HMGXB4 Targets Sleeping Beauty Transposition to Germinal Stem Cells.

Transposons are parasitic genetic elements that frequently hijack vital cellular processes of their host. HMGXB4 is a known Wnt signaling-regulating HMG-box protein, previously identified as a host-encoded factor of Sleeping Beauty (SB) transposition. Here, we show that HMGXB4 is predominantly maternally expressed, and marks both germinal progenitor and somatic stem cells. SB piggybacks HMGXB4 to activate transposase expression and target transposition to germinal stem cells, thereby potentiating heritable transposon insertions. The HMGXB4 promoter is located within an active chromatin domain, offering multiple looping possibilities with neighboring genomic regions. HMGXB4 is activated by ERK2/MAPK1, ELK1 transcription factors, coordinating pluripotency and self-renewal pathways, but suppressed by the KRAB-ZNF/TRIM28 epigenetic repression machinery, also known to regulate transposable elements. At the post-translational level, SUMOylation regulates HMGXB4, which modulates binding affinity to its protein interaction partners and controls its transcriptional activator function via nucleolar compartmentalization. When expressed, HMGXB4 can participate in nuclear-remodeling protein complexes and transactivate target gene expression in vertebrates. Our study highlights HMGXB4 as an evolutionarily conserved host-encoded factor that assists Tc1/Mariner transposons to target the germline, which was necessary for their fixation and may explain their abundance in vertebrate genomes.

Cytokine physiognomies of MSCs from varied sources confirm the regenerative commitment post-coculture with activated neutrophils.

The interaction of mesenchymal stromal cells (MSCs) with paracrine signals and immunological cells, and their responses and regenerative commitment thereafter, is understudied. In the current investigation, we compared MSCs from the umbilical cord blood (UCB), dental pulp (DP), and liposuction material (LS) on their ability to respond to activated neutrophils. Cytokine profiling (interleukin-1α [IL-1α], IL-2, IL-4, IL-6, IL-8, tumor necrosis factor-α [TNF-α], interferon-γ [IFN-γ], transforming growth factor-ß [TGF-ß]), cellular proliferation and osteogenic differentiation patterns were assessed. The results showed largely comparable cytokine profiles with higher TNF-α and IFN-γ levels in LSMSCs owing to their mature cellular phenotype. The viability and proliferation between LS/DP/UCB MSCs were comparable in the coculture group, while direct activation of MSCs with lipopolysaccharide (LPS) showed comparable proliferation with significant cell death in UCB MSCs and slightly higher cell death in the other two types of MSC. Furthermore, when MSCs post-neutrophil exposure were induced for osteogenic differentiation, though all the MSCs devoid of the sources differentiated, we observed rapid and significant turnover of DPMSCs positive of osteogenic markers rather than LS and UCB MSCs. We further observed a significant turnover of IL-1α and TGF-ß at mRNA and cytokine levels, indicating the commitment of MSCs to differentiate through interacting with immunological cells or bacterial products like neutrophils or LPS, respectively. Taken together, these results suggest that MSCs have more or less similar cytokine responses devoid of their anatomical niche. They readily switch over from the cytokine responsive cell phenotype at the immunological microenvironment to differentiate and regenerate tissue in response to cellular signals.

Sleeping Beauty transposon-based system for cellular reprogramming and targeted gene insertion in induced pluripotent stem cells.

The discovery of direct cell reprogramming and induced pluripotent stem (iPS) cell technology opened up new avenues for the application of non-viral, transposon-based gene delivery systems. The Sleeping Beauty (SB) transposon is highly advanced for versatile genetic manipulations in mammalian cells. We established iPS cell reprogramming of mouse embryonic fibroblasts and human foreskin fibroblasts by transposition of OSKM (Oct4, Sox2, Klf4 and c-Myc) and OSKML (OSKM + Lin28) expression cassettes mobilized by the SB100X hyperactive transposase. The efficiency of iPS cell derivation with SB transposon system was in the range of that obtained with retroviral vectors. Co-expression of the miRNA302/367 cluster together with OSKM significantly improved reprogramming efficiency and accelerated the temporal kinetics of reprogramming. The iPS cells displayed a stable karyotype, and hallmarks of pluripotency including expression of stem cell markers and the ability to differentiate into embryoid bodies in vitro. We demonstrate Cre recombinase-mediated exchange allowing simultaneous removal of the reprogramming cassette and targeted knock-in of an expression cassette of interest into the transposon-tagged locus in mouse iPS cells. This strategy would allow correction of a genetic defect by site-specific insertion of a therapeutic gene construct into 'safe harbor' sites in the genomes of autologous, patient-derived iPS cells.

