Redifferentiation Of Cells Research Articles

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.

Galectin-8 counteracts folic acid-induced acute kidney injury and prevents its transition to fibrosis

Acute kidney injury (AKI), characterized by a sudden decline in kidney function involving tubular damage and epithelial cell death, can lead to progressive tissue fibrosis and chronic kidney disease due to interstitial fibroblast activation and tissue repair failures that lack direct treatments. After an AKI episode, surviving renal tubular cells undergo cycles of dedifferentiation, proliferation and redifferentiation while fibroblast activity increases and then declines to avoid an exaggerated extracellular matrix deposition. Appropriate tissue recovery versus pathogenic fibrotic progression depends on fine-tuning all these processes. Identifying endogenous factors able to affect any of them may offer new therapeutic opportunities to improve AKI outcomes. Galectin-8 (Gal-8) is an endogenous carbohydrate-binding protein that is secreted through an unconventional mechanism, binds to glycosylated proteins at the cell surface and modifies various cellular activities, including cell proliferation and survival against stress conditions. Here, using a mouse model of AKI induced by folic acid, we show that pre-treatment with Gal-8 protects against cell death, promotes epithelial cell redifferentiation and improves renal function. In addition, Gal-8 decreases fibroblast activation, resulting in less expression of fibrotic genes. Gal-8 added after AKI induction is also effective in maintaining renal function against damage, improving epithelial cell survival. The ability to protect kidneys from injury during both pre- and post-treatments, coupled with its anti-fibrotic effect, highlights Gal-8 as an endogenous factor to be considered in therapeutic strategies aimed at improving renal function and mitigating chronic pathogenic progression.

SETMAR Facilitates the Differentiation of Thyroid Cancer by Regulating SMARCA2-Mediated Chromatin Remodeling.

Thyroid cancer is the most common type of endocrine cancer, and most patients have a good prognosis. However, the thyroid cancer differentiation status strongly affects patient response to conventional treatment and prognosis. Therefore, exploring the molecular mechanisms that influence the differentiation of thyroid cancer is very important for understanding the progression of this disease and improving therapeutic options. In this study, SETMAR as a key gene that affects thyroid cancer differentiation is identified. SETMAR significantly regulates the proliferation, epithelial-mesenchymal transformation (EMT), thyroid differentiation-related gene expression, radioactive iodine uptake, and sensitivity to MAPK inhibitor-based redifferentiation therapies of thyroid cancer cells. Mechanistically, SETMAR methylates dimethylated H3K36 in the SMARCA2 promoter region to promote SMARCA2 transcription. SMARCA2 can bind to enhancers of the thyroid differentiation transcription factors (TTFs) PAX8, and FOXE1 to promote their expression by enhancing chromatin accessibility. Moreover, METTL3-mediated m6A methylation of SETAMR mRNA is observed and showed that this medication can affect SETMAR expression in an IGF2BP3-dependent manner. Finally, the METTL3-14-WTAP activator effectively facilitates the redifferentiation of thyroid cancer cells via the SETMAR-SMARCA2-TTF axis utilized. The research provides novel insights into the molecular mechanisms underlying thyroid cancer dedifferentiation and provides a new approach for therapeutically promoting redifferentiation.

IL-17-Producing Regulatory T lymphocytes – Suppressors or Effectors?

Regulatory T (Treg) lymphocytes are a T helper population that controls the intensity of the immune response and maintains immune tolerance by selectively suppressing the activity of effector T lymphocytes. Treg cells are unstable and show high plasticity towards effector T helper populations, and the most common variant is Treg redifferentiation into T helpers producing interleukin-17 (IL-17 (Th17)). Although their formation has been confirmed by many studies in vivo and in vitro, Treg cells remain poorly understood in terms of their functional activity. The latter is yet fundamentally important for two major reasons. Firstly, an increase in the population of IL-17-producing Tregs has been identified for a number of diseases, raising the question of how these cells are involved in the development of pathologies. Secondly, understanding and predicting the behavior of Tregs in a pro-inflammatory environment promotes their therapeutic use. This review article analyzes the functional consequences of the redifferentiation of Treg cells into Th17.

