The management of bone defect using long non-coding RNA as a potential biomarker for regulating the osteogenic differentiation process

Tissue engineered bone brings hope to the treatment of bone defects, and the osteogenic differentiation of stem cells is the key link. Inducing osteogenic differentiation of stem cells may be a potential approach to promote bone regeneration. In recent years, lncRNA has been studied in the field increasingly, which is believed can regulate cell cycle, proliferation, metastasis, differentiation and immunity, participating in a variety of physiology and pathology processes. At present, it has been confirmed that certain lncRNAs regulate the osteogenesis of stem cells and take part in mediating signaling pathways including Wnt/β-catenin, MAPK, TGF-β/BMP, and Notch pathways. Here, we provided an overview of lncRNA, reviewed its researches in the osteogenic differentiation of stem cells, emphasized the importance of lncRNA in bone regeneration, and focused on the roles of lncRNA in signaling pathways, in order to make adequate preparations for applying lncRNA to bone tissue Engineering, letting it regulate the osteogenic differentiation of stem cells for bone regeneration.

Digital Differentiation in Gifted Education

Gifted students are a group who has unique needs. These unique needs require differentiation in their education. With the technological developments, it becomes necessary to use digital tools within or outside the classroom which can enhance the differentiation process. Digital differentiation emerges because of all these reasons that answer students' needs with the usage of technology. The purpose of this chapter is to introduce digital differentiation as a strategy used in gifted education. Digital differentiation can be carried out in traditional classrooms by preparing digital differentiation packages which includes three types of different content or in online environments by using learning management systems besides digital differentiation packages. In this chapter, strategies used in gifted education will be explained and then digital differentiation will be presented and the tools that can be used exemplified. Then how the digital differentiation can be carried out in both traditional classrooms and online environments will be clarified.

Kościoły chrześcijańskie a globalizacja – perspektywa europejska

In a globalized reality, Christian churches must rediscover its place. The task is extremely difficult as it’s impossible to know where the congregation’s spiritual needs are heading. Migration and mass media rise as well as problematic identities and roles influence the deepening of the differentiation process, which makes it difficult to define unambiguous scenarios regarding the future of the religious market. On the other hand, the prolificacy of the presented theories proves religion’s unusually complex nature and context. The article’s aim was to present the most significant forecasts on where Christian churches will stand in the today’s global village.

MicroRNA-202 Induces Myoblast to Myocyte Differentiation through Targeting Rock-1

The expression patterns of microRNAs (small non-coding RNAs) are altered in many biological processes such as myogenesis. In this study, we aimed to investigate the impact of predicted miR-202, its target genes Akt2 and Rock-1 as a potential regulator of myoblast in the myocyte differentiation process using the C2C12 cell line. After confirmation of the differentiation process induced by 3% horse serum, the expression level of miRNA and its targets were evaluated. In the following, a luciferase assay was conducted to approve the effect of miRNA on its target. Our results indicated that miR-202 and Akt2 were significantly up-regulated during differentiation, while Rock-1 was downregulated. Co-transfection of miRNA with psiCHECK2-Rock-1 significantly presented that Rock-1 was directly targeted by miR-202. On the contrary, miR-202 has failed to enforce its inhibitory effect on Akt2 expression. In particular, miR-202 seems to be a regulator of muscle differentiation pathway thought targeting Rock-1.

Proteogenomic Analysis Reveals Proteins Involved in the First Step of Adipogenesis in Human Adipose-Derived Stem Cells