Transposon-mediated transgenesis, transgenic rescue, and tissue-specific gene expression in rodents and rabbits.

Germline transgenesis is an important procedure for functional investigation of biological pathways, as well as for animal biotechnology. We have established a simple, nonviral protocol in three important biomedical model organisms frequently used in physiological studies. The protocol is based on the hyperactive Sleeping Beauty transposon system, SB100X, which reproducibly promoted generation of transgenic founders at frequencies of 50-64, 14-72, and 15% in mice, rats, and rabbits, respectively. The SB100X-mediated transgene integrations are less prone to genetic mosaicism and gene silencing as compared to either the classical pronuclear injection or to lentivirus-mediated transgenesis. The method was successfully applied to a variety of transgenes and animal models, and can be used to generate founders with single-copy integrations. The transposon vector also allows the generation of transgenic lines with tissue-specific expression patterns specified by promoter elements of choice, exemplified by a rat reporter strain useful for tracking serotonergic neurons. As a proof of principle, we rescued an inborn genetic defect in the fawn-hooded hypertensive rat by SB100X transgenesis. A side-by-side comparison of the SB100X- and piggyBac-based protocols revealed that the two systems are complementary, offering new opportunities in genome manipulation.

Chronic metabolic and induced stress impacts mesenchymal stromal cell differentiation and modulation of dental origin in-vitro.

The impact of induced (smoking) and metabolic stress (diabetes) on dental stem cells with respect to pre-impact consideration on differentiation and bone formation were investigated. The progenitor stem cells isolated from dental pulp, follicle and gingival tissues were phenotyped and subjected to nicotine and high glucose stress mimicking the smoking and diabetic condition in-vitro. The results showed that the cellular viability post treatment with 100 µM nicotine and 10uM glucose was about 86% to 89% respectively in all the three cell types while about 73% in combined nicotine and glucose treatment. No variation in the expression of pro-inflammatory TNF-α, IL-1ß and IL-12 in all the three cell types were noticed. The observed viability in nicotine treated cells were due to elevated IL-6, IL-10 while in glucose was due to brain derived neurotropic factor (BDNF). Higher expression of IL-4, IL-6, IL-10, TGF-ß and heme oxygenase -1 (HO-1) were found high in both stressors treated cells. Differentiation and mineralization markers Alkaline phosphatase (ALP), Collagenase I (COL1), Osteocalcin, Runt related transcription factor 2 (RUNX2), Osteopontin and Bone sialoprotein were expressed in the dental pulp stem cells (DPSCs) and gingival mesenchymal stem cells (GMSCs) at varying levels post nicotine or glucose treatment while not significantly observed in dental follicular stem cells (DFSCs). Therefore, it is evident that the stem cells of varied dental origin responded to the stress are more or less uniform with physiological delay in differentiation into osteoblast. It is evident from the study that, the metabolic or induced stress subverts the process of regenerative healing by mesenchymal stromal cells with their anatomical niche.

Glucose and oleic acid mediate cellular alterations in GLP-1-induced insulin-positive differentiating UCBMSCs.

