The MADS-box genes SOC1 and AGL24 antagonize XAL2 functions in Arabidopsis thaliana root development.

MADS-domain transcription factors play pivotal roles in numerous developmental processes in Arabidopsis thaliana. While their involvement in flowering transition and floral development has been extensively examined, their functions in root development remain relatively unexplored. Here, we explored the function and genetic interaction of three MADS-box genes (XAL2, SOC1 and AGL24) in primary root development. By analyzing loss-of-function and overexpression lines, we found that SOC1 and AGL24, both critical components in flowering transition, redundantly act as repressors of primary root growth as the loss of function of either SOC1 or AGL24 partially recovers the primary root growth, meristem cell number, cell production rate, and the length of fully elongated cells of the short-root mutant xal2-2. Furthermore, we observed that the simultaneous overexpression of AGL24 and SOC1 leads to short-root phenotypes, affecting meristem cell number and fully elongated cell size, whereas SOC1 overexpression is sufficient to affect columella stem cell differentiation. Additionally, qPCR analyses revealed that these genes exhibit distinct modes of transcriptional regulation in roots compared to what has been previously reported for aerial tissues. We identified 100 differentially expressed genes in xal2-2 roots by RNA-seq. Moreover, our findings revealed that the expression of certain genes involved in cell differentiation, as well as stress responses, which are either upregulated or downregulated in the xal2-2 mutant, reverted to WT levels in the absence of SOC1 or AGL24.

DNA methylation analysis identifies key transcription factors involved in mesenchymal stem cell osteogenic differentiation.

BACKGROUND: Knowledge about regulating transcription factors (TFs) for osteoblastogenesis from mesenchymal stem cells (MSCs) is limited. Therefore, we investigated the relationship between genomic regions subject to DNA-methylation changes during osteoblastogenesis and the TFs known to directly interact with these regulatory regions. RESULTS: The genome-wide DNA-methylation signature of MSCs differentiated to osteoblasts and adipocytes was determined using the Illumina HumanMethylation450 BeadChip array. During adipogenesis no CpGs passed our test for significant methylation changes. Oppositely, during osteoblastogenesis we identified 2462 differently significantly methylated CpGs (adj. p < 0.05). These resided outside of CpGs islands and were significantly enriched in enhancer regions. We confirmed the correlation between DNA-methylation and gene expression. Accordingly, we developed a bioinformatic tool to analyse differentially methylated regions and the TFs interacting with them. By overlaying our osteoblastogenesis differentially methylated regions with ENCODE TF ChIP-seq data we obtained a set of candidate TFs associated to DNA-methylation changes. Among them, ZEB1 TF was highly related with DNA-methylation. Using RNA interference, we confirmed that ZEB1, and ZEB2, played a key role in adipogenesis and osteoblastogenesis processes. For clinical relevance, ZEB1 mRNA expression in human bone samples was evaluated. This expression positively correlated with weight, body mass index, and PPARγ expression. CONCLUSIONS: In this work we describe an osteoblastogenesis-associated DNA-methylation profile and, using these data, validate a novel computational tool to identify key TFs associated to age-related disease processes. By means of this tool we identified and confirmed ZEB TFs as mediators involved in the MSCs differentiation to osteoblasts and adipocytes, and obesity-related bone adiposity.

Grafting of maleic anhydride on poly(lactic acid)/hydroxyapatite composites augments their ability to support osteogenic differentiation of human mesenchymal stem cells.

Implantation of bone substitutes is the treatment of choice for bone defects exceeding a critical size, when self-healing becomes impossible. The use of 3D printing techniques allows the construction of scaffolds with customized properties. However, there is a lack of suitable materials for bone replacement. In this study, maleic anhydride-grafted poly (lactic acid) (MAPLA) was investigated as a potential compatibilizer agent for 3D-printed polylactic acid (PLA)/hydroxyapatite (HA) composites, in order to enhance the physicochemical and biological properties of the scaffolds. The grafting process was performed by reactive processing in a torque rheometer, with the evaluation of the use of different concentrations of maleic anhydride (MA). The success of the grafting reaction was confirmed by titration of acid groups and spectroscopic analyses, indicating the presence of succinic anhydride groups on the PLA chain. Morphological analysis of the PLA/HA 3D scaffolds, using SEM, revealed that the use of the compatibilizer resulted in a structure free from voids and holes. The compatibilization also increased the degradation process. On the other hand, TGA and DSC analyses revealed that the use of a compatibilizer had little effect on the thermal properties of the composite. Most importantly, the samples with compatibilizer were demonstrated to have a minimal cytotoxic effect on human mesenchymal stem cells (MSCs), promoting the osteogenic differentiation of these cells in a medium without the addition of classical osteogenic factors. Therefore, the grafting of PLA/HA composites improved their physicochemical and biological properties, especially the induction of MSC osteogenic differentiation, demonstrating the potential of these scaffolds for bone tissue replacement.

