Apoptosis Of Progenitor Cells Research Articles

Distal-Less Homeobox 5 Is a Therapeutic Target for Attenuating Hypertrophy and Apoptosis of Mesenchymal Progenitor Cells.

Chondrocyte hypertrophy is a hallmark of osteoarthritis (OA) pathology. In the present study, we elucidated the mechanism underlying the relationship between the hypertrophy/apoptotic phenotype and OA pathogenesis in bone marrow-derived mesenchymal stem cells (BM-MSCs) via gene targeting of distal-less homeobox 5 (DLX5). Our primary objectives were (1) to determine whether DLX5 is a predictive biomarker of cellular hypertrophy in human osteoarthritic tissues; (2) To determine whether modulating DLX5 activity can regulate cell hypertrophy in mesenchymal stem/progenitor cells from marrow and cartilage. Whole transcriptome sequencing was performed to identify differences in the RNA expression profile between human-cartilage-derived mesenchymal progenitors (C-PCs) and bone-marrow-derived mesenchymal progenitors (BM-MSCs). Ingenuity Pathway Analysis (IPA) software was used to compare molecular pathways known to regulate hypertrophic terminal cell differentiation. RT-qPCR was used to measure DLX5 and hypertrophy marker COL10 in healthy human chondrocytes and OA chondrocytes. DLX5 was knocked down or overexpressed in BM-MSCs and C-PCs and RT-qPCR were used to measure the expression of hypertrophy/terminal differentiation markers following DLX5 modulation. Apoptotic cell activity was characterized by immunostaining for cleaved caspase 3/7. We demonstrate that DLX5 and downstream hypertrophy markers were significantly upregulated in BM-MSCs, relative to C-PCs. DLX5 and COL10 were also significantly upregulated in cells from OA knee joint tissues, relative to normal non-arthritic joint tissues. Knocking down DLX5 in BM-MSCs inhibited cell hypertrophy and apoptotic activity without attenuating their chondrogenic potential. Overexpression of DLX5 in C-PCs stimulated hypertrophy markers and increased apoptotic cell activity. Modulating DLX5 activity regulates cell hypertrophy and apoptosis in BM-MSCs and C-PCs. These findings suggest that DLX5 is a biomarker of OA changes in human knee joint tissues and confirms the DLX5 mechanism contributes to hypertrophy and apoptosis in BM-MSCs.

Circ-ADAM9 targeting PTEN and ATG7 promotes autophagy and apoptosis of diabetic endothelial progenitor cells by sponging mir-20a-5p

Dysfunction of endothelial progenitor cells (EPCs) is a key factor in vascular complications of diabetes mellitus. Although the roles of microRNAs and circular RNAs in regulating cell functions have been thoroughly studied, their role in regulating autophagy and apoptosis of EPCs remains to be elucidated. This study investigated the roles of mir-20a-5p and its predicted target circ-ADAM9 in EPCs treated with high glucose (30 mM) and in a diabetic mouse hind limb ischemia model. It is found that Mir-20a-5p inhibited autophagy and apoptosis of EPCs induced by high-concentration glucose. Further, mir-20a-5p could inhibit the expression of PTEN and ATG7 in EPCs, and promote the phosphorylation of AKT and mTOR proteins under high-glucose condition. Investigation of the underlying mechanism revealed that circ-ADAM9, as a miRNA sponges of mir-20a-5p, promoted autophagy and apoptosis of EPCs induced by high-concentration glucose. Circ-ADAM9 upregulated PTEN and ATG7 in interaction with mir-20a-5p, and inhibited the phosphorylation of AKT and mTOR to aggravate autophagy and apoptosis of EPCs under high glucose. In addition, silencing of circ-ADAM9 increased microvessel formation in the hind limbs of diabetic mice. Our findings disclose a novel autophagy/apoptosis-regulatory pathway that is composed of mir-20a-5p, circ-ADAM9, PTEN, and ATG7. Circ-ADAM9 is a potential novel target for regulating the function of diabetic EPCs and angiogenesis.

Perinatal Exposure to Triclosan Results in Abnormal Brain Development and Behavior in Mice.

