Redifferentiation Process Research Articles

Label-free long-term measurements of adipocyte differentiation from patient-driven fibroblasts and quantitative analyses of in situ lipid droplet generation

The visualization and tracking of adipocytes and their lipid droplets (LDs) during differentiation are pivotal in developmental biology and regenerative medicine studies. Traditional staining or labeling methods, however, pose significant challenges due to their labor-intensive sample preparation, potential disruption of intrinsic cellular physiology, and limited observation timeframe. This study introduces a novel method for long-term visualization and quantification of biophysical parameters of LDs in unlabeled adipocytes, utilizing the refractive index (RI) distributions of LDs and cells. We employ low-coherence holotomography (HT) to systematically investigate and quantitatively analyze the 42-day redifferentiation process of fat cells into adipocytes. This technique yields three-dimensional, high-resolution refractive tomograms of adipocytes, enabling precise segmentation of LDs based on their elevated RI values. Subsequent automated analysis quantifies the mean concentration, volume, projected area, and dry mass of individual LDs, revealing a gradual increase corresponding with adipocyte maturation. Our findings demonstrate that HT is a potent tool for non-invasively monitoring live adipocyte differentiation and analyzing LD accumulation. This study, therefore, offers valuable insights into adipogenesis and lipid research, establishing HT and image-based analysis as a promising approach in these fields.

Insights into the bZIP gene family in Osmanthus fragrans and the role of OfbZIP98 in the callus and flower

Osmanthus fragrans has important ornamental and economic value. As the callus re-differentiation process is difficult, a genetic transformation system has not been successfully established in O. fragrans. The basic leucine zipper (bZIP) transcription factors play an important role in plant growth and development. A total of 116 OfbZIP genes were identified in O. fragrans using bioinformatics methods. According to the evolutionary relationships, these genes were divided into 12 subfamilies and were found to be unevenly distributed on 23 chromosomes of O. fragrans. The quantitative real-time PCR results showed that the expression trend of OfbZIP98 was consistent with the proliferation of O. fragrans callus. The subcellular localization and yeast self-activation results showed that OfbZIP98 was localized in the nucleus and had self-activation activity. Further, phenotypic observation of over-expressed tobacco showed that compared with the wild type (WT), the transgenic strain formed callus 4 d earlier, and callus re-differentiation began on day 15. Analysis of the callus differentiation rate showed that after 20 d of culture, the differentiation rate of the leaf callus of the transgenic strain was about twice that of the WT plants. In addition, measurements of corolla diameter, corolla circumference, corolla area, and corolla tube diameter indicated that the petals of the transgenic strain were significantly larger than the WT plants. Our results showed that OfbZIP98 has the important function of promoting callus growth, re-differentiation, and flower organ enlargement. These findings provide new insights into the potential role of OfbZIP98 in the growth and development of O. fragrans.

Mdka produced by the activated HSCs drives bipotential progenitor cell redifferentiation during zebrafish biliary-mediated liver regeneration.

After extensive hepatocyte loss or impaired hepatocyte proliferation, liver regeneration occurs through trans-differentiation of biliary epithelial cells (BECs), which involves dedifferentiation of biliary epithelial cells into bipotential progenitor cells (BP-PCs) and subsequent redifferentiation of BP-PCs into nascent hepatocytes and biliary epithelial cells. Despite several studies on the redifferentiation process of BP-PCs into nascent hepatocytes, the contributions of nonparenchymal cells in this process remain poorly understood. Using the zebrafish severe liver injury model, we observed specific expression of midkine a (Mdka) in the activated HSCs through single-cell analyses and fluorescence in situ hybridization. Genetic mutation, pharmacological inhibition, whole-mount in situ hybridizations, and antibody staining demonstrated an essential role of mdka in the redifferentiation of BP-PCs during liver regeneration. Notably, we identified Nucleolin (Ncl), the potential receptor for Mdka, specifically expressed in BP-PCs, and its mutant recapitulated the mdka mutant phenotypes with impaired BP-PC redifferentiation. Mechanistically, the Mdka-Ncl axis drove Erk1 activation in BP-PCs during liver regeneration. Furthermore, overexpression of activated Erk1 partially rescued the defective liver regeneration in the mdka mutant. The activated HSCs produce Mdka to drive the redifferentiation process of BP-PCs through activating Erk1 during the biliary-mediated liver regeneration, implying previously unappreciated contributions of nonparenchymal cells to this regeneration process.