DNA methylation analysis identifies key transcription factors involved in mesenchymal stem cell osteogenic differentiation

BACKGROUND: Knowledge about regulating transcription factors (TFs) for osteoblastogenesis from mesenchymal stem cells (MSCs) is limited. Therefore, we investigated the relationship between genomic regions subject to DNA-methylation changes during osteoblastogenesis and the TFs known to directly interact with these regulatory regions. RESULTS: The genome-wide DNA-methylation signature of MSCs differentiated to osteoblasts and adipocytes was determined using the Illumina HumanMethylation450 BeadChip array. During adipogenesis no CpGs passed our test for significant methylation changes. Oppositely, during osteoblastogenesis we identified 2462 differently significantly methylated CpGs (adj. p < 0.05). These resided outside of CpGs islands and were significantly enriched in enhancer regions. We confirmed the correlation between DNA-methylation and gene expression. Accordingly, we developed a bioinformatic tool to analyse differentially methylated regions and the TFs interacting with them. By overlaying our osteoblastogenesis differentially methylated regions with ENCODE TF ChIP-seq data we obtained a set of candidate TFs associated to DNA-methylation changes. Among them, ZEB1 TF was highly related with DNA-methylation. Using RNA interference, we confirmed that ZEB1, and ZEB2, played a key role in adipogenesis and osteoblastogenesis processes. For clinical relevance, ZEB1 mRNA expression in human bone samples was evaluated. This expression positively correlated with weight, body mass index, and PPARγ expression. CONCLUSIONS: In this work we describe an osteoblastogenesis-associated DNA-methylation profile and, using these data, validate a novel computational tool to identify key TFs associated to age-related disease processes. By means of this tool we identified and confirmed ZEB TFs as mediators involved in the MSCs differentiation to osteoblasts and adipocytes, and obesity-related bone adiposity.

Photobiomodulation treatments drive osteogenic versus adipocytic fate of bone marrow mesenchymal stem cells reversing the effects of hyperglycemia in diabetes.

Diabetes mellitus (DM) is a chronic metabolic disease that affects bone metabolism, which can be related to a reduced osteogenic potential of bone marrow mesenchymal stem cells (BM-MSCs). MSCs from diabetic rats (dBM-MSC) have shown a tendency to differentiate towards adipocytes (AD) instead of osteoblasts (OB). Since photobiomodulation (PBM) therapy is a non-invasive treatment capable of recovering the osteogenic potential of dBM-MSCs, we aimed to evaluate whether PBM can modulate MSC's differentiation under hyperglycemic conditions. BM-MSCs of healthy and diabetic rats were isolated and differentiated into osteoblasts (OB and dOB) and adipocytes (AD and dAD). dOB and dAD were treated with PBM every 3 days (660 nm; 5 J/cm2; 0.14 J; 20 mW; 0.714 W/cm2) for 17 days. Cell morphology and viability were evaluated, and cell differentiation was confirmed by gene expression (RT-PCR) of bone (Runx2, Alp, and Opn) and adipocyte markers (Pparγ, C/Ebpα, and C/Ebpß), production of extracellular mineralized matrix (Alizarin Red), and lipid accumulation (Oil Red). Despite no differences on cell morphology, the effect of DM on cells was confirmed by a decreased gene expression of bone markers and matrix production of dOB, and an increased expression of adipocyte and lipid accumulation of dAD, compared to heatlhy cells. On the other hand, PBM reversed the effects of dOB and dAD. The negative effect of DM on cells was confirmed, and PBM improved OB differentiation while decreasing AD differentiation, driving the fate of dBM-MSCs. These results may contribute to optimizing bone regeneration in diabetic patients.