Triclosan (TCS) is one of the most common endocrine-disrupting chemicals (EDCs) present in household and personal wash products. Recently, concerns have been raised about the association between abnormal behavior in children and exposure to EDC during gestation. We hypothesized that exposure to TCS during gestation could affect brain development. Cortical neurons of mice were exposed in vitro to TCS. In addition, we examined in vivo whether maternal TCS administration can affect neurobehavioral development in the offspring generation. We determined that TCS can impair dendrite and axon growth by reducing average length and numbers of axons and dendrites. Additionally, TCS inhibited the proliferation of and promoted apoptosis in neuronal progenitor cells. Detailed behavioral analyses showed impaired acquisition of spatial learning and reference memory in offspring derived from dams exposed to TCS. The TCS-treated groups also showed cognition dysfunction and impairments in sociability and social novelty preference. Furthermore, TCS-treated groups exhibited increased anxiety-like behavior, but there was no significant change in depression-like behaviors. In addition, TCS-treated groups exhibited deficits in nesting behavior. Taken together, our results indicate that perinatal exposure to TCS induces neurodevelopment disorder, resulting in abnormal social behaviors, cognitive impairment, and deficits in spatial learning and memory in offspring.

1134-P: The Secretome of Visceral Adipose Cells Induces Apoptosis of Cardiac Progenitor Cells in Human Obesity: Protective Effects of the SGLT2 Inhibitor Dapagliflozin

This study aimed to assess whether the secretome from abdominal visceral (AV) and epicardial (E) adipose stem cells (ASC), ASC-derived mature adipocytes, and isolated AV mature adipocytes may affect the viability of cardiac progenitor cells (CPC) in human obesity, and whether dapagliflozin (DAPA) may reduce the proapoptotic effects in injured CPC. ASC were isolated from AV and E adipose tissue biopsies of nondiabetic subjects with varying levels of BMI and used before and after differentiation into adipocytes in vitro. CPC were isolated from right auricle biopsies of non-obese (Ob) subjects, preincubated in the presence or absence of 10 uM DAPA, and exposed or not to conditioned media (CM) of AV-ASC, E-ASC, AV-ASC-derived adipocytes and isolated AV mature adipocytes. The CM of AV-ASC and differentiated adipocytes from Ob subjects induced CPC apoptosis after 4-8-24 h (p<0.05). In addition, exposure of CPC to CM of Ob AV-ASC and mature adipocytes resulted in marked inhibition of insulin-stimulated Akt phosphorylation (Akt-Ph) (p<0.05). In contrast, the CM of AV-ASC and differentiated adipocytes from non-Ob subjects did not induce apoptosis nor impaired insulin signaling. Similarly, the CM of AV mature adipocytes isolated from Ob subjects induced a marked apoptosis of CPC (p<0.05) and inhibited insulin-stimulated Akt-Ph, whereas no effects were observed when CPC were exposed to CM of mature adipocytes isolated from non-Ob subjects. Of note, the CM of E-ASC isolated from Ob subjects also induced CPC apoptosis and impaired insulin-stimulated Akt-Ph (p<0.05), while these effects were not observed when CPC were exposed to CM of E-ASC from non-Ob subjects. The proapoptotic effects of the CM in the CPC were inhibited when these cells were pretreated with DAPA (p<0.05). In conclusion, the secretome of adipose cells from Ob subjects impairs insulin action and viability of the CPC, and these effects can be counteracted by DAPA. Disclosure S. Porro: None. S. Perrini: None. C. Caccioppoli: None. R. Doria: None. V. Genchi: None. R. Schipani: None. A. Cignarelli: None. A. Natalicchio: Consultant; Self; AstraZeneca, Novo Nordisk Inc., Sanofi-Aventis. L. Laviola: Advisory Panel; Self; Abbott, AstraZeneca, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Novo Nordisk Inc., Roche Diabetes Care, Sanofi-Aventis. Speaker’s Bureau; Self; Abbott, Medtronic. F. Giorgino: Advisory Panel; Self; AstraZeneca, Eli Lilly and Company, Novo Nordisk Inc., Roche Diabetes Care, Sanofi. Consultant; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., LifeScan, Inc., MedImmune, Merck Sharp & Dohme Corp., Roche Diabetes Care, Sanofi. Research Support; Self; Eli Lilly and Company, LifeScan, Inc., Takeda Development Centre Europe Ltd.

Flow Cytometry Definition of Rat Mammary Epithelial Cell Populations and Their Distinct Radiation Responses.

Breast tissue is very susceptible to radiation-induced carcinogenesis, and mammary stem/progenitor cells are potentially important targets of this. The mammary epithelium is maintained as two mostly independent lineages of luminal and basal cells. To elucidate their immediate radiation responses, we analyzed the mammary glands of female Sprague-Dawley rats, a radiation carcinogenesis model, using colony formation, flow cytometry and immunofluorescence. The results revealed that flow cytometry successfully fractionates rat mammary cells into CD49fhi CD24lo basal, CD49fmed CD24hi luminal progenitor, and CD49flo CD24hi mature luminal populations, resembling human breast, rather than mouse tissues. The colony-forming ability of the basal cells was more radiosensitive than the luminal progenitor cells. Flow cytometry and immunofluorescence showed more efficient cell cycle arrest, γ-H2AX responses, and apoptosis in the irradiated luminal progenitor cells, than in the basal cells. These results provide important insights into the early phase of radiation-induced breast cancer.

