparation and characteristics of gelatin sponges crosslinked by microbial transglutaminase.

Microbial transglutaminase (mTG) was used as a crosslinking agent in the preparation of gelatin sponges. The physical properties of the materials were evaluated by measuring their material porosity, water absorption, and elastic modulus. The stability of the sponges were assessed via hydrolysis and enzymolysis. To study the material degradation in vivo, subcutaneous implantations of sponges were performed on rats for 1-3 months, and the implanted sponges were analyzed. To evaluate the cell compatibility of the mTG crosslinked gelatin sponges (mTG sponges), adipose-derived stromal stem cells were cultured and inoculated into the scaffold. Cell proliferation and viability were measured using alamarBlue assay and LIVE/DEAD fluorescence staining, respectively. Cell adhesion on the sponges was observed by scanning electron microscopy (SEM). Results show that mTG sponges have uniform pore size, high porosity and water absorption, and good mechanical properties. In subcutaneous implantation, the material was partially degraded in the first month and completely absorbed in the third month. Cell experiments showed evident cell proliferation and high viability. Results also showed that the cells grew vigorously and adhered tightly to the sponge. In conclusion, mTG sponge has good biocompatibility and can be used in tissue engineering and regenerative medicine.

[Effect of different electrical stimulation waves on orientation and alignment of adipose derived mesenchymal stem cells].

Objective: To investigate the effect of different electrical stimulation waves on orientation and alignment of adipose derived mesenchymal stem cells (ADSCs). Methods: ADSCs were isolated from 5-week-old Sprague Dawley rats (weight, 100-150 g) and cultivated. The cells at passages 3-5 were inoculated to prepare cell climbing slices, subsequently was exposed to direct-current electrical stimulations (ES) at electric field strengths of 1, 2, 3, 4, 5, and 6 V/cm on a homemade electric field bioreactor (groups A1, A2, A3, A4, A5, and A6); at electric field strength of 6 V/cm, at 50% duty cycle, and at frequency of 1 and 2 Hz (groups B1 and B2) of square wave ES; at electric field strength of 6 V/cm, at pulse width of 2 ms, and at frequency of 1 and 2 Hz (groups C1 and C2) of biphasic pulse wave ES; and no ES was given as a control (group D). The changes of cellular morphology affected by applied ES were evaluated by time-lapse micropho-tography via inverted microscope. The cell alignment was evaluated via average orientation factor ( OF). The cytoske-leton of electric field treated ADSCs was characterized by rhodamine-phalloidin staining. The cell survival rates were assessed via cell live/dead staining and intracellular calcium activities were detected by calcium ion fluorescent staining. Results: The response of ADSCs to ES was related to the direct-current electric field intensity. The higher the direct-current electric field intensity was, the more cells aligned perpendicular to the direction of electric field. At each time point, there was no obvious cell alignment in groups B1, B2 and C1, C2. The average OF of groups A5 and A6 were significantly higher than that of group D ( P<0.05), but no significant difference was found between other groups and group D ( P>0.05). The cytoskeleton staining showed that the cells of groups A5 and A6 exhibited a compact fascicular structure of cytoskeleton, and tended to be perpendicular to the direction of the electric field vector. The cellular survival rate of groups A4, A5, and A6 were significantly lower than that of group D ( P<0.05), but no significant difference was found between other groups and group D ( P>0.05). Calcium fluorescence staining showed that the fluorescence intensity of calcium ions in groups A4, A5, and A6 was slightly higher than that in group D, and no significant difference was found between other groups and group D. Conclusion: The direct-current electric field stimulations with physiological electric field strength (5 V/cm and 6 V/cm) can induce the alignment of ADSCs, but no cell alignment is found under conditions of less than 5 V/cm direct-current electric field, square wave, and biphasic pulse wave stimulation. The cellular viability is negatively correlated with the electric field intensity.

Obtaining spontaneously beating cardiomyocyte-like cells from adipose-derived stromal vascular fractions cultured on enzyme-crosslinked gelatin hydrogels.

Heart failure often develops after acute myocardial infarction because the injured myocardial tissue fails to recover or regenerate. Stem cell transplantation using adult cell sources, such as adipose-derived stromal vascular fraction (SVF), draws extensive attention. In this study, SVF cells were isolated from rat adipose tissue and cultivated on enzyme-crosslinked gelatin hydrogels. Morphological features of cell development and spontaneous beating behavior from these cells were observed and recorded. Cardiac phenotypes were characterized via immunofluorescence staining, and the expression of cardiac-specific genes was measured via RT-PCR. The functional assessment of SVF-derived cardiomyocyte-like cells (SVF-CMs) was performed by detecting cellular calcium transient activities and pharmacological responses. Results showed that most SVF-CMs exhibited elongated myotubule shapes and expressed cardiac troponin I strongly. SVF-CMs expressed cardiac-specific RNA (including transcription factors GATA binding protein 4) and myocyte enhancer factor 2c, as well as the structural proteins, namely, sarcomere actinin alpha 2, cardiac troponin I type 3, cardiac troponin T type 2, and cardiac gap junction protein alpha 1. Their beating mode, calcium activities, and pharmacological responses were similar to those of native CMs. Spontaneously beating SVF-CMs can be derived from adipose tissue-derived SVFs, and enzyme-crosslinked gelatin hydrogel promoted the cardiac differentiation of SVF cells.

[THREE-DIMENSIONAL CULTURED ADIPOSE-DERIVED STEM CELLS BASED ON MICROBIAL TRANSGLUTAMINASE ENZYME CROSSLINKED GELATIN HYDROGEL].

OBJECTIVE: To study the growth of adipose-derived stem cells (ADSCs) planted in three-dimensional (3D) materials, a 3D cultured ADSCs system based on microbial transglutaminase (mTG) enzyme crosslinked gelatin hydrogel was constructed. METHODS: ADSCs were isolated from the subcutaneous adipose tissue of a Sprague Dawley rat by collagenase digestion and centrifugation, and were cultured for passage. The mTG enzyme crosslinked gelatin hydrogel was firstly synthesized by mixing gelatin and mTG, and then the ADSCs were encapsulated in situ (2D environment) and cultured in the 3D materials (3D environment). The morphology and adhesion of cells were observed by inverted phase contrast microscope. In addition, HE staining and Masson staining were carried out to observe the distribution of cells in the material. Living and death situation of ADSCs in the materials was observed by fluorescence microscope and laser scanning confocal microscopy. Scanning electron microscopy was used to observe the adhesion of ADSCs on hydrogel surface. Alamar-Blue method was used to detect the proliferation of ADSCs in the hydrogel. Moreover, the results were compared between the cells cultured in 2D environment and those in 3D environment. RESULTS: The result of 2D culture showed that ADSCs grew well on the hydrogel surface with normal functioning and had good adhesion. The results of 3D culture showed that ADSCs grew well in 3D cultured mTG enzyme crosslinked gelatin hydrogel, and presented 3D shape. Cells obviously extended in all directions. The number of apoptotic cells was very small. The cells of 3D culture at each time point was significantly less than that of the conventional culture cells, difference was statistically significant (P<0.05). But after 8 days culture, the proliferation of the cells cultured in the mTG enzyme crosslinked gelatin hydrogel increased more quickly. CONCLUSIONS: ADSCs can grow well with good adhesion and show high viability in 3D culture system constructed by mTG enzyme crosslinked gelatin hydrogel.