"BDSM" refers to various forms of sexuality that incorporate restraint, pressure, sensation, training, and elements of both erotic and non-erotic power exchange between the involved parties. BDSM's ...ideas and development were not accepted by the majority and are often associated with mental illnesses. However, recent western research suggested that BDSM practices may benefit its practitioners. This study aimed to unearth the life wisdom of three female kinky in Hong Kong. It employed a narrative inquiry informed by narrative therapy techniques. Results concluded four themes based on the development of their life wisdom through engaging as a submissive in a D/s relationship. The four themes were: (1) enriching identity in BDSM relationships, (2) shaping a preferred locus of control, (3) gaining sexual autonomy through BDSM and (4) healing with BDSM interactions. This study provided insights, implementations, and suggestions for social work practice and research in BDSM contexts.
Animal and epidemiological studies demonstrated association of persistent exposure of TCDD, an endocrine disrupting chemical, to susceptibility of type 2 diabetes (T2D). High doses of TCDD were ...commonly employed in experimental animals to illustrate its diabetogenic effects. Data linking the epigenetic effects of low doses of TCDD on embryonic cells to T2D susceptibility risks is very limited. To address whether low dose exposure to TCDD would affect pancreatic development, hESCs pretreated with TCDD at concentrations similar to human exposure were differentiated towards pancreatic lineage cells, and their global DNA methylation patterns were determined. Our results showed that TCDD-treated hESCs had impaired pancreatic lineage differentiation potentials and altered global DNA methylation patterns. Four of the hypermethylated genes (PRKAG1, CAPN10, HNF-1B and MAFA) were validated by DNA bisulfite sequencing. PRKAG1, a regulator in the AMPK signaling pathway critical for insulin secretion, was selected for further functional study in the rat insulinoma cell line, INS-1E cells. TCDD treatment induced PRKAG1 hypermethylation in hESCs, and the hypermethylation was maintained after pancreatic progenitor cells differentiation. Transient Prkag1 knockdown in the INS-1E cells elevated glucose stimulated insulin secretions (GSIS), possibly through mTOR signaling pathway. The current study suggested that early embryonic exposure to TCDD might alter pancreatogenesis, increasing the risk of T2D.
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•TCDD impaired differentiation of ESCs to early pancreatic lineage cells.•TCDD induced global DNA methylation changes in hESCs.•TCDD treatment induced PRKAG1 hypermethylation in hESCs.•TCDD-induced PRKAG1 hypermethylation was detected during early differentiation.•Prkag1 knockdown in INS-1E cells activated mTOR pathway and increased GSIS.
STUDY QUESTION
Can human embryonic stem cell-derived trophoblastic spheroids be used to study the early stages of implantation?
SUMMARY ANSWER
We generated a novel human embryonic stem cell-derived ...trophoblastic spheroid model mimicking human blastocysts in the early stages of implantation.
WHAT IS KNOWN ALREADY
Both human embryos and choriocarcinoma cell line derived spheroids can attach onto endometrial cells and are used as models to study the early stages of implantation. However, human embryos are limited and the use of cancer cell lines for spheroid generation remains sub-optimal for research.
STUDY DESIGN, SIZE, DURATION
Experimental induced differentiation of human embryonic stem cells into trophoblast and characterization of the trophoblast.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Trophoblastic spheroids (BAP-EB) were generated by inducing differentiation of a human embryonic stem cell line, VAL3 cells with bone morphogenic factor-4, A83-01 (a TGF-β inhibitor), and PD173074 (a FGF receptor-3 inhibitor) after embryoid body formation. The expressions of trophoblastic markers and hCG levels were studied by real-time PCR and immunohistochemistry. BAP-EB attachment and invasion assays were performed on different cell lines and primary endometrial cells.