MicroRNA-384-5p Promotes Endothelial Progenitor Cell Proliferation and Angiogenesis in Cerebral Ischemic Stroke through the Delta-Likeligand 4-Mediated Notch Signaling Pathway

Background: MicroRNAs (miRs) have a crucial regulatory role in endothelial cell function and tumor angiogenesis by inhibiting the expressions of their target genes. The participation of microRNA-384-5p (miR-384-5p) has been prominently reported in various ischemia-induced diseases such as myocardial ischemia and atherosclerosis. Hence, the present study aimed at exploring the effect of miR-384-5p on proliferation, apoptosis, and angiogenesis of endothelial progenitor cells (EPCs) in cerebral ischemic stroke and investigating the associated underlying mechanism. Methods: A middle cerebral artery occlusion (MCAO) mouse model was established, with determination of the expression of cluster of differentiation 31 (CD31) and vascular endothelial growth factor (VEGF) proteins. Next, the MCAO mice and EPCs separated from MCAO mice were injected or transfected with mimics or inhibitors of miR-384-5p, or small interference RNA Delta-likeligand 4 (si-DLL4) in order to evaluate their effect on brain infarct size, cell proliferation, apoptosis, and angiogenesis. The relationship among miR-384-5p, DLL4, and the Notch signaling pathway was then verified by a series of experiments. Results: In MCAO mice, an increased brain infarct size and cell apoptosis in brain tissues were evident, with decreased expression of miR-384-5p, VEGF, and CD31, as well as increased DLL4 expression. After miR-384-5p mimic or si-DLL4 treatment, the brain infarct size and cell apoptosis in the brain tissues were reduced in compliance with an increased expression of VEGF and CD31. Our findings demonstrated that miR-384-5p negatively regulated the expression of DLL4, which further downregulated the Notch signaling pathway. When miR-384-5p was overexpressed or DLL4 silenced, the cell proliferation and angiogenesis of EPCs were promoted and cell apoptosis was inhibited. Conclusions: Our study demonstrated that overexpressed miR-384-5p targeting DLL4 could stimulate proliferation and angiogenesis, while inhibiting apoptosis of EPCs in mice with cerebral ischemic stroke through the Notch signaling pathway.

Congenital pituitary hypoplasia model demonstrates hypothalamic OTX2 regulation of pituitary progenitor cells.

Pituitary develops from oral ectoderm in contact with adjacent ventral hypothalamus. Impairment in this process results in congenital pituitary hypoplasia (CPH); however, there have been no human disease models for CPH thus far, prohibiting the elucidation of the underlying mechanisms. In this study, we established a disease model of CPH using patient-derived induced pluripotent stem cells (iPSCs) and 3D organoid technique, in which oral ectoderm and hypothalamus develop simultaneously. Interestingly, patient iPSCs with a heterozygous mutation in the orthodenticle homeobox 2 (OTX2) gene showed increased apoptosis in the pituitary progenitor cells, and the differentiation into pituitary hormone-producing cells was severely impaired. As an underlying mechanism, OTX2 in hypothalamus, not in oral ectoderm, was essential for progenitor cell maintenance by regulating LHX3 expression in oral ectoderm via FGF10 expression in the hypothalamus. Convincingly, the phenotype was reversed by the correction of the mutation, and the haploinsufficiency of OTX2 in control iPSCs revealed a similar phenotype, demonstrating that this mutation was responsible. Thus, we established an iPSC-based congenital pituitary disease model, which recapitulated interaction between hypothalamus and oral ectoderm and demonstrated the essential role of hypothalamic OTX2.

Twist1 Plays an Anti-apoptotic Role in Mutant Huntingtin Expression Striatal Progenitor Cells.