Background. Obesity is characterized as a disease that directly affects the whole-body metabolism and is associated with excess fat mass and several related comorbidities. Dynamics of adipocyte hypertrophy and hyperplasia play an important role in health and disease, especially in obesity. Human adipose-derived stem cells (hASC) represent an important source for understanding the entire adipogenic differentiation process. However, little is known about the triggering step of adipogenesis in hASC. Here, we performed a proteogenomic approach for understanding the protein abundance alterations during the initiation of the adipogenic differentiation process. Methods. hASC were isolated from adipose tissue of three donors and were then characterized and expanded. Cells were cultured for 24 hours in adipogenic differentiation medium followed by protein extraction. We used shotgun proteomics to compare the proteomic profile of 24 h-adipogenic, differentiated, and undifferentiated hASC. We also used our previous next-generation sequencing data (RNA-seq) of the total and polysomal mRNA fractions of hASC to study posttranscriptional regulation during the initial steps of adipogenesis. Results. We identified 3420 proteins out of 48,336 peptides, of which 92 proteins were exclusively identified in undifferentiated hASC and 53 proteins were exclusively found in 24 h-differentiated cells. Using a stringent criterion, we identified 33 differentially abundant proteins when comparing 24 h-differentiated and undifferentiated hASC (14 upregulated and 19 downregulated, respectively). Among the upregulated proteins, we shortlisted several adipogenesis-related proteins. A combined analysis of the proteome and the transcriptome allowed the identification of positive correlation coefficients between proteins and mRNAs. Conclusions. These results demonstrate a specific proteome profile related to adipogenesis at the beginning (24 hours) of the differentiation process in hASC, which advances the understanding of human adipogenesis and obesity. Adipogenic differentiation is finely regulated at the transcriptional, posttranscriptional, and posttranslational levels.

Comprehensive epigenomic profiling of human alveolar epithelial differentiation identifies key epigenetic states and transcription factor co-regulatory networks for maintenance of distal lung identity

Background Disruption of alveolar epithelial cell (AEC) differentiation is implicated in distal lung diseases such as chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and lung adenocarcinoma that impact morbidity and mortality worldwide. Elucidating underlying disease pathogenesis requires a mechanistic molecular understanding of AEC differentiation. Previous studies have focused on changes of individual transcription factors, and to date no study has comprehensively characterized the dynamic, global epigenomic alterations that facilitate this critical differentiation process in humans. Results We comprehensively profiled the epigenomic states of human AECs during type 2 to type 1-like cell differentiation, including the methylome and chromatin functional domains, and integrated this with transcriptome-wide RNA expression data. Enhancer regions were drastically altered during AEC differentiation. Transcription factor binding analysis within enhancer regions revealed diverse interactive networks with enrichment for many transcription factors, including NKX2–1 and FOXA family members, as well as transcription factors with less well characterized roles in AEC differentiation, such as members of the MEF2, TEAD, and AP1 families. Additionally, associations among transcription factors changed during differentiation, implicating a complex network of heterotrimeric complex switching in driving differentiation. Integration of AEC enhancer states with the catalog of enhancer elements in the Roadmap Epigenomics Mapping Consortium and Encyclopedia of DNA Elements (ENCODE) revealed that AECs have similar epigenomic structures to other profiled epithelial cell types, including human mammary epithelial cells (HMECs), with NKX2–1 serving as a distinguishing feature of distal lung differentiation. Conclusions Enhancer regions are hotspots of epigenomic alteration that regulate AEC differentiation. Furthermore, the differentiation process is regulated by dynamic networks of transcription factors acting in concert, rather than individually. These findings provide a roadmap for understanding the relationship between disruption of the epigenetic state during AEC differentiation and development of lung diseases that may be therapeutically amenable.

Induced Pluripotent Stem Cell-Derived Gamma Delta CAR-T Cells for Cancer Immunotherapy