Coordinated effects of glucose and oleic acid on glucagon-like peptide-1 (GLP-1) mediated differentiation of insulin-positive differentiating umbilical cord mesenchymal stromal cells (dUCBMSCs) was studied using a co-culture of NCI-H716 (GLP-1+) and UCBMSCs (insulin+). The addition of 2.5 mM glucose increased the proliferation of NCI-H716 cells by 30% and induced transformation of UCBMSCs into insulin-secreting cells in 18 days as compared to 22 days in control cells. Oleic acid (25 µM) showed decrease in cell proliferation, autophagy, and apoptosis in NCI-H716 cells while no effect was observed in dUCBMSCs. Prolonged glucose and oleic acid resulted in apoptosis and cell cycle changes in dUCBMSCs after day 18 while higher concentrations resulted in cell death. Additionally, the expression of FAS and ACC mRNA was observed in NCI-H716 and dUCBMSCs post 24-hr addition of glucose and/or oleic acid. Absorption of oleic acid was high in NCI-H716 compared to dUCBMSCs. Taken together, optimal concentrations of glucose and oleic acid could be a key factor in stimulating intrinsic GLP-1, which in turn stimulates differentiating MSCs in a glucose-dependent manner. PRACTICAL APPLICATIONS: The aim of this article was to study whether differentiating or differentiated MSCs after mobilization or post-transplant would require optimal glucose and oleic acid to naturally stimulate intrinsic GLP-1, or otherwise, the high or long-term overload of glucose or oleic acid could result in inhibition of differentiated cells resulting in failure of insulin secretion.

High altitude as a possible factor for dysbiosis of salivary microbiome in orthodontic patients.

BACKGROUND: External stressors such as high altitude and low oxygen are known to affect the human microbiome, and in light of the increased occurrence of dental caries and periodontitis in orthodontic patients, the effect of high altitude and the altered oral environment in orthodontic patients on the oral salivary microbiome was researched. MATERIALS & METHODS: 31 orthodontic patients from high altitude, Aseer region and 25 orthodontic patients, residing at sea level, as controls were included. DNA isolation was done from the saliva collected from the study participants. V3 area of 16s RNA was targeted by universal primers through PCR to decipher the salivary microbiome in both the groups. RESULTS: A total of 11 genera belonging to 4 phyla of bacteria were identified in both groups. The most abundant microbiome at the phylum level was: Firmicutes, Bacteroidetes Proteobacteria, and Cyanobacteria. The salivary microbiome was more diverse in sea level controls compared to that of the orthodontic patients at high altitude wherein the presence of only two main phyla: Firmicutes and Proteobacteria were seen. The controls revealed Firmicutes, Proteobacteria, Bacteroidetes and Cyanobacteria. CONCLUSIONS: The findings of the study suggest that the biodiversity of the salivary microbiome is severely perturbed under the cumulative influences of high altitude and presence of fixed orthodontic appliance. Under these circumstances, a strict and meticulous oral hygiene regimen should be recommended and followed to avoid harmful effects on the periodontal tissues.

Proteomic profiling of various human dental stem cells - a systematic review.

BACKGROUND: The proteomic signature or profile best describes the functional component of a cell during its routine metabolic and survival activities. Additional complexity in differentiation and maturation is observed in stem/progenitor cells. The role of functional proteins at the cellular level has long been attributed to anatomical niches, and stem cells do not deflect from this attribution. Human dental stem cells (hDSCs), on the whole, are a combination of mesenchymal and epithelial coordinates observed throughout craniofacial bones to pulp. AIM: To specify the proteomic profile and compare each type of hDSC with other mesenchymal stem cells (MSCs) of various niches. Furthermore, we analyzed the characteristics of the microenvironment and preconditioning changes associated with the proteomic profile of hDSCs and their influence on committed lineage differentiation. METHODS: Literature searches were performed in PubMed, EMBASE, Scopus, and Web of Science databases, from January 1990 to December 2018. An extra inquiry of the grey literature was completed on Google Scholar, ProQuest, and OpenGrey. Relevant MeSH terms (PubMed) and keywords related to dental stem cells were used independently and in combination. RESULTS: The initial search resulted in 134 articles. Of the 134 full-texts assessed, 96 articles were excluded and 38 articles that met the eligibility criteria were reviewed. The overall assessment of hDSCs and other MSCs suggests that differences in the proteomic profile can be due to stem cellular complexity acquired from varied tissue sources during embryonic development. However, our comparison of the proteomic profile suffered inconsistencies due to the heterogeneity of various hDSCs. We believe that the existence of a heterogeneous population of stem cells at a given niche determines the modalities of regeneration or tissue repair. Added prominences to the differences present between various hDSCs have been reasoned out. CONCLUSION: Systematic review on proteomic studies of various hDSCs are promising as an eye-opener for revisiting the proteomic profile and in-depth analysis to elucidate more refined mechanisms of hDSC functionalities.