The Twist basic helix-loop-helix transcription factor 1 (Twist1) has been implicated in embryogenesis and carcinogenesis, due to its effects on cell proliferation and anti-apoptosis signaling. Interestingly, a connection between Twist1 and neurotoxicity was recently made in mutant huntingtin (mHtt)-expressing primary cortical neurons; however, the role of Twist1 in Huntington's disease (HD)-affected striatal neurons remains undescribed. In this study, we evaluated the expression and function of Twist1 in the R6/2 HD mouse model, which expresses the polyQ-expanded N-terminal portion of human HTT protein, and a pair of striatal progenitor cell lines (STHdhQ109 and STHdhQ7), which express polyQ-expanded or non-expanded full-length mouse Htt. We further probed upstream signaling events and Twist1 anti-apoptotic function in the striatal progenitor cell lines. Twist1 was increased in mHtt-expressing striatal progenitor cells (STHdhQ109) and was correlated with disease progression in striatum and cortex brain regions of R6/2 mice. In the cell model, downregulation of Twist1 induced death of STHdhQ109 cells but had no effect on wild-type striatal progenitor cells (STHdhQ7). Twist1 knockdown stimulated caspase-3 activation and apoptosis. Furthermore, we found that signal transducer and activator of transcription 3 (STAT3) were increased in HD striatal progenitor cells and acted as an upstream regulator of Twist1. As such, inhibition of STAT3 induced apoptosis in HD striatal progenitor cells. Our results suggest that mHtt upregulates STAT3 to induce Twist1 expression. Upregulated Twist1 inhibits apoptosis, which may protect striatal cells from death during disease progression. Thus, we propose that Twist1 might play a protective role against striatal degeneration in HD.

Recreation of t(7;12)(q36;p13) in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9 Generates a Useful Model for Studying Acute Myeloid Leukemia

Introduction Acute myeloid leukemia (AML) is the result of aberrant hematopoietic processes, such as enhanced proliferation, blocked differentiation, and dysregulated apoptosis of hematopoietic stem and progenitor cells, and frequently these changes are initiated by chromosomal translocations in leukemia. Efficient methods for modelling leukemia and to recreate leukemia-associated genetic aberrations, such as chromosome translocations, are therefore crucial for investigating how leukemia is initiated. Today, most such models are murine and usually based on introduction of fusion gene transcripts of interest under the control of a constitutive active promoter using lenti- or retroviral transduction rather than the chromosomal translocation itself. The aim of the current project was to create a human cellular model of a chromosomal translocation that is typically found in AML in children under 24 months of age, the translocation t(7;12)(q36;p13). This translocation has been associated with poor prognosis, and leads to a gene fusion MNX1-ETV6 but also aberrant MNX1 gene expression. Its mechanism for leukemia initiation is so far unknown, mainly due to lack of a suitable experimental model. Material and methods CRISPR/Cas9 was used to reconstruct the genetics of the t(7;12)(q36;p13) rearrangement in human induced pluripotent stem cells (iPSC) while maintaining the genomic architecture and regulatory elements. Ribonucleoprotein (RNP) complex was delivered by lipofection (Nucleofection, Amaxa 4D system) into undifferentiated iPSC (ChiPSC 22, Cellartis). An ATTO550 tag on tracrRNA/RNP complex was used to sort out positive cells by flow cytometry and then seeded as single-cells in 96-well plates. Genomic DNA from the single-cell derived iPSC clones were screened by PCR for the presence of the translocation and positive clones were verified with a FISH probe specific for t(7;12)(q36;p13) (Double Fusion Break Apart probe, Metasystem). RT-qPCR was used to detect and quantify the expression of MNX1-ETV6 fusion and MNX1 transcripts. Differentiation potential was tested with the Trilineage Differentiation and Hematopoietic Kits (STEMdiff, STEMCELL Technologies). Results Using CRISPR/Cas9, we could successfully generate iPSC with the t(7;12)(q36;p13) translocation. The translocation was confirmed using conventional karyotyping and FISH and the mRNA expression of the fusion was confirmed with RT-qPCR. No additional chromosomal aberrations were seen. The t(7;12)(q36;p13) iPSC showed similar growth and differentiation properties as the parental iPSC. They showed propensity to differentiate into all three germ layers, confirming their pluripotent stem cell properties. The potential for differentiation into hematopoietic progenitor cells was shown by expression of CD34+, CD43+ and CD45+. In AML with t(7;12)(q36;p13), MNX1 mRNA expression is increased and this may play a role for leukemia development. In the t(7;12)(q36;p13) iPSC, RT-qPCR indeed showed increased expression of MNX1 expression compared with iPSC without the translocation. This increase of MNX1 was not seen in murine adult bone marrow or fetal liver cells transduced with retrovirus expressing the MNX1-ETV6 fusion. Further characterization of the t(7;12)(q36;p13) iPSC, e.g. whole exome and transcriptome sequencing and engraftment potential in immunocompromised mice (NSG-SGM), is ongoing. Conclusion In summary, we have using CRISPR/Cas9 successfully created a t(7;12)(q36;p13) iPSC line with potential to differentiate into hematopoietic progenitor cells and with gene expression pattern similar to what is seen in human AML samples with the t(7;12)(q36;p13). The introduction of the MNX1-ETV6 fusion in its correct genomic context could recapitulate local gene regulation, making it superior to models based on lenti- or retroviral introduction of fusion genes transcripts. In conclusion, this created cell line will be a valuable tool to study the mechanisms behind t(7;12)(q36;p13) AML, a severe form of AML associated with poor prognosis. Disclosures No relevant conflicts of interest to declare.