Cardiomyocyte Maturation During Regeneration

The interplay between calcium signaling and sarcomeres plays a crucial role in directing cardiomyocyte maturation during cardiac regeneration. During cardiac regeneration, various processes occur to restore damaged or lost cardiac tissue, including the proliferation and differentiation of precursor cells into functional cardiomyocytes. These newly formed cardiomyocytes undergo a maturation process to acquire the structural and functional characteristics of adult cardiomyocytes. Nguyen and colleagues found that the cardiac dyad, a structure that regulates calcium handling and excitation-contraction coupling, played a key role in the redifferentiation process. A component of the cardiac dyad called leucine-rich repeat-containing 10 (Lrrc10) acted as a negative regulator of proliferation, prevented cardiomegaly, and induced redifferentiation. They found that its function was conserved in mammalian cardiomyocytes. Their study highlights the importance of the underlying mechanisms required for heart regeneration and their application to the generation of fully functional cardiomyocytes. Science. 2023 May 19;380(6646):758-764. doi: 10.1126/science.abo6718.

Identification of Th1-polarized Th17 cells: solving the problem

Helper T cells producing IL-17 (Th17) have high plasticity: restimulation of lymphocytes in an inflammatory environment can induce their transformation into cells with another phenotype, and a shift towards Th1 is the most common. The result of this transformation is the appearance of cells expressing along with the classical markers of Th17 cells key Th1-associated molecules. In its most general form, this population is represented by CD4+CD161+CCR6+CXCR3+IL-17+IFNγ+Т cells, and in the current literature it is most often referred to as Th17.1. Some Th17.1 cells can completely lose the production of IL-17, while maintaining the expression of other Th17-associated molecules; these are the so-called ex-Th17 cells (CD4+CD161+CCR6+CXCR3+IL-17- IFNγ+Т cells). Consequently, the population of Th1-polarized Th17 includes Th17.1, ex-Th17 cells and a number of additional transitional forms. It has unique functional properties – an increased pro-inflammatory potential and the ability to overcome histohematic barriers. It is these cells that are currently assigned a key role in the pathogenesis of many autoimmune diseases, and the process of Th17 redifferentiation into Th1 is considered as a promising therapeutic target. However, the development of this direction is complicated by the weak comparability of data on the size of such a population. The analysis of methods for determining Th1-polarized Th17 in vivo and in vitro, carried out in this work, made it possible to resolve these contradictions and develop optimal approaches to identifying this population. In most studies, especially clinical ones, it is identified by co-expression of key cytokines (IL-17/IFNγ) or chemokine receptors (CCR6/CXCR3), rarely by their combination. In this approach, co-expression of CCR6/ CXCR3 marks the total population of Th1-like Th17, including both Th17.1 and ex-Th17, while co-expression of IL-17/IFNγ cytokines identifies only Th17.1 cells, and the subpopulation of ex-Th17 is misclassified as classic Th1 in this case. Such “underestimation” of the ex-Th17 subpopulation significantly marks down the results, since it is ex-Th17 that accounts for the bulk of Th1-like Th17. And only a simultaneous assessment of the co-expression of cytokines and Th17-associated membrane molecules allows identification Th17.1 and exTh17 cells separately, which is important to consider when interpreting data on the problem and when planning clinical trials.