A cell transcriptomic profile provides insights into adipocytes of porcine mammary gland across development

BackgroundStudying the composition and developmental mechanisms in mammary gland is crucial for healthy growth of newborns. The mammary gland is inherently heterogeneous, and its physiological function dependents on the gene expression of multiple cell types. Most studies focused on epithelial cells, disregarding the role of neighboring adipocytes.ResultsHere, we constructed the largest transcriptomic dataset of porcine mammary gland cells thus far. The dataset captured 126,829 high-quality nuclei from physiological mammary glands across five developmental stages (d 90 of gestation, G90; d 0 after lactation, L0; d 20 after lactation, L20; 2 d post natural involution, PI2; 7 d post natural involution, PI7). Seven cell types were identified, including epithelial cells, adipocytes, endothelial cells, fibroblasts cells, immune cells, myoepithelial cells and precursor cells. Our data indicate that mammary glands at different developmental stages have distinct phenotypic and transcriptional signatures. During late gestation (G90), the differentiation and proliferation of adipocytes were inhibited. Meanwhile, partly epithelial cells were completely differentiated. Pseudo-time analysis showed that epithelial cells undergo three stages to achieve lactation, including cellular differentiation, hormone sensing, and metabolic activation. During lactation (L0 and L20), adipocytes area accounts for less than 0.5% of mammary glands. To maintain their own survival, the adipocyte exhibited a poorly differentiated state and a proliferative capacity. Epithelial cells initiate lactation upon hormonal stimulation. After fulfilling lactation mission, their undergo physiological death under high intensity lactation. Interestingly, the physiological dead cells seem to be actively cleared by immune cells via CCL21-ACKR4 pathway. This biological process may be an important mechanism for maintaining homeostasis of the mammary gland. During natural involution (PI2 and PI7), epithelial cell populations dedifferentiate into mesenchymal stem cells to maintain the lactation potential of mammary glands for the next lactation cycle.ConclusionThe molecular mechanisms of dedifferentiation, proliferation and redifferentiation of adipocytes and epithelial cells were revealed from late pregnancy to natural involution. This cell transcriptomic profile constitutes an essential reference for future studies in the development and remodeling of the mammary gland at different stages.

Extended lifespan and improved genome stability in HepaRG-derived cell lines through reprogramming by high-density stress

The characteristics and fate of cancer cells partly depend on their environmental stiffness, i.e., the local mechanical cues they face. HepaRG progenitors are liver carcinoma cells exhibiting transdifferentiation properties; however, the underlying mechanisms remain unknown. To evaluate the impact of external physical forces mimicking the tumor microenvironment, we seeded them at very high density for 20 h, keeping the cells round and unanchored to the substrate. Applied without corticoids, spatial confinement due to very high density induced reprogramming of HepaRG cells into stable replicative stem-like cells after replating at normal density. Redifferentiation of these stem-like cells into cells very similar to the original HepaRG cells was then achieved using the same stress but in the presence of corticoids. This demonstrates that the cells retained the memory required to run the complete hepatic differentiation program, after bypassing the Hayflick limit twice. We show that physical stress improved chromosome quality and genomic stability, through greater efficiency of DNA repair and restoration of telomerase activity, thus enabling cells to escape progression to a more aggressive cancer state. We also show the primary importance of high-density seeding, possibly triggering compressive stress, in these processes, rather than that of cell roundness or intracellular tensional signals. The HepaRG-derived lines established here considerably extend the lifespan and availability of this surrogate cell system for mature human hepatocytes. External physical stress is a promising way to create a variety of cell lines, and it paves the way for the development of strategies to improve cancer prognosis.

Significant expansion of the donor pool achieved by utilizing islets of variable quality in the production of allogeneic "Neo-Islets", 3-D organoids of Mesenchymal Stromal and islet cells, a novel immune-isolating biotherapy for Type I Diabetes.