Dual Inhibition of MDM2 and XPO1 Synergizes to Induce Apoptosis in Acute Myeloid Leukemia Progenitor Cells with Wild-Type TP53 through Nuclear Accumulation of p53 and Suppression of c-Myc

Dual Inhibition of MDM2 and XPO1 Synergizes to Induce Apoptosis in Acute Myeloid Leukemia Progenitor Cells with Wild-Type TP53 through Nuclear Accumulation of p53 and Suppression of c-Myc

Efficient Elimination of Acute Myeloid Leukemia Cells through Inhibition of KDM4A in Combination with PARP Inhibition

Epigenetic therapies are emerging as a promising therapeutic strategy for acute myeloid leukemia (AML), exemplified by advances in the development of inhibitors targeting DNMT3A, DOT1L and LSD1. We identified an essential role for the H3K9me3 histone demethylase, KDM4A, in maintaining AML cell survival with genetic depletion of KDM4A having no effect on normal hematopoiesis. Therefore, we hypothesise KDM4A inhibition may represent a novel and effective strategy to treat AML. To address this, we developed a series of novel KDM4A inhibitors (KDM4Ai), based on the structure of pan inhibitor IOX1, and fully characterised their functional potential in AML cells representing major molecular subtypes, and primary patient blasts in comparison with healthy donor cells, as single agents or in combination with other anti-cancer drugs. To evaluate these compounds in physiological conditions, we utilised a stromal co-culture system mimicking the bone marrow microenvironment. Furthermore, we carried out global transcriptomic profiling by RNA-seq to elucidate the molecular consequences responsible for KDM4Ai induced leukemic killing. As a mono-therapy, KDM4Ai induced leukemic cell differentiation and apoptosis in a broad spectrum of human AML cells, with an IC50 of 3.2µM ± 0.2 in MLL-AF9 driven THP1 cells after 48hr treatment (n=3), similar efficacy was observed in other human AML cell lines (n= ≤3) including Kasumi1 (2.69µM ± 0.1) , OCI-AML3 (4.9 µM ± 1.0) and MOLM13 (1.7 µM ± 0.7) and in primary patient blasts (3.8µM). A complete removal of colony forming potential was observed upon treatment (n = 2). The global expression of KDM4A's established substrate, H3K9me3 was upregulated by immunofluorescence and transcriptional changes in a 9-gene signature identified previously as direct KDM4A downstream targets, is indicative of an on-target effect by KDM4Ai. Importantly, KDM4Ai specifically reduced CD34+leukemic stem cell enriched population ( reduced by 6%). In contrast, a non significant reduction was observed on donor CD34+hematopoietic stem and progenitor cells proliferation and apoptosis suggesting a therapeutic window. Cytoprotection provided by stromal co-culture in both AML cell lines and primary samples resulted in a 50% decrease in apoptotic cells with maintenance of the CD34+compartment . Taking these results into account we identify importance of the microenvironment in drug mechanism and resistance. To better understand the mechanism driving this selective anti-leukemic effect, we performed transcriptomic analysis on KDM4Ai treated THP1 cells (n=3). Corroborating the differentiation phenotype, pathway analysis showed an enrichment of IL4 & IL13 signalling (Enrichment score (ES) = - 0.53, q value = 0.025), and neutrophil degranulation (Enrichment score (ES) = - 0.51, q value = 0.025), this was accompanied by significant up-regulation of DNA damage response pathways (ES = 0.66, q value = 0.025) . These results were confirmed in AML cell lines, displaying accumulation > 20% of γH2AX by intracellular flow cytometry and PARP cleavage by western blot following treatment. Based on these results we hypothesised that KDM4Ai may sensitise leukemic cells to DNA damage pathway inhibitors, such as PARP inhibitors (PARPi) (n = ≤3). While standard chemotherapies, such as cytarabine and azacitidine, in combination with KDM4Ai showed a largely additive effect, a dual inhibition of KDM4Ai at 3mM with 5mM olaparib (PARPi), exhibited a Combination Index of ~0.69 with a decrease in proliferation (>15% reduction) and increased apoptosis (>20%) in MLLr-AML cell lines compared with KDM4Ai alone (p=0.005). This effect was corroborated ex vivo using cells isolated from a patient derived xenograft model of MLL-AF10 (n=3). Taken together these results suggest a synergistic leukemic cell killing and have subsequently been subjected to global RNA-seq to confirm the detailed molecular mechanism underlying the synergistic effect. Pharmacological inhibition of KDM4A using novel compounds effectively eliminated leukemic cells sparing normal hematopoietic cells with a synthetic lethality observed through combination with PARPi offering a promising therapeutic strategy in AML. Our data further support the essential role for KDM4A in myeloid oncogenesis, promoting future clinical evaluation of KDM4Ai its associated downstream targets as potential tractable therapeutic vulnerabilities in AML. Disclosures No relevant conflicts of interest to declare.