Interplay between calcium and sarcomeres directs cardiomyocyte maturation during regeneration

Zebrafish hearts can regenerate by replacing damaged tissue with new cardiomyocytes. Although the steps leading up to the proliferation of surviving cardiomyocytes have been extensively studied, little is known about the mechanisms that control proliferation and redifferentiation to a mature state. We found that the cardiac dyad, a structure that regulates calcium handling and excitation-contraction coupling, played a key role in the redifferentiation process. A component of the cardiac dyad called leucine-rich repeat–containing 10 (Lrrc10) acted as a negative regulator of proliferation, prevented cardiomegaly, and induced redifferentiation. We found that its function was conserved in mammalian cardiomyocytes. This study highlights the importance of the underlying mechanisms required for heart regeneration and their application to the generation of fully functional cardiomyocytes.

Tel2 regulates redifferentiation of bipotential progenitor cells via Hhex during zebrafish liver regeneration.

Upon extensive hepatocyte loss or impaired hepatocyte proliferation, liver regeneration occurs via biliary epithelial cell (BEC) transdifferentiation, which includes dedifferentiation of BECs into bipotential progenitor cells (BP-PCs) and then redifferentiation of BP-PCs to nascent hepatocytes and BECs. This BEC-driven liver regeneration involves reactivation of hepatoblast markers, but the underpinning mechanisms and their effects on liver regeneration remain largely unknown. Using a zebrafish extensive hepatocyte ablation model, we perform an N-ethyl-N-nitrosourea (ENU) forward genetic screen and identify a liver regeneration mutant, liver logan (lvl), in which the telomere maintenance 2 (tel2) gene is mutated. During liver regeneration, the tel2 mutation specifically inhibits transcriptional activation of a hepatoblast marker, hematopoietically expressed homeobox (hhex), in BEC-derived cells, which blocks BP-PC redifferentiation. Mechanistic studies show that Tel2 associates with the hhex promoter region and promotes hhex transcription. Our results reveal roles of Tel2 in the BP-PC redifferentiation process of liver regeneration by activating hhex.

SOX9 keeps growth plates and articular cartilage healthy by inhibiting chondrocyte dedifferentiation/osteoblastic redifferentiation

Cartilage is essential throughout vertebrate life. It starts developing in embryos when osteochondroprogenitor cells commit to chondrogenesis, activate a pancartilaginous program to form cartilaginous skeletal primordia, and also embrace a growth-plate program to drive skeletal growth or an articular program to build permanent joint cartilage. Various forms of cartilage malformation and degeneration diseases afflict humans, but underlying mechanisms are still incompletely understood and treatment options suboptimal. The transcription factor SOX9 is required for embryonic chondrogenesis, but its postnatal roles remain unclear, despite evidence that it is down-regulated in osteoarthritis and heterozygously inactivated in campomelic dysplasia, a severe skeletal dysplasia characterized postnatally by small stature and kyphoscoliosis. Using conditional knockout mice and high-throughput sequencing assays, we show here that SOX9 is required postnatally to prevent growth-plate closure and preosteoarthritic deterioration of articular cartilage. Its deficiency prompts growth-plate chondrocytes at all stages to swiftly reach a terminal/dedifferentiated stage marked by expression of chondrocyte-specific (Mgp) and progenitor-specific (Nt5e and Sox4) genes. Up-regulation of osteogenic genes (Runx2, Sp7, and Postn) and overt osteoblastogenesis quickly ensue. SOX9 deficiency does not perturb the articular program, except in load-bearing regions, where it also provokes chondrocyte-to-osteoblast conversion via a progenitor stage. Pathway analyses support roles for SOX9 in controlling TGFβ and BMP signaling activities during this cell lineage transition. Altogether, these findings deepen our current understanding of the cellular and molecular mechanisms that specifically ensure lifelong growth-plate and articular cartilage vigor by identifying osteogenic plasticity of growth-plate and articular chondrocytes and a SOX9-countered chondrocyte dedifferentiation/osteoblast redifferentiation process.