MAIN RESULTS AND THE ROLE OF CHANCE
After 48 h of induced differentiation, the BAP-EB resembled early implanting human embryos in terms of size and morphology. The spheroids derived from embryonic stem cells (VAL3), but not from several other cell lines studied, possessed a blastocoel-like cavity. BAP-EB expressed several markers of trophectoderm of human blastocysts on Day 2 of induced differentiation. In the subsequent days of differentiation, the cells of the spheroids differentiated into trophoblast-like cells expressing trophoblastic markers, though at levels lower than that in the primary trophoblasts or in a choriocarcinoma cell line. On Day 3 of induced differentiation, BAP-EB selectively attached onto endometrial epithelial cells, but not other non-endometrial cell lines or an endometrial cell line that had lost its epithelial character. The attachment rates of BAP-EB was significantly higher on primary endometrial epithelial cells (EEC) taken from 7 days after hCG induction of ovulation (hCG+7 day) when compared with that from hCG+2 day. The spheroids also invaded through Ishikawa cells and the primary endometrial stromal cells in the co-culture.
LIMITATIONS, REASONS FOR CAUTION
The attachment rates of BAP-EB were compared between EEC obtained from Day 2 and Day 7 of the gonadotrophin stimulated cycle, but not the natural cycles.
WIDER IMPLICATIONS OF THE FINDINGS
BAP-EB have the potential to be used as a test for predicting endometrial receptivity in IVF cycles and provide a novel approach to study early human implantation, trophoblastic cell differentiation and trophoblastic invasion into human endometrial cells.
STUDY FUNDING/COMPETING INTEREST(S)
This study was supported in part by a General Research Fund (grant number: 17111414) from the Research Grants Council of Hong Kong. The authors declare no conflicts of interest.
TRIAL REGISTRATION NUMBER
Nil.
The prevalence of type 2 diabetes (T2D) is rapidly increasing across the globe. Fetal exposure to maternal diabetes was correlated with higher prevalence of impaired glucose tolerance and T2D later ...in life. Previous studies showed aberrant DNA methylation patterns in pancreas of T2D patients. However, the underlying mechanisms remained largely unknown. We utilized human embryonic stem cells (hESC) as the in vitro model for studying the effects of hyperglycemia on DNA methylome and early pancreatic differentiation. Culture in hyperglycemic conditions disturbed the pancreatic lineage potential of hESC, leading to the downregulation of expression of pancreatic markers PDX1, NKX6−1 and NKX6−2 after in vitro differentiation. Genome-wide DNA methylome profiling revealed over 2000 differentially methylated CpG sites in hESC cultured in hyperglycemic condition when compared with those in control glucose condition. Gene ontology analysis also revealed that the hypermethylated genes were enriched in cell fate commitment. Among them, NKX6−2 was validated and its hypermethylation status was maintained upon differentiation into pancreatic progenitor cells. We also established mouse ESC lines at both physiological glucose level (PG-mESC) and conventional hyperglycemia glucose level (HG-mESC). Concordantly, DNA methylome analysis revealed the enrichment of hypermethylated genes related to cell differentiation in HG-mESC, including Nkx6−1. Our results suggested that hyperglycemia dysregulated the epigenome at early fetal development, possibly leading to impaired pancreatic development.
Forced-expression of transcription factors can reprogram somatic cells into induced pluripotent stem cells (iPSC). Recent studies show that the reprogramming efficiency can be improved by inclusion ...of small molecules that regulate chromatin modifying enzymes. We report here that sirtuin 1 (SIRT1), a member of the sirtuin family of NAD(+)-dependent protein deacetylases, is involved in iPSC formation. By using an efficient mouse secondary fibroblast reprogramming system with doxycycline (DOX) inducible Yamanaka's transcription factors delivered by piggyBac (PB) transposition (2°F/1B MEF), we show that SIRT1 knockdown decreased while resveratrol (RSV) increased the efficiency of iPSC formation. The treatments were associated with altered acetylated p53 and its downstream Nanog but not p21 expression. The stimulatory effect was also confirmed by SIRT1 over-expression, which stimulated the formation of colonies with induced Nanog and reduced p21 expression. Furthermore, the effects of RSV and SIRT1 knockdown on reprogramming were most pronounced during the initiation phase of reprogramming. MicroRNA-34a is a known regulator of SIRT1. Its inhibitor increased, while its mimics reduced iPSC formation. The stimulatory effect of SIRT1 during reprogramming was also confirmed in the primary MEF. RSV increased while tenovin-6, a small molecule that activates p53 through SIRT1 inhibition, suppressed reprogramming. In conclusion, SIRT1 enhances iPSC generation, in part, through deacetylation of p53, inhibition of p21 and enhancement of Nanog expression.