Introduction Allogenic CAR-T cell therapies for cancer provide a new option to reduce barriers faced by autologous cell therapies, but several challenges remain. One challenge is the risk of graft versus host disease (GvHD) caused by the infused T cells. A potential solution is the use of a subset of gamma delta (γδ) CAR-T cells whose T cell receptors (TCRs) recognize invariant antigens rather than hypervariable MHC molecules. Here we describe an off-the-shelf, induced pluripotent stem cell (iPSC)-derived γδ CAR-T (γδ CAR-iT) for treatment of cancer and a process for deriving such cells. Methods T cell-derived iPSCs (TiPSC) are generated by reprogramming γδ T cells to yield pluripotent stem cells. For proof-of-concept studies, TiPSC were engineered using CRISPR gene editing to deliver a CD19 CAR transgene. TiPSC are then subjected to a two-stage differentiation process. First, TiPSC are differentiated into CD34-expressing hematopoietic progenitor cells (HPCs). HPCs are then exposed to a feeder-free differentiation process that results in uniform γδ CAR-iT cells. The purity and identity of γδ CAR-iT cells were assessed by flow cytometry and the ability of γδ CAR-iT cells to respond to homeostatic growth factors was determined by intracellular staining of phosphorylated signaling proteins and mRNA transcriptome analysis. Cytokine production by CAR-iT cells was measured by immunoassays following stimulation of the CAR. Tumor cell killing by γδ CAR-iT cells was performed using IncuCyte cytotoxicity assays. In vivo control of tumors by γδ CAR-iT in immunodeficient mice was determined using a NALM-6 B cell lymphoblastic xenograft model. Results A research-grade γδ TiPSC line was used to develop an iT differentiation process. This γδ TiPSC line was engineered to express a CD19 CAR molecule and then subjected to the differentiation process after which >95% of cells were CD3 + γδ TCR + CAR + iT cells. These γδ CAR-iT cells responded to IL-2 and IL-15. STAT5 phosphorylation levels were similar but STAT3 phosphorylation levels were greater in response to IL-15 compared to IL-2 at equimolar concentrations of cytokine. IL-2 and IL-15 elicited qualitatively similar transcriptional responses, but the magnitude of cytokine-induced gene expression was generally greater in IL-15-treated cells. Upon activation, γδ CAR-iT cells released markedly less IFN-γ and other inflammatory cytokines than conventional blood-derived ab CAR-T cells. In an IncuCyte serial killing assay, γδ CAR-iT cells exhibited sustained killing of NALM-6 tumor cells for at least one week in the presence of IL-15. In vivo, γδ CAR-iT cells caused a significant reduction in NALM-6 tumor burden with a single dose of γδ CAR-iT resulting in >95% tumor growth inhibition. To establish an efficient method for derivation of clinical grade γδ TiPSC lines, we investigated methods to isolate, expand, and reprogram human γδ T cells. When γδ T cells were expanded by exposure to the chemical zoledronic acid (zoledronate) and IL-2, we found a large disparity between donors; some donors exhibit robust expansion while others are seemingly resistant to zoledronate. In order to enhance γδ T cell expansion we screened dozens of activation conditions and eventually established a universal activation protocol that can elicit robust expansion of γδ T cells from all donors tested. When expanded γδ T cells were subjected to reprogramming conditions, dozens to hundreds of individual TiPSC colonies were obtained from each donor. The identity of the rearranged γδ TCR locus was confirmed using molecular assays. New γδ TiPSC lines were engineered with a CD19 CAR molecule and killing activity was confirmed in an in vitro serial killing assay. Conclusions γδ CAR-iT cells provide a new opportunity to treat cancers with an off-the-shelf universal T cell platform without the risk for GvHD. γδ CAR-iT cells are readily manufacturable, and we have derived an end-to-end process that enables new TiPSC line reprogramming, genetic modification of TiPSC lines, and feeder-free differentiation. γδ CAR-iT cells exhibit potent antigen-specific tumor killing and they release less inflammatory cytokine than conventional CAR-T cells, potentially reducing the risk for cytokine-mediated toxicities. We believe that this off-the-shelf platform will enable safer and more accessible allogenic cell therapies for hematologic and solid cancers. Disclosures Wallet: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Nishimura: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Del Casale: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Lebid: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Salantes: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Santostefano: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Bucher: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Mendonca: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Beqiri: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Thompson: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Morse: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Millar Quinn: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Borges: Century Therapeutics: Current Employment, Current equity holder in publicly-traded company.