Novel biotherapies for Type 1 Diabetes that provide a significantly expanded donor pool and that deliver all islet hormones without requiring anti-rejection drugs are urgently needed. Scoring systems have improved islet allotransplantation outcomes, but their use may potentially result in the waste of valuable cells for novel therapies. To address these issues, we created "Neo-Islets" (NIs), islet-sized organoids, by co-culturing in ultralow adhesion flasks culture-expanded islet (ICs) and Mesenchymal Stromal Cells (MSCs) (x 24 hrs, 1:1 ratio). The MSCs exert powerful immune- and cyto-protective, anti-inflammatory, proangiogenic, and other beneficial actions in NIs. The robust in vitro expansion of all islet hormone-producing cells is coupled to their expected progressive de-differentiation mediated by serum-induced cell cycle entry and Epithelial-Mesenchymal Transition (EMT). Re-differentiation in vivo of the ICs and resumption of their physiological functions occurs by reversal of EMT and serum withdrawal-induced exit from the cell cycle. Accordingly, we reported that allogeneic, i.p.-administered NIs engraft in the omentum, increase Treg numbers and reestablish permanent normoglycemia in autoimmune diabetic NOD mice without immunosuppression. Our FDA-guided pilot study (INAD 012-0776) in insulin-dependent pet dogs showed similar responses, and both human- and canine-NIs established normoglycemia in STZ-diabetic NOD/SCID mice even though the utilized islets would be scored as unsuitable for transplantation. The present study further demonstrates that islet gene expression profiles (α, β, γ, δ) in human "non-clinical grade" islets obtained from diverse, non-diabetic human and canine donors (n = 6 each) closely correlate with population doublings, and the in vivo re-differentiation of endocrine islet cells clearly corresponds with the reestablishment of euglycemia in diabetic mice. Conclusion: human-NIs created from diverse, "non-clinical grade" donors have the potential to greatly expand patient access to this curative therapy of T1DM, facilitated by the efficient in vitro expansion of ICs that can produce ~ 270 therapeutic NI doses per donor for 70 kg recipients.

Markers of Th1 polarized Th17 cells (literature review)

T helpers (Th) producing IL-17 (Th17) have high plasticity and under the influence of external conditions are able to redifferentiate into cells with a different phenotype, primarily in Th1-lymphocytes, forming a population that combines the characteristics of both Th17 and Th1 and has a high pro-inflammatory potential, as well as a unique ability to overcome histohematic barriers. These cells are currently assigned a key role in the pathogenesis of many inflammatory diseases, including autoimmune ones: they account for up to half of the lymphocytes present in infiltrates of inflamed tissues. The paper discusses the reasons for the increased plasticity of Th17 cells in comparison with the main T helper populations (Th1 and Th2) and considers in detail the mechanisms of formation of IFNγ producing Th17, taking into account not only the redifferentiation of mature Th17, but also possible alternative pathways, in particular, Th1 cell redifferentiation or naive CD4+T lymphocytes direct differentiation into cells with an intermediate Th1/Th17 phenotype. The main inducers of differentiation of IFNγ producing Th17 cells and the reversibility of this process are also discussed. Particular attention is paid to the methods for identifying Th1 polarized Th17 cells: this population is heterogeneous, and its size significantly depends on the type of markers used to characterize these cells – Th1/Th17-associated transcription factors, key cytokines, as well as chemokine receptors and other membrane molecules. As a result, the data in the works on this problem are poorly comparable with each other. The unification of approaches to identifying a population of Th1 like Th17 cells will solve this problem and make it possible to use an assessment of the size and activity of such a population as diagnostic or prognostic markers.