Cellular and morphological changes during leaf explant dedifferentiation and plant regeneration of Tagetes erecta

Plant tissue culture allows the maintenance of species of interest under controlled conditions. There have been few studies related to the morphological changes that take place during callus formation and redifferentiation processes. The aim of this work was to analyze the morphological changes during dedifferentiation and redifferentiation processes in leaf explants of marigold (Tagetes erecta L.) using transmission electron microscopy, digital image analysis and concepts of fractal dimension. Leaf explants were sown in MS medium supplemented with 2.0 mg L−1 of benzyladenine and 2.0 mg L−1 of 2,4-dichlorophenoxyacetic acid to induce dedifferentiation (callus formation). The redifferentiation process (bud formation) was obtained with explants sown in MS medium supplemented with 3.0 mg L−1 indoleacetic acid and 0.5 mg L−1 benzyladenine. During the first 8 days, the dedifferentiation process showed gradual changes in terms of the area and perimeter of the explants. The fractal dimension of the perimeter corresponding to the projected image of the calli changed from smooth to rough at day 20. These features indicate variations in tissue morphometry as a result of the callus formation process and further formation of cellular aggregates. Structural observations during the dedifferentiation process showed changes in shape and cellular organization as callus formation became evident. Round-shaped cells were observed in the callus, with few organelles and a low degree of differentiation. Indirect organogenesis was achieved over a period of 10 days; in addition, plantlets developed from the obtained buds in 20 days in growth regulator-free MS medium.

Dynamic Transcriptome Analysis Reveals Uncharacterized Complex Regulatory Pathway Underlying Dose IBA-Induced Embryogenic Redifferentiation in Cotton.

The somatic embryogenesis (SE) process of plants is regulated by exogenous hormones. During the SE, different genes sensitively respond to hormone signals through complex regulatory networks to exhibit plant totipotency. When cultured in indole-3-butyric acid (IBA) concentration gradient medium supplemented with 0 mg dm−3, 0.025 mg dm−3, and 0.05 mg dm−3 IBA, the callus differentiation rate first increased then decreased in cotton. To characterize the molecular basis of IBA-induced regulating SE, transcriptome analysis was conducted on embryogenic redifferentiation. Upon the examination of the IBA’s embryogenic inductive effect, it was revealed that pathways related to plant hormone signal transduction and alcohol degradation were significantly enriched in the embryogenic responsive stage (5 days). The photosynthesis, alcohol metabolism and cell cycle pathways were specifically regulated in the pre-embryonic initial period (20 days). Upon the effect of the IBA dose, in the embryogenic responsive stage (5 days), the metabolism of xenobiotics by the cytochrome P450 pathway and secondary metabolism pathways of steroid, flavonoid, and anthocyanin biosynthesis were significantly enriched. The phenylpropanoid, brassinosteroid, and anthocyanin biosynthesis pathways were specifically associated in the pre-embryonic initial period (20 days). At different developmental stages of embryogenic induction, photosynthesis, flavonoid biosynthesis, phenylpropanoid biosynthesis, mitogen-activated protein kinase (MAPK) signaling, xenobiotics metabolism by cytochrome P450, and brassinosteroid biosynthesis pathways were enriched at low a IBA concentration. Meanwhile, at high IBA concentration, the carbon metabolism, alcohol degradation, circadian rhythm and biosynthesis of amino acids pathways were significantly enriched. The results reveal that complex regulating pathways participate in the process of IBA-induced redifferentiation in cotton somatic embryogenesis. In addition, collections of potential essential signaling and regulatory genes responsible for dose IBA-induced efficient embryogenic redifferentiation were identified. Quantitative real-time PCR (qRT-PCR) was performed on the candidate genes with different expression patterns, and the results are basically consistent with the RNA-seq data. The results suggest that the complicated and concerted IBA-induced mechanisms involving multiple cellular pathways are responsible for dose-dependent plant growth regulator-induced SE. This report represents a systematic study and provides new insight into molecular signaling and regulatory basis underlying the process of dose IBA-induced embryogenic redifferentiation during SE.