Reprogramming and differentiation-dependent transcriptional alteration of DNA damage response and apoptosis genes in human induced pluripotent stem cells.

Pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have a dual capability to self-renew and differentiate into all cell types necessary to develop an entire organism. Differentiation is associated with dynamic epigenetic alteration and transcriptional change, while self-renewal depends on maintaining the genome DNA accurately. Genome stability of PSCs is strictly regulated to maintain pluripotency. However, the DNA damage response (DDR) mechanism in PSCs is still unclear. There is accumulating evidence that genome stability and pluripotency are regulated by a transcriptional change in undifferentiated and differentiated states. iPSCs are ideal for analyzing transcriptional regulation during reprogramming and differentiation.This study aimed to elucidate the transcriptional alteration surrounding genome stability maintenance, including DNA repair, cell cycle checkpoints and apoptosis in fibroblasts, iPSCs and neural progenitor cells (NPCs) derived from iPSCs as differentiated cells. After ionizing radiation exposure, foci for the DNA double-stranded break marker γ-H2AX increased, peaking at 0.5 h in all cells (>90%), decreasing after 4 h in fibroblasts (32.3%) and NPCs (22.3%), but still remaining at 52.5% (NB1RGB C2 clone) and 54.7% (201B7 cells) in iPSCs. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells were detected, indicating that iPSCs’ apoptosis increases. In addition, RNA sequencing (RNA-Seq) analysis showed high expression of apoptosis genes (TP53, CASP3 and BID) in iPSCs. Results suggested that increased apoptosis activity maintains accurate, undifferentiated genome DNA in the cell population.

Role of Erythropoietin in Cerebral Glioma: An Innovative Target in Neuro-Oncology

Erythropoietin (EPO) is a cytokine primarily involved in the regulation of erythropoiesis. In response to hypoxia-ischemia, hypoxia-inducible factor 1 induces EPO production, which, in turn, inhibits apoptosis of erythroid progenitor cells. By the same mechanism and acting through other signaling pathways, EPO exerts neuroprotective effects. Increased resistance to hypoxia and decreased apoptosis are thought to be important mechanisms for tumor progression, including malignant glioma. Because recent studies have demonstrated that EPO and its receptor (EPOR) are expressed in several tumors and can promote tumor growth, in the present study, we investigated EPO and EPOR expression in human glioma and the effect of EPO administration in a rat model of glioma implantation. Using Western blotting and immunohistochemical analysis, we examined the expression of EPO, EPOR, platelet endothelial cell adhesion molecule, and Ki-67 in human glioma specimens and experimentally induced glioma in rats. In the experimental setting, a daily dose of recombinant human EPO (rHuEPO) or saline solution were administered for 21 days in Fischer rats subjected to 9L cell line implantation. In both human and animal specimens, we found an increase in EPOR expression as long as the lesion presented with an increasing malignant pattern. A significant direct correlation was found between the expression of EPOR and Ki-67 and EPOR and platelet endothelial cell adhesion molecule in low- and high-grade gliomas. The rats treated with rHuEPO presented with significantly larger tumor spread compared with the saline-treated rats. The results of our study have shown that the EPO/EPOR complex might play a significant role in the aggressive behavior of high-grade gliomas. The larger tumor spread in rHuEPO-treated rats suggests a feasible role for EPO in the aggressiveness and progression of malignant glioma.

Metabolic Regulation of Hippocampal Neuroprogenitor Apoptosis After Irradiation.

The tumor suppressor p53 is an important regulator of cell fate response after DNA damage. Cell fate response following metabolic stresses has also been linked to p53-dependent pathways. In this study, we asked if 5'-adenosine monophosphate-activated protein kinase (AMPK), the master sensor of cellular energy balance, played a role in p53-dependent apoptosis of neural progenitor cells (NPCs) in the hippocampus after irradiation. Adult mice with targeted disruption of p53 or prkaa2 (gene that encodes AMPKα) in the brain were used to determine the role of p53 and AMPK, respectively, in radiation-induced apoptosis of NPCs in the hippocampus. The p53-dependent apoptosis of NPCs was associated with an increase in phospho-AMPK expression in the dentate gyrus at 8 hours after irradiation. Activation of AMPK was seen in granule neurons and subgranular NPCs. Compared with wildtype mice, apoptosis of NPCs was significantly attenuated in AMPK deficient (nestinCre: prkaa2fl/fl) mice after irradiation. AMPK deficiency did not however alter p53 activation in NPCs after irradiation. We conclude that AMPK may regulate apoptosis of hippocampal NPCs after irradiation. These findings suggest that cellular metabolism may play a role in determining cell fate response such as apoptosis after DNA damage in NPCs.