Stem cells as a potential therapy for diabetes mellitus: a call-to-action in Latin America.

Latin America is a fast-growing region that currently faces unique challenges in the treatment of all forms of diabetes mellitus. The burden of this disease will be even greater in the coming years due, in part, to the large proportion of young adults living in urban areas and engaging in unhealthy lifestyles. Unfortunately, the national health systems in Latin-American countries are unprepared and urgently need to reorganize their health care services to achieve diabetic therapeutic goals. Stem cell research is attracting increasing attention as a promising and fast-growing field in Latin America. As future healthcare systems will include the development of regenerative medicine through stem cell research, Latin America is urged to issue a call-to-action on stem cell research. Increased efforts are required in studies focused on stem cells for the treatment of diabetes. In this review, we aim to inform physicians, researchers, patients and funding sources about the advances in stem cell research for possible future applications in diabetes mellitus. Emerging studies are demonstrating the potential of stem cells for ß cell differentiation and pancreatic regeneration. The major economic burden implicated in patients with diabetes complications suggests that stem cell research may relieve diabetic complications. Closer attention should be paid to stem cell research in the future as an alternative treatment for diabetes mellitus.

SVCT2 Is Expressed by Cerebellar Precursor Cells, Which Differentiate into Neurons in Response to Ascorbic Acid.

Ascorbic acid (AA) is a known antioxidant that participates in a wide range of processes, including stem cell differentiation. It enters the cell through the sodium-ascorbate co-transporter SVCT2, which is mainly expressed by neurons in the adult brain. Here, we have characterized SVCT2 expression in the postnatal cerebellum in situ, a model used for studying neurogenesis, and have identified its expression in granular precursor cells and mature neurons. We have also detected SVCT2 expression in the cerebellar cell line C17.2 and in postnatal cerebellum-derived neurospheres in vitro and have identified a tight relationship between SVCT2 expression and that of the stem cell-like marker nestin. AA supplementation potentiates the neuronal phenotype in cerebellar neural stem cells by increasing the expression of the neuronal marker ß III tubulin. Stable over-expression of SVCT2 in C17.2 cells enhances ß III tubulin expression, but it also increases cell death, suggesting that AA transporter levels must be finely tuned during neural stem cell differentiation.

Caracterización morfológica, génica y protéica en la diferenciación de células madre embrionarias de ratón a células pancreáticas tempranas / Morphological, genetic and protein characterization in the differentiation of embryonic stem cells to early pancreatic cells

Debido al auge de la medicina regenerativa, las Células Madre (SC) representan una fuente de reemplazo celular para cualquier tejido, decidiendo emprender este trabajo de investigación con el objetivo de diferenciar células madre embrionarias de ratón (mESC) a células pancreáticas tempranas, realizando su caracterización génica y morfológica. Primeramente se cultivaron y arrestaron en su ciclo celular fibroblastos embrionarios de ratón (MEF) con mitomicina, posteriormente se expandieron las mESC y se sometieron a un protocolo de diferenciación de 21 días hacía células pancreáticas tempranas, evaluándose durante la diferenciación su morfología y expresión relativa de los genes sox-17, pdx-1, ins-1 e ins-2, determinando además la producción de las proteínas insulina y glucagón mediante inmunocitoquímica y citometría de flujo. Se obtuvieron cuerpos embrionarios (EBs) a partir de mESC, con características morfológicas diferentes de acuerdo a su diferenciación, los cuales expresaron genes de la línea germinal endodérmica (sox-17 y pdx-1) a los días 0, 11 y 17 de diferenciación, gen inductor del desarrollo embrionario pancreático (pdx-1) al día 11 de diferenciación y, genes de expresión pancreática (ins-1 e ins-2) a los días 17 y 21 de diferenciación. Finalmente se detectó la producción de proteínas insulina y glucagón en los EBs al día 21 de diferenciación. Se logró diferenciar mESC. El análisis morfológico evidenció cúmulos celulares tridimensionales correspondientes a EBs. Con el análisis de los patrones de expresión génica, se distinguieron inicialmente células con características genéticas de endodermo y posteriormente a partir del día 17 células pancreáticas tempranas, las cuales al día 21 de diferenciación expresaron las proteínas insulina y glucagón...