Abstract 460: Catalase Overexpression Modulates Plaque Fibrosis And Vascular Contractile Function In Aged Hyperlipidemic Mice

Background: Plaque fibrosis and re-differentiation of vascular smooth muscle cells (VSMCs) from a contractile to a “synthetic” phenotype are hallmarks of advanced atherosclerosis. While in vitro studies demonstrated a clear role of oxidative stress in the loss of VSMC contractile phenotypes, the roles of specific antioxidant mechanisms and their therapeutic utility remain unclear. Here we test the hypothesis that overexpression of catalase protects against excess plaque fibrosis and losses in contractile phenotypes in vivo . Methods: We used littermate-matched, hypercholesteremic (ldlr -/- /ApoB 100/100 ; LA) mice lacking a transgenic construct (LA-CAT 0/0 ) or harboring a single copy of the human catalase gene (LA-CAT Tg/0 ), which were fed a Western Diet for 3 or 12 months. Isolated organ bath chambers, histopathological analyses, and quantitative RT-PCR were used to assess vasomotor function, structure, and molecular changes in aorta. Results: Oxidative stress increased with disease progression in aortic tissue from LA-CAT 0/0 mice and was modestly reduced in LA-CAT Tg/0 mice. Collagen deposition and fiber thickness increased with disease progression in LA-CAT 0/0 mice, and these increases in fiber thickness were attenuated in LA-CAT Tg/0 mice at 12 months and more pronounced in male mice. In parallel, we observed favorable rightward shifts in the vascular stress-strain relationship in LA-CAT Tg/0 mice that were more pronounced in aged male mice. Interestingly, these structural changes were associated with improved contractile responses to either PGF 2α or serotonin in male LA-CAT Tg/0 mice at 12 months. Consistent with these changes, the contractile marker calponin-1 was profoundly reduced with increasing age in LA-CAT 0/0 mice, with these changes being partially attenuated in aging, male LA-CAT Tg/0 mice. In vitro, treatment of VSMCs with PEG-catalase attenuated BMP2-dependent contractile protein expression. Conclusion: These results demonstrate that moderate overexpression of catalase can attenuate deleterious fibrotic and contractile changes associated with prolonged progression of atherosclerosis. Future mechanistic investigation exploring the context-dependence of these changes (e.g., sex dependence) is warranted.

Modulation of EZH2 Activity Induces an Antitumoral Effect and Cell Redifferentiation in Anaplastic Thyroid Cancer.

Anaplastic thyroid cancer (ATC) is a rare and lethal form of thyroid cancer that requires urgent investigation of new molecular targets involved in its aggressive biology. In this context, the overactivation of Polycomb Repressive Complex 2/EZH2, which induces chromatin compaction, is frequently observed in aggressive solid tumors, making the EZH2 methyltransferase a potential target for treatment. However, the deregulation of chromatin accessibility is yet not fully investigated in thyroid cancer. In this study, EZH2 expression was modulated by CRISPR/Cas9-mediated gene editing and pharmacologically inhibited with EZH2 inhibitor EPZ6438 alone or in combination with the MAPK inhibitor U0126. The results showed that CRISPR/Cas9-induced EZH2 gene editing reduced cell growth, migration and invasion in vitro and resulted in a 90% reduction in tumor growth when EZH2-edited cells were injected into an immunocompromised mouse model. Immunohistochemistry analysis of the tumors revealed reduced tumor cell proliferation and less recruitment of cancer-associated fibroblasts in the EZH2-edited tumors compared to the control tumors. Moreover, EZH2 inhibition induced thyroid-differentiation genes' expression and mesenchymal-to-epithelial transition (MET) in ATC cells. Thus, this study shows that targeting EZH2 could be a promising neoadjuvant treatment for ATC, as it promotes antitumoral effects in vitro and in vivo and induces cell differentiation.

Experimental conditions and protein markers for redifferentiation of human coronary artery smooth muscle cells.