Alterations in characteristics of canine articular chondrocytes in non-passaged long-term monolayer culture: Matter of differentiation, dedifferentiation and redifferentiation.

This study investigated the effects of culture time on phenotype stability of canine articular chondrocytes (CACs) in non-passaged long-term monolayer culture. Third passage (P3) CACs isolated from four cartilage samples were seeded at three different initial seeding densities (0.2 × 104, 1.0 × 104 and 5.0 × 104 cells/cm2) and maintained in monolayer condition up to 8 weeks without undergoing subculture after confluence. The characteristic changes of chondrocytes during the culture period were evaluated based on the cell morphology, cell proliferation, glycosaminoglycans (GAGs) content, DNA quantification, mRNA expression and ultrastructure of chondrocytes. Chondrocytes maintained under post-confluence condition exhibited a capability to grow and proliferate up to 4 weeks. Alcian blue staining and Dimethylmethylene blue (DMMB) assay revealed that the extracellular matrix (ECM) synthesis was increased in a time-dependent manner from 2 to 8 weeks. The chondrocyte mRNA expression profile was dramatically affected by prolonged culture time, with a significant downregulation of collagen type I, whereas the expression of collagen type II, aggrecan, Sox9 and matrix metalloproteinase 13 (MMP-13) were significantly upregulated. In addition, transmission electron microscopy (TEM) result indicated dilation of rough endoplasmic reticulum (RER) in these long-term monolayer cultured chondrocytes. These findings demonstrate that the chondrocytes phenotype could be partially redifferentiated through the spontaneous redifferentiation process in long-term cultures using standard culture medium without the addition of chondrogenic supplements or tissue-culture scaffolds.

Electron microscopic observation of human auricular chondrocytes transplanted into peritoneal cavity of nude mice for cartilage regeneration.

Restoration of damaged cartilage tissue has been deemed futile with current treatments. Although there have been many studies on cartilage regeneration thus far, there is no report that chondrocytes were completely re-differentiated in vitro. The clarification of cellular composition and matrix production during cartilage regeneration must be elucidated to fabricate viable mature cartilage in vitro. In order to achieve this aim, the chondrocytes cultured on coverslips were transplanted into the peritoneal cavities of mice. At different time points post-transplantation, the cartilage maturation progression and cells composing the regeneration were examined. Cartilage regeneration was confirmed by hematoxylin & eosin (HE) and toluidine blue staining. The maturation progression was carefully examined further by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). At the first and second week time points, various cell shapes were observed using SEM. Chronologically, by the third week, the number of fibers increased, suggesting the progression of extracellular matrix (ECM) maturation. Observation through TEM revealed the chondrocytes located in close proximity to various cells including macrophage-like cells. On the second week, infiltration of lymphocytes and capillary vessels were observed, and after the third week, the chondrocytes had matured and were abundantly releasing extracellular matrix. Chronological observation of transplanted chondrocytes by electron microscopy revealed maturation of chondrocytes and accumulation of matrix during the re-differentiation process. Emerging patterns of host-derived cells such as macrophage-like cells and subsequent appearance of lymphocytes-like cells and angiogenesis were documented, providing crucial context for the identification of the cells responsible for in vivo cartilage regeneration.

Dedifferentiation, Proliferation, and Redifferentiation of Adult Mammalian Cardiomyocytes After Ischemic Injury.