Due to the boom in regenerative medicine, Stem Cells (SC) represent a source of cell replacement to any tissue, we decided to undertake this research with the objective of differentiating mouse embryonic stem cells (mESC) to early pancreatic cells, developing their genetic and morphological characterization. Initially Mouse embryonic fibroblasts (MEF) were grown and arrested in their cell cycle with mitomycin, subsequently mouse embryonic SC (mESC) were expanded and subjected in to a pancreatic cell differentiation protocol of 21 days. During differentiation, morphology and the relative expression of sox-17, pdx-1, Ins-1 and Ins-2 genes were assessed, also the production of insulin and glucagon proteins was determinated by fluorescence microscopy and flow cytometry. Embryoid bodies (EBs) were obtained from mESC, with different morphological characteristics according to their differentiation, which expressed endodermal germ line genes (sox-17 y pdx-1) at days 0, 11 and 17 of differentiation, an inductor gene of embryonic pancreas development (pdx-1) was detected at day 11 of differentiation. Pancreas genes (ins-1 e ins-2) were expressed at day 17 and 21 of differentiation. Finally the production of insulin and glucagon proteins was detected on the EBS at day 21 of differentiation. In conclusion, the mESC differentiation was achieved. The morphological analysis evidenced three-dimensional cell clusters corresponding to EBs. Analysis of the gene expression patterns in the differentiation process, cells initially showed genetic characteristics of endoderm and thereafter from day 17 of differentiation characteristics of early pancreatic cells which by day 21 of differentiation expressed insulin and glucagon proteins...

Variation in neuronal differentiation of a newly isolated mouse embryonic stem cell line: a detailed immunocytochemistry study.

Neural precursor differentiation from mouse ES (embryonic stem) cells have been demonstrated using EB (embryoid body), co-culture on stromal feeder layers, and in the absence of external inducing signals. Most of available mouse ES cell original research articles have worked with only six different cell lines. Our goals were to isolate one new mouse ES lineage, and perform a detailed immunocytochemistry study during neural differentiation, making use of an EB strategy protocol following the generation of neural progenitors, glial cells and postmitotic neurons. The dynamics of differentiation of ES cell derived neuronal precursors into differentiated glia cells and neurons were followed in vitro and correlated to exposure to specific elements of feeder medium. Morphological aspects of generated cellular types, including its immunocytochemical expression of differentiation markers were studied. Immuno-positivity against ß-III tubulin, PGP and TH (tyrosine hydroxylase) was observed from stage I. Approximately 80% of cells were positive for TH at stage I. The first glial cell type appears in stage III. TH, PGP or ß-III tubulin-positive cells with neuronal typical morphology only being seen in stage III when TH-positive cells corresponded to approximately 12% of total cells. Variations among other literature findings can be explained by the choice we made to use a newly isolated ES cell line. As colonies may behave differently during neuronal differentiation, it reinforces the necessity of studying original ES cell lines.

HOX gene analysis in the osteogenic differentiation of human mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (hMSCs) have the capacity to differentiate into osteoblasts during osteogenesis. Several studies attempted to identify osteogenesis-related genes in hMSCs. Although HOX genes are known to play a pivotal role in skeletogenesis, their function in the osteogenesis of hMSCs has not yet been investigated in detail. Our aim was to characterize the expression of 37 HOX genes by multiplex RT-PCR to identify the ones most probably involved in osteogenic differentiation. The results showed that the expression patterns of four HOX genes were altered during this process. In particular, the expression levels of HOXC13 and HOXD13 were dramatically changed. Real-time PCR and Western blot analysis were performed in order to further analyze the expression of HOXC13 and HOXD13. The qRT-PCR results showed that transcription of HOXC13 was up-regulated by up to forty times, whereas that of HOXD13 was down-regulated by approximately five times after osteogenic differentiation. The Western blot results for the HOXC13 and HOXD13 proteins also corresponded well with the real-time PCR result. These findings suggest that HOXC13 and HOXD13 might be involved in the osteogenic differentiation of hMSCs.