Pluripotent stem cells (PSCs) hold great promise in cell-based therapy because of their pluripotent property and the ability to proliferate indefinitely. Embryonic stem cells (ESCs) derived from ...inner cell mass (ICM) possess unique cell cycle control with shortened G1 phase. In addition, ESCs have high expression of homologous recombination (HR)-related proteins, which repair double-strand breaks (DSBs) through HR or the non-homologous end joining (NHEJ) pathway. On the other hand, the generation of induced pluripotent stem cells (iPSCs) by forced expression of transcription factors (Oct4, Sox2, Klf4, c-Myc) is accompanied by oxidative stress and DNA damage. The DNA repair mechanism of DSBs is therefore critical in determining the genomic stability and efficiency of iPSCs generation. Maintaining genomic stability in PSCs plays a pivotal role in the proliferation and pluripotency of PSCs. In terms of therapeutic application, genomic stability is the key to reducing the risks of cancer development due to abnormal cell replication. Over the years, we and other groups have identified important regulators of DNA damage response in PSCs, including
,
and
. They function through transcription regulation of downstream targets (
, CDK1) that are involved in cell cycle regulations. Here, we review the fundamental links between the PSC-specific HR process and DNA damage response, with a focus on the roles of
and
on maintaining genomic integrity.
Aberrations of circulating nucleic acid integrity have been observed in cancer patients. However, the clinical significance of such changes has not been completely elucidated. In this study, we ...investigated the plasma DNA integrity in nasopharyngeal carcinoma (NPC) patients and its association with patients' survival after radiotherapy.
Plasma DNA integrity was analyzed for 105 NPC patients before and after curative-intent radiotherapy and for 40 healthy controls. The plasma DNA concentration of each sample was measured by two real-time PCRs targeting the leptin gene. The amplicon sizes of the two assays were 105 and 201 bp. The integrity index was calculated as the ratio of the two concentrations (201 bp/105 bp). More intact circulating DNA would give a higher integrity index.
The plasma DNA integrity index of the NPC patients was significantly higher than that of the healthy controls (median, 0.356 versus 0.238; P < 0.001). After radiotherapy, a reduction in plasma DNA integrity index was observed in 70% NPC patients. Patients with persistent aberrations of plasma DNA integrity had significantly poorer survival probability than those with reduced DNA integrity after treatment (P < 0.001, Kaplan-Meier).
NPC is associated with disturbances in the integrity of circulating cell-free DNA. The persistence of DNA integrity aberrations after radiotherapy is associated with reduced probability of disease-free survival. Therefore, the measurement of plasma DNA integrity may serve as a useful marker for the detection and monitoring of malignant diseases.
Human expanded potential stem cells (hEPSC) have been derived from human embryonic stem cells and induced pluripotent stem cells. Here direct derivation of hEPSC from human pre‐implantation embryos ...is reported. Like the reported hEPSC, the embryo‐derived hEPSC (hEPSC‐em) exhibit a transcriptome similar to morula, comparable differentiation potency, and high genome editing efficiency. Interestingly, the hEPSC‐em show a unique H3 lysine‐4 trimethylation (H3K4me3) open chromatin conformation; they possess a higher proportion of H3K4me3 bound broad domain (>5 kb) than the reported hEPSC, naive, and primed embryonic stem cells. The open conformation is associated with enhanced trophoblast differentiation potency with increased trophoblast gene expression upon induction of differentiation and success in derivation of trophoblast stem cells with bona fide characteristics. Hippo signaling is specifically enriched in the H3K4me3 broad domains of the hEPSC‐. Knockout of the Hippo signaling gene, YAP1 abolishes the ability of the embryo‐derived EPSC to form trophoblast stem cells.
hEPSC‐em show enhanced trophoblast differentiation potency through a unique H3 lysine‐4 trimethylation (H3K4me3) open chromatin conformation. YAP1 is specifically enriched in the H3K4me3 broad domains of the hEPSC‐em. YAP1 knockout hindered trophoblast differentiation through disruption of cell polarity.
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