ROCK inhibitor increases proacinar cells in adult salivary gland organoids.

Salisphere-derived adult epithelial cells have been used to improve saliva production of irradiated mouse salivary glands. Importantly, optimization of the cellular composition of salispheres could improve their regenerative capabilities. The Rho Kinase (ROCK) inhibitor, Y27632, has been used to increase the proliferation and reduce apoptosis of progenitor cells grown in vitro. In this study, we investigated whether Y27632 could be used to improve expansion of adult submandibular salivary epithelial progenitor cells or to affect their differentiation potential in different media contexts. Application of Y27632 in medium used previously to grow salispheres promoted expansion of Kit+ and Mist1+ cells, while in simple serum-containing medium Y27632 increased the number of cells that expressed the K5 basal progenitor marker. Salispheres derived from Mist1CreERT2; R26TdTomato mice grown in salisphere media with Y27632 included Mist1-derived cells. When these salispheres were incorporated into 3D organoids, inclusion of Y27632 in the salisphere stage increased the contribution of Mist1-derived cells expressing the proacinar/acinar marker, Aquaporin 5 (AQP5), in response to FGF2-dependent mesenchymal signals. Optimization of the cellular composition of salispheres and organoids can be used to improve the application of adult salivary progenitor cells in regenerative medicine strategies.

Notch and EGFR regulate apoptosis in progenitor cells to ensure gut homeostasis in Drosophila

The regenerative activity of adult stem cells carries a risk of cancer, particularly in highly renewable tissues. Members of the family of inhibitor of apoptosis proteins (IAPs) inhibit caspases and cell death, and are often deregulated in adult cancers; however, their roles in normal adult tissue homeostasis are unclear. Here, we show that regulation of the number of enterocyte‐committed progenitor (enteroblast) cells in the adult Drosophila involves a caspase‐mediated physiological apoptosis, which adaptively eliminates excess enteroblast cells produced by intestinal stem cells (ISCs) and, when blocked, can also lead to tumorigenesis. Importantly, we found that Diap1 is expressed by enteroblast cells and that loss and gain of Diap1 led to changes in enteroblast numbers. We also found that antagonistic interplay between Notch and EGFR signalling governs enteroblast life/death decisions via the Klumpfuss/WT1 and Lozenge/RUNX transcription regulators, which also regulate enteroblast differentiation and cell fate plasticity. These data provide new insights into how caspases drive adult tissue renewal and protect against the formation of tumours.

Downregulation of microRNA-129-5p increases the risk of intervertebral disc degeneration by promoting the apoptosis of nucleus pulposus cells via targeting BMP2.

miR-129-5p is implicated in many diseases, such as laryngeal cancer and breast cancer. In this study, we studied the mechanism underlying the role of BMP2 in intervertebral disc degeneration (IDD). We used a luciferase assay system to determine the relationship between BMP2 and miR-129-5 expression. In addition, Western blot and real-time PCR were used to confirm the regulatory relationship between miR-129-5p and its targets, while flow cytometry was used to evaluate the effect of miR-129-5p on the apoptosis of neural progenitor cells (NPCs). BMP2 was confirmed as a direct target of miR-129-5p. Furthermore, the expression of miR-129 was downregulated along with upregulated BMP2 expression in IDD patients. Meanwhile, BMP2 was validated as the target of miR-129-5p in cells transfected with miR-129-5p and BMP2 siRNA. Also, compared with NPCs transfected with blank/scramble controls or miR-129-5p inhibitors, the NPCs treated with miR-129-5p mimics or BMP2 siRNA exhibited evidently elevated viability and inhibited apoptosis. The data demonstrated that miR-129-5p was poorly expressed in IDD patients, and the dysregulation of miR-129-5p might contribute to the development of IDD by targeting BMP2 expression.