A phenotype switch from contractile type to proliferative type of arterial smooth muscle cells is known as dedifferentiation, but to the best of our knowledge, little is known about redifferentiation of coronary artery smooth muscle cells. The purpose of the present study was to determine in vitro culture conditions for inducing redifferentiation of coronary artery smooth muscle cells. In addition, the present study aimed to determine protein markers for detection of redifferentiated arterial smooth muscle cells. Human coronary artery smooth muscle cells (HCASMCs) were cultured in the presence or absence of growth factors, including epidermal growth factor, fibroblast growth factor-B and insulin. Protein expression and migration activity of HCASMCs were evaluated using western blotting and migration assay, respectively. In HCASMCs 5 days after 100% confluency, expression levels of α-smooth muscle actin (α-SMA), calponin, caldesmon and SM22α were significantly increased, while expression levels of proliferation cell nuclear antigen (PCNA) and S100A4 and migration activity were significantly decreased, compared with the corresponding levels just after reaching 100% confluency, indicating that redifferentiation occurred. Redifferentiation was also induced in a low-density culture of HCASMCs in the medium without growth factors. When the culture medium for confluent cells was replaced daily with fresh medium, the expression levels of α-SMA, caldesmon, SM22α, PCNA and S100A4 and migration activity were not significantly different but the calponin expression was significantly increased compared with the levels in dedifferentiated cells just after reaching 100% confluency. Thus, redifferentiation was induced in HCASMCs by deprivation of growth factors from culture medium. The results suggested that α-SMA, caldesmon and SM22α, but not calponin, are markers of redifferentiation of HCASMCs.

Harmonization and standardization of nucleus pulposus cell extraction and culture methods.

In vitro studies using nucleus pulposus (NP) cells are commonly used to investigate disc cell biology and pathogenesis, or to aid in the development of new therapies. However, lab-to-lab variability jeopardizes the much-needed progress in the field. Here, an international group of spine scientists collaborated to standardize extraction and expansion techniques for NP cells to reduce variability, improve comparability between labs and improve utilization of funding and resources. The most commonly applied methods for NP cell extraction, expansion, and re-differentiation were identified using a questionnaire to research groups worldwide. NP cell extraction methods from rat, rabbit, pig, dog, cow, and human NP tissue were experimentally assessed. Expansion and re-differentiation media and techniques were also investigated. Recommended protocols are provided for extraction, expansion, and re-differentiation of NP cells from common species utilized for NP cell culture. This international, multilab and multispecies study identified cell extraction methods for greater cell yield and fewer gene expression changes by applying species-specific pronase usage, 60-100 U/ml collagenase for shorter durations. Recommendations for NP cell expansion, passage number, and many factors driving successful cell culture in different species are also addressed to support harmonization, rigor, and cross-lab comparisons on NP cells worldwide.

A GRIP-1-EZH2 switch binding to GATA-4 is linked to the genesis of rhabdomyosarcoma through miR-29a.

Terminal differentiation failure is an important cause of rhabdomyosarcoma genesis, however, little is known about the epigenetic regulation of aberrant myogenic differentiation. Here, we show that GATA-4 recruits polycomb group proteins such as EZH2 to negatively regulate miR-29a in undifferentiated C2C12 myoblast cells, whereas recruitment of GRIP-1 to GATA-4 proteins displaces EZH2, resulting in the activation of miR-29a during myogenic differentiation of C2C12 cells. Moreover, in poorly differentiated rhabdomyosarcoma cells, EZH2 still binds to the miR-29a promoter with GATA-4 to mediate transcriptional repression of miR-29a. Interestingly, once re-differentiation of rhabdomyosarcoma cells toward skeletal muscle, EZH2 was dispelled from miR-29a promoter which is similar to that in myogenic differentiation of C2C12 cells. Eventually, this expression of miR-29a results in limited rhabdomyosarcoma cell proliferation and promotes myogenic differentiation. We thus establish that GATA-4 can function as a molecular switch in the up- and downregulation of miR-29a expression. We also demonstrate that GATA-4 acts as a tumor suppressor in rhabdomyosarcoma partly via miR-29a, which thus provides a potential therapeutic target for rhabdomyosarcoma.