Adult mammalian hearts have a limited ability to generate new cardiomyocytes. Proliferation of existing adult cardiomyocytes (ACMs) is a potential source of new cardiomyocytes. Understanding the fundamental biology of ACM proliferation could be of great clinical significance for treating myocardial infarction (MI). We aim to understand the process and regulation of ACM proliferation and its role in new cardiomyocyte formation of post-MI mouse hearts. β-Actin-green fluorescent protein transgenic mice and fate-mapping Myh6-MerCreMer-tdTomato/lacZ mice were used to trace the fate of ACMs. In a coculture system with neonatal rat ventricular myocytes, ACM proliferation was documented with clear evidence of cytokinesis observed with time-lapse imaging. Cardiomyocyte proliferation in the adult mouse post-MI heart was detected by cell cycle markers and 5-ethynyl-2-deoxyuridine incorporation analysis. Echocardiography was used to measure cardiac function, and histology was performed to determine infarction size. In vitro, mononucleated and bi/multinucleated ACMs were able to proliferate at a similar rate (7.0%) in the coculture. Dedifferentiation proceeded ACM proliferation, which was followed by redifferentiation. Redifferentiation was essential to endow the daughter cells with cardiomyocyte contractile function. Intercellular propagation of Ca2+ from contracting neonatal rat ventricular myocytes into ACM daughter cells was required to activate the Ca2+-dependent calcineurin-nuclear factor of activated T-cell signaling pathway to induce ACM redifferentiation. The properties of neonatal rat ventricular myocyte Ca2+ transients influenced the rate of ACM redifferentiation. Hypoxia impaired the function of gap junctions by dephosphorylating its component protein connexin 43, the major mediator of intercellular Ca2+ propagation between cardiomyocytes, thereby impairing ACM redifferentiation. In vivo, ACM proliferation was found primarily in the MI border zone. An ischemia-resistant connexin 43 mutant enhanced the redifferentiation of ACM-derived new cardiomyocytes after MI and improved cardiac function. Mature ACMs can reenter the cell cycle and form new cardiomyocytes through a 3-step process: dedifferentiation, proliferation, and redifferentiation. Intercellular Ca2+ signal from neighboring functioning cardiomyocytes through gap junctions induces the redifferentiation process. This novel mechanism contributes to new cardiomyocyte formation in post-MI hearts in mammals.

Technical Note: A comparison among adipogenic induction protocols for dedifferentiated fat (DFAT) cells obtained from subcutaneous fat of pigs

In the current study we have performed two experiments to evaluate the effects of adipogenic induction media on dedifferentiated fat (DFAT) cells redifferentiation into mature adipocytes. In experiment 1, we aimed to evaluate whether it is necessary to use insulin in the induction media to allow DFAT cells differentiation into mature adipocytes by establishing two experimental treatments where insulin was either withdrawn from the culture medium, after 72h of a normal induction period (Treatment 1: Insulin-) or kept in culture media (Treatment 2: Insulin+) for 16 d. In experiment 2, we aimed to evaluate if the lack of 3-isobutyl-1-methylxanthine (IBMX) in the induction medium would affect the differentiation of DFAT cells into mature adipocytes. For that, DFAT cells were induced to differentiate into lipid assimilating adipocytes using an induction medium containing IBMX (Treatment 1: IBMX+) or without IBMX (Treatment 2: IBMX-) during the first 72h of induction. In both experiments we have evaluated the mRNA expression of lipid metabolism markers and cell morphology through Oil-Red staining as indicators of differentiation of DFAT cells into lipid-assimilating cells. The results of Experiment 1 revealed no differences in mRNA expression for any of the lipid metabolism markers with exception of GLUT4 (P=0.02), which was greater in Insulin- compared to Insulin+ treatment. Similarly, no differences were observed for mRNA expression of adipogenic markers between IBMX+ and IBMX- treatments with exception of FABP4 (P=0.01), which was greater for the IBMX- compared to IBMX+ treatment. In both experiments we did not observed any differences in cell morphology among treatments. Our results suggest that neither insulin nor IBMX are required to accelerate redifferentiation process of pig-derived DFAT cells.

Generation of Human Lens Epithelial-Like Cells From Patient-Specific Induced Pluripotent Stem Cells.