HERVH-derived lncRNAs negatively regulate chromatin targeting and remodeling mediated by CHD7

Chd7 encodes an ATP-dependent chromatin remodeler which has been shown to target specific genomic loci and alter local transcription potentially by remodeling chromatin structure. De novo mutations in CHD7 are the major cause of CHARGE syndrome which features multiple developmental defects. We examined whether nuclear RNAs might contribute to its targeting and function and identified a preferential interaction between CHD7 and lncRNAs derived from HERVH loci in pluripotent stem cells. Knockdown of HERVH family lncRNAs using LNAs or knockout of an individual copy of HERVH by CRISPR-Cas9 both resulted in increased binding of CHD7 and increased levels of H3K27ac at a subset of enhancers. Depletion of HERVH family RNAs led to the activation of multiple genes. CHD7 bound HERVH RNA with high affinity but low specificity and this interaction decreased the ability of CHD7 to bind and remodel nucleosomes. We present a model in which HERVH lncRNAs act as a decoy to modulate the dynamics of CHD7 binding to enhancers in pluripotent cells and the activation of numerous genes that might impact the differentiation process.

Differentiation of mouse embryonic stem cells into cells with spermatogonia-like morphology with chemical intervention-dependent increased gene expression of LIM homeobox 1 (Lhx1).

During the development of the male germline, spermatogonial stem cells (SSCs) originate from gonocytes that differentiate from primordial germ cells (PGCs). In the developing and regenerating mouse testis, expression of the gene LIM homeobox 1 (Lhx1) marks the most undifferentiated SSCs. However, an enrichment of Lhx1 expression in spermatogonia-like cells generated in vitro has not been reported so far. Previously, it was shown that a chemical intervention in male mouse embryonic stem (ES) cells in serum culture, including a timed combination of the SIRT1 inhibitor Ex-527, the DNA methyltransferase inhibitor RG-108 and the electrophilic redox cycling compound tert-butylhydroquinone (tBHQ), was associated with molecular markers of the PGC to gonocyte differentiation process. Here, we report the in vitro differentiation of male mouse ES cells, cultured under dual chemical inhibition of GSK3β and MEK (termed 2i) with leukemia inhibitory factor (LIF) (termed 2iL) and serum, into cells with spermatogonia-like morphology (CSMs) and population-averaged expression of spermatogonia-specific genes. This was achieved by the removal of 2iL and a specific schedule of 2 partial medium replacements per day with alternating 8-hour and 16-hour intervals over a period of 32 days. Combination of this new cell culture protocol with the previously reported chemical intervention in ES cells changed the population-averaged expression of spermatogonia- and gonocyte-specific genes during the differentiation process and increased the population-averaged gene expression of Lhx1 in the resulting CSMs compared to CSMs without chemical intervention. Furthermore, we detected single CSMs with a strong nuclear LHX1/5 protein signal only in the chemical intervention group. Our results provide the first experimental evidence for the generation of CSMs with an enrichment of Lhx1 expression in vitro. We propose that further investigation of the CSMs generated with this in vitro system may provide new insights into male germline and stem cell development.

Serotonin 5-HT2A receptor activity mediates adipocyte differentiation through control of adipogenic gene expression

Serotonin 5-HT2 receptors are expressed in many tissues and play important roles in biological processes. Although the 5-HT2A receptor is primarily known for its role in central nervous system, it is also expressed in peripheral tissues. We have found that 5-HT2A receptor antagonists inhibit human subcutaneous primary adipocyte differentiation. We also show that siRNA knockdown of the 5-HT2A receptor blocks differentiation. Using gene expression analysis in combination with receptor antagonists we found that activity of 5-HT2A receptors is necessary very early in the differentiation process to mediate expression of adipogenic genes, including peroxisome proliferator-activated receptor gamma (ppar-γ), adipocyte protein 2 (aP2), adiponectin, and serine/threonine-protein kinase 1 (sgk1). We show here for the first time that 5-HT2A receptor activity is necessary for differentiation of human primary subcutaneous preadipocytes to adipocytes, and that 5-HT2A receptor activity mediates key genes related to adipogenesis during this process. Importantly, this work contributes to a greater understanding of the adipocyte differentiation process, as well as to the role of 5-HT2A receptors in peripheral tissues, and may be relevant to the development of novel therapeutic strategies targeting this receptor for the treatment of obesity related diseases.