48 (PB038) - NMS-173, a potent, covalent second generation IDH1/IDH2 inhibitor

Background: IDH1 and IDH2 (isocitrate dehydrogenase 1 and 2) are metabolic enzymes mutated in subsets of solid and hematological tumors. Mutations in IDH1 or IDH2 generate high levels of the 2-HG (2-hydroxyglutarate) oncometabolite that promotes de-differentiation, stemness and tumorigenesis. Inhibition of mutant IDH1or IDH2 decreases 2-HG production and induces re-differentiation of tumor cells. First generation IDH inhibitors received FDA approval for the treatment of subsets of AML and cholangiocarcinoma patients, but extent and duration of response are limited in these patients and no targeted options are available for the other IDH1/ IDH2 mutant solid tumors.

Biomimetic hydrogel blanket for conserving and recovering intrinsic cell properties

BackgroundCells in the human body experience different growth environments and conditions, such as compressive pressure and oxygen concentrations, depending on the type and location of the tissue. Thus, a culture device that emulates the environment inside the body is required to study cells outside the body.MethodsA blanket-type cell culture device (Direct Contact Pressing: DCP) was fabricated with an alginate-based hydrogel. Changes in cell morphology due to DCP pressure were observed using a phase contrast microscope. The changes in the oxygen permeability and pressure according to the hydrogel concentration of DCP were analyzed. To compare the effects of DCP with normal or artificial hypoxic cultures, cells were divided based on the culture technique: normal culture, DCP culture device, and artificial hypoxic environment. Changes in phenotype, genes, and glycosaminoglycan amounts according to each environment were evaluated. Based on this, the mechanism of each culture environment on the intrinsic properties of conserving chondrocytes was suggested.ResultsChondrocytes live under pressure from the surrounding collagen tissue and experience a hypoxic environment because collagen inhibits oxygen permeability. By culturing the chondrocytes in a DCP environment, the capability of DCP to produce a low-oxygen and physical pressure environment was verified. When human primary chondrocytes, which require pressure and a low-oxygen environment during culture to maintain their innate properties, were cultured using the hydrogel blanket, the original shapes and properties of the chondrocytes were maintained. The intrinsic properties could be recovered even in aged cells that had lost their original cell properties.ConclusionsA DCP culture method using a biomimetic hydrogel blanket provides cells with an adjustable physical pressure and a low-oxygen environment. Through this technique, we could maintain the original cellular phenotypes and intrinsic properties of human primary chondrocytes. The results of this study can be applied to other cells that require special pressure and oxygen concentration control to maintain their intrinsic properties. Additionally, this technique has the potential to be applied to the re-differentiation of cells that have lost their original properties.

Sinomenine Hydrochloride Promotes TSHR-Dependent Redifferentiation in Papillary Thyroid Cancer.

Radioactive iodine (RAI) plays an important role in the diagnosis and treatment of papillary thyroid cancer (PTC). The curative effects of RAI therapy are not only related to radiosensitivity but also closely related to the accumulation of radionuclides in the lesion in PTC. Sinomenine hydrochloride (SH) can suppress tumor growth and increase radiosensitivity in several tumor cells, including PTC. The aim of this research was to investigate the therapeutic potential of SH on PTC cell redifferentiation. In this study, we treated BCPAP and TPC-1 cells with SH and tested the expression of thyroid differentiation-related genes. RAI uptake caused by SH-pretreatment was also evaluated. The results indicate that 4 mM SH significantly inhibited proliferation and increased the expression of the thyroid iodine-handling gene compared with the control group (p < 0.005), including the sodium/iodide symporter (NIS). Furthermore, SH also upregulated the membrane localization of NIS and RAI uptake. We further verified that upregulation of NIS was associated with the activation of the thyroid-stimulating hormone receptor (TSHR)/cyclic adenosine monophosphate (cAMP) signaling pathway. In conclusion, SH can inhibit proliferation, induce apoptosis, promote redifferentiation, and then increase the efficacy of RAI therapy in PTC cells. Thus, our results suggest that SH could be useful as an adjuvant therapy in combination with RAI therapy in PTC.

Modeling braf-induced thyroid cancer development and cell redifferentiation using pluripotent stem cell-derived organoids

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Chordin-like 2 influences the differentiation fate of retinal pigment epithelium cells by dynamically regulating BMP pathway.