Cataractogenesis begins from the dynamic lens epithelial cells (LECs) and adjacent fiber cells. LECs derived from cell lines cannot maintain the crystalline expression as the primary LECs. The current study aimed to efficiently generate large numbers of human LECs from patient-specific induced pluripotent stem cells (iPSCs). Anterior lens capsules were collected from cataract surgery and were used to culture primary hLECs. iPSCs were induced from these primary hLECs by lentiviral transduction of Oct4, Sox2, Klf4, and c-Myc. Then, the generated iPSCs were re-differentiated into hLECs by the 3-step addition of defined factor combinations (Noggin, BMP4/7, bFGF, and EGF) modified from an established method. During the re-differentiation process, colonies of interest were isolated using a glass picking tool and cloning cylinders based on the colony morphology. After two steps of isolation, populations of LEC-like cells (LLCs) were generated and identified by the expression of lens marker genes by qPCR, western blot and immunofluorescence staining. The study introduced a modified protocol to isolate LLCs from iPSCs by defined factors in a short time frame. This technique could be useful for mechanistic studies of lens-related diseases. J. Cell. Physiol. 231: 2555-2562, 2016. © 2016 Wiley Periodicals, Inc.

MicroRNA 302/367 Cluster Effectively Facilitates Direct Reprogramming from Human Fibroblasts into Functional Neurons.

Recent studies suggest that mature somatic cells can be reprogrammed to become induced pluripotent stem cells by overexpressing specific transcription factors or microRNAs (miRNAs). Theoretically, this technique could provide a wide array of cells for therapeutics. However, the process of redifferentiation after cell reprogramming to pluripotency is inefficient and time restricted. We proposed that the differentiation of somatic cells into specific cells of another germ layer can be induced and accelerated with appropriate miRNAs and culture conditions. In human fibroblasts, we found that overexpression of pluripotency stem cell-specific miRNA-302/367 cluster, together with two other neuron-specific miRNAs (miRNA-9/9* and miRNA-124) induced fibroblasts conversion into neurons. The cells assumed neuron morphology, were positive for several neuron markers, and exhibited neuronal membrane potential feature. Moreover, concentrated expression of synaptic markers were observed in these cells in vitro and in vivo in nude mice brain, suggesting possible connectivity. To achieve efficient reprogramming, miRNA-302/367 cluster, miRNA-9/9*, and miRNA-124 were all required. The combination of the proved pluripotency-inducing miRNA-302/367 cluster and cell-specific miRNAs provides a unique strategy for one-step cellular conversion that could have important implications for studies of neuron development and neurological disease therapy.

Are surface antigens suited to verify the redifferentiation potential and culture purity of human chondrocytes in cell-based implants.

Cell expansion in vitro is a prequisite to obtain a sufficient quantity of cells for cell-based cartilage repair of articular cartilage lesions. During this process verification of redifferentiation potential of highly expanded chondrocytes is required. Furthermore, cellular impurities of chondrocyte cultures have to be excluded. For this purpose, redifferentiation of expanded human chondrocytes in passage 3 or 5 was initiated in bioresorbable polyglycolic acid-fibrin (PGA-fibrin) scaffolds and selected potential markers were analysed during the process of cell expansion and redifferentiation. Chondrocyte expansion was accompanied by a decrease of collagen type II and COMP and an increase of collagen type I expression indicating cell dedifferentiation. Redifferentiation of chondrocytes in PGA-fibrin scaffolds was accompanied by an increase of collagen II/I ratio. Flow cytometric analyses revealed that in contrast to CD44 and CD49e, CD63 and CD166 showed significant changes in the number of positive cells during redifferentiation. CD14 and CD45 are not expressed by chondrocytes and are therefore possible candidates to detect specifically monocytes or haematopoetic cells in chondrocyte cultures. Characterization of surface antigen expression revealed two promising candidates (CD63 and CD166) to describe the process of redifferentiation, while CD14 and CD45 are suitable markers to exclude impurities by monocytes or haematopoetic cells.