Transcriptomic and Epigenomic Profiling of Histone Deacetylase Inhibitor Treatment Reveals Distinct Gene Regulation Profiles Leading to Impaired Neutrophil Development

The clinical use of histone deacetylase inhibitors (HDACi) for the treatment of bone marrow failure and hematopoietic malignancies has increased dramatically over the last decades. Nonetheless, their effects on normal myelopoiesis remain poorly evaluated. Here, we treated cord blood derived CD34+ progenitor cells with two chemically distinct HDACi inhibitors MS-275 or SAHA and analyzed their effects on the transcriptome (RNA-seq), epigenome (H3K27ac ChIP-seq) and functional and morphological characteristics during neutrophil development. MS-275 (entinostat) selectively inhibits class I HDACs, with a preference for HDAC1, while SAHA (vorinostat) is a non-selective class I/II HDACi. Treatment with individual HDACi resulted in both overlapping and distinct effects on both transcriptome and epigenome, whereas functional effects were relatively similar. Both HDACi resulted in reduced expansion and increased apoptosis in neutrophil progenitor cells. Morphologically, HDACi disrupted normal neutrophil differentiation what was illustrated by decreased percentages of mature neutrophils. In addition, while SAHA treatment clearly showed a block at the promyelocytic stage, MS-275 treatment was characterized by dysplastic features and skewing towards the monocytic lineage. These effects could be mimicked using shRNA-mediated knockdown of HDAC1. Taken together, our data provide novel insights into the effects of HDAC inhibition on normal hematopoietic cells during neutrophil differentiation. These findings should be taken into account when considering the clinical use of MS-275 and SAHA, and can be potentially utilized to tailor more specific, hematopoietic-directed HDACi in the future.

Influence of BMP4 on Regulation of Cell Cycle and Apoptosis of Hematopoietic Stem Cells/Progenitor Cells and Its Mechanism in Chemotherapy-Induced Myelosuppression

To explore the effect of bone morphogenetic protein 4(BMP4) on the cell cycle and apoptosis of hemaropoictic stem and progenitor cells (HSPC) in conditions of 5-fluorouracil (5-FU)-inducing bone marrow suppression and stress hemogenesis, and its possible mechanism. The C57BL transgenic mice with BMP4 overexpression were established and were enrolled in transgenic group (BMP4 group), at the same time the wild type mice matching in age, sex and body weight were selected and were enrolled in control group (WT group). The bone marrow suppression was induced by injection with 5-FU in dose of 150 mg/kg, then the nucleated cells were isolated from bone marrow. After the HSPCs were markered with C-kit/sca-1 fluorescent antibodies, the changes of cell cycle and apoptosis of HSPC were detected by Aunexin V/PI and Ki67/DAPI double staining; the cell cycle-essociated hemotopoietic regulatory factors were detected by RT-qPCR. Under physiologic status, there were no significant differences in cell cycle and apoptotic rate of HSPC between WT group and BMP-4 group. After the bone marrow was suppressed, the ratio of HSPC at G0 phase in BMP4 group significantly decreased(P＜0.05); the apoptosis rate of HSPC significantly increased(P＜0.05); the mRNA expression levels of hypoxia-inducing factor Hif-1α and chemotactic factor CXCL12 in stroma of BMP4 group were down-regulated significanfly(P＜0.05). Under non-physiologic conditions such as stress hemogenesis or bone marrow suppression, the up-regulation of BMP4 can promote HSPC into cell cycle and apoptosis of HSPC, moreover, the BMP4 may play a regulatory role for cell cycle of HSPC through direct or indirect down-regulation of Hif-1α and CXCL-12 expressions.

Aluminum affects neural phenotype determination of embryonic neural progenitor cells.

Aluminum (Al) is a neurotoxin and is associated with the etiology of neurodegenerative diseases, such as Alzheimer's disease (AD). The Al-free ion (Al3+) is the biologically reactive and toxic form. However, the underlying mechanisms of Al toxicity in the brain remain unclear. Here, we evaluated the effects of Al3+ (in the chloride form-AlCl3) at different concentrations (0.1-100µM) on the morphology, proliferation, apoptosis, migration and differentiation of neural progenitor cells (NPCs) isolated from embryonic telencephalons, cultured as neurospheres. Our results reveal that Al3+ at 100µM reduced the number and diameter of neurospheres. Cell cycle analysis showed that Al3+ had a decisive function in proliferation inhibition of NPCs during neural differentiation and induced apoptosis on neurospheres. In addition, 1µM Al3+ resulted in deleterious effects on neural phenotype determination. Flow cytometry and immunocytochemistry analysis showed that Al3+ promoted a decrease in immature neuronal marker β3-tubulin expression and an increase in co-expression of the NPC marker nestin and glial fibrillary acidic protein. Thus, our findings indicate that Al3+ caused cellular damage and reduced proliferation and migration, resulting in global inhibition of NPC differentiation and neurogenesis.