Transcriptional activities of the Sleeping Beauty transposon and shielding its genetic cargo with insulators.

The Sleeping Beauty (SB) transposable element shows efficient transposition in human cells, and provides long-term transgene expression in preclinical animal models. Random chromosomal insertion of SB vectors represents a safety issue in human gene therapeutic applications, due to potential genotoxic effects associated with transposon integration. We investigated the transcriptional activities of SB in order to assess its potential to alter host gene expression upon integration. The untranslated regions (UTRs) of the transposon direct convergent, inward-directed transcription. Transcription from the 5'-UTR of SB is upregulated by the host-encoded factor high-mobility group 2-like 1 (HMG2L1), and requires a 65-base pair (bp) region not present in commonly used SB vectors. The SB transposase antagonizes the effect of HMG2L1, suggesting that natural transposase expression is under a negative feedback regulation. SB transposon vectors lacking the 65-bp region associated with HMG2L1-dependent upregulation exhibit benign transcriptional activities, at a level up to 100-times lower than that of the murine leukemia virus (MLV) long terminal repeat (LTR). Incorporation of chicken beta-globin HS4 insulator sequences in SB-based vectors reduces the transactivation of model promoters by transposon-borne enhancers, and thus may lower the risk of transcriptional activation of host genes situated close to a transposon insertion site.

Mesenchymal Stem Cells (MSCs) Coculture Protects [Ca2+]i Orchestrated Oxidant Mediated Damage in Differentiated Neurons In Vitro.

Cell-therapy modalities using mesenchymal stem (MSCs) in experimental strokes are being investigated due to the role of MSCs in neuroprotection and regeneration. It is necessary to know the sequence of events that occur during stress and how MSCs complement the rescue of neuronal cell death mediated by [Ca2+]i and reactive oxygen species (ROS). In the current study, SH-SY5Y-differentiated neuronal cells were subjected to in vitro cerebral ischemia-like stress and were experimentally rescued from cell death using an MSCs/neuronal cell coculture model. Neuronal cell death was characterized by the induction of proinflammatory tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and -12, up to 35-fold with corresponding downregulation of anti-inflammatory cytokine transforming growth factor (TGF)-ß, IL-6 and -10 by approximately 1 to 7 fold. Increased intracellular calcium [Ca2+]i and ROS clearly reaffirmed oxidative stress-mediated apoptosis, while upregulation of nuclear factor NF-κB and cyclo-oxygenase (COX)-2 expressions, along with ~41% accumulation of early and late phase apoptotic cells, confirmed ischemic stress-mediated cell death. Stressed neuronal cells were rescued from death when cocultured with MSCs via increased expression of anti-inflammatory cytokines (TGF-ß, 17%; IL-6, 4%; and IL-10, 13%), significantly downregulated NF-κB and proinflammatory COX-2 expression. Further accumulation of early and late apoptotic cells was diminished to 23%, while corresponding cell death decreased from 40% to 17%. Low superoxide dismutase 1 (SOD1) expression at the mRNA level was rescued by MSCs coculture, while no significant changes were observed with catalase (CAT) and glutathione peroxidase (GPx). Interestingly, increased serotonin release into the culture supernatant was proportionate to the elevated [Ca2+]i and corresponding ROS, which were later rescued by the MSCs coculture to near normalcy. Taken together, all of these results primarily support MSCs-mediated modulation of stressed neuronal cell survival in vitro.