Pathology of ischemia reperfusion induced acute kidney injury in rats

Ischemia reperfusion induced acute kidney injury (I/R AKI) by bilateral renal artery occlusion-reperfusion method in male SD rats were performed to study progressive pathological events after acute kidney injury. Serum creatinine level rose up to the level of 4–4.6 mg/dL, 24h after I/R AKI episode. Serum creatinine, urine volume, urine osmolality, urinary protein, gross pathology, histopathology and morphometry were studied at periodical interval from day 1 to day 28 after I/R AKI along with sham control group. Increased serum creatinine, decreased urine volume and urine osmolality were recorded at day 1. These changes returned to the normal level by day 7 of I/R AKI except slight increase in urine volume on day 28 of I/R AKI. Histopathological changes mainly affecting the tubules of outer stripe of medulla occasionally involving the cortex and pelvis were observed. These changes comprised complete loss of cellular detail of tubules as acute tubular necrosis followed by process of regeneration and redifferentiation in the form of tubules, which represented almost near to normal structure by day 14 of I/R AKI. However, presence of tubulo-interstitial foci on day 28 of I/R AKI with increased interstitial index and collagen fibers indicated that a portion of tubules did not recover completely and may be responsible for the further deterioration of kidney function.

MTOR Inhibition Promotes TTF1-Dependent Redifferentiation and Restores Iodine Uptake in Thyroid Carcinoma Cell Lines

Redifferentiation of thyroid carcinoma cells has the potential to increase the efficacy of radioactive iodine therapy in treatment-refractory, nonmedullary thyroid carcinoma (TC), leading to an improved disease outcome. Mammalian target of rapamycin (mTOR) is a key regulator of cell fate affecting survival and differentiation, with autophagy and inflammation as prominent downstream pathways. The effects of mTOR inhibition were studied for its redifferentiation potential of the human TC cell lines BC-PAP, FTC133, and TPC1 by assessment of mRNA and protein expression of thyroid-specific genes and by performance of iodine uptake assays. In thyroid transcription factor 1 (TTF1)-expressing cell lines, mTOR inhibition promoted redifferentiation of TC cells by the up-regulation of human sodium-iodine symporter mRNA and protein expression. Furthermore, these cells exhibited markedly elevated iodine uptake capacity. Surprisingly, this redifferentiation process was not mediated by autophagy induced during mTOR inhibition or by inflammatory mediators but through transcriptional effects at the level of TTF1 expression. Accordingly, small interfering RNA inhibition of TTF1 completely abrogated the induction of human sodium-iodine symporter by mTOR inhibition. The present study has identified the TTF1-dependent molecular mechanisms through which the inhibition of mTOR leads to the redifferentiation of TC cells and subsequently to increased radioactive iodine uptake.

Electron tomography and cryo-SEM characterization reveals novel ultrastructural features of host-parasite interaction during Chlamydia abortus infection

Chlamydia (C.) abortus is a widely spread pathogen among ruminants that can be transmitted to women during pregnancy leading to severe systemic infection with consecutive abortion. As a member of the Chlamydiaceae, C. abortus shares the characteristic feature of an obligate intracellular biphasic developmental cycle with two morphological forms including elementary bodies (EBs) and reticulate bodies (RBs). In contrast to other chlamydial species, C. abortus ultrastructure has not been investigated yet. To do so, samples were fixed by high-pressure freezing and processed by different electron microscopic methods. Freeze-substituted samples were analysed by transmission electron microscopy, scanning transmission electron microscopical tomography and immuno-electron microscopy, and freeze-fractured samples were analysed by cryo-scanning electron microscopy. Here, we present three ultrastructural features of C. abortus that have not been reported up to now. Firstly, the morphological evidence that C. abortus is equipped with the type three secretion system. Secondly, the accumulation and even coating of whole inclusion bodies by membrane complexes consisting of multiple closely adjacent membranes which seems to be a C. abortus specific feature. Thirdly, the formation of small vesicles in the periplasmic space of RBs in the second half of the developmental cycle. Concerning the time point of their formation and the fact that they harbour chlamydial components, these vesicles might be morphological correlates of an intermediate step during the process of redifferentiation of RBs into EBs. As this feature has also been shown for C. trachomatis and C. pneumoniae, it might be a common characteristic of the family of Chlamydiaceae.