To explore the functions of Chordin-like 2, which is encoded by CHRDL2, in the process of retinal pigmented epithelium (RPE) differentiation and damage repair. The fetal RPE cells (fRPE) was obtained from aborted fetus which obeyed medical ethics. Real-time quantitative polymerase chain reaction was used to measure expression quantity of CHRDL2 and other functional genes expression. Knocking down and overexpression was used to analyze the functions about Chordin-like 2. Enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion of bone morphogenetic proteins 4 (BMP4). Flow cytometry was used to analyze cell cycle. Cell morphology was observed by phase contrast microscope (PCM). In normal RPE cells, CHRDL2 was firstly upregulated and followed a downregulation but eventually, it was expressed higher than the cells which undergone epithelial-mesenchymal transition (EMT). After knocking down CHRDL2, the secretion of BMP4 was decreased, RPE-related genes (OTX2, MITF, RPE65) were downregulated while EMT-related genes (SNAI1, VIM) were upregulated. However, the expression of these related genes after overexpression of CHRDL2 had contrary results. Chordin-like 2 also regulated the cell cycle by regulating BMP pathway. When CHRDL2 was knocked down, more fRPE cells stayed in S phase of cell cycle, while adding BMP4 reduced the proportion of the cells in S phase. However, overexpression of CHRDL2 increased more BMP4 secretion, this effect decreased the number of cells in S phase, but exogenous BMP inhibitor also could change this effect. At last, in the process of RPE cells differentiation, adding BMP4 at early stage could intervene normal RPE differentiation. Compared with BMP4, inhibiting BMP pathway had no significant negative effect at early stage, but suppressed differentiation at late stage. BMP pathway can be activated in a correct temporal order, otherwise, the cells have incorrect differentiation orientation. And Chordin-like 2 plays a role in dynamic regulation of BMP pathway and it also regulates the differentiation of RPE cells. Therefore, this research enlightens a new direction to inhibit EMT and promote cell redifferentiation after injury.

Retention of epigenetic and transcriptional memory among reprogrammed T cells (T-iPSCs) imparts selective advantage for T cell re-differentiation.

Abstract Various somatic cell types reprogramed into induced pluripotent stem cells (iPSCs) can be differentiated into T cells. It remains unclear what impact the input cells reprogramed into iPSCs have on T cell differentiation. We hypothesized that preservation of T cell genetic and/or epigenetic memory would bias iPSC clones toward T cell differentiation. CD8 naïve T cells (TN), CD8 stem memory T cells (TSM), and fibroblasts (FB) were reprogramed into iPSCs using nonintegrating Sendai virus to deliver Yamanaka factors. Input cells and iPSC clones were subjected to RNA-seq and ATAC-seq to assess whether epigenetic and transcriptomic memory are retained after reprogramming. Pluripotent associated genes (NANOG, LIN28A/B, SOX2) had increased expression in both T cell− and FB-iPSCs compared to their cells of origin. Of interest, gene enrichment analysis of fibroblasts and FB-iPSCs identified fibroblast related annotations, including migration and growth factors, in reprogramed iPSCs. Among CD8 TN− and TSM-iPSCs as a group, expression of only 41 genes and 6 genomic regions were retained, but these included known mature T cell genes, CD3ɛ, ZBTB7B, and CD95 as well as a region crucial for T cell signaling. Therefore, although much of the transcriptome and accessible genome is overridden in TN/TSM-iPSCs, there exists maintenance of both the transcriptome and chromatin accessibility after reprogramming. CD8 T-iPSCs were superior to FB-iPSCs in producing T progenitors in vitro. Highlighted here is the importance of starting populations for reprogramming and differentiation into T lineage cells. Leveraging the benefits of epigenetic and transcriptomic memory of input T cells could provide a more robust source of T cells for adoptive cell therapy. Supported in part by funds from NIH P01 CA065493 and R37 AI34495 from the National Cancer Institute and National Institute of Allergy and Infectious disease, respectively, the Childrens' Cancer Research Fund, and Chan-Zuckerberg Biohub.

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.